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.
Highlights
Is produced during late exponential phase, or in response to stress, to modify global gene transcription and to allow stationary phase survival and general stress resistance[3,4,5]
D135 and E137, were not able to interact with Crl in bacterial two hybrid (BACTH) assays (Supplementary Fig. S1), confirming that the DPE motif in σ SSTM is involved in Crl binding
The DPE motif is conserved in σ S of P. aeruginosa (σ SPA) that does not have crl[22], and the sequence of the helix α 2 differs from σ SSTM by only four residues (Fig. 1c,d and Supplementary Fig. S2), prompting us to examine whether σ SPA can be activated by Crl. σ SPA activity and its response to Crl were evaluated in a S
Summary
Is produced during late exponential phase, or in response to stress, to modify global gene transcription and to allow stationary phase survival and general stress resistance[3,4,5]. Domain 2 (σ 2), the most highly conserved domain of σ factors[19,20], is the only σ S domain involved in Crl binding, and two regions of σ S2 are required for interaction[16,21]. Many bacterial species containing rpoS do not harbour crl in their genome, such as Pseudomonas aeruginosa[22], in which σ S is involved in stress resistance and in the production of virulence factors[8]. The output models show that two specific salt bridges can be formed between Crl and σ S, in agreement with our previous biophysical data suggesting that σ S-Crl complex formation is driven by electrostatic interactions[18]
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