Unit: Transcriptional Regulations - CNRS URA 2172

Director: Kolb, Annie

Our aim is to analyse the reprogramming of genetic expression in response to stress and starvation. σ S, the stationary phase sigma factor of RNA polymerase, encoded by the rpoS gene is the master regulator of the general stress response and controls Salmonella virulence. Our studies are focussed on both a functional analysis of the rpoS regulon in Salmonella with the isolation and characterisation of mutants in σS-responsive genes and a mechanistic analysis of σS-dependent promoter recognition.

The σS factor of RNA polymerase

RNA polymerase is responsible for the first step of gene expression and the target of numerous regulations. The bacterial holoenzyme consists of the core enzyme E and a σ factor that enables the specific

recognition of promoters and transcription initiation. The Enterobacteria Escherichia coli and Salmonella enterica have seven σ factors which compete for core binding. σ 70, the "house-keeping" σ factor, controls the transcription of all the essential functions of the bacteria. As cells reach the end of the exponential phase or are subjected to various environmental stresses, σS, also called σ38, comes into play. σS is responsible for the transcription of more than 100 genes, important for acquisition of the generalised stress resistance status and for survival in the stationary phase. σS is involved in resistance to osmotic, acid and oxidative stresses, biofilm formation and expression of virulence factors. Up to now, the exact function of nearly half of the genes regulated by σS is still unknown.

Identification and functional analysis of the genes of the rpoS regulon in Salmonella (F. Norel, V. Robbe-Saule)

σS is essential for virulence and persistence of the facultative intracellular pathogen Salmonella enterica serotype Typhimurium which causes severe infections in man and animals. Our goal is to analyse the functions of the rpoS regulon in the physiology and pathogenesis of Salmonella and to identify the key functions of this regulon important for its survival in the environment, including its hosts. To perform a functional analysis of the rpoS regulon in Salmonella, we isolated σ S-activated lacZ gene fusions from a bank of S. Typhimurium mutants. One-third of the fusions mapped to Salmonella DNA regions not present in E. coli K-12. This suggests that the composition of the rpoS regulon differs markedly in the two species. Most of the fusions mapped in genes of unidentified function. One of these, the katN gene, was shown to encode a non-haem catalase. katN is not present in E. coli K-12 but is conserved in enterohemorrhagic E. coli O157, Klebsiella pneumoniae and Pseudomonas aeruginosa. Most of the remaining genes are likely to possess metabolic functions involved in adaptation of Enterobacteria under suboptimal growth conditions in natural environments.

Promoter recognition by EσS and Eσ70 RNA polymerases (A. Kolb)

Among the 6 alternative σ factors of Enterobacteria, σS is the most similar to σ 70. The similarities are strikingly strong in the promoter binding domains (regions 2.4 and 4.2 of the protein that recognise the -10 and -35 hexamers respectively). Numerous promoters can be recognised in vitro and in vivo by both Eσ70 and EσS holoenzymes, with, however, different affinities and kinetics. The discrimination mechanism is still not clearly understood: σS-dependent promoters generally lack a discernible -35 consensus sequence but possess the same optimal -10 hexamer as Eσ70 -dependent promoters (TATAA/CT). However unlike Eσ70, EσS might recognise both C and T as first nucleotide in the -10 hexamer and has a strong preference for a cytosine at position-13. Using site directed mutagenesis and suppression genetics in collaboration with C. Gutierrez' group in Toulouse, we show that two aminoacids of σS within region 2.4 (Q152 and E155) participate to the preference for a C at position -13 of its target promoters. Interestingly the homologous residues in Eσ70 (Q437 and T440) are also involved in the strong preference for a nucleotide in the -10 region, the first T of the -10 hexamer located at -12 at σ70-dependent promoters. The substitutions Q437H and T440E allow Eσ70 to better recognise the aidB promoter a σS-dependent promoter, under study with the group of P. Landini (Dubendorf-Switzerland) suggesting that two corresponding aminoacids in EσS and Eσ70 might recognise different, albeit adjacent, promoter base pairs.

