Kehrenberg Corinna, Schwarz Stefan, Jacobsen Lene, Hansen Lykke H, Vester Birte
Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft, Höltystrasse 10, 31535 Neustadt-Mariensee, Germany.
Mol Microbiol. 2005 Aug;57(4):1064-73. doi: 10.1111/j.1365-2958.2005.04754.x.
The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant. Comparative investigation of E. coli with and without a plasmid-encoded Cfr showed a decreased drug binding to ribosomes in the presence of Cfr. As chloramphenicol/florfenicol and clindamycin have partly overlapping drug binding sites on the ribosome, the most likely explanation is that Cfr modifies the RNA in the drug binding site. This hypothesis was supported by drug footprinting data that showed both a decreased drug binding and an enhanced reverse transcriptase stop at position 2504, which corresponds to a modification at position A2503 at the drug binding site. A 45 n long RNA fragment containing the appropriate region was isolated and MALDI-TOF mass spectrometry in combination with tandem mass spectrometry showed an additional methylation at position A2503. Moreover, reduced methylation was detected at nucleotide C2498. The results show that Cfr is an RNA methyltransferase that targets nucleotide A2503 and inhibits ribose methylation at nucleotide C2498, thereby causing resistance to chloramphenicol, florfenicol and clindamycin.
松鼠葡萄球菌中cfr的基因产物赋予葡萄球菌属和大肠杆菌对氯霉素、氟苯尼考和克林霉素的抗性。Cfr与任何其他已知的氯霉素抗性决定因素都不相似。对携带和不携带质粒编码Cfr的大肠杆菌进行的比较研究表明，在存在Cfr的情况下，药物与核糖体的结合减少。由于氯霉素/氟苯尼考和克林霉素在核糖体上有部分重叠的药物结合位点，最可能的解释是Cfr修饰了药物结合位点中的RNA。药物足迹数据支持了这一假设，该数据显示药物结合减少，并且在对应于药物结合位点A2503位置修饰的2504位处逆转录酶终止增强。分离出一个包含适当区域的45个核苷酸长的RNA片段，基质辅助激光解吸电离飞行时间质谱结合串联质谱显示在A2503位置有额外的甲基化。此外，在核苷酸C2498处检测到甲基化减少。结果表明，Cfr是一种靶向核苷酸A2503并抑制核苷酸C2498处核糖甲基化的RNA甲基转移酶，从而导致对氯霉素、氟苯尼考和克林霉素的抗性。
