EVOLUTION OF DNA UPTAKE IN HAEMOPHILUS AND NEISSERIA

描述(改编自申请者摘要)：本项目的中心是 流感嗜血杆菌DNA摄取序列分析。它包括一种混合物 模型实验、基因组分析和实验工作。建模将会 试图回答这样一个问题，即 从其他细菌中获得核苷酸足以证明 细菌DNA转化能力的进化。在一个单独的 方法，将开发一种模型来测试DNA的受体 带有序列偏差的转换将导致USS的积累 基因组中的序列。建模还将检查USS的分布 并试图确定它是否是随机的。基因组学 将对放线杆菌的基因组序列进行分析 伴生放线菌、淋球菌和脑膜炎奈瑟菌 确定USS的相同组织模式是否适用于这些 微生物对流感嗜血杆菌的影响。这些分析将区分单个 以及USS的重复副本及其在ORF内部和之间的分布。这个 还将扫描基因组，以确定它们是否包含 跨越物种障碍的异乎寻常的同源性。实验工作将检验 DNA转化致死是否仅限于流感嗜血杆菌Rd株 以确定致命性是否是普遍现象。粘液中含有 DNA，因此分析将决定DNA是否可以从 以及流感嗜血杆菌是否正常生活在粘液中 抑制或不抑制变换。USS的专一性将通过以下方式进行测试 合成的50聚体USS在27个位置中的每个位置都与 一致性测序和检测它们对DNA的摄取和抑制能力 领悟。序列不同的较大DNA片段也将通过以下方式获得 并进行了PCR扩增和检测。美国航空母舰对DNA摄取的绝对必要性 将使用比以前使用的更长的孵化时间进行测试。尝试将 被用来鉴定细菌细胞表面的USS结合蛋白，并 在细胞内。如果一种蛋白质能够被识别，它将被测序。尝试 还将确定USS是否在类核中受到保护。

DESCRIPTION (Adapted from the Applicant's Abstract): This project centers on DNA uptake sequences (USS) in Haemophilus influenzae. It includes a mixture of modeling experiments, genomic analyses and experimental work. Modeling will attempt to answer the question whether the benefits expected from the acquisition of nucleotides from other bacteria are sufficient to justify the evolution of bacterial competence for DNA transformation. In a separate approach, a model will be developed to test whether a receptor for DNA transformation with a sequence bias will result in the accumulation of USS sequences in the genome. Modeling will also examine the distribution of USS around the genome and attempt to determine whether it is random or not. Genomic analyses will be performed with genome sequences of Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae and Neisseria meningitidis to determine whether the same patterns of organization of USS apply to these organisms as to H. influenzae. These analyses will distinguish between single and repeated copies of USS and their distribution within and between ORFs. The genomes will also be scanned to determine whether they contain genes that are unusually homologous across species barriers. Experimental work will test whether killing by DNA transformation is restricted to H. influenzae strain Rd in order to determine whether lethality is a common phenomenon. Mucus contains DNA and therefore analyses will determine whether DNA can be taken up from mucus or not and whether the mucus that H. influenzae normally lives in inhibits transformation or not. The specificity of the USS will be tested by synthesizing 50mer USS that differ by one bp at each of 27 positions from the consensus sequence and testing them for uptake and ability to inhibit DNA uptake. Larger DNA fragments that differ in sequence will also be obtained by PCR amplification and tested. The absolute necessity of an USS for DNA uptake will be tested using longer incubation times than formerly used. Attempts will be made to identify a USS-binding protein on the bacterial cell surface and intracellularly. If a protein can be identified, it will be sequenced. Attempts will also be made to determine whether the USS are protected in nucleoids.