Physiological response of the moderately halophilic psychrotolerant strain Chromohalobacter sp. N1 to salinity change and low temperature.

The available information on de novo synthesized compatible solutes in response to high medium salinity by bacteria of the Chromohalobacter genus is limited to studies of the mesophilic moderately halophilic strain Chromohalobacter salexigens DSM 3043T. Therefore, there is a need for studies of representatives of other species of the Chromohalobacter genus of the Halomonadaceae family. A moderately halophilic psychrotolerant bacterium, strain N1, closely related to the species Chromohalobacter japonicus was isolated from the salt crust of a rock salt waste pile in Berezniki, Perm Krai, Russia. An intracellular pool of compatible solutes of strain N1 was investigated by NMR spectroscopy. Cells grown in the presence of 5% NaCl at optimal growth temperature (28 °C) accumulated ectoine, glutamate, N(4)-acetyl-l-2,4-diaminobutyrate (NADA), alanine, trehalose, hydroxyectoine, and valine. Such a combination of compatible solutes is unique and distinguishes the strain from C. salexigens DSM 3043T. Hyperosmotic stress induced by 15% NaCl caused the accumulation of ectoine, NADA, and hydroxyectoine but led to a decrease in the amount of alanine, valine, and trehalose. The intracellular pool of glutamate was not significantly changed. A reduction of the growth temperature from 28 to 5 °C led to an increase in the amount of ectoine, NADA, trehalose, and hydroxyectoine. Ectoine was the major compatible solute.

Interplay between iron homeostasis and the osmotic stress response in the halophilic bacterium Chromohalobacter salexigens.

In this study, the connection between iron homeostasis and the osmostress response in the halophile Chromohalobacter salexigens was investigated. A decrease in the requirement for both iron and histidine and a lower level of siderophore synthesis were observed at high salinity, and these findings were correlated with a lower protein content in salt-stressed cells. A six-gene operon (cfuABC-fur-hisI-orf6 operon) located downstream of the ectABC ectoine synthesis genes was characterized. A fur strain (in which the ferric iron uptake regulator Fur was affected) had the Mn resistance phenotype typical of fur mutants, was deregulated for siderophore production, and displayed delayed growth under iron limitation conditions, indicating that fur encodes a functional iron regulator. hisI was essential for histidine synthesis, which in turn was necessary for siderophore production. Fur boxes were found in the promoters of the cfuABC-fur-hisI-orf6 and ectABC operons, suggesting that Fur directly interacts with DNA in these regions. Fur mediated the osmoregulated inhibition of cfuABC-fur-hisI-orf6 operon expression by iron and functioned as a positive regulator of the ectABC genes under high-salinity conditions, linking the salt stress response with iron homeostasis. Excess iron led to a higher cytoplasmic hydroxyectoine content, suggesting that hydroxyectoine protects against the oxidative stress caused by iron better than ectoine. This study provides the first evidence of involvement of the iron homeostasis regulator Fur as part of the complex circuit that controls the response to osmotic stress in halophilic bacteria.

Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens.

BACKGROUND: Osmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions. RESULTS: A C. salexigens Tn1732-induced mutant (CHR95) showed a delayed growth with glucose at low and optimal salinities, could not grow at high salinity, and was able to use ectoines as carbon sources at low salinity. CHR95 was affected in the transport and/or metabolism of glucose, and showed a deregulated ectoine uptake at any salinity, but it was not affected in ectoine metabolism. Transposon insertion in CHR95 caused deletion of three genes, Csal0865-Csal0867: acs, encoding an acetyl-CoA synthase, mntR, encoding a transcriptional regulator of the DtxR/MntR family, and eupR, encoding a putative two-component response regulator with a LuxR_C-like DNA-binding helix-turn-helix domain. A single mntR mutant was sensitive to manganese, suggesting that mntR encodes a manganese-dependent transcriptional regulator. Deletion of eupR led to salt-sensitivity and enabled the mutant strain to use ectoines as carbon source at low salinity. Domain analysis included EupR as a member of the NarL/FixJ family of two component response regulators. Finally, the protein encoded by Csal869, located three genes downstream of eupR was suggested to be the cognate histidine kinase of EupR. This protein was predicted to be a hybrid histidine kinase with one transmembrane and one cytoplasmic sensor domain. CONCLUSIONS: This work represents the first example of the involvement of a two-component response regulator in the osmoadaptation of a true halophilic bacterium. Our results pave the way to the elucidation of the signal transduction pathway involved in the control of ectoine transport in C. salexigens.

Genome-scale reconstruction of metabolic network for a halophilic extremophile, Chromohalobacter salexigens DSM 3043.

BACKGROUND: Chromohalobacter salexigens (formerly Halomonas elongata DSM 3043) is a halophilic extremophile with a very broad salinity range and is used as a model organism to elucidate prokaryotic osmoadaptation due to its strong euryhaline phenotype. RESULTS: C. salexigens DSM 3043's metabolism was reconstructed based on genomic, biochemical and physiological information via a non-automated but iterative process. This manually-curated reconstruction accounts for 584 genes, 1386 reactions, and 1411 metabolites. By using flux balance analysis, the model was extensively validated against literature data on the C. salexigens phenotypic features, the transport and use of different substrates for growth as well as against experimental observations on the uptake and accumulation of industrially important organic osmolytes, ectoine, betaine, and its precursor choline, which play important roles in the adaptive response to osmotic stress. CONCLUSIONS: This work presents the first comprehensive genome-scale metabolic model of a halophilic bacterium. Being a useful guide for identification and filling of knowledge gaps, the reconstructed metabolic network iOA584 will accelerate the research on halophilic bacteria towards application of systems biology approaches and design of metabolic engineering strategies.