摘要
牛支原体(M.bovis)感染与生存依赖于外界微环境提供的各类营养代谢因子.开展M.bovis外源生物素摄取的机制研究,将对M.bovis防控具有极其重要的意义.通过对M.bovis PG45 MbBioY(MBOVPG45_0349)基因进行系统发育分析、分子对接分析,筛选M.bovis PG45 MbBioY突变株,比较PG45和PG45ΔMbBioY的生物素敏感性,异源构建MbBioY功能性克隆验证其功能.结果表明,PG45染色体上存在一种崭新的生物素膜转运蛋白,全长996 bp,GC含量30.95％,蛋白长度为332个氨基酸,是由7个跨膜结构域组成的能量偶合因子(ECF)转运蛋白S成分MbBioY.进一步验证后发现,当M.bovis缺失MbBioY会影响其生长,表现出生物素代谢营养缺陷的表型.通过异源敲入生物素代谢营养缺陷大肠杆菌MG1655,表明MbBioY单独存在时,就能够恢复生物素代谢营养缺陷大肠杆菌缺失株的生理功能.初步证明,MbBioY编码的是能量偶合因子(ECF)转运蛋白的S成分具有摄取外源生物素的能力,该研究结果将补充细菌生物素代谢调控的多样性和复杂性,为M.bovis的有效防控提供独特视角.
Abstract
M.bovis infection and survival depend on various nutritional and metabolic factors pro-vided by the external microenvironment.The study of the mechanism of exogenous biotin uptake by M.bovis will be of substantial significance to M.bovis prevention and control.An examina-tion of the M.bovis PG45 MbBioY(MBOVPG45_0349)gene was used for phylogenetic analysis and molecular docking analysis;Screening of M.bovis PG45 ΔMbBioY mutant;to compare the biotin sensitivity of PG45 and PG45 ΔMbBioY the function of M.bovis was further verified by heterologous construction of MbBioY functional clone.The results showed that a novel biotin membrane transporter MbBioY was found on the chromosome of PG45.It was 996 bp in length,30.95％GC content,and 332 amino acids in length.The protein consists of seven transmem-brane domains.Further verification revealed that MbBioYloss affected M.bovis growth and showed phenotypic biotin metabolism deficiencies.By heterologous knock-in of biotin-metaboliz-ing nutrient-deficient E.coli MG1655,it was shown that MbBioY alone could restore the physi-ological function of the nutrient-deficient E.coli strain with biotin metabolism.It has been pre-liminarily demonstrated that the S component of the energy coupling factor(ECF)transporter encoded by MbBioY can absorb exogenous biotin.This research result will contribute to the di-versity and complexity of bacterial biotin metabolism regulation.M.bovis prevention and control will be provided from an innovative perspective.
基金项目
中国农业科学院科技创新工程(CAAS-ASTIP-JBGS-20210701)
国家自然科学基金面上项目(32172857)
甘肃省科技重大专项(22ZD6NA001)
国家科技期刊平台
NSTL
万方数据