bluuebunny/crossref_metadata_2025 huggingface dataset:
A protein that binds to the regulatory region of the Escherichia coli ilvIH operon
Authors: Ricca E , Aker D A , Calvo J M
Published: November 2016

The ilvIH operon of Escherichia coli encodes acetohydroxyacid synthase III, an isoenzyme involved in branched-chain amino acid biosynthesis. Transcription of the ilvIH operon is repressed by growing cells in the presence of leucine (C.H. Squires, M. DeFelice, S.R. Wessler, and J.M. Calvo, J. Bacteriol. 147:797-804, 1981). A protein in crude extracts of E. coli, termed the ilvIH-binding (IHB) protein, bound specifically in vitro to DNA upstream of the ilvIH operon. The binding protein, partially purified by Polymin precipitation, gel filtration, and phosphocellulose chromatography, has a native molecular weight of 43,000 and is composed of two subunits of identical size. As determined by protection against lambda exonuclease and DNase I, the protein binds within a region -190 to -260 relative to the start point of transcription. In addition, the IHB protein binds to a site between positions -100 and -40. The following evidence suggests that binding of this protein to the region upstream of ilvIH is related to the regulation of this operon by leucine. Binding of the IHB protein to the ilvIH regulatory region in vitro was reduced by leucine but not by isoleucine, valine, or threonine. In a mutant strain isolated by M.V. Ursini, P. Arcari, and M. DeFelice (Mol. Gen. Genet. 181:491-496, 1981), transcription was not repressed by leucine. A protein in extracts of this mutant strain bound to the ilvIH regulatory region, but the complex migrated through agarose gels with a mobility different from that of the complex formed by wild-type protein. Furthermore, a concentration of leucine that substantially reduced binding of the wild-type to DNA did not affect binding of the protein from the mutant strain. A simple model consistent with these findings is that transcription from the ilvIH promoter is stimulated by binding the IHB protein to one or more sites upstream of the promoter and that leucine interferes with this binding.
Sequence similarities in the genes encoding polychlorinated biphenyl degradation by Pseudomonas strain LB400 and Alcaligenes eutrophus H850
Authors: Yates J R , Mondello F J
Published: November 2016

DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to LB400 in PCB-degrading capability. These two organisms showed a strong conservation of restriction sites in the region of DNA encoding PCB metabolism. No other sequence similarities were detected in the two genomes. DNA from the other PCB-degrading strains showed no hybridization to the probe, which demonstrated the existence of at least two distinct classes of genes encoding PCB degradation.
Two chromosomal loci involved in production of exopolysaccharide in Agrobacterium tumefaciens
Authors: Kamoun S , Cooley M B , Rogowsky P M , Kado C I
Published: November 2016

The chromosomal locus pscA (exoC) of Agrobacterium tumefaciens LBA4301 has been cloned by complementation of the avirulent and exopolysaccharide (EPS)-deficient mutant LBA4301 pscA. We have also identified a new locus, termed psdA (polysaccharide depression) and located 16 kilobases from pscA in the A. tumefaciens chromosome, that negatively affects EPS production when it is present in more than one copy in A. tumefaciens LBA4301. Subcloning, transposon mutagenesis, and transcriptional analysis have been conducted for both loci and indicate that pscA and psdA are transcribed in the same orientation. Acidic-EPS assays showed that psdA depresses succinoglycan production and that its negative effect increases with the copy number of the gene. Virulence tests of psdA transconjugants on Datura stramonium showed no visible alteration in virulence, while LBA4301 pscA was totally avirulent.
Structure of an acidic exopolysaccharide of Pseudomonas marginalis HT041B
Authors: Osman S F , Fett W F
Published: November 2016

The exopolysaccharide of Pseudomonas marginalis HT041B has been characterized as a 1,3-linked galactoglucan in which galactose and glucose are in the alpha- and beta-anomeric configurations, respectively. The polysaccharide is substituted with pyruvate at the 4 and 6 positions of galactose and with succinic acid at either the 2 or 4 position of glucose. This polysaccharide has been given the trivial name marginalan.
Regions of toxin A involved in toxin A excretion in Pseudomonas aeruginosa
Authors: Hamood A N , Olson J C , Vincent T S , Iglewski B H
Published: November 2016

