FIG. 4.

Construction and characterization of M. smegmatis ald null mutants. (A) Open reading frame organization at the ald locus in both strain mc²155 (wild type) and the ald null mutant strain (Δald::aph). Relevant restriction enzyme sites and the PCR fragment used as the ald probe are indicated. (B) Southern blot analysis of genomic DNA from wild-type and ald null mutant strains. Genomic DNA was digested with ApaI (lanes 1 to 5) or PstI (lanes 6 to 10). The strains examined are wild-type strain mc²155 (lanes 1 and 6) and ald mutants GPM267 (lanes 2 and 7), GPM268 (lanes 3 and 8), GPM269 (lanes 4 and 9), and GPM270 (lanes 5 and 10). Blots were hybridized with the radiolabeled 1.1-kb PCR fragment containing the ald gene and washed under stringent conditions as described in Materials and Methods. MW, molecular weight. (C) Analysis of l-alanine dehydrogenase activities in M. smegmatis wild-type, mutant, and recombinant strains. Extracts from organisms grown in both M-ADC-TW and M-ADC-TW supplemented with l-alanine were prepared and analyzed. Ald specific activities are expressed in micromoles of l-alanine per milligram per minute. The values are means ± standard deviations of triplicate measurements. A mock assay was also carried out with bovine serum albumin (BSA) in place of equivalent amounts of the cell extracts.