Synthesis and fractionation properties of SpoIIGA, a protein essential for pro-sigma E processing in Bacillus subtilis.
sigma E, a major sporulation-specific sigma factor of Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma E). The formation of sigma E from pro-sigma E requires the products of several stage II genes, including spoIIGA, a gene that is cotranscribed with the pro-sigma E coding region (spoIIGB, or sigE). SpoIIGA has been hypothesized to be both a membrane-bound protein and the protease which converts pro-sigma E into sigma E. to learn more of its properties, we joined the Escherichia coli lacZ gene to the 3' end of spoIIGA as a translational fusion, creating a gene whose product was found to contain both beta-galactosidase and SpoIIGA activities. Assaying for the beta-galactosidase activity of the chimeric protein as a measure of its abundance, we determined that the spoIIGA::lacZ product accumulated to approximately 10% the level of a spoIIGB::lacZ fusion protein. Using differential centrifugation to fractionate B. subtilis extracts that contained beta-galactosidase fusion proteins, we observed that the beta-galactosidase activity of the spoIIGA::lacZ fusion protein was preferentially associated with a Triton X-100-sensitive, fast-sedimenting portion of the extract, while the beta-galactosidase activity of the spoIIGB::lacZ fusion protein remained primarily in the supernatant fraction. If the properties of the fusion proteins are assumed to be representative of those of the products of the genes to which lacZ is joined, these results support the hypothesis that SpoIIGA is a membrane-bound protein that acts catalytically in the processing of pro-sigma E into sigma E.
Peters
H K
HK
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.
Haldenwang
W G
WG
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002458:Cell Fractionation; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor; D014157:Transcription Factors
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C063202:sporulation-specific sigma factors; D004247:DNA; D010447:Peptide Hydrolases; C071976:SpoIIGA protein, Bacillus subtilis