Enzyme studies and salt sensitive mutant characterization in relation to the halotolerance of Halomonas elongata

The cytoplasmic enzyme alanine dehydrogenase (EC 1.4.1.1) and the extracellular enzymes alkaline and acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2, respectively) from Halomonas elongata were partially purified from cells grown in an L-alanine defined medium with NaCl at 0.05, 1.37 and 3.4 M. The activity profile for alanine dehydrogenase isolated from the three cell samples showed optimal activity at a NaCl concentration corresponding to the internal sodium concentration for each sample. Enzyme isolated from 0.05 M NaCl cells had highest activity at 0-20 mM NaCl; while enzyme isolated from 1.37 and 3.4 M NaCl cells had optimal activity at 340 and 500-600 mM NaCl respectively. Enzyme activity decreased when KCl or LiCl was substituted for NaCl. Polyacrylamide gel electrophoresis followed by histochemical staining revealed that 0.05 and 1.37 M NaCl cell extracts had two bands of activity (isozymes). Alkaline (AlP) and acid (AcP) phosphatases were cytochemically localized on the cell envelope. Enzyme assays were conducted at the pH 9.0 (AlP) and 5.0 (AcP) with varying concentrations of NaCl, KCl, or LiCl in the assay buffer. Results show higher acid phosphatase activity compared to that of alkaline phosphatase and all enzyme activities were optimal at NaCl concentrations similar to the medium NaCl concentrations in which the cells were grown. Enzyme activities decreased significantly (with exceptions) when KCl or LiCl was substituted for NaCl. Polyacrylamide gel electrophoresis followed by histochemical staining for the phosphatases showed only one band for both enzymes from each cell sample grown at different NaCl concentrations. Salt sensitive mutants of H. elongata were generated by frame shift mutagens and isolated by selecting for the inability to grow at high NaCl concentrations. Salt tolerance studies revealed that each mutant tested had the same salt tolerance profile. The salt tolerance of one mutant (Hm1) could be extended by the presence of large amounts of tryptophan and glycerol. Tryptophan pathway studies demonstrated that Hm1 had a salt sensitive conditional mutation in the trp operon, possibly in the trp B gene region. Transport experiments using ($\sp{14}$C) tryptophan and ($\sp{14}$C) aminoisobutyric acid showed that Hm1 had a diminished uptake of these compounds compared to the parent. (Abstract shortened with permission of author.