This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Feng, Z. Articles by Barletta, R. G. Search for Related Content PubMed PubMed Citation Articles by Feng, Z. Articles by Barletta, R. G.

 Previous Article  |  Next Article 

Journal of Bacteriology, September 2002, p. 5001-5010, Vol. 184, No. 18
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.18.5001-5010.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mycobacterium smegmatis L-Alanine Dehydrogenase (Ald) Is Required for Proficient Utilization of Alanine as a Sole Nitrogen Source and Sustained Anaerobic Growth

Zhengyu Feng,¹ Nancy E. Cáceres,² Gautam Sarath,² and Raúl G. Barletta^1*

Department of Veterinary and Biomedical Sciences,¹ Center for Biotechnology, University of Nebraska, Lincoln, Nebraska 68583-0905²

Received 18 March 2002/ Accepted 13 June 2002

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.

---

* Corresponding author. Mailing address: Department of Veterinary and Biomedical Sciences, 211 VBS, Fair Street and East Campus Loop, University of Nebraska, Lincoln, NE 68583-0905. Phone: (402) 472-8543. Fax: (402) 472-9690. E-mail: rbarletta{at}unl.edu.

A contribution of the University of Nebraska Agricultural Research Division, Lincoln. Journal Series no. 13651.

---

Journal of Bacteriology, September 2002, p. 5001-5010, Vol. 184, No. 18
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.18.5001-5010.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Parish, T., Smith, D. A., Roberts, G., Betts, J., Stoker, N. G. (2003). The senX3-regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence. Microbiology 149: 1423-1435 [Abstract] [Full Text]