Purification and characterization of the spoOA protein of Bacillus subtilis from an overproducing strain of Escherichia coli.
The spoOA gene of Bacillus subtilis is essential for the earliest stage of sporulation. To purify and characterize the product of the spoOA gene, we constructed a fusion plasmid in which the spoOA coding region was placed under the control of the Ptac promoter. When expression of the spoOA gene was induced in Escherichia coli cells by derepression of Ptac, the SpoOA protein constituted 15% of total cellular protein. The SpoOA protein was purified to homogeneity from these cells. We found that the NH2-terminal amino acid sequence of the purified protein was essentially the same as that of the SpoOA protein (spoOA-cat protein) from B. subtilis, and that the NH2-terminal methionine of the SpoOA protein from E. coli was formylated presumably because of insufficient amounts of the deformylating enzyme. The T signal [Ganoza, M. C., Marliere, P., Kofoid, E. C. and Louis, B. G. (1985) Proc. Natl Acad. Sci. USA 82, 4587-4591], in addition to the Shine-Dalgarno signal to determine the initiation codon of the spoOA gene, is considered to function in E. coli as well as in B. subtilis. We also found that the purified SpoOA protein had a DNA-binding activity. It was preferentially bound to the 175-bp BclI fragment of phi 105 DNA, and was released in the presence of 0.3 M KCl.
D001412:Bacillus subtilis / Q000032:analysis*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003062:Codon; D004279:DNA, Viral / Q000378:metabolism; D004926:Escherichia coli / Q000032:analysis*; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D014176:Protein Biosynthesis; D013171:Spores, Bacterial; D014157:Transcription Factors
D001426:Bacterial Proteins; D003062:Codon; D004279:DNA, Viral; C080950:Spo0A protein, Bacillus subtilis; D014157:Transcription Factors