Molecular Genetics of Mycobacteriophages.

Mycobacteriophages have provided numerous essential tools for mycobacterial genetics, including delivery systems for transposons, reporter genes, and allelic exchange substrates, and components for plasmid vectors and mutagenesis. Their genetically diverse genomes also reveal insights into the broader nature of the phage population and the evolutionary mechanisms that give rise to it. The substantial advances in our understanding of the biology of mycobacteriophages including a large collection of completely sequenced genomes indicates a rich potential for further contributions in tuberculosis genetics and beyond.

Related papers:

title: A Bacteriophage for Mycobacterium smegmatis. ∗
abstract: A bacteriophage speciric for M. smegmatis was isolated from sarnples of compost and soil by specific enrichment with a heavy washed suspension of M. smegmatis. The method of enrichment employed may be useful in the isolation of other phages.

title: Two bacteriophages for Mycobacterium smegmatis.
abstract: rpHE first definite demonstration of a bacteriophage for a mycobacterium was made by Gardner and Weiser (1947). Previously, Steenken (1935 and 1938) had described "spontaneous lysis" in a culture of tubercle bacilli (H37Rv) with the subsequent formation of "resistant" colonies which were unable to induce progressive disease in guinea pigs. Three to four months' incubation was necessary before softening and liquefaction occurred. Filtrates of the lysed material induced a similar change in recent subcultures. The lytic phenomenon was not, however, referred to as a bacteriophage. Gardner and Weiser employed a soil-enrichment procedure using M . smegmatis to inoculate samples of leaf compost. Seven-day cultures of the micro-organism were washed, resuspended in saline, and added to the compost twice weekly. After three weeks, filtrates of the compost were found to contain a phage which was active on the strain of M. smegmatis used as the inoculum, but which was not active on another smegmatis strain nor on a phlei strain on which it was tested. In subsequent experiments it was shown that this phage could be isolated from 2 of 6 samples of enriched compost and 1 sample of soil, but could not be demonstrated in any of 8 samples of unenriched compost, nor in 4 samples of soil. Apparently the enrichment technique employed was necessary for the recovery of phage. Garden soil was the source of a phage isolated by Haudroy and Rosset (1948). By incubating broth with the soil, filtering, and adding the filtrate to broth cultures of M. paratuberculosis and reincubating, they were able to observe an altered turbidity in the cultures. After three or four passages, complete inhibition of growth was obtained.  isolated five phages from earth obtained in the proximity of a manure pile. It had been demonstrated previously that this earth was rich in acid-fast micro-organisms. The phages were recovered by adding sterile distilled water to the soil and filtering. The filtrate was then tested on the micro-organisms which had been isolated from the soil, by spreading the filtrate over plates of each strain. The phages found were named after the microorganisms on which they were isolated, viz., Phagus pellegrini, Phagus phlei, Phagus lacticola, Phagus rabinozvitschi, and Phagus smegmatis. The classification of these phages was based on their activity, morphology as shown by the electron microscope, and their antigenic differences. By applying these methods to the phage of Haudroy and Rosset, the phage of Gardner and Weiser and Phagus smegmatis, Penso and Ortali (1949b)

title: The genetics of mycobacteria and mycobacteriophages - a review.
abstract: The genus Mycobacterium, despite its medical importance, has so far received relatively little attention from bacterial geneticists. Nevertheless examples of the transfer of genes from one strain to another by means of transformation, phage-mediated transduction and direct cellular contact have been reported. The modification of strains by experimental or naturally occurring lysogeny has been studied in some detail and there is evidence that phage may contribute significantly to variation within species. Mycobacteriophages have been the subject of genetic analyses and have proved of value in the study of certain aspects of host-induced phage modification. In addition phages are being used to develop typing systems for use in epidemiological studies. It is evident that much of interest and value awaits discovery by means of genetic analysis of the mycobacteria and their phages and hopefully the next decade or so will bring great advances in this subject.

title: Bacteriophage typing of Mycobacterium tuberculosis cultures from incidents of suspected laboratory cross-contamination.
abstract: Bacteriophage typing was performed on 235 Mycobacterium tuberculosis cultures submitted from 31 laboratories. In each instance, either the attending physician questioned the misdiagnosis of tuberculosis or the laboratory supervisor suspected that laboratory cross-contamination had occurred. Phage typing data confirmed these suspicions. Phage typing is a useful adjunct in the investigation of suspected cross-contamination of laboratory cultures of M. tuberculosis.

title: The usefulness of phage typing Mycobacterium tuberculosis isolates.
abstract: Mycobacteriophage typing of Mycobacterium tuberculosis isolates was used as an epidemiologic aid in investigating the transmission of tuberculosis in community, industrial, and institutional outbreaks. The technique was also useful in other situations, e.g., documenting congenital transmission of infection and distinguishing exogenous reinfection from endogenous reactivation. Additional studies are indicated to further explore the value of phage typing for tracking the transmission of tuberculosis in the community.

title: Bacteriophage typing of strains of Mycobacterium tuberculosis from Nepal.
abstract: One hundred strains of Mycobacterium tuberculosis isolated from cases of pulmonary tuberculosis in Nepalese villagers were typed with the World Health Organization set of bacteriophages. The number of strains in the 3 major phage types A, I, and B were 19, 53, and 28 respectively. This distribution is significantly different from those described in other geographical regions. In particular there was a relatively low incidence of type A strains and, in common with South India, there was a high proportion of type I strains. All the strains in this study were resistant to 5-Furan-2 carbonic acid hydrazine and 34 were highly resistant to isoniazid. Representative strains of the 3 phage types, including 4 isoniazid-resistant type I strains, did not differ in their virulence in the guinea pig; thus the type I strains found in Nepal may not be of the same origin as those of this phage type from India which are usually attenuated in the guinea pig. This study provides further evidence for the existence of geographical differences in the types of tubercle bacilli.

title: Phage typing of mycobacteria using paper discs.
abstract: The suitability of phage-impregnated paper discs for the phage typing of mycobacteria was studied. The relevance of the routine test dilution, the volume of the phage used, the mode of incubation, and the effect of prolonged storage of phage-impregnated paper discs were considered. By using paper discs, each impregnated with one of 5 different mycobacteriophages (BG1, BK1, G37, CRI-3 and LG) that lyse Mycobacterium smegmatis 607B, it was determined that 100 x the routine test dilution in a volume of at least 20 microliter was required for phage lysis. Soaked and dried paper discs produced larger areas of lysis than those with 20-microliter volumes. Soaked discs were found to be stable even after storage for 8 weeks at 4 degrees C.

title: Lysogeny in mycobacteria. I. Conversion of colony morphology, nitrate reductase activity, and tween 80 hydrolysis of Mycobacterium sp. ATCC 607 associated with lysogeny.
abstract: Colony changes from rough to smooth were observed in a rough strain of mycobacterium ATCC 607 after exposure to phages D29 and B4. The colonies surviving after exposure to these two phages were both smooth and lysogenic. Increased nitrate reductase and Tween 80 hydrolytic activities accompanied lysogenization. Loss of lysogeny was accompanied by conversion to the rough colony type and a decrease in nitrate reductase and Tween 80 hydrolytic activities. The rapid loss and gain of these multiple characteristics suggested that the genetic control lies in a plasmid of mycobacteria.

title: Lysogenic mycobacteria: phage variations and changes in host cells.
abstract: Lysogenic conversion of mycobacteria by phage Roy results in reciprocal changes in the phase and in the host cells. The temperate phage released after lysogenization differs from the converting phage in its host range. The lysogenic derivatives show marked changes in their colony morphology and in their sensitivity to phagolysis. Some, but not all, of the lysogenic bacteria have suffered losses in enzymic activity. An attempt is made to distinguish between host-determined and phage-determined variations.

title: The nature and incidence of lysogeny in Mycobacterium fortuitum.
abstract: Ten of 28 strain of Mycobacterium fortuitum (ranae) were found to be associated with bacteriophage; three were pseudolysogenic, one liberated a phage that lysed a sensitive indicator strain, two liberated morphologically complete phages that did not lyse any of the strains used in this study and four liberated morphologically defective phages. The lysogenic and defectively lysogenic strains showed anomalies in cultural, biochemical and antigenic properties and in susceptibility to superinfecting phages. In view of the high frequency of lysogeny found in M. fortuitum, the role of bacteriophage in the variation of properties, including pathogenicity, of mycobacteria of greater clinical importance merits further consideration.

title: Lysogeny associated with mucoid variation in Mycobacterium kansasii.
abstract: Ten of 200 strains of Mycobacterium kansasii were found to produce very mucoid growth on Löwenstein-Jensen medium. By electronmicroscopy these 10 strains were found to be lysogenic, whereas no phage was observed in cultures of 30 non-mucoid strains. The cultural and biochemical properties of the lysogenic strains are compared with those of non-lysogenic strains, and the morphology of the phages is described.

title: Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA
abstract: Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl2, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From 32P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).

title: Infection of Competent Mycobacterium smegmatis with Deoxyribonucleic Acid Extracted from Bacteriophage B1
abstract: A relatively competent state of Mycobacterium smegmatis for infection with deoxyribonucleic acid (DNA) extracted from phage B1 was found in the late log phase of bacterial growth. This state of the culture was used in quantitative studies on the infectivity of the DNA. The buoyant density of B1 DNA was 1.728 g/cc in CsCl, and 1 μg of the DNA produced 84 infective centers, the phage equivalent of which was 1.5 × 10−8. The infectivity was destroyed by catalytic amounts of deoxyribonuclease but not by specific B1 antiserum. Tween 80, which prevents phage adsorption, did not prevent DNA infection. The response of plaque-forming ability to DNA concentration suggested that two or more molecules are required to initiate an infective center. The low efficiency of DNA infection in mycobacteria was considered to be caused by a limiting population of competent cells in the culture employed; in this experiment less than 10−5 of the cells were infected with DNA. A typical cycle of infection was observed, although the latent period was prolonged and the burst size reduced after DNA infection. The transition of B1 DNA infection to deoxyribonuclease insensitivity had a lag period of about 10 min, and increased linearly with a velocity of about 0.24 infective centers per min per μg of DNA. Half of the infective titer was inactivated by heating at 92 C for 15 min. The melting temperature was about 96 C. Species barriers were not crossed by B1 DNA; however, the DNA was infectious for a B1-resistant mutant of the host.

title: Properties of Mycobacteriophage DS6A I. Immunogenicity in Rabbits∗
abstract: The antigenicity of mycobacteriophage DS6A was determined in rabbits. The concentration of antibody produced was low (K values 10 or less). The amount of antibody produced appears to be dependent on the method of immunization. Bacteriophage DS6A appears to be serologically unrelated to mycobacteriophages D29, R1, and Leo by neutralization tests.

title: Phage-induced alteration of enzymic activity in lysogenic Mycobacterium smegmatis strains.
abstract: Changes in the amidase, nitrate reductase and phenolphthalein sulphatase activity of Mycobacterium smegmatis were observed following B1-lysogeny. These changes were more consistent in lysogenic derivatives of strains sn2 and sn14 than in those of strain sn10. Strains sn2 and sn14 appeared to differ from sn 10 only in that the former possessed a functional salicylamidase locus while the latter did not. Emergence of various phenotypes in the same strain and during the same lysogenic event were observed. Multiple sites of phage integration may have accounted for the variation of sn10 (B1) phenotypes.

title: Inhibition by fifampin of mycobacteriophage D29 replication in its drug-resistant host, Mycobacterium smergmatis ATCC 607.
abstract: The study of cell viability during replication of mycobacteriophage D29 in Mycobacterium smegmatis demonstrated that the host cells survived the phage burst. The results of the host survival experiments suggested that the release of the phage was followed by an unknown repair mechanism. The antimicrobial drug rifampin inhibited the phage production at all times during the life cycle of the virus. Because the host bacteria were shown to be naturally resistant to rifampin, the results of the study suggest that the replication of mycobacteriophage D29 requires the new synthesis of a deoxyribonucleic acid-dependent ribonucleic acid polymerase. The eclipse period of D29 was established as 87 minutes.

title: Inactivation of phages DS6A and D29 by acetone extracts of Mycobacterium tuberculosis and Mycobacterium bovis.
abstract: The adsorption rate constant for mycobacteriophage DS6A on clumped cells of Mycobacterium tuberculosis strain H37Rv was found to be 6.33 x 10(-13) ml per min per colony former. Acetone-extracted cells of H37Rv did not absorb phage DS6A. Acetone extracts of cells of H37Rv and of Mycobacterium bovis strain BCG inhibited plaque formation of mycobacteriophage DS6A and of phage D29. Suspensions of these extracts in heart infusion broth with 0.002 M calcium chloride and 1 per cent dioxane required 20 hours of incubation (activation) at 37 degrees C with stirring before they were capable of inactivating phage DS6A, but did not require activation for inhibition of phage D29. Sonication of water mixtures of these extracts for 45 sec yielded suspensions that were highly active against phage DS6A and independent of the 20-hour lag period. Such preparations were also active against phage D29. The extract prepared from cells of BCG was more active than the extract from cells of H37Rv. Water washing of chloroform solutions of each extract did not remove significant amounts of solid material or reduce the phage-inactivating activites of either extract.

title: Chemical and physical properties of mycobacteriophage D29.
abstract: Mycobacteriophage D29 has a head of uniform size (average diameter 65 nm) and regular shape and a tail of variable length. The stability of the bacteriophage is optimal between pH 9 and 10. The virus contain double-stranded DNA and six structural polypeptides, three major and three minor. The molecular weights of these six polypeptides, as determined by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, are 150 000, 138 000, 13 000, 66 000 and 24 000. The virus contains no lipids as shown by (a) the lack of structural changes after inactivation of the bacteriophage with chloroform, (b) the absence of lipids containing [32P]phosphate or [35S]sulfate in labeled virus, and (c) the absence of an electron paramagnetic resonance spectrum in bacteriophage which had been incubated with a nitroxide-containing fatty acid.

title: Methylated nucleic acid bases in Mycobacterium and mycobacteriophage DNA.
abstract: Methylated bases of the DNA of two mycobacteria (Mycobacterium phlei and Mycobacterium smegmatis var. butyricum) and two mycobacteriophages (Phage phlei and Phage butyricum) have been studied. In both the bacterial and the phage DNAs 5-methyl-cytosine and 6-methyl-aminopurine could be detected. Using L-(methyl-H3)-methionine as methyl donor not only the methylated bases of bacterium and phage DNA proved to be radioactive, but also the non-methylated purine residues and thymine. Possible pathways of this phenomenon are discussed.

title: Presence of random single-strand gaps in mycobacteriophage I3 DNA.
abstract: The genomic double-stranded DNA of mycobacteriophage I3, when denatured with alkali, heat, formamide or dimethylsulfoxide, breaks down to heterogeneous-sized single-strand (ss) fragments smaller than the expected intact unit genome length suggesting the presence of random ss interruptions on both the strands. The occurrence of the interruptions at random is also demonstrated by two-dimensional gel electrophoresis of the restriction fragments of I3 DNA. These interruptions have no adverse effect on the phage infectivity or DNA transfectivity. Studies with nuclease BAL 31 and end-labeling analysis confirm the presence of random interruptions. Detailed analysis using T4 DNA ligase, nuclease S1 and DNA polymerase I Klenow fragment revealed that the interruptions are in the form of small gaps rather than single phosphodiester bond breaks. The average length of the gap is about 10 nucleotides long and there are 13 to 14 such gaps per DNA molecule.

title: Bacterial protoplast fusion: Recombination in fused protoplasts of Streptomyces coelicolor
abstract: SummaryNumerous recombinants arose when protoplasts of S. coelicolor were treated with polyethylene glycol and regenerated on non-selective solid medium. In six-factor crosses, recombination frequencies of more than 10% (up to 17%) were routinely observed. This recombination did not require either of the known sex factors, SCP1 and SCP2.The proportion of multiple crossover classes was much higher than amongst recombinants produced by conjugation between mycelia. Analysis of the spatial distribution of crossovers in double and quadruple crossover recombinants showed only a slight tendency for crossovers to occur closer together than randomly on the complete linkage group. This suggests that genomes brought together by protoplast fusion are complete, or nearly so (in conjugation, in contrast, one genome is represented by a comparatively short fragment). Individual colonies arising from fused protoplasts did not contain different parental genomes without recombinants, but recombinants often occurred without parentals. Several recombinant genotypes often occurred in the same colony, showing a segregation of some, only, of the parental alleles. Complementary genotypes, parental or recombinant, did not occur in the same colony. It is postulated that complete genomes of fused protoplasts usually become fragmented and that crossing-over, often repeated, occurs between the fragments, to generate haploid recombinants.Analysis of fusions between protoplasts of four different genotypes indicated that the average number of protoplasts fusing together was low, but nevertheless appreciable numbers of fusions involved three or four genomes. Crossing-over between them produced recombinants inheriting markers from three or four parents.The generation of nearly random populations of recombinants between two or more parent strains by protoplast fusion under the conditions described appears to have simple applications in industrial and academic strain construction.

