Inactivation of ahpC on the
chromosome of M. smegmatis. (A) Schematic
representation of the recombinational events between chromosomal
ahpC and ahpC::Kmr
on a 2.5-kb PstI fragment introduced into M.
smegmatis mc2 155 by electroporation. B,
BglII; P, PstI; Bs, BspEI.
Balloon, 1.2-kb Kmr cassette. Lines I and II correspond to
the wild-type BglII and PstI fragments,
respectively. (B) Southern blot hybridization analysis of
the insertional inactivation of ahpC in the strain
VD1865-6. Lanes: 1 and 3, M. smegmatis mc2
155 ahpC+; 2 and 4, M.
smegmatis VD1865-6. I, 1.7-kb BglII fragment
(wt); II, 1.3-kb PstI fragment (wt). I+Kmr
(2.9 kb) and II+Kmr (2.5 kb) are the corresponding bands
hybridizing with the ahpC probe in the strain VD1865-6.
(C) Western blot analysis of AhpC expression in the parent
strain M. smegmatis mc2 155 (wt) and its
mutant derivative VD1865-6
(ahpC::Kmr). Equal amounts of
protein from crude extracts separated by SDS/PAGE were probed with
anti-DirA antibody.