Toxin A is excreted by Pseudomonas aeruginosa as a mature 66,583-dalton protein. In this study, we used molecular cloning and deletion analysis to define specific regions of the toxin molecule involved in its excretion. Subclones that express either the amino terminus, the carboxy terminus, or toxin A molecules with internal deletions were constructed. The hypotoxigenic mutant PAO-T1 was used as a host for the expression of the toxin constructs. When overexpressed (by the presence of extra copies of the toxin A-positive regulatory gene, regA, in trans), toxin A-cross-reactive materials produced by most of these constructs were detected in the supernatant of PAO-T1. The supernatant of P. aeruginosa PAO-T1 contained proteolytic activity that degraded toxin A-derived products but not the intact toxin molecule. A single SalI intragenic deletion (coding for the leader peptide, the first 30 amino acids, and the last 305 amino acids of the toxin) resulted in a relatively stable product in the supernatant of PAO-T1. The product of the carboxy terminus construct (which codes for the last 305 amino acids of the toxin) was detected in the lysate of PAO-T1 only. The data suggest that the amino terminus region of toxin A (the leader peptide plus the first 30 amino acid of the mature protein) is sufficient for its excretion, and that a second region, amino acids 309 through 413, protects an internally truncated toxin A molecule from the proteolytic activity in the supernatant of P. aeruginosa PAO-T1.
Mobilization of the relaxable Staphylococcus aureus plasmid pC221 by the conjugative plasmid pGO1 involves three pC221 loci
Authors: Projan S J , Archer G L
Published: November 2016

The Staphylococcus aureus plasmid pC221, a 4.6-kilobase multicopy chloramphenicol resistance plasmid that forms plasmid-protein relaxation complexes, was mobilized for transfer by the conjugative plasmid pGO1. Two open reading frames on the pC221 genome, now designated mobA and mobB, as well as a cis-acting locus, the putative oriT, were shown to be in involved in pC221 mobilization. The mobA (but not mobB) and oriT loci were required for pC221 relaxation, and relaxation was necessary but not sufficient for pC221 mobilization by pGO1. oriT was cloned onto a pE194 derivative and complemented in trans for both relaxation and mobilization. Mobilization of relaxable plasmids in S. aureus appears to be analogous to mobilization by donation observed in gram-negative bacteria.
Deletion hot spots in chimeric Escherichia coli plasmids
Authors: Michel B , D'Alençon E , Ehrlich S D
Published: November 2016

Deletions form frequently in chimeric plasmids composed of M13mp2, pBR322, and pC194 (B. Michel and S. D. Ehrlich, Proc. Natl. Acad. Sci. USA 83:3386-3390, 1986). They are generated by joining of the nucleotide neighboring the nick site in the M13 replication origin to a nonadjacent nucleotide. This nucleotide is most often located within particular short plasmid regions, named deletion hot spots. Three natural hot spots were present in the chimeric plasmids. Two were active only when the DNA replication initiated at the M13 origin was allowed to progress; the third was active only in the presence of wild-type amounts of DNA ligase. Three artificial hot spots were generated by creating palindromic sequences in the plasmids.
Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae
Authors: Taylor R K , Manoil C , Mekalanos J J
Published: November 2016

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.
Identification of a gene linked to Rhizobium meliloti ntrA whose product is homologous to a family to ATP-binding proteins
Authors: Albright L M , Ronson C W , Nixon B T , Ausubel F M
Published: November 2016

The ntrA gene of Rhizobium meliloti has recently been identified and shown to be required for a diverse set of metabolic functions (C. W. Ronson, B. T. Nixon, L. M. Albright, and F. M. Ausubel, J. Bacteriol. 169:2424-2431, 1987). As a result of sequencing the ntrA gene and its flanking regions from R. meliloti, we identified an open reading frame directly upstream of ntrA, ORF1, whose predicted product is homologous to a superfamily of ATP-binding proteins involved in transport, cell division, nodulation, and DNA repair. The homology of ORF1 to this superfamily and its proximity to ntrA led us to investigate its role in symbiosis by mutagenesis and expression studies. We were unable to isolate an insertion mutation in ORF1, suggesting that ORF1 may code for an essential function. We identified the start of transcription for the ntrA gene in vegetative cells and bacteroids and showed that ORF1 and ntrA are transcriptionally unlinked. ORF1 appears to be in an operon with one or more upstream genes.
Structure of the cutinase gene and detection of promoter activity in the 5'-flanking region by fungal transformation
Authors: Soliday C L , Dickman M B , Kolattukudy P E
Published: November 2016

