Cooperation and cheating in Pseudomonas aeruginosa: the roles of the las, rhl and pqs quorum-sensing systems

Abstract

Pseudomonas aeruginosa coordinates the transcription of hundreds of genes, including many virulence genes, through three hierarchically arranged quorum-sensing (QS) systems, namely las, rhl and pqs. Each system consists of genes involved in autoinducer synthesis, lasI, rhlI and pqsABCDH, as well as cognate-regulatory genes, lasR, rhlR and pqsR. In this study, we analyzed the social behavior of signal-blind (ΔlasR, ΔrhlR, ΔpqsR) and signal-negative (ΔlasI, ΔrhlI, ΔpqsA) mutants from each QS system. As each system controls extracellular common goods but differs in the extent of regulatory control, we hypothesized that all signal-blind mutants can behave as cheaters that vary in their ability to invade a QS-proficient population. We found that lasR and pqsR, but not rhlR, mutants evolve from a wild-type ancestor in vitro under conditions that favor QS. Accordingly, defined lasR and pqsR mutants enriched in wild-type co-culture, whereas rhlR and all signal-negative mutants did not. Both lasR and pqsR mutants enriched with negative frequency dependence, suggesting social interactions with the wild type, although the pqsR mutant also grew well on its own. Taken together, the lasR mutant behaved as a typical cheater, as reported previously. However, the pqsR and rhlR mutants exhibited more complex behaviors, which can be sufficiently explained by positive and negative pleiotropic effects through differential regulation of pqs gene expression in the interconnected QS network. The evolutionary approach adopted here may account for the prevalence of naturally occurring QS mutants.

Figure 1

Emergence of QS-deficient isolates during in vitro evolution of the P. aeruginosa wild type. Deficiencies in skim-milk proteolysis (solid squares), growth on adenosine (solid triangles), rhamnolipid production (solid circles) and AQ production (solid diamonds) are shown. CFU ml⁻¹ (open squares) are indicated on the right y axis. Skim-milk proteolysis and growth on adenosine are las dependent, rhamnolipid production is rhl dependent and AQ production is pqs dependent. The discrepancy in the number of adenosine and protease-negative isolates is due to the fact that some lasR mutants regain the ability to degrade skim milk (Sandoz et al., 2007). Data are averages of two independent in vitro evolution experiments.

Figure 2

Complementation analysis of AQ-deficient in vitro evolution isolates. AQ production of P. aeruginosa AQ-deficient variants containing pJN105 (black), pJN105.pqsR-H controlling pqsR from a heterologous promoter (dark gray) or pJN105.pqsR-N controlling pqsR from the native P. aeruginosa promoter (light gray). ‘PQS' and ‘HHQ' are synthetic signal controls. Data are given as percentage of PAO1/pJN105. Error bars indicate s.d. of the mean of three replicates.

Figure 3

Single-culture growth of signal-blind P. aeruginosa strains. Growth of the wild-type (black), lasR (dark gray), rhlR (light gray) and pqsR (open) strains in M9-CAA and in M9-caseinate media (circle and square symbols, respectively). Error bars indicate s.d. of the mean of three replicates, and are too small to be visible in some cases.

Figure 4

Caseinolytic activity of signal-blind P. aeruginosa strains. Degradation of FITC-casein by the indicated strains grown in either M9-CAA (light gray bars) or M9-caseinate (dark gray bars). Data are given as the percentage of the wild type grown in M9-caseinate. Error bars indicate s.d. of the mean of three replicates. Statistical significance of the data was determined using a two-tailed unpaired t-test with ‘^*' indicating P-values <0.05. Protease production of each mutant was compared with that of the wild type in the respective media.

Figure 5

Enrichment of defined QS mutant strains in co-culture. Cultures were grown in M9-CAA (light gray bars) and M9-caseinate (dark gray bars) media with initial mutant frequencies of ∼1%. Values above each bar indicate fold enrichment (the ratio of final frequency versus initial frequency). (a) The lasR or lasI mutants in wild-type co-culture. (b) The pqsR or pqsA mutants in wild-type co-culture. (c) The rhlR or rhlI mutants in wild-type co-culture. (d) The lasR, pqsR and rhlR mutants combined (each at an initial frequency of 1%) in wild-type co-culture. (e) The lasR or rhlR mutants (each at an initial frequency of 1%) in pqsR mutant co-culture. Error bars indicate s.d. of the mean of three replicates. Statistical significance of the data was determined using a two-tailed unpaired t-test, with ‘^**' indicating P-values <0.05 and ‘^*' indicating P-values <0.1. For each individual condition, the initial frequency was compared with the final frequency. For between-condition comparisons, fold change was compared. For panels a–c, brackets indicate P-values from comparisons between growth in M9-caseinate versus M9-CAA, as well as growth of the signal-blind versus signal-negative strains.

Figure 6

Frequency-dependent relative fitness of signal-blind strains. Relative fitness of (a) the lasR mutant, (b) the pqsR mutant and (c and d) the rhlR mutant calculated as the comparison of initial and final mutant frequencies (v, left panels) or as the ratio of mutant and wild-type Malthusian growth parameters (w, right panels). Data in the left panels a–c are on a double logarithmic scale and are fitted with either a power regression line (panels a and c) or an exponential regression line (panel b). Data in the right panels a–c are on a semi-logarithmic scale and are fitted with either a logarithmic regression line (panels a and c) or an exponential regression line (panel b). Panel d includes initial rhlR mutant frequencies of 90 and 99% (in addition to 1, 10 and 50%) plotted on either a semi-logarithmic scale (left) or a linear scale (right), resulting in a unimodal regression. Goodness of fit is indicated by R². Fitness trends were considered significant by one-way ANOVA (using log-transformed values for v); P-values are indicated. Assays were performed in quadruplicate.