NhaR, LeuO and H-NS are part of an expanded regulatory network for ectoine biosynthesis expression

Abstract

AbstractBacteria accumulate compatible solutes, to maintain cellular turgor pressure when exposed to high salinity. In the marine halophileVibrio parahaemolyticus,the compatible solute ectoine is biosynthesizedde novo, which is energetically more costly than uptake; therefore, tight regulation is required. To uncover novel regulators of the ectoine biosynthesisectABC-asp_ectoperon, a DNA affinity pulldown of proteins interacting with theectABC-asp_ectregulatory region was performed. Mass spectrometry analysis identified, amongst others, three regulators: LeuO, NhaR, and the nucleoid associated protein H-NS. In-frame non-polar deletions were made for each gene and PectA-gfppromoter reporter assays were performed in exponential and stationary phase cells. PectA-gfpexpression was significantly repressed in the ΔleuOmutant and significantly induced in the ΔnhaRmutant compared to wild type, suggesting positive and negative regulation, respectively. In the Δhnsmutant, PectA-gfpshowed 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 ΔleuO/Δhnsmutant, PectA-gfpshowed reduced expression, but significantly more than ΔleuOsuggesting H-NS and LeuO interact to regulate ectoine expression. Whereas ΔnhaR/Δhnshad no additional effect as compared to ΔnhaRsuggesting NhaR regulation is independent of H-NS. To examineleuOregulation further, a PleuO-gfpreporter analysis was examined that showed significantly increased expression in the ΔleuO, Δhnsand ΔleuO/Δhnsmutants as 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.ImportanceEctoine 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. Thede novobiosynthesis 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 also 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.