Unit: Microbial Biochemistry
Director: Patrick TRIEU-CUOT
The Unit of Microbial Biochemistry studied the regulation of genetic expression in the model Gram-positive bacteria (Bacillus, Streptomyces) as well as the study of virulence factors and their expression in pathogenic bacteria (Listeria, Streptococcus, Staphylococcus). This Unit was closed on December 31, 2003.
Response to the environment in Gram-positive pathogenic bacteria
(T. Msadek, A. Chastanet, S. Dubrac, J. Fert, C. Cyncynatus, J. Bignon, M. Débarbouillé et B. Fournier)
Our research relates to stress response and signal transduction involving two-component regulatory systems in Gram-positive pathogenic bacteria (Listeria monocytogenes and Staphylococcus aureus).
Analysis of the complete genome of L. monocytogenes revealed the presence of several new Clp ATPases. One of them presents strong similarities with the ClpB protein of E. coli, but is absent from the genome of the Gram-positive bacterium model, Bacillus subtilis. A clpB mutant of L. monocytogenes was constructed and its virulence was found to be strongly impaired in a murine model of infection (collaboration with S. Naïr-Bedouelle, Faculty of Medicine Necker-Enfants Malades). Interestingly, this effect does not seem due to a defect in stress resistance. This is the first example of a role for a ClpB ATPase in virulence in a Gram-positive bacterium.
Whereas the response regulator DegU is present in the genome of L. monocytogenes, the gene coding the associated kinase DegS is missing. In B. subtilis, the DegU regulator is particularly interesting because it is extremely pleiotropic, controlling the synthesis of exocellular enzymes (proteases, saccharases, alpha-amylase) as well as motility and competence for transformation by exogenic DNA. A degU mutant of L. monocytogenes was constructed in order to show that DegU controls its own synthesis negatively. A transcriptome analysis of the degU mutant of L. monocytogenes was carried out and we identified 22 genes whose expression is controlled by DegU. Among the genes whose expression is activated, several were involved in transport and metabolism of sugars. Gel retardation and DNAse I protection experiments have shown a specific binding of purified DegU to a region located downstream from the degU promoter, which confirms the fact that it represses its own synthesis.
Gram-positive bacteria possess a two-component regulatory system which appears essential to their survival, the YycG/YycF system. Gel retardation and DNAse I protection experiments revealed a specific binding of the purified YycF protein of Staphylococcus aureus to the regulatory region of the ssaA gene which encodes a major antigen involved in resistance to macrolides. We also showed that YycF binds specifically to the promoter regions of the genes coding for the IsaA antigen and the peptidoglycane LytM hydrolase. This observation is in agreement with the role suggested for the YycG/YycF system in the control of virulence and cell-wall metabolism.
The two-component system ArlS-ArlR is involved in the virulence of S. aureus by down-regulating the expression of virulence genes such as protein A or alpha-toxin. A collaboration with the proteomic plate-forme of Institut Pasteur showed that the production of 3 cytoplasmic proteins involved in cell physiology is regulated by this system.
Role and expression of the PlcR regulon in bacteria of the "Bacillus cereus" group.
(D. Lereclus, M. Gominet, V. Sanchis, L. Slamti, J. Brillard, S. Fedhila et C. Nielsen-Leroux)
PlcR is a pleiotropic regulator which activates the transcription of many virulence factors in bacteria belonging to the "Bacillus cereus" group. Analysis of the sequence of the genome of B. cereus and of the extracellular proteome indicate that more than 50 genes are potentially controlled by PlcR. The deletion of the plcR gene in these bacteria leads to a reduction of their pathogenicity in the insect and the mouse, but also in a rabbit model of endophthalmitis. We showed that a metalloprotease, whose gene belongs to the PlcR regulon, plays a major role in the virulence of the bacteria in insects.
A gene (papR), also regulated by PlcR, specifies the synthesis of a peptide of 48 amino acids carrying an export signal sequence. A part of this peptide is thus diffusible in the extracellular medium and is perhaps reimported in the bacterial cell, in the form of a pentapeptide (LPFEF), via the Opp transport system. This pentapeptide interacts with the PlcR protein thus allowing its binding to the target DNA sequences. There is a specificity of PlcR-PapR interaction. Indeed, three types of pentapeptides (LPFEF, MPFEF, VPFEF) exist in the bacteria composing the "B. cereus" group. It is thus possible to divide this vast bacterial family into three groups based on the specificity of the PlcR-PapR pairs. This molecular signalling system ensures a specific control making it possible for bacteria of the same group to produce virulence factors when their density in the medium is sufficient (quorum sensing).
Differentiation of Streptomyces, role of proteases and chaperones.
(P. Mazodier, A. Bellier, C. Lavire)
Streptomyces are filamentous Gram-positive bacteria found in the soil. They display remarkable morphological (Hyphas, Sporulation) and physiological (production of antibiotics) differentiation. We are studying the global regulation of these phenomena.
The proteases generally involved in the degradation of regulatory proteins are ATP-dependent proteases, such as the Clp proteins. In Streptomyces, the inactivation of the clpP1-clpP2 operon blocks morphological differentiation. The overproduction of ClpX accelerates or activates the antibiotic production. We showed that the transcriptional activators PopR and ClgR are implied in the regulation of the expression of several clp genes.
Translational regulation plays an important role in the differentiation of Streptomyces. Expression of genes containing rare codons is controlled at the translational level and can involve the availability of the corresponding tRNAs. In Escherichia coli, the presence of rare codons in the mRNA is sufficient to recruit the tmRNA tagging system. This incited us to study the role of tmRNA (ssrA) in Streptomyces.