Expanding the Pathways of Manganese Homeostasis: Role of a Small Manganese Chaperone Protein, MntS. Escherichia coli str. K-12 substr. MG1655

Escherichia coli possesses >65 small proteins of <50 amino acids, many of which are uncharacterized. We have identified a new small protein, MntS, involved in manganese homeostasis. Manganese is a critical micronutrient, serving as an enzyme cofactor and protecting against oxidative stress. Yet manganese is toxic in excess and little is known about its function in cells. Bacteria carefully control intracellular manganese levels using the transcription regulator MntR. Before this work, mntH, which encodes a manganese importer, was the only gene known to respond to manganese via MntR repression in E. coli K12. We demonstrated that mntS is another member of the MntR manganese regulon. We also identified yebN, which encodes a putative manganese efflux pump, as the first gene positively regulated by MntR in Enterobacteria. Since MntS is expressed when manganese levels are low, causes manganese sensitivity when overexpressed, and binds manganese, we propose that MntS may be a manganese chaperone. This study reveals new factors involved in manganese regulation and metabolism and expands our knowledge of how small proteins function. Overall design: Two E. coli strains, MG1655 (wild type) and GSO458 (Delta-mntR) were grown to OD600 ~ 0.5 in M9 glucose media at 37 ºC and treated with 10 microM MnCl2. In the first experiment, this incubation with 10 microM MnCl2 was for 60 min and in the second experiment, it was for 10 min. RNA was extracted using the hot phenol method and cDNA prepared and hybridized according the manufacturer's instructions (Affymetrix).

大肠杆菌（Escherichia coli）基因组编码超过65种长度小于50个氨基酸的小蛋白，其中多数尚未被功能注释。本研究鉴定出一种新型小蛋白MntS，其参与锰离子稳态（manganese homeostasis）调控。锰离子是关键的微量营养素，既可作为酶的辅因子，又能抵御氧化应激；但过量的锰离子会产生细胞毒性，目前学界对其在细胞内的功能仍知之甚少。细菌借助转录调控因子MntR严格调控细胞内的锰离子水平。在本研究开展前，大肠杆菌K12菌株中仅有一种已知基因mntH——其编码一种锰离子转运蛋白——可通过MntR的转录抑制响应锰离子信号。本研究证实，mntS是MntR调控的锰离子调节子（manganese regulon）的另一成员。此外，本研究还鉴定出yebN——其编码一种假定的锰离子外排泵——为肠杆菌科中首个受MntR正向调控的基因。鉴于MntS在锰离子水平较低时表达、过量表达时会导致细胞对锰离子敏感且可结合锰离子，我们推测MntS可能是一种锰离子分子伴侣（manganese chaperone）。本研究揭示了参与锰离子调控与代谢的新型因子，拓展了学界对小蛋白功能机制的认知。
实验整体设计：选取两种大肠杆菌菌株——MG1655（野生型）与GSO458（Δ-mntR缺失株）——在37℃条件下于M9葡萄糖培养基中培养至OD600约为0.5，随后用10 μM氯化锰（MnCl2）处理。首次实验中，菌株经10 μM MnCl2孵育60分钟；第二次实验则孵育10分钟。采用热酚法提取总RNA，按照Affymetrix公司的试剂盒说明书制备cDNA并完成杂交实验。