Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi

Abstract

In a process called quorum sensing, bacteria communicate using extracellular signal molecules termed autoinducers. Two parallel quorum-sensing systems have been identified in the marine bacterium Vibrio harveyi. System 1 consists of the LuxM-dependent autoinducer HAI-1 and the HAI-1 sensor, LuxN. System 2 consists of the LuxS-dependent autoinducer AI-2 and the AI-2 detector, LuxPQ. The related bacterium, Vibrio cholerae, a human pathogen, possesses System 2 (LuxS, AI-2, and LuxPQ) but does not have obvious homologues of V. harveyi System 1. Rather, System 1 of V. cholerae is made up of the CqsA-dependent autoinducer CAI-1 and a sensor called CqsS. Using a V. cholerae CAI-1 reporter strain we show that many other marine bacteria, including V. harveyi, produce CAI-1 activity. Genetic analysis of V. harveyi reveals cqsA and cqsS, and phenotypic analysis of V. harveyi cqsA and cqsS mutants shows that these functions comprise a third V. harveyi quorum-sensing system that acts in parallel to Systems 1 and 2. Together these communication systems act as a three-way coincidence detector in the regulation of a variety of genes, including those responsible for bioluminescence, type III secretion, and metalloprotease production.

FIG. 1.

Models of the V. harveyi and V. cholerae quorum-sensing systems. (A) V. harveyi has three parallel quorum-sensing systems that regulate the genes encoding bioluminescence (Lux), TTS, a secreted metalloprotease (VhpA), and other quorum-sensing-activated and -repressed genes. (B) V. cholerae possesses the CAI-1-CqsS system and the AI-2-LuxPQ system. V. cholerae also has an unidentified third circuit (System 3) that acts through LuxO. Symbols: pentagons, HAI-1; diamonds, AI-2; circles, CAI-1. Dotted lines denote targets for which regulation has not been shown to be direct. In both panels, phosphate flows toward LuxO at low cell density, and the flow of phosphate is reversed at high cell density (double-headed arrows). Details of the phosphorelay mechanism are provided in the text. Abbreviations: H, histidine; D, aspartate; HTH, helix-turn-helix; IM, inner membrane; OM, outer membrane.

FIG. 2.

Analysis of cell-free culture fluids for CAI-1 activity. (A) Cell-free culture fluids were prepared from the strains shown and added at 30% (vol/vol) to V. cholerae MM920. Light production by MM920 was measured. Strain names are as follows (left to right): KSK1052, MM893, BB120, JAF633, KM387, KM413, JMH603, JMH598, DH5α, MM1467. (B) CAI-1 production over the growth curve is shown for WT V. harveyi strain BB120. Samples were collected every 2 h, the OD₆₀₀s were measured, and cell-free culture fluids were prepared and assayed for CAI-1 activity using MM920. CAI-1 activity is reported as the fold induction of light production in MM920.

FIG. 3.

The cqs locus in V. harveyi. (A) CAI-1 activity is shown for recombinant E. coli DH5α strains carrying either the cosmid pLAFR or pLAFR containing vibrio DNA. pJMH17F7 carries 25 kb of V. harveyi genomic DNA containing the gene responsible for CAI-1 activity. pJMH159, pJMH163, and pJMH164 contain Tn5lacZ insertions in pJMH17F7. pJMH273 carries only the cqsA_Vh gene, and pJMH282 carries only the cqsS_Vh gene. (B) Map of the cqs locus of V. harveyi and V. cholerae. Black triangles indicate the sites of Tn5lacZ insertions in pJMH163, pJMH164 and pJMH159. The cqsS and cqsA genes are shown in black and white, respectively. The downstream ORF in V. harveyi has amino acid homology to VCA0528 and is shown in gray. This gene was not fully sequenced. The downstream ORF in V. cholerae, denoted VCA0524, is shown in stripes.

FIG. 4.

V. harveyi single, double, and triple autoinducer synthase mutant phenotypes. (A) Light production per cell (RLU) was measured from high-cell-density V. harveyi cultures. The V. harveyi strains are (from left to right) BB120 (WT), JMH603 (cqsA_Vh), KM387 (luxS), JAF633 (luxM), JMH605 (luxM cqsA_Vh), JMH606 (luxS cqsA_Vh), KM413 (luxM luxS), and JMH634 (luxM luxS cqsA_Vh), and the rightmost two bars are JMH603 (cqsA_Vh). In these latter two cases, JMH603 (cqsA_Vh) was grown in the presence of 30% V. harveyi or V. cholerae WT culture fluids. The asterisks denote exogenously added autoinducer. (B) CqsA_Vh and CqsS_Vh epistasis analysis. RLU were measured at high cell density in the following strains (left to right): BB120 (WT), JMH603 (cqsA_Vh), JMH598 (cqsS_Vh), JMH763 (cqsA_Vh cqsS_Vh). Relative light units are defined as counts min⁻¹ ml⁻¹ × 1,000/CFU ml⁻¹.

FIG. 5.

LuxU and LuxO are epistatic to CqsA. RLU were measured from high-cell-density V. harveyi cultures of the following strains (left to right): BB120 (WT), JMH603 (cqsA_Vh), JAF536 (luxU), JAF483 (luxOD47A), JMH621 (luxU cqsA_Vh), JMH774 (luxOD47A cqsA_Vh).

FIG. 6.

All three autoinducers are required for quorum-sensing-controlled gene regulation. β-Galactosidase assays were performed on V. harveyi recipient strains (high cell density) grown overnight in the presence of 10% cell-free culture fluids containing various autoinducers. Cell-free culture fluids were prepared from the following strains (left to right): BB120 (WT), JMH603 (cqsA_Vh), KM387 (luxS), JAF633 (luxM), JMH606 (luxS cqsA_Vh), JMH605 (luxM cqsA_Vh), KM413 (luxM luxS), and JMH634 (luxM luxS cqsA_Vh). (A) The recipient strain was V. harveyi JMH670 (vhpA::Tn5lacZ luxM luxS cqsA_Vh). (B) The recipient strain was JMH674 (vcrD::Tn5lacZ luxM luxS cqsA_Vh). β-Galactosidase units are reported as (V_max × dilution factor)/OD₆₀₀.

FIG. 7.

V. harveyi single, double, and triple autoinducer sensor mutant phenotypes. Density-dependent bioluminescence assays were performed on (A) BB120 (WT), BB721 (luxO), BB170 (luxN), BB886 (luxQ), and JMH598 (cqsS_Vh); (B) BB120 (WT), BB721 (luxO), JMH612 (luxQ cqsS_Vh), JMH597 (luxN cqsS_Vh), JMH625 (luxN luxQ), and JMH626 (luxN luxQ cqsA_Vh); and (C) BB120 (WT), BB721 (luxO), JMH625 (luxN luxQ), and JMH628 (luxN luxQ cqsS_Vh). Relative light units are reported as counts min⁻¹ ml⁻¹ × 1,000/CFU ml⁻¹. In panels A and B, cells were diluted 1:5,000, and in panel C, cells were diluted 1:500,000 prior to the start of the experiment.