MRC/NHLS/UCT Molecular Mycobacteriology Research Unit and DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, University of Cape Town, Observatory, Cape Town 7925, South Africa.
Open Biol. 2013 Feb 13;3(2):120175. doi: 10.1098/rsob.120175.
Vitamin B₁₂-dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis, an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B₁₂ in vitro, it is uncertain whether the organism is able to scavenge B₁₂ during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B₁₂ transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B₁₂. A small proportion of these mapped to Rv1314c, identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B₁₂ assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B₁₂ acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B₁₂ and related corrinoids in vitro. Our results establish an alternative to the canonical BtuCD-type system for B₁₂ uptake in M. tuberculosis, and elucidate a role in B₁₂ metabolism for an ABC protein implicated in chronic mycobacterial infection.
依赖维生素 B₁₂ 的酶在结核分枝杆菌（一种专性病原体）的核心生化途径中发挥作用，其体内代谢过程知之甚少。尽管结核分枝杆菌可以在体外获取维生素 B₁₂，但目前尚不清楚该生物体是否能够在宿主感染期间摄取 B₁₂。这个问题对于代谢功能的预测至关重要，但由于结核分枝杆菌基因组中缺乏迄今为止描述的唯一细菌 B₁₂ 转运系统 BtuFCD 的直接同源物，因此解决这个问题变得复杂。我们应用全基因组转座子诱变来鉴定无法使用外源性 B₁₂ 的结核分枝杆菌突变体。这些突变体中的一小部分映射到 Rv1314c，这表明假定的 PduO 型 ATP:co(I)rrinoid 腺苷转移酶对于 B₁₂ 的同化是必需的。然而，最值得注意的是，Rv1819c 中的插入主导了突变体库，揭示了一种意想不到的功能，即一种以前被研究为分枝杆菌 BacA 同源物的 ATP 结合盒（ABC）型蛋白在 B₁₂ 获取中的作用。此外，Rv1819c 的靶向缺失消除了结核分枝杆菌在体外运输 B₁₂ 和相关钴胺素的能力。我们的研究结果确立了一种替代经典 BtuCD 型系统的方法，用于结核分枝杆菌中 B₁₂ 的摄取，并阐明了在慢性分枝杆菌感染中涉及的 ABC 蛋白在 B₁₂ 代谢中的作用。
