bluuebunny/crossref_metadata_2025 huggingface dataset:
How a mutation in the gene encoding sigma 70 suppresses the defective heat shock response caused by a mutation in the gene encoding sigma 32
Authors: Zhou Y N , Gross C A
Published: November 2016

In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters. sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells. Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response. Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response. This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70. We have determined the mechanism of suppression. In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift. Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.
Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes
Authors: Walter K A , Bennett G N , Papoutsakis E T
Published: November 2016

A 4-kb segment of DNA containing two previously cloned butanol dehydrogenase (BDH) isozyme genes (D. Petersen, R. Welch, F. Rudolph, and G. Bennett, J. Bacteriol. 173:1831-1834, 1991) was sequenced. Two complete open reading frames (ORFs) were identified (bdhA and bdhB), along with a third truncated ORF (ORF1). The translation products of bdhA and bdhB corresponded to the N-terminal sequences of the purified BDH I and BDH II proteins, respectively. The two isozymes had a high amino acid identity (73%) and showed homology to a newly described class of alcohol dehydrogenases. Northern blots revealed that bdhA and bdhB did not form an operon. Primer extension experiments located single transcriptional start sites 37 and 58 bp upstream of the start codons of bdhA and bdhB, respectively. The -10 and -35 promoter regions for these genes were almost identical. bdhA and bdhB were found to be induced or derepressed immediately prior to significant butanol production in controlled pH 5.0 batch fermentations.
A locus that contributes to colonization of the intestinal tract by Bacteroides thetaiotaomicron contains a single regulatory gene (chuR) that links two polysaccharide utilization pathways
Authors: Cheng Q , Hwa V , Salyers A A
Published: November 2016

Previously, we isolated two Tn4351-generated mutants of Bacteroides thetaiotaomicron (46-1 and CS3) that were unable to grow either on heparin or on chondroitin sulfate. This phenotype was unexpected, since the heparin and chondroitin sulfate utilization pathways had appeared from earlier studies to be independent of each other. Mutants 46-1 and CS3 were also of interest because both were unable to compete successfully with wild-type B. thetaiotaomicron in the intestinal tracts of germfree mice. Thus, both appeared to have a colonization defect. We have now cloned the chromosomal locus in which the transposon insertions in 46-1 and CS3 occurred. Southern blot analysis showed that the Tn4351 insertions in 46-1 and CS3 were about 100 bp apart. Using complementation and insertional mutagenesis, we localized the region affected by the 46-1 and CS3 insertions to within 2.5 kbp. This DNA segment was sequenced and found to contain a 401-codon open reading frame (ORF1) and the N-terminal segment of a second open reading frame (ORF2), which was downstream of ORF1 and transcribed in the same direction. The deduced amino acid sequence of ORF1 showed significant homology to that of a putative positive regulator of an arylsulfatase gene in Klebsiella aerogenes. ORF2 was at least 381 amino acids long and did not exhibit homology to any proteins in the data bases searched. Transposon insertions in both mutants 46-1 and CS3 disrupted ORF1. The results of insertional mutagenesis and complementation experiments indicated that ORF2 was not essential for growth on chondroitin sulfate or heparin. Thus, the chondroitin sulfate-negative and heparin-negative phenotypes of 46-1 and CS3 appear to be due to the interruption of a regulatory gene encoded by ORF1 and not to a polar effect of the insertions on a downstream gene(s). The gene encoding ORF1 has been designated chuR, for regulation of chondroitin sulfate and heparin utilization. Transcriptional fusion studies showed that the expression of chuR occurred at the same level under inducing and noninducing conditions, in contrast to the regulated expression of structural genes of the chondroitin sulfate utilization system. chuR was not autoregulated, nor was its expression affected by a mutation (46-4) that eliminated the expression of all chondroitin sulfate utilization genes but did not affect the utilization of heparin.
Characterization of the Escherichia coli membrane domain responsible for binding oriC DNA
Authors: Chakraborti A , Gunji S , Shakibai N , Cubeddu J , Rothfield L
Published: November 2016

It has previously been shown that hemimethylated DNA from the Escherichia coli replication origin (oriC) binds with high specificity to membrane fractions isolated from disrupted cells. In this article, the membrane localization of oriC-binding activity was studied by subjecting crude membrane preparations to successive cycles of sedimentation and flotation gradient analysis. This revealed that approximately two-thirds of the membrane-associated oriC-binding activity of the cell was not associated with the outer membrane fraction as previously suggested but was recovered instead in a unique membrane fraction (OCB1) whose buoyant density and protein profile differed from those of both inner and outer membranes. The specific activity of oriC binding in OCB1 was approximately fivefold higher than the activity of the isolated outer membrane peak. It is likely that membrane fraction OCB1 includes the membrane domain responsible for the binding of hemimethylated oriC to the cell envelope in intact cells.
Multiple forms of bile salt hydrolase from Lactobacillus sp. strain 100-100
Authors: Lundeen S G , Savage D C
Published: November 2016