title: Formation and reversion of Streptomycete protoplasts: cultural condition and morphological study.
abstract: Cultural conditions for forming and stabilizing protoplasts of Streptomyces griseus and S. venezuelae were studied by reference to the number of protoplasts formed, leakage from protoplasts and reversion rate. Effective formation and stabilization of the protoplasts was accomplished by using a hypertonic medium containing 10 mM-MgCl2 and 25 mM-CaCl2. Electron microscopy showed that the protoplasts of S. griseus, when prepared in the above medium, formed vesicles on the cytoplasmic membrane or out of the protoplasts, but when prepared in a medium with 3 mM-MgCl2 and 3 mM-CaCL they provided few such vesicles. The reversion of protoplasts to the normal filamentous state was examined by the growth on various synthetic agar media. A high reversion rate was obtained by incubating the protoplasts on a hypertonic agar medium containing 20 or 50 mM-MgCl2, 50 or 20 mM-CaCl2, 0·44 or 0·22 mM-phosphate and 0·01 % Casamino acids. Combinations of appropriate concentrations of MgCl2 and CaCl2 were significantly effective in the reversion as well as in the stability of protoplasts. The concentration of phosphate should be adjusted to 0·44 or 0·22 mM. Casamino acids enhanced both growth and reversion rates. The reversion of protoplasts of Streptomyces griseus was followed by using phase-contrast microscopy. The protoplasts incubated on medium R1 enlarged to about 40 μ and then generated filamentous hyphae from the periphery.

title: Genetic systems for mycobacteria.
abstract: Publisher Summary This chapter discusses genetic systems for mycobacteria. The ability to perform genetic analyses on bacteria has provided powerful tools and experimental systems to unravel fundamental biological processes. The advances of recombinant DNA technologies have ignited the development of genetic systems for bacteria that are difficult to work with. The genus Mycobacterium contains a set of the most difficult bacterial species to manipulate experimentally. The tuberculosis vaccine strain, bacille Calmette Guerin (BCG) has been used to vaccinate more individuals than any other live bacterial vaccine, yet little is known about mycobacterial gene structure and expression. The recent development of phage, plasmid, and gene replacement systems for the introduction of recombinant DNA into mycobacteria has opened up a new era of research on members of the genus Mycobacterium .

title: Lysogeny and transformation in mycobacteria: stable expression of foreign genes.
abstract: Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes. Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers. First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E. coli and phage capable of lysogenizing the mycobacterial host. These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome. When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E. coli was introduced into M. smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained. Second, to develop a plasmid transformation system in mycobacteria, M. fortuitum/E. coli hybrid plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed. When introduced into M. smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var. Bacille-Calmette-Guérin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants. These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines.

title: Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis
abstract: Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow‐growing, Mycobacterium smegmatis is a fast‐growing, non‐pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 104 to 105 times greater than those of the parent strain, yielding more than 105 transformants per μg of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.

title: Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis.
abstract: Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30 degrees C but not at 37 degrees C (TM4) or 38.5 degrees C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn10(kan) occurred in a site-specific fashion in M. smegmatis whereas Tn5367 transposed apparently randomly in M. phlei, Bacille Calmette-Guérin (BCG), and M. tuberculosis. Sequence analysis of the M. tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M. tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M. phlei, BCG, and M. tuberculosis.

title: Genome organization and characterization of mycobacteriophage Bxb1
abstract: Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow‐growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.

title: Genome structure of mycobacteriophage D29: implications for phage evolution.
abstract: Mycobacteriophage D29 is a lytic phage that infects both fast and slow-growing mycobacterial species. The complete genome sequence of D29 reveals that it is a close relative of the temperate mycobacteriophage L5, whose sequence has been described previously. The overall organization of the D29 genome is similar to that of L5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of D29 to form lysogens. Comparison of the two genomes shows that they are punctuated by a large number of insertions, deletions, and substitutions of genes, consistent with the genetic mosaicism of lambdoid phages.

title: Origins of Highly Mosaic Mycobacteriophage Genomes
abstract: Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.

title: Exploring the Mycobacteriophage Metaproteome: Phage Genomics as an Educational Platform
abstract: Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 “phamilies” of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three—encoding tape-measure proteins, lysins, and minor tail proteins—are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.

title: Bacteriophage genomics.
abstract: The past three years have seen an escalation in the number of sequenced bacteriophage genomes with more than 500 now in the NCBI phage database, representing a more than threefold increase since 2005. These span at least 70 different bacterial hosts, with two-thirds of the sequenced genomes of phages representing only eight bacterial hosts. Three key features emerge from the comparative analysis of these genomes. First, they span a very high degree of genetic diversity, suggesting early evolutionary origins. Second, the genome architectures are mosaic, reflecting an unusually high degree of horizontal genetic exchange in their evolution. Third, phage genomes contain a very high proportion of novel genetic sequences of unknown function, and probably represent the largest reservoir of unexplored genes. With an estimated 10(31) bacterial and archael viruses in the biosphere, our view of the virosphere will draw into sharper focus as further bacteriophage genomes are characterized.

title: Mycobacteriophages: genes and genomes.
abstract: Viruses are powerful tools for investigating and manipulating their hosts, but the enormous size and amazing genetic diversity of the bacteriophage population have emerged as something of a surprise. In light of the evident importance of mycobacteria to human health--especially Mycobacterium tuberculosis, which causes tuberculosis--and the difficulties that have plagued their genetic manipulation, mycobacteriophages are especially appealing subjects for discovery, genomic characterization, and manipulation. With more than 70 complete genome sequences available, the mycobacteriophages have provided a wealth of information on the diversity of phages that infect a common bacterial host, revealed the pervasively mosaic nature of phage genome architectures, and identified a huge number of genes of unknown function. Mycobacteriophages have provided key tools for tuberculosis genetics, and new methods for simple construction of mycobacteriophage recombinants will facilitate postgenomic explorations into mycobacteriophage biology.

title: Complete Genome Sequences of 138 Mycobacteriophages
abstract: Bacteriophages are the most numerous biological entities in the biosphere, and although their genetic diversity is high, it remains ill defined. Mycobacteriophages—the viruses of mycobacterial hosts—provide insights into this diversity as well as tools for manipulating Mycobacterium tuberculosis. We report here the complete genome sequences of 138 new mycobacteriophages, which—together with the 83 mycobacteriophages previously reported—represent the largest collection of phages known to infect a single common host, Mycobacterium smegmatis mc2 155.

title: Molecular Genetics of Mycobacteriophages.
abstract: Mycobacteriophages have provided numerous essential tools for mycobacterial genetics, including delivery systems for transposons, reporter genes, and allelic exchange substrates, and components for plasmid vectors and mutagenesis. Their genetically diverse genomes also reveal insights into the broader nature of the phage population and the evolutionary mechanisms that give rise to it. The substantial advances in our understanding of the biology of mycobacteriophages including a large collection of completely sequenced genomes indicates a rich potential for further contributions in tuberculosis genetics and beyond.

title: Mycobacteriophages and Tuberculosis
abstract: This chapter focuses on a discussion of recent developments in mycobacteriophage research with an emphasis on how these have advanced one’s understanding of tuberculosis. Mycobacteriophages have been isolated from rather exotic locations, such as the zebra pits at the Bronx zoo and the grounds of the tuberculosis hospital in Chennai, but they can be isolated from most soil and compost samples. The overall diversity of the mycobacteriophage population can also be examined by assessing the proportion of gene products that are unique and do not match any other mycobacteriophage genes. It is sometimes stated that the average genome size for double-stranded DNA (dsDNA) tailed phages is about 50 kbp, perhaps based largely on the well characterized and much utilized phage lambda. One of the most evident features of the mycobacteriophage genomes is their pervasive mosaicism, a common feature of phage genomes. This mosaicism is found in other phage genomes but is particularly pervasive in the mycobacteriophages; it is notable in that the mosaic elements are frequently single genes rather than large sets of genes. Examination of mycobacteriophage tape measure protein (Tmp) reveals the presence of small sequence motifs that are related to Mycobacterium tuberculosis proteins. Investigators may be discouraged from using phage-based delivery tools for mycobacterial genetics due to the perceived complexity of constructing the necessary recombinant phages.

title: The secret lives of mycobacteriophages.
abstract: The study of mycobacteriophages provides insights into viral diversity and evolution, as well as the genetics and physiology of their pathogenic hosts. Genomic characterization of 80 mycobacteriophages reveals a high degree of genetic diversity and an especially rich reservoir of interesting genes. These include a vast number of genes of unknown function that do not match known database entries and many genes whose functions can be predicted but which are not typically found as components of phage genomes. Thus many mysteries surround these genomes, such as why the genes are there, what do they do, how are they expressed and regulated, how do they influence the physiology of the host bacterium, and what forces of evolution directed them to their genomic homes? Although the genetic diversity and novelty of these phages is full of intrigue, it is a godsend for the mycobacterial geneticist, presenting an abundantly rich toolbox that can be exploited to devise new and effective ways for understanding the genetics and physiology of human tuberculosis. As the number of sequenced genomes continues to grow, their mysteries continue to thicken, and the time has come to learn more about the secret lives of mycobacteriophages.

title: Bacteriophages as tools for vaccine development.
abstract: The construction of live recombinant bacterial vaccines requires a reasonably sophisticated genetic system for the introduction, stabilization and expression of foreign antigen genes. Bacteriophages offer a rich collection of tools that can be used for vaccine construction, including site-specific integration-proficient vectors, non-antibiotic selectable markers and signals for efficient transcription and translation of foreign genes. We describe the characterization of a temperate phage of the mycobacteria, mycobacteriophage L5, and application of these phage studies for the construction of recombinant BCG vaccines.

title: Comparative genomics of the mycobacteriophages: insights into bacteriophage evolution.
abstract: The recognition of the vast numbers of bacteriophages in the biosphere has prompted a renewal of interest in understanding their morphological and genetic diversity, and elucidating the evolutionary mechanisms that give rise to them. We have approached these questions by isolating and characterizing a collection of mycobacteriophages that infect a common bacterial host, Mycobacterium smegmatis. Comparative genomic analysis of 50 mycobacteriophages shows that they are highly diverse, although not uniformly so, that they are pervasively mosaic with a multitude of single gene modules, and that this mosaicism is generated through illegitimate recombination.

title: Bacteriophages and their genomes.
abstract: Bacteriophages occupy a unique position in biology, representing an absolute majority of all organisms in the biosphere. Because their genomes are relatively small, elucidating the genetic diversity of the phage population, deciphering their origins, and identifying the evolutionary mechanisms that shape the population would seem readily feasible. And yet the pace of phage genome characterization has slowed over the past three years, reflecting in part a need to transition from sequencing known and well-characterized bacteriophages to the isolation and comparative analysis of new isolates. The current state of bacteriophage genomics shows that the genetic diversity of the population is very high, that phages have been actively evolving for billions of years with active engagement of horizontal genetic exchange, and that their genomes are consequently pervasively mosaic in their architectures. But we have barely scratched the surface and the next years of phage genome exploration promise to be especially revealing.

title: Mycobacteriophages: Cornerstones of Mycobacterial Research
abstract: This chapter describes the ways that a systematic use of phages led to the development of novel cloning vectors and ultimately transformation of mycobacteria. In addition, it describes how the detailed characterization of one phage, L5, has provided an abundance of novel insights into and genetic tools for mycobacterial molecular genetics. Lastly, it also describes how the combination of a reporter gene with a mycobacteriophage has breathed new life into rapid assessment of drug susceptibilities in clinical samples of Mycobacterium tuberculosis and is also providing a useful tool for screening novel antituberculosis compounds. Mycobacteriophage L5 is the best characterized of the mycobacteriophages. L5 lysogens of M. smegmatis contain a copy of the L5 prophage integrated site specifically into the bacterial chromosome. The introduction of the BACTEC system for clinical analysis of M. tuberculosis has considerably shortened the time needed for drug susceptibility determination. Luciferase reporter mycobacteriophages have the potential of greatly reducing the time needed for M. tuberculosis analysis in a simple and relatively inexpensive assay. The attractions in using luciferase reporter phages for drug screening are many. First, they can provide a readout of results in as little as 2 h. Second, they can be easily adapted to an automated system that uses microtiter plates. The finding of even a single new antituberculosis drug could have an important impact on the control of tuberculosis, and the authors believe that the search for such a drug warrants efforts to develop luciferase reporter antibiotic screening systems for M. tuberculosis.

title: Mycobacteriophages as versatile tools for genetic manipulation of mycobacteria and development of simple methods for diagnosis of mycobacterial diseases.
abstract: Tuberculosis, caused by Mycobacterium tuberculosis, is responsible for over two million deaths per year worldwide. Due to its long doubling time (18 h), the microbiological detection of M. tuberculosis by conventional methods takes up to one month, unless the number of bacilli in the biological sample is high enough. Thus, drug resistance assessment requires at least one month for obtaining the primary culture and another month to determine its susceptibility to antimycobacterial drugs. Moreover, for a long time, the lack of genetic tools for mycobacteria has been a barrier for undertaking studies aimed at understanding the mechanisms of drug resistance and drug target identification, being all these topics of utmost importance considering the increase in the number of drug-resistant clones and the few therapeutic options available. Mycobacteriophages are promising as a novel source of genetic elements for mycobacteria manipulation, as well as for the development of versatile, simple, fast and cheap methods for drug resistance assessment of M. tuberculosis clinical isolates. We herein describe the background related to the use of mycobacteriophages, with emphasis placed on their utilization for drug resistance analysis in our country.

title: TB: the return of the phage. A review of fifty years of mycobacteriophage research.
abstract: The first mycobacteriophage was isolated in 1947, and since that time over 250 of these viruses have been identified. Phages have made a significant contribution to our knowledge of mycobacteria over the past fifty years, and following the development of typing techniques in the 1960s and 1970s they were widely used in epidemiological studies of tuberculosis. Unfortunately, attempts to use lytic phages therapeutically during tuberculosis infection have so far failed to elicit cure in experimentally infected animals. During the past decade phages have become important in molecular studies of mycobacteria, both in terms of studying phage biology and as tools in recombinant DNA technology, thus facilitating the investigation of mycobacterial pathogenesis. Today their potential as diagnostics reagents is also being realised with the development of exciting new techniques for rapid bacterial detection and drug susceptibility testing. This review outlines the history of these remarkable organisms, from their discovery fifty years ago to the current developments in rapid diagnostic techniques.

title: Mycobacteriophage and their application to disease control
abstract: The resurgence of tuberculosis and emergence of drug resistant disease has stimulated fresh research into mycobacteriophage. Studies are currently underway to develop phage-based tools for therapeutic and diagnostic use. Previous attempts at mycobacteriophage therapy in experimentally infected animals were not successful and alternative strategies of phage delivery that enable killing of intracellular bacteria are required. Replication of mycobacteriophage provides a simple means of detecting viable bacteria and good progress has been made towards the development of new phage-based diagnostic tools. When screening isolates for resistance to the major antituberculosis drug rifampicin phage-based tests have been shown to have high sensitivity. For the diagnosis of pulmonary tuberculosis evaluation studies indicate that current phage tests are not as sensitive as traditional culture methods. Further trials are needed to determine whether they might have a role in the detection of smear negative tuberculosis. A second generation of phage tests are under development following the construction of luciferase reporter phage. Preliminary data suggests they may offer rapid detection of mycobacteria and simple screening for drug resistance. The potential of mycobacteriophage to detect and treat other mycobacterial diseases remains largely unexplored. 2.

