Modulation of Pseudomonas aeruginosa surface-associated group behaviors by individual amino acids through c-di-GMP signaling

Abstract

To colonize the cystic fibrosis lung, Pseudomonas aeruginosa establishes sessile communities referred to as biofilms. Although the signaling molecule c-di-GMP governs the transition from motile to sessile growth, the environmental signal(s) required to modulate biofilm formation remain unclear. Using relevant in vivo concentrations of the 19 amino acids previously identified in cystic fibrosis sputum, we demonstrated that arginine, ornithine, isoleucine, leucine, valine, phenylalanine and tyrosine robustly promoted biofilm formation in vitro. Among the seven biofilm-promoting amino acids, only arginine also completely repressed the ability of P. aeruginosa to swarm over semi-solid surfaces, suggesting that arginine may be an environmental cue favoring a sessile lifestyle. Mutating two documented diguanylate cyclases required for biofilm formation (SadC and RoeA) reduced biofilm formation and restored swarming motility on arginine-containing medium. Growth on arginine increased the intracellular levels of c-di-GMP, and this increase was dependent on the SadC and RoeA diguanylate cyclases. Strains mutated in sadC, roeA or both also showed a reduction in biofilm formation when grown with the other biofilm-promoting amino acids. Taken together, these results suggest that amino acids can modulate biofilm formation and swarming motility, at least in part, by controlling the intracellular levels of c-di-GMP.

Fig. 1

Amino acids impact surface-associated group behaviors of P. aeruginosa. (A) 24-h biofilms of P. aeruginosa PA14 grown in M63 minimal medium supplemented with 0.2% glucose (Glc), 0.2% glucose/0.5% casamino acids (Glc/CAA) or 0.4% arginine (Arg). Arginine was used at 0.4% in this experiment based on our previous study (Caiazza and O’Toole, 2004). The bar graph represents the quantification of biofilms formed in the wells of a microtiter plate stained with crystal violet (CV), the CV solubilized in glacial acetic acid (30% v/v) and the CV solution measured at 550 nm. Shown are CV-stained wells (top) and quantification of biofilms (bottom). Data represent the mean ± standard error of the mean (SEM) of four replicates normalized to 0.2% glucose values. The dotted line represents the mean value of biofilms formed by P. aeruginosa PA14 when grown on glucose (0.2%) shown for comparison. The asterisks indicate values significantly different from 0.2% glucose-grown biofilms by one-tailed unpaired t test: *P = 0.001 and **P ≤ 0.0001. (B) Ability of P. aeruginosa PA14 to swarm over semi-solid surfaces (0.5% agar) containing 0.2% glucose, G/CAA, or 0.4% arginine. (C) Repression of P. aeruginosa PA14 swarming motility by arginine in a dose-dependent manner. Swarm motility plates contained 0.2% glucose/0.1% CAA with various concentrations of arginine (from 0 to 0.4%). In this experiment, the concentration of CAA was reduced from 0.5% to 0.1% to better discern the repressive effects of arginine. Shown are pictures of swarm plates incubated for 16–18 h at 37 °C.

Fig. 2

P. aeruginosa biofilm formation in response to individual amino acids. Shown is the quantification of 24 h biofilms formed in the wells of a microtiter plate stained with CV, the CV solubilized in glacial acetic acid (30% v/v) and the CV solution measured at 550 nm. The biofilm data shown here are normalized to their respective their planktonic growth (OD₆₀₀) on each amino acid used as sole carbon and energy source (see Supplemental Fig. S3), and represent the mean ± SEM of at least five replicates. The dotted line represents the mean value of biofilms formed by P. aeruginosa PA14 when grown on glucose (0.2%) shown for comparison. Amino acids that significantly boosted biofilm formation as sole carbon and energy source when compared to growth on 0.2% glucose are shown in white bars. Also shown are growth-normalized biofilm formation data for the amino acids that supported biofilm formation to an extent not significantly different from glucose (black bars). Amino acids were used at the following concentrations (mM): proline (27.2), glutamate (24), histidine (8), aspartate (12.8), isoleucine (17.6), arginine (4.8), leucine (25.6), phenylalanine (8), ornithine (11.2), tyrosine (3.2) and valine (17.6). These amino acid concentrations represent 16-fold the value previously quantified in CF sputum (Palmer et al., 2007), except for tyrosine that is used at 4-fold over the average measured sputum concentration. These concentrations were selected for the seven biofilm-promoting amino acids because they supported robust biofilm formation when they were provided as sole carbon and energy source (Fig. S1). Asterisk indicates values significantly different from 0.2% glucose by one-way analysis of variance (ANOVA) corrected with the Dunnett’s multiple comparison Post test (P < 0.05).

Fig. 3

The impact of biofilm-promoting amino acids on swarming motility. Swarming motility of P. aeruginosa PA14 in response to the seven biofilm-promoting amino acids. Shown are pictures of swarm plates after 16–18 h of incubation at 37 °C. Glc/CAA was used as a positive control for swarming motility. In these studies, the medium contained 0.2% glucose supplemented with individual amino acids at the following concentrations (mM): isoleucine (17.6), leucine (25.6), valine (17.6), arginine (4.8), ornithine (11.2), phenylalanine (8), and tyrosine (3.2). These concentrations are the same as those evaluated for the biofilm formation assay represented in Fig. 2.

Fig. 4

The role of c-di-GMP on biofilm formation and swarming repression. (A) 24 h biofilms of P. aeruginosa PA14 (WT) and the sadCroeA double mutant on the indicated growth substrate. The biofilm data shown are normalized to their respective final planktonic growth (OD₆₀₀) and represent the mean ± SEM of at least three replicates. (B) 24 h biofilms of single sadC or roeA mutants on the indicated growth substrate and the values normalized relative to the WT strain. Data represent the mean ± SEM of at least three replicates. The asterisks indicate values significantly different from the WT (panel A) or sadC mutant (panel B) using two-way ANOVA corrected with the Bonferroni Post test: *P < 0.05; **P < 0.01; ***P ≤ 0.001. (C) Shown are images of swarm plates for P. aeruginosa PA14 (WT) and the sadCroeA double mutant taken after 16–18 h of incubation at 37 °C. Swarming assays performed on medium supplemented with either glucose/CAA (G/CAA), as previously reported (Merritt et al., 2010) or glucose 0.2% supplemented with arginine (G/Arg, 0.3 or 4.8 mM Arg, as indicated). (D) Swarming assays performed on medium containing 0.2% glucose supplemented with either isoleucine, leucine, phenylalanine, ornithine, tyrosine or valine using the same concentrations described in Fig. 3.

Fig. 5

Quantification of the intracellular level of c-di-GMP. (A) Shown is the relative concentration of the total extracted c-di-GMP level for the WT strain of P. aeruginosa PA14 grown on glucose (0.2%) plus the indicated concentration of arginine. The dotted line represents the mean value of total c-di-GMP extracted from P. aeruginosa PA14 when grown on glucose (0.2%) shown for comparison. The asterisk indicates a value significantly different from the glucose-only condition by the two-tailed unpaired t test (*P < 0.001). (B) Shown is the relative concentration of the total extracted c-di-GMP level for P. aeruginosa PA14 (WT) and the sadCroeA double mutant grown on glucose (0.2%) plus 4.8 mM arginine. The asterisk indicates a value significantly different from WT P. aeruginosa PA14 by two-tailed unpaired t test (*P < 0.001). Data represent the mean ± SEM of three replicates.