Production of the antimicrobial secondary metabolite indigoidine contributes to competitive surface colonization by the marine roseobacter Phaeobacter sp. strain Y4I

Abstract

Members of the Roseobacter lineage of marine bacteria are prolific surface colonizers in marine coastal environments, and antimicrobial secondary metabolite production has been hypothesized to provide a competitive advantage to colonizing roseobacters. Here, we report that the roseobacter Phaeobacter sp. strain Y4I produces the blue pigment indigoidine via a nonribosomal peptide synthase (NRPS)-based biosynthetic pathway encoded by a novel series of genetically linked genes: igiBCDFE. A Tn5-based random mutagenesis library of Y4I showed a perfect correlation between indigoidine production by the Phaeobacter strain and inhibition of Vibrio fischeri on agar plates, revealing a previously unrecognized bioactivity of this molecule. In addition, igiD null mutants (igiD encoding the indigoidine NRPS) were more resistant to hydrogen peroxide, less motile, and faster to colonize an artificial surface than the wild-type strain. Collectively, these data provide evidence for pleiotropic effects of indigoidine production in this strain. Gene expression assays support phenotypic observations and demonstrate that igiD gene expression is upregulated during growth on surfaces. Furthermore, competitive cocultures of V. fischeri and Y4I show that the production of indigoidine by Y4I significantly inhibits colonization of V. fischeri on surfaces. This study is the first to characterize a secondary metabolite produced by an NRPS in roseobacters.

Fig 1

Qualitative screen for V. fischeri inhibition by Phaeobacter sp. strain Y4I. The wild type (WT) and Tn5 insertional mutants are shown on a lawn of V. fischeri. igiD encodes the indigoidine NRPS, clpA encodes a regulatory protein protease, and luxR encodes a regulatory protein involved in quorum sensing.

Fig 2

Phaeobacter sp. strain Y4I indigoidine biosynthesis operon. The organization of the genes within the operon (RBY4I_313, RBY4I_1554, RBY4I_3713, RBY4I_2890, RBY4I_618, and RBY4I_1719) was obtained from the published genome sequence. The genes are represented by page arrows, which indicate orientation and are shown to scale. Gene product names are given in Table 2.

Fig 3

Chemical analyses of purified pigment. (A) Mass spectrum analysis by high-resolution MS-DART. The peak m/z at 249.06110 is consistent with protonated indigoidine (C₁₀H₉N₄O₄). (B) ¹H-NMR spectrum shows peaks at 11.29 (NH), 8.18 (CH), and 6.47 (NH₂), which is consistent with the published spectrum of indigoidine (55).

Fig 4

Proposed mechanism of indigoidine biosynthesis by Phaeobacter sp. strain Y4I. (Indigoidine and leucoindigoidine structures are derived from references and 55).

Fig 5

Phenotypic characterization of Phaeobacter sp. strain Y4I and mutants with alterations in indigoidine biosynthesis. (A) Viable cell counts of planktonic Y4I variants after exposure to 250 mM hydrogen peroxide for 0 (No HOOH), 10, and 60 min. The asterisk denotes cell counts that are the same (P < 0.001); all others are statistically different from each other (P ≤ 0.05). (B) Viable counts of wild-type (WT) Y4I and the igiD::Tn5 strain as they colonize a glass surface over time. (C) Viable cell counts of attached Y4I (WT) and the igiD::Tn5 strain after exposure to 75 mM hydrogen peroxide from 0 to 10 min. (D) Diameter of swim zone of Y4I (WT) and igiD::Tn5 strains on soft-agar motility plates. Viable counts of competitions between planktonic Y4I variants and V. fischeri in broth culture (E) and in biofilms (F) are shown. Black bars, V. fischeri, light gray bars, Y4I (WT); white bars, igiD::Tn5; dark gray bars, clpA::Tn5. Error bars represent the standard deviation of biological and technical triplicates.

Fig 6

igiD gene expression of Phaeobacter sp. strain Y4I (wild type [WT]) and the clpA::Tn5 mutant under different culture conditions. Expression levels are normalized to three reference genes and relative to late-exponential (16.5 h) wild-type planktonic cells. Error bars represent the standard error of the mean. Images show the phenotype of each strain in the culture condition measured.