Four isozymes of bile salt hydrolase (BSH) have been purified from the cytosol of cells of Lactobacillus sp. strain 100-100. The four proteins were designated BSH A, B, C, and D. They eluted from anion-exchange high-pressure liquid chromatography columns at 0.15, 0.18, 0.21, and 0.25 M NaCl, respectively. They are catalytically similar, except that the Vmax of BSH D is about 10-fold lower than those of the other three isozymes. All four proteins consist of one or two polypeptides. The peptides have molecular weights of 42,000 and 38,000 and are designated alpha and beta, respectively. The approximate native molecular weights of BSH A, B, C, and D are 115,000, 105,000, 95,000, and 80,000, respectively. The native proteins are probably trimers; the four isozymes are the array of possible subunit combinations alpha 3, alpha 2 beta 1, alpha 1 beta 2, and beta 3 for A, B, C, and D, respectively. The two subunits are antigenically distinct. Polyclonal antibodies raised against BSH A (all alpha peptide) react in Western blots (immunoblots) only with proteins containing the alpha peptide; such antibodies raised against BSH D (all beta peptide) react only with proteins containing the beta peptide. The amino acid compositions of the two peptides differ. This is the first report of a bacterium that makes four BSH isozymes.
Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome
Authors: Stettler R , Leisinger T
Published: November 2016

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.
Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes
Authors: Brahamsha B , Haselkorn R
Published: November 2016

The sigA gene of Anabaena sp. strain PCC 7120, encoding the principal RNA polymerase sigma factor, and the complement of the rpoD oligonucleotide (K. Tanaka, T. Shiina, and H. Takahashi, Science 242:1040-1042, 1988) were used as probes to isolate two genes, sigB and sigC, which encode two putative sigma factors exhibiting high degrees of similarity to SigA, to HrdA, -B, -C, and -D of Streptomyces coelicolor, and to KatF of Escherichia coli. sigB and sigC code for polypeptides of 332 and 416 amino acids with predicted molecular weights of 38,431 and 47,459, respectively. sigB and sigC mRNAs are detectable only under nitrogen-limiting conditions. Insertional inactivation of sigB and sigC indicates that neither gene alone is essential for nitrogen fixation or heterocyst differentiation.
Regulation of partitioned sterol biosynthesis in Saccharomyces cerevisiae
Authors: Casey W M , Keesler G A , Parks L W
Published: November 2016

Using yeast strains with null mutations in structural genes which encode delta-aminolevulinic acid synthetase (HEM1), isozymes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG1 and HMG2), squalene epoxidase (ERG1), and fatty acid delta 9-desaturase (OLE1), we were able to determine the effect of hemes, sterols, and unsaturated fatty acids on both sterol production and the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in Saccharomyces cerevisiae. We found that the HMGR isozymes direct essentially equal amounts of carbon to the biosynthesis of sterols under heme-competent conditions, despite a huge disparity (57-fold) in the specific activities of the reductases. Our results demonstrate that palmitoleic acid (16:1) acts as a rate-limiting positive regulator and that ergosterol acts as a potent inhibitor of sterol production in strains which possess only the HMGR1 isozyme (HMG1 hmg2). In strains which contain only the HMGR2 isozyme (hmg1 HMG2), sterol production was inhibited by oleic acid (18:1) and to a lesser degree by ergosterol. The specific activities of the two reductases (HMGR1 and HMGR2) were found to be differentially regulated by hemes but not by ergosterol, palmitoleic acid, or oleic acid. The disparate effects of unsaturated fatty acids and sterols on these strains lead us to consider the possibility of separate, compartmentalized isoprenoid pathways in S. cerevisiae.
The terminal reductases for selenate and nitrate respiration in Thauera selenatis are two distinct enzymes
Authors: Rech S A , Macy J M
Published: November 2016

