Evolutionary divergence of the Wsp signal transduction system in β- and γ-proteobacteria

Abstract

Bacteria rapidly adapt to their environment by integrating external stimuli through diverse signal transduction systems. Pseudomonas aeruginosa, for example, senses surface-contact through the Wsp signal transduction system to trigger the production of cyclic di-GMP. Diverse mutations in wsp genes that manifest enhanced biofilm formation are frequently reported in clinical isolates of P. aeruginosa, and in biofilm studies of Pseudomonas spp. and Burkholderia cenocepacia. In contrast to the convergent phenotypes associated with comparable wsp mutations, we demonstrate that the Wsp system in B. cenocepacia does not impact intracellular cyclic di-GMP levels unlike that in Pseudomonas spp. Our current mechanistic understanding of the Wsp system is entirely based on the study of four Pseudomonas spp. and its phylogenetic distribution remains unknown. Here, we present the first broad phylogenetic analysis to date to show that the Wsp system originated in the β-proteobacteria then horizontally transferred to Pseudomonas spp., the sole member of the γ-proteobacteria. Alignment of 794 independent Wsp systems with reported mutations from the literature identified key amino acid residues that fall within and outside annotated functional domains. Specific residues that are highly conserved but uniquely modified in B. cenocepacia likely define mechanistic differences among Wsp systems. We also find the greatest sequence variation in the extracellular sensory domain of WspA, indicating potential adaptations to diverse external stimuli beyond surface-contact sensing. This study emphasizes the need to better understand the breadth of functional diversity of the Wsp system as a major regulator of bacterial adaptation beyond B. cenocepacia and select Pseudomonas spp.

Identification of cyclic-di-GMP-modulating protein residues by bi-directionally evolving a social trait in Pseudomonas fluorescens

“…Simultaneously, WspE phosphorylates the methyl-esterase WspF, which de-methylates WspA and terminates the signaling cascade (Bantinaki et al, 2007). Significant work has been carried out to characterize the biochemical functions of WspE and WspR in particular, and WspE is a common mutational target in a clinical setting (Kessler et al, 2021). More recently, studies have begun to explore the functional role of WspA during signal activation and subcellular localization, revealing that both properties are dependent on WspB and WspD proteins (O’Connor et al, 2012; Armbruster et al, 2019).…”

“…The first D strain 2.1 of lineage 2 carries a mutation in wspA that appears to activate WspA through an in-frame deletion of the methylation helices. This mutation likely affects the helical coiled-coil domain of WspA to mimic a permanently methylated state (Kessler et al, 2021), resulting in increased signal transduction and the overproduction of c-di-GMP. The following shift to the M phenotype is associated with a missense mutation in WspD (W90L), which likely disrupts the relay of signal from WspA to WspE.…”

“…This mutation likely weakens or abolishes signal transduction to WspE. In the following D strain 3.3, the missense mutation in WspE (G601V) occurs in the putative WspF/WspR docking site (Kessler et al, 2021). This mutation likely allows WspF and WspR to interact with WspE more easily, thereby increasing the phosphorylation of both proteins.…”

“…Our extensive library of wsp mutations allows us to reinforce the current functional model of the Wsp system, which particularly lacks resolution in WspA, WspB, and WspD (Kessler et al, 2021).…”

“…2). This activation is undone in the follow M strain 5.2 by an in-frame deletion of 3 nucleotides in wspA that removes the amino acid residue S329 of WspA, which falls in the linker region between the methylation sites and the signaling domain (Kessler et al, 2021). This mutation likely interrupts the methylated signal received from WspC and prevents or weakens the transmission of signal to WspE.…”