Unit: Transcriptional Regulations
Director: Annie Kolb
Our aim is to analyse the reprogramming of genetic expression in response to stress and starvation. σS, 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 focused on the expression and function of σS-regulated genes in Salmonella with the characterisation of mutants in σS-responsive genes, the identification of factors modulatingσS expression or activity and a mechanistic analysis of σS-dependent promoter recognition
Our aim is to analyse the reprogramming of genetic expression in response to stress and starvation.σS, 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 focused on the expression and function of σS-regulated genes in Salmonella with the characterisation of mutants in σS-responsive genes, the identification of factors modulatingσS expression or activity 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 sigma factor that enables the specific recognition of promoters and transcription initiation. The Enterobacteriaceae Escherichia coli and Salmonella enterica have seven sigma factors which compete for core binding.σ70, the "house-keeping" sigma 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 calledσ38, comes into play. σS is responsible for the transcription of a few hundred 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.
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 the regulon important for its survival in the environment, including its hosts. To perform a functional analysis of the rpoS regulon in Salmonella, we isolated σS-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.
Modulators of EσS activity (F. Norel, V. Robbe-Saule , C.Talhouarne, H . Mahtout, N. Miroslavova)
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 forσS, 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 or assistσS. 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 σ70 tightly with a dissociation constant in the nM range (as determined by surface plasmon resonance at the Biophysics platform of the Pasteur Institute). In vitro, Rsd down-regulates the activity of some σ70-dependent promoters. Rsd is able to dissociate the Eσ70 holoenzyme and hence to favour indirectly EσS-association. Rsd may also act directly by promoting the binding of RNA polymerase containing σS to promoters. Up to now the rsd null mutant did not show any particular phenotype and the biological function of Rsd remains unknown.
The Crl protein binds directly toσS. Despite its rather low affinity for σS (Kd = 2.5 microM), it has a positive direct effect on σS -mediated transcription in vitro. In S. Typhimurium Crl is required for the development of a typical "rdar" morphotype (so named after the aspect of the colonies on Congo Red "Red, Dry and Rough"). This multicellular behaviour depends on σS and is involved in biofilm formation at low temperature and low osmolarity in stationary phase. It is characterised by the formation of an extracellular matrix consisting of curli fibres and cellulose. Crl is essential for maximal expression of the genes, involved in curli and cellulose biosynthesis (csgD, csgB, adrA and bcsA). Trans-complementation of the crl mutation by the cloned crl gene can be achieved in the crl mutant but not in the crl rpoS double mutant, indicating that σS is required for Crl function. Expression of σS is increased in the crl mutant and thus, Crl appears to function in concert withσS. Crl expression is induced in stationary phase of growth and slightly precedes that ofσS.
Other 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.
Architecture of the σ54-dependent promoter glnAP2 revealed by protein induced DNA bending (Y. Huo, collaboration with Y. Wang, Beijing University)
σ54-RNA polymerase (Eσ54) predominantly contacts one face of the DNA helix in the closed promoter complex. It requires contact with activators bound to upstream enhancer sequences to form an open complex. By creating a protein-induced DNA bend at precise locations between the enhancer and the core promoter of the σ54-dependent glnAp2 promoter without changing the distance between them, we observe enhanced or decreased expression of promoter activity. The locations from which the DNA bending proteins (CRPH159L or IHF) exert their optimal stimulatory effects are all found on the opposite face of the DNA helix with respect to the DNA bound Eσ54 in the closed complex. These results provide evidence that the activator approaches Eσ54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. Modelling of the Eσ54-activator complex fully supports this proposal. Interestingly, in the wild type glnAp2 sequence, a FIS binding site located on the opposite face as Eσ54 (at position -55) is found and activates glnAp2 transcription.
An unusual primary sigma factor in the phyla Bacteroidetes and Chlorobi (D. Vingadassalom, collaboration with E. Collatz and I. Podglajen, INSERM-U655, University Paris VI).
The primary sigma factors that have been analyzed previously share a common organisation with 4 regions involved in specialized functions. They recognize two major promoter sequence elements, the -10 and -35 hexamers with the consensus sequences TATAAT and TTGACA. The primary sigma factors of B. fragilis and other members of the two phyla are devoid of region 1.1 and have a unique signature of 29 conserved amino acids. The representative B. fragilis factor functions only when associated with its own core enzyme and then recognizes the typical Bacteroides promoters with their -10 and -35 sequences, respectively TAnnTTTG and TTTG. It is unable to recognize some classical promoters of E. coli (lacUV5 and RNAI). Sigma factor mutants are presently under study to identify the key residues involved in promoter recognition.
Differential expression of the morphotypes of Salmonella wild type strain ATCC14028 (1) and its mutant derivatives ATCCcrl (2) ATCCrpoS (3) ATCCcrlcsgB (4) and ATCCcrlbcsA (5) after five days growth on CR plates at 28°C. ATCC14028 and ATCCrpoS show typical rdar (red, dry and rough) and saw (smooth and white) morphotypes, respectively. The crl mutant shows a previously undescribed morphotype (rdarcrl), which was affected by abolition of curli production in the crl csgB double mutant strain and by abolition of cellulose production in the crl bcsA double mutant strain.
Model of promoter-DNA, activator and Eσ54 (from Huo et al., Mol. Microbiol., 2006, 59:168-80). The activator (grey ring-shaped oligomer) approaches the Eσ54-holoenzyme from the unbound face of the promoter β is shown in blue and β' in green, α in red, w in yellow and σ54 in magenta. The structure of Eσ54-promoter has been adapted from the crystal structure of Thermus aquaticus EσA-DNA (PDB code 1L9Z)
Keywords: RNA polymerase, Salmonella, stationary phase, sigma, anti-sigma, cyclic AMP Receptor Protein (CRP), MOLECULAR MICROBIOLOGY, BIOCHEMISTRY