A number of approaches have been used to show that a recently isolated selenate-respiring bacterium, Thauera selenatis, is able to synthesize both a selenate reductase (SR) and a nitrate reductase (NR). (i) The pH optimum of the SR was found to be 6.0; that of the NR was 7.0. (ii) The presence of nitrate did not inhibit selenate reduction in selenate-grown cells. (iii) In cell extracts, the highest SR or NR activity was observed in cells grown with the respective electron acceptor. (iv) Mutants that were unable to grow with nitrate as the terminal electron acceptor and lacked NR activity were isolated; these mutants grew normally with selenate and synthesized SR. (v) The SR was found in the periplasmic space of the cell, whereas the NR was present in the cytoplasmic membrane. A hypothetical electron transport system involving the SR is described.
Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa
Authors: Hoyne P A , Haas R , Meyer T F , Davies J K , Elleman T C
Published: November 2016

Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell.
The gene encoding a Prevotella loescheii lectin-like adhesin contains an interrupted sequence which causes a frameshift
Authors: Manch-Citron J N , Allen J , Moos M , London J
Published: November 2016

We cloned and sequenced the Prevotella loescheii gene plaA, which encodes a lectin-like adhesin that mediates the coaggregation of P. loescheii 1295 with Streptococcus oralis 34. A probe derived from the N-terminal amino acid sequence of the purified adhesin was used to identify the plaA gene from a P. loescheii genomic library constructed in lambda GEM-11. Sequence analysis of plaA indicates that the initial translation product contains a 22-amino-acid leader. The reading frame of the plaA gene is interrupted after amino acid 28 of the mature protein by a TAA termination codon. Amplification of the P. loescheii genomic DNA in the region surrounding this codon by the polymerase chain reaction followed by DNA sequencing of the cloned DNA fragment established that this stop codon was not an experimental artifact. A frameshift beginning 29 bp downstream of the ochre terminator was required to access the only large open reading frame in the gene. Amino acid sequences of six purified peptides derived by limited proteolysis of adhesin with endoproteinase Lys-C matched the downstream amino acid sequence derived by translation of the large open reading frame. The gene coding sequence of 2.4 kb contains sufficient information for the synthesis of an 89-kDa protein. A putative rho-independent terminator (delta G = -25.5 kcal/mol [ca. -107 kJ/mol]) was detected 38 bp downstream from the plaA stop codon.
The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus
Authors: Kusian B , Yoo J G , Bednarski R , Bowien B
Published: November 2016

Several genes (cfx genes) encoding Calvin cycle enzymes in Alcaligenes eutrophus are organized in two highly homologous operons comprising at least 11 kb. One cfx operon is located on the chromosome; the other is located on megaplasmid pHG1 of the organism (B. Bowien, U. Windhövel, J.-G. Yoo, R. Bednarski, and B. Kusian, FEMS Microbiol. Rev. 87:445-450, 1990). Corresponding regions of about 2.7 kb from within the operons were sequenced. Three open reading frames, designated cfxX (954 bp), cfxY (765 bp), and cfxE (726 bp), were detected at equivalent positions in the two sequences. The nucleotide identity of the sequences amounted to 94%. Heterologous expression of the subcloned pHG1-encoded open reading frames in Escherichia coli suggested that they were functional genes. The observed sizes of the gene products CfxX (35 kDa), CfxY (27 kDa), and CfxE (25.5 kDa) closely corresponded to the values calculated on the basis of the sequence information. E. coli clones harboring the cfxE gene showed up to about 19-fold-higher activities of pentose-5-phosphate 3-epimerase (PPE; EC 5.1.3.1) than did reference clones, suggesting that cfxE encodes PPE, another Calvin cycle enzyme. These data agree with the finding that in A. eutrophus, PPE activity is significantly enhanced under autotrophic growth conditions which lead to a derepression of the cfx operons. No functions could be assigned to CfxX and CfxY.
Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone
Authors: Nakahigashi K , Miyamoto K , Nishimura K , Inokuchi H
Published: November 2016

Cells with a novel mutation that is lethal when the cells are exposed to visible light were isolated from Escherichia coli K-12. The mutation was mapped at 63 min on the linkage map of the E. coli chromosome, and the gene, designated visB, was cloned and sequenced. From its map position and the evidence that the gene product VisB exhibits homology with flavin monooxygenase of Pseudomonas fluorescens, the visB gene was deduced to be identical to the ubiH gene, which is a gene required for the biosynthesis of ubiquinone and is thought to be similar to the gene for flavin monooxygenase. The photosensitive phenotype appears to be due to the accumulation of the substrate for the reaction catalyzed by the visB (ubiH) gene product because other mutations that block earlier steps in the biosynthesis of ubiquinone can reverse the photosensitivity. The accumulated intermediates may produce active species of oxygen in the mutant bacteria upon illumination by visible light, and these active oxygen species may cause the death of the cells by a mechanism similar to that associated with mutations in visA (hemH).
A-signalling and the cell density requirement for Myxococcus xanthus development
Authors: Kuspa A , Plamann L , Kaiser D
Published: November 2016

