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 Mikroo rganismen 3043 accumulated ectoine, hydroxyectoine, glutamate, and glu tamine in response to osmotic stress (3 M NaCl), Two Tn1732-induced mu tants, CHR62 and CHR63, that were severely affected in their salt tole rance were isolated, Mutant CHR62 could not grow above 0.75 at NaCl, a nd CHR63 did not grow above 1.5 M NaCl. These mutants did not synthesi ze ectoine but accumulated ectoine precursors, as shown by C-13 NMR an d mass spectroscopy, Mutant CHR62 accumulated low levels of diaminobut yric acid, and mutant CHR63 accumulated high concentrations of N-gamma -acetyl-diaminobutyric acid, These results suggest that strain CHR62 c ould be defective in the gene for diaminobutyric acid acetyltransferas e (ectB), and strain CHR63 could be defective in the gene for the ecto ine 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 coul d be partially repaired by the addition of extracts of strain CHR63, w hich contained N-gamma-acetyldiaminobutyric acid, This is the first ev idence for the role of N-gamma-acetyldiaminobutyric acid as osmoprotec tant, Finally, a cosmid from the H, elongata genomic library was isola ted which complemented the Ect(-) phenotype of both mutants, indicatin g that it carried at least the genes ectB and ectC of the biosynthetic pathway of ectoine.