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. 2012 Jun;80(6):1958–1968. doi: 10.1128/IAI.00021-12

Fig 1.

Fig 1

Generation of M. tuberculosis strains lacking all cyclopropanation or all mycolic acid methyltransferases. (A) Deletion of mmaA1-mmaA2-mmaA3-mmaA4 locus from a merodiploid strain of M. tuberculosis Erdman with attB::mmaA1-4. When digested with EcoRI and probed with the 1,000-bp piece immediately upstream of mmaA1, the WT gives a 6.4-kb band, whereas the strain with the mmaA1-4 deletion gives a 3.1-kb band. The strain marked with an asterisk was chosen for further manipulation and was named MGM1953. (B) Deletion of pcaA/umaA1 from Δmma1-4::loxP attB::mmaA1-4 strain. The Southern blot confirms the deletion of pcaA and umaA1 from a strain previously deleted of mmaA1-4 (after removal of the loxP-hyg-loxP marker with Cre recombinase). When digested with SmaI and probed with the 600-bp piece immediately upstream of umaA1, the WT produces a 1.8-kb band, whereas the pcaA/umaA1 deletion strain produces a 4.2-kb band. The strain marked with an asterisk was designated MGM1960 and used for further genetic manipulation. (C) Deletion of cmaA2 from ΔmmaA1-4::loxp ΔpcaA/umaA1::Hyg attB::mmaA3-4 strain (MGM1961) and ΔmmaA1-4::loxp ΔpcaA/umaA1::Hyg attB::Strepr strain (MGM1962) via transduction of ΔcmaA2::zeo (phDB10). The Southern blot confirms the deletion of cmaA2 from MGM1961 or MGM1962. When digested with HindIII and probed with the 600-bp piece immediately upstream of cmaA2, the WT produces a 16.4-kb band, whereas the ΔcmaA2 strain produces a 3.5-kb band. The strains marked with asterisks were designated MGM1990 and MGM1991 and were characterized further. (D) Two-dimensional TLC of mycolic acid methyl esters of the cell wall of MGM1990. All three mycolic acid classes are present (black arrowhead, alpha class; black dashed arrow, methoxy class; and black arrow, keto class), and all are fully unsaturated. Essentially no mature cyclopropanated mycolic acids are made, as shown by the trivial amounts marked by the gray arrows (methoxy and keto classes) or gray arrowhead (alpha class). (E) Two-dimensional TLC of mycolic acid methyl esters of the cell wall of MGM1991. No oxygenated mycolates are present (no methoxy- or ketomycolates), and the remaining alpha mycolate is almost fully unsaturated. Small amounts of alpha mycolate with a single cyclopropane ring are visible (gray arrow).