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Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine.

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.

SUBMITTER: Gregory GJ

PROVIDER: S-EPMC6821967 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine.

Gregory Gwendolyn J GJ Morreale Daniel P DP Carpenter Megan R MR Kalburge Sai S SS Boyd E Fidelma EF

Applied and environmental microbiology 20191030 22

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 d ...[more]

PMID: 31519665

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Project description:BackgroundVibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2-4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood.Methodology/principal findingsThe bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2-4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promoter analysis indicated the above regulatory circuits were shared by several other closely related Vibrios but with slight exceptions.Conclusions/significanceThis study gave a comprehensive computational and characterization of the direct transcriptional regulation of five target genes, opaR, qrr2-4 and ahpA, by OpaR in V. parahaemolyticus. These characterized regulatory circuits were conserved in V. harveyi and V. parahaemolyticus.

Project description:By using natural-abundance (13)C-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography (HPLC) analysis we have investigated the types of compatible solutes that are synthesized de novo in a variety of Bacillus species under high-osmolality growth conditions. Five different patterns of compatible solute production were found among the 13 Bacillus species we studied. Bacillus subtilis, B. licheniformis, and B. megaterium produced proline; B. cereus, B. circulans, B. thuringiensis, Paenibacillus polymyxa, and Aneurinibacillus aneurinilyticus synthesized glutamate; B. alcalophilus, B. psychrophilus, and B. pasteurii synthesized ectoine; and Salibacillus (formerly Bacillus) salexigens produced both ectoine and hydroxyectoine, whereas Virgibacillus (formerly Bacillus) pantothenticus synthesized both ectoine and proline. Hence, the ability to produce the tetrahydropyrimidine ectoine under hyperosmotic growth conditions is widespread within the genus Bacillus and closely related taxa. To study ectoine biosynthesis within the group of Bacillus species in greater detail, we focused on B. pasteurii. We cloned and sequenced its ectoine biosynthetic genes (ectABC). The ectABC genes encode the diaminobutyric acid acetyltransferase (EctA), the diaminobutyric acid aminotransferase (EctB), and the ectoine synthase (EctC). Together these proteins constitute the ectoine biosynthetic pathway, and their heterologous expression in B. subtilis led to the production of ectoine. Northern blot analysis demonstrated that the ectABC genes are genetically organized as an operon whose expression is strongly enhanced when the osmolality of the growth medium is raised. Primer extension analysis allowed us to pinpoint the osmoregulated promoter of the B. pasteurii ectABC gene cluster. HPLC analysis of osmotically challenged B. pasteurii cells revealed that ectoine production within this bacterium is finely tuned and closely correlated with the osmolality of the growth medium. These observations together with the osmotic control of ectABC transcription suggest that the de novo synthesis of ectoine is an important facet in the cellular adaptation of B. pasteurii to high-osmolarity surroundings.

Project description:BackgroundCompatible solutes are natural substances that are known to stabilize cellular functions. Preliminary ex vivo and in vivo studies demonstrated that the compatible solute ectoine restores natural apoptosis rates of lung neutrophils and contributes to the resolution of lung inflammation. Due to the low toxicity and known compatibility of the substance, an inhalative application as an intervention strategy for humans suffering from diseases caused by neutrophilic inflammation, like COPD, had been suggested. As a first approach to test the feasibility and efficacy of such a treatment, we performed a population-based randomized trial.ObjectiveThe objective of the study was to test whether the daily inhalation of the registered ectoine-containing medical device (Ectoin® inhalation solution) leads to a reduction of neutrophilic cells and interleukin-8 (IL-8) levels in the sputum of persons with mild symptoms of airway disease due to lifelong exposure to environmental air pollution.MethodsA double-blinded placebo-controlled trial was performed to study the efficacy and safety of an ectoine-containing therapeutic. Prior to and after both inhalation periods, lung function, inflammatory parameters in sputum, serum markers, and quality-of-life parameters were determined.ResultsWhile the other outcomes revealed no significant effects, sputum parameters were changed by the intervention. Nitrogen oxides (nitrate and nitrite) were significantly reduced after ectoine inhalation with a mean quotient of 0.65 (95% confidence interval 0.45-0.93). Extended analyses considering period effects revealed that the percentage of neutrophils in sputum was significantly lower after ectoine inhalation than in the placebo group (P=0.035) even after the washout phase.ConclusionThe current study is the first human trial in which the effects of inhaled ectoine on neutrophilic lung inflammation were investigated. Besides demonstrating beneficial effects on inflammatory sputum parameters, the study proves the feasibility of the therapeutic approach in an aged study group.

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Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine.

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Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine.

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