The cutinase gene from Fusarium solani f. sp. pisi (Nectria hematococa) was cloned and sequenced. Sau3A fragments of genomic DNA from the fungus were cloned in a lambda Charon 35 vector. When restriction fragments generated from the inserts were screened with 5' and 3' probes from cutinase cDNA, a 5.5-kilobase SstI fragment hybridized with both probes, suggesting the presence of the entire cutinase gene. A 2,818-base pair segment was sequenced, revealing a 690-nucleotide open reading frame that was identical to that found in the cutinase cDNA with a single 51-base pair intron. Transformation vectors were constructed containing a promoterless gene for hygromycin resistance, which was translationally fused to flanking sequences of the cutinase gene. When protoplasts and mycelia were transformed with these vectors, hygromycin-resistant transformants were obtained. Successful transformation was assessed by Southern blot analysis by using radiolabeled probes for the hygromycin resistance gene and the putative promoter. The results of Southern blot analysis indicated that the plasmid had integrated into the Fusarium genome and that the antibiotic resistance was a manifestation of the promoter activity of the cutinase flanking sequences. Transformation of Colletotrichum capsici with the same construct confirmed the promoter activity of the flanking region and the integration of the foreign DNA. Transformation and deletion analysis showed that promoter activity resided within the 360 nucleotides immediately 5' to the cutinase initiation codon.
Evidence that the transcription activator encoded by the Pseudomonas putida nahR gene is evolutionarily related to the transcription activators encoded by the Rhizobium nodD genes
Authors: Schell M A , Sukordhaman M
Published: November 2016

The nahR gene of the 83-kilobase naphthalene degradation plasmid NAH7 of Pseudomonas putida encodes a 34-kilodalton polypeptide which binds to the nah and sal promoters to activate transcription of the degradation genes in response to the inducer salicylate. The DNA sequence of the nahR gene was determined, and a derived amino acid sequence of the NahR protein was obtained. A computer search for homologous proteins showed that within the first 124 amino-terminal residues, NahR has approximately 35% identity with the transcriptional activator proteins encoded by the nodD genes of Rhizobium species. Allowing for ultraconservative amino acid substitutions, greater than 47% overall similarity was found between NahR and NodD, while 32% similarity was found between NahR and another transcription activator, LysR of Escherichia coli. The region of greatest similarity among all three proteins contained a probable helix-turn-helix DNA-binding motif as suggested by homology with the proposed consensus sequence for Cro-like DNA-binding domains. The high level of amino acid identity between NahR and NodD, in conjunction with the observations that nahR and nodD are 45% homologous in DNA sequence, are divergently transcribed from homologous promoters near the structural genes they control, and have similar DNA-binding sites, strongly suggests that these two genes evolved from a common ancestor.
Release of flagellar filament-hook-rod complex by a Salmonella typhimurium mutant defective in the M ring of the basal body
Authors: Okino H , Isomura M , Yamaguchi S , Magariyama Y , Kudo S , Aizawa S I
Published: November 2016

A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.
The Escherichia coli cell division mutation ftsM1 is in serU
Authors: Leclerc G , Sirard C , Drapeau G R
Published: November 2016

The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.
Nucleotide sequence analysis and expression of the minimum REPI replication region and incompatibility determinants of pColV-K30
Authors: Perez-Casal J F , Gammie A E , Crosa J H
Published: November 2016

We sequenced the minimum REPI replication region and the incompatibility determinants of pColV-K30. The minimum replication region contains an open reading frame which corresponds to a 35-kilodalton (kDa) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis with maxicells transformed with a number of deletion derivatives demonstrated that this replication region encodes a 39-kDa protein and also established the direction of transcription of the RepI protein gene. The 39-kDa polypeptide was identified as the trans-acting factor essential for replication of REPI-containing plasmids. A translated region of the nucleotide sequence of the RepI protein gene showed homology with the helix-turn-helix binding domains of a number of DNA-binding proteins and also with other plasmid replication proteins. Further nucleotide analysis of the REPI region revealed the presence of direct and inverted repeat sequences in the incE, incF, and ori regions. The REPI ori also contained a perfect DnaA-binding site in addition to a high frequency of occurrence of the DNA adenine methylation (dam) site 5'GATC3'.
Use of gene replacement to construct Escherichia coli strains carrying mutations in two genes required for stability of multicopy plasmids
Authors: Flinn H , Burke M , Stirling C J , Sherratt D J
Published: November 2016

Escherichia coli mutants completely defective in ColE1 cer-mediated site-specific recombination have been mapped to two genes, xerA and xerB. In this study, xerA xerB double mutants were constructed by gene replacement with a lambda dv plasmid and were shown to be both viable and defective in ColE1 site-specific recombination.
In vitro proteolytic cleavage of the Escherichia coli Ada protein by the ompT gene product
Authors: Sedgwick B
Published: November 2016

Down regulation of the adaptive response to alkylation damage in Escherichia coli has been proposed to occur by proteolytic cleavage of the regulatory Ada protein. In this paper, it is shown that proteolysis of the Ada protein as observed in cell extracts is caused by the ompT gene product. This protease, however, was not involved in switching off the adaptive response in vivo.
Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism
Authors: Bukau B , Walker G C
Published: November 2016

