Summary: Electroporation with shuttle plasmids carrying a kanamycin resistance gene as a selectable marker failed to generate transformants in two mycobacterial species currently being used in human vaccine trials (Mycobacterium w and Mycobacterium vaccae). In contrast, efficient transformation [103-105 transformants (μg DNA)−1] was obtained using novel vectors with selection based on expression of resistance to hygromycin. The hygromycin resistance vector was also found to be more efficient than kanamycin resistance vectors for transformation of Mycobacterium smegmatis and Mycobacterium bovis BCG. The hygromycin resistance vector was used to overexpress superoxide dismutase of Mycobacterium tuberculosis in M. vaccae in a form suitable for detailed structural analysis. The potential use of this approach for generation of novel recombinant mycobacterial vaccines is discussed.
CooperJ.B.,
DriessenH.P.C.,,
WoodS.P.,
ZhangY.,
YoungJ. Crystallization and preliminary X-ray analysis of the superoxide dismutase from Mycobacterium tuberculosis. J Mol Bio1994 in press;
GarbeT.,
HarrisD.,
VordermeierM.,
LathigraR.,
IvanyiJ.,
YoungD. Expression of the Mycobacterium tuberculosis 19—kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation.. Infect Immun(1993); 61:260–267
LydiateD.J.,
AshbyA.M.,
HendersonD.J.,
KieserH.M.,
HopwoodD.A. Physical and genetic characterization of chromosomal copies of the Streptomyces coelicolor mini-circle. J Gen Microbiol(1989); 135:941–955
RanesM.G.,
RauzierJ.,
LagranderieM.,
GeorghiouM.,
GicquelB. Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a ‘ mini ’ Mycobacterium- Escherichia coli shuttle vector. J Bacteriol(1990); 172:2293–2297
StoverK.C.,
De la Cruz,
VF,
FuerstT.R.,
BurleinJ.E.,
BensonL.A.,
BennetL.T.,
BansalG.P.,
YoungJ.F.,
LeeM.H.,
HatfullG.F.,
SnapperS.B.,
BarlettaR.G.,
JacobsW.R.,
BloomB.R. New use of BCG for recombinant vaccines. Nature(1991); 351:456–460
TalwarG.P.,
ZaheerS.A.,
MukherjeeR.,
WaliaR.,
MisraR.S.,
SharmaA.K. Immunotherapeutic effects of a vaccine based on a saprophytic cultivable mycobacterium, Mycobacterium w, in multibacillary leprosy patients. Vaccine(1990); 8:121–129
TowbinH.,
StaehelinT.,
GordonG. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA (1979); 76:4350–4354
ZhangY.,
GarbeT.,
YoungD. Transformation with katG restores isoniazid-sensitivity in Mycobacterium tuberculosis isolates resistant to a range of drug concentrations. Mol Microbiol(1993); 8:521–524