bluuebunny/crossref_metadata_2025 huggingface dataset:
The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat
Authors: Parent R , Roy P H
Published: November 2016

We have sequenced the gene coding for the chloramphenicol acetyltransferase of Tn2424 of plasmid NR79. This gene codes for a protein of 23,500 Da, and the derived protein sequence is similar to those of the chromosomal chloramphenicol acetyltransferases of Agrobacterium tumefaciens and Pseudomonas aeruginosa and of unidentified open reading frames, which may encode chloramphenicol acetyltransferases, adjacent to the ermG macrolide-lincosamide-streptogramin resistance gene of Bacillus sphaericus and the vgb virginiamycin resistance gene of Staphylococcus aureus. Weaker similarity to the LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) proteins of Escherichia coli and the NodL protein of Rhizobium leguminosarum is also observed. There is no significant similarity to any other chloramphenicol acetyltransferase genes, such as that of Tn9. The Tn2424 cat gene is part of a 4.5-kb region which also contains the aacA1a aminoglycoside-6'-N-acetyltransferase gene; Tn2424 is similar to Tn21 except for the presence of this region. Sequences flanking the cat gene are typical of those flanking other genes inserted into pVS1-derived "integrons" by a site-specific recombinational mechanism.
Genetics of the white-opaque transition in Candida albicans: demonstration of switching recessivity and mapping of switching genes
Authors: Chu W S , Rikkerink E H , Magee P T
Published: November 2016

Spheroplast fusion has been used to analyze the genetics of the reversible phenotypic transition, white-opaque, in Candida albicans WO-1. This transition involves changes in cell shape, permeability, and colony morphology. Fusion of switching with nonswitching cells gave nonswitching fusants, suggesting that the white-opaque phenotype is recessive. Chromosome loss induced by heat shock gave segregants of the fusants which were able to undergo the transition, indicating that the repressor function is genetically defined and probably limited to one or two chromosomes. Chromosomes R, 1, 3, 4, and 7 were eliminated as unique sites for the repressor, leaving 2, 5, and 6 as possible locations. When a ura3 (chromosome 3) nonswitching strain was fused with a switching strain, all ura3 segregants induced by heat shock were incapable of the phenotypic transition. Therefore, some or all of the genes (called SWI genes) essential for the transition are located on chromosome 3. UV irradiation-induced recombination did give rise to Ura- switching progeny, showing that the failure to switch was not due to a side effect of the pyrimidine requirement. The failure to isolate normally switching ura3 progeny generated by UV irradiation suggests a close linkage between the two genes.
The lemA gene required for pathogenicity of Pseudomonas syringae pv. syringae on bean is a member of a family of two-component regulators
Authors: Hrabak E M , Willis D K
Published: November 2016

The lemA gene of the plant pathogen Pseudomonas syringae pv. syringae is required for disease lesion formation on bean plants. Cosmid clones that complemented a lemA mutant in trans were isolated previously. The lemA gene was localized by subcloning and transposon mutagenesis. The lemA region and flanking DNA were sequenced, and an open reading frame of 2.7 kb was identified. The nucleotide and predicted amino acid sequences of the lemA gene showed sequence similarity to a family of prokaryotic two-component regulatory proteins. Unlike most of the previously described two-component systems, the lemA gene product contained homology to both components in one protein. Mutations introduced upstream and downstream of the lemA gene failed to locate a gene for a second protein component but identified the putative cysM gene of P. syringae pv. syringae. The cysM gene was located upstream of the lemA gene and was divergently transcribed. The lemA gene product was expressed at low levels in P. syringae pv. syringae and appeared to be positively auto-regulated.
Regulation of tabtoxin production by the lemA gene in Pseudomonas syringae
Authors: Barta T M , Kinscherf T G , Willis D K
Published: November 2016

Pseudomonas syringae pv. coronafaciens, a pathogen of oats, was mutagenized with Tn5 to generate mutants defective in tabtoxin production. From a screen of 3,400 kanamycin-resistant transconjugants, seven independent mutants that do not produce tabtoxin (Tox-) were isolated. Although the Tn5 insertions within these seven mutants were linked, they were not located in the previously described tabtoxin biosynthetic region of P. syringae. Instead, all of the insertions were within the P. syringae pv. coronafaciens lemA gene. The lemA gene is required by strains of P. syringae pv. syringae for pathogenicity on bean plants (Phaseolus vulgaris). In contrast to the phenotype of a P. syringae pv. syringae lemA mutant, the Tox- mutants of P. syringae pv. coronafaciens were still able to produce necrotic lesions on oat plants (Avena sativa), although without the chlorosis associated with tabtoxin production. Northern (RNA) hybridization experiments indicated that a functional lemA gene was required for the detection of a transcript produced from the tblA locus located in the tabtoxin biosynthetic region. Marker exchange mutagenesis of the tblA locus resulted in loss of tabtoxin production. Therefore, both the tblA and lemA genes are required for tabtoxin biosynthesis, and the regulation of tabtoxin production by lemA probably occurs at the transcriptional level.
Isolation and characterization of kikA, a region on IncN group plasmids that determines killing of Klebsiella oxytoca
Authors: Hengen P N , Denicourt D , Iyer V N
Published: November 2016

