Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa

Abstract

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.

FIG. 1.

Twitching motility phenotypes of quorum-sensing and other mutants. Assays were carried out by the subsurface agar stab method (3, 35). Twitching motility is observed as the large zone of Coomassie-stained cells radiating from the point of inoculation at the interstitial surface between the agar and the bottom of the petri dish. (A) P. aeruginosa PAO1 quorum-sensing mutants. (B) P. aeruginosa PAK quorum-sensing mutants, twitching-defective variants, and variants complemented with wild-type vfr and algR cloned on the multicopy vector pUCPSK (abbreviated pUCP) (44). Bar, 1 cm; wt, wild type.