bluuebunny/crossref_metadata_2025 huggingface dataset:
Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules
Authors: Swift S , Karlyshev A V , Fish L , Durant E L , Winson M K , Chhabra S R , Williams P , Macintyre S , Stewart G S
Published: November 2016

Spent culture supernatants from both Aeromonas hydrophila and Aeromonas salmonicida activate a range of biosensors responsive to N-acylhomoserine lactones (AHLs). The genes for a quorum sensing signal generator and a response regulator were cloned from each Aeromonas species and termed ahyRI and asaRI, respectively. Protein sequence homology analysis places the gene products within the growing family of LuxRI homologs. ahyR and asaR are transcribed divergently from ahyI and asaI, respectively, and in both Aeromonas species, the genes downstream have been identified by DNA sequence and PCR analysis. Downstream of both ahyI and asaI is a gene with close homology to iciA, an inhibitor of chromosome replication in Escherichia coli, a finding which implies that in Aeromonas, cell division may be linked to quorum sensing. The major signal molecule synthesized via both AhyI and AsaI was purified from spent culture supernatants and identified as N-(butanoyl)-L-homoserine lactone (BHL) by thin-layer chromatography, high-pressure liquid chromatography analysis, and mass spectrometry. In addition, a second, minor AHL, N-hexanoyl-L-homoserine lactone, was identified. Transcriptional reporter studies with ahyI::luxCDABE fusions indicate that AhyR and BHL are both required for ahyI transcription. For A. salmonicida, although the addition of exogenous BHL gives only a small stimulation of the production of serine protease with comparison to the control culture, the incorporation of a longer-chain AHL, N-(3-oxodecanoyl)-L-homoserine lactone, reduced the final level (by approximately 50%) and delayed the appearance (from an A650 of 0.9 in the control to an A650 of 1.2 in the test) of protease in the culture supernatant. These data add A. hydrophila and A. salmonicida to the growing family of gram-negative bacteria now known to control gene expression through quorum sensing.
A homoserine lactone autoinducer regulates virulence of an insect-pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae)
Authors: Dunphy G , Miyamoto C , Meighen E
Published: November 2016

N-beta-Hydroxybutanoyl homoserine lactone (HBHL), the autoinducer of the luminescent system of Vibrio harveyi, has been identified as the first small compound to restore virulence to avirulent mutants of Xenorhabdus nematophilus. HBHL stimulated the level of lipase activity excreted by avirulent X. nematophilus and lowered the phenoloxidase activity in the hemolymph of insects infected with X. nematophilus, parameters that are both associated with insect pathogenesis. Moreover, mortality of the insects infected with avirulent X. nematophilus was restored upon injection with HBHL. Chloroform extraction of medium conditioned with wild-type but not avirulent X. nematophilus led to the isolation of a compound with the same chromatographic mobility as HBHL as well as the ability to stimulate the luminescence of a dim autoinducer-dependent mutant of V. harveyi. Transfer of the V. harveyi lux operon into avirulent and wild-type X. nematophilus generated dim and bright luminescent strains, respectively, which responded to HBHL and an agonist and antagonist in a manner analogous to their effects on the luminescence of dim autoinducer-deficient and bright wild-type strains of V. harveyi, indicating that similar HBHL-dependent regulatory systems exist in these two bacterial species.
Purification and characterization of an arginine regulatory protein, ArgR, from Pseudomonas aeruginosa and its interactions with the control regions for the car, argF, and aru operons
Authors: Park S M , Lu C D , Abdelal A T
Published: November 2016