Mutations in any of three asg (A-signalling) loci cause fruiting body development of Myxococcus xanthus to arrest at about the 2-h stage. Development can be restored to asg mutants by the addition of conditioned buffer in which wild-type cells have been developing or of A-factor purified from the conditioned buffer. Two forms of A-factor have been identified: heat-stable A-factor, which is composed of amino acids and peptides, and heat-labile A-factor, which consists of at least two proteases. A-factor is found in conditioned buffer in rough proportion to the cell density. As decreasing amounts of either form of A-factor are added, the developmental response of asg cells decreases until a threshold concentration is reached, below which no response is detected. In addition, wild-type cells fail to develop when their density is decreased below the point at which the level of A-factor is predicted to fall short of this threshold. The development of low-density asg+ cells can, however, be restored by the addition of either form of A-factor. These experiments show that A-factor is important for the development of wild-type cells. Moreover, the development of an asgB mutant that produces 5 to 10% the wild-type level of A-factor can be restored when the cell density is increased 10-fold above the standard density. We propose that the A-signal is used by M. xanthus to specify the minimum cell density required for the initiation of development. Differences in the response to A-factor between different asg mutants suggest that the different asg loci govern A-factor production in diverse ways.
Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis
Authors: Arfman N , Worrell V , Ingram L O
Published: November 2016

The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase II (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobilis by using a broad-host-range vector containing the tac promoter and the lacIq repressor gene. Maximal IPTG (isopropyl-beta-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z. mobilis resulted in a 35-fold increase in alcohol dehydrogenase I activity, a 16.7-fold increase in alcohol dehydrogenase II activity, and a 6.3-fold increase in pyruvate decarboxylase activity. Small changes in the activities of these enzymes did not affect glycolytic flux in cells which are at maximal metabolic activity, indicating that flux under these conditions is controlled at some other point in metabolism. Expression of adhA, adhB, or pdc at high specific activities (above 8 IU/mg of cell protein) resulted in a decrease in glycolytic flux (negative flux control coefficients), which was most pronounced for pyruvate decarboxylase. Growth rate and flux are imperfectly coupled in this organism. Neither a twofold increase in flux nor a 50% decline from maximal flux caused any immediate change in growth rate. Thus, the rates of biosynthesis and growth in this organism are not limited by energy generation in rich medium.
Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes
Authors: Yanai K , Takaya N , Kojima N , Horiuchi H , Ohta A , Takagi M
Published: November 2016

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.
The lethal phenotype caused by null mutations in the Escherichia coli htrB gene is suppressed by mutations in the accBC operon, encoding two subunits of acetyl coenzyme A carboxylase
Authors: Karow M , Fayet O , Georgopoulos C
Published: November 2016

Insertion mutations in the Escherichia coli htrB gene result in the unique phenotype of not affecting growth at temperatures below 32.5 degrees C but leading to a loss of viability at temperatures above this in rich media. When htrB bacteria growing in rich media were shifted to the nonpermissive temperature of 42 degrees C, they continued to grow at a rate similar to that at 30 degrees C but they produced phospholipids at the rate required for growth at 42 degrees C. This led to the accumulation of more than twice as much phospholipid per milligram of protein compared with that in wild-type bacteria. Consistent with HtrB playing a role in phospholipid biosynthesis, one complementation group of spontaneously arising mutations that suppressed htrB-induced lethality were mapped to the accBC operon. This operon codes for the biotin carboxyl carrier protein and biotin carboxylase subunits of the acetyl coenzyme A carboxylase enzyme complex, which catalyzes the first step in fatty acid biosynthesis. Four suppressor mutations mapped to this operon. Two alleles were identified as mutations in the accC gene, the third allele was identified as a mutation in the accB gene, and the fourth allele was shown to be an insertion of an IS1 transposable element in the promoter region of the operon, resulting in reduced transcription. The suppressor mutations caused a decrease in the rate of phospholipid biosynthesis, restoring the balance between the biosynthesis of phospholipids and growth rate, thus enabling htrB bacteria to grow at high temperatures.
Microcin 25, a novel antimicrobial peptide produced by Escherichia coli
Authors: Salomón R A , Farías R N
Published: November 2016

Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system.
Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans
Authors: Debussche L , Couder M , Thibaut D , Cameron B , Crouzet J , Blanche F
Published: November 2016

