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PubMed1998

Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants.

Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to L-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to L-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to L-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to L-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in L-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of L-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis. The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.

Adenosine↗

PubMed1999

A response regulator that represses transcription of several virulence operons in the group A streptococcus.

A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209-219, 1998), caused the strain to produce mucoid colonies and to increase transcription of hasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase), sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo (streptolysin O), mga (multiple gene regulator of GAS), emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system in Escherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for "control of virulence genes" in GAS. Transcription of the covR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen.

Adhesins, Bacterial↗

PubMed1999

Mutational analysis of the cbb operon (CO2 assimilation) promoter of Ralstonia eutropha.

PL promoters direct the transcription of the duplicated cbb operons from the facultative chemoautotroph Ralstonia eutropha H16. The operons encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle required for CO2 assimilation. Their transcription depends on the activator protein CbbR. Structure-function relationships in the cloned chromosomal promoter region were analyzed by site-directed mutagenesis. PL was altered in its presumed hexameric -35 and/or -10 box or in the spacer region between the boxes to achieve a greater or lesser resemblance to the structure of the sigma70 consensus promoter of Escherichia coli. PL::lacZ transcriptional fusions of various promoter variants were assayed in transconjugant strains of R. eutropha as well as in corresponding cbbR deletion mutants. Mutations increasing the similarity of the -35 and/or -10 box to the consensus sequence stimulated PL activity to various extents, whereas mutations deviating from the consensus decreased the activity. The length of the spacer region also proved to be critical. The conversion of the boxes, either individually or simultaneously, into the consensus sequences resulted in a highly active PL. All improved PL mutants, however, retained the activation under inducing or derepressing growth conditions, although the full-consensus promoter was nearly constitutive. They were also activated in the cbbR mutants. The activity of the overlapping, divergently oriented cbbR promoter was less affected by the mutations. The half- and full-consensus PL mutants were comparably active in E. coli. Two major conclusions were drawn from the results: (i) the location and function of PL were verified, and (ii) indirect evidence was obtained for the involvement of another regulator(s), besides CbbR, in the transcriptional control of the R. eutropha cbb operons.

Bacterial Proteins↗

PubMed1999

Autogenous regulation of the Bacillus anthracis pag operon.

Protective antigen (PA) is an important component of the edema and lethal toxins produced by Bacillus anthracis. PA is essential for binding the toxins to the target cell receptor and for facilitating translocation of the enzymatic toxin components, edema factor and lethal factor, across the target cell membrane. The structural gene for PA, pagA (previously known as pag), is located on the 182-kb virulence plasmid pXO1 at a locus distinct from the edema factor and lethal factor genes. Here we show that a 300-bp gene located downstream of pagA is cotranscribed with pagA and represses expression of the operon. We have designated this gene pagR (for protective antigen repressor). Two pagA mRNA transcripts were detected in cells producing PA: a short, 2.7-kb transcript corresponding to the pagA gene, and a longer, 4.2-kb transcript representing a bicistronic message derived from pagA and pagR. The 3' end of the short transcript mapped adjacent to an inverted repeat sequence, suggesting that the sequence can act as a transcription terminator. Attenuation of termination at this site results in transcription of pagR. A pagR mutant exhibited increased steady-state levels of pagA mRNA, indicating that pagR negatively controls expression of the operon. Autogenous control of the operon may involve atxA, a trans-acting positive regulator of pagA. The steady-state level of atxA mRNA was also increased in the pagR mutant. The mutant phenotype was complemented by addition of pagR in trans on a multicopy plasmid.

Amino Acid Sequence↗

PubMed1999

Cloning and analysis of the rnc-era-recO operon from Pseudomonas aeruginosa.

The rnc operon from Pseudomonas aeruginosa has been cloned and characterized. The three genes comprising this operon, rnc, era, and recO, are arranged similarly to those in some other gram-negative bacteria. Multicopy plasmids carrying the rnc operon of P. aeruginosa functionally complement mutations of the rnc, era, and recO genes in Escherichia coli. In particular, the P. aeruginosa era homolog rescues the conditional lethality of era mutants in E. coli, and the presumptive protein has 60% identity with the Era of E. coli. We discuss these data and evidence suggesting that a GTPase previously purified from P. aeruginosa and designated Pra is not an Era homolog.

