Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid
Open Access
- 1 February 2012
- journal article
- Published by Microbiology Society in Microbiology
- Vol. 158 (2), 308-318
- https://doi.org/10.1099/mic.0.053983-0
Abstract
Thymidylate synthase (TS) enzymes catalyse the biosynthesis of deoxythymidine monophosphate (dTMP or thymidylate), and so are important for DNA replication and repair. Two different types of TS proteins have been described (ThyA and ThyX), which have different enzymic mechanisms and unrelated structures. Mycobacteria are unusual as they encode both thyA and thyX, and the biological significance of this is not yet understood. Mycobacterium tuberculosis ThyX is thought to be essential and a potential drug target. We therefore analysed M. tuberculosis thyA and thyX expression levels, their essentiality and roles in pathogenesis. We show that both thyA and thyX are expressed in vitro, and that this expression significantly increased within murine macrophages. Under all conditions tested, thyA expression exceeded that of thyX. Mutational studies show that M. tuberculosis thyX is essential, confirming that the enzyme is a plausible drug target. The requirement for M. tuberculosis thyX in the presence of thyA implies that the essential function of ThyX is something other than dTMP synthase. We successfully deleted thyA from the M. tuberculosis genome, and this deletion conferred an in vitro growth defect that was not observed in vivo. Presumably ThyX performs TS activity within M. tuberculosis ΔthyA at a sufficient rate in vivo for normal growth, but the rate in vitro is less than optimal. We also demonstrate that thyA deletion confers M. tuberculosis p-aminosalicylic acid resistance, and show by complementation studies that ThyA T202A and V261G appear to be functional and non-functional, respectively.Keywords
This publication has 43 references indexed in Scilit:
- Forkhead-associated (FHA) Domain Containing ABC Transporter Rv1747 Is Positively Regulated by Ser/Thr Phosphorylation in Mycobacterium tuberculosisJournal of Biological Chemistry, 2011
- The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of actionNucleic Acids Research, 2011
- Thr202Ala in thyA Is a Marker for the Latin American Mediterranean Lineage of the Mycobacterium tuberculosis Complex Rather than Para -Aminosalicylic Acid ResistanceAntimicrobial Agents and Chemotherapy, 2010
- Activity-Based Metabolomic Profiling of Enzymatic Function: Identification of Rv1248c as a Mycobacterial 2-Hydroxy-3-oxoadipate SynthaseCell Chemical Biology, 2010
- Development of a repressible mycobacterial promoter system based on two transcriptional repressorsNucleic Acids Research, 2010
- Molecular Genetics of para -Aminosalicylic Acid Resistance in Clinical Isolates and Spontaneous Mutants of Mycobacterium tuberculosisAntimicrobial Agents and Chemotherapy, 2009
- An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX geneNature, 2009
- Global Transcriptional Profile of Mycobacterium tuberculosis during THP-1 Human Macrophage InfectionInfection and Immunity, 2008
- Silencing Essential Protein Secretion in Mycobacterium smegmatis by Using Tetracycline RepressorsJournal of Bacteriology, 2007
- An adaptation of the LMS method to determine expression variations in profiling dataNucleic Acids Research, 2007