MOLECULAR ANALYSIS OF A POTENTIAL TRANSPORT OPERON IN AA

伴放线放线杆菌(Aa)是一种主要病原菌。 与多种口腔和非口腔感染有关。多数 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方面进行了表征 细菌。例如，百日咳波氏杆菌毒素 解脱(PTL)操纵子与根癌农杆菌毒力 (VIRB)操纵子是两个特征良好的、同源的运输 歌剧。然而，AA运输物质的机制 从细胞内环境到细胞外环境还没有 已澄清。我们已经确定了七个开放阅读框架(ORF) 编码的推定蛋白质与 分离的AA质粒pVT745上的PTL和VIRB运输操纵子 来自AA菌株VT745(最初指定为JP2)。Southern印迹 分析表明，这些pVT745序列中的许多也是 存在于AA株VT747的染色体上。DNA序列 这些染色体编码的基因中的一种 VT747在DNA水平上与该病毒有95%的同源性 相应的pVT745编码基因。其氨基酸序列 这两个基因的预测蛋白质分别为58%和56%。 分别与B.PtlC预测蛋白同源。 百日咳。和47%和50%的同源性，分别与 预测了根癌农杆菌的VirB4蛋白。据推测， 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的运输操纵子，并且该基因 在AA中，产物可能负责大分子的运输。 为解决这一假设，提出了以下具体目标： 1.利用重组DNA技术进行插入灭活 (A)AA株染色体编码的ptlC/virB4基因同源物 VT747和(B)相应的质粒编码基因 VT745，以证明它们在AA运输中的重要性。2.至 (A)确定存在于AA染色体上的其他基因 VT747与潜在的运输基因具有同源性 质粒pVT745，和(B)确定是否鉴定出 染色体和质粒基因分别转录。 或者作为歌剧的一部分。3.克隆并测序 染色体编码的AA基因在特定的目的2中被确定。 大分子分泌相关基因的鉴定 来自AA的可能导致旨在 削弱这种病原微生物的毒力潜力。

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.