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

Abstract

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.

Regulators H-NS and LeuO inversely control swarming motility and biofilm formation inVibrio parahaemolyticus

“…We wanted to determine the role of LeuO and H-NS in controlling swarming motility and biofilm formation, mutually exclusive behaviors in V. parahaemolyticus . To accomplish this, in-frame non-polar deletion of hns (VP1133) and leuO (VP0350) and a double deletion mutant leuO/hns constructed using SOE PCR and allelic exchange were used (48). To ensure that none of the deletions in V. parahaemolyticus leads to a growth defect, growth was examined in LB broth with no growth differences observed among strains (data not shown).…”

“…LeuO in V. parahaemolyticus is positively regulated by the global regulator ToxR and is required for acid tolerance (47). Also, a recent study showed an epistatic relationship between LeuO and H-NS in the control of osmotic stress in this species (48).…”

Ectoine Globally Hypomethylates DNA in Skin Cells and Suppresses Cancer Proliferation

“…Among these, substantial and naturally produced compatible solutes/osmolytes, including some amino acids and their derivatives, have been proven to be critical in protecting organisms from increased environmental abiotic and biotic stresses [2]. In 1985, Galinski et al characterized the wellestablished flagship and novel amino acid derivative ectoine (1,4,5,6-tetrahydro-2-methyl-4pyrimidine carboxylic acid) osmolyte as a new component of present in marine microorganisms such as the extremely halophilic bacterial genus Ectothiorhodospira [3][4][5][6][7][8]. Ectoine binds strongly to water and forms a protective shield against the hydration of DNA, proteins, and other biomolecules [9,10].…”