bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosomal supercoiling in Escherichia coli
Authors: Miller† William G. , Simons Robert W.
Published: October 2006

The Escherichia coli chromosome is compacted into 40‐50 negatively supercoiled domains. It has been proposed that these domains differ in superhelical density. Here, we present evidence that this is probably not the case. A modified Tn10 transposable element was inserted at a number of locations around the E. coli chromosome. This element, mTn10‐plac‐lacZ+, contains the lac operon promoter, plac, whose activity increases with increasing superhelical density, fused to a lacZ+ reporter gene. Although mTn10‐plac‐lacZ+ fusion expression varies as much as approximately threefold at different insertion sites, the relative levels of expression from these elements are unaffected by replacing plac with the gyrA promoter, pgyrA, which has a reciprocal response to changes in superhelical density. Importantly, topoisomerase mutations and coumermycin, which inhibits DNA gyrase activity, alter mTn10‐plac‐lacZ+ and mTn10‐pgyrA‐lacZ+ fusion expression in expected ways, showing that the elements remain responsive to supercoiling and that topoisomerase activity is required for maintaining superhelical density. Fusion expression is not affected by anaerobic growth or osmotic shock, two physiological conditions thought to alter supercoiling. The approximately threefold difference in mTn10‐plac‐lacZ‐ and mTn10‐pgyrA‐lacZ+ fusion expression observed at different sites may be explained by regional differences in chromosomal copy number that arise from bidirectional replication. Together, these results strongly suggest that the E. coli chromosomal domains do not differ in functional superhelical density.
Novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation
Authors: Facius Dirk , Meyer Thomas F.
Published: October 2006

A novel genetic determinant (comA) has been identified and found to be required for the transformation of piliated Neisseria gonorrhoeae. Mutants in comA of strain MS11 grow normally and are DNA‐uptake proficient but blocked in the translocation of DNA into the cytoplasm. Here we show by site‐specific mutagenesis and genetic complementation that only one of two open reading frames identified in comA is essential for competence: it encodes a protein (ComA) with a predicted size of 74kDa. The comAgene maps upstream of the iga locus and is transcribed in the opposite orientation, probably under the control of a putative σ;54 type promoter. While DNA probes specific for the N. gonorrhoeae iga locus reveal only a little cross‐reactivity with commensal Neisseria species, the neighbouring comA gene appears to be present in most of them. ComA fusion proteins were obtained by in vitro translation. The synthesized gene products migrated atypically in SDS gels indicating its strong hydrophobicity. Several transmembrane α‐helices were predicted from the amino acid sequence of ComA which, in the context of an observed sequence similarity with other inner membrane proteins, suggests a location for the protein in the inner membrane. Using piliated and non‐piliated comA mutants the consequences of transformation deficiency on pilin phase variation were assessed. We show that the comA defect affects some but not all types of DNA rearrangements associated with pilE variation. The results are in agreement with previous observations supporting the notion that multiple recombination pathways contribute to the variability of pilE.
Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly
Authors: Stanley Peter L. D. , Diaz Pilar , Bailey Marc J. A. , Gygi Daniel , Juarez Antonio , Hughes Coiin
Published: October 2006

A transposon mutant of Escherichia coli 5K was isolated which reduced 10‐ to 50‐fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within the rfaP gene (required for attachment of phosphate‐containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene. The toss in cytolytic activity of the secreted HlyA protein was not related to the HlyC‐directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate‐zonal centrifugation the mutant‐secreted HlyA migrated as a larger species than the wild type. The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide‐independent secretion process.
Mode and origin of replication of pSAM2, a conjugative integrating element of Streptomyces ambofaciens
Authors: Hagège Juliette , Pernodet Jean‐Luc , Mann Annick Fried , Guérineau Michel
Published: October 2006

pSAM2 is an 11 kb integrating element from Streptomyces ambofaciens that is capable of replication. It generates single‐stranded DNA during replication, and is therefore the first Streptomyces integrating element to be described that may belong to the family of elements, called the ssDNA elements, that replicate by a rolling‐circle mechanism. The direction of replication has been identified. The plus origin (ori) of replication and minus origin (M‐O) have been located. Streptomyces lividans harbouring replicating pSAM2 also contain numerous small covalently closed circular DNA molecules (scm) derived from pSAM2. These scm contain ori and extend on both sides of the putative nick site. Sequences at the junction points of these scm are heterogeneous but short direct repeats were always found in the vicinity of these junctions.
Construction of chimeric proteins from the σ;N‐associated transcriptional activators VnfA and AnfA of Azotobacter vinelndii shows that the determinants of promoter specificity lie outside the‘recognition’helix of the HTH motif in the C‐terminal domain
Authors: Jacob Jansen , Drummond Martin
Published: October 2006

