In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.
The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.
Takamatsu
H
H
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Fuma
S
S
Nakamura
K
K
Sadaie
Y
Y
Shinkai
A
A
Matsuyama
S
S
Mizushima
S
S
Yamane
K
K
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005838:Genotype; D007700:Kinetics; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010957:Plasmids; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C075517:DIV+ protein, Bacillus subtilis; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000516:alpha-Amylases; D001618:beta-Lactamases; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins