VqmA and VqmR are not produced in strain 882.

(A) Schematic of the vqmR-vqmA loci of V. parahaemolyticus RIMD2210633 and strain 882. The dotted pattern signifies that strain 882 harbors an 897 bp deletion encompassing a portion of a neighboring upstream gene (VPA1076, encoding a putative transcriptional regulator of a proline metabolic operon), a putative gene of unknown function (VPA1077), the vqmR promoter, the vqmR coding sequence, and a portion of the non-coding region upstream of vqmA882. (B) Representative western blot showing VqmARIMD-3XFLAG in V. parahaemolyticus RIMD2210633, VqmA882-3XFLAG in strain 882, and VqmA882-3XFLAG in strain 882 with the non-coding region upstream of vqmA882 restored (i.e., this strain is 882 vqmA882+). RpoA was used as the loading control. (C) Consensus sequences of the intergenic regions between vqmR and vqmA for the two designated groups. Gray “u” letters indicate ≤50% agreement. Black nucleotides with clear backgrounds indicate >50% but Left: schematic showing exchange of regions between V. parahaemolyticus RIMD2210633 and V. cholerae at the vqmR-vqmA loci. Genes and non-coding regions from V. parahaemolyticus RIMD2210633 and V. cholerae are colored cyan and red, respectively. Right: representative western blot of VqmA-3XFLAG produced by V. parahaemolyticus RIMD2210633 and V. cholerae from their native promoters (Parent) and following exchange of their promoters (SWAP). RpoA was used as the loading control. Data are representative of three independent experiments (B) and two independent experiments (D).</p

Phage VP882 is more virulent in QS-competent <i>V</i>. <i>parahaemolyticus</i> than in QS-deficient <i>V</i>. <i>parahaemolyticus</i>.

(A) Quantitation of viral particles collected from the indicated 882 strains carrying arabinose-inducible vqmAPhage grown in medium lacking (black bars) or containing 0.02% arabinose (white bars). Relative viral load is the amount of phage VP882-specific DNA (gp69) relative to non-phage DNA (hfq) measured by qPCR. (B) Growth of the 882 parent (cyan), 882 vqmR-vqmA882+ (green), 882 luxO+ (orange), and 882 vqmR-vqmA882+ luxO+ (red) strains carrying arabinose-inducible vqmAPhage and grown in medium lacking (diamonds) or containing (circles) 0.02% arabinose. Data are represented as means ± std with n = 3 biological replicates and n = 4 technical replicates (A), and as means ± std with n = 3 biological replicates (B).</p

Data points used to make plots that appear in this study.

Data points used to make plots that appear in this study.</p

Strains used in this study.

Quorum sensing (QS) is a chemical communication process that bacteria use to track population density and orchestrate collective behaviors. QS relies on the production, accumulation, and group-wide detection of extracellular signal molecules called autoinducers. Vibriophage 882 (phage VP882), a bacterial virus, encodes a homolog of the Vibrio QS receptor-transcription factor, called VqmA, that monitors the Vibrio QS autoinducer DPO. Phage VqmA binds DPO at high host-cell density and activates transcription of the phage gene qtip. Qtip, an antirepressor, launches the phage lysis program. Phage-encoded VqmA when bound to DPO also manipulates host QS by activating transcription of the host gene vqmR. VqmR is a small RNA that controls downstream QS target genes. Here, we sequence Vibrio parahaemolyticus strain O3:K6 882, the strain from which phage VP882 was initially isolated. The chromosomal region normally encoding vqmR and vqmA harbors a deletion encompassing vqmR and a portion of the vqmA promoter, inactivating that QS system. We discover that V. parahaemolyticus strain O3:K6 882 is also defective in its other QS systems, due to a mutation in luxO, encoding the central QS transcriptional regulator LuxO. Both the vqmR-vqmA and luxO mutations lock V. parahaemolyticus strain O3:K6 882 into the low-cell density QS state. Reparation of the QS defects in V. parahaemolyticus strain O3:K6 882 promotes activation of phage VP882 lytic gene expression and LuxO is primarily responsible for this effect. Phage VP882-infected QS-competent V. parahaemolyticus strain O3:K6 882 cells lyse more rapidly and produce more viral particles than the QS-deficient parent strain. We propose that, in V. parahaemolyticus strain O3:K6 882, constitutive maintenance of the low-cell density QS state suppresses the launch of the phage VP882 lytic cascade, thereby protecting the bacterial host from phage-mediated lysis.</div

Numerical values, and associated p-values, for heatmaps in Fig 4A.

