The synthesis of the compatible solute ectoine, mediated by the ectABC gene products, is the main mechanism used by the halophilic bacterium Chromohalobacter salexigens to cope with osmotic stress. Evidence was found that this process is regulated at the transcriptional level. S1 protection analyses performed with RNA extracted from cells grown in minimal medium at low (0?75 M NaCl) or high (2?5 M NaCl) osmolarity suggested the existence of four promoters upstream of ectA. Two of these (PectA1 and PectA2) might be recognized by the main vegetative sigma factor s 70 , and one (PectA3) might be dependent on the general stress sigma factor s S .The S1 protection assays suggest that PectA1 and PectA3 may be osmoregulated promoters. In addition, an internal promoter showing sequences homologous to promoters dependent on the heat-shock sigma factor s 32 was found upstream of ectB. Transcription from PectA in C. salexigens followed a pattern typical of s S -dependent promoters, and was reduced by 50 % in an E. coli rpoS background. These data strongly suggest the involvement of the general stress sigma factor s S in ectABC transcription in C. salexigens. Expression of PectA-lacZ and PectB-lacZ trancriptional fusions was very high at low salinity, suggesting that ectABC may be a partially constitutive system. Both transcriptional fusions were induced during continuous growth at high temperature and their expression was reduced in cells grown in the presence of osmoprotectants (ectoine or glycine betaine) or the DNA gyrase inhibitor nalidixic acid. Moreover, PectA-lacZ expression was negatively modulated in cells grown with an excess of iron (FeCl 3 ). Measurement of ectoine levels in the presence of glycine betaine at different NaCl concentrations suggests that an additional post-transcriptional control may occur as well.
International audienceAims: To investigate the catabolism of ectoine and hydroxyectoine, which are the major compatible solutes synthesized by Chromohalobacter salexigens. Methods and Results: Growth curves performed in M63 minimal medium with low (0·75 mol l-1 NaCl), optimal (1·5 mol l-1 NaCl) or high (2·5 mol l-1 NaCl) salinity revealed that betaine and ectoines were used as substrate for growth at optimal and high salt. Ectoine transport was maximal at optimal salinity, and showed 3- and 1·5-fold lower values at low and high salinity respectively. The salt-sensitive ectA mutant CHR62 showed an ectoine transport rate 6·8-fold higher than that of the wild type. Incubation of C. salexigens in a mixture of glucose and ectoine resulted in a biphasic growth pattern. However, CO2 production due to ectoine catabolism was lower, but not completely abolished, in the presence of glucose. When used as the sole carbon source, glycine betaine effectively inhibited ectoine and hydroxyectoine synthesis at any salinity. Conclusions: The catabolic pathways for ectoine and hydroxyectoine in C. salexigens operate at optimal and high (although less efficiently) salinity. Endogenous ectoine(s) may repress its own transport. Ectoine utilization was only partially repressed by glucose. Betaine, when used as carbon source, suppresses synthesis of ectoines even under high osmolarity conditions. Significance and Impact of the Study: This study is a previous step to the subsequent isolation and manipulation of the catabolic genes, so as to generate strains with enhanced production of ectoine and hydroxyectoin
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.
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