DnaK is a major heat shock protein of Escherichia coli and has been previously reported to be essential for growth at high temperatures. We systematically investigated the role of DnaK in cellular metabolism at a wide range of growth temperatures by analyzing cellular defects caused by deletion of the dnaK gene (delta dnaK52). At intermediate temperatures (30 degrees C), introduction of the delta dnaK52 allele into wild-type cells caused severe defects in cell division, slow growth, and poor viability of the cells. delta dnaK52 mutants were genetically unstable at 30 degrees C and frequently acquired secondary mutations. At high (42 degrees C) and low (11 and 16 degrees C) temperatures the delta dnaK52 allele could only be introduced into the subpopulation of wild-type cells that had duplicated the dnaK region of their chromosome. delta dnaK52 mutants isolated at 30 degrees C were cold sensitive as well as temperature sensitive for growth. Cell division defects of delta dnaK52 mutants at 30 degrees C were largely suppressed by overproduction of the FtsZ protein, which is normally required for septation during cell division; however, slow growth and poor viability at 30 degrees C and cold sensitivity and temperature sensitivity of growth were not suppressed, indicating that delta dnaK52 mutants had additional defective cellular functions besides cell division.
Bacillus licheniformis alpha-amylase gene, amyL, is subject to promoter-independent catabolite repression in Bacillus subtilis
Authors: Laoide B M , Chambliss G H , McConnell D J
Published: November 2016

Expression of the Bacillus licheniformis alpha-amylase gene, amyL, was temporally activated and subject to catabolite repression both in its natural host and when cloned on a 3.55-kilobase fragment in Bacillus subtilis. A subclone from which the promoter region of amyL and sequences upstream from the promoter were deleted had a low level of amylase activity. Expression of the promoterless gene was still subject to repression by glucose when the gene was present either on a multicopy plasmid or integrated into the B. subtilis chromosome. Catabolite repression occurred independently of the amylase promoter and irrespective of the distance of the promoterless amyL gene from the promoter which transcribed it. The transcriptional start sites of amyL activated by its own promoter and by a vector sequence promoter were determined by S1 mapping. alpha-Amylase-specific mRNA levels were measured in repressing and nonrepressing media, and catabolite repression was found to act at the level of transcription.
Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA
Authors: Sen P , Pazour G J , Anderson D , Das A
Published: November 2016

The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.
Exchange of chromosomal and plasmid alleles in Escherichia coli by selection for loss of a dominant antibiotic sensitivity marker
Authors: Russell C B , Dahlquist F W
Published: November 2016

Transfer of an allele from a donor DNA to a recipient DNA molecule was selected by the loss of a dominant conditional lethal mutation previously incorporated ito the gene of interest in the recipient DNA. Both the Escherichia coli chromosome and plasmids carrying E. coli genes were used successfully as donor molecules. Recipient molecules for these exchanges were constructed in vitro by using the rpsL gene, which confers sensitivity to streptomycin, to replace segments of specific E. coli genes located either on multicopy plasmids or in the E. coli chromosome. Plasmids carrying such replacements were capable of acquiring chromosomal alleles of the gene(s) of interest, and strains carrying rpsL replacements in the chromosome were capable of acquiring plasmid-encoded alleles at the sight of the rpsL replacement. In both situations, these allele transfers resulted in loss of the rpsL gene from the recipient DNA molecule. The desired transfer events constituted a large percentage of these events, which gave rise to viable colonies when appropriate donor-recipient pairs were subjected to streptomycin selection. Thus, this is a useful approach for transferring alleles of interest from plasmids to the E. coli chromosome and vice versa.
Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature
Authors: Strauch K L , Johnson K , Beckwith J
Published: November 2016

The degP gene, required for proteolysis in the cell envelope of Escherichia coli, maps at approximately 3.5 min on the chromosome. Null mutations in degP result in temperature-sensitive growth. In certain genetic backgrounds, expression of abnormal periplasmic or inner membrane proteins (protein fusions or proteins with internal deletions) enhances the temperature-sensitive phenotype. Such growth defects were used as a selection for cloning the degP gene into Mud4042 and pACYC184 plasmid vectors, and a restriction map was determined. Analysis of deletion and insertion mutations on one of these plasmids showed that the degP gene is approximately 1.5 kilobases in size. The plasmid-encoded DegP protein had an apparent molecular weight of 50,000, as determined by maxicell analysis. Protein fusions between DegP and alkaline phosphatase had high alkaline phosphatase enzymatic activity, indicating that DegP is a periplasmic or membrane protein.
Microaerophilic growth and induction of the photosynthetic reaction center in Rhodopseudomonas viridis
Authors: Lang F S , Oesterhelt D
Published: November 2016

