Quorum sensing regulates type III secretion in Vibrio harveyi and Vibrio parahaemolyticus

Abstract

In a process known as quorum sensing, bacteria communicate with one another by producing, releasing, detecting, and responding to signal molecules called autoinducers. Vibrio harveyi, a marine pathogen, uses two parallel quorum-sensing circuits, each consisting of an autoinducer-sensor pair, to control the expression of genes required for bioluminescence and a number of other target genes. Genetic screens designed to discover autoinducer-regulated targets in V. harveyi have revealed genes encoding components of a putative type III secretion (TTS) system. Using transcriptional reporter fusions and TTS protein localization studies, we show that the TTS system is indeed functional in V. harveyi and that expression of the genes encoding the secretion machinery requires an intact quorum-sensing signal transduction cascade. The newly completed genome of the closely related marine bacterium Vibrio parahaemolyticus, which is a human pathogen, shows that it possesses the genes encoding both of the V. harveyi-like quorum-sensing signaling circuits and that it also has a TTS system similar to that of V. harveyi. We show that quorum sensing regulates TTS in V. parahaemolyticus. Previous reports connecting quorum sensing to TTS in enterohemorrhagic and enteropathogenic Escherichia coli show that quorum sensing activates TTS at high cell density. Surprisingly, we find that at high cell density (in the presence of autoinducers), quorum sensing represses TTS in V. harveyi and V. parahaemolyticus.

FIG. 1.

Quorum sensing in V. harveyi. V. harveyi has two parallel quorum-sensing systems that regulate bioluminescence (luxCDABE) and other target genes. System 1 is composed of an autoinducer, HAI-1 (pentagons), and its cognate sensor LuxN. System 2 includes the autoinducer AI-2 (diamonds) and the sensor LuxPQ. The HAI-1 and AI-2 synthases are LuxM and LuxS, respectively. Information from both sensors is transduced to LuxU, and LuxU transmits the signal to LuxO. LuxO indirectly represses luxCDABE expression through a putative repressor we call X that appears to act at the level of luxR expression. LuxR is a transcriptional activator required for expression of luxCDABE. LuxR also controls TTS gene expression; however, in this case the action of LuxR is negative. We do not know whether this activity is direct or indirect. Details of the phosphorelay mechanism are given in the text. H and D stand for histidine and aspartate, which are the sites of phosphorylation. HTH denotes helix-turn-helix.

FIG. 2.

The LuxN-LuxO circuit controls the HAI-1-regulated targets. β-Gal assays were performed to measure the activity from the fusion in JMH70 (vopN::mini-MulacZ) with the following in trans alleles: vector alone (JMH97), luxN D771A (JMH276), and luxO D47E (JMH127). Cultures were supplemented with 10% V. harveyi MM77 (HAI-1⁻, AI-2⁻) cell-free culture fluids (black bars) or V. harveyi MM30 (HAI-1⁺, AI-2⁻) cell-free culture fluids (white bars). All experiments were performed in triplicate. JMH70 produces wild-type levels of AI-2.

FIG. 3.

Organization of the TTS loci in V. harveyi and V. parahaemolyticus. The organizations of the loci encoding components of TTS systems in V. harveyi (accession number AY524044) and V. parahaemolyticus are shown. Transposon insertions obtained in V. harveyi in TTS system genes are denoted by black triangles and labeled as follows: KM114 (vopB::mini-MulacZ), JMH70 (vopN::mini-MulacZ), and KM96 (vscP::mini-MulacZ). Three homologous TTS system gene clusters found in both Vibrio species are indicated with black and gray shading and stripes and are aligned in the figure. Adjacent genes in V. parahaemolyticus that do not appear to encode TTS system components are shown with white boxes. Boxes indicating additional V. parahaemolyticus genes that encode proteins with homology to TTS functions that have not been identified in V. harveyi are marked with black dots. A horizontal arrow indicates the predicted direction of transcription of each operon.

FIG. 4.

Quorum-sensing regulation of TTS system genes in V. harveyi. The activity levels of the vopN::mini-MulacZ, vscP::mini-MulacZ, and vopB::mini-MulacZ fusions were measured in V. harveyi containing luxO D47E on the chromosome to simulate low cell density (black bars) and in the wild-type V. harveyi background at high cell density (white bars). Strains were grown 14 h in AB medium. Experiments were performed in triplicate.

FIG. 5.

The TTS protein VopD is produced and secreted under low-cell-density conditions. VopD present in whole-cell extracts (left panels) and concentrated cell-free culture fluids (right panels) was assayed by Western blotting using polyclonal antibodies directed against VopD. The strains of V. harveyi used in this analysis were grown in low-calcium, EGTA-containing medium and were as follows: BB120 (wild type), BB721 (luxO), JAF548 (luxO D47E), JAF633 (luxM), MM30 (luxS), MM77 (luxM, luxS), and KM357 (luxR). The nitrocellulose membranes were probed with anti-LuxS antibody as a control for leakage of cytoplasmic protein into the cell-free culture fluids. Gels containing identical samples of concentrated cell-free culture fluids were stained with Coomassie blue to visualize the BSA protein precipitation control.

FIG. 6.

Transposon insertions in genes encoding the TTS system abolish secretion but not production of VopD in V. harveyi. (A) Whole-cell extracts (left panels) and concentrated cell-free culture fluids (right panels) of V. harveyi TTS fusions in the luxO D47E strain background were analyzed by Western blotting to visualize VopD and LuxS protein. The strain designated the wild-type TTS system is JAF548 (luxO D47E). The mini-MulacZ (Mini-Mu) insertion strains in vscP, vopB, and vopN in the luxO D47E background are KM470, KM476, and JMH401, respectively. The Coomassie-stained portion of the gel containing BSA is shown as a control for protein loading and precipitation. (B) Coomassie-stained gel containing cell-free culture fluids used for the Western blot shown in panel A.

FIG. 7.

Quorum sensing controls TTS in V. parahaemolyticus. Whole-cell extracts (left panels) and concentrated cell-free culture fluids (right panels) prepared from the V. parahaemolyticus wild type (strain LM5312) and the opaR null mutant (LM4437) were analyzed by Western blotting and probed with antibodies to V. harveyi VopD and LuxS. As described for Fig. 5 and 6A, the BSA control is shown.