Functional chimeras have been generated from the transcriptional activators VnfA and AnfA, which control expression of the alternative nitrogenases in Azotobacter vinelandii. The activation profiles of the native and chimeric proteins have been determined using lacZ fusions to A. vinelandii anf and vnf promoters in Klebsiella pneumoniae. Repiacing the C‐terminal domain of AnfA with that of VnfA gives a protein with the promoter specificity of VnfA, confirming that the C‐terminal domain contains the determinants of promoter specificity. However, substituting the VnfA sequence from the turn in the helix‐turn‐helix motif to the C‐terminus does not alter the promoter specificity of AnfA. These changes in promoter specificity were reflected in changes in affinity for a VnfA‐binding site, as measured by an in vivo repression assay using a lacZ fusion to a synthetic promoter. This supports the assumption that promoter recognition is determined by activator binding to enhancer‐like sequences, and shows that the principal determinants of specific DNA‐binding lie outside the‘recognition’helix. This may be a general feature of transcriptional activators dependent on σ;N (σ54). The chimera with the promoter specificity of VnfA retained the dependence on nitrogenase Fe protein characteristic of AnfA, indicating that this property is not related to particular promoter sequences, but is a function of the central or N‐terminal domains of AnfA.
In a class of its own — the RNA polymerase sigma factor σ;54 (σN)
Authors: Merrick M. J.
Published: October 2006

Bacteria synthesize a number of different sigma factors which allow the co‐ordinate expression of groups of genes owing to the ability of sigma to confer promoter‐specific transcription initiation on RNA polymerase. In nearly all cases these sigmas belong to a single family of proteins which appear to be related structurally and functionally to the major Escherichia coli sigma factor, σ;70. A clear exception is the Sigma factor σ;54 (σN), encoded by rpoN, which represents a second family of sigmas that is widely distributed in prokaryotes. Studies of σ;54 (σN) have demonstrated that this sigma is quite distinct both structurally and functionally from the σ;70 family and the mode of transcription initiation which it mediates may have more in common with that found in eukaryotes than that which occurs with σ;70 and its relatives.
Crystalline bacterial cell surface layers
Authors: Sleytr Uwe B. , Messner Paul , Pum Dietmar , Sára Margit
Published: October 2006

Crystalline arrays of proteinaceous subunits forming surface layers (S‐layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram‐positive and Gram‐negative archaeobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S‐layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S‐layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram‐negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad‐application potential in biotechnology, vaccine development and molecular nanotechnology.
Construction and use of integrative vectors to express foreign genes in mycobacteria
Authors: Dellagostin Odir A. , Wall Sue , Norman Elizabeth , O'Shaughnessy Tina , Dale Jeremy W. , McFadden JohnJoe
Published: October 2006

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin‐resistance gene into a 'suicide’vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot‐and‐mouth disease virus VP1 140‐160 epitope were successfully cloned into the 18kDa gene and expression in M. smegmatis was obtained.
A novel ompC mutation of Escherichia coli K‐12 that reduces OmpC and OmpF levels in the outer membrane
Authors: Misra Rajeev
Published: October 2006

A novel ompC mutation was isolated that not only lowered the amount of its own product, OmpC27, but also reduced the level of OmpF present in the outer membrane. ompC27 codes for a mutant OmpC protein that contains two non‐native cysteine residues. The ompC27 allele confers phage resistance by lowering the level of OmpC present in the outer membrane. This effect on OmpC27 was manifested at the level of assembly as a result of disulphide bond formation between the two cysteine residues. This disulphide bonding in OmpC27 also produced a novel phenotype by specifically influencing OmpF levels. The effect of OmpC27 on OmpF was partly a result of a lowering of ompF transcription, and partly a result of an effect at the post‐transcription level. The transcriptional effect is likely to be brought about by a defective membrane as a result of the insertion of the disulphide bond containing OmpC27. The post‐transcriptional effect of OmpC27 on OmpF could be due to interference at the assembly level. In a dsbA::kan1 background where the in vivo disulphide bonding ability was dramatically reduced, the OmpC27‐mediated effects were also curtailed.
Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains
Authors: Sela Shiomo , Aviv Adi , Tovi Aviva , Burstein Israel , Caparon Michael G. , Hanski Emanuel
Published: October 2006

The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C‐terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V‐terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N‐terminal domain was mixed in a binding reaction with a protein extract containing the C‐terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C‐terminal domain, blocked the binding partially. A clone exclusively expressing the C‐terminal domain, completely blocked the binding of the 30 kDa N‐terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N‐terminal domain failed to block the binding of this FN fragment. Thus, the two FN‐binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed.
A physical and genetic map of the Stigmatella aurantiaca DW4/3.1 chromosome
Authors: Neumann Björn , Pospiech Andreas , Schairer Hans Ulrich
Published: October 2006