Rhodopseudomonas viridis was grown in liquid culture at 30 degrees C anaerobically in light (generation time, 13 h) and under microaerophilic growth conditions in the dark (generation time, 24 h). The bacterium could be cloned at the same temperature anaerobically in light (1 week) and aerobically in the dark (3 to 4 weeks) if oxygen was limited to 0.1%. Oxygen could not be replaced by dimethyl sulfoxide, potassium nitrate, or sodium nitrite as a terminal electron acceptor. No growth was observed anaerobically in darkness or in the light when air was present. A variety of additional carbon sources were used to supplement the standard succinate medium, but enhanced stationary-phase cell density was observed only with glucose. Conditions for induction of the photosynthetic reaction center upon the change from microaerophilic to phototrophic growth conditions were investigated and optimized for a mutant functionally defective in phototrophic growth. R. viridis consumed about 20-fold its cell volume of oxygen per hour during respiration. The MICs of ampicillin, kanamycin, streptomycin, tetracycline, 1-methyl-3-nitro-1-nitrosoguanidine, and terbutryn were determined.
Function of alpha-D-glucosyl monophosphorylpolyprenol in biosynthesis of cell wall teichoic acids in Bacillus coagulans
Authors: Shimada A , Tamatukuri J , Ito E
Published: November 2016

D-[alpha-14C]]glucosyl phosphorylpolyprenol ([ 14C]Glc-P-prenol) was formed from UDP-D-[14C]glucose in each of the membrane systems obtained from Bacillus coagulans AHU 1631 and AHU 1634 and two Bacillus megaterium strains. Membranes of these B. coagulans strains, which possess beta-D-glucosyl branches on the repeating units in their major cell wall teichoic acids, were shown to catalyze the transfer of the glucose residue from [14C]Glc-P-prenol to endogenous polymer. On the other hand, membranes of B. coagulans AHU 1366, which has no glucose substituents in the cell wall teichoic acid, exhibited neither [14C]Glc-P-prenol synthetase activity nor the activity of transferring glucose from [14C]Glc-P-prenol to endogenous acceptor. The enzyme which catalyzes the polymer glycosylation in the former two B. coagulans strains was most active at pH 5.5 and in the presence of the Mg2+ ion. The apparent Km for [14C]Glc-P-prenol was 0.6 microM. Hydrogen fluoride hydrolysis of the [14C]glucose-linked polymer product yielded a major fragment identical to D-galactosyl-alpha(1----2)(D-glucosyl-beta(1----1/3)) glycerol, the dephosphorylated repeating unit in the major cell wall teichoic acids of these B. coagulans strains. This result, together with the behavior of the radioactive polymer in chromatography on Sepharose CL-6B, DEAE-Sephacel, and Octyl-Sepharose CL-4B, led to the conclusion that [14C]Glc-P-prenol serves as an intermediate in the formation of beta-D-glucosyl branches on the polymer chains of cell wall teichoic acids in B. coagulans.
Identification of an Escherichia coli gene homologous to virR, a regulator of Shigella virulence
Authors: Hromockyj A E , Maurelli A T
Published: November 2016

Virulence in Shigella spp., as well as in strains of enteroinvasive Escherichia coli, is regulated by growth temperature. Previously, virR had been identified as the gene controlling the temperature-regulated expression of Shigella virulence. Since Shigella spp. and E. coli are also known to share greater than 90% DNA sequence homology, we sought to determine if nonpathogenic E. coli K-12 C600 contains a gene homologous to the Shigella flexneri 2a gene virR. Through the use of transduction and molecular cloning of strain C600 chromosomal DNA we have shown that E. coli K-12 does indeed contain a gene functionally homologous to the virR of S. flexneri.
Derepression of conjugal transfer of the antibiotic resistance plasmid R100 by antisense RNA
Authors: Dempsey W B
Published: November 2016

Conjugal transfer of the normally repressed antibiotic resistance plasmid R100 was derepressed by fragments of R100 that carried the traJ promoter and the traJ leader but lacked the finP promoter.
Concentration-dependent repression of the soluble and membrane components of the Streptococcus mutans phosphoenolpyruvate: sugar phosphotransferase system by glucose
Authors: Hamilton I R , Gauthier L , Desjardins B , Vadeboncoeur C
Published: November 2016