Pseudomonas aeruginosa ArgR, a regulatory protein that plays a major role in the control of certain biosynthetic and catabolic arginine genes, was purified to homogeneity. ArgR was shown to be a dimer of two equal subunits, each with a molecular mass of 37,000 Da. Determination of the amino-terminal amino acid sequence showed it to be identical to that predicted from the derived sequence for the argR gene. DNase I footprinting showed that ArgR protects a region of 45 to 47 bp that overlaps the promoters for the biosynthetic car and argF operons, indicating that ArgR exerts its negative control on the expression of these operons by steric hindrance. Studies were also carried out with the aru operon, which encodes enzymes of the catabolic arginine succinyl-transferase pathway. Quantitative S1 nuclease experiments showed that expression of the first gene in this operon, aruC, is initiated from an arginine-inducible promoter. Studies with an aruC::lacZ fusion showed that this promoter is under the control of ArgR. DNase I experiments indicated that ArgR protects two 45-bp binding sites upstream of aruC; the 3' terminus for the downstream binding site overlaps the -35 region for the identified promoter. Gel retardation experiments yielded apparent dissociation constants of 2.5 x 10(-11), 4.2 x 10(-12), and 7.2 x 10(-11) M for carA, argF, and aruC operators, respectively. Premethylation interference and depurination experiments with the car and argF operators identified a common sequence, 5'-TGTCGC-3', which may be important for ArgR binding. Alignment of ArgR binding sites reveals that the ArgR binding site consists of two half-sites, in a direct repeat arrangement, with the consensus sequence TGTCGCN8AAN5.
Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB)
Authors: Hoang T T , Schweizer H P
Published: November 2016

The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemophilus influenzae. Chromosomal fabA and fabB mutants were isolated; the mutants were auxotrophic for unsaturated fatty acids. A temperature-sensitive fabA mutant was obtained by site-directed mutagenesis of a single base that induced a G101D change; this mutant grew normally at 30 degrees C but not at 42 degrees C, unless the growth medium was supplemented with oleate. By physical and genetic mapping, the fabAB genes were localized between 3.45 and 3.6 Mbp on the 5.9-Mbp chromosome, which corresponds to the 58- to 59.5-min region of the genetic map.
Attachment of Agrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide
Authors: Reuhs B L , Kim J S , Matthysse A G
Published: November 2016

An early step in crown gall tumor formation involves the attachment of Agrobacterium tumefaciens to host plant cells. A. tumefaciens C58::A205 (C58 attR) is a Tn3HoHo1 insertion mutant that was found to be avirulent on Bryophyllum daigremontiana and unable to attach to carrot suspension cells. The mutation mapped to an open reading frame encoding a putative protein of 247 amino acids which has significant homology to transacetylases from many bacteria. Biochemical analysis of polysaccharide extracts from wild-type strain C58 and the C58::A205 mutant showed that the latter was deficient in the production of a cell-associated polysaccharide. Anion-exchange chromatography followed by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry analyses showed that the polysaccharide produced by strain C58 was an acetylated, acidic polysaccharide and that the polysaccharide preparation contained three sugars: glucose, glucosamine, and an unidentified deoxy-sugar. Application of the polysaccharide preparation from strain C58 to carrot suspension cells prior to inoculation with the bacteria effectively inhibited attachment of the bacteria to the carrot cells, whereas an identical preparation from strain C58::A205 had no inhibitory effect and did not contain the acidic polysaccharide. Similarly, preincubation of Arabidopsis thaliana root segments with the polysaccharide prevented attachment of strain C58 to that plant. This indicates that the acidic polysaccharide may play a role in the attachment of A. tumefaciens to host soma plant cells.
A new type of illegitimate recombination is dependent on restriction and homologous interaction
Authors: Kusano K , Sakagami K , Yokochi T , Naito T , Tokinaga Y , Ueda E , Kobayashi I
Published: November 2016

Illegitimate (nonhomologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a delta rac recBC recG ruvC strain. Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination. The recombining sites themselves shared, at most, a few base pairs of homology. Many of the recombination events joined a site in one of the repeats with a site in another repeat. In two of the products, one of the recombining sites was at the end of one of the repeats. Removal of one of the EcoKI sites resulted in decreased recombination. We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination. The possible roles and consequences of this type of homologous interaction are discussed.
Regulation of expression of the Lactobacillus pentosus xylAB operon
Authors: Lokman B C , Heerikhuisen M , Leer R J , van den Broek A , Borsboom Y , Chaillou S , Postma P W , Pouwels P H
Published: November 2016