Transfer of the IncN group plasmid pCU1 from Escherichia coli to Klebsiella oxytoca by conjugation kills a large proportion (90 to 95%) of the recipients of plasmid DNA, whereas transfer to E. coli or even to the closely related Enterobacter aerogenes does not. Two regions, kikA and kikB, have been identified on pCU1 that contribute to the Kik (killing in klebsiellas) phenotype. We have localized the kikA region to 500 bp by deletion analysis and show by DNA-DNA hybridization that kikA is highly conserved among the plasmids of incompatibility group N. The expression in K. oxytoca of kikA under the control of the strong inducible E. coli tac promoter results in loss of cell viability. The nucleotide sequence showed two overlapping open reading frames (ORFs) within the kikA region. The first ORF codes for a putative polypeptide of 104 amino acids (ORF104). The second ORF codes for a 70-amino-acid polypeptide (ORF70). The properties of the putative protein encoded by ORF104 and gene fusions of kikA to alkaline phosphatase by using TnphoA suggest that killing may involve an association with the bacterial membrane; however, we could not rule out the possibility that ORF70 plays a role in the Kik phenotype.
The Escherichia coli mutant requiring D-glutamic acid is the result of mutations in two distinct genetic loci
Authors: Dougherty T J , Thanassi J A , Pucci M J
Published: November 2016

D-Glutamic acid is an essential component of bacterial cell wall peptidoglycan in both gram-positive and gram-negative bacteria. Very little is known concerning the genetics and biochemistry of D-glutamate production in most bacteria, including Escherichia coli. Evidence is presented in this report for the roles of two distinct genes in E. coli WM335, a strain which is auxotrophic for D-glutamate. The first gene, which restores D-glutamate independence in WM335, was mapped, cloned, and sequenced. This gene, designated dga, is a previously reported open reading frame, located at 89.8 min on the E. coli map. The second gene, gltS, is located at 82 min. gltS encodes a protein that is involved in the transport of D- and L-glutamic acid into E. coli, and the gltS gene of WM335 was found to contain two missense mutations. To construct D-glutamate auxotrophs, it is necessary to transfer sequentially the mutated gltS locus, and then the mutated dga locus into the recipient. The sequences of the mutant forms of both dga and gltS are also presented.
Oxygen-dependent transcriptional regulation of cytochrome aa3 in Bradyrhizobium japonicum
Authors: Gabel C , Maier R J
Published: November 2016

Cytochrome aa3 is one of two terminal oxidases expressed in free-living Bradyrhizobium japonicum but not symbiotically in bacteroids. Difference spectra (dithionite reduced minus ferricyanide oxidized) for membranes from cells incubated with progressively lower O2 concentrations showed a concomitant decrease in the A603, the absorption peak characteristic of cytochrome aa3. The level of N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity, a measure of cytochrome aa3 activity, was also found to depend on the O2 level. Dot blots of total RNA isolated from cells grown at various O2 levels were probed with a fragment of the coxA gene from B. japonicum; a sixfold reduction in transcription from the highest (250 microM) to the lowest (12.5 microM) O2 concentration was observed. Bacteroids had even less coxA message, approximately 19% that in the 12.5 microM O2-incubated cells. Primer extension analysis established the transcription initiation site of the coxA gene at 72 bases upstream of the putative translational start codon. Sequence analysis of the region upstream of the transcription initiation site revealed no homology with previously reported B. japonicum promoters.
Detection of a nitrous oxide reductase structural gene in Rhizobium meliloti strains and its location on the nod megaplasmid of JJ1c10 and SU47
Authors: Chan Y K , Wheatcroft R
Published: November 2016

