Campos-Pardos Elena, Sanz-Asensio Laura, Pérez-Jiménez Sandra, Yruela Inmaculada, Contreras-Moreira Bruno, Toledo-Arana Alejandro, Gonzalo-Asensio Jesús
Grupo de Genética de Micobacterias, Departamento de Microbiología, Facultad de Medicina, Universidad de Zaragoza, IIS Aragón, Zaragoza, Spain.
CIBER Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain.
Microb Biotechnol. 2025 Jun;18(6):e70176. doi: 10.1111/1751-7915.70176.
Vitamin B12 metabolism differs among members of the Mycobacterium genus. While non-tuberculous mycobacterial species are B12 producers, tuberculous mycobacteria lack endogenous production and rely on the host supply of this vitamin. Here, we hypothesise that this discrepant phenotype might impact the function of B12-dependent enzymes. We specifically focused on methionine synthases MetH and MetE. Both enzymes showed genetic differences in the Mycobacterium genus, resulting in a clear divergence between tuberculous and non-tuberculous species. Unexpectedly, the dependency of MetH on B12 was indistinguishable between M. tuberculosis and M. smegmatis, assayed as representative members of tuberculous and non-tuberculous species, respectively. However, MetE showed robust phenotypic differences between these species, displaying a finely tuned B12 regulation in M. tuberculosis, in contrast to a more permissive regulation in M. smegmatis. Both orthologs differ in the vitamin isoform specifically recognised, and the B12 threshold level required for MetE regulation. Since the B12 regulatory element in the metE gene is an RNA riboswitch, we analysed the polymorphisms in this region, with a special focus on loss-of-function mutations identified after in vitro selection. We used this information to engineer a whole-cell B12 biosensor in the genetically fastidious Mycobacterium genus, being able to detect vitamin B12 concentration in the range of micrograms per millilitre.
维生素B12的代谢在分枝杆菌属成员中存在差异。非结核分枝杆菌能够产生B12，而结核分枝杆菌缺乏内源性B12的产生，依赖于宿主提供这种维生素。在此，我们推测这种不同的表型可能会影响依赖B12的酶的功能。我们特别关注甲硫氨酸合酶MetH和MetE。这两种酶在分枝杆菌属中表现出基因差异，导致结核分枝杆菌和非结核分枝杆菌之间存在明显差异。出乎意料的是，分别作为结核分枝杆菌和非结核分枝杆菌的代表性成员进行检测时，结核分枝杆菌和耻垢分枝杆菌中MetH对B12的依赖性没有区别。然而，MetE在这些物种之间表现出明显的表型差异，在结核分枝杆菌中表现出精细调节的B12调控，而在耻垢分枝杆菌中则表现出更宽松的调控。这两种直系同源物在特异性识别的维生素异构体以及MetE调控所需的B12阈值水平上存在差异。由于metE基因中的B12调控元件是一种RNA核糖开关，我们分析了该区域的多态性，特别关注体外筛选后鉴定出的功能丧失突变。我们利用这些信息在遗传特性要求严格的分枝杆菌属中构建了一种全细胞B12生物传感器，能够检测每毫升微克范围内的维生素B浓度。