title: Mycobacteriophage vector systems.
abstract: Successful application of molecular genetic approaches to the study of mycobacteria necessitates the introduction of recombinant DNA molecules into mycobacterial cells. Efficient methods of introducing DNA into Mycobacterium smegmatis protoplasts have been developed, and the construction of mycobacteriophage recombinant DNA vectors has been initiated. Novel Escherichia coli-Mycobacterium shuttle vectors, termed shuttle phasmids, have been constructed. These vectors were constructed by inserting E. coli cosmids into nonessential regions of mycobacteriophage DNAs. Shuttle phasmids are multifunctional vectors that replicate in E. coli as plasmids and replicate in mycobacteria as phage. The presence of the bacteriophage lambda cos sequences permits the use of the lambda in vitro packaging system for efficient cloning of additional genes into these vectors. Temperate shuttle phasmids have been constructed that can infect and lyse mycobacterial cells or lysogenize mycobacterial cells to stably integrate and express cloned DNA into mycobacterial genomes. Shuttle phasmids can be transduced into a wide variety of mycobacterial species and thus should permit the development of molecular genetic systems for the mycobacteria.

title: Isolation of and studies on bacteriophage active against mycobacteria.
abstract: More than forty bacteriophages lytic for saprophytic mycobacterial strains have been isolated from soil. Seven were isolated without enrichment, one following enrichment with ATCC No. 607 and Mycob...

title: Mycobacterial phages isolated from stool specimens of patients with pulmonary disease.
abstract: Although most of the bacteriophages with activity on mycobacteria have been isolated from soil, many have been obtained from other sources. Piguet (1) isolated phage that lysed saprophytic mycobacteria from the gut of a mosquito larva. Hikawa (2) and Uyeda and Ito (3) found phage that lysed several strains of acidfast bacilli in animal and chicken feces. Several temperate phages have been isolated (4) from lysogenic mycobacteria. The demonstration by Mankiewicz (5) in 1961 that bacteriophage active on mycobacteria could be isolated from stool specimens of tuberculous and nontuberculous patients opened up a new source of phages for study. Unsuccessful attempts to isolate phage from stool specimens had been made previously by Hnatko (6) and others. In view of Mankiewicz's success there appeared to be possibilities of isolating, from stool specimens of patients producing sputum containing unclassified mycobacteria, strains of phage that might possess more or less specific lytic action on the bacteria of these groups. Examination of 17 stool specimens from patients with pulmonary disease has yielded five phages with lytic action on mycobacteria. Some preliminary studies on these phages are recorded in this paper.

title: Induction of bacteriophage from members of the Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum serocomplex.
abstract: Bacteriophages have been induced from strains in the Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum serocomplex by exposure of cultures to UV light or treatment with mitomycin C. One-sixth of the strains examined, representing all but one of the 31 authenticated serotypes, were found to possess phages lytic for a Mycobacterium smegmatis indicator strain. Four single-plaque isolated phages, TM4, TM9, TM10 and TM20, were purified and shown to have a similar morphology on electron micrographs. They had an isometric head of diameter 50-58 nm and a flexible non-contractile tail about 170 nm in length with a terminal bulb. All had an identical buoyant density in CsCl of 1.521 g cm-3 and extreme sensitivity to chloroform. The induced phages differed in host range and possessed the unique ability to lyse other members of the serocomplex. Interest in these phages centres on a possible role in mediating genetic interrelationships between members of the serocomplex.

title: The First Structure of a Mycobacteriophage, the Mycobacterium abscessus subsp. bolletii Phage Araucaria
abstract: The unique characteristics of the waxy mycobacterial cell wall raise questions about specific structural features of their bacteriophages. No structure of any mycobacteriophage is available, although ∼3,500 have been described to date. To fill this gap, we embarked in a genomic and structural study of a bacteriophage from Mycobacterium abscessus subsp. bolletii, a member of the Mycobacterium abscessus group. This opportunistic pathogen is responsible for respiratory tract infections in patients with lung disorders, particularly cystic fibrosis. M. abscessus subsp. bolletii was isolated from respiratory tract specimens, and bacteriophages were observed in the cultures. We report here the genome annotation and characterization of the M. abscessus subsp. bolletii prophage Araucaria, as well as the first single-particle electron microscopy reconstruction of the whole virion. Araucaria belongs to Siphoviridae and possesses a 64-kb genome containing 89 open reading frames (ORFs), among which 27 could be annotated with certainty. Although its capsid and connector share close similarity with those of several phages from Gram-negative (Gram−) or Gram+ bacteria, its most distinctive characteristic is the helical tail decorated by radial spikes, possibly host adhesion devices, according to which the phage name was chosen. Its host adsorption device, at the tail tip, assembles features observed in phages binding to protein receptors, such as phage SPP1. All together, these results suggest that Araucaria may infect its mycobacterial host using a mechanism involving adhesion to cell wall saccharides and protein, a feature that remains to be further explored.

title: Genome Analysis of Mycobacterium massiliense Strain M172, Which Contains a Putative Mycobacteriophage
abstract: The genome of Mycobacterium massiliense M172, isolated from a human sputum sample, was sequenced using Illumina GA IIX technology and found to contain 5,204,460 bp, including putative genes for virulence and antibiotic resistance as well as a 92-kb genomic region most likely to correspond to a mycobacteriophage.

title: Complete Genome Sequence of the Frog Pathogen Mycobacterium ulcerans Ecovar Liflandii
abstract: In 2004, a previously undiscovered mycobacterium resembling Mycobacterium ulcerans (the agent of Buruli ulcer) was reported in an outbreak of a lethal mycobacteriosis in a laboratory colony of the African clawed frog Xenopus tropicalis. This mycobacterium makes mycolactone and is one of several strains of M. ulcerans-like mycolactone-producing mycobacteria recovered from ectotherms around the world. Here, we describe the complete 6,399,543-bp genome of this frog pathogen (previously unofficially named “Mycobacterium liflandii”), and we show that it has undergone an intermediate degree of reductive evolution between the M. ulcerans Agy99 strain and the fish pathogen Mycobacterium marinum M strain. Like M. ulcerans Agy99, it has the pMUM mycolactone plasmid, over 200 chromosomal copies of the insertion sequence IS2404, and a high proportion of pseudogenes. However, M. liflandii has a larger genome that is closer in length, sequence, and architecture to M. marinum M than to M. ulcerans Agy99, suggesting that the M. ulcerans Agy99 strain has undergone accelerated evolution. Scrutiny of the genes specifically lost suggests that M. liflandii is a tryptophan, tyrosine, and phenylalanine auxotroph. A once-extensive M. marinum-like secondary metabolome has also been diminished through reductive evolution. Our analysis shows that M. liflandii, like M. ulcerans Agy99, has the characteristics of a niche-adapted mycobacterium but also has several distinctive features in important metabolic pathways that suggest that it is responding to different environmental pressures, supporting earlier proposals that it could be considered an M. ulcerans ecotype, hence the name M. ulcerans ecovar Liflandii.

title: Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence
abstract: Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

title: Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis.
abstract: Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.

title: Genome analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of the etiologic agent of tuberculosis
abstract: Global spread and limited genetic variation are hallmarks of M. tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology are restricted to East Africa. Here, we sequenced and analyzed the whole genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4–5× coverage), 454/Roche (13–18× coverage) and/or Illumina DNA sequencing (45–105× coverage). We show that STB isolates are highly recombinogenic and evolutionarily early branching, with larger genome sizes, higher rates of genetic variation, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse infection experiments showed that STB strains are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral STB-like pool of mycobacteria by gain of persistence and virulence mechanisms, and we provide insights into the molecular events involved.

title: Comparative genomic analysis of 60 Mycobacteriophage genomes: genome clustering, gene acquisition, and gene size.
abstract: Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of 60-all infecting a common bacterial host-provides further insight into their diversity and evolution. Of the 60 phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, 5 of which can be further divided into subclusters; 5 genomes do not cluster with other phages. The sequence diversity between genomes within a cluster varies greatly; for example, the 6 genomes in Cluster D share more than 97.5% average nucleotide similarity with one another. In contrast, similarity between the 2 genomes in Cluster I is barely detectable by diagonal plot analysis. In total, 6858 predicted open-reading frames have been grouped into 1523 phamilies (phams) of related sequences, 46% of which possess only a single member. Only 18.8% of the phams have sequence similarity to non-mycobacteriophage database entries, and fewer than 10% of all phams can be assigned functions based on database searching or synteny. Genome clustering facilitates the identification of genes that are in greatest genetic flux and are more likely to have been exchanged horizontally in relatively recent evolutionary time. Although mycobacteriophage genes exhibit a smaller average size than genes of their host (205 residues compared with 315), phage genes in higher flux average only 100 amino acids, suggesting that the primary units of genetic exchange correspond to single protein domains.

title: Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes
abstract: Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution.

title: Classroom-Based Science Research at the Introductory Level: Changes in Career Choices and Attitude
abstract: Our study, focused on classroom-based research at the introductory level and using the Phage Genomics course as the model, shows evidence that first-year students doing research learn the process of science as well as how scientists practice science. A preliminary but notable outcome of our work, which is based on a small sample, is the change in student interest in considering different career choices such as graduate education and science in general. This is particularly notable, as previous research has described research internships as clarifying or confirming rather than changing undergraduates’ decisions to pursue graduate education. We hypothesize that our results differ from previous studies of the impact of engaging in research because the students in our study are still in the early stages of their undergraduate careers. Our work builds upon the classroom-based research movement and should be viewed as encouraging to the Vision and Change in Undergraduate Biology Education movement advocated by the American Association for the Advancement of Science, the National Science Foundation, and other undergraduate education stakeholders.

title: The Genomics Education Partnership: Successful Integration of Research into Laboratory Classes at a Diverse Group of Undergraduate Institutions
abstract: Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning approach after participation in GEP projects. They show knowledge gains on pre- and postcourse quizzes about genes and genomes and in bioinformatic analysis. Participating faculty also report professional gains, increased access to genomics-related technology, and an overall positive experience. We have found that using a genomics research project as the core of a laboratory course is rewarding for both faculty and students.

title: Bioactive Endophytes Warrant Intensified Exploration and Conservation
abstract: A key argument in favor of conserving biodiversity is that as yet undiscovered biodiversity will yield products of great use to humans. However, the link between undiscovered biodiversity and useful products is largely conjectural. Here we provide direct evidence from bioassays of endophytes isolated from tropical plants and bioinformatic analyses that novel biology will indeed yield novel chemistry of potential value.  We isolated and cultured 135 endophytic fungi and bacteria from plants collected in Peru. nrDNAs were compared to samples deposited in GenBank to ascertain the genetic novelty of cultured specimens. Ten endophytes were found to be as much as 15–30% different than any sequence in GenBank. Phylogenetic trees, using the most similar sequences in GenBank, were constructed for each endophyte to measure phylogenetic distance. Assays were also conducted on each cultured endophyte to record bioactivity, of which 65 were found to be bioactive.  The novelty of our contribution is that we have combined bioinformatic analyses that document the diversity found in environmental samples with culturing and bioassays. These results highlight the hidden hyperdiversity of endophytic fungi and the urgent need to explore and conserve hidden microbial diversity. This study also showcases how undergraduate students can obtain data of great scientific significance.

title: Linguistic Analysis of Project Ownership for Undergraduate Research Experiences
abstract: We used computational linguistic and content analyses to explore the concept of project ownership for undergraduate research. We used linguistic analysis of student interview data to develop a quantitative methodology for assessing project ownership and applied this method to measure degrees of project ownership expressed by students in relation to different types of educational research experiences. The results of the study suggest that the design of a research experience significantly influences the degree of project ownership expressed by students when they describe those experiences. The analysis identified both positive and negative aspects of project ownership and provided a working definition for how a student experiences his or her research opportunity. These elements suggest several features that could be incorporated into an undergraduate research experience to foster a student's sense of project ownership.

title: Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage.
abstract: We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage phiC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, phiflu, in the Haemophilus influenzae genome, and two small prophage-like elements, phiRv1 and phiRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.

title: DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics
abstract: Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast‐ and slow‐growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic‘toolbox’for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.

title: Mycobacteriophage Marvin: a New Singleton Phage with an Unusual Genome Organization
abstract: Mycobacteriophages represent a genetically diverse group of viruses that infect mycobacterial hosts. Although more than 80 genomes have been sequenced, these still poorly represent the likely diversity of the broader population of phages that can infect the host, Mycobacterium smegmatis mc2155. We describe here a newly discovered phage, Marvin, which is a singleton phage, having no previously identified close relatives. The 65,100-bp genome contains 107 predicted protein-coding genes arranged in a noncanonical genomic architecture in which a subset of the minor tail protein genes are displaced about 20 kbp from their typical location, situated among nonstructural genes anticipated to be expressed early in lytic growth. Marvin is not temperate, and stable lysogens cannot be recovered from infections, although the presence of a putative xis gene suggests that Marvin could be a relatively recent derivative of a temperate parent. The Marvin genome is replete with novel genes not present in other mycobacteriophage genomes, and although most are of unknown function, the presence of amidoligase and glutamine amidotransferase genes suggests intriguing possibilities for the interactions of Marvin with its mycobacterial hosts.

title: Structural transitions during bacteriophage HK97 head assembly.
abstract: Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.

title: Conserved translational frameshift in dsDNA bacteriophage tail assembly genes.
abstract: A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.

title: A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells
abstract: The predominant morphotype of mycobacteriophage virions has a DNA‐containing capsid attached to a long flexible non‐contractile tail, features characteristic of the Siphoviridae. Within these phage genomes the tape measure protein (tmp) gene can be readily identified due to the well‐established relationship between the length of the gene and the length of the phage tail – because these phages typically have long tails, the tmp gene is usually the largest gene in the genome. Many of these mycobacteriophage Tmp's contain small motifs with sequence similarity to host proteins. One of these motifs (motif 1) corresponds to the Rpf proteins that have lysozyme activity and function to stimulate growth of dormant bacteria, while the others (motifs 2 and 3) are related to proteins of unknown function, although some of the related proteins of the host are predicted to be involved in cell wall catabolism. We show here that motif 3‐containing proteins have peptidoglycan‐hydrolysing activity and that while this activity is not required for phage viability, it facilitates efficient infection and DNA injection into stationary phase cells. Tmp's of mycobacteriophages may thus have acquired these motifs in order to avoid a selective disadvantage that results from changes in peptidoglycan in non‐growing cells.

title: Antimicrobial drug discovery through bacteriophage genomics
abstract: Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

title: Superinfection immunity of mycobacteriophage L5: applications for genetic transformation of mycobacteria
abstract: Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. We show here that the 183‐amino‐acid product of L5 gene 71 confers immunity to L5 superinfection, is required for maintenance of the lysogenic state and contains a helix‐turn‐helix DNA‐binding motif—properties associated with repressors of temperate phages. We have utilized these observations to demonstrate the use of L5 gene 71 as a selectable marker for genetic transformation of the mycobacteria. Significantly, the use of L5 gene 71 as a selectable gene avoids the requirement for antibiotic‐resistance genes providing an important tool for manipulation of the pathogens Mycobacterium tuberculosis and Mycobacterium avium, and for the construction of recombinant BCG vaccines.

title: Identification of three cytotoxic early proteins of mycobacteriophage L5 leading to growth inhibition in Mycobacterium smegmatis.
abstract: Mycobacteriophage L5 is a temperate phage with a broad host range among the fast- and slow-growing mycobacteria such as Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium ulcerans. L5 switches off host protein synthesis during the early stage of lytic growth, as was previously shown by protein expression profiling. Also, lethal genetic elements have been identified in L5 based on the fact that transformants could not be obtained with these genes. Using an inducible mycobacterial shuttle vector, we have identified three ORFs within an early operon of mycobacteriophage L5 which encode gene products (gp) toxic to the host M. smegmatis when expressed. These ORFs, coding for gp77, gp78 and gp79, presumably function as shut-off genes during early stages of phage replication. There is evidence that cell division is affected by one of the proteins (gp79). The transcription of the cytotoxic polypeptides is directed by a promoter situated in ORF83 and transcription control is achieved through the phage repressor gp71, which is shown by co-expression of this protein. The findings presented here should provide useful tools for the molecular genetics of mycobacteria. Further analysis of these and other mycobacteriophage-derived toxic polypeptides, together with the identification of their cellular targets, might provide a tool for the rapid identification of promising drug targets in emerging and re-emerging mycobacterial pathogens.