Hydrogenobyrinic acid a,c-diamide was shown to be the substrate of cobaltochelatase, an enzyme that catalyzes cobalt insertion in the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrificans. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of M(r) 140,000 and 450,000, which were purified to homogeneity. The 140,000-M(r) component was shown to be coded by cobN, whereas the 450,000-M(r) component was composed of two polypeptides specified by cobS and cobT. Each component was inactive by itself, but cobaltochelatase activity was reconstituted upon mixing CobN and CobST. The reaction was ATP dependent, and the Km values for hydrogenobyrinic acid a,c-diamide, Co2+, and ATP were 0.085 +/- 0.015, 4.2 +/- 0.2, and 220 +/- 36 microM, respectively. Spectroscopic data revealed that the reaction product was cob(II)yrinic acid a,c-diamide, and experiments with a coupled-enzyme incubation system containing both cobaltochelatase and cob(II)yrinic acid a,c-diamide reductase (F. Blanche, L. Maton, L. Debussche, and D. Thibaut, J. Bacteriol. 174:7452-7454, 1992) confirmed this result. This report not only provides the first evidence that hydrogenobyrinic acid and its a,c-diamide derivative are indeed precursors of adenosylcobalamin but also demonstrates that precorrin-6x, precorrin-6y, and precorrin-8x, three established precursors of hydrogenobyrinic acid (D. Thibaut, M. Couder, A. Famechon, L. Debussche, B. Cameron, J. Crouzet, and F. Blanche, J. Bacteriol. 174:1043-1049, 1992), are also on the pathway to cobalamin.
Determination of genome sizes of Rickettsia spp. within the spotted fever group, using pulsed-field gel electrophoresis
Authors: Roux V , Drancourt M , Raoult D
Published: November 2016

The chromosome lengths of six spotted fever group Rickettsia species (Rickettsia rickettsii, R. conorii, R. rhipicephali, R. sibirica, R. australis, and R. akari) were estimated by pulsed-field gel electrophoresis. The genome size of R. rickettsii was about 2,100 kb, but the chromosome lengths of the five other species were, surprisingly, much lower and ranged between 1,200 and 1,300 kb.
Sequence of the S-layer gene of Thermus thermophilus HB8 and functionality of its promoter in Escherichia coli
Authors: Faraldo M M , de Pedro M A , Berenguer J
Published: November 2016

The nucleotide sequence of the slpA gene, which is responsible for the synthesis of the S-layer protein of Thermus thermophilus HB8, is described. This gene is transcribed as a unit in which the coding region is preceded by a 127-base-long leader mRNA sequence. The promoter region is also recognized by the RNA polymerase of Escherichia coli because of the presence of homologous -35 and -10 boxes. Homologies with other promoters from Thermus spp. are also presented.
A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules
Authors: Weinstein M , Roberts R C , Helinski D R
Published: November 2016

We demonstrate for the first time that the broad-host-range stabilization loci from plasmid RK2 cause total retention of plasmids in cells of Rhizobium meliloti during symbiosis with alfalfa. Two derivatives of plasmid RK2, pRK290 and a 7.3-kb mini-RK2 plasmid, were stabilized in R. meliloti cells isolated from root nodules by the insertion of a 3.2-kb DNA fragment or a smaller 0.8-kb DNA fragment derived from the RK2 stabilization region.
Hydrolysis of urea by Ureaplasma urealyticum generates a transmembrane potential with resultant ATP synthesis
Authors: Smith D G , Russell W C , Ingledew W J , Thirkell D
Published: November 2016

When urea is added to Ureaplasma urealyticum, it is hydrolysed internally by a cytosolic urease. Under our measuring conditions, and at an external pH of 6.0, urea hydrolysis caused an ammonia chemical potential equivalent to almost 80 mV and, simultaneously, an increase in proton electrochemical potential (delta p) of about 24 mV with resultant de novo ATP synthesis. Inhibition of the urease with the potent inhibitor flurofamide abolished both the chemical potential and the increase of delta p such that ATP synthesis was reduced to approximately 5% of normally obtained levels. Uncouplers of electrochemical gradients had little or no effect on these systems. The electrochemical parameters and ATP synthesis were measured similarly at three other external pH values. Any change in delta p was primarily via membrane potential (delta psi), and the level of de novo ATP synthesis was related to the increase in delta p generated upon addition of urea and more closely to the ammonia chemical potential. Although the organisms lack an effective mechanism for internal pH homeostasis, they maintained a constant delta pH. The data reported are consistent with, and give evidence for, the direct involvement of a chemiosmotic mechanism in the generation of around 95% of the ATP by this organism. Furthermore, the data suggest that the ATP-generating system is coupled to urea hydrolysis by the cytosolic urease via an ammonia chemical potential.
Cloning and genetic characterization of the Helicobacter pylori and Helicobacter mustelae flaB flagellin genes and construction of H. pylori flaA- and flaB-negative mutants by electroporation-mediated allelic exchange
Authors: Suerbaum S , Josenhans C , Labigne A
Published: November 2016

