The Global Posttranscriptional Regulator RsmA Modulates Production of Virulence Determinants and N- Acylhomoserine Lactones in Pseudomonas aeruginosa

Abstract

Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a Pseudomonas aeruginosa gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of rsmA resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of rsmA in P. aeruginosa had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on N-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL) produced by the rsmA mutant and the rsmA-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using lasI and rhlI translational fusions. These data also highlighted the temporal expression control of the lasI gene, which was induced much earlier and to a higher level during the exponential growth phase in an rsmA mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control, hcn translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (hcnABC). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the hcnA ribosome-binding site. This suggests that, in P. aeruginosa, RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner

“…The hcnA gene encodes part of the hydrogen cyanide synthase enzyme that is implicated in virulence (44,45). RsmA was shown to previously negatively affect a leader fusion using the tac promoter and the ribosome-binding site region of hcnA (45).…”

“…RsmA was shown to previously negatively affect a leader fusion using the tac promoter and the ribosome-binding site region of hcnA (45). An hcnA leader fusion was constructed by annealing 33 nucleotides, including one of the predicted RsmA-binding sites, the ribosome-binding site, and the translational start codon of hcnA.…”

Cell-signalling repression in bacterial quorum sensing

“…The retarding effect of RsmA on P. aeruginosa is demonstrated in Figure 4A of Pessi et al [21], whereby the up regulation of QS activity is initiated significantly earlier in a rsmA − mutant than in the wild-type. Chugani et al [6] observed similar results in their experiments comparing wild-type and qscR − strains; however, the repressive action of qscR is currently uncertain.…”

“…Discoveries in recent years have shown that a few species have evolved means of bypassing this problem, by repressing certain components of the QS process. The most well studied examples are those of P. aeruginosa [6,7,8,21] and A. tumefaciens [4,23]. The repression mechanisms investigated in this paper are summarised below, the first two being known to be relevant for P. aeruginosa and the last to A. tumefaciens.…”

“…"Background" Inhibition (BI) of QSM output by constitutively produced regulator proteins which interfere in some way with the production or action of the QSE (which catalyses the reaction of the QSM precursors). Two such proteins have been identified in P. aeruginosa, namely QscR [6] and RsmA [21], which are produced independently of QS; see Figure 1. RsmA is believed to destroy messenger-RNA transcripts for many proteins [13], including that for the enzyme LasI, the QSE for the QSM 3-oxo-C12-HSL in P. aeruginosa (see Figure 2); how QscR inhibit QSM output is currently unknown.…”