title: The cytotoxic early protein 77 of mycobacteriophage L5 interacts with MSMEG_3532, an L‐serine dehydratase of Mycobacterium smegmatis
abstract: Mycobacteriophage L5 is a temperate phage infecting a broad range of mycobacterial species. Upon induction of lytic growth, L5 rapidly switches off host protein synthesis. We have recently identified the mycobacteriophage L5 early protein gp77 as a host shut‐off protein that acts growth inhibitory in the mycobacterial host when expressed through the corresponding phage promoter. Here we present data showing that this purified phage protein of unknown function specifically binds to protein MSMEG_3532 when incubated with cell lysates of Mycobacterium smegmatis. This interaction was confirmed by pull‐down assays using purified MSMEG_3532 as bait which co‐purified with gp77. The amino acid sequence of MSMEG_3532 is nearly identical to that of threonine dehydratases, serine dehydratases and an L‐threo‐3‐hydroxyaspartate dehydratase. An enzymatic assay identified this host protein as a pyridoxal‐5′‐phosphate‐dependent L‐serine dehydratase (SdhA) which converts L‐serine to pyruvate. This is the first biochemical characterization of a SdhA derived from mycobacteria. Though the addition of purified gp77 to the established in vitro assay had no influence on SdhA activity at a saturating L‐serine concentration, the specific interaction of phage protein and dehydratase in vivo may well have a role in altering the amino acid pool or the products of amino acid metabolism in favour of phage maturation.

title: Synonymous Codon Usage Analysis of Thirty Two Mycobacteriophage Genomes
abstract: Synonymous codon usage of protein coding genes of thirty two completely sequenced mycobacteriophage genomes was studied using multivariate statistical analysis. One of the major factors influencing codon usage is identified to be compositional bias. Codons ending with either C or G are preferred in highly expressed genes among which C ending codons are highly preferred over G ending codons. A strong negative correlation between effective number of codons (Nc) and GC3s content was also observed, showing that the codon usage was effected by gene nucleotide composition. Translational selection is also identified to play a role in shaping the codon usage operative at the level of translational accuracy. High level of heterogeneity is seen among and between the genomes. Length of genes is also identified to influence the codon usage in 11 out of 32 phage genomes. Mycobacteriophage Cooper is identified to be the highly biased genome with better translation efficiency comparing well with the host specific tRNA genes.

title: Comparison of synonymous codon distribution patterns of bacteriophage and host genomes.
abstract: Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available.

title: Synonymous codon usage analysis of the mycobacteriophage Bxz1 and its plating bacteria M. smegmatis: identification of highly and lowly expressed genes of Bxz1 and the possible function of its tRNA species.
abstract: The extent of codon usage in the protein coding genes of the mycobacteriophage, Bxz1, and its plating bacteria, M. smegmatis, were determined, and it was observed that the codons ending with either G and / or C were predominant in both the organisms. Multivariate statistical analysis showed that in both organisms, the genes were separated along the first major explanatory axis according to their expression levels and their genomic GC content at the synonymous third positions of the codons. The second major explanatory axis differentiates the genes according to their genome type. A comparison of the relative synonymous codon usage between 20 highly- and 20 lowly expressed genes from Bxz1 identified 21 codons, which are statistically over represented in the former group of genes. Further analysis found that the Bxz1- specific tRNA species could recognize 13 out of the 21 over represented synonymous codons, which incorporated 13 amino acid residues preferentially into the highly expressed proteins of Bxz1. In contrast, seven amino acid residues were preferentially incorporated into the lowly expressed proteins by 10 other tRNA species of Bxz1. This analysis predicts for the first time that the Bxz1-specific tRNA species modulates the optimal expression of its proteins during development.

title: Mycobacteriophages BPs, Angel and Halo: comparative genomics reveals a novel class of ultra-small mobile genetic elements.
abstract: Mycobacteriophages BPs, Angel and Halo are closely related viruses isolated from Mycobacterium smegmatis, and possess the smallest known mycobacteriophage genomes, 41,901 bp, 42,289 bp and 41,441 bp, respectively. Comparative genome analysis reveals a novel class of ultra-small mobile genetic elements; BPs and Halo each contain an insertion of the proposed mobile elements MPME1 and MPME2, respectively, at different locations, while Angel contains neither. The close similarity of the genomes provides a comparison of the pre- and post-integration sequences, revealing an unusual 6 bp insertion at one end of the element and no target duplication. Nine additional copies of these mobile elements are identified in a variety of different contexts in other mycobacteriophage genomes. In addition, BPs, Angel and Halo have an unusual lysogeny module in which the repressor and integrase genes are closely linked. The attP site is located within the repressor-coding region, such that prophage formation results in expression of a C-terminally truncated, but active, form of the repressor.

title: Expanding the Definition of Class 3 Inteins and Their Proposed Phage Origin
abstract: Inteins are the protein equivalent of introns. Their protein splicing activity is essential for the host protein's maturation and function. Inteins are grouped into three classes based on sequence signature and splicing mechanism. The sequence signature of the recently characterized class 3 inteins is a noncontiguous Trp-Cys-Thr (WCT) motif and the absence of the standard class 1 Cys1 or Ser1 N-terminal nucleophile. The intein N-terminal Cys1 or Ser1 residue is essential for splicing in class 1 inteins. The mycobacteriophage Catera Gp206, Nocardioides sp. strain JS614 TOPRIM, and Thermobifida fusca YX Tfu2914 inteins have a mixture of class 1 and class 3 motifs. They carry the class 3 Trp-Cys-Thr motif and have the standard class 1 N-terminal Ser1 or Cys1. This study determined which class the mycobacteriophage Catera Gp206 and Nocardioides sp. JS614 TOPRIM inteins belong to based on catalytic mechanism. The mycobacteriophage Catera Gp206 intein (starting with Ser1) is a class 3 intein, and its Ser1 residue is not required for splicing. Based on phylogenetic analysis, we propose that class 3 inteins arose from a single mutated intein that was spread by phage into predominantly helicase genes in various phages and their hosts.

title: Host range of 14 mycobacteriophages in Mycobacterium ulcerans and seven other mycobacteria including Mycobacterium tuberculosis--application for identification and susceptibility testing.
abstract: The host range of well-characterized mycobacteriophages, such as D29 and TM4, has been determined, together with that of more recently isolated mycobacteriophages, in Mycobacterium ulcerans, Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium fortuitum and Mycobacterium chelonae. Here, a set of virulent phages for M. ulcerans, a pathogen with a dramatic increase of incidence over the last decade, is demonstrated. In this work, a mycobacteriophage replication assay was adapted for the identification and rifampicin-susceptibility testing of M. ulcerans. Mycobacteriophages have generated a number of useful tools and enabled insights into mycobacterial genetics. With regard to the neglected pathogen M. ulcerans, the findings presented in this work allow the application of a large range of phage-based vectors and markers. The potential of phage therapy can now be evaluated for this extracellular pathogen.

title: Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles
abstract: Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions – such as virion proteins and repressors – cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non‐essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non‐coding RNA in a lysogen and in late lytic growth, although it is non‐essential for lytic growth and does not alter lysogeny.

title: Defects in glycopeptidolipid biosynthesis confer phage I3 resistance in Mycobacterium smegmatis.
abstract: Mycobacteriophages have played an important role in the development of genetic tools and diagnostics for pathogenic mycobacteria, including Mycobacterium tuberculosis. However, despite the isolation of numerous phages that infect mycobacteria, the mechanisms of mycobacteriophage infection remain poorly understood, and knowledge about phage receptors is minimal. In an effort to identify the receptor for phage I3, we screened a library of Mycobacterium smegmatis transposon mutants for phage-resistant strains. All four phage I3-resistant mutants isolated were found to have transposon insertions in genes located in a cluster involved in the biosynthesis of the cell-wall-associated glycopeptidolipid (GPL), and consequently the mutants did not synthesize GPLs. The loss of GPLs correlated specifically with phage I3 resistance, as all mutants retained sensitivity to two other mycobacteriophages: D29 and Bxz1. In order to define the minimal receptor for phage I3, we then tested the phage sensitivity of previously described GPL-deficient mutants of M. smegmatis that accumulate biosynthesis intermediates of GPLs. The results indicated that, while the removal of most sugar residues from the fatty acyl tetrapeptide (FATP) core of GPL did not affect sensitivity to phage I3, a single methylated rhamnose, transferred by the rhamnosyltransferase Gtf2 to the FATP core, was critical for phage binding.

title: Nature of the Receptor Substance of Mycobacterium smegmatis for D4 Bacteriophage Adsorption
abstract: The acetone-soluble fraction extracted from lyophilized cells of Mycobacterium smegmatis ATCC 607 inhibited D4, a species-specific phage active against M. smegmatis. Evidence is presented indicating that the D4 inhibition was caused by the phage receptor substance(s) contained in this fraction. A fraction eluted from silicic acid with chloroform-methanol (95:5, v/v) showed the strongest inhibition of D4 phage. This fraction contained sugars and amino acids, and its infrared absorption spectrum was practically identical with those of the mycoside C isolated from the other species of mycobacteria. Further fractionation revealed that the active material was a mixture of several closely related peptidoglycolipids all of which showed, more or less, the phage inhibition. One of the compounds was purified and partially characterized; it contains three different amino acids, allo-threonine, alanine, and phenylalanine, at a molar ratio of 1:1:1, and also three different deoxyhexoses, probably 6-deoxytalose, 3,4-di-o-methylrhamnose, and 2,3,4-tri-o-methylrhamnose. A tentative name of “mycoside Csm” is proposed for this substance which possesses a slightly different structure from the known types of mycoside C and is probably specific for the species of M. smegmatis. A fraction extracted from the D4-resistant mutant of M. smegmatis ATCC 607 by acetone and then by chloroform-methanol (95:5, v/v) showed no phage inhibition and had no sugar component. In addition, this fraction contained lysine, serine, and a small amount of both glycine and an unidentified amino acid.

title: A study on the receptor for a mycobacteriophage : phage phlei.
abstract: From Mycobacterium phlei, glycolipid fractions have been isolated which inactivate phage Phlei. On the basis of the characteristics of the inactivation (specificity, kinetics, requirement for Ca++) typical of the phage-host cell system, it was concluded that these fractions contain the receptor sites for phage Phlei ; this conclusion was supported by electron microscopic studies. All the active fractions contain four kinds of components : fatty acids, glycerol, sugars (D-lyxose, 6-0-methyl-D-glucose, and low amounts of glucose and mannose), and water-soluble acids. These acids are isolated by degradation of the receptor fractions as oxalic and pyruvic acids. Variations of the ratio oxalic acid/pyruvic acid according to the mode of degradation and the absence of the peak characteristic of the protons of a pyruvic acid residue in the NMR spectrum, suggest that these acids might arise from the splitting of oxaloacetic acid. A tentative structure of the receptor is proposed, in many monoglycerides are linked through keto-acid to a polysaccharide core.

title: Chemistry of the Lyxose-Containing Mycobacteriophage Receptors of Mycobacterium phlei/Mycobacterium smegmatis†
abstract: Mycobacterium phlei (strain Timothy) (Mycobacterium smegmatis ATCC 19249) is characterized by the presence of a family of alkali-labile glycolipids, reminiscent of the trehalose-containing lipooligosaccharide class of antigens but lacking the nonreducing trehalose core. Through a combination of methylation analyses, 1H and 13C NMR, two-dimensional 1H/1H and 1H/13C NMR, fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and other analytical techniques, these new structures were shown to possess three distinct features. Firstly, they contained the pentose D-lyxose (Lyx), rarely found in biology, but an epimer of D-arabinose, a key component of the mycobacterial cell wall arabinogalactan and lipoarabinomannnan. Thus, it was apparent that these glycolipids are the same as those described by Bisso et al. and attributed with phage receptor properties [Bisso, G., Castelnuovo, G., Nardelli, M.-G., Orefici, G., Arancia, G., Laneelle, G., Asselineau, C., & Asselineau, J. (1976) Biochemie 58, 87-97]. Secondly, the complex oligosaccharides within the glycolipids contain the repeating units Lyxn(6-O-CH3-Glc)m and Lyxn(6-O-CH3-Glc)mMan1, where n+m equal to approximately 16 glycosyl residues. Thirdly, the M. phlei glycolipids were found to be heavily O-acylated, such that every D-Lyx residue invariably possesses an acyl function at position -2 and, in some instances, at both positions -2 and -4. The chemical characterization of these glycolipids, not feasible 20 years ago, clearly demonstrates that they are distinct from the type- and species-specific glycopeptidolipids, lipooligosaccharides, phenolic glycolipids, and the genus-specific phosphatidylinositol-based lipoglycans of mycobacteria. This present and previous studies begin to define the precise structural requirements responsible for the attachment of mycobacteriophage to the host cell wall.

title: CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes
abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.

title: The phage abortive infection system, ToxIN, functions as a protein–RNA toxin–antitoxin pair
abstract: Various mechanisms exist that enable bacteria to resist bacteriophage infection. Resistance strategies include the abortive infection (Abi) systems, which promote cell death and limit phage replication within a bacterial population. A highly effective 2-gene Abi system from the phytopathogen Erwinia carotovora subspecies atroseptica, designated ToxIN, is described. The ToxIN Abi system also functions as a toxin–antitoxin (TA) pair, with ToxN inhibiting bacterial growth and the tandemly repeated ToxI RNA antitoxin counteracting the toxicity. TA modules are currently divided into 2 classes, protein and RNA antisense. We provide evidence that ToxIN defines an entirely new TA class that functions via a novel protein-RNA mechanism, with analogous systems present in diverse bacteria. Despite the debated role of TA systems, we demonstrate that ToxIN provides viral resistance in a range of bacterial genera against multiple phages. This is the first demonstration of a novel mechanistic class of TA systems and of an Abi system functioning in different bacterial genera, both with implications for the dynamics of phage-bacterial interactions.

title: Comparative genomic structures of Mycobacterium CRISPR‐Cas
abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) are inheritable genetic elements of many archaea and bacteria, conferring acquired immunity against invading nucleic acids. CRISPR might be indicative of the bacterial niche adaptation and evolutionary. Mycobacterium is an important genus occupying diverse niches with profound medical and environmental significance. To present a comparative genomic landscape of the Mycobacterium CRISPR, the feature of mycobacterium CRISPR structures with sequenced complete genomes were bioinformatically analyzed. The results show that CRISPR structures can be found among 14 mycobacteria, and all loci are chromosomally located. Long CRISPRs present in three species, namely M. tuberculosis, M. bovis, and M. avium. Integrated CRISPR‐Cas system can only be found in M. tuberculosis and M. bovis, with highly conserved repeat sequences, very short leaders, and promoterless. M. tuberculosis and M. bovis repeat sequences cannot form stable RNA secondary structure, consistent with a Cas6‐binding sequence. M. avium repeat sequences can form classical stem‐loop structure. A three‐step model of M. tuberculosis CRISPR‐Cas system action was put forward based on the composition and function of cas genes cluster. M. tuberculosis and M. bovis CRISPRs might interfere with the invading nucleic acids, but have somehow lost the capacity to incorporate new spacers and  with corresponding mycobacteriophages.  2464–2473, 2012.

title: Isolation of the Genome Sequence Strain Mycobacterium avium 104 from Multiple Patients over a 17-Year Period
abstract: The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated from an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genotypic identity to strain 104. This process was facilitated by the use of a novel two-step approach. In the first step, all 208 strains in the sample were subjected to a high-throughput, large sequence polymorphism (LSP)-based genotyping test, in which DNA from each strain was tested by PCR for the presence or absence of 4 hypervariable genomic regions. Nineteen isolates exhibited an LSP type that resembled that of strain 104. This subset of 19 isolates was then subjected to high-resolution repetitive sequence-based PCR typing, which identified 10 isolates within the subset that were genotypically identical to strain 104. These isolates came from 10 different patients at 5 clinical sites in the western United States, and they were isolated over a 17-year time span. Therefore, the sequenced genome of M. avium strain 104 has been associated with disease in multiple patients in the western United States. Although M. avium is known for its genetic plasticity, these observations also show that strains of the pathogen can be genotypically stable over extended time periods.

