Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes

The moderate halophile Halomonas elongata Deustche Sammlung fur 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 at NaCl, and CHR63 did not grow above 1.5 M NaCl. These mutants did not synthesize ectoine but accumulated ectoine precursors, as shown by C-13 NMR and mass spectroscopy, Mutant CHR62 accumulated low levels of diaminobutyric acid, and mutant CHR63 accumulated high concentrations of N-gamma-acetyl-diaminobutyric 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-gamma-acetyldiaminobutyric acid, This is the first evidence for the role of N-gamma-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.