8] Quorum sensing, gene expression, and Pseudomonas biofilms

Abstract

Quorum sensing has been shown to be important for the development of a normal P. aeruginosa biofilm, and it follows that other microorganisms may employ a similar mechanism in the development of mature biofilms. To methods for detecting the presence of AI activity in biofilms are presented that employ an AI-responsive reporter strain harboring a lacZ fusion. Method 1 involves detection of AI activity in crude biofilms, whereas Method 2 employs an AI purification procedure. By using multiple indicator strains activated by AIs various acyl chain lengths, a wide range of AI molecules can be detected. Chromosomal knockout mutants are extremely useful for examining the contribution of a given gene to a specific phenotype. For quorum-sensing gene expression studies, mutants deficient in the production of AI offer more versatility than R-protein mutants. The main advantage of the AI mutants is that they can be complemented by either the AI synthase gene or the AI itself. Complementation with the AI circumvents having to grow the cells in the presence of antibiotics and allows experimental parameters such as AI concentration and time of addition to be manipulated easily. Finally, three reporter systems suitable for monitoring gene expression in P. aeruginosa biofilms are summarized in T Table II. The choice of reporter fusion depends mainly on whether in vivo analysis is required, whether temporal gene expression is to be examined, and the availability of equipment. In the case of P. aeruginosa, expression of quorum-sensing genes can be monitored either directly, by examining fusions of the R genes or AI synthase genes, or indirectly, by analyzing expression of genes controlled by these quorum-sensing systems.