bluuebunny/crossref_metadata_2025 huggingface dataset:
Variations of Candida albicans electrophoretic karyotypes
Authors: Rustchenko-Bulgac E P
Published: November 2016

We previously described 14 rare spontaneous morphological mutants of Candida albicans that were associated with chromosomal aberrations (E. P. Rustchenko-Bulgac, F. Sherman, and J. B. Hicks, J. Bacteriol. 172:1276-1283, 1990). Improved conditions for separation of chromosomes, as well as hybridization probes, were used to investigate the variation of karyotypes of clinical isolates and additional morphological mutants. All 23 newly analyzed morphological mutants, representing frequently occurring and highly unstable colonial forms, had a variety of altered karyotypes. All chromosomal changes were similar to those previously observed in mutants m1 to m14. In this study, I particularly noted that the most frequent changes involved the long chromosome VIII, which carries ribosomal DNA cistrons. Two rates of instability were uncovered by analyzing the progenies from two highly unstable mutants. An unstable mutant proved to be able to continuously produce a large number of altered karyotypes that could result in a wide variety of different phenotypes. Furthermore, all four independent clinical isolates, FC18, C9, 3153A, and WO-1, common laboratory strains, revealed different electrophoretic karyotypes and distinct colonial morphologies on a synthetic medium, similar to spontaneous mutants. The differences of electrophoretic karyotypes observed among clinical isolates resembled the changes found among different kinds of spontaneous morphological mutants. These findings contribute to the understanding of natural karyotypic variability and are in agreement with the hypothesis that chromosomal alterations observed spontaneously under laboratory conditions provide this amictic species with genetic variability in nature.
Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51
Authors: van der Meer J R , Zehnder A J , de Vos W M
Published: November 2016

Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp. strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280. Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position. When a 12-kb HindIII fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, Tn5280 was found to transpose into the genome at random and in single copy. The insertion elements IS1066 and IS1067 differed in a single base apir located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei. The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways.
Recombination in Escherichia coli and the definition of biological species
Authors: Dykhuizen D E , Green L
Published: November 2016

The DNA sequence of part of the gnd (6-phosphogluconate dehydrogenase) gene was determined for eight wild strains of Escherichia coli and for Salmonella typhimurium. Since a region of the trp (tryptophan) operon and the phoA (alkaline phosphatase) gene have been sequenced from the same strains, the gene trees for these three regions were determined and compared. Gene trees are different from species or strain trees in that a gene tree is derived from a particular segment of DNA, whereas a species or strain tree should be derived from many such segments and is the tree that best represents the phylogenetic relationship of the species or strains. If there were no recombination in E. coli, the gene trees for different genes would not be statistically different from the strain tree or from each other. But, if the gene trees are significantly different, there must have been recombination. Methods are proposed that show these gene trees to be statistically different. Since the gene trees are different, we conclude that recombination is important in natural populations of E. coli. Finally, we suggest that gene trees can be used to create an operational means of defining bacterial species by using the biological species definition.
Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene
Authors: Kuroda A , Sekiguchi J
Published: November 2016

A major Bacillus subtilis 168S autolysin (N-acetylmuramoyl-L-alanine amidase [EC 3.5.1.28]) was purified and then cleaved with cyanogen bromide. The N-terminal amino acid sequence of one of the resultant peptides was determined in order to make synthetic oligonucleotides. A 2.5-kb EcoRI fragment was cloned into Escherichia coli JM109 and detected by colony hybridization by using the oligonucleotides as probes. Sequencing of the insert showed the presence of an open reading frame (designated cwlB), starting at a UUG codon, which encodes a polypeptide of 496 amino acids with a molecular mass of 52,623 Da. CWLB had a presumed signal peptide which is processed after Ala at position 24. Insertional inactivation of the cwlB gene of the B. subtilis chromosome led to an approximately 90% decrease in the total cell wall hydrolytic activity of stationary-phase cells and extraordinary resistance to cell lysis, even after 6 days of incubation at 37 degrees C. No apparent changes in cell morphology, motility, competence, sporulation, or germination were observed.
Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase
Authors: Gosalbes M J , Pérez-González J A , González R , Navarro A
Published: November 2016

Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtilis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows alpha-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O. Grépinet, M. C. Chebrou, and P. Béguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids.
The Escherichia coli mraY gene encoding UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase
Authors: Ikeda M , Wachi M , Jung H K , Ishino F , Matsuhashi M
Published: November 2016