The gene encoding a denitrification enzyme, nitrous oxide reductase (EC 1.7.99.6), in Rhizobium meliloti and other gram-negative bacteria was detected by hybridization to an internal 1.2-kb PstI fragment of the structural gene (nosZ) cloned from Pseudomonas stutzeri Zobell (W.G. Zumft, A. Viebrock-Sambale, and C. Braun, Eur. J. Biochem. 192:591-599, 1990). Homology to the probe was detected in the DNAs of two N2-fixing strains of P. stutzeri, two denitrifying Pseudomonas species, one Alcaligenes eutrophus strain, and 36 of 56 R. meliloti isolates tested. Except for two isolates of R. meliloti, all showed nitrous oxide reduction activity (Nos+). Therefore, at least part of the nosZ sequence appears to be conserved and widely distributed among denitrifiers, which include free-living and symbiotic diazotrophs. By using Agrobacterium tumefaciens transconjugants harboring different megaplasmids of R. meliloti JJ1c10 and SU47, sequence homology with the nosZ probe was unequivocally located on the nod megaplasmid. A cosmid clone of JJ1c10 in which nosZ homology was mapped on a 4.2-kb BamHI fragment was selected. This cosmid, which conferred Nos+ activity to the R. meliloti wild-type strains ATCC 9930 and Balsac (Nos- and nondenitrifying, respectively) also restored Nos+ activity in the mutants of JJ1c10 and SU47 in which the 4.2-kb BamHI segment was deleted. Therefore, this segment contains sequences essential for nos gene expression in JJ1c10 and SU47 and thus confirms that the nod megaplasmid in JJ1c10 and SU47 which carries genes essential for symbiotic dinitrogen fixation also carries genes involved in the antagonistic process of denitrification.
Characterization of two phosphate transport systems in Acinetobacter johnsonii 210A
Authors: Van Veen H W , Abee T , Kortstee G J , Konings W N , Zehnder A J
Published: November 2016

The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.
A mutation in a new gene of Escherichia coli, psu, requires secondary mutations for survival: psu mutants express a pleiotropic suppressor phenotype
Authors: Ephrati-Elizur E
Published: November 2016

A mutation in an apparently new gene of Escherichia coli, psu, maps close to ara (1.3 min). psu mutants express a pleiotropic suppressor phenotype in which several auxotrophic requirements and some deletion mutations are suppressed. psu cloned in pBR322 can be maintained by the transformed cell only in the presence of several secondary mutations which accumulate in cultures of psu mutants and have an apparently compensatory role. The accumulation of secondary mutations is not due to mutator activity. The secondary mutations can each act as a suppressor of an auxotrophic requirement in the absence of psu, while suppression of deletions requires the presence of psu. Thus, the suppressor phenotype of psu mutants is due to both psu and the secondary mutations. The functions of psu and the secondary mutations are not known. However, two observations suggest an association with DNA gyrase and with DNA supercoiling. (i) psu mutants are highly resistant to oxolinic acid, the gyrase A inhibitor, while the secondary mutants vary from being very sensitive to more resistant than the wild-type strain. (ii) Novobiocin, which decreases the level of DNA supercoiling, significantly stimulates suppression of auxotrophy in some secondary mutants.
Osmotic repression of anaerobic metabolic systems in Escherichia coli
Authors: Gouesbet G , Abaibou H , Wu L F , Mandrand-Berthelot M A , Blanco C
Published: November 2016

The influence of the osmolarity of the growth medium on anaerobic fermentation and nitrate respiratory pathways was analyzed. The levels of several enzymes, including formate dehydrogenase, hydrogenase, and nitrate reductase, plus a nickel uptake system were examined, as was the expression of the corresponding structural and regulatory genes. While some functions appear to be only moderately affected by an increase in osmolarity, others were found to vary considerably. An increase in the osmolarity of the medium inhibits both fermentation and anaerobic respiratory pathways, though in a more dramatic fashion for the former. fnr expression is affected by osmolarity, but the repression of anaerobic gene expression was shown to be independent of FNR regulatory protein, at least for hyd-17 and fdhF. This repression could be mediated by the intracellular concentration of potassium and is reversed by glycine betaine.
Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences
Authors: Tiboni O , Cammarano P , Sanangelantoni A M
Published: November 2016

The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA gene as the probe. An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp. The encoded protein was 49% identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family. The P. woesei GSI was aligned with available homologs from other archaea (S. solfataricus, M. voltae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria. Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments. In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny. On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles. One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G + C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G + C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation). The GSIs of the Thermotoga and the Bacillus-Clostridium lineages shared a direct common ancestor with that of P. woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S. coelicolor. The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism. However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained.
The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity
Authors: Doublet P , van Heijenoort J , Bohin J P , Mengin-Lecreulx D
Published: November 2016

The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.
Cloning, sequencing, and transcriptional analysis of the gene coding for the vegetative sigma factor of Agrobacterium tumefaciens
Authors: Segal G , Ron E Z
Published: November 2016

The sigA gene of Agrobacterium tumefaciens was cloned and sequenced. Comparison with previously analyzed sigA genes revealed a high degree of similarity in nucleotide and amino acid sequences of regions two, three, and four of vegetative sigma factors. However, the upstream regulatory region shows no sequence homology with the Escherichia coli heat shock (sigma 32) promoters. It also does not contain the hairpin-loop structure (inverted repeat sequence) that was found in the upstream region of the groE operon in A. tumefaciens. The transcription initiation site of the gene was determined and found to be at the same position during normal growth and under heat shock conditions. Furthermore, no heat shock activation was observed at the transcriptional level.
Binding of an Escherichia coli double-stranded DNA virus PRD1 to a receptor coded by an IncP-type plasmid
Authors: Kotilainen M M , Grahn A M , Bamford J K , Bamford D H
Published: November 2016

