Diguanylate Cyclases and Phosphodiesterases Required for Basal-Level c-di-GMP in Pseudomonas aeruginosa as Revealed by Systematic Phylogenetic and Transcriptomic Analyses

Abstract

Cyclic diguanosine monophosphate (c-di-GMP) is an important second messenger involved in bacterial switching from motile to sessile lifestyles. In the opportunistic pathogen Pseudomonas aeruginosa, at least 40 genes are predicted to encode proteins for the making and breaking of this signal molecule. However, there is still paucity of information concerning the systemic expression pattern of these genes and the functions of uncharacterized genes. In this study, we analyzed the phylogenetic distribution of genes from P. aeruginosa that were predicted to have a GGDEF domain and found five genes (PA5487, PA0285, PA0290, PA4367, and PA5017) with highly conserved distribution across 52 public complete pseudomonad genomes. PA5487 was further characterized as a typical diguanylate cyclase (DGC) and was named dgcH. A systemic analysis of the gene expression data revealed that the expression of dgcH is highly invariable and that dgcH probably functions as a conserved gene to maintain the basal level of c-di-GMP, as reinforced by gene expression analyses. The other four conserved genes also had an expression pattern similar to that of dgcH. The functional analysis suggested that PA0290 encoded a DGC, while the others functioned as phosphodiesterases (PDEs). Our data revealed that there are five DGC and PDE genes that maintain the basal level of c-di-GMP in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in animals, humans, and plants. The formation of biofilms by P. aeruginosa is the central mode of action to persist in hosts and evade immune and antibiotic attacks. Cyclic-di-GMP (c-di-GMP) is an important second messenger involved in the regulation of biofilm formation. In P. aeruginosa PAO1 strain, there are around 40 genes that encode enzymes for making and breaking this dinucleotide. A major missing piece of information in this field is the phylogeny and expression profile of those genes. Here, we took a systemic approach to investigate this mystery. We found that among 40 c-di-GMP metabolizing genes, 5 have well-conserved phylogenetic distribution and invariable expression profiles, suggesting that there are enzymes required for the basal level of c-di-GMP in P. aeruginosa. This study thus provides putative therapeutic targets against P. aeruginosa infections.

A library of promoter-gfpfusion reporters for studying systemic expression pattern of cyclic-di-GMP metabolism-related genes inPseudomonas aeruginosa

“…In addition, there are four genes, PA2870, PA3343 ( hsbD ), PA5487 ( dgcH ) and PA5442 (marked with blue color), whose promoter activities were only reduced in M63 and kept the same level in the other three media. PA5487 ( dgcH ) was reported to be a conserved DGC that its expression is highly invariable, which is consistent with our results (14).…”

“…Fluorescence of GFP was measured on a Synergy H4 hybrid reader (BioTek, USA) at excitation wavelength at 488 nm and emission wavelength of 520 nm with gain 50. Biomass was determined at 600 nm with gain 20 as scattered light (14). Fluorescence signal value was normalized to OD 600 , and fluorescence derived from pPROBE-AT' was subtracted to account for background fluorescence.…”

Traditional Chinese Medicine Tanreqing Inhibits Quorum Sensing Systems in Pseudomonas aeruginosa

“…As a negative control, we used the previously characterized dgcH promoter fusion, which showed invariable under various environmental conditions and genetic backgrounds (Wei et al, 2019b). No significant change was observed in gene expression level ( Figure 6G), consistent with its original RNA-seq data (see GEO dataset GSE141753).…”

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

“…The subsequent M progeny strain 1.4 carries a frameshift mutation in wspE , which should terminally shut down the Wsp system, and the c-di-GMP level was reduced as predicted. C-di-GMP production was restored in this lineage once again in the following D strain 1.5 through a missense mutation in DgcH (V459G), a bioinformatically predicted DGC in P. aeruginosa PAO1 that impacts biofilm formation (Wei et al, 2019). While DgcH shares a namesake with the DGCs DgcA, DgcB, and DgcC, no known interactions exist between these proteins and DgcH.…”