Growth of Streptococcus mutans Ingbritt in continuous culture (pH 7.0, dilution rate of 0.1 h-1) at medium glucose concentrations above 2.6 mM resulted in repression of the sugar-specific membrane components, enzyme IIGlc (EIIGlc) and EIIMan, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In one experiment, significant repression (27-fold) was observed with 73 mM glucose when the glycolytic capacity of the cells was reduced by only 2-fold and when the culture was still glucose limited. In a more comprehensive experiment in which cells were grown in continuous culture at eight glucose concentrations from 2.6 to 304 mM, in addition to repression of specific EII activities for glucose, mannose, 2-deoxyglucose, and fructose, synthesis of the general protein, EI, was repressed at all glucose levels above 2.6 mM to a maximum of 4-fold at 304 mM glucose when the culture was growing with excess glucose (i.e., nitrogen limited). The other PTS general protein, HPr, was less sensitive to the exogenous glucose level but was nevertheless repressed fourfold under glucose-excess conditions. The Km for glucose for EIIGlc increased from 0.22 mM during growth at 3.6 mM glucose (glucose limited) to 0.48 mM at 271 mM glucose (glucose excess). The shift from heterofermentation to homofermentation during growth with increasing glucose levels suggests the involvement of glycolytic intermediates, ATP, or another high-energy phosphate metabolite in regulation of the synthesis of the PTS components in S. mutans.
DNA-binding properties of the transcription activator (OmpR) for the upstream sequences of ompF in Escherichia coli are altered by envZ mutations and medium osmolarity
Authors: Forst S A , Delgado J , Inouye M
Published: November 2016

Expression in Escherichia coli of the genes that encode the major outer membrane porin proteins (OmpF and OmpC) is regulated by the transcription activator protein OmpR and the receptorlike protein EnvZ, which is located in the inner membrane. Using synthesized oligonucleotide fragments containing the OmpR-binding site of ompF, we show that soluble extracts and partially purified OmpR derived from both the parent strain grown in nutrient broth plus 20% sucrose and the envZ11 strain grown in nutrient broth produced high-affinity DNA-binding activity, whereas soluble extracts from the parent strain grown in nutrient broth produced low-affinity binding. We also show that the soluble extracts from the envZ22(Am) strain grown in nutrient broth did not produce detectable bound forms of the ompF fragments, but low levels of DNA binding were detected with soluble extracts of the envZ22 strain grown in nutrient broth plus sucrose. In addition, the time course of the repression of OmpF synthesis produced by a shift to high-osmolarity growth medium was correlated with an increase in the DNA-binding affinity of soluble extracts to the ompF fragment. These results provide evidence that envZ function influences the DNA-binding activity of OmpR and suggest that high-affinity binding of OmpR to the upstream sequences of ompF is correlated with the repression of OmpF production.
Transport of glutamine by Streptococcus bovis and conversion of glutamine to pyroglutamic acid and ammonia
Authors: Chen G J , Russell J B
Published: November 2016

Streptococcus bovis JB1 cells energized with glucose transported glutamine at a rate of 7 nmol/mg of protein per min at a pH of 5.0 to 7.5; sodium had little effect on the transport rate. Because valinomycin-treated cells loaded with K and diluted into Na (pH 6.5) to create an artificial delta psi took up little glutamine, it appeared that transport was driven by phosphate-bond energy rather than proton motive force. The kinetics of glutamine transport by glucose-energized cells were biphasic, and it appeared that facilitated diffusion was also involved, particularly at high glutamine concentrations. Glucose-depleted cultures took up glutamine and produced ammonia, but the rate of transport per unit of glutamine (V/S) by nonenergized cells was at least 1,000-fold less than the V/S by glucose-energized cells. Glutamine was converted to pyroglutamate and ammonia by a pathway that did not involve a glutaminase reaction or glutamate production. No ammonia production from pyroglutamate was detected. S. bovis was unable to take up glutamate, but intracellular glutamate concentrations were as high as 7 mM. Glutamate was produced from ammonia via a glutamate dehydrogenase reaction. Cells contained high concentrations of 2-oxoglutarate and NADPH that inhibited glutamate deamination and favored glutamate formation. Since the carbon skeleton of glutamine was lost as pyroglutamate, glutamate formation occurred at the expense of glucose. Arginine deamination is often used as a taxonomic tool in classifying streptococci, and it had generally been assumed that other amino acids could not be fermented. To our knowledge, this is the first report of glutamine conversion to pyroglutamate and ammonia in streptococci.
Cloning, sequencing, and overexpression of mvaA, which encodes Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase
Authors: Beach M J , Rodwell V W
Published: November 2016

