WIDE VARIATION IN ANTIBIOTIC RESISTANCE PROTEINS IDENTIFIED BY FUNCTIONAL METAGE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 土壤微生物中存在的大多数抗生素耐药性基因仍未被探索，因为大多数环境微生物无法培养。 直到最近，通过使用不依赖于培养的方法来鉴定这些基因才变得可行。 我们在大肠杆菌宿主中筛选了土壤宏基因组DNA文库中可以赋予对卡那霉素、庆大霉素、利福平、甲氧苄啶、氯霉素或四环素抗性的基因。 筛选发现了41个基因，编码8个蛋白质家族的新型蛋白质变体，包括氨基糖苷乙酰转移酶、利福平ADP核糖基转移酶、二氢叶酸还原酶和转运蛋白。 同一家族的几种蛋白质彼此相差很大，但具有相当的抗性。 例如，在成对比较中共享至多44%氨基酸序列同一性的五种二氢叶酸还原酶在赋予甲氧苄啶抗性方面是等同的。 我们确定了氨基糖苷类乙酰转移酶和转运蛋白的变体，这些变体在它们赋予耐药性的药物的特异性方面不同。 我们还发现了蛋白质结构的广泛变化。 两种形式的利福平ADP-核糖基转移酶，一种是另一种的两倍大小，在赋予利福平抗性方面同样有效，尽管短形式的表达水平低得多。 功能性宏基因组筛选提供了对抗生素抗性蛋白序列的大变异性的深入了解，揭示了保留蛋白质功能的不同变体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Most genes for antibiotic resistance present in soil microbes remain unexplored because most environmental microbes cannot be cultured. Only recently has the identification of these genes become feasible through the use of culture-independent methods. We screened a soil metagenomic DNA library in an Escherichia coli host for genes that can confer resistance to kanamycin, gentamicin, rifampin, trimethoprim, chloramphenicol, or tetracycline. The screen revealed 41 genes that encode novel protein variants of eight protein families, including aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, and transporters. Several proteins of the same family deviate considerably from each other, yet confer comparable resistance. For example, five dihydrofolate reductases sharing at most 44% amino acid sequence identity in pairwise comparisons were equivalent in conferring trimethoprim resistance. We identified variants of aminoglycoside acetyltransferases and transporters that differ in the specificity of the drugs for which they confer resistance. We also found wide variation in protein structure. Two forms of rifampin ADP-ribosyltransferases, one twice the size of the other, were similarly effective at conferring rifampin resistance, although the short form was expressed at much lower level. Functional metagenomic screening provides insight into the large variability in antibiotic resistance protein sequences, revealing divergent variants that preserve protein function.