FIG. 2.
Construction of Δadl-ogt mutants of M. tuberculosis. (A) Genetic organization at the Rv1317c-Rv1316c locus of M. tuberculosis H37Rv, where rrf and rrl are the 5S and 23S rRNA genes, respectively (5). Intergenic distances (base pairs) are shown beneath the map. Rv1317c is annotated in the EMBL and Tuberculist databases as “alkA” (5). However, Rv1317c is chimeric (9, 16), comprising 5′-terminal adaA (18, 24) and 3′-terminal alkA components, and we have thus designated this gene “adl” to denote this fact. (B) Restriction maps and genotypes of the wild-type and mutant alleles (not drawn to scale). The Δadl-ogt::hyg allele was delivered on the vector pSDVM, which is a subclone of pGEM3Zf(+) that also contained a lacZ gene, whereas the Δadl-ogt::aph allele was delivered on pBSEB, which is a subclone of ptrpA-1-rpsL (25). Allelic exchange in M. tuberculosis H37Rv to form the SID-H mutant was performed by using UV-pretreated pSDVM (20), whereas that in streptomycin-resistant H37Rv to form the BS-SK mutant was carried out with pBSEB by using previously described methods (27). The flanking homologous sequences carried on the delivery vectors are shown as thick horizontal lines of the indicated sizes (base pairs). The mutant alleles differed in that an additional 246 bp of the ogt gene were deleted in Δadl-ogt::aph compared to Δadl-ogt::hyg. The selectable markers are shown as stippled boxes, and the thick dashed line in the H37Rv map represents the 1,592-bp KpnI-NotI fragment used as the probe for the Southern blot shown in panel C. Shown above the wild-type allele is the 4,207-bp KpnI fragment carrying the adl-ogt operon and flanking sequences that was used to genetically complement the SID-H mutant by single-copy insertion at the attB locus via the integrative delivery vector pAINT (1). Relevant restriction sites are shown and abbreviated as follows: E, EcoRI; K, KpnI; N, NotI, Ns, NsiI; S, SacI; Sm, SmaI; Sp, SpeI. (C) Genotypic confirmation of the SID-H mutant by Southern blot analysis. Genomic DNA was digested with SmaI and probed by using the 1,592-bp KpnI-NotI fragment. WT, wild type.