The xylose cluster of Lactobacillus pentosus consists of five genes, two of which, xylAB, form an operon and code for the enzymes involved in the catabolism of xylose, while a third encodes a regulatory protein, XylR. By introduction of a multicopy plasmid carrying the xyl operator and by disruption of the chromosomal xylR gene, it was shown that L. pentosus xylR encodes a repressor. Constitutive expression of xylAB in the xylR mutant is repressed by glucose, indicating that glucose repression does not require XylR. The xylR mutant displayed a prolonged lag phase compared to wild-type bacteria when bacteria were shifted from glucose to xylose medium. Differences in the growth rate in xylose medium at different stages of growth are not correlated with differences in levels of xylAB transcription in L. pentosus wild-type or xylR mutant bacteria but are positively correlated in Lactobacillus casei with a plasmid containing xylAB. Glucose repression was further investigated with a ccpA mutant. An 875-bp internal fragment of the ccpA gene of L. pentosus was isolated by PCR and used to construct a ccpA knockout mutant. Transcription analysis of L. pentosus xylA showed that CcpA is involved in glucose repression. CcpA was also shown to be involved in glucose repression of the alpha-amylase promoter of Lactobacillus amylovorus by demonstrating that glucose repression of the chloramphenicol acetyltransferase gene under control of the alpha-amylase promoter is strongly reduced in the L. pentosus ccpA mutant strain.
Mutational characterization of promoter regions recognized by the Salmonella dublin virulence plasmid regulatory protein SpvR
Authors: Grob P , Kahn D , Guiney D G
Published: November 2016

The virulence plasmid-encoded spv regulon is essential for virulence of Salmonella dublin in mice. The spvR gene product belongs to the LysR family of transcriptional regulator proteins. SpvR induces the expression of the spvABCD operon and positively regulates its own expression. DNase I protection analysis with purified SpvR fusion proteins identified SpvR binding sites within the spvA and spvR promoters (P. Grob and D. G. Guiney, J. Bacteriol. 178:1813-1820, 1996). We have used PCR mutagenesis, combined with functional selection for reduced SpvR affinity, to define the DNA elements essential for SpvR binding. For the spvR promoter fragment, a screen for reduced expression was also applied. Sequence analysis of the resulting mutant fragments reveals that the base pair changes are clustered in distinct regions. Determination of the apparent dissociation constants of SpvR for the mutant promoters showed that the spvA LysR-type motif and the upstream palindromic sequences of both promoters play an important role in SpvR recognition.
In vitro phosphorylation study of the arc two-component signal transduction system of Escherichia coli
Authors: Georgellis D , Lynch A S , Lin E C
Published: November 2016

The ArcB and ArcA proteins constitute a two-component signal transduction system that plays a broad role in transcriptional regulation. Under anoxic or environmentally reducing conditions, the sensor kinase (ArcB) is stimulated to autophosphorylate at the expense of ATP and subsequently transphosphorylates the response regulator (ArcA). ArcB is a complex, membrane-bound protein comprising at least three cytoplasmic domains, an N-terminal transmitter domain with a conserved His292 residue (H1), a central receiver domain with a conserved Asp576 residue (D1), and a C-terminal alternative transmitter domain with a conserved His717 residue (H2). To study the phosphoryl transfer pathways of the Arc system, we prepared the following His-tagged proteins: H1, D1, H2, H1-D1, D1-H2, H1-D1-H2, and ArcA. Incubations of various combinations of Arc proteins with [gamma-32P]ATP indicated that H1, but not D1 or H2, catalyzes autophosphorylation; that H1-P transfers the phosphoryl group to D1 much more rapidly than to ArcA; and that D1 accelerates the transphosphorylation of H2. Finally, ArcA is phosphorylated much more rapidly by H2-P than by H1-P. Available data are consistent with a signal transduction model in which (i) reception of a membrane signal(s) triggers autophosphorylation of H1 at His292, (ii) the phosphoryl group can migrate to D1 at Asp576 and subsequently to H2 at His717, and (iii) ArcA receives the phosphoryl group from either His292 or His717, the relative contribution of which is regulated by cytosolic effectors.
Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp. strain JW/SL YS485: a beta-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity
Authors: Lorenz W W , Wiegel J
Published: November 2016

