title: Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study.
abstract: To analyse the proteome of Bacillus subtilis extracellular proteins, extracellular protein samples were prepared from culture media (minimal medium containing 0.4% glucose) of parental B. subtilis 168, a secA-temperature sensitive mutant and an ffh conditional mutant, and examined by two-dimensional gel electrophoresis. Approximately 100 to 110 spots were visualized in a gel of B. subtilis 168 extracellular proteins. Over 90% and 80% of these disappeared in the absence of SecA and Ffh, respectively. Thirty-eight obvious spots on the gel of the B. subtilis 168 preparation were selected and compared with spots obtained under SecA- or Ffh-deficient conditions. The appearance of 36 of these 38 spots depended on SecA and Ffh. Nineteen additional extracellular proteins were detected in cultures maintained in cellobiose, maltose and soluble starch. Among 23 proteins of which the N-terminal amino acid sequences were determined, 17 were extracellular proteins having signal peptides in their precursor form. Two membrane proteins, Yfnl and YflE, were cleaved behind 226Ala-Tyr-Ala228 and 213Ala-Leu-Ala215, respectively, and of which products seemed to be liberated into the culture medium. The production of Yfnl and YflE were also dependent on SecA and Ffh. These results indicate that most extracellular proteins target to and translocate across the cytoplasmic membrane by co-operation between the signal-recognition particle and Sec protein-secretion pathways. In contrast, a spot for Hag appeared independent from SecA and Ffh. Intracellular proteins Gap, SodA and KatA were identified in the extracellular protein samples. On the basis of these results and computer searches, it was predicted that B. subtilis produces 150 to 180 proteins extracellularly.
authors.affiliation: Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1.
authors.forename: Isao
authors.initials: I
authors.lastname: Hirose
authors.affiliation: Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Sano
authors.affiliation: Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1.
authors.forename: Izumi
authors.initials: I
authors.lastname: Shioda
authors.affiliation: Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kumano
authors.affiliation: Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1.
authors.forename: Kouji
authors.initials: K
authors.lastname: Nakamura
authors.affiliation: Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1.
authors.forename: Kunio
authors.initials: K
authors.lastname: Yamane
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D001483:Base Sequence; D002244:Carbon / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D003470:Culture Media; D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D020543:Proteome / Q000032:analysis / Q000737:chemistry*; D051381:Rats; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D018271:Signal Recognition Particle / Q000235:genetics / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D020543:Proteome; D000069816:SEC Translocation Channels; D018271:Signal Recognition Particle; D002244:Carbon; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins