Characterization of quorum sensing genes and N-acyl homoserine lactones in Citrobacter amalonaticus strain YG6

In the phylum of Proteobacteria, quorum sensing (QS) system is widely driven by synthesis and response of N-acyl homoserine lactone (AHL) signalling molecules. AHL is synthesized by LuxI homologue and sensed by LuxR homologue. Once the AHL concentration achieves a threshold level, it triggers the regulation of target genes. In this study, QS activity of Citrobacter amalonaticus strain YG6 which was isolated from clams was investigated. In order to characterise luxUR homologues, the genome of C. amalonaticus strain YG6 (4.95 Mbp in size) was sequenced using Illumina MiSeq sequencer. Through in silico analysis, a pair of canonical luxl/R homologues and an orphan luxR homologue were identified and designated as caml, carnR, and camR2, respectively. A putative lux box was identified at the upstream of caml. The car& gene was cloned and overexpressed in E. coli BL21 (DE3) pLysS. High-resolution triple quadrupole liquid chromatography mass spectrometry (LC-MS/MS) analysis verified that the Caml is a functional AHL synthase which produced multiple AHL species, namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-tetradecanoyl-L-homoserine lactone (C14-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL) in C. amalonaticus strain YG6 and carol gene in recombinant E. colt BL21(DE3)pLysS. To our best knowledge, this is the first functional study report of cams as well as the first report describing the production of C14-HSL by C. amalonaticus.