Numerical values, and associated p-values, for heatmaps in Fig 4A.</p

Simplified schematics of the vibriophage VP882 and <i>Vibrio</i> QS circuits at HCD and mechanisms driving phage VP882 host-cell lysis.

(A) Multiple autoinducer-receptor pairs control QS in V. parahaemolyticus. AI-1 (circles), AI-2 (triangles), and CAI-1 (squares) are detected by LuxN, LuxPQ, and CqsS, respectively. At LCD, the receptors are kinases and funnel phosphate to LuxO (via a protein LuxU, not shown). LuxO~P activates transcription of genes encoding the Qrr sRNAs. The Qrr sRNAs post-transcriptionally repress opaR, the master regulator of group behaviors. At HCD (shown in the cartoon), the liganded receptors act as phosphatases, reversing the phosphorylation cascade. Consequently, production of Qrr sRNAs ceases, and opaR translation is derepressed, OpaR is produced, and the cells undertake group behaviors. In parallel, at HCD, the DPO autoinducer (hexagons) interacts with the VqmA transcription factor. The complex activates vqmR expression and the VqmR sRNA controls QS behaviors. Phage VP882 carries vqmAPhage. Binding of VqmAPhage to host-produced DPO activates expression of a gene encoding an antirepressor called Qtip, which drives the phage lytic pathway. (B) Two pathways control phage VP882 lysis-lysogeny transitions. Upper: DPO-bound VqmAPhage activates expression of qtip. Qtip sequesters the phage cIVP882 repressor of lysis leading to phage replication and host-cell killing. Lower: the phage cIVP882 repressor is proteolyzed in response to host SOS/DNA damage via a host RecA-dependent mechanism. The consequence is phage replication and host-cell killing.</p

V. parahaemolyticus strain 882 is the only sequenced Vibrio that lacks vqmR and harbors vqmA, vqmA expression is not auto-regulated in V. parahaemolyticus, and Vibrio vqmA promoters cluster into two distinct classes.

(A) Percentage of strains possessing (gray) or lacking (turquoise) vqmR-vqmA pairs (left), and number of strains analyzed (right) for the designated species. Vas: Vibrio aestuarianus. Val: Vibrio alginolyticus. Van: Vibrio anguillarum. Vbr: Vibrio breoganii. Vcm: Vibrio campbellii. Vch: Vibrio cholerae. Vcrl: Vibrio coralliilyticus. Vcrr: Vibrio coralliirubri. Vcr: Vibrio crassostreae. Vcy: Vibrio cyclitrophicus. Vdb: Vibrio diabolicus. Vdz: Vibrio diazotrophicus. Vfl: Vibrio fluvialis. Vfr: Vibrio furnissii. Vhr: Vibrio harveyi. Vjs: Vibrio jasicida. Vkn: Vibrio kanaloae. Vln: Vibrio lentus. Vmd: Vibrio mediterranei. Vmtc: Vibrio metoecus. Vmts: Vibrio metschnikovii. Vmm: Vibrio mimicus. Vnv: Vibrio navarrensis. Vmg: Vibrio nigripulchritudo. Vow: Vibrio owensii. Vpch: Vibrio paracholerae. Vpr: Vibrio parahaemolyticus. Vrt: Vibrio rotiferianus. Vsp: Vibrio splendidus. Vts: Vibrio tasmaniensis. Vvl: Vibrio vulnificus. (B) Left: Relative PvqmARIMD-lux output from E. coli carrying arabinose-inducible vqmA882-3XFLAG. The treatments—and + VqmA882 refer to water and 0.2% arabinose, respectively. RLU as in Fig 2B. Right: representative western blot of VqmA882-3XFLAG produced by the E. coli in the left panel. RpoA was used as the loading control. (C) Multiple DNA sequence alignment of the intergenic regions between vqmR and vqmA for the V. cholerae clade (Clade 1) and the V. parahaemolyticus clade (Clade 2). A representative strain (as designated) was chosen for each species in each clade. Thick gray or red bars indicate, respectively, nucleotides that are identical with or different from the consensus (>50% agreement among aligned sequences). Thin gray lines indicate gaps in the sequence alignments. Scale bar indicates 50 bp. Data in B are represented as means ± std with n = 3 biological replicates (left) and representative of two independent experiments (right). (TIF)</p

Primers and gBlocks used in this study.

