The complete nucleotide sequence of the glutamine synthetase gene (glnA) of Bacillus subtilis

Abstract

The glutamine synthetase (GS) gene from Bacillus subtilis PCI 219 was cloned in Escherichia coli using the vector pBR329. A plasmid, pSGS2, was isolated from a glnA+ transformant and the cloned GS gene was found to be located in a 3.6 kb DNA fragment. The nucleotide sequence of a 1.8 kb segment encoding the GS was determined. This segment showed an open reading frame which would encode a polypeptide of 444 amino acids. The amino acid sequence of this GS gene product has higher homology with that of the Clostridium acetobutylicum GS than that of the E. coli GS.

Purification and characterization of glutamine synthetase from alkalophilic Bacillus No. 170.

“…The results shown in Fig. 1 are in good agreement with those of B. subtilis glutamine synthetase which was 444 amino acids long (50,205 Da), compared with 469 for E, coli (51,823 Da) deduced from the nucleotide sequence of the cloned gene (18,22). The molecular weight of alkalophilic Bacillus No.…”

“…5. Although the analysis was limited, the sequence of alkalophilic Bacillus glutamine synthetase was in agreement with the sequence from two strains of B, subtilis and B. cereus glutamine synthetase (16,18,22 ), and demonstrated high homology with the enzyme from Clostridium acetobutylicum (7). The sequence of Bacillus glutamine synthetase is not similar to those from the three other Grampositive bacteria, such as Streptomyces (25) and Mycobacterium (10), nor to those from Gram-negative bacteria.…”

“…In contrast, there is no evidence that the Bacillus subtilis glutamine synthetase is regulated by reversible adenylylation although it appears that the mechanisms involved are different from those in E. coli and other enterics. In earlier papers, we reported the isolation and characterization of the glutamine synthetase from both Bacillus cereus and Bacillus subtilis (12,13), the putative amino acid sequence from the DNA sequence of each gene (16,18), and the determination of the substrate binding sites by chemical modification (15,17,24) . To confirm the unique characteristic of the glutamine synthetase from these spore-forming bacteria, we have been investigating nitrogen metabolism in alkalophilic Bacillus.…”

“…Regulation by sulfhydryl group modification in vivo is difficult to establish, however Bacillus glutamine synthetases had a unique, reactive cysteine residue. The modified cysteine residue of B. cereus was Cys 306 (15) that is conserved in Bacillus glutamine synthetase (16,18,22).…”

Expression and Efficient Export of Enzymatically ActiveMycobacterium tuberculosis Glutamine Synthetase in Mycobacterium smegmatis and Evidence That the Information for Export is Contained within the Protein

“…1) and to E. coli glutamine synthetase residues 317-329 (NSYKRLVPGYEAP; antisense, 5Ј 3 3Ј: CGGSGCCTCGTAGCCSGGSACSAGSCGCTTGTA-SGAGTT) (primer 2), were designed based on our N-terminal amino acid sequence analysis of native glutamine synthetase and published DNA sequences of several bacterial glutamine synthetases (7). A polymerase chain reaction amplification product was obtained after 40 cycles at 94 to 37 to 72°C using these primers and M. tuberculosis Erdman genomic DNA as a template, cloned into pCR II (Invitrogen), verified by DNA sequencing across the vector/insert junctions with M13 and SP6 primers (pCR II kit, Invitrogen), and used as a probe for a Southern blot of M. tuberculosis DNA restricted with PstI.…”