ABSTRACT To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus , a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae , was found to repress ect expression in V. parahaemolyticus . In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR , while AphA is an indirect activator of cosR . Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness. IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus . We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR , which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.
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CosR is a global regulator of the osmotic stress response with widespread distribution among bacteria
Bacteria accumulate small, organic compounds, called compatible solutes, via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine ( ectABCasp_ect ) and glycine betaine ( betIBAproXWV ), four betaine-carnitine-choline transporters ( bcct1-bcct4 ) and a second ProU transporter ( proVWX). All of these systems are osmotically inducible with the exception of bcct2. Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABCasp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBAproXWV , bcct1 , bcct3 , bcct4 and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed expression of these systems is de-repressed in the mutant at low salinity compared to wild-type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli GFP reporter assays, we demonstrated that CosR directly represses transcription of betIBAproXWV , bcct3 , and proVWX . Similar to V. harveyi , we showed betIBAproXWV was directly activated by the quorum sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family and bioinformatics analysis showed widespread distribution among Gamma-Proteobacteria in general. Incidentally, in Aliivibrio fischeri, A. finisterrensis, A. sifiae and A. wodanis , an unrelated MarR-type regulator named ectR was clustered with ectABC-asp , which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria. IMPORTANCE Vibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gamma - Proteobacteria . Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.
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- Award ID(s):
- 1656688
- NSF-PAR ID:
- 10147663
- Date Published:
- Journal Name:
- Applied and Environmental Microbiology
- Volume:
- 86
- Issue:
- 10
- ISSN:
- 0099-2240
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/AEM.00120-20