A physical map of the myxobacterium Stigmatella aurantiaca DW4/3.1 chromosome was constructed by pulsed‐field gel (PFG) long‐range mapping. One‐and two‐dimensional pulsed‐field gel analyses were used together with reciprocal double‐restriction, cross‐hybridization and hybridization fingerprint analysis. These PFG results were confirmed by Smith‐Birnstiel analysis, by Southern hybridization using linking clones and clones of a λ genomic library for the determination of adjacent restriction fragments and by transposon insertion mapping using defined genomic sequences for hybridization. It was thus possible to construct a circular restriction map of the single 9.35 Mbp chromosome of S. aurantiaca based on the endonucleases Asel and Spel. Genetic loci as well as the replication origin were located on the physical map by Southern hybridization using heterologous (derived from Myxococcus xanthus, Escherichia coli and Streptomyces lividans) and homologous probes that are mainly involved in development and ceil motility.
Isolation and characterization of full‐length chromosomes from non‐culturable plant‐pathogenic Mycoplasma‐like organisms
Authors: Neimark Harold , Kirkpatrick Bruce C.
Published: October 2006

We describe the isolation and characterization of full‐length chromosomes from non‐culturable plant‐pathogenic, mycoplasma‐like organisms (MLOs). MLO chromosomes are circular and their sizes (640 to 1185kbp) are heterogeneous. Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents. MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non‐culturability. Hybridization with cloned MLO‐specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO ‘type’ strains. Large DNA molecules that appear to be MLO megaplasmids were also demonstrated. The ability to characterize full‐length chromosomes from virtually any non‐culturable prokaryote should greatly facilitate the molecular and genetic analysis of these difficult bacteria.
SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane
Authors: Oliver Donald B.
Published: October 2006

Recent insight into the biochemical mechanism of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent translocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein‐SecB complex, binding to inner‐membrane anionic phospholipids, insertion into the membrane biiayer and association with the preprotein translocator, SecY/SecE. ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate‐determining step in protein secretion, SecA synthesis is co‐ordinated with the activity of the protein export pathway. This form of negative reguiation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA‐dependent catalysis of protein export and the details of secA regulation appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways.
Translocation of precytochrome C2 into intracytoplasmic membrane vesicles of Rhodobacter capsulatus requires a peripheral membrane protein
Authors: Wieseler Beate , Müller Matthias
Published: October 2006

Rhodobacter capsulatus is a member of the group α‐purple bacteria which are closely related to the ancestral endosymbiont that gave rise to mitochondria. It has therefore been hypothesized that the molecular mechanisms governing protein export in α‐purple bacteria have been conserved during the evolution of mitochondria. To enable analysis of protein export in α‐purple bacteria we describe here the development of a homologous cell‐free synthesis/export system consisting entirely of components of R. capsulatus. Translocation of precytochrome C2 into intracytoplasmic membrane vesicles of this organism was found to require the proton‐motive force and proceed at a significantly higher efficiency when membranes were present during protein synthesis. Furthermore, we show that, in this cell‐free system, translocation depends on a preparation of peripheral membrane proteins Which do not possess detectable SecA‐ and SecB‐like actvities.
Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions
Authors: Huckle James W. , Morby Andrew P. , Turner Jennifer S. , Robinson Nigel J.
Published: October 2006

In eukaryotes, metallothioneins (MTs) are involved in cellular responses to elevated concentrations of certain metal ions. We report the isolation and analysis of a prokaryotic MT locus from Synechococcus PCC 7942. The MT locus (smt) includes smtA, which encodes a class II MT, and a divergently transcribed gene, smtB. The sites of transcription initiation of both genes have been mapped and features within the smt operator‐promoter region identified. Elevated concentrations of the ionic species of Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn elicited an increase in the abundance of smtA transcripts. There was no detectable effect of elevated metal (Cd) on smtA transcript stability. Sequences upstream of smtA, fused to a promoterless lacZ gene, conferred metal‐dependent β‐galactosidase activity in Synechococcus PCC 7942 (strain R2‐PIM8). At maximum permissive concentrations, Zn was the most potent elicitor in vivo, followed by Cu and Cd with slight induction by Co and Ni. The deduced SmtB polypeptide has similarity to the ArsR and CadC proteins involved in resistance to arsenate/arsenite/antimonite and to Cd, contains a predicted helix‐turn‐helix DNA‐binding motif and is shown to be a repressor of transcription from the smtA operator‐promoter.
Deletion within the metallothionein locus of cadmium‐tolerant Synechococcus PCC 6301 involving a highly iterated palindrome (HIP1)
Authors: Gupta Amit , Morby Andrew P. , Turner Jennifer S. , Whitton Brian A. , Robinson Nigel J.
Published: October 2006

