Isolation and Characterization of Salt-sensitive Mutants of the Moderate Halophile Halomonas elongata and Cloning of the Ectoine Synthesis Genes

Abstract

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.

Osmotically Regulated Synthesis of the Compatible Solute Ectoine in Bacillus pasteurii and Related Bacillus spp

“…The five glutamate-accumulating Bacillus species are the most salt-sensitive species that we have studied (Table 1); this suggests that proline and ectoine synthesis is a more effective osmoadaptive measure than glutamate synthesis alone. Indeed, disruption of the osmoregulatory proline biosynthetic pathway in B. subtilis (J. Brill and E. Bremer, unpublished results) or of the ectoine biosynthetic genes in the moderate halophilic bacteria C. salexigens and H. elongata (12,22) strongly impairs the ability of these microorganisms to cope effectively with hyperosmotic growth conditions. Ectoine was originally discovered as a compatible solute in the extremely halophilic phototropic sulfurbacterium Ectothiorhodospira halochloris (19).…”

Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

“…Like ectoine, 5-hydroxyectoine serves as a compatible solute in vivo, functions as an osmoprotectant, and exhibits protein stabilizing properties in vitro (22)(23)(24)(25). The ability of a given microorganism to synthesize 5-hydroxyectoine invariably depends on its ability to produce ectoine, suggesting that 5-hydroxyectoine formation occurs either directly from ectoine (26,27) or from one of its biosynthetic intermediates (17). The most straightforward route to produce 5-hydroxyectoine would be the direct hydroxylation of ectoine via a substrate-specific hydroxylase (Fig.…”

“…Disruption of these genes in Halomonas elongata, Chromohalobacter salexigens, or Vibrio cholerae results in the loss of the ectoine biosynthetic capacity (17)(18)(19). Ectoine biosynthesis is mediated by a three-step enzymatic reaction (16,20) that converts the precursor L-aspartate-␤-semialdehyde, an intermediate in amino acid metabolism, into ectoine; L-2,4-diaminobutyrate and N ␥ -acetyl-L2,4-diaminobutyrate are the intermediates in this process (Fig.…”

The ectD Gene, Which Is Involved in the Synthesis of the Compatible Solute Hydroxyectoine, Is Essential for Thermoprotection of the Halophilic Bacterium Chromohalobacter salexigens

“…On the other hand, its temperature range in complex SW-10 medium is 15 to 45°C, with optimal growth at 37°C (2). Osmoadaptation is mainly achieved by de novo synthesis of ectoine and hydroxyectoine (13). In addition, C. salexigens accumulates externally supplied osmoprotectants, such as glycine betaine, which is taken up from the medium or synthesized from choline (11,12).…”

“…Ectoine was first discovered in the extremely halophilic phototrophic sulfobacterium Ectothiorhodospira halochloris (17), whereas hydroxyectoine was originally discovered in the actinomycin D producer Streptomyces parvulus (28). The ectABC gene cluster involved in the synthesis of ectoine has been isolated and characterized for a number of strains belonging to the above bacterial groups (10,13,21,22,32,34,46). In addition, the ectoine hydroxylase gene (thpD), responsible for the conversion of ectoine to hydroxyectoine, has been described for the actinomycin D producer Streptomyces chrysomallus as forming part of the thpABCD (ectABCD) gene cluster (22).…”