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

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Charles Henry Leach II Fund National Institute of General Medical Sciences (NIGMS)

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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.

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Data collection was executed through Jupyter Notebook running Python and R scripts to retrieve GenBank and RefSeq data from the National Center for Biotechnology Information (NCBI).

Scripts were executed locally on a performance desktop with an AMD Ryzen Threadripper 24-core processor, a NVIDIA GeForce RTX 2060 gpu, and 128 GB of RAM.


Applied and Environmental Microbiology



File Format


Data S1.pdf (7863 kB)
MultiGeneBlast metadata for the P. fluorescens Pf0-1 wsp operon.

Data S2.pdf (7292 kB)
MultiGeneBlast metadata for the B. cenocepacia HI2424 wsp gene cluster.

File S1.pdf (216 kB)
Python script executed on Data S1-S2.

File S2.pdf (122 kB)
R script executed by File S1 to download RefSeq genome assemblies.

File S3.pdf (67 kB)
R script executed by File S1 to parse File S2 output for Bac120 set creation.

File S4.pdf (55 kB)
R script executed by File S1 to parse File S3 output and finalize Bac120 dataset.