Genomic rearrangements involving amplification of metallothionein (MT) genes have been reported in metal‐tolerant eukaryotes. Similarly, we have recently observed amplification and rearrangement of a prokaryotic MT locus, smt, in cells of Synechococcus PCC 6301 selected for Cd tolerance. Following the characterization of this locus, the altered smt region has now been isolated from a Cd‐tolerant cell line, C3.2, and its nucleotide sequence determined. This has identified a deletion within smtB, which encodes a trans‐acting repressor of smt transcription. Two identical palindromic octanucleotides (5′‐GCGATC‐GC‐3′) traverse both borders of the excised element. This palindromic sequence is highly represented in the smt locus (7 occurrences in 1326 nucleotides) and analysis of the GenBank/EMBL /DDB J DNA Nucleotide Sequence Data Libraries reveals that this is a highly iterated palindrome (HIP1) in other known sequences from Synechococcus strains (estimated to occur at an average frequency of once every c. 664 bp). HIP1 is also abundant in the genomes of other cyanobacteria. The functional significance of smtB deletion and the possible role of HIP1 in genome plasticity and adaptation in cyanobacteria are discussed.
Genetic analysis of the φC31 ‐specific phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)
Authors: Laity Carole , Chater Keith F. , Lewis Cinzia G. , Buttner Mark J.
Published: October 2006

The phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) was shown to be specific to φC31 homo‐immune phages, and to be absent from the closely related strain Streptomyces Iividans. A 16 kb fragment of S. coelicolor A3(2) DNA was isolated which complemented the Pgl− phenotype of J1501, a pgl mutant derivative of the PgltsS. coelicolor strain M130. The cloned DNA complemented only half of the available pgl mutants, which therefore represented at least two groups, designated Pgl class A and class B strains. It follows that more than one kind of high‐frequency genetic event can lead to the Pgl− phenotype. Crosses between class A and class B strains yielded high frequencies of Pgl+ recombinants. Crosses between strains of the same class gave no Pgl+ recombinants. The cloned DNA was altered by deletion or apparent point mutation upon passage through the two class B strains tested, such that it was no longer capable of complementing class A strains. This accumulation of mutations might suggest that the expression of the cloned DNA is toxic to at least some class B strains. The nature of the genetic instability associated with the Pgl system was not detectable by Southern blot analysis.
Transition‐state regulators: sentinels of Bacillus subtilis post‐exponential gene expression
Authors: Strauch Mark A. , Hoch James A.
Published: October 2006

When Bacillus subtilis encounters a nutrient‐depleted environment, it expresses a wide variety of genes that encode functions in alternative pathways of metabolism and energy production. Expression of these genes first occurs during the transition from active growth into stationary phase and is controlled by a class of proteins termed transition‐state regulators. In several instances, a given gene is redundantly controlled by two or more of these regulators and many of these regulators control genes in numerous different pathways. The AbrB, Hpr and Sin proteins are the best‐studied examples of these regulatory molecules. Their role is to prevent inappropriate and possibly detrimental functions from being expressed during exponential growth when they are not needed. They serve as elements integrating sporulation with ancillary stationary‐phase phenomena and appear to participate in the timing of early sporulation events and in fine‐tuning the magnitude of gene expression in response to specific environmental conditions.
Protein HU binds specifically to kinked DNA
Authors: Pontiggia Andrea , Negri Armando , Beltrame Monica , Bianchi Marco E.
Published: October 2006

We have purified the main four‐way junction DNA‐binding protein of Escherichia coli, and have found It to be the well‐known HU protein. HU protein recognizes with high‐affinity one of the angles present in the junction, a molecule with the shape of an X. Other DNA structures characterized by sharp bends or kinks, like bulged duplex DNAs containing unpaired bases, are also bound. HU protein appears to inhibit cruciform extrusion from supercoiled inverted repeat (palindromic) DNA, either by constraining supercoiling or by trapping a metastable interconversion intermediate. All these properties are analogous to the properties of the mammalian chromatin protein HMG1. We suggest that HU is a prokaryotic HMG1‐like protein rather than a histone‐like protein.
Isolation of dnaJ, dnaK, and grpE homologues from Borrelia burgdorferi and complementation of Escherichia coli mutants
Authors: Tilly Kit , Hauser Robyn , Campbell Jim , Ostheimer Gerard J.
Published: October 2006

