Study of the sigma 54 regulon of B. subtilis (M. Débarbouillé, N. Ould Ali, M. Arnaud, J. Bignon)
The sigma L gene encodes a sigma factor homologous to the sigma 54 factor from Gram-negative bacteria. The sigma L factor is required for the utilization of certain amino acids as nitrogen sources (arginine, ornithine, isoleucine and valine) and sugars as carbon sources (such as fructose). Genes of specific regulators involved in these pathways have been identified. They activate transcription by binding to sequences located either upstream (UAS) or downstream (DAS) from specific 12/-24 promoters. The acoABCL operon involved in the catabolism of acetoin, the major compound from glucose degradation, was analyzed in detail as well as its regulator, AcoR. Moreover, the expression of the operon is highly repressed by glucose through the CcpA transcriptional regulator which binds a specific target sequence, CRE, located upstream from the acoR gene.
Stress responses (T. Msadek, A. Chastanet, I. Derré)
We have shown that heat shock genes encoding the catalytic and regulatory subunits of the ATP-dependent Clp protease are under the control of the CtsR repressor. A comparative genome analysis indicates that the CtsR protein and its target sequences are highly conserved among Gram-positive bacteria of low G + C content, including pathogens (Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis).
Experiments carried out in vivo (gene fusions in B. subtilis) and in vitro (gel retardation, DNAse I footprinting) indicate that the operon encoding the GroESL chaperones from S. pneumoniae are controlled by both CtsR and another global regulator, HrcA. In S. aureus, the whole HrcA regulon comprising the groESL and dnaK operons, is included in the CtsR regulon which in addition controls the clpP and clpB genes and the clpC operon.
Studies on the regulation by CtsR of clp genes from L. monocytogenes, performed in collaboration with P. Berche and coll. (Faculty of Medicine, Necker, Paris) showed that CtsR is involved in the resistance to different stresses. Its role was tested in a murine model. The presence of ctsR in multicopy in Listeria led to a significant decrease of virulence, likely due to the repression of clp genes.
Signal transduction (T. Msadek, E. Guédon, B. Fournier, P. Mazodier, J. Viala, A. Sobczyk)
A transcriptome analysis of the two-component systems (TCS) of B. subtilis is in progress with DNA macro-arrays corresponding to the complete genome (4200 genes). About 80 genes were found to be controlled by DegS/DegU, among which the majority, organized in operons are repressed. It has been previously shown that DegS/DegU controls the synthesis of degradative enzymes and competence (integration of DNA).
Another TCS of B. subtilis, LytS/LytT, analogous to that of S. aureus, was also studied. In contrast to S. aureus, this system is not involved in the expression of autolysins. An operon of two genes adjacent to the lyt locus, was shown to be controlled by this TCS. The identification of other genes belonging to the same regulon is under way by transcriptome analysis.
In S. aureus, a TCS, ArlS/ArlR, implicated in virulence, was also studied. Deletion of the arl locus led to a strong increase of protein A and of other secreted proteins : a-toxin, b-haemolysin, lipase, coagulase, serine protease. The synthesis of ArlS/ArlR is positively regulated by the 2 major virulence loci of S. aureus, sarA and agr. Conversely, the TCS activates the expression of sarA and represses that of agr.
In Streptomyces, the chaperone proteins GroES, GroEL1 and GroEL2 are dependent, as in B. subtilis, upon the HrcA repressor which acts on target sequences called CIRCE. Two new regulators were characterized in S. albus : HspR which represses the expression of the dnaK operon and that of the clpB and lon genes, and RheA which represses the expression of the major heat shock protein, Hsp18. Their target sequences were defined as specific inverted repeats of 7 bp. It was also shown that RheA is reversibly inactivated by heat and acts as a cellular thermometer.
In addition, the genes encoding the ClpP, ClpX and ClpC subunits were characterized. In S. lividans, 4 clpP genes are present, clpP1 and clpP2 being organized in tandem, while clpP3 and clpP4 are included in an operon under the control of a new activator, PopR which acts on a palindromic target of 6 bp. The Clp system is involved in the morphological differentiation of S. lividans, S. albus and S. coelicolor, while ClpX controls the production of the antibiotic actinorhodin in S. lividans and S. coelicolor.
Study on ATP-dependent proteases was extended to the characterization of the Lon protein. It was shown that Lon is not involved in the regulation of differentiation, in contrast to the Clp complex. Obtention of a lon mutant will be exploited for the production of heterologous proteins, in the framework of a European project.
Regulation of gene expression involved in virulence in B. thuringiensis and B. cereus cereus(D. Lereclus, L. Slamti, M. Gominet, S. Fedhila-Hamza)
The PlcR protein is a pleiotropic regulator of the expression of virulence factors in these two microorganisms (an insect pathogen and a human pathogen, respectively). It activates the transcription of at least 15 genes encoding extracellular proteins (phospholiphases C, proteases, haemolysin, enterotoxins). Disruption of this gene strongly decreases the haemolytic and cytolytic properties of B. thuringiensis and B. cereus. In the susceptible insects (Lepidoptera), the disruption of the plcR gene reduces the synergistic effect of the Bacillus spores on the insecticidal activity of the crystal proteins from B. thuringiensis. In mice, the mortality caused by nasal instillation of spores was strongly diminished or totaly abolished using the plcR mutant.
The expression of plcR, which takes place at the onset of stationary phase, is autoregulated and negatively controlled by spo0A, the master gene involved in the triggering of sporulation. Expression of plcR also depends upon a quorum sensing system of the ABC transporter type which is involved in the transport of peptides (Opp). Studies on the nature of the peptides involved in the intercellular activity and their mechanism of action are in progress.
In addition, 2 clpP genes were characterized in B. thuringiensis. These genes were disrupted, and their role in sporulation and in virulence is presently studied.
Regulation of expression of secreted insecticidal toxins from Bacillus thuringiensis (V. Sanchis, S. Espinasse)
Analysis by insertional mutagenesis of an overproducingb-exotoxin strain, an ATP analogue which inhibits RNA polymerase, allowed the identification of an ABC-transporter and of a putative sigma factor of extracytoplasmic function (ECF) which could be involved in the expression and export of theb-exotoxin. Disruption of the corresponding genes led to an avirulent phenotype. The expression of the ECF gene itself is auto-regulated and its transcription is higher in the overproducing strain compared to the non toxic wild-type strain. The search of other genes controlled by this sigma factor is in progress ; this could allow the identification and determination of the function of genes which are not expressed in standard conditions of culture but are coordinately induced in specific ecological situations of the bacterium.
Transcriptome analysis of the B. subtilis DegS/DegU regulon. All 4107 open reading frames of the B. subtilis genome are present on a high density macroarray. Red spots correspond to genes whose expression is repressed by DegU, green spots are genes activated by DegU, yellow spots are those whose expression is not dependent on DegU and black spots are genes not expressed under the assay conditions.