IncP plasmid RP1 Tra regions are needed to assemble the receptor for lipid-containing double-stranded DNA bacteriophage PRD1 on the cell surface. Using radioactively labeled phage and electron microscopic techniques, we showed that the surfaces of Salmonella typhimurium(RP1) and Escherichia coli(RP1) cells contained approximately 50 and 20 PRD1 binding sites, respectively. Expression of the receptor was growth phase dependent and was highest at late logarithmic or early stationary phase. The PRD1-resistant RP1 transposon mutants isolated were all Tra-, and the transposons were located in both the Tra1 and Tra2 regions.
Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens
Authors: Wirawan I G , Kang H W , Kojima M
Published: November 2016

A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated by transposon (Tn5) mutagenesis. The mutant exhibited growth rates on L agar and minimal medium (AB) plates similar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid). The mutant was avirulent on all host plants tested: Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana. The mutant was not impaired in attachment ability to carrot cells. The mutant had one insertion of Tn5 in its chromosome. The avirulent phenotype of B119 was shown to be due to the Tn5 insertion in the chromosome by the marker exchange technique. A wild-type target chromosomal segment (3.0 kb) which included the site of mutation was cloned and sequenced. Two open reading frames, ORF-1 (468 bp) and ORF-2 (995 bp), were identified in the 3.0-kb DNA segment. Tn5 was inserted in the middle of ORF-2 (acvB gene). Introduction of the acvB gene into the mutant B119 strain complemented the avirulent phenotype of the strain. Homology search found no genes homologous to acvB, although it had some similarity to the open reading frame downstream of the virA gene on the Ti plasmid. Thus, the acvB gene identified in this study seems to be a new chromosomal virulence gene of A. tumefaciens.
Specific in vitro guanylylation of a 43-kilodalton membrane-associated protein of Streptomyces coelicolor
Authors: Obaya A J , Guijarro J
Published: November 2016

Incubation of [alpha-32P]GTP with cellular extracts or membranes of Streptomyces coelicolor labels a protein of 43 kDa, which was also labeled with [8,5'-3H]GTP but not with [alpha-32P]ATP or [gamma-32P]GTP. Radioactivity remained associated with this protein after boiling in 0.1 N NaOH, but it was dissociated after incubation in 0.1 N HCl or hydroxylamine. Chromatographic analysis of the HCl-dissociated compound showed that GMP was the covalently bound nucleotide. Furthermore, guanylylation appeared to be reversible and to take place by a pyrophosphorylytic mechanism. Guanylylation was more efficient at low temperatures. Several Streptomyces species showed a guanylylated protein with a similar molecular mass.
Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis
Authors: Michel G P
Published: November 2016

The DnaK protein of Zymomonas mobilis (DnaKz) was identified and found to be 80% identical to the DnaK protein of Escherichia coli on the basis of the sequence of the N-terminal 21 amino acids. The dnaKz gene was cloned and found to be expressed in a thermosensitive dnaK mutant of Escherichia coli. Expression of the foreign gene restored a thermoresistant phenotype but failed to modulate the heat shock response in E. coli.
Change in direction of flagellar rotation in Escherichia coli mediated by acetate kinase
Authors: Dailey F E , Berg H C
Published: November 2016

Strains of Escherichia coli lacking all cytoplasmic chemotaxis proteins except CheY swim smoothly under most conditions. However, they tumble when exposed to acetate. Acetate coenzyme A synthetase (EC 6.2.1.1) was thought to be essential for this response. New evidence suggests that the tumbling is mediated instead by acetate kinase (EC 2.7.2.1), which might phosphorylate CheY via acetyl phosphate. In strains that were wild type for chemotaxis, neither acetate coenzyme A synthetase, acetate kinase, nor phosphotransacetylase (EC 2.3.1.8) (and thus acetyl phosphate) was required for responses to aspartate, serine, or sugars sensed by the phosphotransferase system. Thus, acetate-induced tumbling does not appear to play an essential role in chemotaxis in wild-type cells.
Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage
Authors: Abshire K Z , Neidhardt F C
Published: November 2016

Salmonella typhimurium is a facultative intracellular pathogen, able both to invade and to survive within eukaryotic cells and to grow in various extracellular environments. To compare the bacterial responses to these disparate environments and to shed light on the nature of the intracellular environment, we have examined the pattern of protein synthesis by two-dimensional polyacrylamide gel electrophoresis. The levels of approximately 40 proteins were observed to increase during growth within macrophage-like U937 cells, while approximately 100 proteins exhibited levels that were repressed relative to those of an extracellular control culture. To aid in the interpretation of these results, the patterns of proteins made by S. typhimurium exposed to various environmental conditions in the laboratory were determined. The intracellular protein pattern was then compared with each of these benchmark protein patterns. This analysis revealed that, as expected, the intracellular environment appears to impose numerous stresses on the bacteria, but unexpectedly, the macrophage-induced response was not a simple sum of the individual stress responses displayed during extracellular growth.
Cloning and characterization of ftsN, an essential cell division gene in Escherichia coli isolated as a multicopy suppressor of ftsA12(Ts)
Authors: Dai K , Xu Y , Lutkenhaus J
Published: November 2016

