Development of Genetic Tools for Clostridium botulinum

描述（由申请人提供）：尽管肉毒杆菌作为人类和动物病原体的重要性及其在生物恐怖主义中的潜在作用，但该生物在细胞过程及其在发病机制中的作用方面仍然非常缺乏特征。这几乎完全是无效遗传系统的结果，特别是整合工具。这种不能轻易产生稳定突变体（定向或随机）是所有梭菌物种的共同特点，严重阻碍了科学界在假设驱动的研究中充分利用梭菌基因组信息的能力。该项目的目标是开发必要的技术来克服这一障碍。目的是：(1)建立整合载体，引入肉毒杆菌基因的靶向突变；(2)寻找随机诱变的有效转座子；(3)评估反义RNA策略对肉毒杆菌基因表达的调节和分析。这些目标将通过美国和英国实验室的互补知识和专业知识来实现。所开发的遗传工具将不仅为肉毒杆菌基因组的深入分析铺平道路，也为其他梭菌基因组的深入分析铺平道路。例如，它们未来的应用将有助于阐明控制生长和毒素产生的生理因素、发病机制以及梭状芽孢杆菌和相关生物特有的发育过程，包括孢子内形成、抗性、萌发和生长。

DESCRIPTION (provided by applicant): Despite the importance of Clostridium botulinum as a human and animal pathogen, and its potential role in bioterrorism, the organism remains extremely poorly characterized with regard to cellular processes and their role in pathogenesis. This is almost entirely a consequence of ineffective genetic systems, and in particular integrational tools. This inability to easily generate stable mutants (directed or random) is common to all clostridial species, and severely hinders the ability of the scientific community at large to fully exploit clostridial genome information in hypothesis driven research. The goal of this project is specifically to develop the necessary enabling technology to overcome this impediment. The aims are: (1) to develop integrational vectors to introduce targeted mutations in genes of C. botulinum; (2) to identify effective transposons for random mutagenesis and (3) to assess antisense RNA strategies for modulation and analysis of C. botulinum gene expression. These aims will be accomplished through the complementary knowledge and expertise of a USA and UK laboratory. The developed genetic tools will pave the way for in depth analysis of not only the C. botulinum genome, but other clostridial genomes too. Their future deployment will, for example, lead to the elucidation of physiological factors that control growth and toxin production, pathogenesis, and developmental processes unique to clostridia and related organisms including endospore formation, resistance, germination and outgrowth.