Investigations of dimethylglycine (DMG), glycine betaine and ectoine uptake by a BCCT family transporter with broad substrate specificity inVibriospecies

Abstract

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.

The organosulfur compound dimethylsulfoniopropionate (DMSP) is utilized as an osmoprotectant byVibriospecies

“…The V. parahaemolyticus ectB deletion mutant that cannot grow in high NaCl growth conditions in the absence of exogenous osmolytes, as was previously shown was used as a positive control (34,36). We examined growth of wild type and the ectB mutant in M9 minimal media supplemented with glucose and 6% NaCl (M9G 6%NaCl), in the presence and absence of DMSP.…”

“…We examined growth of wild type and the ectB mutant in M9 minimal media supplemented with glucose and 6% NaCl (M9G 6%NaCl), in the presence and absence of DMSP. Growth pattern analysis after 24 h showed that the lag phase of the wild-type strain without exogenous compatible solutes is approximately six hours with a growth rate of 0.042 h -1 , while the ΔectB mutant did not grow (36) (Fig. 1).…”

“…One of the key responses of Vibrio species to fluctuations in osmolarity is the accumulation of compatible solutes, either through biosynthesis of the osmolytes ectoine and glycine betaine or the rapid uptake of these osmolytes from the environment using as many as six osmolyte transporters (33)(34)(35). Vibrio parahaemolyticus, a halophile ubiquitous in the marine environment, biosynthesizes glycine betaine from exogenously supplied choline, and ectoine from aspartic acid de novo and can uptake at least 14 different compatible solutes (33)(34)(35)(36). This species contains two osmolyte ATP Binding Cassette (ABC)-family transporters, ProU1 (VP1726-VP1729) and ProU2 (VPA1112-VPA1114), and four Betaine Carnitine Choline Transporter (BCCT) family transporters, BccT1 (VP1456), BccT2 (VP1723), BccT3 (VP1905), and BccT4 (VPA0356) (33).…”

“…vulnificus, V. cholerae and Aliivibrio fischeri contain only a homolog of BccT3 (33). We have previously demonstrated that BCCTs can transport glycine betaine, choline, dimethylglycine (DMG), ectoine, and proline, with some redundancy in the substrates transported by each BCCT (35,36).…”