Helicobacter pylori is one of the most common human pathogens. It causes chronic gastritis and is involved in the pathogenesis of gastroduodenal ulcer disease and possibly gastric carcinoma. Helicobacter mustelae is a bacterium closely related to H. pylori that causes gastritis and ulcer disease in ferrets and is therefore considered an important animal model of gastric Helicobacter infections. Motility, even in a viscous environment, is conferred to the bacteria by several sheathed flagella and is regarded as one of their principal virulence factors. The flagellar filament of H. pylori consists of two different flagellin species expressed in different amounts. The gene (flaA) encoding the major flagellin has recently been cloned and sequenced. Here we report the cloning and sequencing of two highly homologous new flagellin genes from H. pylori 85P and H. mustelae NCTC 12032. The nucleotide sequence of the H. pylori gene proved that it encoded the second flagellin molecule found in H. pylori flagellar filaments. The genes were named flaB. The H. mustelae and H. pylori flaB genes both coded for proteins with 514 amino acids and molecular masses of 54.0 and 53.9 kDa, respectively. The proteins shared 81.7% identical amino acids. The degree of conservation between H. pylori FlaB and the H. pylori FlaA major flagellin was much lower (58%). Both flaB genes were preceded by sigma 54-like promoter sequences. Mapping of the transcription start site for the H. pylori flaB gene by a primer extension experiment confirmed the functional activity of the sigma 54 promoter. To evaluate the importance of both genes for motility, flaA- and flaB-disrupted mutants of H. pylori N6 were constructed by electroporation-mediated allelic exchange and characterized by Western blot (immunoblot) analysis and motility testing. Both mutations selectively abolished the expression of the targeted gene without affecting the synthesis of the other flagellin molecule. Whereas flaA mutants were completely nonmotile, flaB mutants retained motility.
A new putative sigma factor of Myxococcus xanthus
Authors: Apelian D , Inouye S
Published: November 2016

A third putative sigma factor gene, sigC, has been isolated from Myxococcus xanthus by using the sigA gene (formerly rpoD of M. xanthus) as a probe. The nucleotide sequence of sigC has been determined, and an open reading frame of 295 residues (M(r) = 33,430) has been identified. The deduced amino acid sequence of sigC exhibits the features which are characteristic of other bacterial sigma factors. The characterization of a sigC-lacZ strain has demonstrated that sigC expression is induced immediately after cells enter into the developmental cycle and is dramatically reduced at the onset of sporulation. A deletion mutant of sigC grows normally in vegetative culture and is able to develop normally. However, in contrast to the wild-type cells, the sigC deletion mutant cells became capable of forming fruiting bodies and myxospores on semirich agar plates. This suggests that sigC may play a role in expression of genes involved in negatively regulating the initiation of fruiting body formation.
Adhesion and growth rate of Clostridium cellulolyticum ATCC 35319 on crystalline cellulose
Authors: Gelhaye E , Petitdemange H , Gay R
Published: November 2016

The rate of tritiated-thymidine incorporation into DNA was used to estimate Clostridium cellulolyticum H10 growth rates on Avicel cellulose, taking into consideration both the unattached cells and the cells adhered to the substrate. The generation time on cellobiose calculated from the data on cell density (4.5 h) agreed well with the generation time calculated by tritiated-thymidine incorporation (3.8 h). Growth on Avicel cellulose occurred when bacteria were adhered to their substrate; 80% of the biomass was detected on the cellulose. Taking into consideration attached and free bacteria, the generation time as determined by thymidine incorporation was about 8 h, whereas by bacterial-protein estimation it was about 13 h. In addition to the growth rate of the bacteria on the cellulose, the release of adhered cells constituted an important factor in the efficiency of the cellulolysis. The stage of growth influenced adhesion of C. cellulolyticum; maximum adhesion was found during the exponential phase. Under the conditions used, the end of growth was characterized by an acute release of biomass and cellulase activity from the cellulose. An exhaustion of the accessible cellulose could be responsible for this release.
Flagellin gene transcription in Bordetella bronchiseptica is regulated by the BvgAS virulence control system
Authors: Akerley B J , Miller J F
Published: November 2016

