Deletion of a mycobacterial gene encoding a reductase leads to an altered cell wall containing β-oxo-mycolic acid analogues, and the accumulation of long-chain ketones related to mycolic acids

and specialized transduction were performed as described earlier (Bardarov et al., 2002). Allelic
exchange in hygromycin-resistant transductants was confirmed by Southern blot.

Construction of complemented strains

MSMEG4722 was PCR amplified from M. smegmatis mc²155 genomic DNA using the primers
MS4722-U (5'-GCAGGATCCAATGAGCCGCATGCCAGTACCCG-3') and MS4722-D (5'-
GCAGAATTCCTAACCGCCGCCGAGCTTCTTG-3') and cloned into the E. coli-
Mycobacterium shuttle plasmid pMV261 using the primer incorporated BamHI and EcoRI sites
to yield the recombinant plasmid pMV261-MSMEG4722. In a similar fashion, the plasmid
pMV261-Rv2509 was constructed using Rv2509 which was PCR amplified from M. tuberculosis
H37Rv genomic DNA using the primers Rv2509-U     (5'-

GCAGGATCCAATGCCGATACCCGCGCCC-3') and Rv2509-D      (5'-

GCAGAATTCCTAGCTGCCCCCAAGCCTC-3'). The complemented strains ∆MSMEG4722-
C and ∆MSMEG4722-CRv were obtained by selecting kanamycin-resistant transformants
following electroporation of the mutant strain ∆MSMEG4722 with pMV261-MSMEG4722 or
pMV261-Rv2509 respectively. Electroporation was done as described earlier (Snapper et al.,
1990).

Lipid and mycolic acid extraction and analysis

Polar and apolar lipids were extracted from M. smegmatis strains and analyzed as described
earlier (Dobson et al., 1985). For extraction of mycolic acid methyl esters (MAMEs), both the

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