Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg.

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.

Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168.

A beta-D-fructofuranosidase -- called levanase -- capable of the hydrolysis of sucrose, inulin and levans has been identified in Bacillus subtilis Marburg. This enzyme can not be detected in strain 168. However, sacL mutations -- mapped on the chromosome of strain 168 between the pheA and aroD reference markers -- lead to constitutive levanase synthesis. This synthesis is repressed by carbon sources such as glucose, glycerol or sucrose.

The project of sequencing the entire Bacillus subtilis genome.

The results obtained during the first year of the project involving the sequencing of the Bacillus subtilis genome are presented. Different gene libraries using a yeast artificial chromosome vector and bacteriophage vectors, lambda FixII and phi 105J124, have been constructed. A total of 300 kbp have been cloned using the lambda FixII vector, 68 kbp of which have been fully sequenced. Several open reading frames showing homologies with genes of other organisms were found. Two genes, previously unknown in this organism, have been identified.

A gene encoding a tyrosine tRNA synthetase is located near sacS in Bacillus subtilis.

Within the frame of an attempt to sequence the whole Bacillus subtilis genome, a region of 5.5 kbp of the B. subtilis chromosome near the sacS locus has been sequenced. It contains five complete coding sequences, including the sequence of sacY, three unknown CDS and a sequence coding for a tyrosine tRNA synthetase. That the corresponding CDS encodes a functional synthetase has been demonstrated by complementation of an Escherichia coli mutant possessing a thermosensitive tRNA synthetase. Insertion of a kanamycin resistance cassette in the B. subtilis chromosome at the corresponding locus resulted, however, in no apparent phenotype, demonstrating that this synthetase is dispensable. Finally phylogenetic relationships between known tyrosine and tryptophan tRNA synthetases are discussed.

Whole-genome comparison between Photorhabdus strains to identify genomic regions involved in the specificity of nematode interaction.

The bacterium Photorhabdus establishes a highly specific association with Heterorhabditis, its nematode host. Photorhabdus strains associated with Heterorhabditis bacteriophora or Heterorhabditis megidis were compared using a Photorhabdus DNA microarray. We describe 31 regions belonging to the Photorhabdus flexible gene pool. Distribution analysis of regions among the Photorhabdus genus identified loci possibly involved in nematode specificity.

Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis.

Growth under conditions of salt stress has important effects on the synthesis of degradative enzymes in Bacillus subtilis. Salt stress strongly stimulates the expression of sacB, encoding levansucrase (about ninefold), and downregulates the expression of aprE, encoding alkaline protease (about sixfold). It is suggested that the DegS-DegU two-component system is involved in sensing salt stress. Moreover, it has been shown that the level of sacB expression strongly depends on the growth conditions; its expression level is about eightfold higher in cells grown on agar plates than in cells grown in liquid medium.

MecB of Bacillus subtilis, a member of the ClpC ATPase family, is a pleiotropic regulator controlling competence gene expression and growth at high temperature.

The Bacillus subtilis DegS-DegU histidine kinase-response regulator pair controls the expression of genes encoding degradative enzymes such as levansucrase (sacB) and of genes involved in genetic competence. The mecA and mecB mutations were previously isolated as allowing competence gene expression in complex media. We have shown that the mec mutations also lead to overexpression of sacB, bypassing the DegS-DegU requirement. This expression was shown to be entirely dependent upon ComK, a positive regulator of competence gene expression. The mecB gene was cloned and its nucleotide sequence was determined. The predicted MecB protein show very high similarity over its entire length with members of the ClpC family of ATPases (60% identity). MecB is essential for growth of B. subtilis at high temperature. MecB also acts as a negative regulator of ComK synthesis, thus preventing late competence gene expression. We suggest that under these conditions MecB may interact with MecA to sequester or otherwise inactivate ComK. In response to an unknown signal, active ComK would accumulate through a positive feedback loop, leading to expression of competence genes allowing DNA uptake.

Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis. Identity of the sacUh, amyB and pap mutations.

The sacUh, amyB and pap mutations are identical with respect to their pleiotropic phenotype and their genetic location. Strains bearing these mutations overproduce several exocellular enzymes: alpha amylase, lavansucrase and proteases, they are poorly or not at all transformable and most of them are devoid of flagella. These mutations are tightly linked to the sacU- mutations by transformation and therefore lie between the hisA1 and gtaB290 markers. It is possible that the sacUh, amyB and pap mutations on one hand and the sacU- mutations on the other are two different classes of alterations of the same regulatory gene controlling the synthesis of some exocellular enzymes and several other cellular functions. Furthermore an amy- mutation, leading to the lack of alpha-amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.

The Bacillus subtilis sigL gene encodes an equivalent of sigma 54 from gram-negative bacteria.

The levanase operon in Bacillus subtilis is expressed from a -12, -24 promoter and transcription is stimulated by the regulator LevR, which contains a domain homologous with the central domain of the NifA and NtrC family of regulators. We isolated mutants defective in the expression of the levanase operon. These strains contain mutations that define a gene, called sigL, located between cysB and sacB on the genetic map. The sigL gene was cloned and sequenced. It encodes a polypeptide containing 436 residues with a molecular weight of 49,644. The amino acid sequence of SigL is homologous with all sigma 54 factors from Gram-negative bacteria, including Rhizobium meliloti (32% identity) and Klebsiella pneumoniae (30% identity). B. subtilis sigL mutants have a pleiotropic phenotype: (i) the transcription of the levanase operon is strongly reduced and (ii) in minimal medium lacking ammonia, sigL mutants cannot grow when arginine, ornithine, isoleucine, or valine is the sole nitrogen source. These results indicate that the sigL gene encodes an equivalent of the sigma 54 factor in B. subtilis, to our knowledge, the first of this type to be identified in Gram-positive bacteria.

DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ.

Production of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis is regulated at the transcriptional level by a signal transduction pathway which includes the DegS-DegU two-component system and at least two additional regulatory genes, degQ and degR, encoding polypeptides of 46 and 60 amino acids, respectively. Expression of degQ was shown to be controlled by DegS-DegU. This expression is decreased in the presence of glucose and increased under any of the following conditions: growth with poor carbon sources, amino acid deprivation, phosphate starvation, and growth in the presence of decoyinine, a specific inhibitor of GMP synthetase. In addition, expression of degQ is shown to be positively regulated by the ComP-ComA two-component system. Separate targets for regulation of degQ gene expression by DegS-DegU and ComP-ComA were located by deletion analysis between positions -393 and -186 and between positions -78 and -40, respectively. Regulation of degQ expression by amino acid deprivation was shown to be dependent upon ComA. Regulation by phosphate starvation, catabolite repression, and decoyinine was independent of the two-component systems and shown to involve sequences downstream from position -78. The ComP-ComA and DegS-DegU two-component systems seem to be closely related, sharing several target genes in common, such as late competence genes, as well as the degQ regulatory gene. Sequence analysis of the degQ region revealed the beginning of an open reading frame directly downstream from degQ. Disruption of this gene, designated comQ, suggests that it also controls expression of degQ and is required for development of genetic competence.

Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase.

The DegS-DegU protein kinase-response regulator pair controls the expression of genes encoding degradative enzymes as well as other cellular functions in Bacillus subtilis. Both proteins were purified. The DegS protein was autophosphorylated and shown to transfer its phosphate to the DegU protein. Phosphoryl transfer to the wild-type DegU protein present in crude extracts was shown by adding 32P-labeled DegS to the reaction mixture. Under similar conditions, the modified proteins encoded by the degU24 and degU31 alleles presented a stronger phosphorylation signal compared with that of the wild-type DegU protein. This may suggest an increased phosphorylation of these modified proteins, responsible for the hyperproduction of degradative enzymes observed in the degU24 and degU31 mutants. However, the degU32 allele, which also leads to hyperproduction of degradative enzymes, encodes a modified DegU response regulator which seems not to be phosphorylatable. The expression of the hyperproduction phenotype of the degU32 mutant is still dependent on the presence of a functional DegS protein. DegS may therefore induce a conformational change of the degU32-encoded response regulator enabling this protein to stimulate degradative enzyme synthesis. Two alleles, degU122 and degU146, both leading to deficiency of degradative enzyme synthesis, seem to encode phosphorylatable and nonphosphorylatable DegU proteins, respectively.

The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis.

Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations). We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase. We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for degradative enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of degradative enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype. We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of degradative enzymes in vivo.

Structure of cloned ribosomal DNA cistrons from Bacillus thuringiensis.

A library of B. thuringiensis DNA has been prepared by using the plasmid pBR322 as a cloning vehicle and E. coli as a host cell. By screening this collection with specific probes, 17 clones were identified whose hybrid plasmids contain rRNA genes of B. thuringiensis. Several of these plasmids have been mapped with restriction endonucleases and by DNA-RNA hybridization. By using maps of overlapping fragments, we have been able to establish an overall map of the ribosomal gene cluster.

Characterization of a novel member of the DegS-DegU regulon affected by salt stress in Bacillus subtilis.

As a soil bacterium also found in estuarine and marine habitats, Bacillus subtilis has evolved various sensing and adaptation systems in order to face salt stress conditions. Among these regulatory mechanisms is the DegS-DegU signal transduction system, which was previously shown to be stimulated by high salt concentrations. A search for promoters regulated in response to salt stress led to the identification of wapA, encoding a wall-associated protein, which is strongly expressed at low salt concentrations and almost completely repressed in the presence of 0.7 M disodium succinate. Repression of wapA transcription by salt stress was shown to require the phosphorylated form of DegU. Moreover, DegU-mediated repression of wapA occurred only in high-salt medium. Alignment between the control region of wapA and other DegU-regulated promoters allowed the identification of a putative DegU target sequence, AGAAN(11)TTCAG. Mutation/deletion analyses of the wapA promoter region confirmed the role of the putative DegU control site in repression of wapA transcription at high salt concentrations and revealed a second site of repression located downstream from the transcription start site. Since residual negative control was observed at this second site in the absence of DegU, it seems likely that an additional repressor acts on the wapA control region to further downregulate wapA transcription under salt stress conditions.

Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis.

The sequence and properties of the comQ gene are described. comQ was predicted to encode a 34,209-Da protein, and the product of comQ was shown to be required for the development of genetic competence. The apparent transcriptional initiation and termination sites of comQ were mapped, and the location of a likely E sigma A promoter was inferred. The expression of comQ was maximal early in growth and declined as the cells approached the stationary phase. This expression was not dependent on any of the competence regulatory genes tested (comA, comP, sin, abrB, degU, and spo0A). Disruption of comQ in the chromosome prevented the development of competence as well as the transcription of comG, a late competence operon. This disruption also decreased the expression of srfA, a regulatory operon needed for the expression of competence. These and other results suggest a role for ComQ early in the hierarchy of competence regulatory genes, probably as a component of a signal transduction system.