Amplification of the mraY gene, previously called open reading frame Y (ORF-Y, 1,080 bp), at 2 min in the chromosome map of Escherichia coli enhanced the activity of UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase (EC 2.7.8.13). This enzyme catalyzes the formation of undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide from UDP-N-acetylmuramoyl-pentapeptide and undecaprenyl-phosphate, the first step in the lipid cycle reactions in biosynthesis of bacterial cell wall peptidoglycans. The enhanced enzyme activity was sensitive to tunicamycin, and the amino tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase of Saccharomyces cerevisiae. Very probably mraY is the structural gene for the above enzyme.
Identification of three amino acid residues in the B subunit of Shiga toxin and Shiga-like toxin type II that are essential for holotoxin activity
Authors: Perera L P , Samuel J E , Holmes R K , O'Brien A D
Published: November 2016

Shiga toxin of Shigella dysenteriae type I and Shiga-like toxins I and II (SLT-I and SLT-II, respectively) of enterohemorrhagic Escherichia coli are functionally similar protein cytotoxins. These toxin molecules have a bipartite molecular structure which consists of an enzymatically active A subunit that inhibits protein synthesis in eukaryotic cells and an oligomeric B subunit that binds to globotriaosylceramide glycolipid receptors on eukaryotic cells. Regionally directed chemical mutagenesis of the B subunit of SLT-II was used to identify amino acids in the B subunit that are critical for SLT-II holotoxin cytotoxic activity. Three noncytotoxic mutants were isolated, and their mutations were mapped. The substitutions of arginine with cysteine at codon 32, alanine with threonine at codon 42, and glycine with aspartic acid at codon 59 in the 70-amino-acid mature SLT-II B polypeptide resulted in the complete abolition of cytotoxicity. The analogous arginine, alanine, and glycine residues were conserved at codons 33, 43, and 60 in the 69-amino-acid mature B polypeptide of Shiga toxin. Comparable mutations induced in the B-subunit gene of Shiga toxin by oligonucleotide-directed, site-specific mutagenesis resulted in drastically decreased cytotoxicity (10(3)- to 10(6)-fold) as compared with that of wild-type Shiga toxin. The mutant SLT-II and Shiga toxin B subunits were characterized for stability, receptor binding, immunoreactivity, and ability to be assembled into holotoxin.
p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol
Authors: Hopper D J , Bossert I D , Rhodes-Roberts M E
Published: November 2016

A bacterium, strain PC-07, previously isolated as part of a coculture capable of growing on p-cresol under anaerobic conditions with nitrate as the acceptor was identified as an Achromobacter sp. The first enzyme of the pathway, p-cresol methylhydroxylase, which converts its substrate into p-hydroxybenzyl alcohol, was purified. The enzyme had an Mr of 130,000 and the spectrum of a flavocytochrome. It was composed of flavoprotein subunits of Mr 54,000 and cytochrome subunits of Mr 12,500. The midpoint redox potential of the cytochrome was 232 mV. The Km and kcat for p-cresol were 21 microM and 112 s-1 respectively, and the Km for phenazine methosulfate, the artificial acceptor used in the assays, was determined to be 1.7 mM. These properties place the enzyme in the same class as the p-cresol methylhydroxylases from aerobically isolated Pseudomonas spp.
Activity of the nicotinamide mononucleotide transport system is regulated in Salmonella typhimurium
Authors: Zhu N , Olivera B M , Roth J R
Published: November 2016

Transport of nicotinamide mononucleotide (NMN) requires two functions, NadI(T) and PnuC. The PnuC protein is membrane associated, as judged by isolation of active TnphoA gene fusions and demonstration that the fusion protein is membrane associated. The PnuC function appears to be the major component of the transport system, since mutant alleles of the pnuC gene permit NMN transport in the absence of NadI(T) function. We present evidence that the activity of the NMN transport system varies in response to internal pyridine levels (presumably NAD). This control mechanism requires NadI(T) function, which is provided by a bifunctional protein encoded by the nadI gene (called nadR by Foster and co-workers [J. W. Foster, Y. K. Park, T. Fenger, and M. P. Spector, J. Bacteriol. 172:4187-4196]). The nadI protein regulates transcription of the nadA and nadB biosynthetic genes and modulates activity of the NMN permease; both regulatory activities respond to the internal pyridine nucleotide level.
High-frequency rearrangements in Rhizobium leguminosarum bv. phaseoli plasmids
Authors: Brom S , García de los Santos A , de Lourdes Girard M , Dávila G , Palacios R , Romero D
Published: November 2016

High-frequency genomic rearrangements affecting the plasmids of Rhizobium leguminosarum bv. phaseoli CFN42 were analyzed. This strain contains six large plasmids ranging in size from 200 to 600 kb. In the absence of any selective pressure, we found 11 strains from 320 analyzed colonies that presented different kinds of plasmid-borne rearrangements, including sequence amplification, deletion, cointegration, and loss of plasmids. These data support the concept that the R. leguminosarum bv. phaseoli genome is a dynamic structure and imply that strains are mixtures of similar but not identical cells.
Molecular characterization of the Escherichia coli K-12 zwf gene encoding glucose 6-phosphate dehydrogenase
Authors: Rowley D L , Wolf R E
Published: November 2016

In Escherichia coli K-12, expression of zwf, the gene for glucose 6-phosphate dehydrogenase, is coordinated with the cellular growth rate and induced by superoxide-generating agents. To initiate the study of the molecular mechanisms regulating its expression, the gene was cloned and its DNA sequence was determined. The 5' ends of zwf mRNA isolated from cells growing in glucose and acetate minimal media were mapped. The map was complex in that transcripts mapped to -45, -52, and -62, with respect to the beginning of the coding sequence. Three analytical methods were used to search the DNA sequence for putative promoters. Only one sequence for a promoter recognized by the sigma 70 form of RNA polymerase was found by all three search routines that could be aligned with a mapped transcript, indicating that the other transcripts arise by processing of the mRNA. A computer-assisted search did not reveal a thermodynamically stable long-range mRNA secondary structure that is capable of sequestering the translation initiation region, which suggests that growth-rate-dependent regulation of glucose 6-phosphate dehydrogenase level may not be carried out by a mechanism similar to the one for the gene (gnd) for 6-phosphogluconate dehydrogenase. The DNA segment between the -10 hexamer and the start point of transcription resembles the discriminator sequence of stable RNA genes, which has been implicated in stringent control and growth-rate-dependent regulation.
Analysis of Mycoplasma hyorhinis DNA in the presence of host cells without growing the mycoplasma axenically
Authors: Schaeffer W I , Olson L D , Barile M F , Sun F W
Published: November 2016

Mycoplasma hyorhinis coisolates with the mitochondria of the cells in which it is carried as an infection. Since both mitochondria and mycoplasmas synthesize DNA by using the prokaryotic DNA polymerase gamma, the use of aphidicolin, which inhibits eukaryotic DNA polymerase alpha, allows for selective synthesis of mycoplasmal and mitochondrial DNA. The restriction patterns of mitochondria and mycoplasmas can easily be differentiated from each other in mixtures of both DNAs. Thus, it is possible to study the molecular biology of this noncultivable mycoplasma in situ rather than after growth in artificial media, with its potential genetic consequences during adjustment to axenic growth.
Freeze-substitution of gram-negative eubacteria: general cell morphology and envelope profiles
Authors: Graham L L , Harris R , Villiger W , Beveridge T J
Published: November 2016

Freeze-substitution was performed on strains of Escherichia coli, Pasteurella multocida, Campylobacter fetus, Vibrio cholerae, Pseudomonas aeruginosa, Pseudomonas putida, Aeromonas salmonicida, Proteus mirabilis, Haemophilus pleuropneumoniae, Caulobacter crescentus, and Leptothrix discophora with a substitution medium composed of 2% osmium tetroxide and 2% uranyl acetate in anhydrous acetone. A thick periplasmic gel ranging from 10.6 to 14.3 nm in width was displayed in E. coli K-12, K30, and His 1 (a K-12 derivative containing the K30 capsule genes), P. multocida, C. fetus, P. putida, A. salmonicida, H. pleuropneumoniae, and P. mirabilis. The other bacteria possessed translucent periplasms in which a thinner peptidoglycan layer was seen. Capsular polysaccharide, evident as electron-dense fibers radiating outward perpendicular to the cell surface, was observed on E. coli K30 and His 1 and P. mirabilis cells. A more random arrangement of fibers forming a netlike structure was apparent surrounding cells of H. pleuropneumoniae. For the first time a capsule, distinct from the sheath, was observed on L. discophora. In all instances, capsular polysaccharide was visualized in the absence of stabilizing agents such as homologous antisera or ruthenium red. Other distinct envelope structures were observed external to the outer membrane including the sheath of L. discophora and the S layers of A. salmonicida A450 and C. crescentus CB15A. We believe that the freeze-substitution technique presents a more accurate image of the structural organization of these cells and that it has revealed complex ultrastructural relationships between cell envelope constituents previously difficult to visualize by more conventional means of preparation.
Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis
Authors: Cheo D L , Bayles K W , Yasbin R E
Published: November 2016

DNA damage-inducible (din) genes in Bacillus subtilis are coordinately regulated and together compose a global regulatory network that has been termed the SOS-like or SOB regulon. To elucidate the mechanisms of SOB regulation, operator/promoter regions from three din loci (dinA, dinB, and dinC) of B. subtilis were cloned. Operon fusions constructed with these cloned din promoter regions rendered reporter genes damage inducible in B. subtilis. Induction of all three din promoters was dependent upon a functional RecA protein. Analysis of these fusions has localized sequences required for damage-inducible expression of the dinA, dinB, and dinC promoters to within 120-, 462-, and 139-bp regions, respectively. Comparison of the nucleotide sequences of these three din promoters with the recA promoter, as well as with the promoters of other loci associated with DNA repair in B. subtilis, has identified the consensus sequence GAAC-N4-GTTC as a putative SOB operator site.
srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis
Authors: Nakano M M , Magnuson R , Myers A , Curry J , Grossman A D , Zuber P
Published: November 2016

The srfA locus of Bacillus subtilis is defined by a transposon Tn917 insertion and is required for production of the peptide secondary metabolite surfactin. The srfA locus was isolated by cloning the DNA flanking srfA::Tn917 insertions followed by chromosome walking. The cloned region is an operon of over 25 kb which covers the transcription initiation region but not the intact 3' end of srfA. csh-293, which was previously identified as a Tn917lac mutation that impairs competence development and causes a conditional defect in sporulation, was known to be located in the vicinity of the srfA locus within the B. subtilis genome. The csh-293::Tn917lac mutation was discovered to cause a defect in surfactin production and was shown to be located in the srfA operon by its cotransformation with srfA mutations and by Southern hybridization analysis. Insertion mutations in srfA, created by the chromosomal integration of plasmids bearing overlapping srfA DNA fragments, were examined for their effects on surfactin production, competence, and sporulation. All three processes were found to require the intact 5' half of the srfA operon, whereas the 3' half of srfA was found to be required for sporulation and surfactin production but not competence. These experiments show that srfA gene products function in B. subtilis cell specialization and differentiation.
MAL11 and MAL61 encode the inducible high-affinity maltose transporter of Saccharomyces cerevisiae
Authors: Cheng Q , Michels C A
Published: November 2016

We have investigated the transport of maltose in a genetically defined maltose-fermenting strain of Saccharomyces cerevisiae carrying the MAL1 locus. Two kinetically different systems were identified: a high-affinity transporter with a Km of 4 mM and a low-affinity transporter with a Km of 70 to 80 mM. The high-affinity maltose transporter is maltose inducible and is encoded by the MAL11 (and/or MAL61) gene of the MAL1 (and/or MAL6) locus. The low-affinity maltose transporter is expressed constitutively and is not related to MAL11 and/or MAL61. Both maltose transporters are subject to glucose-induced inactivation.
Effect of promoter mutations and upstream deletions on the expression of genes coding for small, acid-soluble spore proteins of Bacillus subtilis
Authors: Fajardo-Cavazos P , Tovar-Rojo F , Setlow P
Published: November 2016

The sspB and sspE genes code for major small, acid-soluble proteins of Bacillus subtilis spores and are transcribed during sporulation by RNA polymerase containing sigma G. Analysis of the expression in vivo and the sigma G-dependent transcription in vitro of sspB and sspE genes carrying upstream deletions or point mutations in -10 and -35 promoter regions is consistent with sigma G being the only major transcriptional regulator of these genes. These data also provide information on the residues in -10 and -35 regions which are most important for sigma G recognition.
Function of a relaxed-like state following temperature downshifts in Escherichia coli
Authors: Jones P G , Cashel M , Glaser G , Neidhardt F C
Published: November 2016

Temperature downshifts of Escherichia coli throughout its growth range resulted in transient growth inhibition and a cold shock response consisting of transient induction of several proteins, repression of heat shock proteins, and, despite the growth lag, continued synthesis of proteins involved in transcription and translation. The paradoxical synthesis of the latter proteins, which are normally repressed when growth is arrested, was explored further. First, by means of a nutritional downshift, a natural stringent response was induced in wild-type cells immediately prior to a shift from 37 to 10 degrees C. These cells displayed decreased synthesis of transcriptional and translational proteins and decreased induction of cold shock proteins; also, adaptation for growth at 10 degrees C was delayed, even after restoration of the nutrient supplementation. Next, the contribution of guanosine 5'-triphosphate-3'-diphosphate and guanosine 5'-diphosphate-3'-diphosphate, collectively abbreviated (p)ppGpp, to the alteration in cold shock response was studied with the aid of a mutant strain in which overproduction of these nucleotides can be artificially induced. Induction of (p)ppGpp synthesis immediately prior to shifting this strain from 37 to 10 degrees C produced results differing only in a few details from those described above for nutritional downshift of the wild-type strain. Finally, shifting a relA spoT mutant, which cannot synthesize (p)ppGpp, from 24 to 10 degrees C resulted in a greater induction of the cold shock proteins, increased synthesis of transcriptional and translational proteins, decreased synthesis of a major heat shock protein, and faster adaptation to growth than for the wild-type strain. Our results indicate that the previously reported decrease in the (p)ppGpp level following temperature downshift plays a physiological role in the regulation of gene expression and adaptation for growth at low temperature.
Attachment sites for bacteriophage P2 on the Escherichia coli chromosome: DNA sequences, localization on the physical map, and detection of a P2-like remnant in E. coli K-12 derivatives
Authors: Barreiro V , Haggård-Ljungquist E
Published: November 2016

Integration of bacteriophage P2 into the Escherichia coli genome involves recombination between two attachment sites, attP and attB, one on the phage and one on the host genome, respectively. At least 10 different attB sites have been identified over the years. In E. coli C, one site, called locI, is preferred, being occupied before any of the others. In E. coli K-12, no such preference is seen (reviewed in L. E. Bertani and E. W. Six, p. 73-143, in R. Calendar, ed., The Bacteriophages, vol. 2, 1988). The DNA sequence of locI has been determined, and it shows a core sequence of 27 nucleotides identical to attP (A. Yu, L. E. Bertani, and E. Haggård-Ljungquist, Gene 80:1-12, 1989). By inverse polymerase chain reactions, the prophage-host junctions of DNA extracted from P2 lysogenic strains have been amplified, cloned, and sequenced. By combining the attL and attR sequences, the attB sequences of locations II, III, and H have been deduced. The core sequence of location II had 20 matches to the 27-nucleotide core sequence of attP; the sequences of locations III and H had 17 matches. Thus, the P2 integrase accepts at least up to 37% mismatches within the core sequence. The E. coli K-12 strains examined all contain a 639-nucleotide-long cryptic remnant of P2 at a site with a sequence similar to that of locI but that may have a different map position. The P2 remnant consists of the C-terminal part of gene D, all of gene ogr, and attR. Locations II, III, and H have been located on Kohara's physical map to positions 3670, 1570 to 1575, and 2085, respectively.
Purification and some properties of sulfite:ferric ion oxidoreductase from Thiobacillus ferrooxidans
Authors: Sugio T , Hirose T , Ye L Z , Tano T
Published: November 2016

Sulfite:ferric ion oxidoreductase in the plasma membrane of Thiobacillus ferrooxidans AP19-3 was purified to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 650,000 and was composed of two subunits (M(rs), 61,000 and 59,000) as estimated by sodium sulfate-polyacrylamide gel electrophoresis. The Michaelis constants of sulfite:ferric ion oxidoreductase for Fe3+ and sulfite ions were 1.0 and 0.071 mM, respectively. Sulfite:ferric ion oxidoreductase suffered from end product inhibition by 1 mM Fe2+.
Sugar-glycerol cofermentations in lactobacilli: the fate of lactate
Authors: Veiga da Cunha M , Foster M A
Published: November 2016

The simultaneous fermentation of glycerol and sugar by lactobacillus brevis B22 and Lactobacillus buchneri B190 increases both the growth rate and total growth. The reduction of glycerol to 1,3-propanediol by the lactobacilli was found to influence the metabolism of the sugar cofermented by channelling some of the intermediate metabolites (e.g., pyruvate) towards NADH-producing (rather than NADH-consuming) reactions. Ultimately, the absolute requirement for NADH to prevent the accumulation of 3-hydroxypropionaldehyde leads to a novel lactate-glycerol cofermentation. As a result, additional ATP can be made not only by (i) converting pyruvate to acetate via acetyl phosphate rather than to the ethanol usually found and (ii) oxidizing part of the intermediate pyruvate to acetate instead of the usual reduction to lactate but also by (iii) reoxidation of accumulated lactate to acetate via pyruvate. The conversion of lactate to pyruvate is probably catalyzed by NAD-independent lactate dehydrogenases that are found only in the cultures oxidizing lactate and producing 1,3-propanediol, suggesting a correlation between the expression of these enzymes and a raised intracellular NAD/NADH ratio. The enzymes metabolizing glycerol (glycerol dehydratase and 1,3-propanediol dehydrogenase) were expressed in concert without necessary induction by added glycerol, although their expression may also be influenced by the intracellular NAD/NADH ratio set by the different carbohydrates fermented.
Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons
Authors: Cline S W , Doolittle W F
Published: November 2016

We have stably transformed both Haloarcula vallismortis and Haloarcula hispanica with the halobacterium-Escherichia coli shuttle vectors pWL102 (based on the Haloferax volcanii pHV2 replicon) and pUBP2 (based on the Halobacterium halobium pHH1 replicon). Haloferax volcanii, Halobacterium halobium, and Haloarcula vailismortis are equally distant from one another and span the phylogenetic depth of the halophilic Archaea; thus, these vectors may be generally useful for the halophiles. Both Haloarcula vallismortis and Haloarcula hispanica exhibit previously unreported complex life cycles and are therefore significant as genetically approachable models of cellular differentiation within the Archaea.
Positive and negative control of ompB transcription in Escherichia coli by cyclic AMP and the cyclic AMP receptor protein
Authors: Huang L , Tsui P , Freundlich M
Published: November 2016

The ompB operon encodes OmpR and EnvZ, two proteins that are necessary for the expression and osmoregulation of the OmpF and OmpC porins in Escherichia coli. We have used in vitro and in vivo experiments to show that cyclic AMP and the cyclic AMP receptor protein (CRP) directly regulate ompB. ompB expression in an ompB-lacZ chromosomal fusion strain was increased two- to fivefold when cells were grown in medium containing poor carbon sources or with added cyclic AMP. In vivo primer extension analysis indicated that this control is complex and involves both positive and negative effects by cyclic AMP-CRP on multiple ompB promoters. In vitro footprinting showed that cyclic AMP-CRP binds to a 34-bp site centered at -53 and at -75 in relation to the start sites of the major transcripts that are inhibited and activated, respectively, by this complex. Site-directed mutagenesis of the crp binding site provided evidence that this site is necessary for the in vivo regulation of ompB expression by cyclic AMP. Control of the ompB operon by cyclic AMP-CRP may account for the observed regulation of the formation of OmpF and OmpC by this complex (N. W. Scott and C. R. Harwood, FEMS Microbiol. Lett. 9:95-98, 1980).
Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB
Authors: Karow M , Georgopoulos C
Published: November 2016

Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5 degrees C but not at lower temperatures. In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB. The msbB gene has been mapped to 40.5 min on the E. coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB. The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature. The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles. Mutational analysis of msbB shows that the gene is not essential for E. coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30 degrees C not seen with either of the single mutants. Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances. The addition of quaternary ammonium compounds rescues the temperature-sensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2+ or Ca2+. These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function.
Expression of the Xanthomonas campestris pv. vesicatoria hrp gene cluster, which determines pathogenicity and hypersensitivity on pepper and tomato, is plant inducible
Authors: Schulte R , Bonas U
Published: November 2016

The hrp gene cluster from Xanthomonas campestris pv. vesicatoria determines functions necessary not only for pathogenicity on the host plants pepper and tomato but also for the elicitation of the hypersensitive reaction on resistant host and nonhost plants. Transcriptional orientation and expression of the hrp loci were determined with hrp::Tn3-gus fusions. In addition, expression of the hrp loci was studied by RNA hybridization experiments. Expression of the hrp genes was not detectable after growth of the bacteria in complex medium or in minimal medium. However, high levels of induction of hrp gene expression were measured during growth of the bacteria in the plant. To search for a plant molecule responsible for this induction, we examined a variety of materials of plant origin for their ability to induce hrp gene expression. Filtrates from plant suspension cultures induced hrp genes to levels comparable to those induced in the plant. The inducing molecule(s) was found to be heat stable and hydrophilic and to have a molecular mass of less than 1,000 daltons.
Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12
Authors: Parker C T , Pradel E , Schnaitman C A
Published: November 2016

The rfa locus of Escherichia coli K-12 includes a block of about 10 closely spaced genes transcribed in the same direction which are involved in synthesis and modification of the hexose region of the lipopolysaccharide core. We have sequenced the first three genes in this block. The function of the first of these genes is unknown, but we have designated it rfaQ on the basis of its location and similarity to other rfa genes. Complementation of Salmonella typhimurium rfa mutants with E. coli rfa restriction fragments indicated that the second and third genes in the block were rfaG and rfaP. The deduced sizes of the RfaQ, RfaG, and RfaP proteins are 36,298, 42,284, and 30,872 Da, respectively, and the proteins are basic and lack extensive hydrophobic domains. RfaQ shares regions of homology with proteins RfaC and RfaF, which are involved in synthesis of the heptose region of the core. Proteins RfaB, RfaG, and RfaK share a region of homology, which suggests that they belong to a second family of Rfa proteins which are thought to be hexose transferases.
The expression of virulence genes in Listeria monocytogenes is thermoregulated
Authors: Leimeister-Wächter M , Domann E , Chakraborty T
Published: November 2016