The genes encoding acetyl xylan esterase 1 (axe1) and a beta-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp. strain JW/SL YS485. axe1 is located 22 nucleotides 3' of the xylB sequence. The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1. The xylB gene was identified by expression cloning and by sequence homology to known beta-xylosidases. Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or beta-xylosidase (pXylo-1.1) were constructed in Escherichia coli. Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities. Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant beta-xylosidase. Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum. Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did have strong similarity to cephalosporin C deacetylase from Bacillus subtilis. Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid.
The transmission disadvantage of yeast mitochondrial intergenic mutants is eliminated in the mgt1 (cce1) background
Authors: Piskur J
Published: November 2016

A trans-acting element, MGT1 (also called CCE1), has previously been shown to be required in Saccharomyces cerevisiae for the preferential transmission of petite mitochondrial DNA (mtDNA) molecules over wild-type mtDNA molecules. In the present study a possible role of this nuclear gene in the transmission of mtDNA from various respiration-competent mutants was studied. Several of these mutants, lacking one or the other of two biologically active mitochondrial intergenic sequences, were employed in genetic crosses. When these deletion mutants were crossed to the parental wild-type strain in the MGT1/CCE1 background, the progeny contained predominantly wild-type mtDNA molecules. When crosses were performed in the mgt1/cce1 background, the parental molecules interacted in zygotes and underwent homologous recombination but wild-type and intergenic-deletion alleles were transmitted with equal frequencies.
BglG, the response regulator of the Escherichia coli bgl operon, is phosphorylated on a histidine residue
Authors: Amster-Choder O , Wright A
Published: November 2016

We have shown previously that the activity of BglG, the response regulator of the bgl system, as a transcriptional antiterminator is modulated by the sensor BglF, which reversibly phosphorylates BglG. We show here that the phosphoryl group on BglG is present as a phosphoramidate, based on the sensitivity of phosphorylated BglG to heat, hydroxylamine, and acidic but not basic conditions. By analyzing the products of base-hydrolyzed phosphorylated BglG by thin-layer chromatography, we show that the phosphorylation occurs on a histidine residue. This result supports the notion that the bgl system is a member of a new family of bacterial sensory systems.
Identification and characterization of cell wall-cell division gene clusters in pathogenic gram-positive cocci
Authors: Pucci M J , Thanassi J A , Discotto L F , Kessler R E , Dougherty T J
Published: November 2016

Clusters of peptidoglycan biosynthesis and cell division genes (DCW genes) were identified and sequenced in two gram-positive cocci, Staphylococcus aureus and Enterococcus faecalis. The results indicated some similarities in organization compared with previously reported bacterial DCW gene clusters, including the presence of penicillin-binding proteins at the left ends and ftsA and ftsZ cell division genes at the right ends of the clusters. However, there were also some important differences, including the absence of several genes, the comparative sizes of the div1B and ftsQ genes, and a wide range of amino acid sequence similarities when the genes of the gram-positive cocci were translated and compared to bacterial homologs.
Isolation and analysis of the gene encoding the pyruvate-ferredoxin oxidoreductase of Desulfovibrio africanus, production of the recombinant enzyme in Escherichia coli, and effect of carboxy-terminal deletions on its stability
Authors: Pieulle L , Magro V , Hatchikian E C
Published: November 2016