The heat‐shock proteins DnaJ, DnaK, and GrpE are involved in the replication of various species of DNA in Escherichia coli, in addition to their roles in other processes, including protein disaggregation and export. We have cloned the Borrelia burgdorferi homologues of these genes. DNA sequence analysis revealed an open reading frame encoding a protein that is 62% identical to the E. coli DnaK protein. Genes homologous to the E. coli grpE and dnaJ genes, encoding products 28% and 39% identical to their homologues, are located up‐ and downstream, respectively, of the B. burgdorferi dnaK gene. No obvious promoters were detected in the sequenced DNA, although a potential transcription terminator was found downstream of the dnaJ gene, so these three genes may form an operon, perhaps with a fourth gene located upstream of the grpE gene. The grpE homologue complemented an E. coli grpE mutant and the dnaJ homologue complemented an E. coli dnaJ mutant, whereas the B. burgdorferi dnaK gene did not complement dnaK mutants.
Superinfection immunity of mycobacteriophage L5: applications for genetic transformation of mycobacteria
Authors: Donnelly‐Wu Mary K. , Jacobs William R. , Hatfull Graham F.
Published: October 2006

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.
Iron‐dependent stability of the ferredoxin I transcripts from the cyanobacterial strains Synechococcus species PCC 7942 and Anabaena species PCC 7937
Authors: Bovy Arnaud , de Vrieze Geert , Lugones Luis , van Horssen Peter , van den Berg Claudia , Borrias Mies , Weisbeek Peter
Published: October 2006

The effect of iron on ferredoxin I specific mRNA levels was studied in the cyanobacterial strains Synechococcus sp. PCC 7942 (Anacystis nidulans R2) and Anabaena sp. PCC 7937 (Anabaena variabilis ATCC 29413). In both strains addition of iron to iron‐limited cells resulted in a rapid increase in ferredoxin mRNA levels. To investigate the possible role of the ferredoxin promoter in iron regulation, a vector for promoter analysis in Synechococcus PCC 7942 strain R2‐PIM9 was constructed, which contains the ferredoxin promoter fused to the gene encoding β‐glu‐curonidase (GUS) as reporter. Neither the Synechococcus nor the Anabaena ferredoxin promoter was able to direct iron‐regulated GUS activity in Synechococcus R2‐PIM9, Indicating that transcription initiation is not responsible for the iron‐dependent ferredoxin mRNA levels. Determination of the half‐life of the ferredoxin transcript in iron‐supplemented and iron‐limited cells revealed that, in both strains, the ferredoxin transcript is much more stable in iron‐supplemented cells than in iron‐limited cells. These results lead to the conclusion that in these strains, iron‐regulated expression of the ferredoxin I gene is mediated via differential mRNA stability.
Amplified expression of a transcriptional pattern formed during development of Anabaena
Authors: Wolk C. Peter , Elhai Jeff , Kuritz Tanya , Holland Doron
Published: October 2006

The cyanobacterium Anabaena responds to nitrogen deprivation by producing heterocysts, cells specialized for nitrogen fixation, at well‐spaced intervals along its filaments. The gene hepA, required for heterocyst maturation, is expressed in response to nitrogen deprivation, prior to visible differentiation. A spatial pattern of hepA expression indistinguishable from the eventual pattern of heterocysts was made visible by fusing the hepA promoter to luxAB, which encodes bacterial luciferase. Because the resulting signal did not greatly exceed instrumental background, T7 RNA polymerase was used to increase luminescence. The hepA promoter was fused to the gene for that polymerase, and a promoter recognized by that polymerase was fused to luxAB. Filaments containing these two fusions showed spaced luminescing cells many hours before differentiation became discernible morphologically.
Light‐induced carotenogenesis in Myxococcus xanthus: genetic analysis of the carR region
Authors: Hodgson David A.
Published: October 2006

Carotenogenesis is light‐inducible in the non‐photo‐synthetic, Gram‐negative, bacterium Myxococcus xanthus. We report the characterization of the carR region which controls this phenomenon. Insertion of transposon Tn5 close to the carR region caused a dominant, carotenoid‐constitutive mutation because of the presence of a constitutive, outward‐reading promoter in the IS50L component of Tn5. In wild‐type cells, a powerful, tightly‐regulated, light‐inducible promoter directs the transcription of two genetic functions. One of these functions is to activate transcription of the genetically unlinked carB gene, which is involved in carotenoid synthesis. The second function (carR) regulates the light‐inducible promoter. We also report the mapping of two carotenoid constitutive mutations to the previously characterized car A locus.
Translational frameshifting in the control of transposition in bacteria
Authors: Chandler Michael , Fayet Olivier
Published: October 2006

