NhaR, LeuO, and H-NS Are Part of an Expanded Regulatory Network for Ectoine Biosynthesis Expression.

Bacteria accumulate compatible solutes to maintain cellular turgor pressure when exposed to high salinity. In the marine halophile Vibrio parahaemolyticus, the compatible solute ectoine is biosynthesized de novo , which is energetically more costly than uptake; therefore, tight regulation is required. To uncover novel regulators of the ectoine biosynthesis ectABC-asp_ect operon, a DNA affinity pulldown of proteins interacting with the ectABC-asp_ect regulatory region was performed. Mass spectrometry analysis identified, among others, 3 regulators: LeuO, NhaR, and the nucleoid associated protein H-NS. In-frame non-polar deletions were made for each gene and P ectA - gfp promoter reporter assays were performed in exponential and stationary phase cells. P ectA -gfp expression was significantly repressed in the Δ leuO mutant and significantly induced in the Δ nhaR mutant compared to wild type, suggesting positive and negative regulation, respectively. In the Δ hns mutant, P ectA -gfp showed increased expression in exponential phase cells, but no change compared to wild type in stationary phase cells. To examine whether H-NS interacts with LeuO or NhaR at the ectoine regulatory region, double deletion mutants were created. In a Δ leuO/ Δ hns mutant, P ectA -gfp showed reduced expression, but significantly more than Δ leuO , suggesting H-NS and LeuO interact to regulate ectoine expression. However, Δ nhaR/ Δ hns had no additional effect compared to Δ nhaR , suggesting NhaR regulation is independent of H-NS. To examine leuO regulation further, a P leuO -gfp reporter analysis was examined that showed significantly increased expression in the Δ leuO , Δ hns , and Δ leuO/ Δ hns mutants compared to wild type, indicating both are repressors. Growth pattern analysis of the mutants in M9G 6%NaCl showed growth defects compared to wild type, indicating that these regulators play an important physiological role in salinity stress tolerance outside of regulating ectoine biosynthesis gene expression. IMPORTANCE Ectoine is a commercially used compatible solute that acts as a biomolecule stabilizer because of its additional role as a chemical chaperone. A better understanding of how the ectoine biosynthetic pathway is regulated in natural bacterial producers can be used to increase efficient industrial production. The de novo biosynthesis of ectoine is essential for bacteria to survive osmotic stress when exogenous compatible solutes are absent. This study identified LeuO as a positive regulator and NhaR as a negative regulator of ectoine biosynthesis and showed that, similar to enteric species, LeuO is an anti-silencer of H-NS. In addition, defects in growth in high salinity among all the mutants suggest that these regulators play a broader role in the osmotic stress response beyond ectoine biosynthesis regulation.