Previous studies have shown that the pyruvate-ferredoxin oxidoreductase (POR) of the sulfate-reducing bacterium Desulfovibrio africanus is a homodimer that contains one thiamine pyrophosphate and three [4Fe-4S]2+/1+ centers/subunit. Interestingly, the enzyme isolated from a strictly anaerobic bacterium is highly stable in the presence of oxygen, in contrast to the other PORs characterized in anaerobic organisms (L. Pieulle, B. Guigliarelli, M. Asso, F. Dole, A. Bernadac, and E. C. Hatchikian, Biochim. Biophys. Acta 1250:49-59, 1995). We report here the determination of the nucleotide sequence of the por gene encoding the D. africanus POR. The amino acid sequence deduced from this nucleotide sequence corresponds to the first primary structure of a homodimeric POR from strictly anaerobic bacteria. The subunit of the D. africanus POR contains two ferredoxin-type [4Fe-4S] cluster binding motifs (CX2CX2CX3CP) and four additional highly conserved cysteines belonging to a nontypical motif. These 12 cysteine residues may coordinate the three Fe-S centers present in D. africanus POR. The thiamine pyrophosphate binding domain is located in the C-terminal part of the protein close to the four conserved cysteine residues. The D. africanus enzyme sequence appears homologous to the other POR sequences. However, the enzyme differs from all other PORs by a C-terminal extension of about 60 residues of its polypeptide chain. The two cysteine residues located in this additional region may be involved in the formation of a disulfide bridge associated with the activation process of the catalytic activity. The por gene has been expressed, for the first time, in anaerobically grown Escherichia coli behind the isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, resulting in the production of POR in its active form. The recombinant enzyme is stable toward oxygen during several days, and initial characterization of the recombinant POR showed that its activity increased in the presence of dithioerythritol. These properties indicate that the recombinant POR behaves like the native D. africanus enzyme. The study of carboxy-terminal deletion mutants strongly suggests that deletions in the C-terminal region of D. africanus enzyme can have dramatic effects on the stability of the enzyme toward oxygen.
Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli
Authors: Jeong J Y , Yoon G M , Seo H S , Cho M J , Sakai H , Komano T , Bahk J D
Published: November 2016

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.
Roles of the histidine protein kinase pleC in Caulobacter crescentus motility and chemotaxis
Authors: Burton G J , Hecht G B , Newton A
Published: November 2016

The Caulobacter crescentus histidine kinase genes pleC and divJ have been implicated in the regulation of polar morphogenesis and cell division, respectively. Mutations in pleC also potentiate the cell division phenotype of divJ mutations. To investigate the involvement of the PleC kinase in motility and cell cycle regulation, we carried out a pseudoreversion analysis of the divJ332 allele, which confers a temperature-sensitive motility (Mot-) phenotype. All cold-sensitive pseudorevertants with a Mot+ phenotype at 37 degrees C and a cold-sensitive swarm phenotype in soft agar at 24 degrees C contained extragenic suppressors that were null mutations mapping to pleC. Instead of a cell division defect at the nonpermissive temperature, however, revertants displayed a cold-sensitive defect in chemotaxis (Che-). In addition, the mutant cells were also supermotile, a phenotype previously associated only with mutations in the response regulator gene pleD that block the loss of motility. We also found that the Mot- defect of pleC mutants is suppressed by a pleD301/pleD+ merodiploid and results in a similar, supermotile, cold-sensitive Che- phenotype. These results implicate signal transduction pathways mediated by PleC-DivK and DivJ-PleD in the regulation of chemotaxis as well as motility. We discuss these findings and the observation that although the PleC kinase does not play an indispensable role in cell division, a temperature-sensitive allele of pleC (pleC319) has severely reduced viability under stringent growth conditions.
Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase
Authors: Arnau J , Jørgensen F , Madsen S M , Vrang A , Israelsen H
Published: November 2016

The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL. protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.
The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly
Authors: Ohnishi K , Fan F , Schoenhals G J , Kihara M , Macnab R M
Published: November 2016

The flagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components. They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form. N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide--a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays. A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function. We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.
AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli
Authors: Henderson T A , Young K D , Denome S A , Elf P K
Published: November 2016

