Genetic and Ecological Characterization of Indigoidine Production by Phaeobacter sp. strain Y4I

The Roseobacter clade is a widely distributed, abundant, and biogeochemically active lineage of marine alpha-proteobacteria. Members of the Roseobacter lineage are prolific surface colonizers in marine coastal environments, and antimicrobial secondary metabolite production has been hypothesized to provide a competitive advantage in colonization. In this work, Phaeobacter sp. strain Y4I was found to produce the water soluble, blue pigment indigoidine via a nonribosomal peptide synthase-based biosynthetic pathway encoded by a novel series of genetically linked genes, termed igiBCDFE. Comparison of wildtype, non-pigmented, and hyper-pigmented Y4I insertional mutants demonstrated a perfect correlation between indigoidine production and the inhibition of Vibrio fischeri on agar plates, revealing a previously unrecognized bioactivity of this molecule. Competitive co-cultures of V. fischeri and Y4I showed that the production of indigoidine by Y4I significantly inhibits surface colonization of V. fischeri. Subsequent experiments identified a role for quorum sensing in the production of this secondary metabolite. Y4I has two independent quorum sensing systems, termed pgaIR and phaIR. Transposon insertions in each of the phaIR genes resulted in defects in indigoidine production. A transposon insertion in pgaR confers a null indigoidine phenotype. All of these quorum sensing mutants are unable to inhibit the growth of V. fischeri in competition experiments. These strains also have altered biofilm and motility phenotypes suggesting a role for the quorum sensing systems in regulation of these activities. Identification of the N-acyl homoserine lactone signaling molecules that are produced by Y4I was achieved using a combination of (AHL) bioreporters and mass spectrometry analyses. The two dominant AHLs were found to be N-octanoyl homoserine lactone (C8-HSL) and a putative monounsaturated N-3-hydroxydodecanoyl homoserine lactone (3OHC12:1-HSL) when the strain is grown on a complex medium. Evidence is provided that AHL production is not wholly cell-density dependent in this strain. Finally, a comprehensive analysis of the luxRI-type quorum sensing systems in sequenced roseobacter genomes provide evidence that these genetic systems are closely related among lineage members and likely share a common ancestor.