We have cloned, determined the primary structure of, and overexpressed in Escherichia coli the gene mvaA, which is the 1,287-base structural gene for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [EC 1.1.1.88] of Pseudomonas mevalonii. The amino acid composition of HMG-CoA reductase agreed with that predicted from the nucleotide sequence of mvaA, and DNA-derived sequences were identical to all experimentally determined peptide sequences. Overexpression of mvaA in E. coli yielded quantities of HMG-CoA reductase over 1,500-fold higher than those present in control cultures. Comparison of the primary structure of the P. mevalonii enzyme with the DNA-derived primary structure for a mammalian HMG-CoA reductase revealed two regions of similarity suggestive of functional relatedness. An open reading frame, ORF1, lies on the 3' side of mvaA, and a potential ribosome-binding site for ORF1 overlaps the termination codon of mvaA.
Organization of the Haemophilus influenzae Rd genome
Authors: Lee J J , Smith H O , Redfield R J
Published: November 2016

We present the first complete map of the Haemophilus influenzae genome, consisting of a detailed restriction map with a number of genetic loci. All of the ApaI, SmaI, and RsrII restriction sites (total of 45 sites) were mapped by Southern blot hybridization analysis of fragments separated by pulsed-field gel electrophoresis. Cloned genes were placed on the restriction map by Southern hybridization, and antibiotic resistance loci were also located by transformation with purified restriction fragments. The attachment site of the HP1 prophage was mapped. In addition, the number, locations, and orientations of the six rRNA operons in the H. influenzae chromosome were determined. The positions of conserved restriction sites in these rrn operons confirm that the direction of transcription is 16S to 23S, as in most other bacteria. The widely used strain BC200 appears to contain an unexpected 45-kilobase duplication.
Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon
Authors: Solomon K A , Hsu D K , Brusilow W S
Published: November 2016

We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase. We have used these constructions to measure the relative in vivo expression of these genes. The second and third genes, uncB and uncE, which code for the a and c subunits of the F0 sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ. These rates compared favorably with the relative numbers of a and c subunits (a1:c10) in the purified F1F0 complex. The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB.
Genetic reconstitution of the high-affinity L-arabinose transport system
Authors: Horazdovsky B F , Hogg R W
Published: November 2016

Expression plasmids containing various portions of araFGH operon sequences were assayed for their ability to facilitate the high-affinity L-arabinose transport process in a strain lacking the chromosomal copy of this operon. Accumulation studies demonstrated that the specific induction of all three operon coding sequences was necessary to restore high-affinity L-arabinose transport. Kinetic analysis of this genetically reconstituted transport system indicated that it functions with essentially wild-type parameters. Therefore, L-arabinose-binding protein-mediated transport appears to require only two inducible membrane-associated components (araG and araH) in addition to the binding protein (araF).
Product of the Lactococcus lactis gene required for malolactic fermentation is homologous to a family of positive regulators
Authors: Renault P , Gaillardin C , Heslot H
Published: November 2016

Malolactic fermentation is a secondary fermentation that many lactic acid bacteria can carry out when L-malate is present in the medium. The activation of the malolactic system in Lactococcus lactis is mediated by a locus we call mleR. Induction of the genes necessary to perform malolactic fermentation occurs only in bacteria with a functional copy of mleR. The mleR gene consists of one open reading frame capable of coding for a protein with a calculated molecular mass of 33,813 daltons. The amino acid sequence of the predicted MleR gene product is homologous to that of positive activators in gram-negative bacteria: LysR, IlvY gene products of Escherichia coli, MetR, CysB of Salmonella typhimurium, AmpR of Enterobacter cloacae, NodD of Rhizobium sp., and TrpI of Pseudomonas aeruginosa.
Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti
Authors: Inoue T , Sunagawa M , Mori A , Imai C , Fukuda M , Takagi M , Yano K
Published: November 2016

A genomic library of Acetobacter aceti DNA was constructed by using a broad-host-range cosmid vector. Complementation of a spontaneous alcohol dehydrogenase-deficient mutant resulted in the isolation of a plasmid designated pAA701. Subcloning and deletion analysis of pAA701 limited the region that complemented the deficiency in alcohol dehydrogenase activity of the mutant. The nucleotide sequence of this region was determined and showed that this region contained the full structural gene for the 72-kilodalton dehydrogenase subunit of the alcohol dehydrogenase enzyme complex. The predicted amino acid sequence of the gene showed homology with sequences of methanol dehydrogenase structural genes of Paracoccus denitrificans and Methylobacterium organophilum.
Streptomyces lipmanii expresses two restriction systems that inhibit plasmid transformation and bacteriophage plaque formation
Authors: Matsushima P , Baltz R H
Published: November 2016

