D. Canovas et al., ISOLATION AND CHARACTERIZATION OF SALT-SENSITIVE MUTANTS OF THE MODERATE HALOPHILE HALOMONAS-ELONGATA AND CLONING OF THE ECTOINE SYNTHESIS GENES, The Journal of biological chemistry, 272(41), 1997, pp. 25794-25801
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.