Quorum-sensing-negative (lasR) mutants of Pseudomonas aeruginosa avoid cell lysis and death.

In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37 degrees C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > or = 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh(-) phenotype, and five of these mutants, which were functionally complemented by lasR(+), had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.

Characterization of cell-to-cell signaling-deficient Pseudomonas aeruginosa strains colonizing intubated patients.

Cell-to-cell signaling involving N-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by Pseudomonas aeruginosa. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442 P. aeruginosa pulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct P. aeruginosa strains. Six of these strains produced AIs [N-butanoyl-homoserine lactone or N-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the lasR and rhlR genes, which encode the N-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the lasR gene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.

Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1.

Virulence in the opportunistic human pathogen Pseudomonas aeruginosa is controlled by cell density via diffusible signalling molecules ('autoinducers') of the N-acylhomoserine lactone (AHL) type. Two Bacillus sp. isolates (A23 and A24) with AHL-degrading activity were identified among a large collection of rhizosphere bacteria. From isolate A24 a gene was cloned which was similar to the aiiA gene, encoding an AHL lactonase in another Bacillus strain. Expression of the aiiA homologue from isolate A24 in P. aeruginosa PAO1 reduced the amount of the quorum sensing signal N-oxododecanoyl-L-homoserine lactone and completely prevented the accumulation of the second AHL signal, N-butyryl-L-homoserine lactone. This strongly reduced AHL content correlated with a markedly decreased expression and production of several virulence factors and cytotoxic compounds such as elastase, rhamnolipids, hydrogen cyanide and pyocyanin, and strongly reduced swarming. However, no effect was observed on flagellar swimming or on twitching motility, and aiiA expression did not affect bacterial adhesion to a polyvinylchloride surface. In conclusion, introduction of an AHL degradation gene into P. aeruginosa could block cell-cell communication and exoproduct formation, but failed to interfere with surface colonization.

Novel pyoverdine biosynthesis gene(s) of Pseudomonas aeruginosa PAO.

Conjugational mobilization of a Pseudomonas aeruginosa PAO1 cosmid bank (in pMMB33) into a pyoverdine-deficient (pvd) mutant harbouring a mutation in the 47 min region of the chromosome yielded one clone which restored yellow-green pigmentation and fluorescence when grown on iron-deficient medium. The relevant pMMB33-derivative cosmid, pPYP17, contained a 15.1 kb insert which was subcloned into pKT240 as a 10.8 Sacl-CIal fragment conferring the same phenotype. This derivative, pPYP180, like pPYP17, also conferred an apparent wild-type phenotype on pvd mutants previously shown to map genetically in the 23 min region of the P. aeruginosa PAO chromosomes. Physical mapping indicated that the cloned DNA fragment is located at the 66-70 min region of the PAO chromosome, demonstrating that the restored apparent wild-type phenotype observed for the transconjugants was not the result of a true gene complementation. A gene interruption was obtained by replacing a 0.6 kb BgIll-BgIll region of pPYP180 necessary for the expression of the pigmentation/fluorescence phenotype, by a Hgr interposon (omega Hg). After conjugational transfer and introduction of the mutagenized fragment into the PAO1 chromosome by gene replacement, pyoverdine-deficient mutants were recovered, indicating that the fragment indeed contained at least one gene involved in pyoverdine synthesis. The yellow-green fluorescent compound produced by such cells harbouring plasmids pPYP17 or pPYP180 differed from pyoverdine in several aspects and was consequently named pseudoverdine. Although pseudoverdine was able to complex iron, it was unable to restore growth to pvd mutants in the presence of the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid), or to mediate iron uptake into PAO1. Pseudoverdine lacked a peptide chain but possessed spectral properties similar to pyoverdine, suggesting that it was structurally related to the chromophore of the pyoverdine molecule. The recent structural determination of pseudoverdine as a coumarin derivative confirmed this view and sheds some light on the biosynthetic pathway of the pyoverdine chromophore.