Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581

Abstract

The gene npvM encoding the calcium-dependent extracellular proteinase from Bacillus megatevium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HBlO1. The DNA sequence of the cloned 3.7 kb fragment revealed only one open reading frame consisting of 1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation start site (ATG). A possible promoter sequence (TAGACG for the -35 region and TATAAT for the -10 region) was found about 69 bp upstream of the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide 'pro' sequence of 221 amino acids preceding the putative mature protein of 317 amino acid residues. Amino acid sequence comparison revealed 84.5 % homology between the mature protein and that of a thermolabile neutral protease from B. cereus. It also shares 73% homology with the thermostable neutral proteases of B. thermoproteolyticus and B. stearothermophilus. The zinc-binding sites and the catalytic residues are completely conserved in all four proteases. NprM has a temperature optimum of 58 "C, a pH optimum of between 6.4 and 7-2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.

Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674

“…After staining with Coomassie brilliant blue R-250, protein bands were cut out of the membrane, and the N-terminal amino acid sequence was analyzed by the Edman degradation procedure with an Applied Biosystems model 476A protein/ peptide sequencer (Applied Biosystems Inc., U.S.A.). The N-terminal amino acid sequence of the 36-kDa protease was V-R-D-P-N-A-V-G-T-G-K-G-; this sequence shares a high degree of similarity with the N-terminal sequences of a neutral protease from B. cereus DSM 3101 and B. megaterium ATCC 14581 (Table 2) (Kühn and Fortnagel 1993). The thermolysin-like neutral protease of B. cereus DSM 3101 has 566 amino acid residues and contains a signal peptide of 27 amino acids, a pro-sequence of 222 amino acids, and a mature protein of 317 amino acids (Wetmore et al 1992), whereas the calcium-dependent exoproteinase of B. megaterium ATCC 14581 has 562 amino acid residues and contains a signal peptide of 24 amino acids, a pro-sequence of 221 amino acids, and a mature protein of 317 amino acids (Kühn and Fortnagel 1993).…”

“…The N-terminal amino acid sequence of the 36-kDa protease was V-R-D-P-N-A-V-G-T-G-K-G-; this sequence shares a high degree of similarity with the N-terminal sequences of a neutral protease from B. cereus DSM 3101 and B. megaterium ATCC 14581 (Table 2) (Kühn and Fortnagel 1993). The thermolysin-like neutral protease of B. cereus DSM 3101 has 566 amino acid residues and contains a signal peptide of 27 amino acids, a pro-sequence of 222 amino acids, and a mature protein of 317 amino acids (Wetmore et al 1992), whereas the calcium-dependent exoproteinase of B. megaterium ATCC 14581 has 562 amino acid residues and contains a signal peptide of 24 amino acids, a pro-sequence of 221 amino acids, and a mature protein of 317 amino acids (Kühn and Fortnagel 1993). The thermolysinlike neutral protease of B. cereus DSM 3101 shows the highest sequence similarity to the calcium-dependent exoproteinase of B. megaterium ATCC 14581, with 84.5% sequence similarity.…”

Microbial Genomics and the Periodic Table

“…Recent observations of bound Ca 2ϩ ions in the X-ray structure of E. coli transmembrane transporter MthK initially led to the suggestion that this was a Ca 2ϩ -gated K ϩ channel (56), but since affinities of MthK for Ca 2ϩ are in the millimolar range this conclusion is controversial (14). Historically, calcium has been considered a minor cation, largely involved in coordinating to some extracellular enzymes (66) and in specialized functions like sporulation (19). This perception has been changing.…”

Bacterial community analysis of Indonesian hot springs

“…Thermostable enzymes have been isolated from strains of B. megaterium (e.g. [28,29]), but it is not recorded as a hyperthermophilic species. Our isolates thus potentially represent new extremely thermophilic strains of Bacillus.…”