A new cell division gene, ftsN, was identified in Escherichia coli as a multicopy suppressor of the ftsA12(Ts) mutation. Remarkably, multicopy ftsN suppressed ftsI23(Ts) and to a lesser extent ftsQ1(Ts); however, no suppression of the ftsZ84(Ts) mutation was observed. The suppression of ftsA12(Ts), ftsI23(Ts), and ftsQ1(Ts) suggests that FtsN may interact with these gene products during cell division. The ftsN gene was located at 88.5 min on the E. coli genetic map just downstream of the cytR gene. ftsN was essential for cell division, since expression of a conditional null allele led to filamentation and cell death. DNA sequence analysis of the ftsN gene revealed an open reading frame of 319 codons which would encode a protein of 35,725 Da. The predicted gene product had a hydrophobic sequence near its amino terminus similar to the noncleavable signal sequences found in several other Fts proteins. The presumed extracellular domain was unusual in that it was rich in glutamine residues. A 36-kDa protein that was localized to the membrane fraction was detected in minicells containing plasmids with the ftsN gene, confirming that FtsN was a membrane protein.
Involvement of Escherichia coli FIS protein in maintenance of bacteriophage mu lysogeny by the repressor: control of early transcription and inhibition of transposition
Authors: Bétermier M , Poquet I , Alazard R , Chandler M
Published: November 2016

The Escherichia coli FIS (factor for inversion stimulation) protein has been implicated in assisting bacteriophage Mu repressor, c, in maintaining the lysogenic state under certain conditions. In a fis strain, a temperature-inducible Mucts62 prophage is induced at lower temperatures than in a wild-type host (M. Bétermier, V. Lefrère, C. Koch, R. Alazard, and M. Chandler, Mol. Microbiol. 3:459-468, 1989). Increasing the prophage copy number rendered Mucts62 less sensitive to this effect of the fis mutation, which thus seems to depend critically on the level of repressor activity. The present study also provides evidence that FIS affects the control of Mu gene expression and transposition. As judged by the use of lac transcriptional fusions, repression of early transcription was reduced three- to fourfold in a fis background, and this could be compensated by an increase in cts62 gene copy number. c was also shown to inhibit Mu transposition two- to fourfold less strongly in a fis host. These modulatory effects, however, could not be correlated to sequence-specific binding of FIS to the Mu genome, in particular to the strong site previously identified on the left end. We therefore speculate that a more general function of FIS is responsible for the observed modulation of Mu lysogeny.
Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAK1 isolated from Clostridium acetobutylicum NCIB 6444
Authors: Kim A Y , Blaschek H P
Published: November 2016

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Escherichia coli plasmid pAK102 (AprEmr) and the 6.6-kbp HaeIII-linearized replicative form of the CAK1 viruslike particle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 (11.6 kbp) replicated via the ColE1 replication origin derived from pAK102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed with protein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E. coli DH11S transformants containing pCAK1 in the absence of cell lysis. This suggests that the viral-strand DNA synthesis replication origin of CAK1 and associated gene expression are functional in E. coli DH11S. The single-stranded form of pCAK1 isolated from E. coli supernatant was transformed into E. coli DH5 alpha' or DH11S by electroporation. Isolation of ampicillin-resistant E. coli transformants following transformation suggests that the complementary-strand DNA synthesis replication origin of CAK1 is also functional in E. coli. The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and various solvents (i.e., phenol and chloroform), similar to the results obtained previously with CAK1. Following phagemid construction in E. coli, pCAK1 was transformed into C. acetobutylicum ATCC 824 and C. perfringens 13 by intact cell electroporation. Restriction enzyme analysis of pCAK1 isolated from erythromycin-resistant transformants of both C. acetobutylicum and C. perfringens suggested that it was identical to that present in E. coli transformants.
Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin
Authors: Renna M C , Najimudin N , Winik L R , Zahler S A
Published: November 2016

Acetoin is a major extracellular product of Bacillus subtilis grown on glucose and other fermentable carbon sources. The enzymes responsible for the formation of acetoin, acetolactate synthase, and acetolactate decarboxylase are synthesized in detectable amounts only in cells that have reached stationary phase. We have cloned and sequenced the genes encoding these enzymes, alsS and alsD, as well as a gene, alsR, that regulates their expression. alsS and alsD appear to compose a single operon, while alsR is transcribed divergently from the alsSD operon. AlsR shows significant homology to the LysR family of bacterial activator proteins, and when alsR is disrupted the alsSD operon is not expressed. Transcriptional fusions to alsS and alsR revealed that AlsR is required for the transcription of the alsSD operon, which increases during stationary phase. Two mutations that cause increased expression of the alsSD operon have been isolated, cloned, and sequenced. They each change an amino acid in the AlsR protein.
The Salmonella typhimurium uracil-sensitive mutation use is in argU and encodes a minor arginine tRNA
Authors: Lu C D , Abdelal A T
Published: November 2016

