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



Bioinformatics

Sequence alignments were determined using BLAST or EBI ClustalW (Chenna et al., 2003) and
rendered using the EScript 2.2 web server. Structural predictions were performed using the
@TOME server (Douguet and Labesse, 2001) and modeling performed using the FUGUE web
server (
http://tardis.nibio.go.jp/fugue/align.php). PyMOL (DeLano Scientific) was used to create
POV scenes followed by rendering by POV-Ray.

Construction of a MSMEG4722 null mutant

Approximately 1-kb sequences of the upstream and downstream regions of MSMEG4722 were
PCR amplified from
M. smegmatis mc2155 genomic DNA using the primer pairs MS4722LL
(5’-TTTTTTTTCCATAAATTGGTGGCCAGCAGGT AGTAGACG-3’) and MS4722LR (5’-
TTTTTTTTCCATTTCTTGGAGTTCGGTGGCCAACG CTTC-3’), and MS4722RL (5’-
TTTTTTTTCCATAGATTGGTGGATCGACACCGAGTAC AC-3’) and MS4722RR (5’-
TTTTTTTTCCATCTTTTGGAAACTGATCCGCTCCAAGGG-3’) respectively (all primers
had
Van91I recognition sites incorporated at the 5’ end). The PCR fragments were digested with
Van 91I and directly cloned into Van91I digested p0004S (Gift from T. Hsu and W. R. Jacobs
Jr., Albert Einstein College of Medicine, New York). Recombinant plasmids obtained after
transforming
E. coli TOP-10 cells were digested with Van91I for confirmation and sequenced.
One plasmid, p
MSMEG4722, was linearized by PacI digestion and packaged into the
temperature sensitive mycobacteriophage phAE159 as described (Bardarov et al., 2002) to yield
phasmid DNA of the knockout phage ph
MSMEG4722. Generation of high titre phage particles

17



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