Biosynthesis of the Osmoprotectant Ectoine, but Not Glycine Betaine, Is Critical for Survival of Osmotically Stressed Vibrio parahaemolyticus Cells

Vibrio parahaemolyticus is a halophile present in marine and estuarine environments, ecosystems characterized by fluctuations in salinity and temperature. One strategy to thrive in such environments is the synthesis and/or uptake of compatible solutes. TheV. parahaemolyticus genome contains biosynthesis systems for both ectoine and glycine betaine, which are known to act as compatible solutes in other species. We showed thatV. parahaemolyticus had a 6% NaCl tolerance when grown in M9 minimal medium with 0.4% glucose (M9G) with a >5-h lag phase. By using1 H nuclear magnetic resonance spectroscopy (1 H-NMR) analysis, we determined that cells synthesized ectoine and glutamate in a NaCl-dependent manner. The most effective compatible solutes as measured by a reduction in lag-phase growth in M9G with 6% NaCl (M9G 6% NaCl) were in the order glycine betaine > choline > proline = glutamate > ectoine. However,V. parahaemolyticus could use glutamate or proline as the sole carbon source, but not ectoine or glycine betaine, which suggests that these are bona fide compatible solutes. Expression analysis showed that theectA andbetA genes were more highly expressed in log-phase cells, and expression of both genes was induced by NaCl up-shock. Under all conditions examined, theectA gene was more highly expressed than thebetA gene. Analysis of in-frame deletions inbetA andectB and in a double mutant showed that theectB mutant was defective for growth, and this defect was rescued by the addition of glycine betaine, proline, ectoine, and glutamate, indicating that these compounds are compatible solutes for this species. The presence of both synthesis systems was the predominant distribution pattern among members of theVibrionaceae family, suggesting this is the ancestral state.