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-aspk operon, a DNA affinity pulldown of proteins interacting with the ectABC-aspk regulatory region was performed. Mass spectrometry analysis identified, amongst others, five regulators: LeuO, NhaR, OmpR, TorR, and the nucleoid associated protein H-NS. To determine their role in ectoine biosynthesis, in-frame non-polar deletions were made for each gene and PectA-gfp promoter reporter assays were performed. PectA-gfp expression was significantly repressed in the ΔleuO mutant and significantly induced in the ΔnhaR mutant compared to wild type, suggesting positive and negative regulation, respectively. Both ΔompR and ΔtorR mutants also showed induced expression of PectA-gfp, but not to the same extent as ΔnhaR. The Δhns showed no change compared to wild type. To examine whether H-NS interacts with LeuO or NhaR at the ectoine regulatory region, double deletion mutants were created. In a ΔleuO/Δhns mutant, PectA-gfp showed reduced expression, but significantly more than ΔleuO suggesting H-NS and LeuO interact to regulate ectoine expression. Whereas ΔnhaR/Δhns had no additional effect as compared to ΔnhaR suggesting NhaR regulation is independent of H-NS. Next, a PleuO-gfp reporter analysis was performed in wild type ΔleuO, Δhns, ΔleuO/Δhns and ΔtoxR, which encodes ToxR a positive regulator of leuO. PleuO-gfp showed significantly increased expression in the ΔleuO, Δhns and ΔleuO/Δhns mutants as compared to wild type, indicating both are repressors of leuO, and ΔtoxR showed reduced expression. Growth pattern analysis of the ΔleuO, Δhns, and ΔleuO/Δhns mutants in M9G 6%NaCl all showed growth defects compared to wild type, with ΔleuO/Δhns showing the greatest effect. Overall, the data show that NhaR is a negative regulator and LeuO is a positive regulator of ectoine expression and suggest LeuO is an anti-silencer of H-NS. Importance Ectoine is a commercially used compatible solute that acts as a biomolecule stabilizer because of its additional role as a chemical chaperone. The de novo biosynthesis of ectoine is carried out by approximately 70% of Vibrionaceae species many of which are marine halophiles. This study identified LeuO as a positive regulator of ectoine biosynthesis and also showed that similar to enteric species, LeuO is an anti-silencer of the nucleoid associated protein H-NS. NhaR was a negative regulator of ectoine biosynthesis but growth analysis in high salinity showed that LeuO, NhaR and H-NS are required for optimal growth under these conditions. These data indicate that all three regulators also play a wider role in high NaCl tolerance.