Development of a new host vector system in mycobacteria.
The hybrid plasmid pYT72/pYT92 constructed from an Escherichia coli plasmid pACYC177 and mycobacterial plasmid pMSC262 isolated from Mycobacterium scroflaceum strain W262 transformed both E. coli and BCG. Phage-sensitive mutants S-10 and S-20 isolated from BCG Tokyo strain showed higher frequency of transformation than the wild-type strain. Frequency of transformation was dependent on age of the culture and the electroporation condition used. Several deletion mutants were generated from pYT72/92 to determine the minimum region for the replication in the mycobacteria. A 2.3-kb fragment of pMSC262 was found to contain an essential region. Using this fragment and pACYC177, a small shuttle vector pYT937 containing two drug-resistance markers, kanamycin- and ampicillin-resistance, was constructed. pYT937 contains AatII, BamHI, BbvII, GsuI, HincII, PstI, ScaI and XbaI cloning sites.
Goto
Y
Y
Faculty of Agriculture, Miyazaki University, Japan.
Taniguchi
H
H
Udou
T
T
Mizuguchi
Y
Y
Tokunaga
T
T
D003001:Cloning, Molecular; D004261:DNA Replication; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D009161:Mycobacterium / Q000235:genetics*; D010957:Plasmids; D015183:Restriction Mapping; D014169:Transformation, Bacterial
D004269:DNA, Bacterial