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  André KLIER (aklier@pasteur.fr)


  abstract

 

The Unit of Microbial Biochemistry is continuing the study of the regulation of gene expression in model Gram-positive bacteria (Bacillus, Streptomyces). The Unit extented its research activity in the field of virulence factors of pathogenic bacteria (Listeria, Streptococci, Staphylococci).



  report

cale

Study of the sigma 54 regulon of Bacillus subtilis (M. Débarbouillé, J. Bignon, N. Ould Ali)

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 and its transcription is stimulated by RocR. rocG has no UAS and 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.

RocR was shown to bind 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 has 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, 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.

In Listeria monocytogenes, a collection of mutants was constructed in which genes encoding two-component systems are inactivated. One of these systems, similar to the S. aureus Agr system, is involved in peptide-based cell-cell quorum sensing communication, the only system of this type identified in L. monocytogenes. A transcriptome analysis of the corresponding mutant was carried out using high-density membranes (Génopole Institut Pasteur) and approximately 20 genes were thus identified as being positively controlled by the Lmonocytogenes Agr system. Interestingly, these include most of the known virulence genes of L. monocytogenes, corresponding to the PrfA regulon.

An analysis of the L. monocytogenes transcriptome was also carried out in the presence or absence of the overexpressed sigH gene, which encodes an alternative sigma factor. Among approximately 30 positively controlled genes, many appear to be involved in cell wall synthesis, lipoteichoic acid metabolism, or encode surface proteins anchored to the cell wall by the LPXTG motif, exported proteins involved in virulence, as well as several members of the PrfA virulence regulon.

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 and YycF proteins of Bsubtilis were purified and used in in vitro protein/DNA interaction experiments with the upstream regions of several potential target genes. This allowed us to define a potential consensus sequence, which could constitute the binding site of the YycF regulator.

Pathogenicity of Staphylococcus aureus

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 little is known about the molecular basis of S. aureus pathogenicity.

Construction of efficient molecular tools (M. Débarbouillé, M. Arnaud, J. Bignon)

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.

A promoter library was constructed using DNA fragments in a multiple copy plasmid containing the lacZ gene from E. coli. This library was introduced in S. aureus by electroporation. This bank is currently used to find genes whose expression is regulated in response to various stress or stimuli or in diverse culture conditions. Several promoters of genes of interest are studied in details.

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). 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.

ArlS/ArlR two-component system (B. Fournier)

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 toxin a. The Arl system interacts with SarA (a global virulence regulator) to modulate expression of virulence gene expression. Study of the Arl system carries on in different directions : one of these approaches consists in identifying target genes of this system through a proteomic study. For example, expression of several cell wall-associated proteins such as adhesins is modified when the arl operon is not expressed. Another approach is to identify the signal recognized by arl system.

Regulation of expression of the PlcR regulon in Bacillus cereus cereus (D. Lereclus, M. Gominet, V. Sanchis, L. Slamti, J. Brillard)

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 more than 50 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. At least three types of pentapeptides (LPFEF, MPFEF, VPFEF) exist in the B. cereus group. This signalling system allows the bacteria belonging to a same subgroup to produce various virulence factors in response to quorum sensing.

Differentiation of Streptomyces, role of proteases and chaperones (P. Mazodier, A. Bellier, C. Lavire)

Streptomyces are Gram-positive bacteria with a complex growth cycle. Germinated spores form a basal mycelium. The basal mycelium subsequently differentiates into an aerial mycelium, concomitantly ; antibiotic production usually begins in the basal mycelium. Finally, the aerial mycelium undergoes septation to form compartments, which mature and form spores. We continued the characterization of the ATP-dependent proteases of Streptomyces ; in particular the ClpP system, held all our attention In S. lividans five clpP genes are present. Alteration of the synthesis of the ClpP1 affects morphological and physiological differentiation in Streptomyces. The activator PopR regulates transcription of the clpP3 clpP4 operon. We showed that the ClpP proteases degrade PopR. This ClpP-dependent degradation of PopR allows tightly regulated expression of the clpP3 clpP4 operon in S. lividans. The clpP1clpP2 operon appears to be under control of another activator ClgR. A mutation affecting the regulating subunit ClpX was constructed. The phenotype of this mutant is not identical to the clpP mutants' phenotype. That shows that the ClpC1 and ClpC2 subunits are also likely to be functional. Analysis of the role of these ATPase and of the regulating subunit ClpS is underway. In addition we are studying the ssrA system in Streptomyces. The RNA coded by ssrA has the mixed properties of a tRNA and of a messenger RNA. It allows specific tagging of truncated proteins that are addressed for degradation by proteolysis. Mutation of the ssrA gene or of the gene encoding SmpB a protein required for SsrA activity were constructed. Phenotypes of these mutants do not support the idea that the ssrA system control the differentiation of the Streptomyces. Indeed theoretical considerations on the difficult translation of numerous regulating genes pointed to a role of ssrA in this phenomenon.

We are also studying armX a gene whose expression is induced by heat shock and oxidative stresses. armX encodes a protein which, could be a chaperone of a new type.

Photo 1 - Transcriptome analysis of L. monocytogenes in the presence or absence of overexpressed sigH, which encodes an alternative sigma factor. All the open reading frames of the L. monocytogenes genome are present on a high density macroarray. Red spots correspond to genes whose expression is repressed by SigH, green spots are genes activated by SigH, yellow spots are those whose expression is not dependent on SigH and black spots are genes not expressed under the assay conditions.

Photo 2 - Model of regulation of PlcR regulon expression. The PapR Peptide is secreted into the extracellular medium and is reimported in bacterial cells via the Opp system, propably as a pentapeptide. It then interacts with the PlcR protein and allows it to bind to its target sequence (PlcR Box) and activate the PlcR regulon.

Keywords: Gram-positive bacteria, genetic expression, virulence factors, stress responses, signal transduction



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Christine DUGAST (cdugast@pasteur.fr) DEBARBOUILLE Michel mdebarbo@pasteur.fr

FOURNIER Bénédicte bfournie@pasteur.fr

KLIER André aklier@pasteur.fr

LERECLUS Didier lereclus@pasteur.fr

MAZODIER Philippe mazodier@pasteur.fr

MSADEK Tarek tmsadek@pasteur.fr

NIELSEN-LEROUX Christina cnielsen@pasteur.fr

RAPOPORT Georges rapoport@pasteur.fr

SANCHIS Vincent vsanchis@pasteur.fr
BELLIER Audrey abellier@pasteur.fr

BRILLARD Julien brillard@pasteur.fr

CHASTANET Arnaud achastan@pasteur.fr

CYNCYNATUS Camille ccyncyna@pasteur.fr

DUBRAC Sarah sdubrac@pasteur.fr

LAVIRE Céline clavire@pasteur.fr

SLAMTI Leyla lslamti@pasteur.fr

ARNAUD Maryvonne marnaud@pasteur.fr

BIGNON-TOPALOVIC Joëlle jojo@pasteur.fr

DUGAST Christine cdugast@pasteur.fr

FERT Julie jfert@pasteur.fr

GOMINET Myriam mgominet@pasteur.fr


Activity Reports 2002 - Institut Pasteur
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