Biosynthesis And Uptake Of Ectoine And Hydroxyectoine In The Moderately Halophilic Bacterium Virgibacillus Halodenitrificans PDB-F2

A moderately halophilic bacterium, Virgibacillus halodenitrificans strain PDB-F2, was able to degrade phenol in high-salt environments. The strain has potential applications in high-salt wastewater treatment. The moderately halophilic bacteria usually cope with salinity by synthesizing or taking up compatible solutes. Therefore, the study of biosynthesis and uptake of the typical compatible solutes is important to clarify the salt-tolerance mechanisms of the strain PDB-F2.The main compatible solutes in this strain were ectoine and hydroxyectoine, as determined by’H nuclear magnetic resonance spectroscopy (’H-NMR). A cluster of ectoine biosynthesis genes including three genes in the order of ectA, ectB, and ectC, and the hydroxyectoine biosynthesis gene ectD were identified by polymerase chain reactions. The ectD gene was not part of the ectABC gene cluster. High-performance liquid chromatography (HPLC) and real-time polymerase chain reactions (RT-PCR) showed that ectoine and hydroxyectoine biosynthesis were salinity-dependent, and ectoine was the major solute in response to elevated salinity, while hydroxyectoine was a minor solute. The ectoine content and expression of ectABC reached a maximum at 12% NaCl, while hydroxyectoine content and ectD expression increased up to 15% NaCl. The hydroxyectoine/ectoine ratio increased from 0.04 at 3% NaCl to 0.45 at 15% NaCl, indicating that hydroxyectoine is preferably accumulated at high salinities. Ectoine and hydroxyectoine production were also growth phase-dependent.The hydroxyectoine/ectoine ratio increased from 0.018 in the early exponential phase to 0.11 in the stationary phase at 5% NaCl, indicating hydroxyectoine is preferably accumulated in the stationary phase. When the PDB-F2 strain was subjected to an osmotic upshock from 5 to 10% NaCl, the ectoine concentration increased steadily and reached a maximum 6 h after the upshock, but the hydroxyectoine concentration increased gradually after 8 h and reached a maximum 14 h after the upshock. The ectABC genes were also expressed first, followed by the ectD gene. Exogenously provided hydroxyectoine enhanced the maximum growth of the cells while ectoine only caused a reduction in lag phase. Hydroxyectoine was accumulated preferentially by PDB-F2 strain when both ectoine and hydroxyectoine were provided. Increased external salinity triggered ectoine or hydroxyectoine uptake.