The expression of an increasing number of genes of both prokaryotic and eukaryotic origin has been shown to be regulated at the translational level by programmed (sequence‐specific) ribosomal frame‐shifting. Among these are the bacterial insertion sequences IS1 and two members of the widely distributed IS3‐family, IS150 and IS911. Frameshifting provides a means of specifying several proteins with different functions using a minimum of genetic information. In this review, we survey present understanding of the way in which frameshifting is integrated into the overall control of transposition activity in these elements.
Class 1 outer membrane protein of Neisseria meningitidis: epitope analysis of the antigenic diversity between strains, implications for subtype definition and molecular epidemiology
Authors: McGuinness B. T. , Lambden P. R. , Heckels J. E.
Published: October 2006

The VR1 and VR2 regions of the class 1 protein have been sequenced from a number of meningococcal strains, including non‐subtypable strains and strains of apparently identical subtype. The amino acid sequences have been used to construct synthetic peptides for mapping subtype‐specific epitopes. The majority of epitopes was found to be located in VR2 at the apex of a predicted surface‐exposed loop. A single amino acid change within an epitope, or an amino acid deletion outside an epitope, were both associated with loss of subtype specificity, resulting from a change in the predicted conformation at the apex of the loop structure. Analysis of the sequence information combined with knowledge of defined epitopes also revealed considerable additional information not demonstrated by current subtyping procedures.
Bacterial interspersed mosaic elements (BIMEs) are present in the genome of Klebsiella
Authors: Bachellier Sophie , Perrin David , Hofnung Maurice , Gilson Eric
Published: October 2006

Bacterial interspersed mosaic elements (BIMEs) constitute a family of highly repetitive sequences containing palindromic units (PUs), also called repetitive extragenic palindromes (REPs). BIMEs were originally described in Escherichia coli and Salmonella typhimurium. We show here, by determining the nucleotide sequence of two intergenic regions of Klebsiella pneumoniae, by computer searches, and by hybridization, that sequences with similar characteristics are found in the genome of several Klebsiella species. This reinforces the idea that BIMEs play general and important roles in enterobacteria such as in the organization of the bacterial chromosome.
Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium
Authors: Jin Shouguang , Song Yan‐nong , Pan Shen Q. , Nester Eugene W.
Published: October 2006

Transformation of plants by Agrobacterium tumefaciens is mediated by a set of virulence (vir) genes that are specifically induced by plant signal molecules through the VirA/VirG two‐component regulatory system. The plant signal is transmitted from VirA to VirG by a cascade of phosphorylation reactions followed by the sequence‐specific DNA binding of the VirG protein to the vir gene promoters which then activates their transcription. In this report, we describe a VirG mutant which is able to activate Wr gene expression independently of the VirA molecule and the two plant signal molecules, acetosyringone and monosaccharides. A strain of Agrobacterium containing this virG gene but lacking a functional virA gene was able to induce tumours on all three plants that were tested. A single amino acid change of asparagine (N) to aspartate (D) at position 54, adjacent to the site of VirG phosphorylation, aspartate 52, resulted in this constitutive phenotype. In vitro phosphorylation experiments showed that the mutant protein cannot be phosphorylated by VirA, suggesting that the negative charge resulting from the N to D switch mimics the phosphorylated conformation of the VirG molecule. The same amino acid change in the virG gene of the supervirulent strain A281 also resulted in a constitutive phenotype. However, the vir genes were not induced to high levels when compared with the levels of the constitutive Virg of strain A348.
High‐affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida
Authors: Trust Trevor J. , Kostrzynska Magdalena , Emödy Levente , Wadström Torkel
Published: October 2006

The surface of the fish pathogen Aeromonas salmonicida is covered by a paracrystalline array (the A‐layer) which is a virulence factor for the organism. Quantification of the ability of A. salmonicida cells to bind collagen types I and IV in a 125I‐radiolabelled liquid‐phase assay showed that A‐layer‐positive cells bound high levels of collagen type IV, but significantly lower levels of collagen type I. Collagen type IV binding was confirmed using non‐radiolabelled enzyme‐linked immunosorbent assays. 125I‐Collagen type IV binding was rapid, specific, saturable, high affinity, and essentially irreversible by unlabelled collagen type IV. The A‐layer was responsible for collagen type IV binding because binding was inactivated by selective removal of the A‐layer at pH 2.2, and neither isogenic A‐layer‐deficient A. salmonicida mutants nor strains of Aeromonas hydrophila possessing a morphologically similar paracrystalline array bound this basement membrane protein.
The minCD locus of Bacillus subtilis lacks the minE determinant that provides topological specificity to cell division
Authors: Lee Soonyoul , Price Chester W.
Published: October 2006