The use gene of Salmonella typhimurium was previously identified by a mutation conferring sensitivity to uracil in glucose minimal medium. The use gene was cloned and identified as an allele of argU encoding a tRNA for a minor arginine codon (CGG). The uracil-sensitive phenotype was shown to result from a base substitution in the anticodon stem of this tRNA.
Cloning and sequencing of a gene encoding carminomycin 4-O-methyltransferase from Streptomyces peucetius and its expression in Escherichia coli
Authors: Madduri K , Torti F , Colombo A L , Hutchinson C R
Published: November 2016

Sequence analysis of a portion of the Streptomyces peucetius daunorubicin biosynthetic gene cluster revealed a complete open reading frame (dnrK) that showed DNA and protein sequence homology to several O-methyltransferases. Expression of dnrK in Streptomyces lividans and Escherichia coli was done to show that this gene codes for carminomycin 4-O-methyltransferase. The deduced carminomycin 4-O-methyltransferase protein shows a conserved nucleotide binding site for its S-adenosyl-L-methionine cofactor.
Leptospira genomes are modified at 5'-GTAC
Authors: Ralph D , Que Q , Van Etten J L , McClelland M
Published: November 2016

Genomic DNAs of 14 strains from seven species of the spirochete Leptospira were resistant to cleavage by the restriction endonuclease RsaI (5'-GTAC). A modified base comigrating with m4C was detected by chromatography. Genomic DNAs from other spirochetes, Borrelia group VS461, and Serpulina strains were not resistant to RsaI digestion. Modification at 5'-GTAm4C may occur in most or all strains of all species of Leptospira but not in all genera of spirochetes. Genus-wide DNA modification has rarely been observed in bacteria.
Plasmid pT181-linked suppressors of the Staphylococcus aureus pcrA3 chromosomal mutation
Authors: Iordanescu S
Published: November 2016

Plasmid pT181 replication is affected in hosts carrying the chromosomal pcrA3 mutation, resulting in significantly lower plasmid copy numbers. Mutations suppressing this effect have been isolated and characterized. The suppressor mutations were found to map in the plasmid repC gene and manifested pcrA allele specificity, suggesting the existence of a direct RepC-PcrA interaction.
Bacillus subtilis gtaB encodes UDP-glucose pyrophosphorylase and is controlled by stationary-phase transcription factor sigma B
Authors: Varón D , Boylan S A , Okamoto K , Price C W
Published: November 2016

Transcription factor sigma B of Bacillus subtilis controls a large stationary-phase regulon, but in no case has the physiological function of any gene in this regulon been identified. Here we show that transcription of gtaB is partly dependent on sigma B in vivo and that gtaB encodes UDP-glucose pyrophosphorylase. The gtaB reading frame was initially identified by a sigma B-dependent Tn917lacZ fusion, csb42. We cloned the region surrounding the csb42 insertion, identified the reading frame containing the transposon, and found that this frame encoded a predicted 292-residue product that shared 45% identical residues with the UDP-glucose pyrophosphorylase of Acetobacter xylinum. The identified reading frame appeared to lie in a monocistronic transcriptional unit. Primer extension and promoter activity experiments identified tandem promoters, one sigma B dependent and the other sigma B independent, immediately upstream from the proposed coding region. A sequence resembling a factor-independent terminator closely followed the coding region. By polymerase chain reaction amplification of a B. subtilis genomic library carried in yeast artificial chromosomes, we located the UDP-glucose pyrophosphorylase coding region near gtaB, mutations in which confer phage resistance due to decreased glycosylation of cell wall teichoic acids. Restriction mapping showed that the coding region overlapped the known location of gtaB. Sequence analysis of a strain carrying the gtaB290 allele found an alteration that would change the proposed initiation codon from AUG to AUA, and an insertion-deletion mutation in this frame conferred phage resistance indistinguishable from that elicited by the gtaB290 mutation. We conclude that gtaB encodes UDP-glucose pyrophosphorylase and is partly controlled by sigma B. Because this enzyme is important for thermotolerance and osmotolerance in stationary-phase Escherichia coli cells, our results suggest that some genes controlled by sigma B may play a role in stationary-phase survival of B. subtilis.
Repair of heteroduplex DNA molecules with multibase loops in Escherichia coli
Authors: Carraway M , Marinus M G
Published: November 2016

