Quorum sensing in bacteria associated with marine sponges Mycale laxissima and Ircinia strobilina

Abstract

Sponges can form close associations with microbes that in some cases comprise up to 30% of the biomass of the sponge, which we hypothesized would provide an ideal environment for quorum sensing. I isolated 420 bacterial strains from two marine sponge species and screened these isolates for acyl-homoserine lactone (AHL) production. Results showed that isolates from the Silicibacter- Ruegeria (SR) subclade of the Roseobacter group are the dominant AHL producers. Production of these signaling compounds was consistently observed in isolates obtained from different sponge individuals during different seasons. The SR-type strain Ruegeria sp. KLH11 was isolated from tissue of the sponge Mycale laxissima. Chemical analysis of the AHLs produced by Ruegeria sp. KLH11 showed them to be predominantly composed of a mixture of long chains AHLs with 3-OH substitutions. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated as ssaRI, ssbRI and sscI (sponge-associated symbiont locus A, B and C, luxR or luxI homologue). SsaI directs synthesis of predominantly 3-oxo-AHLs whereas SsbI and SscI specify 3-OH-AHL derivatives. Wild type Ruegeria sp. KLH11 cultures are dominated by SsbI or SscI-specified AHLs. Mutation of either ssaR or ssaI results in loss of swimming motility, flagellar production and flagellin synthesis whereas mutation of ssbR or ssbI had no effect on these characteristics and no detectable phenotype. In wild type cultures, flagella are produced only in late stage growth. The non-essential phosphorelay cckA-chpT-ctrA system acts downstream of ssaRI to control flagellar motility. Mutants of ssaI and ssaR showed increased biofilm formation while mutants of ssbI and ssbR were not affected in biofilm formation, and this is not due solely to the loss of motility. The results showed the presence of AHL molecules similar to those specified by SsaI in sponge tissues and that the ssaI gene is actively expressed in situ, revealed by RT-PCR. We have established Ruegeria sp. KLH11 as a model to study the complex symbiotic relationships between sponges and microbes.