Binding interface between the Salmonella σS/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl

Abstract

In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σS accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σS binding to the Crl protein increases σS activity by favouring its association to E. Taking advantage of evolution of the σS sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σS to the S. Typhimurium σS-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σS and to generate in silico models of the σS-Crl complex constrained by mutational analysis. The σS-Crl models suggest that the identified arginine in σS interacts with an aspartate of Crl that is required for σS binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σS-Crl complex.

Structural basis for transcription activation by Crl through tethering of σS and RNA polymerase

“…Altogether, the interaction between Crl and σ S forms an interfacial area of 785 Å 2 (41) and is completely consistent with previous analyses of Crl-s S interactions (26,34,38,40). In summary, our structure: 1) confirms the s S residues previously proposed to interact with Crl based on genetic and biochemical data; 2) identifies additional residues in s S and Crl involved in the intermolecular interaction; 3) reveals that 6 out of 11 (55%) of the residues of σ s contacting Crl are not conserved in σ 70 (Fig.…”

“…The central role of s S helix α2 confirms previous reports that identified key residues in σ S for its interaction with Crl. Sty σ S R82 is not conserved in Pseudomonas aeruginosa (Pae) s S , and Pae σ S does not interact with Sty Crl in in vivo bacterial two-hybrid assays unless the corresponding amino acid (Leu) is mutated to an arginine (38). Mutations at this site lead to bacterial colony morphology changes, which is consistent with the interaction of Crl and σ S being important for processes like biofilm formation.…”

“…1B): First, expected interactions occurred between σ S 4 and the -35 promoter element, which were not observed in a previously determined crystal structure of a σ S transcription initiation complex due to crystal packing restraints (PDB 5IPL) (33). Second, Crl bound σ S 2 in a manner predicted from the results of previous studies (26,38) and is located at the periphery of the complex near the upstream edge of the transcription bubble. Third, Crl also interacted with the RNAP β' subunit.…”

High-Resolution Crystal Structure of RpoS Fragment including a Partial Region 1.2 and Region 2 from the Intracellular Pathogen Legionella pneumophila

“…The region 1.2 adjacent to region 2 was proposed as a recognition site of the regulatory factor Crl. Salmonella enterica serovar Typhimurium RpoS R82L mutant is defective in Crl binding and activation [28]. The fact that LpRpoS 95-195 uses the corresponding Leu113 as a substitute for Arg82 in StRpoS indicates that LpRpoS 95-195 may not be suitable for Crl binding.…”

Crl activates transcription by stabilizing the active conformation of the master stress factor σS

“…A large collection of genetic and biochemical data have highlighted the importance of Crl in σ S -mediated transcription in bacteria that encode σ S (Cavaliere and Norel, 2016). Crl was demonstrated to directly activate σ S -mediate transcription both in vitro and in vivo (Banta et al, 2013;Banta et al, 2014;Cavaliere et al, 2014;Cavaliere et al, 2015;England et al, 2008;Monteil et al, 2010a;Pratt and Silhavy, 1998;Typas et al, 2007a), and Crl-null Salmonella and E. coli strains displayed impaired biogenesis of curli (important for host cell adhesion and invasion as well as formation of biofilm), increased sensitivity to H 2 O 2 stress, and reduced virulence due to decrease expression of several σ S -regulated genes (Arnqvist et al, 1992;Barnhart and Chapman, 2006;Monteil et al, 2010a;Robbe-Saule et al, 2008;Robbe-Saule et al, 2006).…”