Two proteins that bind penicillin were observed in Escherichia coli infected with lambda phages 141, 142, 650, and 651 from the Kohara genomic library. These phages carry chromosomal DNA fragments that do not contain any known penicillin binding protein (PBP) genes, indicating that unrecognized gene products were exhibiting penicillin binding activity. The genes encoding these proteins were subcloned, sequenced, and identified. One gene was ampC, which encodes a chromosomal class C beta-lactamase. The second gene was located at about 8.5 min on the E. coli genomic map and is a previously uncharacterized open reading frame, here named ampH, that encodes a protein closely related to the class C beta-lactamases. The predicted AmpH protein is similar in length to AmpC, but there are extensive alterations in the amino acid sequence between the SXXK and YXN motifs of the two proteins. AmpH bound strongly to penicillin G, cefoxitin, and cephalosporin C; was temperature sensitive; and disappeared from cells after overnight incubation in stationary phase. Although closely related to AmpC and other class C beta-lactamases, AmpH showed no beta-lactamase activity toward the substrate nitrocefin. Mutation of the ampC and/or ampH genes in E. coli lacking PBPs 1a and 5 produced morphologically aberrant cells, particularly in cell filaments induced by aztreonam. Thus, these two members of the beta-lactamase family exhibit characteristics similar to those of the classical PBPs, and their absence affects cell morphology. These traits suggest that AmpC and AmpH may play roles in the normal course of peptidoglycan synthesis, remodeling, or recycling.
Transcriptional regulation of the Escherichia coli oxyR gene as a function of cell growth
Authors: González-Flecha B , Demple B
Published: November 2016

The oxyR regulon plays a central role in the defense of Escherichia coli against the endogenous oxidative damage associated with active aerobic growth. Here we have studied the transcriptional regulation of oxyR in E. coli growing aerobically in rich medium. Expression of a single-copy oxyR'::lacZ reporter construct varied sixfold along the growth curve, with the highest value at 4 to 6 h of growth (approximately 14 x 10(8) cells x ml(-1)). Direct measurements of oxyR mRNA by primer extension showed the same biphasic expression but with a peak somewhat earlier in cell growth (2 to 3 h; approximately 3.5 x 10(8) cells x ml(-1)). The results of immunoblotting experiments demonstrated that the level of OxyR protein exhibits the same biphasic expression. Mutant strains lacking adenylate cyclase (cya) or Crp protein (crp) failed to increase oxyR expression during exponential growth. On the other hand, an rpoS mutation allowed oxyR expression to continue increasing as the cells entered stationary phase. Consistent with a biological role for increased levels of OxyR during exponential growth, the crp cya strain had lower activities of catalase hydroperoxidase I and glutathione reductase and an increased sensitivity to exogenously added hydrogen peroxide. These results suggest that the Crp-dependent upregulation of oxyR in exponential phase is a component of a multistep strategy to counteract endogenous oxidative stress in actively growing E. coli cells.
Amino acid residues in the alpha-subunit C-terminal domain of Escherichia coli RNA polymerase involved in activation of transcription from the mtr promoter
Authors: Yang J , Murakami K , Camakaris H , Fujita N , Ishihama A , Pittard A J
Published: November 2016

To examine the role of the amino acid residues (between positions 258 and 275 and positions 297 and 298) of the alpha-subunit of RNA polymerase in TyrR-mediated activation of the mtr promoter, we have carried out in vitro transcription experiments using a set of mutant RNA polymerases with a supercoiled mtr template. Decreases in factor-independent transcription in vitro by mutant RNA polymerases L262A, R265A, and K297A suggested the presence of a possible UP element associated with the mtr promoter. Mutational studies have revealed that an AT-rich sequence centered at -41 of the mtr promoter (SeqA) functions like an UP element. In vivo and in vitro analyses using a mutant mtr promoter carrying a disrupted putative UP element showed that this AT-rich sequence is responsible for interactions with the alpha-subunit which influence transcription in the absence of TyrR protein. However, the putative UP element is not needed for activator-dependent activation of the mtr promoter by TyrR and phenylalanine. The results from in vitro studies indicated that the alpha-subunit residues leucine-262, arginine-265, and lysine-297 are critical for interaction with the putative UP element of the mtr promoter and play major roles in TyrR-dependent transcription activation. The residues at positions 258, 260, 261, 268, and 270 also play important roles in TyrR-dependent activation. Other residues, at positions 259, 263, 264, 266, 269, 271, 273, 275, and 298, appear to play less significant roles or no role in activation of mtr transcription.
The major sigma factor (RpoD) from Helicobacter pylori and other gram-negative bacteria shows an enhanced rate of divergence
Authors: Solnick J V , Hansen L M , Syvanen M
Published: November 2016

