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Genomics |
Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1
Author for correspondence: Kunio Yamane. Tel: +81 298 53 6680. Fax: +81 298 53 6680. e-mail: kyamane{at}sakura.cc.tsukuba.ac.jp
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, YfnI 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 YfnI 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.
Keywords: Bacillus subtilis, extracellular proteins, proteome analysis, secA and ffh mutants, two-dimensional gel electrophoresis
Abbreviations: 2D, two dimensional; SRP, signal-recognition particle
The SWISS-PROT accession numbers for the N-terminal amino acid sequences reported in this paper are: P00691 for AmyE; P54507 for CotN; O07921 for Csn; P09124 for Gap; P26901 for KatA; P39116 for Pel; P39824 for PenP; P54375 for SodA; P29141 for Vpr; Q07833 for WapA; P54423 for WprA; P54327 for XkdG; Q45071 for XynD; P94421 for YclQ; O31803 for YcnM; O05512 for YdhT; O34952 for YflE; O06487 for YfnI; O31737 for YlqB; P96740 for YwtD; P42110 for YxaK; P94356 for YxkC.
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