Quorum sensing (QS) is a chemical communication process that bacteria use to track population density and orchestrate collective behaviors. QS relies on the production, accumulation, and group-wide detection of extracellular signal molecules called autoinducers. Vibriophage 882 (phage VP882), a bacterial virus, encodes a homolog of the Vibrio QS receptor-transcription factor, called VqmA, that monitors the Vibrio QS autoinducer DPO. Phage VqmA binds DPO at high host-cell density and activates transcription of the phage gene qtip. Qtip, an antirepressor, launches the phage lysis program. Phage-encoded VqmA when bound to DPO also manipulates host QS by activating transcription of the host gene vqmR. VqmR is a small RNA that controls downstream QS target genes. Here, we sequence Vibrio parahaemolyticus strain O3:K6 882, the strain from which phage VP882 was initially isolated. The chromosomal region normally encoding vqmR and vqmA harbors a deletion encompassing vqmR and a portion of the vqmA promoter, inactivating that QS system. We discover that V. parahaemolyticus strain O3:K6 882 is also defective in its other QS systems, due to a mutation in luxO, encoding the central QS transcriptional regulator LuxO. Both the vqmR-vqmA and luxO mutations lock V. parahaemolyticus strain O3:K6 882 into the low-cell density QS state. Reparation of the QS defects in V. parahaemolyticus strain O3:K6 882 promotes activation of phage VP882 lytic gene expression and LuxO is primarily responsible for this effect. Phage VP882-infected QS-competent V. parahaemolyticus strain O3:K6 882 cells lyse more rapidly and produce more viral particles than the QS-deficient parent strain. We propose that, in V. parahaemolyticus strain O3:K6 882, constitutive maintenance of the low-cell density QS state suppresses the launch of the phage VP882 lytic cascade, thereby protecting the bacterial host from phage-mediated lysis.</div

VqmA882 binds DPO and promoter DNA.

(A) Protein sequence alignment (ClustalW) showing V. parahaemolyticus strain 882 VqmA (VqmA882), V. cholerae VqmA (VqmAVc), and phage VP882 VqmA (VqmAPhage) proteins. Black and gray boxes designate identical and conserved residues, respectively. Numbering indicates amino acid positions. Blue boxes indicate key conserved DPO-binding residues from VqmAVc (F67, F99, and K101). (B) Relative fold activation of PvqmRRIMD-lux or Pqtip-lux from Δtdh E. coli harboring arabinose-inducible vqmA882-3XFLAG. Fold activation was calculated by dividing the RLU of induced cells (0.02% arabinose and 10 μM DPO) by the RLU of uninduced cells. (C) Relative PvqmRRIMD-lux and PvqmRVc-lux from Δtdh E. coli harboring arabinose-inducible vqmA882-3XFLAG (designated VqmA882) or vqmAVc-3XFLAG (designated VqmAVc), respectively. E. coli were treated with either water (black bars) or 10 μM DPO (white bars). All cells were treated with 0.02% arabinose. Data are represented as means ± std with n = 3 biological replicates (B, C). RLU as in Fig 2B (B, C). (TIF)</p

LuxO882-3XFLAG and LuxO+-3XFLAG are functional and 88 Vibrio strains possess the Δ91–102 LuxO deletion.

(A) Relative luxCDABE output over time from the 882 luxO882 (cyan), 882 luxO+ (orange), 882 luxO882-3XFLAG (purple), and 882 luxO+-3XFLAG (pink) strains. Data are represented as means ± std with n = 3 biological replicates. (B) Multiple amino acid sequence alignment of LuxO in Vibrio strains that carry the Δ91–102 luxO mutation. Gray or red vertical bars indicate, respectively, amino acids that are identical to or different from the consensus (>50% agreement among aligned sequences). White boxes indicate the 91–102 amino acid deletion. Blue vertical lines indicate insertions. Teal indicates V. parahaemolyticus strains, green indicates V. cholerae strains, and dark blue indicates V. owensii strains. Scale bar indicates 50 amino acids (abbreviated AA). All sequences are aligned with respect to the LuxO sequences of V. parahaemolyticus RIMD2210633 and V. cholerae C6706, which are shown in the first and second row, respectively. (C) As in (B), except the strains carry the Δ67–87 (top) or Δ114–134 (bottom) luxO mutation. (TIF)</p