title: Characterization of a Mycobacterium smegmatis gene that confers resistance to phages L5 and D29 when overexpressed
abstract: Bacteriophage infection requires a specific interaction with the outer surface of a bacterial host followed by interaction with the cell membrane and phage DNA injection. Phages of the mycobacteria encounter a cell wall that is rich in unusual lipid‐ and sugar‐containing components which form a formidable barrier that must be passed to gain access to the membrane. We describe here a gene of Mycobacterium smegmatis that confers resistance to mycobacteriophages L5 and D29. The phage‐resistance phenotype results not from mutation but from elevated expression of a wild‐type gene. It appears that the product of this multicopy phage‐resistance (mpr) gene may alter the structure of the host cell wall or membrane, thereby inhibiting productive phage DNA injection.

title: In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria.
abstract: mariner family transposons are widespread among eukaryotic organisms. These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence of added cofactors. Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo. We have developed simple transposition systems by using minitransposons, made up of short inverted repeats flanking antibiotic resistance markers. These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter. We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene. With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E. coli and Mycobacterium smegmatis. mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.

title: Bacteriophage lysis: mechanism and regulation.
abstract: Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E.

title: The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes
abstract: Bacteriophage λ has four adjacent genes –S, R, Rz and Rz1– dedicated to host cell lysis. While S, encoding the holin and antiholin, and R, encoding the endolysin, have been intensively studied, the products of Rz and Rz1 have not been characterized at either the structural or functional levels. Rz1 is an outer membrane lipoprotein and our results indicate that Rz is a type II signal anchor protein. Here we present evidence that an Rz–Rz1 complex that spans the periplasm carries out the final step in the process of host lysis. These results are discussed in terms of a model where endolysin‐mediated degradation of the cell wall is a prerequisite for conformational changes in the Rz–Rz1 complex leading to the juxtaposition and fusion of the IM and OM. Fusion of the two membranes removes the last physical barrier to efficient release of progeny virions.

title: The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity.
abstract: dsDNA bacteriophages use the dual system endolysin-holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His(6)-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His(6)-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C(4)-C(18)), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C(16) and C(18)). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 degrees C and pH 7.5-8.0. Activity was increased in the presence of Ca(2+) and Mn(2+). To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.

title: Mycobacteriophage Ms6 LysB specifically targets the outer membrane of Mycobacterium smegmatis.
abstract: LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6'-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.

title: Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase
abstract: Mycobacteriophages encounter a unique problem among phages of Gram‐positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid‐rich outer membrane covalently attached to the arabinogalactan–peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an α/β hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four‐helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles ΔlysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan–peptidoglycan layer.

title: Mycobacteriophage Endolysins: Diverse and Modular Enzymes with Multiple Catalytic Activities
abstract: The mycobacterial cell wall presents significant challenges to mycobacteriophages – viruses that infect mycobacterial hosts – because of its unusual structure containing a mycolic acid-rich mycobacterial outer membrane attached to an arabinogalactan layer that is in turn linked to the peptidoglycan. Although little is known about how mycobacteriophages circumvent these barriers during the process of infection, destroying it for lysis at the end of their lytic cycles requires an unusual set of functions. These include Lysin B proteins that cleave the linkage of mycolic acids to the arabinogalactan layer, chaperones required for endolysin delivery to peptidoglycan, holins that regulate lysis timing, and the endolysins (Lysin As) that hydrolyze peptidoglycan. Because mycobacterial peptidoglycan contains atypical features including 3→3 interpeptide linkages, it is not surprising that the mycobacteriophage endolysins also have non-canonical features. We present here a bioinformatic dissection of these lysins and show that they are highly diverse and extensively modular, with an impressive number of domain organizations. Most contain three domains with a novel N-terminal predicted peptidase, a centrally located amidase, muramidase, or transglycosylase, and a C-terminal putative cell wall binding domain.

title: The mycobacteriophage Ms6 encodes a chaperone‐like protein involved in the endolysin delivery to the peptidoglycan
abstract: Like most double‐stranded (ds) DNA phages, mycobacteriophage Ms6 uses the holin‐endolysin system to achieve lysis of its host. In addition to endolysin (lysA) and holin (hol) genes, Ms6 encodes three accessory lysis proteins. In this study we investigated the lysis function of Gp1, which is encoded by the gp1 gene that lies immediately upstream of lysA. Escherichia coli lysis was observed after coexpression of LysA and Gp1 in the absence of Ms6 holin. Gp1 does not belong to the holin class of proteins, and we provide evidence that it shares several characteristics with molecular chaperones. We show that Gp1 interacts with LysA, and that this interaction is necessary for LysA delivery to its target. In addition, PhoA fusions showed that, in Mycobacterium smegmatis, LysA is exported to the extracytoplasmic environment in the presence of Gp1. We also show that Gp1 is necessary for efficient M. smegmatis lysis, as Ms6 gp1 deletion results in host lysis defects. We propose that delivery of Ms6 endolysin to the murein layer is assisted by Gp1, a chaperone‐like protein, in a holin‐independent manner.

title: The Endolysin-Binding Domain Encompasses the N-Terminal Region of the Mycobacteriophage Ms6 Gp1 Chaperone
abstract: The intermolecular interactions of the mycobacteriophage Ms6 secretion chaperone with endolysin were characterized. The 384-amino-acid lysin (lysin384)-binding domain was found to encompass the N-terminal region of Gp1, which is also essential for a lysis phenotype in Escherichia coli. In addition, a GXXXG-like motif involved in Gp1 homo-oligomerization was identified within the C-terminal region.

title: The Spanin Complex Is Essential for Lambda Lysis
abstract: Phage lysis is a ubiquitous biological process, the most frequent cytocidal event in the biosphere. Lysis of Gram-negative hosts has been shown to require holins and endolysins, which attack the cytoplasmic membrane and peptidoglycan, respectively. Recently, a third class of lysis proteins, the spanins, was identified. The first spanins to be characterized were λ Rz and Rz1, an integral cytoplasmic membrane protein and an outer membrane lipoprotein, respectively. Previous work has shown that Rz and Rz1 form complexes that span the entire periplasm. Phase-contrast video microscopy was used to record the morphological changes involved in the lysis of induced λ lysogens carrying prophages with either the λ canonical holin-endolysin system or the phage 21 pinholin-signal anchor release (SAR) endolysin system. In the former, rod morphology persisted until the instant of an explosive polar rupture, immediately emptying the cell of its contents. In contrast, in pinholin-SAR endolysin lysis, the cell began to shorten and thicken uniformly, with the resultant rounded cell finally bursting. In both cases, lysis failed to occur in inductions of isogenic prophages carrying null mutations in the spanin genes. In both systems, instead of an envelope rupture, the induced cells were converted from a rod shape to a spherical form. A functional GFPΦRz chimera was shown to exhibit a punctate distribution when coexpressed with Rz1, despite the absence of endolysin function. A model is proposed in which the spanins carry out the essential step of disrupting the outer membrane, in a manner regulated by the state of the peptidoglycan layer.

title: Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6
abstract: The mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) bacteriophage which, in addition to the predicted endolysin (LysA)-holin (Gp4) lysis system, encodes three additional proteins within its lysis module: Gp1, LysB, and Gp5. Ms6 Gp4 was previously described as a class II holin-like protein. By analysis of the amino acid sequence of Gp4, an N-terminal signal-arrest-release (SAR) domain was identified, followed by a typical transmembrane domain (TMD), features which have previously been observed for pinholins. A second putative holin gene (gp5) encoding a protein with a predicted single TMD at the N-terminal region was identified at the end of the Ms6 lytic operon. Neither the putative class II holin nor the single TMD polypeptide could trigger lysis in pairwise combinations with the endolysin LysA in Escherichia coli. One-step growth curves and single-burst-size experiments of different Ms6 derivatives with deletions in different regions of the lysis operon demonstrated that the gene products of gp4 and gp5, although nonessential for phage viability, appear to play a role in controlling the timing of lysis: an Ms6 mutant with a deletion of gp4 (Ms6Δgp4) caused slightly accelerated lysis, whereas an Ms6Δgp5 deletion mutant delayed lysis, which is consistent with holin function. Additionally, cross-linking experiments showed that Ms6 Gp4 and Gp5 oligomerize and that both proteins interact. Our results suggest that in Ms6 infection, the correct and programmed timing of lysis is achieved by the combined action of Gp4 and Gp5.

title: Analysis of Novel Mycobacteriophages Indicates the Existence of Different Strategies for Phage Inheritance in Mycobacteria
abstract: Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100–400 nm, genome size in the 50–70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.

title: Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin.
abstract: Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified.

title: Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria
abstract: Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP–int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a tRNALys gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions.

title: The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNA(Ala) gene of Mycobacterium spp.
abstract: Genetic determinants of the temperate mycobacteriophage Ms6 required for chromosomal integration were identified. DNA sequence analysis of an attP-containing fragment revealed an ORF encoding a protein of 372 amino acid residues with a C-terminus similar to other conserved C-terminal regions typical of the phage integrase family. Comparison of the sequences of attP, attB and bacteria-prophage junctions attL and attR showed a 26 bp common core sequence, where recombination takes place, near the 5' end of the integrase gene. Nucleotide sequence analysis of the attB chromosomal region showed that the core site overlaps the 3' end of the tRNA(Ala) gene. An integration-proficient plasmid vector was constructed and efficiently inserted at the tRNA(Ala) gene of Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra. It was demonstrated that Ms6 and D29 integrative systems can be used in conjunction for inserting genes at multiple loci. The site-specific integration system of mycobacteriophage Ms6 is a new tool for mycobacterial genetic analysis and is poorly related to those of the L5 bacteriophage family.

title: Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene
abstract: Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3′ end of the groEL1 gene such that integration results in alteration of the C‐terminal 21 amino acid residues. An integration‐proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1‐integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high‐energy cofactors, divalent metals, DNA supercoiling or additional proteins.

title: GroEL1: A Dedicated Chaperone Involved in Mycolic Acid Biosynthesis during Biofilm Formation in Mycobacteria
abstract: Mycobacteria are unusual in encoding two GroEL paralogs, GroEL1 and GroEL2. GroEL2 is essential--presumably providing the housekeeping chaperone functions--while groEL1 is nonessential, contains the attB site for phage Bxb1 integration, and encodes a putative chaperone with unusual structural features. Inactivation of the Mycobacterium smegmatis groEL1 gene by phage Bxb1 integration allows normal planktonic growth but prevents the formation of mature biofilms. GroEL1 modulates synthesis of mycolates--long-chain fatty acid components of the mycobacterial cell wall--specifically during biofilm formation and physically associates with KasA, a key component of the type II Fatty Acid Synthase involved in mycolic acid synthesis. Biofilm formation is associated with elevated synthesis of short-chain (C56-C68) fatty acids, and strains with altered mycolate profiles--including an InhA mutant resistant to the antituberculosis drug isoniazid and a strain overexpressing KasA--are defective in biofilm formation.

title: Enzymatic Hydrolysis of Trehalose Dimycolate Releases Free Mycolic Acids during Mycobacterial Growth in Biofilms*
abstract: Mycobacterial species, like other microbes, spontaneously form multicellular drug-tolerant biofilms when grown in vitro in detergent-free liquid media. The structure of Mycobacterium tuberculosis biofilms is formed through genetically programmed pathways and is built upon a large abundance of novel extracellular free mycolic acids (FM), although the mechanism of FM synthesis remained unclear. Here we show that the FM in Mycobacterium smegmatis biofilms is produced through the enzymatic release from constitutively present mycolyl derivatives. One of the precursors for FM is newly synthesized trehalose dimycolate (TDM), which is cleaved by a novel TDM-specific serine esterase, Msmeg_1529. Disruption of Msmeg_1529 leads to undetectable hydrolytic activity, reduced levels of FM in the mutant, and retarded biofilm growth. Furthermore, enzymatic hydrolysis of TDM remains conserved in M. tuberculosis, suggesting the presence of a TDM-specific esterase in this pathogen. Overall, this study provides the first evidence for an enzymatic release of free mycolic acids from cell envelope mycolates during mycobacterial growth.

title: Characterization of the mycobacteriophage L5 attachment site, attP.
abstract: Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.

title: Positions of strand exchange in mycobacteriophage L5 integration and characterization of the attB site
abstract: Mycobacteriophage L5 integrates into the genome of Mycobacterium smegmatis via site-specific recombination between the phage attP site and the bacterial attB site. These two sites have a 43-bp common core sequence within which strand exchange occurs and which overlaps a tRNAGly gene at attB. We show here that a 29-bp segment of DNA is necessary and sufficient for attB function and identify the positions of strand exchange.

title: Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins.
abstract: Similarity between the DNA substrates and products of integrase-mediated site-specific recombination reactions results in a single recombinase enzyme being able to catalyze both the integration and excision reactions. The control of directionality in these reactions is achieved through a class of small accessory factors that favor one reaction while interfering with the other. These proteins, which we will refer to collectively as recombination directionality factors (RDFs), play architectural roles in reactions catalyzed by their cognate recombinases and have been identified in conjunction with both tyrosine and serine integrases. Previously identified RDFs are typically small, basic and have diverse amino acid sequences. A subset of RDFs, the cox genes, also function as transcriptional regulators. We present here a compilation of all the known RDF proteins as well as those identified through database mining that we predict to be involved in conferring recombination directionality. Analysis of this group of proteins shows that they can be grouped into distinct sub-groups based on their sequence similarities and that they are likely to have arisen from several independent evolutionary lineages. This compilation will prove useful in recognizing new proteins that confer directionality upon site-specific recombination reactions encoded by plasmids, transposons, phages and prophages.

title: Identification and characterization of mycobacteriophage L5 excisionase
abstract: The well‐characterized mycobacteriophage L5 forms stable lysogens in Mycobacterium smegmatis. Establishment of lysogeny involves integration of the phage genome into the chromosome of its mycobacterial hosts through an integrase‐mediated site‐specific recombination event. As L5 lysogens spontaneously generate free phage particles, prophage excision must also occur, although an L5 excisionase gene had not been identified. We show here that L5 gene 36 encodes the phage excisionase and is a small, heat‐stable 56‐amino‐acid protein that strongly stimulates excisive recombination both in vivo and in vitro. The ability to manipulate the highly directional phage integration and excision reactions will provide powerful tools for the introduction, curing and recovery of foreign genes in recombinant mycobacterial strains.

title: Control of directionality in L5 integrase-mediated site-specific recombination.
abstract: Mycobacteriophage L5 is a temperate phage that forms lysogens in Mycobacterium smegmatis. These lysogens carry an integrated L5 prophage inserted at a specific chromosomal location and undergo subsequent excision during induction of lytic growth. Both the integrative and excisive site-specific recombination events are catalyzed by the phage-encoded tyrosine integrase (Int-L5) and require the host-encoded protein, mIHF. The directionality of these recombination events is determined by a second phage-encoded protein, Excise, the product of gene 36 (Xis-L5); integration occurs efficiently in the absence of Xis-L5 while excision is dependent upon it. We show here that Xis-L5 binds to attR DNA, introduces a DNA bend, and facilitates the formation of an intasome-R complex. This complex, which requires mIHF, Xis-L5 and Int-L5, readily recombines with a second intasome formed by Int-L5, mIHF and attL DNA (intasome-L) to generate the attP and attB products of excision. Xis-L5 also strongly inhibits Int-L5-mediated integrative recombination but does not prevent either the protein-DNA interactions that form the attP intasome (intasome-P) or the capture of attB, but acts later in the reaction presumably by preventing the formation of a recombinagenic synaptic intermediate. The mechanism of action of Xis-L5 appears to be purely architectural, influencing the assembly of protein-DNA structures solely through its DNA-binding and DNA-bending properties.

title: A novel host factor for integration of mycobacteriophage L5.
abstract: Bacterial integration host factors (IHFs) play central roles in the cellular processes of recombination, DNA replication, transcription, and bacterial pathogenesis. We describe here a novel mycobacterial IHF (mIHF) of Mycobacterium smegmatis and Mycobacterium tuberculosis that stimulates integration of mycobacteriophage L5. mIHF is the product of a single gene and is unrelated at the sequence level to other integration host factors. By itself, mIHF does not bind preferentially to attP DNA, although it significantly alters the pattern of integrase (Int) binding, promoting the formation of specific integrase-mIHF-attP intasome complexes.

title: Protein-DNA Complexes in Mycobacteriophage L5 Integrative Recombination
abstract: The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis,Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.

