Mutations in surface‐sensing receptor WspA lock the Wsp signal transduction system into a constitutively active state

Abstract

Pseudomonas aeruginosa rugose small-colony variants (RSCVs) are frequently isolated from chronic infections, yet, they are rarely reported in environmental isolates. Here, during the comparative genomic analysis of two P. aeruginosa strains isolated from crude oil, we discovered a spontaneous inframe deletion, wspA Δ280-307 , which led to hyperbiofilm and RSCV phenotypes. WspA is a homologue of methyl-accepting chemotaxis proteins (MCPs) that senses surfaces to regulate biofilm formation by stimulating cyclic-di-guanosine monophosphate (cdi-GMP) synthesis through the Wsp system. However, the methylation sites of WspA have never been identified. In this study, we identified E280 and E294 of WspA as methylation sites. The wspA Δ280-307 mutation enabled the Wsp system to lock into a constitutively active state that is independent of regulation by methylation. The result is an enhanced production of c-di-GMP. Sequence alignment revealed three conserved repeat sequences within the amino acid residues 280-313 (aa280-313) region of WspA homologues, suggesting that a spontaneous deletion within this DNA encoding region was likely a result of intragenic recombination and that similar mutations might occur in several related bacterial genera. Our results provide a plausible explanation for the selection of RSCVs and a mechanism to confer a competitive advantage for P. aeruginosa in a crude-oil environment.

Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ‐FleN complex in Pseudomonas putida

“…wspR deletion abolishes the function of Wsp system, whereas wspF deletion locks Wsp system in a constantly activated state in which WspR is continuously phosphorylated to produce c‐di‐GMP (Güvener and Harwood, 2007; Huangyutitham et al ., 2013). Previous studies have revealed that surface growth activates Wsp system, thus resulting in biofilm formation (Güvener and Harwood, 2007; O'connor et al ., 2012; López‐Farfán et al ., 2019; Hueso‐Gil et al ., 2020; Xu et al ., 2021). More recently, Wsp system has been reported to mediate c‐di‐GMP heterogeneity during surface sensing, leading to two physiologically distinct cell subpopulations (Armbruster et al ., 2019).…”

Priority changes between biofilm exopolysaccharides synthesis and rhamnolipids production are mediated by a c-di-GMP-specific phosphodiesterase NbdA in Pseudomonas aeruginosa

“…To further explore this assumption, we used a PAO1-derived RSCV mutant, PAO1Δ wspF , 24 to assess RLs production during fermentation. Consistent with KT1115, PAO1Δ wspF showed a considerable period of high biofilm exopolysaccharides production from 0 to 96 h ( Figures 3 A and S1 B).…”

The Wsp chemosensory system modulates c-di-GMP-dependent biofilm formation by integrating DSF quorum sensing through the WspR-RpfG complex in Lysobacter

“…WspR has a REC domain fused to the GGDEF domain, and phosphorylation of this REC domain enhances its DGC activity 17 , 18 . Previous studies found that inactivation of wspF in P. aeruginosa , which encodes an ortholog of E. coli CheB methylesterase, resulted in elevated intracellular c-di-GMP level and the formation of a wrinkly colony phenotype, thereby promoting biofilm formation 17 , 19 , 20 . In the wspF mutant background, this effect is caused by enhanced DGC activity of WspR through WspE phosphorylation 20 , 21 .…”

“…Previous studies found that inactivation of wspF in P. aeruginosa , which encodes an ortholog of E. coli CheB methylesterase, resulted in elevated intracellular c-di-GMP level and the formation of a wrinkly colony phenotype, thereby promoting biofilm formation 17 , 19 , 20 . In the wspF mutant background, this effect is caused by enhanced DGC activity of WspR through WspE phosphorylation 20 , 21 . Therefore, it was found that blockade of the methylesterase WspF artificially alters Wsp signaling from a locked state to an activated state by increasing intracellular c-di-GMP levels 20 , 21 .…”

“…In the wspF mutant background, this effect is caused by enhanced DGC activity of WspR through WspE phosphorylation 20 , 21 . Therefore, it was found that blockade of the methylesterase WspF artificially alters Wsp signaling from a locked state to an activated state by increasing intracellular c-di-GMP levels 20 , 21 . Under natural conditions, activation of the P. aeruginosa Wsp system responds to various environmental stimuli, including signals from solid surfaces or fatty acids 22 .…”