Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyper-osmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded known osmolytes used by V. parahaemolyticus to include N-N dimethylglycine (DMG) amongst others. We showed that V. parahaemolyticus requires a BCCT transporter for DMG uptake, carriers that were not known to transport DMG. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG, which was confirmed by functional complementation in E. coli strain MKH13. BccT1 was unusual in that it could uptake both compounds with methylated head groups (glycine betaine (GB), choline and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating residues for glycine betaine in BccT1. In silico modelling analysis demonstrated that glycine betaine, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four resides mutated resulted in loss of uptake of glycine betaine, DMG and ectoine. We showed three of the four residues were essential for ectoine uptake whereas only one of the residues was essential for glycine betaine uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for coordination of glycine betaine, DMG and ectoine transport.ImportanceVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high salinity conditions. In this study, we identified several novel osmolytes that are utilized by V. parahaemolyticus. We demonstrated that the compound dimethylglycine (DMG), which is abundant in the marine environment, is a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT-family carriers, which have not been shown previously to uptake this compound. BccT1 was a carrier for glycine betaine, DMG and ectoine and we identified the amino acid residues essential for coordination of these compounds. The data suggest that for BccT1, glycine betaine is more easily accommodated than ectoine in the transporter binding pocket.
Dimethylsulfoniopropionate (DMSP) is a key component of the global geochemical sulfur cycle that is a secondary metabolite produced in large quantities by marine phytoplankton and utilized as an osmoprotectant. Bacterial DMSP lyases convert DMSP to the climate active gas dimethylsulfide (DMS). Whether marine bacteria can also accumulate DMSP as an osmoprotectant to maintain the turgor pressure of the cell in response to changes in external osmolarity remains unknown. The marine halophile Vibrio parahaemolyticus, contains at least six osmolyte transporters, four betaine carnitine choline transport (BCCT) carriers BccT1-BccT4 and two ABC-family ProU transporters. In this study, we showed that DMSP is used as an osmoprotectant by V. parahaemolyticus and several other Vibrio species including V. cholerae and V. vulnificus. Using a V. parahaemolyticus proU double mutant, we demonstrated that these ABC transporters are not required for DMSP uptake. However, a bccT null mutant lacking all four BCCTs had a growth defect compared to wild type in high salt media supplemented with DMSP. Using bccT triple mutants, possessing only one functional BCCT, in growth pattern assays, we identified two BCCT-family transporters, BccT1 and BccT2 are carriers of DMSP. Vibrio cholerae and V. vulnificus, only contain a homolog of BccT3 and functional complementation in Escherichia coli MKH13 showed only V. cholerae BccT3 could transport DMSP. In V. vulnificus strains, we identified and characterized an additional BCCT transporter that was also a carrier for DMSP. Phylogenetic analysis uncovered at least 11 distinct BCCT transporters among members of the Harveyi clade, with some species having up to 9 BCCTs as exemplified by V. jasicida.ImportanceDMSP is present in the marine environment, produced in large quantities by marine phytoplankton as an osmoprotectant, and is an important component of the global geosulfur cycle. The bacterial family Vibrionaceae is comprised of marine species, many of which are halophiles such as V. parahaemolyticus, which can utilize a wide range of osmolytes and possesses at least six transporters for the uptake of these compounds. Here, we demonstrated that V. parahaemolyticus and other Vibrio species can accumulate DMSP as an osmoprotectant and show that the BCCT family transporters were required. DMSP was transported by four different BCCT transporters; BccT1, BccT2, BccT3 and BccT5 depending on the species. Bioinformatics and phylogenetics demonstrated that Vibrio species contain a large number of BCCTs and that many of these are associated with different metabolic pathways.
Fis (Factor for Inversion Stimulation) is a global regulator that is highly expressed during exponential growth and undetectable in stationary growth. Quorum sensing (QS) is a global regulatory mechanism that controls gene expression in response to cell density and growth phase. In V. parahaemolyticus, a marine species and a significant human pathogen, the QS regulatory sRNAs, Qrr1 to Qrr5, negatively regulate the high cell density QS master regulator OpaR. OpaR is a positive regulator of capsule polysaccharide (CPS) formation required for biofilm formation and a repressor of swarming motility. In Vibrio parahaemolyticus, we showed, using genetics and DNA binding assays, that Fis bound directly to the regulatory regions of the qrr genes and was a positive regulator of these genes. In the Δfis mutant, opaR expression was induced and a robust CPS and biofilm was produced, while swarming motility was abolished. Expression analysis and promoter binding assays showed that Fis was a direct activator of both the lateral flagellum laf operon and the surface sensing scrABC operon, both required for swarming motility. In in vitro growth competition assays, Δfis was outcompeted by wild type in minimal media supplemented with intestinal mucus, and we showed that Fis directly modulated catabolism gene expression. In in vivo colonization competition assays, Δfis was outcompeted by wild type, indicating Fis is required for fitness. Overall, these data demonstrate a direct role for Fis in QS, motility, and metabolism in V. parahaemolyticus.IMPORTANCEIn this study, we examined the role of Fis in modulating expression of the five-quorum sensing regulatory sRNAs, qrr1 to qrr5, and showed that Fis is a direct positive regulator of QS, which oppositely controls CPS and swarming motility in V. parahaemolyticus. The Δfis deletion mutant was swarming defective due to a requirement for Fis in lateral flagella and surface sensing gene expression. Thus, Fis links QS and surface sensing to control swarming motility and, indirectly, CPS production. Fis was also required for cell metabolism, acting as a direct regulator of several carbon catabolism loci. Both in vitro and in vivo competition assays showed that the Δfis mutant had a significant defect compared to wild type. Overall, our data demonstrates that Fis plays a critical role in V. parahaemolyticus physiology that was previously unexamined.
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. 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). Most of these systems are induced in high salt. CosR, a MarR-type regulator, which is divergently transcribed from bcct3, was previously shown to be a direct repressor of ectABCasp_ect in Vibrio species. In this study, we investigated the role of CosR in glycine betaine biosynthesis and compatible solute transporter gene regulation. Expression analyses demonstrated that betIBAproXWV, bcct1, bcct3, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant shows induced expression of these systems in the mutant at low salinity compared to wild-type. DNA binding assays demonstrate that purified CosR binds directly to the regulatory region of each system. In Escherichia coli GFP reporter assays, we demonstrate that CosR directly represses transcription of betIBAproXWV, bcct3, and proVWX. Similar to V. harveyi, we show betIBAproXWV is positively regulated by the LuxR homolog OpaR. Bioinformatics analysis demonstrates that CosR is widespread within the genus, present in over 50 species. In several species, the cosR homolog was clustered with the betIBAproXWV operon, which again suggests the importance of this regulator in glycine betaine biosynthesis. Incidentally, in four Aliivibrio species that contain ectoine biosynthesis genes, we identified another MarR-type regulator, ectR, clustered with these genes, which suggests the presence of a novel ectoine regulator. Homologs of EctR in this genomic context were present in A. fischeri, A. finisterrensis, A. sifiae and A. wodanis.ImportanceVibrio 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 are repressed. However, the mechanism of this repression is not fully elucidated. CosR plays a major role in the repression of multiple compatible solute systems in V. parahaemolyticus as a direct negative regulator of ectoine and glycine betaine biosynthesis systems and four transporters. Homology analysis suggests that CosR functions in this manner in many other Vibrio species. In Aliivibrio species, we identified a new MarR family regulator EctR that clusters with the ectoine biosynthesis genes.
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