Modulators of EσS activity (V. Jaumouillé, H . Mahtout, N. Miroslavova, F. Norel, V. Robbe-Saule)

Non growing cells contain more than twice as many σ70 molecules as σS molecules. Since core enzyme is limiting and has a much higher affinity for σ70 than for σS, it is unclear how σS can capture sufficient core to assure the expression of σS-dependent genes in stationary phase. The bacteria appear to solve this problem by synthesising different molecules which either can handicap σ70 (anti-σ70) or help σS ("pro-σS"). Our aim is to provide a detailed biochemical and functional characterisation of the factors involved in this process.

The stationary phase anti-σ70 factor Rsd binds region 4 of σ70 as the well-known T4 anti-σ70 factor AsiA, but with a reduced affinity. In contrast to AsiA, Rsd is able to dissociate in vitro the Eσ70 holoenzyme and hence to favour indirectly EσS-association. Its effects on EσS-dependent transcription are under investigation. On the other hand the Crl protein of Salmonella binds directly to σS and has a positive direct effect in vitro on σS-mediated transcription. The specifics of this mechanism remain to be clarified. The regulatory functions of Rsd and Crl in Salmonella are investigated by studying the expression of individual genes by gene fusion experiments and by analysing the phenotypes dependent on σS (virulence and stress resistance). Furthermore, two global approaches including phenotype characterisation (phenotypes arrays Biolog) and proteomics are developed to determine if the rpoS and crl regulons fully overlap. We also show that Crl plays a role in the formation of the Salmonella morphotype called "rdar" (for red dry and rough on Congo red agar), a multicellular behaviour due to the formation of an extracellular matrix consisting of aggregative fimbriae (curli) and cellulose. The extracellular matrix synthesis depends on σS and can be correlated with biofilm development. Crl is expressed at its maximal level at low temperature and low osmolarity, conditions essential for the development of the "rdar" morphotype. The exact role played by Crl in "rdar" morphotype and biofilm development is under study.

Regulation of the σ54-dependent promoter glnAP2 by DNA bending induced by the CRP-cAMP complex (Y. Huo, collaboration with Y-P Wang, Beijing University)

σ54 differs from other σ factors by its sequence and mode of action. Eσ 54 requires contact with upstream Enhancer-like sequence (ES) bound proteins (EBP) mediated by DNA looping to make open complexes. Effective DNA looping depends on the correct orientation of RNA polymerase and EBP binding sites. Up to now the precise face of Eσ54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing DNA-bend induced by CRPH159L at precise locations between the ES and the core promoter of the σ54-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased expression of promoter activity. The relative orientations of Eσ54, EBP and DNA bending protein were determined by in vitro footprinting. Intriguingly the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix as the DNA bound σ54-holoenzyme in the closed complex.

Mechanism of activation of the vancomycin resistance genes Enterococcus faecalis BM454 by VanRB (F. Depardieu, P. Courvalin, Unit of Antibacterial Agents and A. Kolb)

We analyse the transcriptional regulation of the regulatory (vanRBSB) and resistance(vanYBWHBBXB) genes and compare the binding sites of VanRB and phosphorylated VanRB to the PRB and PYB promoters. VanR-P binds at a single site at position -32.5 upstream from the PRB transcriptional start site and at two sites at positions -33.5 and - 55.5 upstream from that of PYB. Dimerisation of VanRB is induced by phosphorylation. VanRB-P binds with higher affinity than VanRB to its target sites and appears more efficient than VanRB in promoting open complex formation at PRB and PYB. The E. coli RNA polymerase Eσ70 supports transcription activation by VanRB-P in vitro.

Figure

Effect of σS and Crl on the multicellular aggregation state of S. Typhimurium ATCC14028, grown on Congo red media in low salt at 28°C. (A) The wild type strain shows the "rdar" morphotype (for "red, dry and rough"); (B) Δ rpoS::cm; (C) Δcrl:: cm

Keywords: RNA polymerase, Salmonella, stationary phase, sigma, anti-sigma, cyclic AMP Receptor Protein (CRP), MOLECULAR MICROBIOLOGY, BIOCHEMISTRY


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