The products of the bvgAS locus activate expression of a majority of the known Bordetella virulence factors but also exert negative control over a class of genes called vrg genes (bvg-repressed genes). BvgAS negatively controls the production of flagella and the phenotype of motility in Bordetella bronchiseptica. In this study flaA, the flagellin gene, was cloned and characterized to facilitate studies of this negative control pathway. An internal flaA probe detected hybridizing sequences on genomic Southern blots of Bordetella pertussis, Bordetella parapertussis, and Bordetella avium, although B. pertussis and B. parapertussis are nonmotile. FlaA is similar to the FliC flagellins of Salmonella typhimurium and Escherichia coli, and flaA complemented an E. coli flagellin mutant. Insertional inactivation of the chromosomal flaA locus eliminated motility, which was restored by complementation with the wild-type locus. Analysis of flaA mRNA production by Northern (RNA) blotting and primer extension indicated that negative regulation by BvgAS occurs at the level of transcription. The transcriptional start site of flaA mapped near a consensus site for the alternative sigma factor, sigma F, encoded by fliA in E. coli and S. typhimurium. Consistent with a role for a fliA analog in B. bronchiseptica, transcriptional activation of a flaA-lacZ fusion in E. coli required fliA and a flaA-linked locus designated frl.frl also efficiently complemented mutations in the flagellar master regulatory locus, flhDC, of E. coli. Our analysis of the motility phenotype of B. bronchiseptica suggests that the Bordetella virulence control system mediates transcriptional control of flaA through a regulatory hierarchy that includes the frl locus and an alternative sigma factor.
Amino-acylation site mutations in amino acid-activating domains of surfactin synthetase: effects on surfactin production and competence development in Bacillus subtilis
Authors: D'Souza C , Nakano M M , Corbell N , Zuber P
Published: November 2016

The part of the srfA operon of Bacillus subtilis that contains the region required for competence development is composed of the first four amino acid-activating domains which are responsible for the incorporation of Glu, Leu, D-Leu, and Val into the peptide moiety of the lipopeptide surfactin. Ser-to-Ala substitutions were made in the amino-acylation site of each domain, and their effects on surfactin production and competence development were examined. All of the mutations conferred a surfactin-negative phenotype, supporting the finding that the conserved Ser in the amino-acylation site is required for peptide synthesis. However, none of the mutations affected significantly competence development or the expression of a lacZ fusion to the late competence operon comG. This, coupled with recent findings that only the fourth, Val-activating, domain is required for competence, suggests that some activity, other than amino-acylation and perhaps unrelated to peptide synthesis, possessed by the fourth domain is involved in the role of srfA in regulating competence development.
Multiple effects of lcrD mutations in Yersinia pestis
Authors: Plano G V , Straley S C
Published: November 2016

Plasmid pCD1 of Yersinia pestis contains a low-calcium response stimulon responsible for the temperature- and calcium-regulated expression and secretion of proteins involved in virulence, which include the V antigen and Yops. We have previously shown that insertional inactivation of the bicistronic lcrDR operon abolished the calcium requirement for growth at 37 degrees C and reduced expression of the V antigen and Yops. In this study, we constructed and characterized three mutants having nonpolar lcrD deletions. All three mutants lost the two main low-calcium response properties: a calcium requirement for growth at 37 degrees C and strong expression of the V antigen and Yops. The effects on virulence gene expression occurred at both the levels of transcription and secretion. The growth, transcription, and secretion defects could be at least partially complemented for two of the lcrD mutants by providing lcrD in trans. A third mutant could not be complemented, and a plasmid carrying this mutation had a dominant negative effect over normal LcrD function. In the three mutants, the amount of mutant LcrD protein detectable in immunoblots was inversely related to the amount of complementation. Taken together, these data indicate that LcrD function involves the interaction of LcrD with another molecule.
Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin
Authors: Sozhamannan S , Chattoraj D K
Published: November 2016

Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have confirmed the roles of the three heat shock proteins in promoting RepA binding to the origin. The defects in both activities could be suppressed by increasing the concentration of wild-type RepA over the physiological level. We also isolated RepA mutants that were effective initiators and repressors without requiring the heat shock proteins. These data suggest that the heat shock proteins facilitate both repression and initiation by promoting only the DNA-binding activity of RepA. In a similar plasmid, F, initiator mutants that confer heat shock protein independence for replication were also found, but they were defective for repression. We propose that the initiator binding involved in repression and the initiator binding involved in initiation are similar in P1 but different in F.
Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli
Authors: Reuhs B L , Carlson R W , Kim J S
Published: November 2016