Bacterial Proteins↗

PubMed1999

Escherichia coli Lrp (leucine-responsive regulatory protein) does not directly regulate expression of the leu operon promoter.

Studies by R. Lin et al. (J. Bacteriol. 174:1948-1955, 1992) suggested that the Escherichia coli leu operon might be a member of the Lrp regulon. Their results were obtained with a leucine auxotroph; in leucine prototrophs grown in a medium lacking leucine, there was little difference in leu operon expression between lrp(+) and lrp strains. Furthermore, when leuP-lacZ transcriptional fusions that lacked the leu attenuator were used, expression from the leu promoter varied less than twofold between lrp(+) and lrp strains, irrespective of whether or not excess leucine was added to the medium. The simplest explanation of the observations of Lin et al. is that the known elevated leucine transport capacity of lrp strains (S. A. Haney et al., J. Bacteriol. 174:108-115, 1992) leads to very high intracellular levels of leucine for strains grown with leucine, resulting in the superattenuation of leu operon expression.

3-Isopropylmalate Dehydrogenase↗

PubMed1999

Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon.

Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility. In the present paper, we show that crp and hns mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a reduced expression in vivo of fliA and fliC, which encode the specific flagellar sigma factor and flagellin, respectively. Overexpression of the flhDC master operon restored, at least in part, motility in crp and hns mutant strains, suggesting that this operon is the main target for both regulators. Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays. In vitro transcription experiments showed that CAP activates flhDC expression while H-NS represses it. In agreement with this observation, the activity of a transcriptional fusion carrying the flhDC promoter was decreased in the crp strain and increased in the hns mutant. In contrast, the activity of a transcriptional fusion encompassing the entire flhDC regulatory region extending to the ATG translational start codon was strongly reduced in both hns and crp mutants. These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complementation of the nonmotile phenotype in a crp mutant by activation-deficient CAP mutated proteins and characterization of cfs, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activates flhDC transcription by binding to its promoter and interacting with RNA polymerase.

Bacterial Proteins↗

PubMed1999

A novel reduced flavin mononucleotide-dependent methanesulfonate sulfonatase encoded by the sulfur-regulated msu operon of Pseudomonas aeruginosa.

When Pseudomonas aeruginosa is grown with organosulfur compounds as sulfur sources, it synthesizes a set of proteins whose synthesis is repressed in the presence of sulfate, cysteine, or thiocyanate (so-called sulfate starvation-induced proteins). The gene encoding one of these proteins, PA13, was isolated from a cosmid library of P. aeruginosa PAO1 and sequenced. It encoded a 381-amino-acid protein that was related to several reduced flavin mononucleotide (FMNH2)-dependent monooxygenases, and it was the second in an operon of three genes, which we have named msuEDC. The MsuD protein catalyzed the desulfonation of alkanesulfonates, requiring oxygen and FMNH2 for the reaction, and showed highest activity with methanesulfonate. MsuE was an NADH-dependent flavin mononucleotide (FMN) reductase, which provided reduced FMN for the MsuD enzyme. Expression of the msu operon was analyzed with a transcriptional msuD::xylE fusion and was found to be repressed in the presence of sulfate, sulfite, sulfide, or cysteine and derepressed during growth with methionine or alkanesulfonates. Growth with methanesulfonate required an intact cysB gene, and the msu operon is therefore part of the cys regulon, since sulfite utilization was found to be CysB independent in this species. Measurements of msuD::xylE expression in cysN and cysI genetic backgrounds showed that sulfate, sulfite, and sulfide or cysteine play independent roles in negatively regulating msu expression, and sulfonate utilization therefore appears to be tightly regulated.

Amino Acid Sequence↗

PubMed1999

Phosphorylation of HPr and Crh by HprK, early steps in the catabolite repression signalling pathway for the Bacillus subtilis levanase operon.

Carbon catabolite repression (CCR) of Bacillus subtilis catabolic genes is mediated by CcpA and in part by P-Ser-HPr. For certain operons, Crh, an HPr-like protein, is also implicated in CCR. In this study we demonstrated that in ptsH1 crh1 and hprK mutants, expression of the lev operon was completely relieved from CCR and that both P-Ser-HPr and P-Ser-Crh stimulated the binding of CcpA to the cre sequence of the lev operon.

