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

Han Ming Gan; Lucas K Dailey; Nigel Halliday; Paul Williams; André O Hudson; Michael A Savka

doi:10.7717/peerj.2332

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

PeerJ. 2016 Aug 30:4:e2332. doi: 10.7717/peerj.2332. eCollection 2016.

Authors

Han Ming Gan¹, Lucas K Dailey², Nigel Halliday³, Paul Williams³, André O Hudson², Michael A Savka²

Affiliations

¹ School of Science, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia; Genomics Facility, Tropical Medicine Biology Platform, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia.
² Thomas H. Gosnell School of School of Life Sciences, Rochester Institute of Technology , Rochester , NY , USA.
³ School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.

Abstract

Background: Members of the genus Novosphingobium have 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., the N-acyl-homoserine lactone (AHL) synthase (luxI) homologs in various Novosphingobium species, to date, no luxI homologs have been experimentally validated.

Methods: In this study, we report the draft genome of the N-(AHL)-producing bacterium Novosphingobium subterraneum DSM 12447 and validate the functions of predicted luxI homologs from the bacterium through inducible heterologous expression in Agrobacterium tumefaciens strain 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 the N. subterraneum DSM 12447 strain.

Results: Three predicted luxI homologs were annotated to the locus tags NJ75_2841 (NovINsub1), NJ75_2498 (NovINsub2), and NJ75_4146 (NovINsub3). Inducible heterologous expression of each luxI homologs 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.

Discussion: This study significantly adds to our current understanding of quorum sensing in the genus Novosphingobium and provide the framework for future characterization of the phylogenetically interesting LuxI homologs from members of the genus Novosphingobium and more generally the family Sphingomonadaceae.

Keywords: Acyl-homoserine lactones; LuxIR; N-acyl-homoserine lactone synthases; Novosphingobium; Phylogenetic; Quorum-sensing; Sphingomonadaceae; Two-dimensional thin-layer chromatography; Whole genome sequencing.

Grants and funding

The American Society for Microbiology (ASM) provided a summer 2015 stipend to LKD and MAS under the ASM Undergraduate Research Fellowship program to LKD. MAS, AOH and HMG received support from Thomas H. Gosnell School of Life Sciences (GSOLS) and the College of Science (COS) at the Rochester Institute of Technology (RIT). HMG received support from the Monash University Malaysia Tropical Medicine and Biology Multidisciplinary Platform. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.