GENETIC SYSTEMS TO STUDY VIRULENCE IN BACTEROIDES

脆弱类杆菌是一种重要的人类病原菌。 尝试评估 并研究可能导致这种病毒毒力的决定因素， 由于缺乏合适的遗传系统， 分析. 我们建议继续发展遗传系统， 这个有机体有两个特定的目标。 调查的决定因素 抗生素耐药性及其在B内的转移。fragilis和B. fragilis到E.杆菌 我们将表征pBFTM 10，一个15 kb的转移因子 在B中编码Clnr。fragilis和Tetr;杆菌 这些阻力 是由转座子Tn 4400携带的，Tn 4400在E.杆菌 研究 被描述为确定该元件的换位特性， 在每一端以直接重复形式存在的IS元件。 研究 将致力于了解“四环素转移因子” 促进质粒和转座子在B之间转移。fragilis菌株 转化为E.杆菌 我们建议的第二个方面是发展一个 遗传系统来分析B中的毒力因子。脆弱的 这将 包括分离选定表型特征的突变体， 通过使用一种或多种分离方法分离编码决定簇的基因， B染色体文库。fragilis克隆于E.杆菌 TN 4400将是 在B中用作诱变剂。fragilis帮助定位染色体基因 怀疑是毒力因子。 需要研究的决定因素是 亚胺培南和四环素耐药性、过氧化氢酶产生和神经氨酸酶 生产 将研究使亚胺培南失活的β-内酰胺酶 先 B. fragilis将是 研究，我们将尝试开发一种探针来研究的机制， Tetr转移。 过氧化氢酶在B. fragilis to 将评估在氧合组织中存活。 最后， 神经氨酸酶在B期间的组织破坏中起作用。脆弱菌感染 将进行调查。 一旦突变体被分离出来， 编码的决定因素，然后重要的问题， 表达可以解决。 这些研究的结果应该提供一个 更清晰地展示了DNA是如何在B中转移的。fragilis和帮助 提供了一个遗传系统，研究在这一重要的毒力因子 病原体

Bacteroids fragilis is an important human pathogen. Attempts to evaluate and investigate determinants that may contribute to the virulence of this organism have been hampered by the absence of suitable systems of genetic analysis. We propose to continue the development of genetic systems in this organism with two specific aims. To investigate the determinants of antibiotic resistance and their transfer within B. fragilis and from B. fragilis to E. coli. We will characterize pBFTM10, a 15 kb transfer factor that encodes Clnr in B. fragilis and Tetr in E. coli. These resistances are carried on a transposon, Tn4400, that functions in E. coli. Studies are described to determine the transposition properties of this element and the IS elements which are present as direct repeats at each end. Studies will be directed towards understanding the "tetracycline transfer factor" that promotes plasmid and transposon transfer between B. fragilis strains and into E. coli. The second aspect of our proposal is to develop a genetic system to analyze virulence factors in B. fragilis. This will involve isolating mutants of selected phenotypic characteristics and then isolating the gene(s) encoding the determinants by making use of a chromosome library of B. fragilis cloned in E. coli. Tn4400 will be employed as a mutagen in B. fragilis to help locate chromosomal genes suspected to be virulence factors. The determinants to be investigated are imipenem and tetracycline resistance, catalase production and neuraminidase production. The beta-lactamase that inactivates imipenem will be studied first. The genes encoding tetracycline resistance in B. fragilis will be studied and we will attempt to develop a probe to study the mechanism of Tetr transfer. The role of catalase in the ability of B. fragilis to survive in oxygenated tissue will be assessed. Finally, the role that neuraminidase plays in tissue destruction during B. fragilis infections will be investigated. Once the mutants have been isolated and the gene(s) encoding the determinant are located then important questions related to expression can be addressed. The results of these studies should give a clearer picture of how DNA is transferred in B. fragilis and help to provide a genetic system to study virulence factors in this important pathogen.