Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides.
A single gene (mas) encodes the multifunctional enzyme that catalyzes the synthesis of very long chain multiple methyl branched fatty acids called mycocerosic acids that are present only in slow-growing pathogenic mycobacteria and are thought to be important for pathogenesis. To achieve a targeted disruption of mas, an internal 2-kb segment of this gene was replaced with approximately the same size hygromycin-resistance gene (hyg), such that hyg was flanked by 4.7- and 1.4-kb segments of mas. Transformation of Mycobacterium bovis BCG with this construct in a plasmid that cannot replicate in mycobacteria yielded hygromycin-resistant transformants. Screening of 38 such transformants by PCR revealed several transformants representing homologous recombination with single crossover and one with double crossover. With primers representing the hyg termini and those representing the mycobacterial genome segments outside that used to make the transformation construct, the double-crossover mutant yielded PCR products expected from either side of hyg. Gene replacement was further confirmed by the absence of the vector and the 2-kb segment of mas replaced by hyg from the genome of the mutant. Thin-layer and radio-gas chromatographic analyses of the lipids derived from [1-14C]propionate showed that the mutant was incapable of synthesizing mycocerosic acids and mycosides. Thus, homologous recombination with double crossover was achieved in a slow-growing mycobacterium with an intron-containing RecA. The resulting mas-disrupted mutant should allow testing of the postulated roles of mycosides in pathogenesis.
Azad
A K
AK
Neurobiotechnology Center, The Ohio State University, Columbus, 43210, USA.
Sirakova
T D
TD
Rogers
L M
LM
Kolattukudy
P E
PE
D000217:Acyltransferases / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002934:Cinnamates; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis / Q000737:chemistry; D018390:Gene Targeting; D005798:Genes, Bacterial; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009163:Mycobacterium bovis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
D000900:Anti-Bacterial Agents; D002934:Cinnamates; D017931:DNA Primers; D004269:DNA, Bacterial; D005227:Fatty Acids; D006921:Hygromycin B; C026273:hygromycin A; C032992:mycocerosic acid; D000217:Acyltransferases; C044110:mycocerosic acid synthase