title: The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB.
abstract: Integration of the mycobacteriophage Bxb1 genome into its host chromosome is catalyzed by a serine-integrase, a member of the transposon-resolvase family of site-specific recombinases. These enzymes use a concerted mechanism of strand exchange involving double-stranded cleavages with two-base extensions, and covalent protein-DNA linkages via phosphoserine bonds. In contrast to the resolvase/invertase recombination systems--where there are strict requirements for a specific synaptic complex within which the catalytic potential of the enzyme is activated--synapsis of attP and attB by Bxb1 integrase is completely promiscuous, aligning the sites with equal proclivity in parallel and antiparallel alignments. Moreover, the catalytic potential of Bxb1 integrase is fully active in either alignment. As a consequence, the nonpalindromic central dinucleotide (5'-GT) at the center of attP and attB is the sole determinant of Bxb1 prophage orientation, and a single base pair substitution in the two sites is sufficient to eliminate orientation control.

title: Synapsis in phage Bxb1 integration: selection mechanism for the correct pair of recombination sites.
abstract: Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase-DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase-DNA complexes.

title: Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading
abstract: Bacteriophage-encoded serine-integrases are members of the large family of serine-recombinases and catalyze site-specific integrative recombination between a phage attP site and a bacterial attB site to form an integrated prophage. Prophage excision involves a second site-specific recombination event, in which the sites generated by integration, attL and attR, are used as substrates to regenerate attP and attB. Excision is catalyzed by integrase but also requires a phage-encoded recombination directionality factor (RDF). The Bxb1 recombination sites, attP and attB, are small (<50 bp), different in sequence, and quasisymmetrical, and they give rise to attL- and attR-recombinant products that are asymmetric but similar to each other, each being composed of B- and P-type half-sites. We show here that the determination of correct excision products is a two-step process, with a presynaptic RDF-dependent step that aligns attL and attR in the correct orientation and a postsynaptic step in which the nonpalindromic central dinucleotide confers identity to attL and attR and prevents each from recombining with itself.

title: The Bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage DNA replication.
abstract: Mycobacteriophage Bxb1 encodes a serine-integrase that catalyzes both integrative and excisive site-specific recombination. However, excision requires a second phage-encoded protein, gp47, which serves as a recombination directionality factor (RDF). The viability of a Bxb1 mutant containing an S153A substitution in gp47 that eliminates the RDF activity of Bxb1 gp47 shows that excision is not required for Bxb1 lytic growth. However, the inability to construct a Δ47 deletion mutant of Bxb1 suggests that gp47 provides a second function that is required for lytic growth, although the possibility of an essential cis-acting site cannot be excluded. Characterization of a mutant prophage of mycobacteriophage L5 in which gene 54 - a homologue of Bxb1 gene 47 - is deleted shows that it also is defective in induced lytic growth, and exhibits a strong defect in DNA replication. Bxb1 gp47 and its relatives are also unusual in containing conserved motifs associated with a phosphoesterase function, although we have not been able to show robust phosphoesterase activity of the proteins, and amino acid substitutions with the conserved motifs do not interfere with RDF activity. We therefore propose that Bxb1 gp47 and its relatives provide an important function in phage DNA replication that has been co-opted by the integration machinery of the serine-integrases to control the directionality of recombination.

title: Transcriptional regulation of repressor synthesis in mycobacteriophage L5
abstract: Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. Lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. We show here that there are three promoters located upstream of gene 71 which are active in an L5 lysogen but which do not require any phage‐encoded proteins. In early lytic growth, gene 71 is also transcribed from a promoter, Pleft, located at the right end of the genome and which appears to be a target of gp71 regulation. A model is given for the regulation of L5 life cycles.

title: Integration-dependent bacteriophage immunity provides insights into the evolution of genetic switches.
abstract: Genetic switches are critical components of developmental circuits. Because temperate bacteriophages are vastly abundant and greatly diverse, they are rich resources for understanding the mechanisms and evolution of switches and the molecular control of genetic circuitry. Here, we describe a new class of small, compact, and simple switches that use site-specific recombination as the key decision point. The phage attachment site attP is located within the phage repressor gene such that chromosomal integration results in removal of a C-terminal tag that destabilizes the virally encoded form of the repressor. Integration thus not only confers prophage stability but also is a requirement for lysogenic establishment. The variety of these self-contained integration-dependent immunity systems in different genomic contexts suggests that these represent ancestral states in switch evolution from which more-complex switches have evolved. They also provide a powerful toolkit for building synthetic biological circuits.

title: L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria
abstract: Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug‐sensitive and drug‐resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.

title: Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA
abstract: A mycobacteriophage Ms6 strong promoter region (Plys) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem σ70-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region Plys drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that β-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.

title: Insights into the function of the WhiB‐like protein of mycobacteriophage TM4 – a transcriptional inhibitor of WhiB2
abstract: WhiB‐like proteins of actinomycetes are known to co‐ordinate iron‐sulfur (Fe‐S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB‐like genes in these viruses. Here we focussed on the WhiB‐like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co‐ordinating a Fe‐S cluster. The UV‐visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe‐2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA‐levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo‐WhiB2 of Mycobacterium tuberculosis and apo‐WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe‐S containing transcriptional regulators with DNA‐binding capability.

title: Transcriptional silencing by the mycobacteriophage L5 repressor
abstract: The success of a temperate bacteriophage is dependent upon its ability to completely shut down expression of its lytic genes during lysogenic growth. Mycobacteriophage L5 accomplishes this by an atypical phage repressor, gp71, which binds to multiple asymmetric DNA sites. L5 gp71 regulates transcription initiation at an early lytic promoter, Pleft, but also affects downstream gene expression at 'stoperator’ sites in the phage genome. The L5 genome is replete with stoperator sites located within short intergenic spaces in both the early and late lytic operons and oriented specifically with respect to transcription. Binding of gp71 to these sites results in a strong orientation‐dependent polar effect on downstream gene expression and global silencing of prophage gene expression.

title: Antagonistic effects Na+ and Mg2+ on the structure, function, and stability of mycobacteriophage L1 repressor.
abstract: Temperate mycobacteriophage L1 encodes an unusual repressor (CI) for regulating its lytic-lysogenic switching and, in contrast to the repressors of most temperate phages, it binds to multiple asymmetric operator DNAs. Here, ions like Na(+), Cl(-), and acetate(-) ions were demonstrated to facilitate the optimal binding of CI to cognate operator DNA, whereas K(+), Li(+), NH4(+), Mg(2+), carbonate(2-), and citrate(3-) ions significantly affected its operator binding activity. Of these ions, Mg(2+) unfolded CI most severely at room temperature and, compared to Mg(2+), Na(+) provided improved thermal stability to CI. Furthermore, the intrinsic tryptophan fluorescence of CI was changed notably upon replacing Na(+) with Mg(2+) and these opposing effects of Mg(2+) and Na(+) were also noticed in their actions on the C-terminal fragment (CTD) of CI. Taken together, Na(+) appeared to be more appropriate than Mg(2+) for maintaining the biologically active conformation of CI needed for its optimal binding to operator DNA.

title: Regions and residues of an asymmetric operator DNA interacting with the monomeric repressor of temperate mycobacteriophage L1.
abstract: Previously, the repressor protein of mycobacteriophage L1 bound to two operator DNAs with dissimilar affinity. Surprisingly, the putative operator consensus sequence, 5'GGTGGa/cTGTCAAG, lacks the dyad symmetry reported for the repressor binding operators of lambda and related phages. To gain insight into the structure of the L1 repressor-asymmetric operator DNA complex, we have performed various in vitro experiments. A dimethyl sulfate protection assay revealed that five guanine bases, mostly distributed in the two adjacent major grooves of the 13 bp operator DNA helix, participate in repressor binding. Hydroxyl radical footprinting demonstrated that interaction between the repressor and operator DNA is asymmetric in nature and occurs primarily through one face of the DNA helix. Genetic studies not only confirmed the results of the dimethyl sulfate protection assay but also indicated that other bases in the 13 bp operator DNA are critical for repressor binding. Interestingly, repressor that weakly induced bending in the asymmetric operator DNA interacted with this operator as a monomer. The tertiary structure of the L1 repressor-operator DNA complex therefore appears to be distinct from those of the lambdoid phages even though the number of repressor molecules per operator site closely matched that of the lambda phage system.

title: Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity
abstract: BackgroundLysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor.ResultsUsing highly purified repressor (CI) of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to O64 and OLtypes of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C.ConclusionRepressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.

title: Effects of physical, ionic, and structural factors on the binding of repressor of mycobacteriophage L1 to its cognate operator DNA.
abstract: To determine the factors influencing the binding of L1 repressor to its cognate operator DNA, several gel shift as well as bioinformatic analyses have been carried out. The data show that time, temperature, salt, and pH each greatly affect the binding. In order to achieve optimum operator binding of L1 repressor in Tris buffer, the minimum requirements of time, temperature, salt, and pH were estimated to be 1 min, 32 degrees C, NaCl (50 mM), and 7.9, respectively. Interestingly Na+ but not NH4+, K+, or Li+ was found to augment significantly the binding activity of CI protein above the basal level. Anions like Cl-, citrate-, acetate-, and H2PO4- do not alter the binding of L1 repressor to its operator. We also show that an in frame deletion mutant of L1 repressor which does not carry the putative HTH motif (at its N-terminal end) fails to bind to its cognate operator DNA even at very high concentrations. The putative HTH motif was found highly conserved and evolutionarily very close to that of regulatory proteins of Y. pestis, H. marismortui, A. tumefaciens, etc. Taken together we suggest that N-terminal end of L1 repressor carries a HTH motif. Further analysis of the putative secondary structures of mycobacteriophage repressors reveals that two common regions encompassing more than 90% of primary sequence are present in all the four repressor molecules studied here. The results suggest that these common regions are utilized for carrying out identical functions.

title: A point mutation at the C-terminal half of the repressor of temperate mycobacteriophage L1 affects its binding to the operator DNA.
abstract: The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42 degrees C. While 40-95% operator-binding activity was shown to be retained at 35 to 42 degrees C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38 degrees C, although the latter showed only 10% less binding compared to that of the former at 32 degrees C. The CIts391 showed almost no binding at 42 degrees C. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42 degrees C. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32 degrees C. Interestingly, the repressor-operator complexes preformed at 0 degrees C have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32 degrees C after preincubation at 42 to 52 degrees C. All these data suggest that the 131(st) proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.

title: Transcriptional regulation and immunity in mycobacteriophage Bxb1
abstract: Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis that shares a similar genome organization to mycobacteriophage L5, although the two phages are heteroimmune. We have investigated the regulatory circuitry of Bxb1 and found that it encodes a repressor, gp69, which regulates at least two promoters, an early lytic promoter, Pleft, and the divergent promoter, Pright. Bxb1 gp69 is 41% identical to the L5 repressor (gp71) and binds to repressor binding sites that conform to a similar, but distinct, 13 bp asymmetric consensus sequence to that for the L5 gp71 binding sites. The two phage repressors have a strong preference for their cognate binding sites, thus accounting for their immunity phenotypes. The Bxb1 genome contains 34 putative repressor binding sites located throughout the genome, but situated within short intergenic spaces and orientated in only one direction relative to the direction of transcription. Comparison with the locations of repressor binding sites within the L5 genome provides insights into how these unusual regulatory systems evolve.

title: Cloning and sequencing analysis of the repressor gene of temperate mycobacteriophage L1.
abstract: The wild-type and temperature-sensitive (ts) repressor genes were cloned from the temperate mycobacteriophage L1 and its mutant L1cIts391, respectively. A sequencing analysis revealed that the 131st proline residue of the wild-type repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative N-terminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor.

title: Isolation, characterization, and mapping of temperature-sensitive mutations in the genes essential for lysogenic and lytic growth of the mycobacteriophage L1.
abstract: Forty temperature-sensitive mutations affecting lytic growth and eight affecting both establishment and maintenance of lysogeny of the temperate mycobacteriophage L1 have been isolated. All of the latter mutations form one complementation group and map within a very short region around the 15% coordinate of the L1 genome; these affect a single gene, cl, coding for the L1 repressor. The former 40 mutations form 28 complementation groups, identifying 28 different genes, G1-G28, essential for the lytic growth of L1. These genes have been mapped using the Gts mutations. Of the 28 Gts mutants, 14 are defective in host lysis at 42 degrees but not at 32 degrees while the other 14 can lyse the host at both temperatures. Among the former 14 Gts mutants, 6 are also defective in L1 DNA synthesis at 42 degrees, and they map in two different clusters, 4 around 65% and 2 around 84% of the L1 genome.

title: Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp
abstract: The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species.

title: Evolution of genetic switch complexity
abstract: The circuitry of the phage λ genetic switch determining the outcome of lytic or lysogenic growth is well-integrated and complex, raising the question as to how it evolved. It is plausible that it arose from a simpler ancestral switch with fewer components that underwent various additions and refinements, as it adapted to vast numbers of different hosts and conditions. We have recently identified a new class of genetic switches found in mycobacteriophages and other prophages, in which immunity is dependent on integration. These switches contain only three genes (integrase, repressor and cro) and represent a major departure from the λ-like circuitry, lacking many features such as xis, cII and cIII. These small self-contained switches represent an unrealized, elegant circuitry for controlling infection outcome. In this addendum, we propose a model of possible events in the evolution of a complex λ-like switch from a simpler integration-dependent switch.

title: Recombineering: A powerful tool for modification of bacteriophage genomes.
abstract: Recombineering, a recently developed technique for efficient genetic manipulation of bacteria, is facilitated by phage-derived recombination proteins and has the advantage of using DNA substrates with short regions of homology. This system was first developed in E. coli but has since been adapted for use in other bacteria. It is now widely used in a number of different systems for a variety of purposes, and the construction of chromosomal gene knockouts, deletions, insertions, point mutations, as well as in vivo cloning, mutagenesis of bacterial artificial chromosomes and phasmids, and the construction of genomic libraries has been reported. However, these methods also can be effectively applied to the genetic modification of bacteriophage genomes, in both their prophage and lytically growing states. The ever-growing collection of fully sequenced bacteriophages raises more questions than they answer, including the unknown functions of vast numbers of genes with no known homologs and of unknown function. Recombineering of phage genomes is central to addressing these questions, enabling the simple construction of mutants, determination of gene essentiality, and elucidation of gene function. In turn, advances in our understanding of phage genomics should present similar recombineering tools for dissecting a multitude of other genetically naïve bacterial systems.

title: BRED: A Simple and Powerful Tool for Constructing Mutant and Recombinant Bacteriophage Genomes
abstract: Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED), in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags.

title: Recombineering mycobacteria and their phages
abstract: Bacteriophages are central components in the development of molecular tools for microbial genetics. Mycobacteriophages have proven to be a rich resource for tuberculosis genetics, and the recent development of a mycobacterial recombineering system based on mycobacteriophage Che9c-encoded proteins offers new approaches to mycobacterial mutagenesis. Expression of the phage exonuclease and recombinase substantially enhances recombination frequencies in both fast- and slow-growing mycobacteria, thereby facilitating construction of both gene knockout and point mutants; it also provides a simple and efficient method for constructing mycobacteriophage mutants. Exploitation of host-specific phages thus provides a general strategy for recombineering and mutagenesis in genetically naive systems.

title: Mycobacteriophage exploit NHEJ to facilitate genome circularization.
abstract: Ku-dependent nonhomologous end joining (NHEJ) is a double-strand break repair process conserved in all branches of cellular life but has not previously been implicated in the DNA metabolic processes of viruses. We identified Ku homologs in Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis. These proteins formed homodimers and bound DNA ends in a manner identical to other Ku's and stimulated joining of ends by the host NHEJ DNA ligase (LigD). Omega and Corndog are unusual in having short 4 base cos ends that would not be expected to self-anneal and would therefore require NHEJ during phage genome circularization. Consistently, M. smegmatis LigD null strains are entirely and selectively unable to support infection by Corndog or Omega, with concomitant failure of genome circularization. These results establish a new paradigm for sequestration of the host cell NHEJ process by bacteriophage and provide a framework for understanding similar transactions in eukaryotic viral infections.

title: Bxz1, a new generalized transducing phage for mycobacteria.
abstract: We have isolated and characterized a new generalized transducing phage, Bxz1, from soil sampling at a neighboring Wildlife Preservation Park. The hosts of the phage, measured by the formation of plaques, include fast growing Mycobacterium smegmatis and Mycobacterium vaccae. Bxz1 is capable of transducing chromosomal markers, point mutations, and plasmids at frequencies ranging from 10(-8) to 10(-6) per plaque forming unit between strains of M. smegmatis. We also demonstrated cotransduction of a transposon insertion linked to a point mutation of the ndh gene.

