Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa

Abstract

Opportunistic pathogen Pseudomonas aeruginosa can cause acute and chronic infections in humans. It is notorious for its resistance to antibiotics due to the formation of biofilms. Cyclic‐di‐GMP is a bacterial second messenger that plays important roles during biofilm development. There are 40 genes in P. aeruginosa predicted to participate in c‐di‐GMP biosynthesis or degradation. It is time‐consuming for the functional characterization of these genes. Here, we cloned 16 genes from P. aeruginosa PAO1 that are predicted to encode diguanylate cyclases (DGCs, responsible for c‐di‐GMP biosynthesis) and constructed their corresponding in‐frame deletion mutants. We evaluated the methods to measure the intracellular c‐di‐GMP concentration by using deletion mutants and PAO1 strains containing a plasmid expressing one of the 16 genes, respectively. Functional outputs of all PAO1‐derived stains were also detected and evaluated, including biofilm formation, production of exopolysaccharide, swimming and swarming motilities. Our data showed that measuring the c‐di‐GMP level only characterized a few DGC by using either pCdrA::gfp as a reporter or LC/MS/MS. Functional output results indicated that overexpression of a DGC gave more pronounced phenotypes than the corresponding deletion mutant and suggested that the swimming motility assay could be a quick way to briefly estimate a predicted DGC for further studies. The overall evaluation suggested 15 out of 16 predicted DGCs were functional DGCs, wherein six were characterized to encode DGCs previously. Altogether, we have provided not only a cloning library of 16 DGC‐encoding genes and their corresponding in‐frame deletion mutants but also paved ways to briefly characterize a predicted DGC.

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

“…Except for PA2818 ( arr ), the fluorescence intensity of stationary phase culture is higher than that of the exponential phase culture (Figure 2A), which is consistent with the stability of Gfp. According to previous publications (15, 75), the two key DGCs, SadC and SiaD showed stronger effects on intracellular c-di-GMP concentration than other DGCs of P. aeruginosa , yet our results indicated their transcriptional level was relatively low among DGCs (Figure 1). We then selected five genes, PA0169 ( siaD ), PA2818 ( arr ), PA4332 ( sadC ), PA4601 ( morA ), and PA4843 ( gcbA) , to detect their transcription by qRT-PCR.…”

“…A systemic tool will be a great help for a deep understanding about how P. aeruginosa regulates the 41 CMR genes in response to various environmental temperatures, signals and antibiotics. A few reports have focused on the comprehensive and systemic features of those c-di-GMP related genes or proteins in bacteria (15,17,(59)(60)(61)(62), while those studies are mainly focused on the functions of genes or enzymes. Therefore, a systemic transcriptional profile of the 41 CMR genes in P. aeruginosa remains lacking.…”

Cyclic di-GMP signaling controlling the free-living lifestyle of alpha-proteobacterial rhizobia

“…Regulation of bacterial motility by c-di-GMP is complex. Low levels proved to be beneficial for this trait, whereas either artificial accumulation or removal of c-di-GMP resulted in motility inhibition (Abel et al 2011;Bhasme et al 2020;Pallegar et al 2020;Yang et al 2016).…”

Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes

“…Yet previous studies of c-di-GMP networks in PA14 (Kulasakara et al, 2006;Ha et al, 2014) or another PAO1 strain (Bhasme et al, 2020) have shown a drastic effect on motility-related behaviors (Figure 10). This re-emphasizes the importance of deciphering the impact that individual members of c-di-GMP networks may have in different P. aeruginosa strains, e.g., PA14 and PAO1.…”