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

FIGURE LEGENDS

Figure 1 Mycolic acid reductases in mycobacteria

(A) Schematic representation of the post-FAS-II steps in mycobacterial mycolic acid
biosynthesis. AccD4 and AccD5 are acyl-CoA carboxylases while FadD32 is an acyl-AMP
ligase. It is yet unclear whether the reduction of the β-oxo group occurs while the mycolic acid
precursor in still attached to Pks13 or after release from Pks13 by a thioesterase. (B) Alignment
of amino acid sequences of Rv2509 and MSMEG4722 with 1cyd (PDB file for the structure of
Mus musculus carbonyl reductase with NADP and 2-propanol). α-helices and β-sheets are
indicated above the residues as coils and arrows respectively. Residues essential for
NAD/NADP binding are indicated by triangles while the active site residue is indicated with a
star.

Figure 2 Generation of a MSMEG4722 null mutant

(A) Map of the MSMEG4722 region in the parental M. smegmatis strain mc²155 and its
corresponding region in the ∆MSMEG4722 mutant. res, γδ resolvase site; hyg, hygromycin
resistance gene from Streptomyces hygroscopicus; sacB, sucrose counterselectable gene from
Bacillus subtilis. Digoxigenin-labelled probes were derived from ~1kb upstream and
downstream flanking sequences that were used to construct the knockout plasmid, and are
indicated by thick lines with square ends. ClaI digested bands expected in a Southern blot are
indicated in roman numerals with sizes in brackets. The inset shows the Southern blot of ClaI
digested genomic DNA from the two strains with expected bands indicated by arrows. (B)

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