title: ϕ2GFP10, a High-Intensity Fluorophage, Enables Detection and Rapid Drug Susceptibility Testing of Mycobacterium tuberculosis Directly from Sputum Samples
abstract: The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (ϕ2GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing ϕ2GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. ϕ2GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of ϕ2GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with ϕ2GFP10. Fluorophage ϕ2GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.

title: Fluoromycobacteriophages for Rapid, Specific, and Sensitive Antibiotic Susceptibility Testing of Mycobacterium tuberculosis
abstract: Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections.

title: Comprehensive identification of conditionally essential genes in mycobacteria
abstract: An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a mariner-based transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of Mycobacterium bovis bacillus Calmette–Guérin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.

title: Genes required for mycobacterial growth defined by high density mutagenesis
abstract: Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non‐essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.

title: A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: Application to Mycobacterium tuberculosis
abstract: We describe a postgenomic in silico approach for identifying genes that are likely to be essential and estimate their proportion in haploid genomes. With the knowledge of all sites eligible for mutagenesis and an experimentally determined partial list of nonessential genes from genome mutagenesis, a Bayesian statistical method provides reasonable predictions of essential genes with a subsaturation level of random mutagenesis. For mutagenesis, a transposon such as Himar1 is suitable as it inserts randomly into TA sites. All of the possible insertion sites may be determined a priori from the genome sequence and with this information, data on experimentally hit TA sites may be used to predict the proportion of genes that cannot be mutated. As a model, we used the Mycobacterium tuberculosis genome. Using the Himar1 transposon, we created a genetically defined collection of 1,425 insertion mutants. Based on our Bayesian statistical analysis using Markov chain Monte Carlo and the observed frequencies of transposon insertions in all of the genes, we estimated that the M. tuberculosis genome contains 35% (95% confidence interval, 28%–41%) essential genes. This analysis further revealed seven functional groups with high probabilities of being enriched in essential genes. The PE-PGRS (Pro-Glu polymorphic GC-rich repetitive sequence) family of genes, which are unique to mycobacteria, the polyketide/nonribosomal peptide synthase family, and mycolic and fatty acid biosynthesis gene families were disproportionately enriched in essential genes. At subsaturation levels of mutagenesis with a random transposon such as Himar1, this approach permits a statistical prediction of both the proportion and identities of essential genes of sequenced genomes.

title: Transposon site hybridization in Mycobacterium tuberculosis.
abstract: Microarray mapping of transposon insertions can be used to quantify the relative abundance of different transposon mutants within a complex pool after exposure to selective pressure. The transposon site hybridization (TraSH) method applies this strategy to the study of Mycobacterium tuberculosis and can be adapted to the study of other microorganisms. This chapter describes the methods used to mutagenize mycobacteria with transposons, extract genomic DNA, amplify genomic DNA adjacent to transposon ends using polymerase chain reaction and T7 transcription, and synthesize labeled cDNA. It also describes methods used to construct an appropriate microarray, hybridize labeled cDNA, and analyze the microarray data. Important considerations involved in the experimental design of the selective pressure, the design of the microarray, and the statistical analysis of collected data are discussed.

title: Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP.
abstract: Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc(2) 155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.

title: Mycobacteriophage L5 integrase-mediated site-specific integration in vitro
abstract: Mycobacteriophage L5, a temperate phage of the mycobacteria, forms stable lysogens in Mycobacterium smegmatis via site-specific integration of the phage genome. Recombination occurs within specific phage and bacterial attachment sites and is catalyzed by the phage-encoded integrase protein in vivo. We describe here the overexpression and purification of L5 integrase and its ability to mediate integrative recombination in vitro. We find that L5 integrase-mediated recombination is greatly stimulated by extracts of M. smegmatis but not by Escherichia coli extracts, purified E. coli integration host factor, or purified HU, indicating the presence of a novel mycobacterial integration host factor.

title: Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence
abstract: Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.

title: inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis.
abstract: Isoniazid (isonicotinic acid hydrazide, INH) is one of the most widely used antituberculosis drugs, yet its precise target of action on Mycobacterium tuberculosis is unknown. A missense mutation within the mycobacterial inhA gene was shown to confer resistance to both INH and ethionamide (ETH) in M. smegmatis and in M. bovis. The wild-type inhA gene also conferred INH and ETH resistance when transferred on a multicopy plasmid vector to M. smegmatis and M. bovis BCG. The InhA protein shows significant sequence conservation with the Escherichia coli enzyme EnvM, and cell-free assays indicate that it may be involved in mycolic acid biosynthesis. These results suggest that InhA is likely a primary target of action for INH and ETH.

title: Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors.
abstract: Integrative vectors expressing foreign genes are used as tools for the development of recombinant vaccines in mycobacteria since it is assumed that these vectors are stably maintained even without antibiotic selection. We here demonstrate that integration-proficient vectors are lost from the mycobacterial genome in high frequency. Loss of integrated vectors occurred in recA+ and in recA-strains, indicating a RecA-independent mechanism. Loss of the integrated vector was prevented when integrase gene function was carried on a separate plasmid that is unable to replicate in mycobacteria, indicating that excision is a function of integrase. By providing attP in cis and integrase function in trans, vectors integrating at the attB site are stably maintained, even when carrying genes that deleteriously affect the host.

title: Site‐specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis
abstract: A method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.

title: Taking phage integration to the next level as a genetic tool for mycobacteria.
abstract: Genes must be stably integrated into bacterial chromosomes for complementation of gene deletion mutants in animal infection experiments or to express antigens in vaccine strains. However, with currently available vectors it is cumbersome to create multiple, stable, unmarked chromosomal integrations in mycobacteria. Here, we have constructed a novel integration vector for mycobacteria that enables expression of genes from a cassette protected from transcriptional interference by bi-directional transcriptional terminators proven to be highly efficient in in vitro transcription termination assays. Removal of the integrase gene by a site-specific recombinase, easily identifiable by loss of a backbone reporter gene, stabilizes the integration cassette and makes this vector ideally suitable for infection experiments. This integration vector can be easily adapted to different mycobacteriophage attachment sites (attB) due to its modular design. Integration of a gfp expression cassette at the L5, Giles and Ms6 attB sites in the chromosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis yielded identical gfp expression levels, indicating that none of these sites are compromised for gene expression. The copy number of pAL5000-based extrachromosomal plasmids is 23 in M. smegmatis as determined by quantitative real-time PCR and accounts for the previously observed drastic reduction of gene expression upon integration of plasmids into the chromosome of mycobacteria. Gfp expression and fluorescence of M. smegmatis and M. tuberculosis strains with multiple integrations of gfp increased concomitantly with the copy number demonstrating that these vectors can be used to generate stronger phenotypes and/or to analyze several genes simultaneously in vivo.

title: Use of the mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality.
abstract: Demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as Mycobacterium tuberculosis. One method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult. L5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. The L5 excisionase has been used in Mycobacterium smegmatis to excise and recover these plasmids from chromosome.



Our aims were to establish whether the L5 excisionase could function in M. tuberculosis to remove an L5-based integrated plasmid and, if so, to use this technology as the basis for an improved method for determining whether a gene is essential.



We took two strains of M. tuberculosis carrying the essential gene glnE integrated into the chromosome on an L5-based plasmid, one of which lacked the functional chromosomal copy of the gene. We transformed these with vectors expressing the L5 excisionase and looked for loss of the integrated plasmid.



We obtained efficient excision of an integrated vector from the wild-type strain. However, when the integrated vector carried the only functional copy of the essential gene glnE, the numbers of colonies recovered were reduced to background levels.



The L5 excisionase does function in M. tuberculosis and can be used to confirm the essentiality of a gene. This technology also allows further analysis of essential genes that is difficult or impossible using current methods.

title: Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis.
abstract: We previously used a mycobacteriophage L5-derived integrating vector to demonstrate that glnE and aroK are essential genes in Mycobacterium tuberculosis by showing that we were unable to excise the integrated vector when it carried the only functional copy of these genes. We tested three systems to replace the integrated copy with alternative alleles. The most efficient method was to transform the strain with a second copy of the integrating vector. Excision of the resident vector and integration of the incoming vector occurred at an extremely high efficiency. This technique will allow us to study the role and functionality of essential genes in this important human pathogen.

title: In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family.
abstract: The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in phiC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.

title: Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells.
abstract: Mammalian Markup Recombination techniques utilizing the Cre and Flp systems are now accepted as standard procedures in genome engineering, particularly in manipulating embryonic stem cells, replacing less flexible restriction enzyme approaches. Notwithstanding their popularity, the frequently lackluster efficiency of integration/recombination is still a confounding factor with these approaches. The use of phage-derived serine recombinases has been an attractive alternative to Cre/Flp tyrosine recombinases, but their frequent requirement for cofac-tors and poor efficacy has reduced their appeal. The Bxb1 integrase, however, has recently proven its worth as a robust serine recombinase that is active with fewer restrictions than other such integrases. In the article on p. 460 of this issue, Russell et al. describe for the first time the use of a codon-optimized Bxb1 integrase in mammalian cells, showing that it is able to catalyze unidirectional recombination between attP and attB sites with high efficiency....

title: Rewritable digital data storage in live cells via engineered control of recombination directionality
abstract: The use of synthetic biological systems in research, healthcare, and manufacturing often requires autonomous history-dependent behavior and therefore some form of engineered biological memory. For example, the study or reprogramming of aging, cancer, or development would benefit from genetically encoded counters capable of recording up to several hundred cell division or differentiation events. Although genetic material itself provides a natural data storage medium, tools that allow researchers to reliably and reversibly write information to DNA in vivo are lacking. Here, we demonstrate a rewriteable recombinase addressable data (RAD) module that reliably stores digital information within a chromosome. RAD modules use serine integrase and excisionase functions adapted from bacteriophage to invert and restore specific DNA sequences. Our core RAD memory element is capable of passive information storage in the absence of heterologous gene expression for over 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data storage. We also demonstrate how programmed stochasticity in RAD system performance arising from bidirectional recombination can be achieved and tuned by varying the synthesis and degradation rates of recombinase proteins. The serine recombinase functions used here do not require cell-specific cofactors and should be useful in extending computing and control methods to the study and engineering of many biological systems.

title: Amplifying Genetic Logic Gates
abstract: Biological Transistor A transistor is a device that amplifies and switches electronic signals. Bonnet et al. (p. 599,  see the Perspective by Benenson) engineered a genetic circuit to behave like a transistor in individual living cells. Instead of regulating messenger RNA levels, which has been used previously in designing such systems, the approach relied on changing the state of double-stranded DNA. Six basic logic gates were designed and constructed that were based on the activity of two serine recombinases. A genetic circuit architecture resembling a transistor can be engineered into individual live cells.  Organisms must process information encoded via developmental and environmental signals to survive and reproduce. Researchers have also engineered synthetic genetic logic to realize simpler, independent control of biological processes. We developed a three-terminal device architecture, termed the transcriptor, that uses bacteriophage serine integrases to control the flow of RNA polymerase along DNA. Integrase-mediated inversion or deletion of DNA encoding transcription terminators or a promoter modulates transcription rates. We realized permanent amplifying AND, NAND, OR, XOR, NOR, and XNOR gates actuated across common control signal ranges and sequential logic supporting autonomous cell-cell communication of DNA encoding distinct logic-gate states. The single-layer digital logic architecture developed here enables engineering of amplifying logic gates to control transcription rates within and across diverse organisms.

title: Genetic engineering using homologous recombination.
abstract: In the past few years, in vivo technologies have emerged that, due to their efficiency and simplicity, may one day replace standard genetic engineering techniques. Constructs can be made on plasmids or directly on the Escherichia coli chromosome from PCR products or synthetic oligonucleotides by homologous recombination. This is possible because bacteriophage-encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 50 base pairs. This technology, termed recombineering, is providing new ways to modify genes and segments of the chromosome. This review describes not only recombineering and its applications, but also summarizes homologous recombination in E. coli and early uses of homologous recombination to modify the bacterial chromosome. Finally, based on the premise that phage-mediated recombination functions act at replication forks, specific molecular models are proposed.

title: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.
abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

title: Phage recombinases and their applications.
abstract: The homologous recombination systems of linear double-stranded (ds)DNA bacteriophages are required for the generation of genetic diversity, the repair of dsDNA breaks, and the formation of concatemeric chromosomes, the immediate precursor to packaging. These systems have been studied for decades as a means to understand the basic principles of homologous recombination. From the beginning, it was recognized that these recombinases are linked intimately to the mechanisms of phage DNA replication. In the last decade, however, investigators have exploited these recombination systems as tools for genetic engineering of bacterial chromosomes, bacterial artificial chromosomes, and plasmids. This recombinational engineering technology has been termed "recombineering" and offers a new paradigm for the genetic manipulation of bacterial chromosomes, which is far more efficient than the classical use of nonreplicating integration vectors for gene replacement. The phage λ Red recombination system, in particular, has been used to construct gene replacements, deletions, insertions, inversions, duplications, and single base pair changes in the Escherichia coli chromosome. This chapter discusses the components of the recombination systems of λ, rac prophage, and phage P22 and properties of single-stranded DNA annealing proteins from these and other phage that have been instrumental for the development of this technology. The types of genetic manipulations that can be made are described, along with proposed mechanisms for both double-stranded DNA- and oligonucleotide-mediated recombineering events. Finally, the impact of this technology to such diverse fields as bacterial pathogenesis, metabolic engineering, and mouse genomics is discussed.

title: Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages
abstract: We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria.

title: Recombineering Using RecTE from Pseudomonas syringae
abstract: In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.

title: Efficient point mutagenesis in mycobacteria using single‐stranded DNA recombineering: characterization of antimycobacterial drug targets
abstract: Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis. We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide‐derived single‐stranded DNA recombineering and mycobacteriophage‐encoded proteins. Phage Che9c gp61‐mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10 000‐fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage λ Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host‐specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin.

title: Reporter phage and breath tests: emerging phenotypic assays for diagnosing active tuberculosis, antibiotic resistance, and treatment efficacy.
abstract: The rapid and accurate diagnosis of active tuberculosis (TB) and its drug susceptibility remain a challenge. Phenotypic assays allow determination of antibiotic susceptibilities even if sequence data are not available or informative. We review 2 emerging diagnostic approaches, reporter phage and breath tests, both of which assay mycobacterial metabolism. The reporter phage signal, Green fluorescent protein (GFP) or β-galactosidase, indicates transcription and translation inside the recipient bacilli and its attenuation by antibiotics. Different breath tests assay, (1) exhaled antigen 85, (2) mycobacterial urease activity, and (3) detection by trained rats of disease-specific odor in sputum, have also been developed. When compared with culture, reporter phage assays shorten the time for initial diagnosis of drug susceptibility by several days. Both reporter phage and breath tests have promise as early markers to determine the efficacy of treatment. While sputum often remains smear and Mycobacterium tuberculosis DNA positive early in the course of efficacious antituberculous treatment, we predict that both breath and phage tests will rapidly become negative. If this hypothesis proves correct, phage assays and breath tests could become important surrogate markers in early bactericidal activity (EBA) studies of new antibiotics.

title: Bacteriophage-based assays for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis.
abstract: To summarize, using meta-analysis, the accuracy of bacteriophage-based assays for the detection of rifampicin resistance in Mycobacterium tuberculosis.



By searching multiple databases and sources we identified a total of 21 studies eligible for meta-analysis. Of these, 14 studies used phage amplification assays (including eight studies on the commercial FASTPlaque-TB kits), and seven used luciferase reporter phage (LRP) assays. Sensitivity, specificity, and agreement between phage assay and reference standard (e.g. agar proportion method or BACTEC 460) results were the main outcomes of interest.



When performed on culture isolates (N=19 studies), phage assays appear to have relatively high sensitivity and specificity. Eleven of 19 (58%) studies reported sensitivity and specificity estimates > or =95%, and 13 of 19 (68%) studies reported > or =95% agreement with reference standard results. Specificity estimates were slightly lower and more variable than sensitivity; 5 of 19 (26%) studies reported specificity <90%. Only two studies performed phage assays directly on sputum specimens; although one study reported sensitivity and specificity of 100 and 99%, respectively, another reported sensitivity of 86% and specificity of 73%.