The fate of heteroduplex molecules containing 5-, 7-, 9-, 192-, 410-, and 514-base loops after transformation of wild-type and various mutant strains of Escherichia coli has been examined. No evidence for repair was obtained for the wild type or for strains with mutations in the following genes: mutS, recA, recBC sbcBC, recD, recF, recJ, recN, recO, recR, recBC sbcBC recF uvrA, recG ruvC, ruvB, lexA3, lexA51, uvrA, nfo xth nth, polA(Ts), or pcnB. These results rule out the involvement of the SOS system and most known recombination and repair pathways. Repair of heteroduplex molecules containing 410- and 514-base loops was observed when a 1-base deletion-insertion mismatch was present nearby. The repair of both the mismatch and the loops was directed by the state of dam methylation of the DNA chains and was dependent on the product of the mutS gene. A high efficiency of repair (95%) was found even when the mismatch and the loops were 1,448 nucleotides apart. We conclude that multibase loops in DNA can be removed only as a consequence of corepair by dam-directed mismatch repair.
Analysis of the structure and subcellular location of filamentous phage pIV
Authors: Russel M , Kaźmierczak B
Published: November 2016

The gene IV protein of filamentous bacteriophages is an integral membrane protein required for phage assembly and export. A series of gene IV::phoA fusion, gene IV deletion, and gene IV missense mutations have been isolated and characterized. The alkaline phosphatase activity of the fusion proteins suggests that pIV lacks a cytoplasmic domain. Cell fractionation studies indicate that the carboxy-terminal half of pIV mediates its assembly into the membrane, although there is no single, discrete membrane localization domain. The properties of gene IV missense and deletion mutants, combined with an analysis of the similarities between pIVs from various filamentous phage and related bacterial export-mediating proteins, suggest that the amino-terminal half of pIV consists of a periplasmic substrate-binding domain that confers specificity to the assembly-export system.
Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion
Authors: McIver K S , Olson J C , Ohman D E
Published: November 2016

Pseudomonas aeruginosa secretes elastase in a multistep process which begins with the synthesis of a preproelastase (53.6 kDa) encoded by lasB, is followed by processing to proelastase (51 kDa), and concludes with the rapid accumulation of mature elastase (33 kDa) in the extracellular environment. In this study, mutants of P. aeruginosa were constructed by gene replacement which expressed lasB1, an allele altered in vitro at an active-site His-223-encoding codon. The lasB1 allele was exchanged for chromosomal lasB sequences in two strain backgrounds, FRD2 and PAO1, through a selectable-cassette strategy which placed a downstream Tn501 marker next to lasB1 and provided the selection for homologous recombination with the chromosome. Two lasB1 mutants, FRD720 and PDO220, were characterized, and their culture supernatants contained greatly reduced proteolytic (9-fold) and elastolytic (14- to 20-fold) activities compared with their respective parental lasB+ strains. This was primarily due to the effect of His-223 substitution on substrate binding by elastase and thus its proteolytic activity. However, the concentration of supernatant elastase antigen was also reduced (five- to sevenfold) in the mutant strains compared with the parental strains. An immunoblot analysis of cell extracts showed a large accumulation of 51-kDa proelastase within lasB1 mutant cells which was not seen in wild-type cell extracts. A time course study showed that production of extracellular elastase was inefficient in the lasB1 mutants compared with that of parental strains. This showed that expression of an enzymatically defective elastase inhibits proper processing of proelastase and provides further evidence for autoproteolytic processing of proelastase in P. aeruginosa. Unlike the parental strains, culture supernatants of the lasB1 mutants contained two prominent elastase species that were 33 and 36 kDa in size. Extracellular 51-kDa proelastase was barely detectable, even though it accumulated to high concentrations within the lasB1 mutant cells. These data suggest that production of an enzymatically defective elastase affects proper secretion because autoproteolytic processing of proelastase is necessary for efficient localization to the extracellular milieu. The appearance of reduced amounts of extracellular elastase and their sizes of 33 and 36 kDa suggest that lasB1-encoded elastase was processed by alternate, less-efficient processing mechanisms. Thus, proelastase must be processed by removal of nearly all of the 18-kDa propeptide before elastase is a protein competent for extracellular secretion.
In vivo expression of the Bacillus subtilis spoVE gene
Authors: Theeragool G , Miyao A , Yamada K , Sato T , Kobayashi Y
Published: November 2016

In vivo expression of the Bacillus subtilis spoVE gene was studied by S1 nuclease mapping and spoVE gene fusion analysis. Transcription of spoVE is induced at about the second hour of sporulation from two closely spaced promoters designated P1 and P2. Examination of the precise transcription initiation site by high-resolution primer extension mapping indicated that the nucleotide sequences of the -10 and -35 regions of both P1 and P2 were similar to those of promoters recognized by E sigma E. Moreover, S1 nuclease mapping and translational spoVE-lacZ fusion studies with various spo mutants suggest that the expression of spoVE P2 requires the spoIIG gene product, sigma E. The sporulation of a wild-type strain was inhibited severely in the presence of a multicopy plasmid, pKBVE, carrying the spoVE promoter, indicating the possible titration of a transcriptional regulatory element(s).
Involvement of the alginate algT gene and integration host factor in the regulation of the Pseudomonas aeruginosa algB gene
Authors: Wozniak D J , Ohman D E
Published: November 2016