The polysaccharide components from cultured cells of Rhizobium fredii USDA205 and Rhizobium meliloti AK631 were extracted with hot phenol-water and separated by repetitive gel filtration chromatography. Polyacrylamide gel electrophoresis, nuclear magnetic resonance spectrometry, and gas chromatography analyses showed that both of these bacterial species produce unique polysaccharides that contain a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). These polysaccharides, which constituted a major portion of the extracted carbohydrate, are not excreted into the growth media (i.e., they are not extracellular polysaccharides) and are structurally distinct from the lipopolysaccharides. The primary structure of the preponderant polysaccharide from R. fredii USDA205 was determined by high-performance anion-exchange liquid chromatography, nuclear magnetic resonance spectrometry, fast atom bombardment-mass spectrometry, and gas chromatography-mass spectrometry; it consists of repeating units of [-->3)-alpha-D-Galp-(1-->5)-beta-D-Kdop-(2-->]n. This molecule is structurally analogous to the constituents of one subgroup of K antigens (capsular polysaccharides) produced by Escherichia coli. Polysaccharides of this type have not previously been identified as components of rhizobial cells. The Kdo-containing polysaccharide from R. meliloti, which has not been completely characterized, appears to be structurally related to that of R. fredii.
Regulation of cohesion-dependent cell interactions in Myxococcus xanthus
Authors: Dana J R , Shimkets L J
Published: November 2016

Myxococcus xanthus has two nearly independent genetic systems, A and S, which appear to mediate adventurous (single-cell) movement and social (group) movement, respectively. In addition to a notable reduction in group movement, social motility mutants exhibit decreased biofilm formation, cell cohesion, dye binding, fibril production, and fruiting body formation. The stk-1907 allele, containing transposon Tn5 insertion omega DK1907, was introduced into wild-type cells and many social motility mutants. This allele, which was epistatic to most social motility mutations, caused wild-type and most mutant cells to exhibit increased group movement, cell cohesion, dye binding, and production of cell surface fibrils. The presence of the stk-1907 allele in dsp mutants, which almost completely lack cell surface fibrils, did not result in these phenotypic changes; therefore, stk-1907 is hypostatic to dsp mutations. Those mutants which exhibited increased group movement and cell cohesion with the stk-1907 allele also had increased fruiting body formation, but no significant changes in spore production were observed. These results suggest that fibrils may mediate cell cohesion, dye binding, and group movement. Additionally, the results suggest that the dsp locus contains genes involved in subunit synthesis, transport, and/or assembly of fibrils. The wild-type and mutant alleles of stk were cloned and studied in merodiploids. The mutant allele is recessive, suggesting that Tn5 omega DK1907 caused a null mutation in a gene which acts as a negative regulator of fibril synthesis. The stk-1907 allele appears to cause utilization of the A motility system for group movement, possibly because of increased fibril production.
Structures of the modified folates in the extremely thermophilic archaebacterium Thermococcus litoralis
Authors: White R H
Published: November 2016

The chemical structures of the two modified folates present in Thermococcus litoralis were established. These compounds, each containing a core structure of 1-[4-[[1-(2-amino-7-methyl- 4-oxo-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-[1-alpha-D- ribofuranosyl]-ribitol, were characterized. The five position of the ribose in this core structure was beta-linked to the C-1 of a poly-beta (1-->4)N-acetylglucosamine having a chain length of four or five N-acetylglucosamine residues. Thus, these compounds are N-acetylglucosamine homologs of the modified folates found in Pyrococcus furiosus.
The Chlamydia trachomatis Mip-like protein is a lipoprotein
Authors: Lundemose A G , Rouch D A , Penn C W , Pearce J H
Published: November 2016

The Mip-like protein of Chlamydia trachomatis is similar to the Mip protein of Legionella pneumophila and may be equally important for the initiation of intracellular infection. This article presents data which identify the chlamydial Mip-like protein as a lipoprotein. The amino acid sequence of the Mip-like protein contains a signal peptidase II recognition sequence, as is seen in procaryotic lipoproteins. Palmitic acid was incorporated into the recombinant chlamydial Mip-like protein. Globomycin, known to inhibit signal peptidase II, inhibited processing of the recombinant Mip-like protein. Labelling of chlamydial organisms with palmitic acid revealed incorporation into the native Mip-like protein.