Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth

Abstract

NAD(H)-dependent L-alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald in Mycobacterium smegmatis, we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence of L- or D-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with L-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.

FIG. 1.

Primer extension analysis of the ald transcript from M. smegmatis. Total RNA (50 μg) from mc²155 cells, grown in either M-ADC-TW or M-ADC-TW supplemented with l-alanine, was annealed with primer SMALDPE and extended as described in Materials and Methods. Lanes A, C, G, and T show a dideoxy sequencing ladder of the ald gene generated with the same primer. The transcriptional start site is indicated by an arrow. The putative −10 box deduced from the consensus of mycobacterial promoter sequences is highlighted (19). The start codon (ATG) is enclosed in a box, and the putative ribosome binding site (GAAGGAG) is indicated by asterisks.

FIG. 2.

Two-dimensional gel electrophoresis analysis of M. smegmatis mc²155 cellular proteins. Cells were grown in M-ADC-TW or M-ADC-TW supplemented with l- or d-alanine as indicated. Cell extracts were prepared as described in Materials and Methods. Approximately 600 μg of total proteins was resolved by two-dimensional gel electrophoresis and stained with Coomassie blue. Isoelectric points (pI) are indicated at the bottom, and the positions of molecular mass size markers are indicated on the right. The location of the Ald enzyme (42 kDa, pI 5.8) in each gel is indicated by an arrowhead.

FIG. 3.

Analysis of M. smegmatis l-alanine dehydrogenase activities. Cells were grown under either aerobic or anaerobic conditions to the exponential or stationary phase in M-ADC-TW or M-ADC-TW supplemented with l- or d-alanine as indicated. The relative Ald specific activities were calculated by assigning a value of 1.0 to the specific activity of the extract prepared from exponentially growing cells incubated under aerobic conditions without alanine. The specific activities are expressed in micromoles of l-alanine per milligram per minute. The values are means ± standard deviations of triplicate measurements.

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.

FIG. 5.

Analysis of l-alanine dehydrogenase and glycine dehydrogenase activities in wild-type, ald null mutant, and complemented strains. M. smegmatis was grown in M-ADC-TW to an optical density at 600 nm of approximately 0.8 under aerobic conditions. Approximately one-half of each culture was harvested, and the remaining cells were shifted to anaerobic growth for 24 h. Crude cell extracts were prepared and assayed for Ald (solid bars) and Gdh (open bars) activities as described in Materials and Methods. Specific activities are expressed in micromoles of substrate per milligram per minute. The values are means ± standard deviations of triplicate measurements.

FIG. 6.

Utilization of nitrogen sources by M. smegmatis wild-type, ald null mutant, and complemented strains. Exponentially growing cells of mc²155 (♦), GPM267 (▴), and GPM276 (▪) were harvested, washed, and inoculated into minimal medium containing ammonium chloride (A) or l-alanine (B) as the sole nitrogen source at an initial density of approximately 2.0 × 10⁶ CFU ml⁻¹. At various times, the number of viable cells was determined.

FIG. 7.

Growth of M. smegmatis wild-type, ald null mutant, and complemented strains under anaerobic conditions. Exponentially growing cells of mc²155 (♦), GPM267 (▴), and GPM276 (▪) were inoculated into M-ADC-TW at an initial density of approximately 2.0 × 10⁶ CFU ml⁻¹ in a GasPack system as described in Materials and Methods. At various times, the number of viable cells was determined.