Bacillus subtilis↗

PubMed2000

X-prolyl dipeptidyl aminopeptidase gene (pepX) is part of the glnRA operon in Lactobacillus rhamnosus.

A peptidase gene expressing X-prolyl dipeptidyl aminopeptidase (PepX) activity was cloned from Lactobacillus rhamnosus 1/6 by using the chromogenic substrate L-glycyl-L-prolyl-beta-naphthylamide for screening of a genomic library in Escherichia coli. The nucleotide sequence of a 3.5-kb HindIII fragment expressing the peptidase activity revealed one complete open reading frame (ORF) of 2,391 nucleotides. The 797-amino-acid protein encoded by this ORF was shown to be 40, 39, and 36% identical with PepXs from Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis, respectively. By Northern analysis with a pepX-specific probe, transcripts of 4.5 and 7.0 kb were detected, indicating that pepX is part of a polycistronic operon in L. rhamnosus. Cloning and sequencing of the upstream region of pepX revealed the presence of two ORFs of 360 and 1,338 bp that were shown to be able to encode proteins with high homology to GlnR and GlnA proteins, respectively. By multiple primer extension analyses, the only functional promoter in the pepX region was located 25 nucleotides upstream of glnR. Northern analysis with glnA- and pepX-specific probes indicated that transcription from glnR promoter results in a 2.0-kb dicistronic glnR-glnA transcript and also in a longer read-through polycistronic transcript of 7.0 kb that was detected with both probes in samples from cells in exponential growth phase. The glnA gene was disrupted by a single-crossover recombinant event using a nonreplicative plasmid carrying an internal part of glnA. In the disruption mutant, glnRA-specific transcription was derepressed 10-fold compared to the wild type, but the 7.0-kb transcript was no longer detectable with either the glnA- or pepX-specific probe, demonstrating that pepX is indeed part of glnRA operon in L. rhamnosus. Reverse transcription-PCR analysis further supported this operon structure. An extended stem-loop structure was identified immediately upstream of pepX in the glnA-pepX intergenic region, a sequence that showed homology to a 23S-5S intergenic spacer and to several other L. rhamnosus-related entries in data banks.

Amino Acid Sequence↗

PubMed2000

Rho-dependent transcription termination in the tna operon of Escherichia coli: roles of the boxA sequence and the rut site.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination. Tryptophan induction prevents Rho-dependent transcription termination in the leader region of the operon. Induction requires translation of a 24-residue leader peptide-coding region, tnaC, containing a single, crucial Trp codon. Studies with a lacZ reporter construct lacking the tnaC-tnaA spacer region suggest that, in the presence of excess tryptophan, the TnaC leader peptide acts in cis on the ribosome translating tnaC to inhibit its release. The stalled ribosome is thought to block Rho's access to the transcript. In this paper we examine the roles of the boxA sequence and the rut site in Rho-dependent termination. Deleting six nucleotides (CGC CCT) of boxA or introducing specific point mutations in boxA results in high-level constitutive expression. Some constitutive changes introduced in boxA do not change the TnaC peptide sequence. We confirm that deletion of the rut site results in constitutive expression. We also demonstrate that, in each constitutive construct, replacement of the tnaC start codon by a UAG stop codon reduces expression significantly, suggesting that constitutive expression requires translation of the tnaC coding sequence. Addition of bicyclomycin, an inhibitor of Rho, to these UAG constructs increases expression, demonstrating that reduced expression is due to Rho action. Combining a boxA point mutation with rut site deletion results in constitutive expression comparable to that of a maximally induced operon. These results support the hypothesis that in the presence of tryptophan the ribosome translating tnaC blocks Rho's access to the boxA and rut sites, thereby preventing transcription termination.

Amino Acid Sequence↗

PubMed2000

Inactivation of the stress- and starvation-inducible gls24 operon has a pleiotrophic effect on cell morphology, stress sensitivity, and gene expression in Enterococcus faecalis.

