title: Expression of the naphthoate synthase gene in Mycobacterium tuberculosis in a self-generated oxygen depleted liquid culture system.
abstract: Mycobacterium tuberculosis has been classified for decades as a strict aerobic species. Whole genome sequencing of the type culture strain H37Rv has revealed the presence of a full set of genes allowing for anaerobic metabolism. Naphthoate synthase (menB) is a key enzyme required for the synthesis of menaquinone, which plays a crucial role in anaerobic electron transport, ultimately resulting in the formation of energy generating intermediates. Interrupting the synthesis of this enzyme will interfere with the production of menaquinone and therefore this enzyme is a potential drug target. This study serves to investigate the role of naphtoate synthase in the survival of M. tuberculosis H37Rv when incubated under oxygen limiting conditions of unagitated liquid culture over 15 weeks. M. tuberculosis H37Rv was grown in Middlebrook 7H9 media. The tubes were kept undisturbed at 37 °C for up to 15 weeks. At selected time points, aliquots of cells were removed and frozen. RNA was simultaneously extracted from all aliquots. The RNA was converted to cDNA for Real-Time PCR on the ABI 7000 SDS. Gene expression was normalized against 16S RNA quantities at each time point. A systematic increase in the expression of the menB gene product was observed over the incubation period with a 4.3-fold increase seen at week 6 (P < 0.001) relative to day 0 and an 85.8-fold increase at week 15 (P < 0.001) relative to day 0. Cells of M. tuberculosis increase menaquinone production during prolonged incubation in broth culture as a mechanism of survival. This study substantiates the menB enzyme to be a putative drug target.
authors.affiliation: University of KwaZulu-Natal, Congella, Durban, South Africa.
authors.forename: Prathna
authors.initials: P
authors.lastname: Ramchandra
authors.forename: A Willem
authors.initials: AW
authors.lastname: Sturm
mesh_terms: D003470:Culture Media / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D020869:Gene Expression Profiling; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D024482:Vitamin K 2 / Q000378:metabolism
chemical_list: D003470:Culture Media; D018076:DNA, Complementary; D012333:RNA, Messenger; D024482:Vitamin K 2; D006836:Hydro-Lyases; C021853:naphthoate synthetase; D010100:Oxygen