|Director : André KLIER (firstname.lastname@example.org)|
The Unit of Microbial Biochemistry is continuing its study on the regulation of gene expression in model Gram-positive bacteria (Bacillus, Streptomyces). The Unit extended its research domain to the study of virulence factors in pathogenic Gram-positive bacteria (Listeria, Streptococci, Staphylococci).
Study of the sigma 54 regulon of Bacillus subtilis (N. Ould Ali, J. Bignon, M. Débarbouillé)
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 carbon source (such as fructose). The RocR protein is a member of the NtrC/NifA family which binds to enhancer type sequences called UAS. RocR controls two operons, rocABC and rocDEF which are involved in the degradation of arginine and ornithine to glutamate. These two operons have SigL-dependent promoters and are induced by arginine and ornithine in the growth medium. The rocG gene encoding a glutamate dehydrogenase is located just upstream of the rocABC operon. rocG transcription is stimulated by RocR. Unlike the cases for other sigma 54-dependent genes, rocG has no UAS, instead its expression depends on a sequence located downstream from the rocG gene. The same sequence called DAS also serves as a UAS for the downstream rocABC operon.
The RocR protein was purified and used in DNaseI foot printing experiments. It was shown that RocR binds DNA on two palindromic sequences containing 17 base-pairs called UAS/DAS. Mutations were introduced by site-directed mutagenesis in each of these two targets. Expression of rocG and rocABC are affected in these mutants. In collaboration with Eric Larquet (Institut Pasteur) a study of the DNA structure in the rocG rocABC region was undertaken. It was shown that a stable bend is located in the UAS/DAS region. This stable bend could explain how RocR bound to the UAS/DAS sequences stimulates the RNA polymerase on the rocABC promoter.
Environmental response in B. subtilis and other Gram-positive bacteria (T. Msadek, A. Chastanet, S. Dubrac, J. Fert, E. Guédon, O. Poupel)
Our research is focused on the modification of gene expression in response to environmental signals, particularly stress response and two-component signal transduction, in Bacillus subtilis and Gram-positive pathogens (Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae and Listeria monocytogenes) as well as the role of these systems in virulence.
We recently characterized the CtsR regulon of Streptococcus pneumoniae and Staphylococcus aureus. In these two bacteria, we have unveiled, by using comparative genomics, novel and original regulatory pathways controlling stress response genes, in particular a class of genes subject to dual negative control by CtsR and a second repressor, HrcA.
These results were confirmed by in vivo and in vitro approaches, indicating that in S. pneumoniae, the expression of the operon encoding the GroESL chaperones is dependent both on CtsR and HrcA. It was also shown that ClpP of S. pneumoniae plays a role in competence gene expression and that ClpE and ClpC are both required for virulence of an encapsulated strain.
In S. aureus, the construction of single or double inactivation mutants (*ctsR, *hrcA, *ctsR *hrcA) as well as an in vitro approach (DNaseI footprinting with both purified repressor proteins, CtsR and HrcA) allowed us to show that the entire HrcA regulon (groESL and dnaK) is embedded within the CtsR regulon, which also includes clpP, clpB and the clpC operon.
We are also involved in the functional analysis of the genomes of three Gram-positive bacteria: Bacillus subtilis, Listeria monocytogenes and Streptococcus agalactiae, in particular the study of two-component system regulons by transcriptome analysis.
In Listeria monocytogenes, we have inactivated a gene encoding a response rgulator of a two-component system which may respond to the presence of an extracellular auto- inducer peptide acting as a quorum-sensing device. Target genes controlled by this system will be identified by transcriptome analysis.
Gram-positive bacteria have a specific two-component system which has been shown to be essential for bacterial survival, the YycG/YycF system. The YycG (histidine kinase) and YycF (response regulator) proteins of Staphylococcus aureus were overproduced and purified. YycG is autophosphorylated in vitro in the presence of [g-32P] ATP, and transfers its phosphate group to the YycF response regulator. YycG was tested in vitro as a target for novel antimicrobial compounds and several molecules which inhibit its protein kinase activity were identified. The analogous proteins from B. subtilis were purified and used in in vitro protein/DNA interaction experiments (gel shift experiments, DNaseI experiments). Studies are in progress to evaluate the potential of these molecules.
Pathogenicity of Staphylococcus aureus (M. Débarbouillé, M. Arnaud, J. Bignon, B. Fournier)
S. aureus is an important human pathogen implicated in a wide spectrum of infections ranging from superficial lesions to systemic and life threatening infections such as osteomyelitis, endocarditis, pneumonia, septicemia and toxic shock syndrome. Despite intensive research efforts in particular on the agr system relatively little is known about the molecular basis of S. aureus pathogenicity including the way the bacterium invades the organism and evades host defenses.
1 - Construction of an efficient vector for gene inactivation
As gene inactivation in S. aureus is relatively inefficient, a novel vector pMAD was developed in the laboratory. This new vector contains a thermosensitive Gram-positive origin of replication and a constitutively expressed b-galactosidase gene allowing rapid and simple identification of double crossover events using a plate-based assay. This vector was tested both in S. aureus and L. monocytogenes and was shown to be highly efficient in gene inactivation.
2 - ABC transporters
Oligopeptide permeases (Opp) are members of the widespread ABC family of transporters. These permeases generally transport peptides of three to eight amino acids, such as CSF (Competence and Sporulation Factor). In B. subtilis CSF accumulates in culture supernatants as cells grow to high density and is involved in controlling expression of genes that are regulated by quorum sensing. Five genes sharing similarities with the oppABCDF operon in B. subtilis are present in S. aureus. The opp-like operon was inactivated and the virulence of the null mutant was studied in the murine model in collaboration with Jean-Michel Alonso (Institut Pasteur). The results obtained indicate that this opp-like operon is not involved in the virulence of S. aureus. A study of the other Opp family members has been undertaken.
