Cloning and expression of the secA gene of a marine bacterium, Vibrio alginolyticus, and analysis of its function in Escherichia coli.
We report the cloning, sequencing and functional characterization of the secA gene of a marine bacterium, Vibrio alginolyticus, which has been suggested to utilize ATP and the sodium motive force for protein translocation. Oligodeoxynucleotides corresponding to highly conserved regions of Escherichia coli secA located in the high affinity ATP binding site were utilized as PCR primers to clone the secA gene of V. alginolyticus. It was shown to encode a 103.3-kDa protein. The deduced amino acid sequence of V. alginolyticus SecA (VaSecA) exhibits a high degree of identity (72.7%) to SecA of E. coli (EcSecA). The secA gene of E. coli forms an operon with upstream orfX, whereas no counterpart is present upstream of V. alginolyticus secA. Azide derepresses the EcSecA translation, whereas the level of VaSecA was unaffected by azide. Expression of VaSecA in E. coli carrying a temperature-sensitive secA mutation restored both growth and protein translocation at a non-permissive temperature. VaSecA was thus able to substitute for EcSecA despite the fact that the energy requirement for protein translocation differs between the two organisms. VaSecA was overproduced in V. alginolyticus and purified to homogeneity for N-terminal sequencing. The endogenous ATPase activity of the purified VaSecA was comparable with that of EcSecA. 1998 Elsevier Science B.V.
Kunioka
E
E
Institute of Molecular, Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Matsuyama
S
S
Tokuda
H
H
D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019810:Sodium Azide / Q000494:pharmacology; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D019810:Sodium Azide; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins