Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus

Abstract

Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.

Keywords: Halobacillus halophilus; compatible solutes; glutamine synthetase; halophile; osmoregulation.

FIGURE 1

Cellular concentration of GlnA1 and GlnA2 in dependence of different salinities in the growth media. Cells of Halobacillus halophilus were cultivated in G10 minimal medium in the presence of varying NaCl concentrations (0.4–3 M). Samples were used to prepare cell-free extracts for SDS-Pages and Western blotting followed by densitometric analysis. All quantifications were carried out in duplicate using two independent cell cultures. A Coomassie blue-stained SDS-Page containing 20 μg protein per lane (A). Western blot analysis using specific antibodies against GlnA1 (B) and GlnA2 (C) showing the cellular concentration of GlnA1 (B) and GlnA2 (C), respectively, in dependence of salinities in the growth media. Averaged values of two independent Western blot analyses are given in the lower panel. The highest signal intensities were set to 100%.

FIGURE 2

Construction of pHHΔglnA2. For construction of the non-replicating plasmid pHHΔglnA2 for the deletion of glnA2 1.0 kbp regions upstream and downstream of glnA2 were amplified and merged by using fusion PCR to produce the fragment ΔglnA2 (A). After digestion with BamHI and XbaHI the ΔglnA2 fragment was cloned into the BamHI and XbaHI sites of pHHΔpro (B).

FIGURE 3

Genotype of Halobacillus halophilus ΔglnA2. PstI/BglI digested genomic DNA from Halobacillus halophilus wild type (lane 1) or Halobacillus halophilus ΔglnA2 (lane 2) was separated by gel electrophoresis, transferred to a nylon membrane and probed with specific DIG-labeled DNA fragments against one flanking region of the mutated glnA2 or the glnA2 gene. Numbers in the middle indicate the migration positions of standard DNA fragments.

FIGURE 4

Relative amounts of solutes in Halobacillus halophilus wild type and ΔglnA2 mutant. Cells were cultivated in G10 medium in the presence of 1.0, 2.0, or 3.0 M NaCl and harvested in the exponential growth phase. Compatible solutes were extracted and concentrations of glutamate (A), glutamine (B), proline (C), and ectoine (D) were determined for wild type (white) and ΔglnA2 mutant (gray) by HPLC. The presented relative quantification of solutes was conducted using the value of “wild type, 1.0 M NaCl” sample as a reference. The values represent the means and the standard deviations of the mean (S.D.s) of at least two physiologically independent parallels.

FIGURE 5

Cellular transcript levels of glnA1 (A), proH (B), and ectA (C) in Halobacillus halophilus wild type and ΔglnA2 mutant. Cellular transcript levels were detected in cells grown in G10 medium containing 1.0 (no fill), 2.0 (squares), or 3.0 (horizontal lines) M NaCl and harvested in the early exponential growth phase. The presented relative quantification of transcript levels was conducted using the value of “wild type, 1.0 M NaCl” sample as a reference. The experiment was repeated in two or three independent parallels to ensure statistical relevance.

FIGURE 6

Glutamine synthetase activity is salt-induced in Halobacillus halophilus wild type and ΔglnA2 mutant. Cells were grown in NB medium with 1 M NaCl, permeabilized and harvested in the late exponential growth phase. Cells were washed in an isoosmolar KCl solution, resuspended in 1.5 M KCl solution and exposed to the activity measurements, in which 0 (no fill), 0.5 (gray), 1.0 (squares), 2.0 (horizontal lines), and 3.0 (vertical lines) M KCl, respectively, were present. The values represent the means and the SEMs of three physiologically independent parallels.

FIGURE 7

Glutamine synthetase activities of purified GlnA1 and GlnA2. Halobacillus halophilus was grown in G10 medium containing 1.5 M NaCl to the late exponential growth phase at 30°C. GlnA1 and GlnA2 were purified from the cytoplasm by two PEG precipitation steps followed by an anion exchanger. The chromatography profile (A) showed six major peak fractions, of which fractions 2–4 mainly contained GlnA1 while GlnA2 eluted in fractions 5 and 6 as shown by Western blotting (B). Subsequently, GlnA1 and GlnA2 were enriched to higher purity by gelfiltrations of fraction 2–4 and 5–6, respectively (C). Glutamine synthetase activities were measured for purified GlnA1 at different KCl concentrations in the assay (D). GlnA2 has not shown any activity at the conditions tested.