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

making it more susceptible to lipophilic antibiotics, such as rifampicin. By extension, loss of
Rv2509, the M. tuberculosis homologue, would be expected to have a bearing not only on
susceptibility to antibiotics, but also on virulence as strains of M. tuberculosis with altered
mycolic acids are highly attenuated (Bhatt et al., 2007; Dubnau et al., 2000; Glickman et al.,
2000), highlighting the potential of Rv2509 as a ‘secondary’ drug target. Our studies also shed
some light on the post FAS-II/Pks13 processing, transport and transfer of mycolic acids to their
location in the cell envelope. The replacement of mycolic acids in the cell wall by the α-alkyl, β-
oxo fatty acid precursors suggested that post-Pks13 reduction of the β-oxo group was not
necessary for the subsequent processing pathways.

EXPERIMENTAL PROCEDURES

Bacterial strains, phages, plasmids and culture conditions

All plasmids, phages and bacterial strains used in this study are shown in Table 1. Strains of
Escherichia coli were cultured in Luria-Bertani Broth (LB; Difco). M. smegmatis strains were
grown in either LB broth or Tryptic Soy Broth (TSB; Difco), each containing 0.05 % Tween80.
TSB-agar was prepared by adding 1.5 % agar to TSB prior to autoclaving. For M. smegmatis
hygromycin (100 μg ml^-1) or kanamycin (20 μg ml^-1) was used for selection while hygromycin
(150 μg ml^-1) or kanamycin (40 μg ml^-1) was used for selecting recombinant E. coli strains.
Determination of minimum inhibitory concentrations (MIC) of antibiotics was done in LB Broth
using the Alamar Blue assay (Franzblau et al., 1998).

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