Early activation of quorum sensing.

More than 30 years ago microbiologists found a way to induce luminescent bacteria to emit light prematurely. By adding cell-free culture fluid from a bright bioluminescent culture at high cell density to a nonluminous low-cell-density culture, they were able to eliminate the characteristic lag in bioluminescence (5, 12, 16). Nealson et al. proved that the “light switch” that controls the bioluminescence genes in the marine bacterium Vibrio (formerly Photobacterium) fischeri was cell density dependent, and they hypothesized that this light switch was controlled by a bacterially produced autoinducer signal (16). Bioluminescence could occur only after the autoinducer accumulated in cultures to a threshold level that was attained at high cell density ( 10 CFU/ml) (16). Eberhard et al. went on to elucidate the structure of the autoinducer molecule that was responsible for this effect: the first known acyl-homoserine lactone, 3-oxo-hexanoyl-homoserine lactone (3-oxo-C6-HSL) (6). 3-oxo-C6-HSL was found to freely diffuse from bacterial cells into the surrounding medium and vice versa (11). This phenomenon of cell-density-dependent autoinduction of specific bacterial genes is now referred to as quorum sensing and involves two conserved regulatory gene products: (i) a LuxItype acyl-HSL synthase and (ii) a LuxR-type transcriptional activator whose activity requires a particular acyl-HSL made by the cognate LuxI enzyme (9; for recent reviews, see references 8 and 28). In addition to the LuxR/LuxI control, a second quorum-sensing system regulates the luminescence (lux) genes in V. fischeri. This second system consists of an acyl-HSL synthase, AinS, which directs the synthesis of octanoyl-HSL (C8HSL) (13). V. fischeri ainS mutants exhibited early luminescence, whereas the addition of C8-HSL delayed luminescence in cultures of wild-type cells (13). The above examples illustrate how the timing of a quorum-sensing-controlled (QSC) process can be advanced merely by early exposure of the cells to a critical concentration of acyl-HSL. QSC phenomena include antibiotic production, virulence gene expression, and other processes in many diverse bacteria. The focus of this commentary will be to highlight a number of recently identified gene products that modulate the timing of QSC gene expression in Pseudomonas aeruginosa. One such regulator, encoded by mvaT, is described in a paper in this issue (4). In most cases these gene products serve to prevent the early activation of quorum sensing. Two intertwined quorum-sensing systems have been shown to be involved in virulence, biofilm development, and many other processes in P. aeruginosa. The first system (Las) was discovered by Iglewski and colleagues and consists of a lasIencoded acyl-HSL synthase and the lasR-encoded transcriptional activator (10, 19). The second system (Rhl) was found by a number of investigators and consists of an rhlI-encoded acylHSL synthase and an rhlR-encoded transcriptional activator (2, 14, 17, 18). The respective quorum-sensing systems each produce and respond to a specific acyl-HSL: LasI directs the synthesis of 3-oxo-dodecanoyl-HSL (3-oxo-C12-HSL) (20), and RhlI directs the synthesis of butyryl-HSL (C4-HSL) (33). Further details of the complex regulation of the two quorumsensing systems and how they are thought to control expression of the several genes in P. aeruginosa has been reviewed elsewhere (22). Recently Whiteley et al. using a P. aeruginosa lasI rhlI double mutant identified nearly 40 qsc (quorum sensing controlled) genes that showed a fivefold or greater response to exogenously added acyl-HSL signals (29). The qsc genes were classified based on the temporal pattern of their responses in cells grown in the presence of the Las signal, 3-oxo-C12-HSL, and/or the Rhl signal, C4-HSL (29). A number of “early” qsc genes were found that responded immediately to exogenously added signals (29), suggesting that these genes behave like the lux genes of V. fischeri (described above) and the carbapenem biosynthesis genes of Erwinia (32). Another group of qsc genes (called “late-response”) were able to respond to the signals only during stationary growth phase (29). Whiteley et al. hypothesized that these genes might be under the control of some unknown control mechanism(s) preventing their “early” expression. Since these seminal observations were made a number of other proteins have been found that support this hypothesis, including the stationary-phase sigma factor RpoS (30), a third LuxR homolog (QscR) (3), a secondary metabolite regulator, RsmA (24), and the stringent response protein RelA (27), all of which are involved in modulating expression of qsc genes. P. aeruginosa rpoS, qscR, and rsmA deletion mutants each abolish the lag in expression of the cyanide biogenesis genes (hcn) and pyocyanin genes (phz) (3, 24, 30). All of these studies have begun to dissect the mechanisms responsible for the observed phenotypes. In the case of RpoS, it was found to negatively regulate the expression of the C4-HSL synthase gene, rhlI (30). QscR was found to negatively regulate expression of both rhlI and of the 3-oxo-C12-HSL synthase gene, lasI (3). P. aeruginosa qscR mutants showed early activation of a number of qsc genes and premature synthesis of both signals C4-HSL and 3-oxo-C12-HSL (3). Overexpression of the rsmA gene product resulted in decreased production of QSC virulence factors and acyl-HSLs, whereas deletion of rsmA led * Mailing address: Microbia, Inc., One Kendall Square, Building 1400W, Cambridge MA 02139. Phone: (617) 456-3619. Fax: (617) 494-0908. E-mail: [email protected].