Bacteriophage host range studies suggested that several beta-lactam-producing streptomycetes express similar restriction-modification systems. Streptomyces lipmanii LE32 expressed two restriction-modification systems, designated SliI and SliII. A mutant strain, PM87, was defective only in SliI restriction but expressed both SliI and SliII modification. Streptomyces sp. strain A57986, a natural isolate partially deficient in the expression of SliI and SliII restriction, nevertheless modified bacteriophage DNA for both SliI and SliII specificities. Protoplasts of PM87 and A57986 were transformed by several plasmids, and the modified plasmids isolated from these strains transformed wild-type S. lipmanii efficiently.
Suppression of dnaE nonsense mutations by pcbA1
Authors: Maki H , Bryan S K , Horiuchi T , Moses R E
Published: November 2016

DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli. The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene. We have reported that E. coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present. Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations. We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C). Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype. We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C.
Characterization of a purF operon mutation which affects colicin V production
Authors: Fath M J , Mahanty H K , Kolter R
Published: November 2016

A mini-Tn10-kan insertion mutation identified a gene in the chromosome of Escherichia coli required for colicin V production from plasmid pColV-K30. With the complete restriction map of E. coli, the mutation was rapidly mapped to 50.0 min, within the purF operon. Sequence analysis showed that the insertion occurred in a gene with no previously known function which is located directly upstream of purF. We designated this gene cvpA for colicin V production. The mutant requires adenine for growth, probably because of a polar effect on purF expression. However, an adenine auxotroph showed no defect in colicin V production, suggesting that the cvpA mutation is responsible for the effect on colicin V production. Two possible models of cvpA1 allele function are discussed.
Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I
Authors: Martin A E , Burgess B K , Iismaa S E , Smartt C T , Jacobson M R , Dean D R
Published: November 2016

Flavodoxin and ferredoxin I have both been implicated as components of the electron transport chain to nitrogenase in the aerobic bacterium Azotobacter vinelandii. Recently, the genes encoding flavodoxin (nifF) and ferredoxin I (fdxA) were cloned and sequenced and mutants were constructed which are unable to synthesize either flavodoxin (DJ130) or ferredoxin I (LM100). Both single mutants grow at wild-type rates under N2-fixing conditions. Here we report the construction of a double mutant (DJ138) which does not synthesize either flavodoxin or ferredoxin I. When plated on ammonium-containing medium, this mutant had a very small colony size when compared with the wild type, and in liquid culture with ammonium, this double mutant grew three times slower than the wild type or single mutant strains. This demonstrated that there is an important metabolic function unrelated to nitrogen fixation that is normally carried out by either flavodoxin or ferredoxin. If either one of these proteins is missing, the other can substitute for it. The double mutant phenotype can now be used to screen site-directed mutant versions of ferredoxin I for functionality in vivo even though the specific function of ferredoxin I is still unknown. The double mutant grew at the same slow rate under N2-fixing conditions. Thus, A. vinelandii continues to fix N2 even when both flavodoxin and ferredoxin I are missing, which suggests that a third as yet unidentified protein also serves as an electron donor to nitrogenase.
Ammonium inhibition of nitrogenase activity in Herbaspirillum seropedicae
Authors: Fu H , Burris R H
Published: November 2016

The effect of oxygen, ammonium ion, and amino acids on nitrogenase activity in the root-associated N2-fixing bacterium Herbaspirillum seropedicae was investigated in comparison with Azospirillum spp. and Rhodospirillum rubrum. H. seropedicae is microaerophilic, and its optimal dissolved oxygen level is from 0.04 to 0.2 kPa for dinitrogen fixation but higher when it is supplied with fixed nitrogen. No nitrogenase activity was detected when the dissolved O2 level corresponded to 4.0 kPa. Ammonium, a product of the nitrogenase reaction, reversibly inhibited nitrogenase activity when added to derepressed cell cultures. However, the inhibition of nitrogenase activity was only partial even with concentrations of ammonium chloride as high as 20 mM. Amides such as glutamine and asparagine partially inhibited nitrogenase activity, but glutamate did not. Nitrogenase in crude extracts prepared from ammonium-inhibited cells showed activity as high as in extracts from N2-fixing cells. The pattern of the dinitrogenase and the dinitrogenase reductase revealed by the immunoblotting technique did not change upon ammonium chloride treatment of cells in vivo. No homologous sequences were detected with the draT-draG probe from Azospirillum lipoferum. There is no clear evidence that ADP-ribosylation of the dinitrogenase reductase is involved in the ammonium inhibition of H. seropedicae. The uncoupler carbonyl cyanide m-chlorophenylhydrazone decreased the intracellular ATP concentration and inhibited the nitrogenase activity of whole cells. The ATP pool was not significantly disturbed when cultures were treated with ammonium in vivo. Possible mechanisms for inhibition by ammonium of whole-cell nitrogenase activity in H. seropedicae are discussed.