Genetic Functions of an Enterococcal R Factor

描述(逐字摘自申请者摘要)：肠球菌已经成为 作为医院感染的原因而流行。耐药性的高发 对最有效的抗生素(如万古霉素) 造成治疗肠球菌感染的主要问题，以及巨大的 肠球菌耐药决定子库作为载体 将这些基因传播到其他更具致病性的细菌属。这项研究 重点剖析了一种水平遗传转移的机制。 粪肠球菌耐药质粒pCF10。最新奇的 该转移系统的特点是含有pCF10的供体细胞 通过感测到一个 受体分泌的小肽信号(一种称为cCF10的性信息素)。 供体细胞只有在以下情况下才表达质粒转移所需的基因 在生长培养基中检测到外源cCF10。供体细胞的反应 外源信息素，以及防止 在没有外源信息素的情况下传递函数的表达式为 已经详细研究过的复杂过程。肠球菌产生一种 多肽信息素的种类，由疏水性多肽7-8氨基组成 酸度长。不同的质粒家族各自编码一个高度特异的 对单一同源信息素的反应。当单个细胞携带多个 信息素质粒暴露于一种信息素，只有相应的质粒 即使所有的感测系统都非常相似 到目前为止检测到的质粒数。在下一个资助期， 实验将在分子和遗传学的基础上进行特异性研究 信息素反应。具体目标是： 1)确定信息素cCF10专一性的分子基础 与PrgZ(胞外pCF10编码的信息素结合)的相互作用 蛋白质)和PrGX(被认为是cCF10的胞内受体 以组成参与细胞内阶段的分子开关 信息素诱导。 2)确定特定取消内源性cCF10的分子基础 PrgY(pCF10编码膜)在含pCF10供体细胞中的活性 蛋白质)和iCF10(cCF10的一种质粒编码的多肽抑制物)。 3)确定一种新的活性和特异性的分子基础 调控RNA、Qa通过抑制未诱导细胞的结合表达 它与PRGX及阳性对照中编码的Qs RNA的相互作用 PCF10的区域。 4)开始对基因和分子基础进行实验分析 信息素诱导过程中下游步骤的特异性。这些步骤包括 转录后激活转移基因的表达和接合 DNA处理。

DESCRIPTION (Verbatim from Applicant's Abstract): The enterococci have become prevalent as causes of nosocomial infections. The high incidence of resistance of these organisms to the most efficacious antibiotics (e.g., vancomycin) causes major problems in treating enterococcal infections, and the tremendous reservoir of enterococcal resistance determinants serves as a vector for the spread of these genes to other, more pathogenic bacterial genera. This research is focused on dissection of a mechanism of horizontal genetic transfer of the antibiotic resistance plasmid pCF10 in Enterococcus faecalis. The most novel feature of this transfer system is that the pCF10-containing donor cell perceives the presence of potential recipients in its vicinity by sensing a small peptide signal (a sex pheromone called cCF10) excreted by the recipients. The donor cell only expresses genes required for plasmid transfer when exogenous cCF10 is detected in the growth medium. Both the donor cells response to exogenous pheromone, and the negative control system that prevents expression of transfer functions in the absence of exogenous pheromone, are complex processes that have been studied in detail. Enterococci produce a variety of peptide pheromones, consisting of hydrophobic peptides 7-8 amino acids in length. Different families of plasmids each encode a highly specific response to a single cognate pheromone. When a single cell carrying multiple pheromone plasmids is exposed to one pheromone, only the corresponding plasmid is transferred, even though the sensing systems are quite similar for all the plasmids examined to date. In the next funding period, the focus of the experiments will be on the molecular and genetic basis for the specificity of the pheromone response. The specific aims are: 1) Determine the molecular basis for specificity of pheromone cCF10 interactions with PrgZ (the extracellular pCF10-encoded pheromone binding protein), and with PrgX (the putative intracellular receptor for cCF10 believed to comprise the molecular switch involved in the intracellular phase of pheromone induction. 2) Determine the molecular basis for specific abolition of endogenous cCF10 activity in pCF10-containing donor cells by PrgY (a pCF10-encoded membrane protein), and by iCF10 (a plasmid-encoded peptide inhibitor of cCF10). 3) Determine the molecular basis for the activity and specificity of a novel regulatory RNA, Qa in blocking expression of conjugation in uninduced cells via its interaction with PrgX and with the Qs RNA encoded in the positive control region of pCF10. 4) Begin an experimental analysis of the genetic and molecular basis for specificity of the downstream steps in pheromone induction. These steps include post-transcriptional activation of transfer gene expression, and conjugative DNA processing.