Current evidence is largely restricted to the use of phage assays for the detection of rifampicin resistance in culture isolates. When used on culture isolates, these assays appear to have high sensitivity, but variable and slightly lower specificity. In contrast, evidence is lacking on the accuracy of these assays when they are directly applied to sputum specimens. If phage-based assays can be directly used on clinical specimens and if they are shown to have high accuracy, they have the potential to improve the diagnosis of MDR-TB. However, before phage assays can be successfully used in routine practice, several concerns have to be addressed, including unexplained false positives in some studies, potential for contamination and indeterminate results.

title: Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria.
abstract: Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.

title: Rapid Film-Based Determination of Antibiotic Susceptibilities ofMycobacterium tuberculosis Strains by Using a Luciferase Reporter Phage and the Bronx Box
abstract: Detecting antibiotic resistance in Mycobacterium tuberculosis is becoming increasingly important with the global recognition of drug-resistant strains and their adverse impact on clinical outcomes. Current methods of susceptibility testing are either time-consuming or costly; rapid, reliable, simple, and inexpensive methods would be highly desirable, especially in the developing world where most tuberculosis is found. The luciferase reporter phage is a unique reagent well-suited for this purpose: upon infection with viable mycobacteria, it produces quantifiable light which is not observed in mycobacterial cells treated with active antimicrobials. In this report, we describe a modification of our original assay, which allows detection of the emitted light with a Polaroid film box designated the Bronx Box. The technique has been applied to 25 M. tuberculosis reference and clinical strains, and criteria are presented which allow rapid and simple discrimination among strains susceptible or resistant to isoniazid and rifampin, the major antituberculosis agents.

title: Detection and drug-susceptibility testing of M. tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria Growth Indicator Tube (MGIT) system.
abstract: Rapid diagnosis of drug-resistant M.tuberculosis (Mtb) is desirable worldwide. We (i) describe a new luciferase reporter phage (LRP), phAE142 for this purpose; (ii) compare it to the automated MGIT 960 for time-to-detection of Mtb in clinical specimens; and (iii) evaluate its use for species confirmation and antibiotic susceptibility testing(AST) of Mtb. Twenty sputum samples were inoculated for testing by LRP, or by MGIT 960. After "positives" were identified by either method, the LRP was used for confirmation of Mtb complex (TBC) and for AST. The LRP method proved comparably efficient to MGIT 960 at detecting Mtb. Using an antibiotic uniquely inhibiting TBC with LRP provided species assignment, concurrently with AST, in a median of 3 days, with a sensitivity of 97%. Overall agreement in susceptibility results was 96%. Reliable susceptibility results and identification of TBC can be completed in a median of 12 days (range 8 to 16d) with LRP applied to sputum samples.

title: Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis
abstract: TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day.

title: Luciferase Reporter Mycobacteriophages for Detection, Identification, and Antibiotic Susceptibility Testing ofMycobacterium tuberculosis in Mexico
abstract: The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing ofMycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence ofp-nitro-α-acetylamino-β-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosiscomplex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.

title: Rapid identification and susceptibility testing of Mycobacterium tuberculosis from MGIT cultures with luciferase reporter mycobacteriophages.
abstract: In a prospective study conducted in a diagnostic laboratory in Mexico City, luciferase reporter mycobacteriophages (LRPs) were evaluated for their utility and performance in identification and antibiotic-susceptibility testing of Mycobacterium tuberculosis complex (MTC) isolates from MGIT-960 cultures. Eighty-four consecutive MGIT cultures recovered from 54 patients were included in this study. The LRPs confirmed mycobacterial growth in 79 (94 %) of 84 MGIT cultures. Failure to confirm growth was due to low inoculum (n = 1) or growth with non-tuberculous mycobacteria (n = 4). The median time to confirmation of MGIT cultures was 1 day (range 1-55). Confirmed cultures were identified with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP), a selective inhibitor of MTC species, and results obtained with LRPs were compared with those obtained by BACTEC-460. The sensitivity and specificity of the LRP NAP test were respectively 97 and 100 %, and the median turnaround time for identification was 3 days with both methods. The accuracy and speed of the LRPs for susceptibility testing with rifampicin, streptomycin, isoniazid and ethambutol were compared with BACTEC-460 and discrepant results were tested by the conventional agar proportion method. In total, 72 MTC cultures were tested. The overall agreement between the LRPs and BACTEC-460 was 98.6 %. Four isolates (5.6 %) were falsely identified as ethambutol-resistant. The median turnaround time for susceptibility testing was 3 days (range 3-57) with the LRPs and 9 days (range 7-29) with BACTEC-460. LRPs offer an accurate and rapid approach for identification and susceptibility testing of M. tuberculosis from MGIT-960 cultures.

title: Evaluation of Fluoromycobacteriophages for Detecting Drug Resistance in Mycobacterium tuberculosis
abstract: We tested a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). After promising results in preliminary studies, the EGFP-phage was used to detect isoniazid (INH), rifampin (RIF), and streptomycin (STR) resistance in 155 strains of M. tuberculosis, and the results were compared to the resazurin microplate technique, with the proportion method serving as the reference standard. The resazurin technique yielded sensitivities of 94% for INH and RIF and 98% for STR and specificities of 97% for INH, 95% for RIF, and 98% for STR. The sensitivity of EGFP-phage was 94% for all three antibiotics, with specificities of 90% for INH, 93% for RIF, and 95% for STR. The EGFP-phage results were available in 2 days for RIF and STR and in 3 days for INH, with an estimated cost of ∼2$ to test the three antibiotics. Using a more stringent criterion for resistance improved the specificity of the EGFP-phage for INH and RIF without affecting the sensitivity. In preliminary studies, the EGFP-phage could also effectively detect resistance to the fluoroquinolones. The EGFP-phage method has the potential to be a valuable rapid and economic screen for detecting drug-resistant tuberculosis if the procedure can be simplified, if it can be adapted to clinical material, and if its sensitivity can be improved.

title: Photographic and Luminometric Detection of Luciferase Reporter Phages for Drug Susceptibility Testing of Clinical Mycobacterium tuberculosis Isolates
abstract: Luciferase reporter phages (LRPs) have proven to be efficient tools for drug susceptibility testing of Mycobacterium tuberculosis. Luminometric detection of LRP activity offers higher sensitivity and quantitative results, while a Polaroid film detection method offers a “low-tech” inexpensive alternative that is called the Bronx box. In this work we evaluated, improved, and compared the performance of the luminometer and the Bronx box formats for drug susceptibility testing with LRPs by using 51 clinical isolates of M. tuberculosis, with the agar proportion method (PM) serving as reference. The sensitivity in detecting resistance to isoniazid and rifampin, antibiotics that define multidrug resistance (MDR), was 100% for both methods. The turnaround time for results was reduced from 3 weeks for PM to 54 or 94 h for luminometry or the Bronx box, respectively. These results support the utility of LRPs as a screening test for the surveillance of MDR tuberculosis.

title: [Rapid rifampicin susceptibility test by using recombinant mycobacteriophages].
abstract: To set up a fast, sensitive and specific way to detect mycobacteria and rifampicin susceptibility to mycobacteria by using recombinant mycobacteriophages and bioluminescent method.



Firstly detected the luciferase activity in different bacteria by using mycobacteriophages, then assessed the best drug concentration of rifampicin for drug susceptibility test in luciferase reporter assay, and lastly used the above conditions to decide rifampicin susceptibilities to different mycobacteria and compare the result with L-J medium culture.



Bacteriophages had high light production specifically in mycobacteria and only very low light production in E. coli, the difference being obvious. The light production of rifampicin-resisitant mycobacterium strains was much higher than that of sensitive strains in culture with rifampicin(P < 0.05). Different drug concentrations of rifampicin were used to optimize drug concentration for rifampicin drug susceptibility test and the optimum concentration was found to be 2 micrograms/ml. The correlation of drug susceptibility test between luciferase reporter phages to L-J medium in standard strains and clinical isolates was the sarce. Temperature sensitive Phage 88 was more sensitive than Phage 40(P < 0.05). The time for drug resistance test was 72 hours.



Luciferase reporter phage can detect mycobacteria specifically and can be used in rifampicin susceptibility test. This assay is a fast, sensitive and specific method to detect mycobacterium strains and their resistance to rifampicin.

title: Generation of Affinity-Tagged Fluoromycobacteriophages by Mixed Assembly of Phage Capsids
abstract: Addition of affinity tags to bacteriophage particles facilitates a variety of applications, including vaccine construction and diagnosis of bacterial infections. Addition of tags to phage capsids is desirable, as modification of the tails can lead to poor adsorption and loss of infectivity. Although tags can readily be included as fusions to head decoration proteins, many phages do not have decoration proteins as virion components. The addition of a small (10-amino-acid) Strep-tag II (STAG II) to the mycobacteriophage TM4 capsid subunit, gp9, was not tolerated as a genetically homogenous recombinant phage but could be incorporated into the head by growth of wild-type phage on a host expressing the capsid-STAG fusion. Particles with capsids composed of wild-type and STAG-tagged subunit mixtures could be grown to high titers, showed good infectivities, and could be used to isolate phage-bacterium complexes. Preparation of a STAG-labeled fluoromycobacteriophage enabled capture of bacterial complexes and identification of infected bacteria by fluorescence.

title: Félix d'Herelle and the origins of molecular biology
abstract: To historians of science, to microbiologists, and to the newer breeds of molecular biologists and geneticists alike, the name of Felix d'Herelle will always bring to mind his coining of the word and concept of bacteriophage. Whether this altogether justifies the bold title of the present biography, perilously similar to that of the classic 1966 volume Phage and the origins of molecular biology, is perhaps a moot question. In this account W C Summers examines in depth his studies of bacteriophage and the controversies surrounding their publication: the author's stubborn and lasting attempts to prove his own priority in "discovery" of bacteriophage in the teeth of F W Twort's paper on the same subject published two years earlier. Priority disputes whatever later generations may or may not think of their importance were characteristic of nineteenthand early twentieth-century science, and were often flavoured by chauvinistic attitudes linked to the many political and military conflicts within nineteenth-century Europe. In the end bacteriophage observations became known collectively as the "Twort-d'Herelle phenomenon"; and advocates of Twort's priority included a number of former colleagues of d'Herelle in and around the Paris Pasteur Institute. Twort subsequently busied himself with other problems at London's Brown Institution; whereas d'Herelle wrestled for the rest of his life with questions of therapeutic uses of bacteriophage. Felix d'Herelle was born in Montreal in 1873, the son of a French-Canadian father and a Dutch mother. The father died when Felix was six years old, and the mother, considerably younger and left in comfortable circumstances, moved to Paris with Felix and his younger brother, where the boys attended a local lycee. On graduation Felix embarked on travels in South America, where he learnt to speak Spanish; on the return trip by ship from Rio de Janeiro he witnessed an outbreak of yellow fever when twenty passengers and crew died. Calm in the face of danger from little known infectious diseases, d'Herelle returned to Paris in 1890 with no clear plans for further education, let alone any firm ideas for a future career. After adventurous travelling, he finally decided to settle in Montreal, and to pursue a career in bacteriology, in 1897. At this point, Summers is at pains to remind us of d'Herelle's early and varied achievements: his studies on fermentation, including commercial attempts to produce alcohol from surplus maple syrup in Quebec, and "banana whiskey" from surplus bananas in Guatemala. Fermentation studies appealed particularly to d'Herelle, increasing his self-regarding sense of emulating Pasteur. During the Spanish-American War in 1898 he was asked to assist Mexican sisal growers and the Yucatafn Department of Agriculture to develop fermentation procedures in order to dispose profitably of surplus sisal, again by using it for the production of alcohol. The lengthy and detailed explanations of the various fermentation studies are hard on the reader, not helped by a less than sparkling style and a number of sentences where verbs and/or prepositions appear to be missing and the meaning is left to the imagination of the reader, who begins to long for a more succinct presentation and details relegated to notes. Towards the end of his stay in Mexico, and during further travels

title: Management of difficult multidrug‐resistant tuberculosis and extensively drug‐resistant tuberculosis: Update 2012
abstract: Multidrug‐resistant (MDR) tuberculosis (TB) denotes bacillary resistance to at least isoniazid and rifampicin. Extensively drug‐resistant (XDR) TB is MDR‐TB with additional bacillary resistance to any fluoroquinolone and at least one second‐line injectable drugs. Rooted in inadequate TB treatment and compounded by a vicious circle of diagnostic delay and improper treatment, MDR‐TB/XDR‐TB has become a global epidemic that is fuelled by poverty, human immunodeficiency virus (HIV) and neglect of airborne infection control. The majority of MDR‐TB cases in some settings with high prevalence of MDR‐TB are due to transmission of drug‐resistant bacillary strains to previously untreated patients. Global efforts in controlling MDR‐TB/XDR‐TB can no longer focus solely on high‐risk patients. It is difficult and costly to treat MDR‐TB/XDR‐TB. Without timely implementation of preventive and management strategies, difficult MDR‐TB/XDR‐TB can cripple global TB control efforts. Preventive strategies include prompt diagnosis with adequate TB treatment using the directly observed therapy, short‐course (DOTS) strategy and drug‐resistance programmes, airborne infection control, preventive treatment of TB/HIV, and optimal use of antiretroviral therapy. Management strategies for established cases of difficult MDR‐TB/XDR‐TB rely on harnessing existing drugs (notably newer generation fluoroquinolones, high‐dose isoniazid, linezolid and pyrazinamide with in vitro activity) in the best combinations and dosing schedules, together with adjunctive surgery in carefully selected cases. Immunotherapy may also have a role in the future. New diagnostics, drugs and vaccines are required to meet the challenge, but science alone is insufficient. Difficult MDR‐TB/XDR‐TB cannot be tackled without achieving high cure rates with quality DOTS and beyond, and concurrently addressing poverty and HIV.

title: Therapy of experimental tuberculosis in guinea pigs with mycobacterial phages DS-6A, GR-21 T, My-327.
abstract: Guinea pigs, weighing 250-350 g, were infected with approximately 5,000 of live germs M- tuberculosis H 37 Rv grown 10 days in deep culture of liquid semisynthetic medium according to Sula. The infection was performed subcutaneously in inquinal region. For the therapy following phages were used: DS-6A, GR-21/T, My-327 injected twice a week subcutaneously in the dose of 10(6)/1 ml of live particles for 10 weeks. The therapeutic effect was expressed by spleen and hilus index. Out of the phages used, phage DS-6A had the highest therapeutic effect with mean spleen index of 0.19, corresponding approximately to the spleen index reached with the most effective tuberculostaticum INH. The exact explanation of the phage therapeutic effect in given experimental conditions, when the phages are not applied locally in order to gain the direct contact with infectious antigens, is not known. It is suggested that there presumably exists an interaction between the released phage nucleic acid and the nucleic acid synthesis needed for the growth of mycobacteria in vivo.

title: In vivo efficacy of phage therapy for Mycobacterium avium infection as delivered by a nonvirulent mycobacterium.
abstract: The emergence of mycobacteria resistant to currently available antimicrobial agents has become an important problem in modern medicine. Mycobacterium avium and M. tuberculosis are intracellular pathogens that replicate and survive within the mononuclear phagocytes. TM4 is a lytic mycobacteriophage that kills both extracellular M. avium and M. tuberculosis. When delivered by M. smegmatis transiently infected with TM4, it kills both M. avium and M. tuberculosis within RAW 264.7 macrophages. To evaluate the treatment of M. avium infection with phage in vivo, C57 BL/6 mice were infected with M. avium 109 and, 7 days later, treated either once or twice with TM4 phage (7.9 x 10(10) PFU/ml), M. smegmatis (4 x 10(8) cFU/ml), or M. smegmatis with TM4 phage delivered intravenously (i.v.). Treatment with TM4 phage alone or M. smegmatis without TM4 did not show a significant decrease in number of intracellular bacteria in the spleen compared with untreated control. In contrast, administration of M. smegmatis-TM4 resulted in a significant decrease in the number of M. avium in the spleen. However, 23% of bacteria recovered from treated mice were resistant to TM4. These in vivo studies confirmed the in vitro findings that an avirulent mycobacterium can be used as a carrier to deliver antimycobacterial phage intracellularly.