Biochemical and molecular characterization of the biosynthesis of glutamine and glutamate, two major compatible solutes in the moderately halophilic bacterium Halobacillus halophilus

Abstract

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.

FIG. 1.

Cellular concentrations of glutamate and glutamine in H. halophilus at different salinities. Cells were grown in glucose mineral salt medium. Compatible solutes were extracted and analyzed by high-performance liquid chromatography. Glutamate concentrations are shown by gray bars, and glutamine concentrations are shown by white bars.

FIG. 2.

Genetic organization of genes potentially involved in the biosynthesis of glutamine and glutamate in H. halophilus. (A) Glutamate dehydrogenase (gdh). Two ORFs encoding a putative glutamate dehydrogenase (gdh-1 and gdh-2) could be identified. The other ORFs shown encode the following proteins: orf1, l-asparaginase; orf2, pyridine nucleotide-disulfide oxidoreductase family protein (thioredoxin reductase); orf3, d-alanyl-d-alanine ligase A; orf4, negative regulator of genetic competence; orf5, unknown; orf6, enoyl-(acyl carrier protein) reductase; and orf7, unknown. (B) Glutamate synthase (glt). The glutamate synthase is a heterodimer encoded by gltA (large subunit) and gltB (small subunit). In the genome of H. halophilus, only one ORF could be identified encoding GltA, whereas two ORFs were found encoding GltB (gltB1 and gltB2). gltB1 and gltA are organized in an operon (data not shown). The other ORFs shown encode the following proteins: orf1, catalase; orf2, l-aminopeptidase/d-esterase; orf3, dihydropyrimidine dehydrogenase; and orf4, beta-alanine synthase. (C) Glutamine synthetase (glnA). The ORFs shown encode the following proteins: orf1, hypothetical conserved protein; glnR, transcriptional regulator; orf2, aluminum resistance protein; orf3, GTP-binding protein; orf4, spore formation protein; orf5, ABC transporter protein; orf6, oxidoreductase; orf7, putative dehydrogenase; orf8, conserved protein; orf9, conserved hypothetical protein/GTPase of unknown function; and orf10, hypothetical protein.

FIG. 3.

Glutamine synthetase activity in H. halophilus cells increases with increasing salinity of the medium. Cells were harvested in the late exponential growth phase, washed in an isosmolar KCl solution, and resuspended in a 1.5 M KCl solution.

FIG. 4.

Induction of glutamine synthetase activity in H. halophilus is stimulated by chloride. Cells were grown with 1 M NaCl, sodium gluconate (Na-gluc), or NaNO₃, harvested in the late exponential growth phase, washed in an iso-osmolar KCl solution, and resuspended in 1.5 M KCl.

FIG. 5.

Glutamine synthetase activity in H. halophilus is dependent on chloride. Cells were cultivated in the presence of 2.5 M NaCl, harvested in the late exponential growth phase, washed in isosmolar KCl solution, and resuspended in 1.5 M KCl. Different salts were used in the assay buffer at a final concentration of 1.5 M, whereas the final concentration of KCl was 188 mM, originating from the cell suspension.

FIG. 6.

Expression of glnA2 but not of glnA1 is salt dependent. H. halophilus cells were cultivated at the indicated salinities. Total RNA was isolated and transcribed into cDNA, and relative transcription levels were measured using real-time PCR analysis. In the diagram, relative quantitation of transcript levels is given. All values were compared to the value at 0.4 M, which was defined as 1. The experiment was repeated in three independent parallels to ensure statistical relevance.

FIG. 7.

Expression of glnA2 is stimulated by chloride. H. halophilus was cultivated in glucose mineral salt medium in the presence of NaCl, sodium gluconate (Na-gluc), or NaNO₃ at a final concentration of 1.0 M. Total RNA was isolated and transcribed into cDNA, and relative transcription levels were measured using real-time PCR analysis. In the diagram, relative quantitation of transcript levels is given. All values were compared to the value for NaCl, which was defined as 1. The experiment was repeated in three independent parallels to ensure statistical relevance.