Enterococcus faecalis induces the synthesis of at least 42 proteins during 24 h of glucose starvation. Because of its induction during carbohydrate and complete starvation (incubation in tap water) and CdCl(2) and bile salts stresses, one of these proteins (Gls24) was qualified as a "general stress protein" and was analyzed at the molecular level. Its corresponding gene, gls24, seems to be the penultimate gene of an operon composed, altogether, of six open reading frames (ORFs). The ORF preceding gls24 (orf4) showed very strong identity with gls24. The deduced polypeptides of these two genes showed similarity with a 20-kDa hypothetical protein from Lactococcus lactis and an alkaline stress protein from Staphylococcus aureus with no previously known biological significance. Data from the operon sequence and Northern analysis led to the conclusions that (i) gls24 possesses its own promoter which is especially induced at the onset of starvation and (ii) the operon promoter is stress inducible in exponential-phase cells. A mutation in the gls24 gene led to a severe reduction of growth rate and reduction of survival against 0.3% bile salts in the 24-h-starved cells compared to the wild-type strain. Moreover, the chain length of the mutant is significantly reduced during growth. These results argue strongly for a role of the protein Gls24 and/or GlsB in morphological changes and in stress tolerance in E. faecalis. Comparison of two-dimensional protein gels from wild-type cells with those from gls24 mutant cells revealed a pleiotropic effect of the mutation on gene expression. At least nine proteins were present in larger amounts in the mutant. For six of them, the corresponding N-terminal microsequence has been obtained. Three of these sequences map in genes coding for L-lactate dehydrogenase, lipoamide dehydrogenase, and pyruvate decarboxylase, all involved in pyruvate metabolism.

Amino Acid Sequence↗

PubMed2000

Inactivation of the ampDE operon increases transcription of algD and affects morphology and encystment of Azotobacter vinelandii.

Transcription of algD, encoding GDP-mannose dehydrogenase, the key enzyme in the alginate biosynthetic pathway, is highly regulated in Azotobacter vinelandii. We describe here the characterization of a Tn5 insertion mutant (AC28) which shows a higher level of expression of an algD::lacZ fusion. AC28 cells were morphologically abnormal and unable to encyst. The cloning and nucleotide sequencing of the Tn5-disrupted locus in AC28 revealed an operon homologous to the Escherichia coli ampDE operon. Tn5 was located within the ampD gene, encoding a cytosolic N-acetyl-anhydromuramyl-L-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments. The ampE gene encodes a transmembrane protein, but the function of the protein is not known. We constructed strains carrying ampD or ampE mutations and one with an ampDE deletion. The strain with a deletion of the ampDE operon showed a phenotype similar to that of mutant AC28. The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.

Alginates↗

PubMed2000

A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication.

As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment.

Agrobacterium tumefaciens↗

PubMed2000

IS1675, a novel lactococcal insertion element, forms a transposon-like structure including the lacticin 481 lantibiotic operon.

Two copies of IS1675, a novel lactococcal insertion element from the IS4 family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon. The whole structure could be a composite transposon designated Tn5721. This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant. We identified five other IS1675 copies not associated with the lacticin 481 operon. The conservation of IS1675 flanking sequences suggested a 24-bp target site.

Amino Acid Sequence↗

PubMed2000

Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of beta oxidation of the intermediate benzylsuccinate.

The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent beta-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in beta oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the beta-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp.

Acetyl-CoA C-Acyltransferase↗

PubMed2000

The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance.

Previous studies in our laboratory have shown that the Staphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the lrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing the lrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the lrgAB mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which the lrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the lrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which lrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.

Amino Acid Sequence↗

PubMed2001

areCBA is an operon in Acinetobacter sp. strain ADP1 and Is controlled by AreR, a sigma(54)-dependent regulator.

The areCBA genes in Acinetobacter sp. strain ADP1, determining growth on benzyl alkanoates, are shown to be transcribed as a single operon and regulated by areR, which encodes a regulatory protein of the NtrC/XylR family. Assays of the Are enzymes and of two insertions of lacZ as a reporter gene have shown that the operon is induced by benzyl acetate, benzyl alcohol, and benzaldehyde, as well as 2- and 4-hydroxybenzyl acetates and benzyl propionate and butyrate. Two adjacent sites of transcriptional initiation were 97 and 96 bp upstream of the start codon for areC, near a sigma(54)-dependent -12, -24 promoter. Inactivation of areR and rpoN (for RNA polymerase sigma(54)) drastically reduced growth rates on the Are substrates and induction of the operon.

Acinetobacter↗