Molecular mechanisms of the salt tolerance response in the halophile Vibrio parahaemolyticus

Ongagna Yhombi, Yvon Serge
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University of Delaware
In order to investigate the functionality of specialized genomic features in the osmotic stress tolerance of Vibrio parahaemolyticus , we divided the research project in three parts: (1) Examine the role of the ectoine and glycine betaine biosynthesis systems in the NaCl stress response, (2) Determine the role of the four putative Betaine Carnitine Choline transporter (BCCT) homologues in the NaCl stress response, and (3) Determine the role of two ProXWV (ProU1 and ProU2) homologues in the NaCl stress response. The role of the two synthesis systems in the NaCl stress response. We demonstrated that V. parahaemolyticus had an extended salt tolerance range up to 10.5% NaCl in complex media and a 6% NaCl tolerance in defined M9 minimal media. We demonstrated that exogenously supplied compatible solutes or their precursors in defined media relieved the growth constraint caused by high NaCl. By using proton Nuclear Magnetic Resonance spectroscopy (1 HNMR) analysis, we determined for the first time the profile of compatible solutes synthesized and accumulated by this organism. Furthermore, we showed that V. parahaemolyticus cells could perform de novo synthesis of ectoine and glutamate in a NaCl dependent manner. By comparative growth analysis in defined media amended to high osmolarity containing exogenously supplied compatible solutes or their precursors, we ranked the most effective compatible solutes in V. parahaemolyticus in the following order: glycine betaine, choline, proline, glutamate, and ectoine. Interestingly, we showed that V. parahaemolyticus could use glutamate or proline as a sole carbon source, but not ectoine and glycine betaine. By expression analysis of the ectoine (ectA ) and glycine betaine ( betA ) biosynthesis genes we determined that both systems were constitutively expressed at a basal level, but upregulated upon NaCl upshock. To examine the essentiality of the biosynthesis systems in V. parahaemolyticus at high osmolarity, comparative growth analysis was performed between V. parahaemolyticus wild type (WT) and mutants harboring deletion mutations in the betaine synthesis (Δ betA ), the ectoine synthesis (ΔectB ), and both systems (ΔbetAectB ). Growth in media of high osmolarity lacking compatible solutes revealed that Δ ectB and deltabetAectB mutant strains were defective compared to the WT and ΔbetA mutant. Furthermore, this result indicated that the ectoine synthesis was critical for growth at high osmolarity. Interestingly, the ΔbetA mutant strain could not grow in high osmolarity media supplied with choline. Irrespective of the mutant strains, growth at high osmolarity was rescued to the wild type level in the presence of glycine betaine. Finally, we performed a phylogenetic survey of the synthesis systems among the members of the genus Vibrio and showed the predominance of both systems in these species. The role of the four putative BCCT homologues in the NaCl stress response. To test the hypothesis that the four Betaine Carnitine Choline Transporters (BCCTs) are functional, we examined the role of these systems at high osmolarity. Expression analysis of the four BCCTs subjected to NaCl upshock showed that three of the BCCTs, VP1456, VP1905 and VPA0356 were induced. We constructed in frame single deletion mutations in all four BCCTs and revealed a behavior similar to wild type, demonstrating redundancy of the systems. However, a triple mutant deltaVP1456VP1723VP1905 was defective in glycine betaine and ectoine uptake. Using Escherichia coli MHK13, a glycine betaine synthesis and compatible solute transporter negative strain, we examined the transport specificity of each BCCT in this background. All four BCCTs could transport glycine betaine, but VP1456 had the most diverse substrate transport ability, uptaking glycine betaine, proline, ectoine, and choline. The role of two ProU homologues in the NaCl stress response. The two ProU transporters identified in V. parahaemolyticus are predicted from bioinformatics to function as compatible solutes transporters [3]. A proU1 mutant was previously constructed and showed no distinguishable phenotype compared to the wild type under high osmolarity [3]. To further understand the role of these systems, we investigated the roles of the ProU in the osmotic stress tolerance of V. parahaemolyticus. We showed that these systems were transcriptionally induced by NaCl upshock. We created several strains carrying deletion mutations in the ProU systems and showed that the ΔproU1ΔproU2 double mutant strain exhibited significant growth defect at high osmolarity relative to the wild type strain. Furthermore, we demonstrated that the ability to uptake glycine betaine was retained in both wild type and double proU mutant strain at high osmolarity, suggesting that the ProU transport systems are not essential for the uptake of glycine betaine. Growth in the presence of choline, ectoine, and proline resulted in a slighter reduction in the lag phase in the double proU mutant compared to the wild type strain. (Abstract shortened by UMI.)