The expression of listeriolysin, a major virulence factor of the gram-positive facultative intracellular pathogen Listeria monocytogenes, is positively regulated by a transcriptional activator, the prfA gene product. We had previously shown that mutations within the prfA gene lead to loss of listeriolysin production. In this communication, the regulation of expression of listeriolysin by a specific environmental condition, namely, temperature, was studied in wild-type strains of Listeria monocytogenes. We found that expression of the hemolysis phenotype was thermoregulated. A lisA::lacZ fusion was constructed, and its expression in the wild-type strain was studied at various growth temperatures. The results showed that the fusion beta-galactosidase activity was expressed only when cultures were grown at temperatures above 30 degrees C. This activity could be either specifically repressed or induced, depending on growth temperature. No change in activity was detected in a strain harboring a control beta-galactosidase fusion at the various growth temperatures tested. Northern (RNA) blot analysis of lisA-specific RNA transcripts showed that thermoregulation is manifested at the level of transcription. We also found that the transcription of other PrfA-regulated virulence genes in L. monocytogenes was similarly affected by growth temperature. Hence, as in other facultative intracellular pathogens, Shigella and Yersinia spp., temperature is an important cue in the induction of expression of virulence genes in L. monocytogenes. Our studies revealed that a higher level of regulation is imposed on the PrfA-mediated activation of virulence genes in pathogenic L. monocytogenes.
Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression
Authors: Gray J , Wang J , Gelvin S B
Published: November 2016

vir regulon expression in Agrobacterium tumefaciens involves both chromosome- and Ti-plasmid-encoded gene products. We have isolated and characterized a new chromosomal gene that when mutated results in a 2- to 10-fold reduction in the induced expression of vir genes by acetosyringone. This reduced expression occurs in AB minimal medium (pH 5.5) containing either sucrose or glucose and containing phosphate at high or low concentrations. The locus was cloned and used to complement A. tumefaciens strains harboring Tn5 insertions in the gene. Sequence analysis of this locus revealed an open reading frame with strong homology to the miaA locus of Escherichia coli and the mod5 locus of Saccharomyces cerevisiae. These genes encode tRNA: isopentenyltransferase enzymes responsible for the specific modification of the A-37 residue in UNN codon tRNA species. The function of the homologous gene in A. tumefaciens was proven by genetic complementation of E. coli miaA mutant strains. tRNA undermodification in A. tumefaciens miaA mutant strains may reduce vir gene expression by causing a reduced translation efficiency. A slight reduction in the virulence of these mutant Agrobacterium strains on red potato plants, but not on tobacco, tomato, kalanchoe, or sunflower plants, was observed.
Characterization of the novel nisin-sucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis
Authors: Rauch P J , De Vos W M
Published: November 2016

A novel, chromosomally located conjugative transposon in Lactococcus lactis, Tn5276, was identified and characterized. It encodes the production of and immunity to nisin, a lanthionine-containing peptide with antimicrobial activity, and the capacity to utilize sucrose via a phosphotransferase system. Conjugal transfer of Tn5276 was demonstrated from L. lactis NIZO R5 to different L. lactis strains and a recombination-deficient mutant. The integration of Tn5276 into the plasmid-free strain MG1614 was analyzed by using probes based on the gene for the nisin precursor (nisA) and the gene for sucrose-6-phosphate hydrolase (sacA). The transposon inserted at various locations in the MG1614 chromosome and showed a preference for orientation-specific insertion into a single target site (designated site 1). By using restriction mapping in combination with field inversion gel electrophoresis and DNA cloning of various parts of the element including its left and right ends, a physical map of the 70-kb Tn5276 was constructed, and the nisA and sacA genes were located. The nucleotide sequences of Tn5276 junctions in donor strain NIZO R5 and in site 1 of an MG1614-derived transconjugant were determined and compared with that of site 1 in recipient strain MG1614. The results show that the A + T-rich ends of Tn5276 are flanked by a direct hexanucleotide repeat in both the donor and the transconjugant but that the element does not contain a clear inverted repeat.
The central domain of Rhizobium leguminosarum DctD functions independently to activate transcription
Authors: Huala E , Stigter J , Ausubel F M
Published: November 2016

Sigma 54-dependent transcriptional activators such as Escherichia coli NtrC, Rhizobium meliloti NifA, and Rhizobium leguminosarum DctD share similar central and carboxy-terminal domains but differ in the structure and function of their amino-terminal domains. We have deleted the amino-terminal and carboxy-terminal domains of R. leguminosarum DctD and have demonstrated that the central domain of DctD, like that of NifA, is transcriptionally competent.
Cloning and sequencing of an Agrobacterium tumefaciens beta-glucosidase gene involved in modifying a vir-inducing plant signal molecule
Authors: Castle L A , Smith K D , Morris R O
Published: November 2016

