The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone

Abstract

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).

FIG. 1

Genetic map of the vanMN locus of V. anguillarum. The vanN gene overlaps the vanM gene by 8 bp. The horizontal arrows indicate the direction of transcription. The open arrowhead indicates where pNQVanN2 was inserted into vanN to make strain DM34. The dotted line indicates the region of vanM that was deleted in frame to make strain DM27. The line labeled P1 indicates the region of vanN that was amplified using the degenerate primers complementary to ainS and luxN. This P1 region of vanN was used as the probe for first screening of the gene library from which pBSVanN10-1 was isolated. The line labeled P2 indicates the region of vanN that was used as a probe in the second screening of the gene library from which pBSVanN10-5 was isolated.

FIG. 2

Protein sequence alignments. (A) The corrected V. harveyi LuxM* protein sequence, the V. fischeri AinS protein sequence, and the V. anguillarum VanM protein sequence were aligned and compared for similarities. The LuxL-LuxM fused protein will be called LuxM (Bassler, personal communication), but for the sake of discussion it is called LuxM* in this report. (B) The V. harveyi LuxN protein sequence, the V. fischeri AinR partial protein sequence, and the V. anguillarum VanN protein sequence were aligned and compared for similarities. Asterisks indicate amino acids that are identical in the V. harveyi and V. anguillarum protein sequences; plus signs indicate amino acids that are identical in all aligned protein sequences. The amino acids within boxes represent the various conserved motifs important for the function of hybrid sensor kinases (10).

FIG. 3

Mass spectra (LC-MS) of two compounds purified from the spent culture supernatant of E. coli JM109(pBSVanMN) (B and D) are indistinguishable from those of synthetic 3-hydroxy-C6-HSL (A) and C6-HSL (C), respectively.

FIG. 3

Mass spectra (LC-MS) of two compounds purified from the spent culture supernatant of E. coli JM109(pBSVanMN) (B and D) are indistinguishable from those of synthetic 3-hydroxy-C6-HSL (A) and C6-HSL (C), respectively.

FIG. 4

Quantification of AHLs produced by V. anguillarum wild type (WT) and vanM (DM27) and vanI (DM21) mutants (M- and I-) in stationary-phase supernatant determined by LC-MS. Each sample was subjected to LC-MS, and the concentration was determined by comparison with a calibration curve constructed for molecular ion abundance using each of the corresponding AHL synthetic standards.

FIG. 5

TLC analyses of the V. anguillarum vanM mutant (DM27) showing the loss of 3-hydroxy-C6-HSL, C6-HSL (A), and 3-oxo-C10-HSL (B) and the restoration of AHL synthesis in the vanM mutant (DM27) complemented with plasmid-borne copies of vanM [DM27(pVanM-2)]. For this assay, stationary-phase, cell-free supernatants together with synthetic standards were analyzed by TLC in conjunction with the AHL biosensor, C. violaceum CV026 (A) or E. coli JM109(pSB1075) (B), for the detection of short or long-chain AHLs, respectively. (A) C6-HSL (lane 1), wild type (lane 2), DM27 (lane 3), DM27(pVanM-2) (lane 4), and 3-hydroxy-C6-HSL (lane 5); (B) 3-oxo-C10-HSL (lane 1), wild type (lane 2), DM27 (lane 3), and DM27(pVanM-2) (lane 4).