Department of Veterans Affairs Medical Center, Long Beach, CA, USA.
PLoS One. 2012;7(9):e45459. doi: 10.1371/journal.pone.0045459. Epub 2012 Sep 18.
Mycobacterium tuberculosis induces nitrate reductase activity in response to decreasing oxygen levels. This is due to regulation of both the transcription and the activity of the nitrate transporter NarK2. A model of NarK2 structure is proposed containing 12 membrane spanning regions consistent with other members of the major facilitator superfamily. The role of the proton gradient was determined by exposing M. tuberculosis to uncouplers. Nitrite production decreased indicating that the importation of nitrate involved an H(+)/nitrate symporter. The addition of nitrite before nitrate had no effect, suggesting no role for a nitrate/nitrite antiporter. In addition the NarK2 knockout mutant showed no defect in nitrite export. NarK2 is proposed to be a Type I H(+)/nitrate symporter. Site directed mutagenesis was performed changing 23 amino acids of NarK2. This allowed the identification of important regions and amino acids of this transporter. Five of these mutants were inactive for nitrate transport, seven produced reduced activity and eleven mutants retained wild type activity. NarK2 is inactivated in the presence of oxygen by an unknown mechanism. However none of the mutants, including those with mutated cysteines, were altered in their response to oxygen levels. The assimilatory nitrate transporter NasA of Bacillus subtilis was expressed in the M. tuberculosis NarK2 mutant. It remained active during aerobic incubation showing that the point of oxygen control is NarK2.
结核分枝杆菌在氧水平降低时诱导硝酸盐还原酶活性。这是由于硝酸盐转运蛋白 NarK2 的转录和活性调节。提出了 NarK2 结构的模型，其中包含 12 个跨膜区域，与主要易化剂超家族的其他成员一致。质子梯度的作用通过使结核分枝杆菌暴露于解偶联剂来确定。亚硝酸盐的产生减少表明硝酸盐的摄取涉及 H(+)/硝酸盐同向转运体。在添加硝酸盐之前添加亚硝酸盐没有效果，表明没有硝酸盐/亚硝酸盐反向转运体的作用。此外，Nark2 敲除突变体在亚硝酸盐出口方面没有缺陷。Nark2 被提议为 I 型 H(+)/硝酸盐同向转运体。进行了定点突变，改变了 NarK2 的 23 个氨基酸。这允许鉴定该转运体的重要区域和氨基酸。其中 5 个突变体对硝酸盐运输无活性，7 个产生活性降低，11 个突变体保留野生型活性。Nark2 被一种未知机制在氧气存在下失活。然而，包括突变半胱氨酸在内的突变体在对氧水平的反应中没有改变。枯草芽孢杆菌的同化硝酸盐转运蛋白 NasA 在 M. tuberculosis NarK2 突变体中表达。它在有氧孵育期间保持活性，表明氧控制的关键点是 NarK2。
