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The halophilic bacterium Chromohalobacter salexigens synthesizes and accumulates compatible solutes in response to salt and temperature stress.13 C-nuclear magnetic resonance analysis of cells grown in minimal medium at the limiting temperature of 45°C revealed the presence of hydroxyectoine, ectoine, glutamate, trehalose (not present in cells grown at 37°C), and the ectoine precursor, N␥-acetyldiaminobutyric acid. High-performance liquid chromatography analyses showed that the levels of ectoine and hydroxyectoine were maximal during the stationary phase of growth. Accumulation of hydroxyectoine was up-regulated by salinity and temperature, whereas accumulation of ectoine was up-regulated by salinity and down-regulated by temperature. The ectD gene, which is involved in the conversion of ectoine to hydroxyectoine, was isolated as part of a DNA region that also contains a gene whose product belongs to the AraC-XylS family of transcriptional activators. Orthologs of ectD were found within the sequenced genomes of members of the proteobacteria, firmicutes, and actinobacteria, and their products were grouped into the ectoine hydroxylase subfamily, which was shown to belong to the superfamily of Fe(II)-and 2-oxoglutarate-dependent oxygenases. Analysis of the ectoine and hydroxyectoine contents of an ectABC ectD mutant strain fed with 1 mM ectoine or hydroxyectoine demonstrated that ectD is required for the main ectoine hydroxylase activity in C. salexigens. Although in minimal medium at 37°C the wild-type strain grew with 0.5 to 3.0 M NaCl, with optimal growth at 1.5 M NaCl, at 45°C it could not cope with the lowest (0.75 M NaCl) or the highest (3.0 M NaCl) salinity, and it grew optimally at 2.5 M NaCl. The ectD mutation caused a growth defect at 45°C in minimal medium with 1.5 to 2.5 M NaCl, but it did not affect growth at 37°C at any salinity tested. With 2.5 M NaCl, the ectD mutant synthesized 38% (at 37°C) and 15% (at 45°C) of the hydroxyectoine produced by the wild-type strain. All of these data reveal that hydroxyectoine synthesis mediated by the ectD gene is thermoregulated and essential for thermoprotection of C. salexigens.

Halophilic microorganisms are found as normal inhabitants of highly saline environments and thus are considered extremophiles. They are mainly represented, but not exclusively, by the halobacteria (extremely halophilic aerobic Archaea), the moderate halophiles (Bacteria and some methanogens) and several eukaryotic algae. These extremophilic microorganisms are already used for some biotechnological processes, for example halobacteria are used for the production of bacteriorhodopsin, and the alga Dunaliella is used in the commercial production of β-carotene. Several other present or potential applications of halophiles are reviewed, including the production of polymers (polyhydroxyalcanoates and polysaccharides), enzymes, and compatible solutes, and the use of these extremophiles in enhanced oil recovery, cancer detection, drug screening and the biodegradation of residues and toxic compounds.

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.

Background:Chromohalobacter salexigens synthesizes and accumulates ectoines. Results: High ratio of the anaplerotic and catabolic fluxes involved in ectoines synthesis supports high biosynthetic fluxes at high salinity and leads to metabolite overflow at low salinity. Conclusion: Evolution optimized the metabolism of C. salexigens to support high production of ectoines. Significance: Metabolic adaptations in a compatible solute-accumulating halophile are described for the first time.

Chromohalobacter salexigens, a Gammaproteobacterium belonging to the family Halomonadaceae, shows a broad salinity range for growth. Osmoprotection is achieved by the accumulation of compatible solutes either by transport (betaine, choline) or synthesis (mainly ectoine and hydroxyectoine). Ectoines can play additional roles as nutrients and, in the case of hydroxyectoine, in thermotolerance. A supplementary solute, trehalose, not present in cells grown at 37°C, is accumulated at higher temperatures, suggesting its involvement in the response to heat stress. Trehalose is also accumulated at 37°C in ectoine-deficient mutants, indicating that ectoines suppress trehalose synthesis in the wild-type strain. The genes for ectoine (ectABC) and hydroxyectoine (ectD, ectE) production are arranged in three different clusters within the C. salexigens chromosome. In order to cope with changing environment, C. salexigens regulates its cytoplasmic pool of ectoines by a number of mechanisms that we have started to elucidate. This is a highly complex process because (i) hydroxyectoine can be synthesized by other enzymes different to EctD (ii) ectoines can be catabolized to serve as nutrients, (iii) the involvement of several transcriptional regulators (σ S , σ 32 , Fur, EctR) and hence different signal transduction pathways, and (iv) the existence of post-trancriptional control mechanisms. In this review we summarize our present knowledge on the physiology and genetics of the processes allowing C. salexigens to cope with osmotic stress and high temperature, with emphasis on the transcriptional regulation.

The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K m ‫؍‬ 3.06 M and V max ‫؍‬ 9.96 nmol of betaine min ؊1 mg of protein ؊1). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13 C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium.

The moderate halophile Halomonas elongata Deustche Sammlung fü r Mikroorganismen 3043 accumulated ectoine, hydroxyectoine, glutamate, and glutamine in response to osmotic stress (3 M NaCl). Two Tn1732-induced mutants, CHR62 and CHR63, that were severely affected in their salt tolerance were isolated. Mutant CHR62 could not grow above 0.75 M NaCl, and CHR63 did not grow above 1.5 M NaCl. These mutants did not synthesize ectoine but accumulated ectoine precursors, as shown by 13 C NMR and mass spectroscopy. Mutant CHR62 accumulated low levels of diaminobutyric acid, and mutant CHR63 accumulated high concentrations of N-␥-acetyldiaminobutyric acid. These results suggest that strain CHR62 could be defective in the gene for diaminobutyric acid acetyltransferase (ectB), and strain CHR63 could be defective in the gene for the ectoine synthase (ectC). Salt sensitivity of the mutants at 1.5-2.5 M NaCl could be partially corrected by cytoplasmic extracts of the wild-type strain, containing ectoine, and salt sensitivity of strain CHR62 could be partially repaired by the addition of extracts of strain CHR63, which contained N-␥-acetyldiaminobutyric acid. This is the first evidence for the role of N-␥-acetyldiaminobutyric acid as osmoprotectant. Finally, a cosmid from the H. elongata genomic library was isolated which complemented the Ect ؊ phenotype of both mutants, indicating that it carried at least the genes ectB and ectC of the biosynthetic pathway of ectoine.