Induction of Agrobacterium tumefaciens virulence genes by plant phenolic compounds is essential for successful T-DNA transfer to a host plant. In Douglas fir needles, the major virulence region inducer is the glycoside coniferin (J. W. Morris and R. O. Morris, Proc. Natl. Acad. Sci. USA 87:3612-3618, 1990). Agrobacterium strains with high beta-glucosidase activity respond to coniferin and infect Douglas fir seedlings, whereas most strains with low beta-glucosidase activity fail to respond to coniferin and are avirulent on this host. We have cloned two beta-glucosidase genes from A. tumefaciens B3/73 and sequenced one of them, cbg1. It appears to be part of a polycistronic unit and shows a high bias for GC-rich codons. When expressed in Escherichia coli, Cbg1 beta-glucosidase hydrolyzes coniferin but not cellobiose. The 88-kDa predicted product of cbg1 is highly similar to one other bacterial beta-glucosidase and several fungal beta-glucosidases. There is little homology between Cbg1 and other bacterial beta-glucosidases, including an Agrobacterium cellobiase.
Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase
Authors: Collis C M , Hall R M
Published: November 2016

Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.
Quantitation of Dam methyltransferase in Escherichia coli
Authors: Boye E , Marinus M G , Løbner-Olesen A
Published: November 2016

An antiserum against Escherichia coli Dam methyltransferase has been developed in rabbits and employed to detect and quantitate the enzyme in immunoblots. A wild-type, rapidly growing E. coli cell (doubling time = 30 min) was found to contain about 130 molecules of Dam methyltransferase.
Hyperhemolytic phenomena associated with insertions of Tn916 into the hemolysin determinant of Enterococcus faecalis plasmid pAD1
Authors: Ike Y , Flannagan S E , Clewell D B
Published: November 2016

Members of the Tn916 family of conjugative transposons are able to insert themselves into Enterococcus faecalis hemolysin/bacteriocin plasmid pAD1 (and related elements) in such a way as to generate hyperexpression of the hemolysin/bacteriocin. To examine this phenomenon in more detail, E. faecalis (pAD1::Tn916) derivatives defective or altered in hemolysin expression were isolated and characterized with respect to production of the L (lytic) or A (activator) component (also known as CylA) and the specific location of the transposon. The mutants fell into five classes. Class 1 strains were nonhemolytic, and the related insertions mapped in a location known to affect expression of the L component. The other four classes varied from an inability to express hemolysin (class 2) to different degrees of hyperhemolytic expression (classes 3 to 5); the insertions in these classes mapped in a similar place within cylA, near the 3' end of the determinant. A previous study provided evidence that CylA is also necessary for bacteriocin immunity; however, these insertions did not destroy this function. (A Tn917 insertion in the 5' half of the determinant eliminates immunity.) In mutant classes 3 to 5, the presence of tetracycline enhanced hemolysin expression. In late-exponential-phase broth cultures, hemolysin could not be detected in supernatants of classes 2 to 5, in contrast to a wild-type control strain; however, different amounts of the L component could be detected, with the lowest in class 2 and greater-than-normal amounts in classes 3 to 5. Although nucleotide sequencing showed that the Tn916 insertions in classes 2 to 5 were at identical sites, the transposon junction sequences differed in some cases. The data indicated that cylA translation into the transposon would result in different truncation sites, and these differences were probably related to phenotype differences.
The dehalogenase gene dehI from Pseudomonas putida PP3 is carried on an unusual mobile genetic element designated DEH
Authors: Thomas A W , Slater J H , Weightman A J
Published: November 2016

As a result of the production of two dehalogenases (DehI and DehII), Pseudomonas putida PP3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2MCPA), as sole sources of carbon and energy. The DehI gene (dehI) was carried on a mobile genetic element (DEH) located on the chromosome of strain PP3. DEH recombined with target plasmid DNAs at high frequencies (e.g. 3.8 x 10(-4) per RP4.5 plasmid transferred). The regulated expression of dehI was detected in P. putida, Pseudomonas aeruginosa, and Escherichia coli strains containing derivative plasmids of RP4.5 and pWW0 recombined with DEH. Movement of DEH from the unstable RP4 derivatives pNJ5000 and pMR5 resulted in the insertion of DEH into the chromosome of RecA+ strains of P. putida but not in RecA+ nor RecA- strains of E. coli. Rescue of DEH from the chromosome of P. putida KT2441 onto plasmid RP4 involved recombination at a frequency (2.7 x 10(-4) per RP4 plasmid transferred) comparable to that observed in strain PP3. The DEH element was not classified as a conventional transposon because it did not move as a discrete DNA fragment: dehI-containing inserts in plasmid DNA targets varied in size between 6 and 13 kb. In addition, DEH exhibited a marked preference for insertion into a specific site on the plasmid pWW0, but its transposition, independent of host recombinational systems, remains to be demonstrated. However, the transposonlike characteristics of DEH included the conservation of restriction endonuclease sites, high-frequency recombination with different target replicons (plasmid and chromosomal DNA), and promiscuous insertion into plasmid RP4-based replicons. Therefore, it is proposed that DEH is an unusual mobile genetic element.