Layers of signaling in a bacterium-host association.

Quorum sensing, the monitoring of population density by bacteria, is used to coordinately control gene expression and therefore particular behaviors under conditions of high cell density. Such group behaviors provide advantages to organisms under certain conditions, such as during pathogenic colonization when virulence traits are induced by a group of bacteria. In the accompanying paper, Lupp and Ruby describe the requirement for a quorum-sensing system at relatively low cell density during colonization by Vibrio fischeri of its symbiotic host, Euprymna scolopes (18). Quorum-sensing systems in Vibrio fischeri and Vibrio harveyi. Quorum sensing is a field built from laboratory-based investigations of luminous marine bacteria, namely, Vibrio fischeri and Vibrio harveyi (25). It was in V. fischeri that the quorum-sensing regulators LuxR (a transcriptional activator) and LuxI (a signal synthase) first were discovered (reviewed in reference 6). The LuxI-produced signaling molecule, an acyl homoserine lactone (acyl-HSL), diffuses out of and then (under conditions of high cell density) back into the cell, where it activates LuxR and thus the lux (luminescence) genes, which are under LuxR control (11, 14). Homologs of these proteins have been found in most gram-negative quorum-sensing bacteria studied to date. Studies of quorum sensing in V. harveyi subsequently revealed a much more complex system for lux control (Fig. 1) (reviewed in reference 28). This organism uses three sensor kinase proteins to detect its three quorum-sensing molecules (one of which is an acyl-HSL) (1, 2, 13). At low cell density, the sensor proteins autophosphorylate. The phosphate is sequentially transferred to LuxU, a phosphotransferase protein, and LuxO, a transcriptional activator. Phosphorylated LuxO activates the transcription of five small RNA (sRNA) genes (16). The resulting sRNAs work in conjunction with the Hfq protein to destabilize the transcript encoding LuxRVH, a transcriptional regulator that is not homologous to V. fischeri LuxR (16). The consequence is that LuxRVH is not synthesized, and thus the lux genes are not transcribed. Increasing population densities sequentially signal the three sensor proteins to switch from kinases to phosphatases. The consequence is dephosphorylation of LuxO, loss of sRNA synthesis, increased translation of LuxR, and thus transcription of lux and production of light. For some time, it has been known that V. fischeri encodes a second acyl-HSL synthase, termed AinS (8). AinS is homologous to LuxM of V. harveyi yet produces a distinct acyl-HSL (8, 15). More recently, homologs of the other V. harveyi quorumsensing components have been identified (Fig. 1) (7, 17, 19, 24). Although only a few of these components have been examined at a molecular level, the results to date suggest that this second V. fischeri system functions like that of V. harveyi. The ain system is integrated with the lux system at LitR, a LuxRVH homolog that is controlled by LuxO and itself controls transcription of luxR (Fig. 1) (7, 24), thereby controlling lux expression. Roles for AinS and LuxO in symbiotic initiation. Lupp and Ruby report the novel finding that the acyl-HSL synthase AinS is required for initiation of symbiotic colonization of the squid E. scolopes by V. fischeri (18). Loss of ainS delayed both colonization and the luminescence emission that results from the symbiotic association (Fig. 2A). No such delay of colonization was observed for lux mutants, suggesting that AinS may control factors other than lux that are required for symbiotic initiation. Surprisingly, a mutation in luxO, which might be expected to counteract the consequences of an ainS mutation, also prevented normal symbiotic initiation. Together, these data suggested that an optimal level of AinS/LuxO-controlled (non-lux) target gene transcription is necessary for symbiotic initiation. To identify potential AinS-controlled genes, Lupp and Ruby used microarray analysis (the first such published for the recently sequenced V. fischeri [27]). Their screen yielded 30 positively and negatively controlled genes, including, notably, negatively controlled motility genes (18). The effect of the AinS signal on motility was supported experimentally by the finding that the ainS mutant migrated through soft agar faster than the wild-type strain. Is the increase in motility sufficient to account for the colonization defect? Perhaps. Previous work has demonstrated that motility is essential for symbiotic colonization: nonmotile bacteria fail to colonize, while hypermotile mutants exhibit a severe delay (9, 22). Interestingly, a luxO mutant also displayed a defect in motility, in this case a decreased rate of migration through soft agar. These data support the idea that mutations in ainS and luxO can differentially unbalance regulation of a downstream target. Besides motility genes, however, there were a number of other, equally interesting, targets identified that could be the cause of the initiation defect observed and which will presumably be the focus of future investigations. Sequential activation of two quorum-sensing systems. In laboratory culture, induction of lux depends primarily upon ainS. Whereas luxI mutants exhibit near-wild-type levels of light emission, no luminescence can be detected from ainS mutants (19, 29). During symbiotic colonization, the opposite is true: luxI mutants fail to produce any detectable light, while * Mailing address: Department of Microbiology and Immunology, 2160 S. First Ave., Bldg. 105, Maywood, IL 60153. Phone: (708) 2160869. Fax: (708) 216-0896. E-mail: [email protected].