Sequence analysis of the Helicobacter pylori major sigma factor (RpoD) shows that it is highly divergent, which may be related to the marked diversity of the H. pylori chromosome. Furthermore, the rate of divergence of RpoD among other gram-negative bacteria is much greater than that among gram-positive bacteria. This suggests that RpoD from gram-negative bacteria is functionally less constrained than that from gram-positive bacteria.
Peptidoglycan structure of Lactobacillus casei, a species highly resistant to glycopeptide antibiotics
Authors: Billot-Klein D , Legrand R , Schoot B , van Heijenoort J , Gutmann L
Published: November 2016

The structure of the peptidoglycan of Lactobacillus casei ATCC 393, a species highly resistant to glycopeptide antibiotics, was examined. After digestion, 23 muropeptides were identified; monomers represented 44.7% of all muropeptides, with monomer tetrapeptides being the major ones. Fifty-nine percent of the peptidoglycan was O-acetylated. The cross-bridge between D-alanine and L-lysine consisted of one asparagine, although aspartate could be found in minor quantities. Since UDP-MurNAc-tetrapeptide-D-lactate is the normal cytoplasmic precursor found in this species, monomer tetrapeptide-lactate was expected to be found. However, such a monomer was found only after exposure to penicillin, suggesting that penicillin-sensitive D,D-carboxypeptidases were very active in normal growing cells.
nfi, the gene for endonuclease V in Escherichia coli K-12
Authors: Guo G , Ding Y , Weiss B
Published: November 2016

Endonuclease V is specific for single-stranded DNA or for duplex DNA that contains uracil or that is damaged by a variety of agents (B. Demple and S. Linn, J. Biol. Chem. 257:2848-2855, 1982). Thus, it may be a versatile DNA repair enzyme. The protein was purified to apparent homogeneity, and from its N-terminal sequence, its gene, nfi, was identified. nfi is immediately downstream of hemE, at kb 4208 (90.4 min) on the current chromosomal map of Escherichia coli K-12. This region was cloned, and plasmid insertion and deletion mutants were used to study its molecular organization. Although nfi is the third of four closely spaced, codirectional genes, it is expressed independently.
Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP
Authors: Eulberg D , Golovleva L A , Schlömann M
Published: November 2016

The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.
Cloning, analysis, and overexpression of the gene encoding isobutylamine N-hydroxylase from the valanimycin producer, Streptomyces viridifaciens
Authors: Parry R J , Li W , Cooper H N
Published: November 2016

The flavoprotein isobutylamine N-hydroxylase (IBAH) catalyzes the oxidation of isobutylamine to isobutylhydroxylamine, a key step in the biosynthesis of the azoxy antibiotic valanimycin. By using oligonucleotide primers designed from peptide sequence information derived from native IBAH, a fragment of the gene (vlmH) encoding IBAH was amplified by PCR from a genomic library of the valanimycin-producing organism, Streptomyces viridifaciens MG456-hF10. The gene fragment was then employed as a probe to clone the entire vlmH gene from an S. viridifaciens genomic library. Overexpression of the vlmH gene in Escherichia coli gave a soluble protein that was purified to homogeneity. The purified protein exhibited the catalytic activity expected for IBAH. The deduced amino acid sequence of IBAH exhibited the greatest similarity to the Sox/DszC protein from Rhodococcus sp. strain IGT38, a flavoprotein involved in the oxidation of dibenzothiophene to the corresponding sulfone. Significant similarities were also found between the amino acid sequence of IBAH and those of the acyl coenzyme A dehydrogenases.
Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae
Authors: Popolo L , Gilardelli D , Bonfante P , Vai M
Published: November 2016