Strains of Pseudomonas aeruginosa causing pulmonary infection in cystic fibrosis patients are often mucoid because of the synthesis of a capsular polysaccharide called alginate. Regulation of alginate biosynthesis includes the algB gene product (AlgB), which belongs to a class of proteins that control gene transcription in response to environmental stimuli. In this study, a homolog of the DNA-binding-and-bending protein integration host factor (IHF) and the positive regulatory gene algT were shown to be involved in algB expression. An algB-cat gene fusion was constructed on a low-copy-number, broad-host-range plasmid. In alginate-producing (Alg+) P. aeruginosa, levels of chloramphenicol acetyltransferase from algB-cat were twofold higher than in spontaneous Alg- or algT::Tn501 mutant strains, indicating that the mucoid status of the cell influences algB transcription. An algB transcription initiation site was identified 286 nucleotides upstream of translation initiation and revealed an Escherichia coli sigma 70-like promoter. Sequences in the algB promoter region were highly similar to the consensus E. coli IHF binding site. In DNA gel band mobility shift assays, a protein present in extracts from IHF+ E. coli strains and IHF purified from E. coli bound specifically to these algB DNA fragments, while extracts prepared from isogenic IHF- E. coli strains failed to alter the mobility of algB DNA fragments containing the consensus IHF binding site. A protein in cell extracts prepared from P. aeruginosa strains also demonstrated binding to algB fragments containing the IHF binding site, and the position of the complex formed with these extracts was identical to that of the complex formed with purified IHF. Moreover, this binding could be inhibited by anti-IHF antibodies. To test the role of the IHF site in algB regulation, site-specific mutations in the algB IHF site, based on changes which severely affect IHF binding in E. coli, were generated. When either purified E. coli IHF or extracts from P. aeruginosa were used in DNA binding studies, the algB mutant DNAs were severely reduced in IHF binding. Mutations affecting IHF binding at the algB promoter were introduced into the algB-cat plasmid, and all resulted in severely impaired transcriptional activity in Alg- and algT mutant strains of P. aeruginosa. However, these mutations resulted in similar or slightly reduced algB-cat transcription in Alg+ and algB::Tn501 mutant strains. Thus, the algT product plays a positive role in the high-level expression of algB in mucoid cells, whereas as protein present in P.aeruginosa extracts which is likely an IHF homolog plays a positive role in maintaining a basal level of algB expression in nonmucoid strains.
Structural requirements of Bacillus subtilis alpha-amylase signal peptide for efficient processing: in vivo pulse-chase experiments with mutant signal peptides
Authors: Sakakibara Y , Tsutsumi K , Nakamura K , Yamane K
Published: November 2016

The Bacillus subtilis alpha-amylase signal peptide consists of 33 amino acids from its translation initiation site. To analyze the structural requirements for efficient processing of the signal peptide, single and repeated Ala-X-Ala sequences and their modifications were introduced into B. subtilis alpha-amylase signal peptides of different lengths and the mature thermostable alpha-amylase. Then the cleavage positions and processing rates of the signal peptides were analyzed by the NH2-terminal amino acid sequences of the exported thermostable alpha-amylases and by in vivo pulse-chase experiments. In B. subtilis, the most efficient cleavage site was located at the peptide bond between Ala-33 and amino acid X at position 34, even though Val-X-Ala and six repeating Ala-X-Ala sequences were present around the cleavage site. However, the cleavage site was shifted to the peptide bond between Ala-31 and amino acid X when Ala-33 was deleted, and it was also shifted to Ala-35 and X when Ala-33 was replaced with Val-33. The shorter signal peptide consisting of 31 amino acids reduced the processing rate and alpha-amylase production. In contrast, those signal peptides were cleaved preferentially at the peptide bond between Ala-31 and amino acid X in Escherichia coli. In addition to the presence of an Ala residue at the -1 amino acid position, the length of the signal peptide was another important requirement for efficient processing.
The INO1 promoter of Saccharomyces cerevisiae includes an upstream repressor sequence (URS1) common to a diverse set of yeast genes
Authors: Lopes J M , Schulze K L , Yates J W , Hirsch J P , Henry S A
Published: November 2016

The INO1 promoter of Saccharomyces cerevisiae includes a copy of an upstream repression sequence (URS1; 5'AGCCGCCGA 3') observed in the promoters of several unrelated yeast genes. Expression of INO1-lacZ and CYC1-lacI'Z, activated by the INO1 UASINO, is significantly decreased by the INO1 URS1.