Substrate Specificity and Function of the Pheromone Receptor AinR in Vibrio fischeri ES114

Abstract

Two distinct but interrelated pheromone-signaling systems, LuxI/LuxR and AinS/AinR, positively control bioluminescence in Vibrio fischeri. Although each system generates an acyl-homoserine lactone (AHL) signal, the protein sequences of LuxI/LuxR and AinS/AinR are unrelated. AinS and LuxI generate the pheromones N-octanoyl-AHL (C 8 -AHL) and N-3-oxo-hexanoyl-AHL (3OC 6 -AHL), respectively. LuxR is a transcriptional activator that responds to 3OC 6 -AHL, and to a lesser extent to C 8 -AHL. AinR is hypothesized to respond to C 8 -AHL and, based on homology to Vibrio harveyi LuxN, to mediate the repression of a Qrr regulatory RNA. However, a ⌬ainR mutation decreased luminescence, which was not predicted based on V. harveyi LuxN, raising the possibility of a distinct regulatory mechanism for AinR. Here we show that ainR can complement a luxN mutant, suggesting functional similarity. Moreover, in V. fischeri, we observed ainR-dependent repression of a P qrr -lacZ transcriptional reporter in the presence of C 8 -AHL, consistent with its hypothesized regulatory role. The system appears quite sensitive, with a half-maximal effect on a P qrr reporter at 140 pM C 8 -AHL. Several other AHLs with substituted and unsubstituted acyl chains between 6 and 10 carbons also displayed an AinR-dependent effect on P qrr -lacZ; however, AHLs with acyl chains of four carbons or 12 or more carbons lacked activity. Interestingly, 3OC 6 -AHL also affected expression from the qrr promoter, but this effect was largely luxR dependent, indicating a previously unknown connection between these systems. Finally, we propose a preliminary explanation for the unexpected luminescence phenotype of the ⌬ainR mutant.

Phaeobacter sp. Strain Y4I Utilizes Two Separate Cell-to-Cell Communication Systems To Regulate Production of the Antimicrobial Indigoidine

“…V. fischeri has two AHL-based quorum sensing systems that synthesize different AHLs at different stages of squid colonization (12,17,65). Moreover, it has been shown that the response regulator that directly controls bioluminescence (LuxR) can respond to the two AHLs at different binding efficiencies, thus inducing bioluminescence at different cell densities and stages of colonization (67,68).…”

“…Sequential and multitiered quorum sensing regulatory networks have been described for several proteobacterial strains, including Vibrio fischeri, in which quorum sensing is used to modulate intermediate and late symbiotic colonization of the Euprymna scolopes light organ and bioluminescence (12,17,65,66). V. fischeri has two AHL-based quorum sensing systems that synthesize different AHLs at different stages of squid colonization (12,17,65).…”

“…At high cell densities, AHL concentrations can reach a threshold concentration, bind to the corresponding cognate cytoplasmic LuxR proteins, and affect transcription of various genetic loci (8)(9)(10)(11). AHLs have also been shown to activate regulators other than the corresponding cognate LuxR, suggesting that cross talk may play an important role in intra-and interspecies interactions (12)(13)(14)(15)(16)(17). Furthermore, AHL concentrations have been shown to change throughout the growth cycle of some bacteria, suggesting that the type and concentration of an AHLs may also convey information about the metabolic state of the population (18,19).…”

Antisocial luxO Mutants Provide a Stationary-Phase Survival Advantage in Vibrio fischeri ES114

“…1) that is conserved in the Vibrionaceae. Much of this PS cascade was elucidated in Vibrio harveyi, and recent studies have verified parallel functions with subtle differences in V. fischeri (17)(18)(19)(22)(23)(24)(25)(26). In the model that has a Genes for drug resistance are as follows: camR, chloramphenicol resistance; ermR, erythromycin resistance; kanR, kanamycin resistance (aph).…”

“…Oligonucleotides and plasmids are listed in Table 1, and the latter were constructed using standard techniques and materials as described previously (22). Genomic DNA (gDNA) for genome resequencing and natural transformation was purified using the Easy-DNA gDNA purification kit (Life Technologies, Grand Island, NY).…”