A key event of the sporulation process in Bacillus subtilis is the asymmetric cell division that divides the developing cell into two unequal compartments. To examine the function of vegetative cell division genes in this developmental division, we isolated and characterized the B. subtilis counterpart to the Escherichia coli minicell operon minB, which governs correct placement of the division septum. Starting from the closely linked spo/VFlocus, we used walking methods to isolate the region of the B. subtilis chromosome proximate to the divlVB minicell locus. DNA sequence analysis found two open reading frames whose predicted products had significant identity to the E. coli MinC cell division inhibitor and the MinD ATPase activator of MinC, and disruption of minCD function generated a minicell phenotype in B. subtilis. Notably, no homologue to the E. coli MinE topological specificity element was found in the B. subtilis minCD region. The B. subtilis min genes were part of an operon transcribed from a major promoter more than 2.5 kb upstream from minC. An internal promoter immediately upstream from minC was dependent on RNA polymerase containing sigma‐H and was active at the onset of sporulation. However, neither minCnor minD function was absolutely required for sporulation and, by implication, for asymmetric septum formation.
PilS and PilR, a two‐component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa
Authors: Hobbs M. , Collie E. S. R. , Free P. D. , Livingston S. P. , Mattick J. S.
Published: October 2006

Transposon mutagenesis was used to identify genes necessary for the expression of Pseudomonas aeruginosa type 4 fimbriae. In a library of 12 700 mutants, 147 were observed to have lost the spreading colony morphology associated with the presence of functional fimbriae. Of these, 28 had also acquired resistance to the fimbrial‐specific bacteriophage P04. The mutations conferring this phage resistance were found to have occurred at at least six different loci, including the three that had been previously shown to be required for fimbrial biosynthesis or function: the structural subunit (pilA) and adjacent genes (pilB,C,D), the twitching motility gene (pilT), and the Sigma 54 RNA polymerase initiation factor gene (rpoN). One novel group of phage‐resistant mutants was identified in which the transposon had inserted near sequences that cross‐hybridized to an oligonucleotide probe designed against conserved domains in regulators of RpoN‐dependent promoters. These mutants had no detectable transcription of pilA and did not produce fimbriae. A probe derived from inverse polymerase chain reaction was used to isolate the corresponding wild‐type sequences from a P. aeruginosa PAO cosmid reference library, and two adjacent genes affected by transposon insertions, pilS and pilR, were located and sequenced. These genes were shown to be capable of complementing the corresponding mutants, both at the level of restoring the phenotypes associated with functional fimbriae and by the restoration of pilA transcription. The pilSR operon was physically mapped to Spel fragment 5 (corresponding to about 72–75/0 min on the genetic map), and shown to be located approximately 25 kb from pilA–D. PilS and PilR clearly belong to the family of two‐component transcriptional regulatory systems which have been described in many bacterial species. PilS is predicted to be a sensor protein which when stimulated by the appropriate environmental signals activates PilR through kinase activity. PilR then activates transcription of pilA, probably by interacting with RNA polymerase containing RpoN. The identification of pilS and pilR makes possible a more thorough examination of the signal transduction systems controlling expression of virulence factors in P. aeruginosa.
Functional analysis of the cya promoter of Bordetella pertussis
Authors: Goyard Sophie , Ullmann Agnes
Published: October 2006

The cyaA gene of Bordetella pertussis and of Bordetella bronchiseptica encodes a toxin which is a bifunctional protein exhibiting adenylate cyclase and haemolytic activities. In Bordetella, virulence factors are synthesized under the control of the bvg regulatory locus, in response to environmental signals. In Escherichia coli the cyaA gene is not expressed, nor is it activated by bvg indicating that the activation of cya by bvg is indirect. To characterize cis‐acting regulatory regions required for the activation of the cyaA gene we constructed cyaA–lacZY fusions containing progressive deletions in the promoter upstream region and isolated promoter mutations by chemical and site‐directed mutagenesis. Deletion analysis shows that a region extending from −569 to −136 bp upstream from the start site of transcription is required for transactivation by bvg, suggesting that multiple binding sites are involved in the activation of the cyaA promoter. No single or double mutations in the promoter upstream region were found which conferred inactive or bvg‐independent Cya phenotype. A double mutation in positions +10 and +13, relative to the transcription start site, rendered the promoter bvg‐independent and functional in E. coli. The constitutive mutations create a new transcription start site, 20 bp downstream from the witd‐type site, by providing new −10 and −35 elements recognized by RNA polymerase alone.
Initiation of mRNA decay in Bacillus subtilis
Authors: Di Mari John F. , Bechhofer David H.
Published: October 2006

