分枝杆菌RpsI序列差异对核糖体结构与功能的影响

核糖体结构存在动态调控，其变化与细菌发育、环境适应等过程密切相关。使用NCBI BLAST比对结核分枝杆菌(Mycobacterium tuberculosis)核糖体蛋白RpsI、RpmI和RpmJ与耻垢分枝杆菌(Mycobacterium smegmatis)相应蛋白的氨基酸序列，发现RpsI N端氨基酸序列存在较大差异。为了探究该N端序列差异对核糖体结构与功能的影响，将表达有结核分枝杆菌rpsI基因(rpsI_Rv)的质粒整合至耻垢分枝杆菌基因组中，并利用同源重组的方法敲除耻垢分枝杆菌rpsI基因，以此构建重组菌株。聚合酶链反应(polymerase chain reaction，PCR)结果表明该重组菌株构建成功。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示0.5 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)于16 ℃可诱导表达RpsI_Rv。用纯化的RpsI_Rv制备特异性多克隆抗体，其效价为 1 600 000。反转录PCR 和蛋白质印迹法(Western blot)显示rpsI_Rv在重组菌株中成功表达。测定重组菌株与空载对照菌株在不同温度下的生长曲线，该重组菌株在不同温度下的生长速率未发生改变。采用通用液体倍比稀释法测定作用于核糖体不同位点的5种抗生素最小抑菌浓度(MIC₉₀)，重组菌株对阿米卡星(作用于核糖体小亚基A位点的抗生素)的敏感性升高，提示分枝杆菌RpsI序列差异导致核糖体小亚基A位点附近的结构发生改变，这为分枝杆菌核糖体结构与功能的机制研究提供了数据。

Impacts of Mycobacterium tuberculosis RpsI on ribosomal structure and function in Mycobacterium smegmatis

Recent studies demonstrate that the structure of ribosome was committed to dynamic regulation during cell development and stress adaptation. We used NCBI BLAST to analyze the sequence similarity of ribosomal protein RpsI, RpmI and RpmJ between Mycobacterium tuberculosis and Mycobacterium smegmatis and found a striking variation in RpsI N terminal. To further study the influence of this variation on ribosomal function, we constructed a M. smegmatis recombinant strain in which the original rpsI was replaced by the counterpart from M. tuberculosis. The genotype of this strain was verified by PCR. We estimated the growth rate of recombinant strain and empty-control strain under different temperatures. Universal serial liquid dilution method was applied to measure the minimum inhibition concentration (MIC₉₀) of five different ribosome-targeting antibiotics. We found that the recombinant strain became more sensitive to amikacin which binds to the A site of ribosome. These results demonstrate that the sequence variation in Mycobacterial RpsI may impact the structure of ribosomal A site.