Genome sequencing-assisted identification and the first functional validation ofN-acyl-homoserine-lactone synthases from the Sphingomonadaceae family

BackgroundMembers of the genusNovosphingobiumhave been isolated from a variety of environmental niches. Although genomics analyses have suggested the presence of genes associated with quorum sensing signal production e.g., theN-acyl-homoserine lactone (AHL) synthase (luxI) homologs in variousNovosphingobiumspecies, to date, noluxIhomologs have been experimentally validated.MethodsIn this study, we report the draft genome of theN-(AHL)-producing bacteriumNovosphingobium subterraneumDSM 12447 and validate the functions of predictedluxIhomologs from the bacterium through inducible heterologous expression inAgrobacterium tumefaciensstrain NTL4. We developed a two-dimensional thin layer chromatography bioassay and used LC-ESI MS/MS analyses to separate, detect and identify the AHL signals produced by theN. subterraneumDSM 12447 strain.ResultsThree predicted luxI homologs were annotated to the locus tags NJ75_2841 (NovINsub1), NJ75_2498 (NovINsub2), and NJ75_4146 (NovINsub3). Inducible heterologous expression of eachluxIhomologs followed by LC-ESI MS/MS and two-dimensional reverse phase thin layer chromatography bioassays followed by bioluminescent ccd camera imaging indicate that the three LuxI homologs are able to produce a variety of medium-length AHL compounds. New insights into the LuxI phylogeny was also gleemed as inferred by Bayesian inference.DiscussionThis study significantly adds to our current understanding of quorum sensing in the genusNovosphingobiumand provide the framework for future characterization of the phylogenetically interesting LuxI homologs from members of the genusNovosphingobiumand more generally the family Sphingomonadaceae.