Vibrio fischeri LuxS and AinS: comparative study of two signal synthases

Abstract

Vibrio fischeri possesses two acyl-homoserine lactone quorum-sensing systems, ain and lux, both of which are involved in the regulation of luminescence gene expression and are required for persistent colonization of the squid host, Euprymna scolopes. We have previously demonstrated that the ain system induces luminescence at cell densities that precede lux system activation. Our data suggested that the ain system both relieves repression and initially induces the lux system, thereby achieving sequential induction of gene expression by these two systems. Analysis of the V. fischeri genome revealed the presence of a putative third system based on the enzyme LuxS, which catalyzes the synthesis of the Vibrio harveyi autoinducer 2 (AI-2). In this study, we investigated the impact of V. fischeri LuxS on luminescence and colonization competence in comparison to that of the ain system. Similar to the ain system, inactivation of the AI-2 system decreased light production in culture, but not in the squid host. However, while an ainS mutant produces no detectable light in culture, a luxS mutant expressed approximately 70% of wild-type luminescence levels. A mutation in luxS alone did not compromise symbiotic competence of V. fischeri; however, levels of colonization of an ainS luxS double mutant were reduced to 50% of the already diminished level of ainS mutant colonization, suggesting that these two systems regulate colonization gene expression synergistically through a common pathway. Introduction of a luxO mutation into the luxS and ainS luxS background could relieve both luminescence and colonization defects, consistent with a model in which LuxS, like AinS, regulates gene expression through LuxO. Furthermore, while luxS transcription appeared to be constitutive and the AI-2 signal concentration did not change dramatically, our data suggest that ainS transcription is autoregulated, resulting in an over 2,000-fold increase in signal concentration as culture density increased. Taken together, these data indicate that V. fischeri LuxS affects both luminescence regulation and colonization competence; however, its quantitative contribution is small when compared to that of the AinS signal.

FIG. 1.

Luminescence of luxS mutants in the symbiotic light organ. Animal luminescence was monitored during the initial stages of E. scolopes colonization by V. fischeri wild-type (solid diamonds), ainS mutant (solid squares), luxS mutant (solid triangles), and ainS luxS mutant (solid circles) strains. For each time point, mean values of 24 animals were calculated and standard errors of the mean are indicated. The experiment was repeated with the same outcome.

FIG. 2.

Colonization competence of quorum-sensing mutants. Colonization levels of the ainS mutant (A), luxS mutant (S), ainS luxS mutant (A S), and ainS-luxS-luxO (A S O) mutant relative to V. fischeri wild type (wt) were measured at 24 and 48 h postinoculation. Each bar represents the mean value of 15 animals with the associated standard errors. The experiment was conducted twice with the same outcome.

FIG. 3.

Relationship between luminescence and quorum-sensing signal activity during growth of V. fischeri in culture. The relative level of LuxS signal activity (solid diamonds) in the culture supernatant is given as a percentage of the level produced by the positive control, an overnight V. harveyi BB152 culture (see Materials and Methods). The concentration of the AinS signal, C8-HSL (solid circles), in the V. fischeri culture was determined with synthetically produced C8-HSL as standard. Luminescence of the culture (crosses) is presented for comparison. Shown is a representative experiment; standard deviation bars are indicated.

FIG. 4.

Transcriptional activity of luxS and ainS in culture. β-Galactosidase activity of V. fischeri wild-type cells carrying either a luxS::lacZ fusion (solid diamonds) or an ainS::lacZ fusion (open diamonds) in trans on low-copy-number plasmids pCL152 and pCL154, respectively, was measured during growth. Shown are the cumulative data of three cultures; standard deviation bars were smaller than the symbols.

FIG. 5.

Conceptual model for the regulation of luminescence and colonization genes by V. fischeri LuxS and AinS. Arrows indicate positive, inducing effects, and bars indicate negative, inhibitory ones. (See Discussion for explanation.)