Distinct regulation of the sprC gene encoding Streptomyces griseus protease C from other chymotrypsin genes in Streptomyces griseus IFO13350.
The sprC gene encodes Streptomyces griseus protease C (SGPC), a bacterial chymotrypsin-like serine protease. Because the published data on sprC was not complete, we cloned and analyzed a new DNA fragment spanning downstream to upstream of the sprC gene from S. griseus IFO13350. The cloned 2.3-kb DNA fragment was placed on a high-copy number plasmid and introduced into Streptomyces lividans TK24. Chymotrypsin activity of the transformant was 8.5 times higher than that of the control after 3 days of cultivation and stably maintained until 9 days of cultivation, which clearly indicated that the cloned 2.3-kb fragment contained the entire sprC gene with its own promoter. When the same construct was introduced in the S. griseus IFO13350 (wild strain) and its two mutant strains in the A-factor regulatory cascade, deltaadpA and HO1, the chymotrypsin activity increased fivefold only in the deltaadpA strain. Transcriptional analysis based on RT-PCR revealed that the sprC gene is normally transcribed in both strains; however, earlier transcription was observed in the wild strain compared with the deltaadpA strain. A gel mobility shift assay showed that the AdpA protein did not bind to the promoter region of sprC. All these data clearly indicate that the expression of sprC is not dependent on the AdpA protein, but is distinctly regulated from other chymotrypsin genes composing an AdpA regulon. Earlier morphological differentiation was observed in S. lividans TK24, and S. griseus IFO13350 and HO1, transformed with the expression vector. The transformant of S. griseus deltaadpA formed markedly larger colonies. Antisense repression of sprC resulted in severe decrease of chymotrypsin activity, down to one-third of the control, and delayed morphological differentiation. All these data suggest that SGPC is related to normal morphogenesis in S. griseus.
Department of Biological Science, Myongji University, Yongin 449-728, Korea.
Eun-Yong
EY
Choi
Eun A
EA
Oh
Jong-Hee
JH
Kim
Dae-Kyung
DK
Kang
Soon-Kwang
SK
Hong
D001483:Base Sequence; D002918:Chymotrypsin / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D016372:RNA, Antisense / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics*; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics*; D048170:Streptomyces lividans / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D016372:RNA, Antisense; D012329:RNA, Bacterial; D012697:Serine Endopeptidases; C088701:protease C; D002918:Chymotrypsin