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Isolation and characterization of the aspartokinase and aspartate semialdehyde dehydrogenase operon from mycobacteria
J D Cirillo1, T R Weisbrod, L Pascopella
1Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461.
Abstract:
Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall. The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic. The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems. Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria. In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated. The M. smegmatis asd gene was isolated by complementation in Escherichia coli. This gene was then used to isolate the asd genes from other mycobacteria. The asd-complementing fragments from BCG and M. smegmatis were sequenced. An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask). Primer extension analysis revealed that the only transcriptional start in this region is found 5' of the ask gene. This observation indicates that the mycobacterial ask and asd genes are in an operon.
Insights
Diaminopimelic acid (DAP) biosynthesis is crucial for mycobacterial cell walls and potential drug targets. Researchers identified and characterized the aspartate semialdehyde dehydrogenase (asd) and aspartokinase (ask) genes, revealing they form an operon in mycobacteria.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Diaminopimelic acid (DAP) is a key component of the mycobacterial cell wall, implicated in virulence and immunogenicity.
- Understanding DAP biosynthesis is vital for developing new antimycobacterial agents.
- The specific biosynthetic pathway for DAP in mycobacteria remains poorly understood.
Purpose of the Study:
- To investigate the poorly understood diaminopimelic acid (DAP) biosynthesis pathway in mycobacteria.
- To isolate and characterize the aspartate semialdehyde dehydrogenase (asd) genes from various mycobacterial species.
- To elucidate the genetic organization and transcriptional regulation of the asd gene in mycobacteria.
Main Methods:
- Isolation of aspartate semialdehyde dehydrogenase (asd) genes from Mycobacterium smegmatis, BCG, Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis.
- Complementation in Escherichia coli to isolate the M. smegmatis asd gene.
- Sequencing of asd-complementing fragments from BCG and M. smegmatis.
- Identification of the upstream aspartokinase (ask) gene and primer extension analysis for transcriptional start sites.
Main Results:
- The aspartate semialdehyde dehydrogenase (asd) genes were successfully isolated from multiple mycobacterial species.
- The M. smegmatis asd gene was identified and used to facilitate the isolation of homologous genes from other mycobacteria.
- An upstream open reading frame was identified as the mycobacterial aspartokinase (ask) gene.
- Primer extension analysis confirmed a single transcriptional start site upstream of the ask gene, indicating co-transcription.
Conclusions:
- The mycobacterial aspartokinase (ask) and aspartate semialdehyde dehydrogenase (asd) genes are organized into an operon.
- This finding provides a foundation for understanding mycobacterial DAP biosynthesis.
- The identified ask-asd operon represents a potential target for novel antimycobacterial drug development.