Deletion mutant library for investigation of functional outputs of cyclic diguanylate metabolism in Pseudomonas aeruginosa PA14

Abstract

We constructed a library of in-frame deletion mutants targeting each gene in Pseudomonas aeruginosa PA14 predicted to participate in cyclic di-GMP (c-di-GMP) metabolism (biosynthesis or degradation) to provide a toolkit to assist investigators studying c-di-GMP-mediated regulation by this microbe. We present phenotypic assessments of each mutant, including biofilm formation, exopolysaccharide (EPS) production, swimming motility, swarming motility, and twitch motility, as a means to initially characterize these mutants and to demonstrate the potential utility of this library.

FIG 1

Predicted domains in GGDEF-only, EAL-only, HD-GYP-only, and dual-domain GGDEF-EAL proteins. Based on the SMART algorithm, putative functional domains were identified in each protein (42, 43). Note that the cartoon depiction of each protein is not drawn to scale. The CBS (cystathionine beta-synthase) domain is predicted to serve as a binding site for adenosine and derivatives, as well as playing a regulatory role in regulating enzyme activity; the GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain is present in cGMP-specific phosphodiesterases, adenylyl and guanylyl cyclases, and phytochromes as well as FhlA, a regulator of nitrogen fixation in bacteria; the CC (coiled-coil) domain is a structural motif in which α-helices intertwine; the PSB (periplasmic substrate binding) domain participates in ligand binding; Signal indicates the protein secretion signal; the TM (transmembrane) domain is the inner membrane transmembrane domain; the CHASE domain (extracellular sensory domain) is a predicted ligand binding domain; the HAMP (histidine kinases, adenylyl cyclases, methyl binding proteins, phosphatases) domain is a signal transduction domain; HD indicates the HD-GYP domain, a domain found in c-di-GMP degrading phosphodiesterases; the EAL (phosphodiesterase) domain is found in c-di-GMP-degrading phosphodiesterases; the GGDEF (diguanylate cyclase) domain is conserved in proteins that produce c-di-GMP from two molecules of GTP; the PAC domain contributes to the PAS domain fold; the PAS domain is a signal transduction domain; the 7TM (7 transmembrane receptors with diverse intracellular signaling modules) domain is a signal transduction domain; the REC (receiver) domain is conserved in response regulators; and Repeat indicates repeat sequences.

FIG 2

Biofilm formation. Shown is an analysis of biofilm formation by P. aeruginosa PA14 strains carrying mutations in genes encoding GGDEF-only, EAL-only, HD-GYP-only, and dual-domain GGDEF-EAL proteins in glucose+CAA (A)- and arginine (B)-supplemented media. The biomass of the attached cells on PVC plates was measured after 24 h of static incubation at 37°C. Final values corresponding to each mutant were normalized to the value for wild-type P. aeruginosa strain PA14, which was set to a value of 1, for ease of comparison. OD₅₅₀, optical density at 550 nm. ∗, P < 0.05.

FIG 3

Congo red assays. Shown are representative images of Congo red binding assays for P. aeruginosa PA14 and strains carrying mutations in the pelA, PA14_16500 (wspR), and PA14_56790 (bifA) genes. As cited in text, the ΔpelA mutant, the ΔPA14_16500 (wspR) mutant, P. aeruginosa PA14 (wild type), and the ΔPA14_56790 (bifA) mutant are representative of scores of 0, 1, 2, and 3 on the score spectrum, respectively (see Table 1 for a complete list). The ΔPA14_56790 (bifA) mutant also forms a wrinkly colony morphology, which is indicated by “W” in Table 1.

FIG 4

Swimming motility. Shown is an analysis of swimming motility of wild-type P. aeruginosa strain PA14 as well as strains carrying mutations in genes encoding GGDEF-only, EAL-only, HD-GYP-only, and dual-domain GGDEF-EAL proteins. The area covered by the swimming motility zone was normalized to that of the wild-type strain, which was set to a value of 1, for ease of comparison. The nonmotile ΔflgK mutant served as a negative control. ∗, P < 0.05.

FIG 5

Swarming motility. Shown is an analysis of swarming motility of wild-type P. aeruginosa PA14 as well as strains carrying mutations in genes encoding GGDEF-only, EAL-only, HD-GYP-only, and dual-domain GGDEF-EAL proteins. (A) The area covered by the swarm and its tendrils was measured and normalized to that of wild-type P. aeruginosa strain PA14, which was set to a value of 1, for ease of comparison. (B) Representative images of swarming motility. Reduced swarm coverage includes reduced tendrils (ΔPA14_60870 [morA]) or tendril-less growth of the colony (ΔPA14_56790 [bifA]). The nonmotile ΔflgK mutant served as a negative control. ∗, P < 0.05.

FIG 6

Twitch motility. Shown is an analysis of twitching motility by wild-type P. aeruginosa strain PA14 as well as strains carrying mutations in genes encoding GGDEF-only, EAL-only, HD-GYP-only, and dual-domain GGDEF-EAL proteins. Twitch zones were measured and normalized to that of wild-type P. aeruginosa strain PA14, which was set to a value of 1, for ease of comparison. The ΔpilA mutant served as a negative control. ∗, P < 0.05.