3 - Role of the HtrA serine protease
HtrA is a heat shock induced envelope-associated protease that was first identified in E. coli where it was shown to be involved in the degradation of abnormal periplasmic proteins. HtrA has also been demonstrated to be involved in virulence in Salmonella typhimurium, Yersinia enterocolitica and Brucella abortis. In S. aureus, two HtrA-like proteases are present and nothing is known concerning the role and the regulation of these genes. We have shown that the corresponding genes are not induced during heat shock. The induction in different stress conditions is in progress, in collaboration with Alexandra Gruss (INRA, Jouy-en-Josas).
4 - ArlS/ArlR two-component system
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 a-toxin. The Arl system interacts with SarA (a global virulence regulator) to modulate expression of virulence gene expression. The study of the Arl system by proteomic analysis of cytoplasmic proteins shows that at least 15 proteins are regulated by this system and most of them are repressed. This study will allow the identification of the members of the ArlS/arlR regulon and contribute to the understanding of the virulence of this bacteria.
Regulation of genes involved in the differentiation of Streptomyces (P. Mazodier, A. Bellier, S. Braud, J. Viala)
Streptomyces are Gram-positive bacteria with a complex growth cycle. Germinated spores form a basal mycelium which subsequently differentiates into an aerial mycelium. Finally, the aerial mycelium undergoes septation to form compartments which mature and form spores. Antibiotic production usually begins in the basal mycelium.
We are continuing the characterization of the ATP-dependent proteases from Streptomyces. First, the Lon protease was studied. Construction of a Lon mutant allowed the obtention of dnaK operon mutants which were previously impossible to select. The proteolytic Clp system was then studied. It includes the ClpP proteolytic subunit associated with the regulatory ClpB, ClpC or ClpX ATPase subunits. In S. lividans five clpP genes are present. Deletion of clpP1 affects morphological differentiation and the production of antibiotics. Transcription of the clpP3 clpP4 operon is regulated by the activator PopR. We have shown that the ClpP proteases degrade PopR which allows tightly regulated expression of the clpP3 clpP4 operon.
A mutation affecting the ClpX ATPase subunit was constructed. The phenotype of this mutant is not identical to that of the ClpP mutants which suggests that the ClpC1 and ClpC2 subunits may also be involved.
In addition, we have begun the analysis of the ssrA system in Streptomyces. The RNA coded by ssrA has the mixed properties of a tRNA and a messenger RNA. It allows specific tagging of truncated proteins that are targeted for proteolytic degradation. In E. coli, the Clp complex is mainly implied in this degradation. Our first results do not support the hypothesis that the ssrA system is involved in the differentiation of the Streptomyces.
Regulation of expression of the PlcR regulon in Bacillus cereus cereus (D. Lereclus, L. Slamti, M. Gominet, V. Sanchis)
The PlcR protein is a pleiotropic regulator of the expression of virulence factors in Bacillus thuringiensis and Bacillus cereus. Analysis of the B. cereus genome and of the extracellular proteome indicate that 42 genes are potentially regulated by PlcR. Disruption of plcR reduces the pathogenicity of the bacteria in insects and mice.
The expression of the PlcR regulon is controlled at two levels :
1 - Expression of plcR, which takes place at the onset of stationary phase, is negatively controlled by Spo0A, the master factor involved in the triggering of sporulation. Spo0A~P binds two sequences flanking the PlcR box, thus preventing activation by PlcR.
2 - A PlcR-regulated gene, papR, encodes a 48 amino acid peptide. This peptide is secreted and can be reimported in the cell as a pentapeptide, via the oligopeptide permease system (Opp). The pentapeptide interacts with PlcR, thus allowing its binding to specific target sequences. This signalling system allows the bacteria to produce various virulence factors in response to quorum sensing. These results provide new information for understanding the regulation of pathogenicity in Gram-positive bacteria.
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
|More informations on our web site|
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
DUGAST Christine, Secrétaire de Direction IP
DEBARBOUILLE Michel, DR2 CNRS, email@example.com
FOURNIER Bénédicte, Chargé de Recherche IP, firstname.lastname@example.org
KLIER André, Professeur Université Paris 7, email@example.com
LERECLUS Didier, DR1 INRA, firstname.lastname@example.org
MAZODIER Philippe, Chef de Laboratoire IP, email@example.com
MSADEK Tarek, Chargé de Recherche IP, firstname.lastname@example.org
RAPOPORT Georges, DR1 CNRS, Professeur IP, email@example.com
SANCHIS Vincent, CR1 INRA, firstname.lastname@example.org
BELLIER Audrey, Etudiante en thèse, email@example.com
BRAUD Sandrine, Chercheur post-doctoral, firstname.lastname@example.org
CHASTANET Arnaud, Etudiant en thèse, email@example.com
DUBRAC Sarah, Chercheur post-doctoral, firstname.lastname@example.org
ESPINASSE Sylvain, Etudiant en thèse
OULD ALI Naima, Etudiante en thèse, email@example.com
SLAMTI Leyla, Etudiante en thèse, firstname.lastname@example.org
VIALA Julie, Etudiante en thèse, email@example.com
ARNAUD Maryvonne, Ingénieur de Recherche IP, firstname.lastname@example.org
BIGNON-TOPALOVIC Joëlle, Technicienne Supérieure IP, email@example.com
DUGAST Christine, Secrétaire de Direction IP , firstname.lastname@example.org
FERT Julie, Technicienne Université Paris 7, email@example.com
GOMINET Myriam, Technicienne Supérieure IP, firstname.lastname@example.org
POUPEL Olivier, Technicien supérieur IP, email@example.com