Novel Plasmids for Porphyromonas Post-Genomic Research

描述：牙龈卟啉单胞菌被认为是牙周炎的主要病原体，牙周炎是一种在成年人中流行的牙齿支持结构的破坏性疾病。对牙龈假单胞菌的分子遗传学研究已经开始为了解该菌如何在口腔定植并引起病理奠定了基础。现在已经确定了牙龈假单胞菌W83基因组的核苷酸序列，这种理解可能会有戏剧性的增加。到目前为止，使用从其他细菌系统改编的一些工具对这种有机体进行遗传分析是可能的。一些基因工具已经被专门用于紫球藻，但如果我们要充分利用这种病原体的基因组核苷酸序列，那么系统的构建质粒载体是至关重要的。我们建议通过开发、测试和分发深入的牙龈卟啉单胞菌基因组后分析所需的质粒构建来实现这一点。具体地说，我们将：1.鉴定和鉴定来自口腔黑色素革兰氏阴性厌氧菌的天然和重组质粒，因为它们能够稳定地支持牙龈假单胞菌的染色体外复制；2.设计、构建、表征和测试几种用于牙龈假单胞菌分析的可靠穿梭载体；3.使用4种不同的非克隆牙龈假单胞菌菌株，评估可能影响它们作为宿主进行分子遗传学分析的细胞和分子变量；4.将这些新的遗传工具和知识分发给研究牙龈卟啉单胞菌和其他黑色色素革兰氏阴性厌氧菌的工作人员。

DESCRIPTION: Porphyromonas gingivalis is recognized as a prime etiological agent in periodontitis, a destructive disease of the supporting structures of the teeth which is prevalent in adults. Molecular genetic research on P. gingivalis has begun to build a foundation of understanding concerning how this organism colonizes the oral cavity and causes pathology. A dramatic increase in this understanding is likely now that the nucleotide sequence of the genome of P. gingivalis W83 has been determined. To date, genetic analyses of this organism have been possible using a number of tools adapted from other bacterial systems. A few genetic tools have been specifically created for use in Porphyromonas sp., but the systematic construction of plasmid vectors is critically needed if we are to fully exploit the genomic nucleotide sequence of this pathogen. We propose to accomplish this by developing, testing, and distributing plasmid constructs needed for the in-depth postgenomic analyses of Porphyromonas gingivalis. Specifically, we shall: 1. Characterize and native and recombinant plasmids from oral black pigmented Gram negative anaerobes for their ability to stably support extrachromosomal replication in P. gingivalis; 2. Design, build, characterize and test several robust shuttle vectors for the analysis of P. gingivalis; 3. Using 4 different non-clonal P. gingivalis strains, evaluate cellular and molecular variables that may affect their suitability as hosts for molecular genetic analyses; 4. Distribute these new genetic tools and knowledge about their use to workers studying Porphyromonas gingivalis and other black pigmented Gram-negative anaerobes.