INDUCIBLE ANTIBIOTIC RESISTANCE

这项研究计划的长期总目标是研究 基因的表达被抗生素诱导或修饰的调控。 重点是一组赋予抗生素抗性的基因。 的 该提案的具体目标是研究 红霉素诱导的对大环内酯类、林可酰胺类的耐药性， 金黄色葡萄球菌赋予链阳菌素B（MLS）抗生素 质粒pE 194。 一种可诱导的23 S rRNA甲基化酶通过以下方式赋予抗性： 甲基化23 S rRNA中的特定腺嘌呤残基，并且其合成是 红霉素诱导的。 MLS控制的因素 耐药性可以根据它们是否作用于以下方面进行分类：（1） 信息合成的速率（转录控制），（2） 信息的降解（mRNA稳定性），（3） 消息的利用率（翻译控制），以及（4） 可用于转录信息的质粒DNA拷贝 （质粒拷贝数对照）。 这个问题的两个新方面， 作为本工作的重点包括：（1）研究 ErmC甲基化酶，酶家族的一员， 赋予MLS抗性，其23 S rRNA底物;和（2） pE 194 Cop蛋白与其推定结合位点之间的相互作用 在质粒DNA中，一种被认为调节 通过控制质粒拷贝数对红霉素产生耐药性。 上 在这种情况下，我们将试图区分结构方面的 甲基化酶的氨基酸序列，负责的能力， 识别23 S rRNA底物，并将其与其他潜在的 底物，如16 S rRNA，以及鉴定的部分， 负责催化的序列（即，甲基化酶）活性。 在 第二种情况下，我们将研究最近的一个 鉴定的质粒pE 194特异性Cop蛋白及其推测的靶点 质粒pE 194 DNA -具体地问，Cop蛋白结合在哪里， 这种结合最终如何有助于控制 质粒DNA？

The long-term general objective of this research program is to study regulation of genes whose expression is induced or modified by antibiotics. The emphasis is on a group of genes that confer antibiotic resistance. The specific aims of this proposal are to study regulation of erythromycin-inducible resistance to the macrolide, lincosamide, and streptogramin B (MLS) antibiotics conferred by Staphylococcus aureus plasmid pEl94. An inducible 23S rRNA methylase confers resistance by methylating a specific adenine residue in 23S rRNA, and its synthesis is induced by erythromycin. Factors that enter into the control of MLS resistance can be classified according to whether they act on: (1) the rate of synthesis of the message (transcriptional control), (2) the rate of degradation of the message (mRNA stability), (3) the efficiency of utilization of the message (translational control), and (4) the number of plasmid DNA copies that are available from which to transcribe the message (plasmid copy number control). Two new aspects of the problem that will serve as the focus of the present work include: (1) a study of the interaction between the ErmC methylase, a member of the family of enzymes that confer MLS resistance, with its 23S rRNA substrate; and (2) a study of the interaction between the pEl94 Cop protein and its putative binding site in plasmid DNA, a reaction that is proposed to regulate the expression of erythromycin resistance by controlling plasmid copy number. In the first case, we shall attempt to distinguish between structural aspects of the methylase amino acid sequence that are responsible for the ability to recognize the 23S rRNA substrate and distinguish it from other potential substrates such as 16S rRNA as well as to identify the parts of the sequence that are responsible for catalytic (i.e., methylase) activity. In the second case, we shall study the interaction between a recently identified Cop protein specified by plasmid pEl94 and its presumed target plasmid pEl94 DNA - asking specifically, where does Cop protein bind and how does this binding ultimately contribute to control the concentration of plasmid DNA?