WIDE VARIATION IN ANTIBIOTIC RESISTANCE PROTEINS IDENTIFIED BY FUNCTIONAL METAGE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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然没有被发现，因为大多数环境微生物不能培养。直到最近，通过使用与培养无关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元基因组DNA文库，以寻找能够对卡那霉素、庆大霉素、利福平、甲氧苄啶、氯霉素或四环素产生抗性的基因。筛选出41个基因，编码8个蛋白家族的新蛋白变体，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之间有很大的差异，但具有类似的抗性。例如，五种二氢叶酸还原酶在两两比较中氨基酸序列同源性最高为44%，在赋予甲氧普林耐药性方面是相同的。我们确定了氨基糖苷乙酰转移酶和转运体的变种，它们在导致耐药的药物的特异性上有所不同。我们还发现蛋白质结构有很大的差异。两种形式的利福平ADP-核糖基转移酶，一种大小是另一种的两倍，在诱导利福平耐药性方面同样有效，尽管短形式的表达水平要低得多。功能元基因组筛选提供了对抗生素耐药蛋白序列的巨大变异性的洞察，揭示了保存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.