Ribosome stalling in the leader region of ermC mRNA results in a 10–15‐fold increase in ermC mRNA half‐life in Bacillus subtilis. Fusion of the ermC 5′ regulatory region to several B. subtilis coding sequences resulted in induced stability of the fusion RNAs, showing that the ermC 5′ region acts as a general ‘5′ stabilizer’. RNA products of an ermC–lacZ transcriptional fusion were inducibly stable in the complete absence of translation and included a small RNA that is likely to be a decay product arising by blockage of a 3′‐to‐5′ exoribonuclease activity. Insertion of sequences that encode endonucleolytic cleavage sites into the ermC coding sequence resulted in cleavage products whose stability depended on the nature of their 5′ and 3′ ends. It can be concluded from this study that initiation of mRNA decay in B. subtilis generally occurs at or near the 5′ terminus.
A 55 kDa antigen of Pneumocystis carinii: analysis of the cellular immune response and characterization of the gene
Authors: Smulian A. G. , Theus S. A. , Denko N. , Walzer P. D. , Stringer J. R.
Published: October 2006

Rat‐derived Pneumocystis carinii contains a major antigen complex of 45–55 kDa. The fusion protein of a cDNA encoding the 3′ portion of the 55 kDa antigen, which had previously been shown to be recognized by serum antibodies of exposed subjects, was investigated for its ability to stimulate a cellular immune response. Rats exposed to P. carinii via the environment exhibited a vigorous proliferative response to the antigen whereas unexposed rats did not. The full‐length cDNA for a 55 kDa antigen was cloned and found to contain a 1245 bp open reading frame capable of encoding a 414‐amino‐acid peptide. The gene encoding this protein contained a single 39 bp intron and transcribed a 1.45 kb RNA message. The cloning and characterization of the 55 kDa antigen gene will allow production of the specific immunological reagents necessary to characterize this molecule and study its role in the biology and pathogenesis of P. carinii.
Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi
Authors: Py Béatrice , Chippaux Marc , Barras Frédéric
Published: October 2006

Extracellular secretion of endoglucanase Z (EGZ) from Erwinia chrysanthemi is mediated by the so‐called Out general secretion pathway and, presumably, involves recognition of EGZ‐carried structural information by one or more of the Out proteins. Investigating the relationships between structure and secretability of EGZ was the purpose of the present work. EGZ is made of two independent domains, located at the N‐and C‐proximal sides, separated by a Ser/Thr‐rich region, which are responsible for catalysis and cellulose‐binding, respectively. The existence of a secretion region (‘targeting signal’) was investigated by studying the secretability of modified EGZ derivatives. These resulted from deletion or peptide insertion and were designed by using the domain organization cited above as a guide. Catalytic and/or cellulose‐binding tests showed that all proteins exhibited at least a functional EGZ domain while immunoblot analyses confirmed that neither the insertions nor the deletions led to grossly misfolded proteins. In contrast, all of the proteins lost their secretability in E. chrysanthemi. This suggested that at least two secretion motifs existed, one lying within each functional domain. The role of the Ser/Thr‐rich linker region was subsequently tested. Accordingly, two proteins containing a linker region whose length was increased by the addition of 8 and 18 additional residues and one protein lacking the linker region were studied. All three exhibited endoglucanase activity and cellulose‐binding ability, confirming the independence of the domains within the context of EGZ/polysaccharide interaction. In contrast, none was secreted by E. chrysanthemi. Collectively, our results with EGZ (i) suggest the occurrence of multiple secretion‐related sites either acting sequentially or forming a single three‐dimensional secretion signal, (ii) show that secretability is not determined by either one of the two functional domains alone, and (iii) reveal that the linker region plays a role in secretion. We propose that all EGZ derivatives were impaired in the recognition step, the nature of which is discussed.
TraM of plasmid R1 regulates its own expression
Authors: Schwab Margit , Reisenzein Helga , Högenauer Gregor
Published: October 2006

Regulation of the traM gene, which encodes a factor essential for conjugation of resistance plasmid R1, was studied in vivo using translational gene fusions. tram″lacZ fusion constructs were transferred to the chromosome via the recombinant phage λRZ5. The level of β‐galactosidase expressed by the lysogens indicates that the traM promoters are very active. Expression of traM was diminished five‐ to sixfold when the single‐copy plasmids R1 or R1‐19 were present in trans. When recombinant plasmids carrying traM were present at higher copy numbers, traM expression was reduced as much as 45‐fold. The negative effect of R1 plasmids on traM expression in trans, which we interpret as autoregulation, was observed regardless of whether the plasmids were conjugatjvely repressed or derepressed. Site‐specific mutagenesis of the region encoding the N‐terminus of the TraM protein eliminated the autoregulative effect indicating that the N‐terminal amino acids of the protein are important to its DNA‐binding function. The autoregulatory behaviour of TraM is allele specific. R1‐ or P307‐encoded TraM molecules were found to recognize only the cognate DNA.