Faculty of Health Sciences, DST/NRF Centre of Excellence for Biomedical TB Research, University of the Witwatersrand, National Health Laboratory Service, Johannesburg, South Africa.
PLoS One. 2021 Jan 20;16(1):e0245745. doi: 10.1371/journal.pone.0245745. eCollection 2021.
Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller, Mycobacterium smegmatis is able to assimilate nitrate and herein we set out to confirm the genetic basis for this by probing NR activity in mutants defective for putative nitrate reductase (NR) encoding genes. In addition to the annotated narB and narGHJI, bioinformatics identified three other putative NR-encoding genes: MSMEG_4206, MSMEG_2237 and MSMEG_6816. To assess the relative contribution of each, the corresponding gene loci were deleted using two-step allelic replacement, individually and in combination. The resulting strains were tested for their ability to assimilate nitrate and reduce nitrate under aerobic and anaerobic conditions, using nitrate assimilation and modified Griess assays. We demonstrated that narB, narGHJI, MSMEG_2237 and MSMEG_6816 were individually dispensable for nitrate assimilation and for nitrate reductase activity under aerobic and anaerobic conditions. Only deletion of MSMEG_4206 resulted in significant reduction in nitrate assimilation under aerobic conditions. These data confirm that in M. smegmatis, narB, narGHJI, MSMEG_2237 and MSMEG_6816 are not required for nitrate reduction as MSMEG_4206 serves as the sole assimilatory NR.
细菌将硝酸盐还原为亚硝酸盐是氮循环中的一个重要步骤，由多种硝酸盐还原酶（NR）酶催化。土壤居民分枝杆菌能够同化硝酸盐，在此我们通过探测突变体中假定的硝酸盐还原酶（NR）编码基因的 NR 活性来确认这一遗传基础。除了注释的 narB 和 narGHJI 外，生物信息学还鉴定了另外三个假定的 NR 编码基因：MSMEG_4206、MSMEG_2237 和 MSMEG_6816。为了评估每个基因的相对贡献，使用两步等位基因替换法单独和组合删除了相应的基因座。使用硝酸盐同化和改良的格里斯测定法，在有氧和厌氧条件下测试了产生的菌株同化硝酸盐和还原硝酸盐的能力。我们证明，narB、narGHJI、MSMEG_2237 和 MSMEG_6816 单独对于硝酸盐同化和有氧及厌氧条件下的硝酸盐还原酶活性都是可有可无的。只有删除 MSMEG_4206 才会导致有氧条件下硝酸盐同化的显著减少。这些数据证实，在分枝杆菌中，narB、narGHJI、MSMEG_2237 和 MSMEG_6816 不需要硝酸盐还原，因为 MSMEG_4206 是唯一的同化性 NR。
