MOLECULAR ANALYSIS OF A POTENTIAL TRANSPORT OPERON IN AA

AA 中潜在转运操纵子的分子分析

• 批准号: 6379806
• 负责人: Karen F. Novak
• 金额: $ 0.92万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2001-06-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379806
• 关键词: Actinobacillus actinomycetemcomitans; SDS polyacrylamide gel electrophoresis; bacterial genetics; genetic mapping; genetic strain; intracellular transport; molecular cloning; nucleic acid sequence; open reading frames; operon; plasmids; protein transport; recombinant DNA; transposon /insertion element; virulence

Actinobacillus actinomcetemcomitans (Aa) is a primary pathogen associated with a variety of oral and non-oral infections. Most pathogenic microorganisms release into the host environment virulence factors that enhance their pathogenicity. The molecular basis for this protein export has been characterized in some of these bacteria. For example, the Bordetella pertussis pertussis toxin liberation (ptl) operon and the Agrobacterium tumefaciens virulence (virB) operon are two well characterized, homologous transport operons. However, a mechanism by which Aa transports substances from the intracellular to the extracellular environment has not been elucidated. We have identified seven open reading frames (ORFs) encoding putative proteins homologous with those predicted by the ptl and virB transport operons on the Aa plasmid, pVT745, isolated from Aa strain VT745 (originally designated JP2). Southern blot analyses showed that many of these pVT745 sequences also are present in the chromosome of Aa strain VT747. The DNA sequence of one of one of these chromosomally-encoded genes from strain VT747 shared 95% homology at the DNA level with the corresponding pVT745-encoded gene. The amino acid sequences of the predicted proteins of these two genes showed 58% and 56% homology respectively, with the predicted PtlC protein of B. Pertussis. And 47% and 50% homology, respectively, with the predicted VirB4 protein of A. tumefaciens. It is hypothesized that these Aa plasmid and chromosomal genes are parts of operons similar to the ptl or virB transport operons, and that the gene products may be responsible for macromolecular transport in Aa. The following Specific Aims are proposed to address this hypothesis: 1. To use recombinant DNA technology to insertionally inactivate (a) the chromosomally-encoded ptlC/virB4 gene homolog in Aa strain VT747 and (b) the corresponding plasmid-encoded gene in strain VT745 to demonstrate their importance in transport in Aa. 2. To (a) identify additional genes present on the chromosome of Aa VT747 that share homology with the potential transport genes on the plasmid, pVT745, and (b) to determine whether the identified chromosomal and plasmid genes are transcribed, either individually or as part of an operon. 3. To clone and sequence the chromosomally-encoded Aa genes identified in Specific Aim 2. Identification of genes involved in the secretion of macromolecules from Aa may lead to the development of strategies aimed at diminishing the virulence potential of this pathogenic microorganism.

伴放线放线杆菌（Actinobacillusactinomcetemcomitans，Aa）是引起该病的主要病原菌 与各种口腔和非口腔感染有关。 最 病原微生物释放到宿主环境中 增强其致病性的毒力因子。 分子 这种蛋白质出口的基础已经在其中一些 细菌 例如，百日咳杆菌百日咳毒素 释放操纵子与根癌农杆菌毒力 （virB）操纵子是两个充分表征的同源转运 操纵子 然而，Aa运输物质的机制 从细胞内环境到细胞外环境， 阐明。 我们已经确定了七个开放阅读框架（ORF） 编码推定的蛋白质与那些预测的同源 分离的Aa质粒pVT 745上的ptl和virB转运操纵子 来自Aa菌株VT 745（最初命名为JP 2）。 Southern印迹 分析表明，这些pVT 745序列中的许多也是 存在于Aa菌株VT 747的染色体中。 的DNA序列 这些染色体编码的基因之一， VT 747在DNA水平上与GenBank中的GenBank登录号为： pVT 745编码的基因。 的氨基酸序列 这两个基因的预测蛋白分别为58%和56%， 与预测的B的PtlC蛋白同源。 百日咳与原核生物的同源性分别为47%和50% 预测的A.根瘤菌 它是假设 这些Aa质粒和染色体基因是操纵子的一部分 类似于ptl或virB转运操纵子， 产物可能负责Aa中的大分子运输。 为解决这一假设，提出了以下具体目标： 1. 利用重组DNA技术， (a)Aa株中染色体编码ptlC/virB 4基因同源物 VT 747和（B）菌株中相应的质粒编码基因 VT 745证明了它们在Aa运输中的重要性。 2. 到 (a)鉴定Aa染色体上存在的其他基因 VT 747，其与细胞膜上的潜在转运基因共享同源性。 质粒，pVT 745，和（B）以确定鉴定的 染色体和质粒基因被单独转录， 或者作为操纵子的一部分。 3.为了克隆和测序 染色体编码的Aa基因在特定目标2中鉴定。 参与大分子分泌的基因的鉴定 从Aa可能导致战略的发展， 降低了这种病原微生物的毒力潜力。