Quorum sensing regulates the osmotic stress response in Vibrio harveyi

Abstract

Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress. Here we show that LuxR activates expression of the glycine betaine operon betIBA-proXWV, which enhances growth recovery under osmotic stress conditions. BetI, an autorepressor of the V. harveyi betIBA-proXWV operon, activates the expression of genes encoding regulatory small RNAs that control quorum-sensing transitions. Connecting quorum-sensing and glycine betaine pathways presumably enables V. harveyi to tune its execution of collective behaviors to its tolerance to stress.

FIG 1

Model for quorum-sensing regulation of the betIBA-proXWV operon in V. harveyi. Phosphorylated LuxO (LuxO∼P) activates transcription of the genes encoding the Qrr sRNAs at LCD. The Qrr sRNAs repress the translation of AphA and activate the translation of LuxR. LuxR activates the transcription of the betIBA-proXWV operon. BetI activates expression of the genes encoding the Qrr sRNAs.

FIG 2

LuxR activates expression of betIBA-proXWV in V. harveyi. (A) Chromosomal regions encoding the bet and pro genes in E. coli and V. harveyi. V. harveyi gene numbers are indicated below each gene. The positions of the LuxR binding sites (bp −338 and −41, denoted LuxR BS1 and BS2, respectively) and BetI binding site (bp −276, denoted BetI BS) are indicated relative to the translation start site of betI. (B) Transcript levels of V. harveyi genes were determined by qRT-PCR from wild-type V. harveyi (BB120), ΔluxR V. harveyi (KM669), the ΔluxR strain containing a control plasmid (pJV036), and the ΔluxR strain carrying the plasmid expressing luxR (pluxR; pJV239). Error bars represent standard errors for three biological samples, and the data represent those from three independent experiments. (C) EMSAs for reaction mixtures containing 0, 10, 100, or 1,000 nM LuxR incubated with a radiolabeled DNA substrate corresponding to the predicted LuxR binding sites (BS1 or BS2) upstream of betI.

FIG 3

BetI represses the betIBA-proXWV operon. (A) The transcript levels of the betIBA-proXWV genes were assayed by qRT-PCR in the following V. harveyi strains induced with 1 mM IPTG: a ΔluxO strain carrying a control vector (BB721::pJV298) and a ΔluxO strain carrying a vector expressing betI from an IPTG-inducible promoter (BB721::pJV299). Error bars represent the standard errors of measurements from three biological replicates, and these data represent those from at least two independent experiments. (B, C) EMSAs for reaction mixtures containing 0, 250 nM, 500 nM, or 1,000 nM His-BetI incubated with a radiolabeled DNA substrate corresponding to the betI promoter region (PbetI; 359-bp promoter fragment, positioned immediately upstream of the start codon) or the recA ORF (300 bp) (B) or with the 45-bp fragment corresponding to the BetI binding site (BetI BS [see substrate C in Fig. S1C in the supplemental material], positioned 276 bp upstream of the start codon) (C).

FIG 4

BetI regulates quorum-sensing genes. The transcript levels of Qrr1 to Qrr5 in V. harveyi strains induced with 1 mM IPTG were assayed by qRT-PCR. The strains tested were a luxO D47E strain carrying a control vector (white bars; JAF548::pJV298) and a luxO D47E strain carrying a vector expressing betI from an IPTG-inducible promoter (black bars; JAF548::pJV299). Error bars represent the standard errors of measurement from three biological replicates, and these data represent those from two independent experiments.

FIG 5

Osmotic stress response in V. harveyi. (A) Wild-type V. harveyi (BB120) cultures were grown in LOM with 1 mM choline (Ch.) containing 0.2 M, 0.3 M, 0.5 M, or 1 M NaCl. (B) Wild-type V. harveyi (BB120) cultures were grown in LOM containing 0.2 M NaCl in the presence or absence of 1 mM choline. At 6.5 h, either 1 M NaCl or 0.2 M NaCl (final concentrations) was added to the cultures (designated with the arrow specifying salt shock). In panels A and B, data show the mean and standard error from three biological replicates and represent those from three independent experiments. (C) V. harveyi strains were grown in LOM with 1 mM choline and 1 M NaCl. The strains tested were wild-type V. harveyi carrying a control plasmid (BB120::pLAFR2), V. harveyi ΔbetI carrying a control vector (JC2212::pLAFR2), and V. harveyi ΔbetI carrying a plasmid encoding betIBA-proXWV (JC2212::pJV302). The data shown are the means and standard errors from three biological replicates and represent those from three independent experiments. (D, E) The transcript levels of betIBA-proXWV (D) and Qrr4, luxR, and luxC (E) were assayed by qRT-PCR 15 min following salt shock. Test cultures of BB120 received salt shock with 1 M NaCl, and control cultures received an equal volume of 0.2 M NaCl (final concentrations). The data shown are the means and standard errors from three biological replicates and represent those from three independent experiments.

FIG 6

Quorum sensing regulates the osmotic stress response in V. harveyi. (A, B) V. harveyi strains were grown in LOM with 1 mM choline and 0.2 M (A) or 1 M NaCl (B). The strains are tested were wild-type V. harveyi carrying a control plasmid (BB120::pLAFR2), V. harveyi ΔluxR carrying a control vector (KM669::pLAFR2), and V. harveyi ΔluxR carrying a plasmid encoding luxR (KM669::pKM699). The data shown are the means and standard errors from three biological replicates and represent those from at least three independent experiments.