Role of SigmaS in bacterial fitness and adaptation to the environment – SIGMADAPT

We will use approaches in structural biology, biochemistry, biophysics, microbiology, genetics and functional genomics to answer three important biological questions relevant to the function of SigmaS in bacterial fitness. This multidisciplinary approach is required to deeply investigate the molecular mechanisms involved and to integrate data into a biologically relevant adaptation model.

- Our results suggest that Crl family members share a common mechanism of SigmaS binding in which the flexible arms of Crl might play a dynamic role (Biochem J 2014).
- Our work also confirms the large regulatory scope of SigmaS and provides insights into the physiological functions of SigmaS in Salmonella. Extensive regulation by SigmaS of genes involved in metabolism, oxidative stress resistance and membrane composition, and down-regulation of the respiratory chain functions, are important features of the SigmaS effects on gene transcription that might confer fitness advantages to bacterial cells and/or populations under starving conditions.Importantly, the SigmaS-controlled downstream network includes small RNAs that might endow SigmaS with post-transcriptional regulatory functions (PLoS One 2014).

The identification of novel molecular mechanisms orchestrated by SigmaS could be a breakthrough and a major advance in terms of fundamental knowledge and might lead to new collaborative projects. The SIGMADAPT project will provide cues for bacterial fitness that might be useful for the development of new antimicrobial strategies.

- Lévi-Meyrueis C, Monteil V, Sismeiro O, Dillies MA, Monot M, Jagla B, Coppée JY, Dupuy B, Norel F. 2014. Expanding the RpoS/sS-network by RNA sequencing and identification of sS-controlled small RNAs in Salmonella. PLoS One. 9(5):e96918.

- Cavaliere P, Levi-Acobas F, Mayer C, Saul FA, England P, Weber P, Raynal B, Monteil V, Bellalou J, Haouz A, Norel F. 2014. Structural and functional features of Crl proteins and identification of conserved surface residues required for interaction with the RpoS/sS subunit of RNA polymerase. Biochem J. 463(2):215-24.

In eubacteria, a single RNA polymerase core enzyme (E) associates with sigma factors to initiate transcription at specific promoters. SigmaS, encoded by rpoS, is produced during late exponential phase or in response to stress, and controls the expression of a large regulon involved in stationary phase survival and stress resistance in many Gram-negative bacteria. It is also involved in biofilm formation and virulence of Salmonella enterica serovar Typhimurium, a wide-host range pathogen that is a leading cause of human gastroenteritis and food-borne diseases. However, the precise function of nearly half of the genes regulated by sigmaS remains unknown.
The SIGMADAPT project addresses three aspects regarding the molecular mechanisms of action of sigmaS in bacterial fitness and adaptation to the environment:
i) Structural and biophysical insights into the mechanism of action of the unique chaperone-like protein Crl. Crl is a non-conventional regulatory protein that regulates gene expression by increasing the performance of sigmaS. Unlike classical regulators of transcription, Crl binds sigmaS instead of DNA. Proteins that bind sigma factors typically attenuate sigma factor function by restricting its access to E. In contrast, Crl interacts with sigmaS to enhance its association rate to E, thereby facilitating RNAP holoenzyme E-sigmaS formation. Modulation of sigmaS activity by Crl is thus a unique mechanism, where Crl likely promotes a conformational change in sigmaS to favor its binding to E. We will investigate the mechanism of action of Crl at the molecular level, by a variety of structural and biophysical approaches and identify environmental signals that might be transmitted to sigmaS by Crl.
ii) Characterization of downstream effectors of sigmaS in the negative control of gene expression. Sigma factors compete for a limited amount of E in the cells. The house-keeping sigma factor, sigma70, is needed for vegetative growth, whereas sigmaS switches the cell to stress resistance, and has a negative effect on the expression of sigma70- dependent genes involved in metabolism and membrane permeability. The present day dogma is that gene repression by sigmaS is mainly a passive phenomenon, due to competition between sigmaS and sigma70. rpoS mutations would alleviate sigma factor competition and increase sigma70-dependent gene expression. In contrast, our preliminary results in Salmonella suggest that negative regulation by sigmaS requires its binding to DNA, and therefore is likely an active mechanism that may involve putative sigmaS-dependent repressors still to be identified. We will explore this hypothesis further by using transcriptomic approaches and by identifying regulatory pathways responsible for the repression by sigmaS of the ompD gene, encoding the major porin of Salmonella, and the sdh operon required for succinate metabolism.
iii) Characterization of a family of small proteins (of less than 10 kDa) tightly controlled by sigmaS (SCSPs). Small proteins have been largely neglected in bacteria but they might play important cellular and regulatory functions, as adaptors or chaperones to regulate the activity or stability of proteins. By connecting the stability and/or expression of these small proteins to environmental parameters, bacteria might rapidly integrate multiple stress signals downstream of sigmaS itself and might ensure fast and reversible post-transcriptional controls, a key feature for bacterial competitiveness and adaptation to environment. Systematic studies will be conducted at the molecular, physiological and biochemical levels to identify the function in Salmonella fitness of the SCSPs and their putative binding partners.