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.
Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120
Authors: Frías J E , Flores E , Herrero A
Published: November 2016

A region of the genome of the filamentous, nitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 that contains a cluster of genes involved in nitrate assimilation has been identified. The genes nir, encoding nitrite reductase, and nrtABC, encoding elements of a nitrate permease, have been cloned. Insertion of a gene cassette into the nir-nrtA region impaired expression of narB, the nitrate reductase structural gene which together with nrtD is found downstream from nrtC in the gene cluster. This indicates that the nir-nrtABCD-narB genes are cotranscribed, thus constituting an operon. Expression of the nir operon in strain PCC 7120 is subjected to ammonium-promoted repression and takes place from an NtcA-activated promoter located 460 bp upstream from the start of the nir gene. In the absence of ammonium, cellular levels of the products of the nir operon are higher in the presence of nitrate than in the absence of combined nitrogen.
Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis
Authors: Tobisch S , Glaser P , Krüger S , Hecker M
Published: November 2016

A new catabolic system in Bacillus subtilis involved in utilization of beta-glucosidic compounds has been investigated. It consists of five genes encoding phosphotransferase system (PTS) enzyme II (licB and licC) and enzyme IIA (licA), a presumed 6-phospho-beta-glucosidase (licH), as well as a putative regulator protein (licR). The genes map around 334 degrees of the B. subtilis chromosome, and their products are involved in the uptake and utilization of lichenan degradation products. These five genes are organized in two transcriptional units. A weak promoter precedes gene licR, and transcription is obviously terminated at a secondary structure immediately downstream of the reading frame, as shown by Northern RNA blot analysis. Genes licB, licC, licA, and licH constitute an operon. Initiation of transcription at the promoter in front of this operon presumably requires activation by the gene product of licR. The LicR protein shows an unusual domain structure, i.e., similarities to (i) the conserved transcriptional antiterminator BgIG family signature and (ii) PTS enzyme II. Using RNA techniques and transcriptional lacZ fusions, we have shown that the expression of the licBCAH operon is inducible by products of lichenan hydrolysis, lichenan and cellobiose. The presence of excess glucose prevents the induction of this operon, indicating the control by carbon catabolite repression. Moreover, the expression of the operon requires the general PTS components and seems to be negatively controlled by the specific lic PTS enzymes.
The Caulobacter heat shock sigma factor gene rpoH is positively autoregulated from a sigma32-dependent promoter
Authors: Wu J , Newton A
Published: November 2016

Sigma factor sigma32, encoded by rpoH, is required for the recognition of heat shock genes during normal growth conditions and in response to heat shock and other stresses. Unlike the well-studied Escherichia coli rpoH gene, which is transcribed from four promoters recognized by either a sigma70 (sigmaD)- or sigma24 (sigmaE)-containing RNA polymerase, the Caulobacter crescentus rpoH gene is transcribed from two promoters, P1 and P2. In this study, we have examined the structure and expression of these promoters and shown that the rpoH P2 promoter is sigma32 dependent. We present evidence here that P2 is specifically recognized and transcribed by the reconstituted C. crescentus Esigma32 RNA polymerase holoenzyme. We show that site-directed mutations within either the -10 or the -35 regions of P2 have substantial effects on the levels of transcription by the Esigma32 polymerase predicted from the sigma32 promoter consensus sequence. The mutations have similar effects in vivo as assayed with rpoH-lacZ transcription fusions. Analysis of the rpoH P1 promoter provided evidence that it is sigma70 dependent. S1 nuclease protection assays of rpoH P1- and P2-specific expression after heat shock at 42 or 50 degrees C and during synchronous cell division cycles under normal growth conditions showed that the two promoters are differentially regulated. Mutations within the rpoH P2 promoter consensus sequences abolished the response to heat shock induction in C. crescentus. We conclude from these results that, unlike rpoH genes studied previously in other bacteria, the major transcriptional response of the C. crescentus rpoH gene to heat shock depends on positive autoregulation of the sigma32-dependent promoter.