Transformation using in vivo and in vitro methylation in Streptomyces griseus.
Streptomyces griseus does not readily take up foreign DNA isolated from other Streptomyces species or Escherichia coli, presumably due to its unique restriction-modification systems that function as a barrier for interspecific DNA transfer. To efficiently transform S. griseus by avoiding the restriction barriers, we methylated incoming DNA in vivo and in vitro and treated protoplasts with heat prior to transformation. Whereas heat treatment of protoplasts or methylation of the E. coli-Streptomyces shuttle vectors (pXE4 and pKK1443) did not prominently improve the transformation efficiency, HpaII methylation of the vectors from any E. coli strains tested in this study highly increased the transformation efficiency. The highest transformation efficiency was observed when the shuttle vectors were isolated from the dam, hsd strain of E. coli (GM161) and methylated by AluI and HpaII methyltransferases, and the efficiency was approximately the same as that of the vectors from S. griseus. We identified several restriction-modification systems that decrease the transformation efficiency. This research also led us to understand methylation profiles and restriction-modification systems in S. griseus.
Kwak
Jangyul
J
Korea Research Institute of Biology and Biotechnology, 52 Oun-Dong, Yusong-Ku, Taejon 305-333, South Korea. kwak91@hotmail.com
Jiang
Hao
H
Kendrick
Kathleen E
KE
D019175:DNA Methylation; D004269:DNA, Bacterial / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics*; D014170:Transformation, Genetic / Q000502:physiology*
D004269:DNA, Bacterial; C029641:DNA modification methylase HpaII; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; C024669:endodeoxyribonuclease AluI; D015252:Deoxyribonucleases, Type II Site-Specific