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Brun, Yves V. (Ed.)ABSTRACT Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic 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 the known osmolytes used by V. parahaemolyticus to include N , N -dimethylglycine (DMG), among others. Growth pattern analysis of four triple- bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB 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 the coordination of GB, DMG, and ectoine transport. IMPORTANCE Vibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus . We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.more » « less
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The marine diazotroph
Crocosphaera watsonii provides fixed carbon (C) and nitrogen (N) to open‐ocean regimes, where nutrient deficiency controls productivity. The growth ofCrocosphaera can be limited by low phosphorus (P) concentrations in these oligotrophic environments. Biomarkers such as the high‐affinityABC transporter phosphate‐binding gene,pstS , are commonly used to monitor when such organisms are under P stress; however, transcriptional regulation of these markers is often complex and not well‐understood. In this study, we interrogated changes inpstS transcript levels inC. watsonii cells under P starvation, and in response to added dissolved inorganic phosphorus (DIP ), dissolved organic phosphorus (DOP ), and changing light levels. We observed elevated relativepstS transcript levels inC. watsonii WH 8501 atDIP concentrations below 60 and above 20 nmol · L−1. Transcript levels were suppressed by both inorganic and bioavailable organic phosphorus; however, the P stress response was more sensitive toDIP thanDOP sources. Increasing light intensity resulted in increased relativepstS transcript abundances independently of low external P, and seemed to exacerbate the physiological effects of P stress. The variable response to different P compounds and rapid and transient influence of high light onpstS transcript abundances suggests thatpstS is an indicator of internal P status inCrocosphaera .
