Development of Genetic Tools for Clostridium botulinum

描述（由申请人提供）：尽管肉毒梭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，但该生物体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。这几乎完全是无效的遗传系统，特别是整合工具的结果。这种不能容易地产生稳定突变体（定向或随机）的情况对于所有梭菌物种都是常见的，并且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克服这一障碍的必要技术。目的是：（1）构建整合载体，将突变基因导入C.肉毒杆菌毒素;（2）鉴定用于随机诱变的有效转座子和（3）评估用于调节和分析C.肉毒杆菌基因表达。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。开发的遗传工具将为深入分析不仅是C。肉毒杆菌基因组，但其他梭菌基因组也是如此。例如，它们的未来部署将导致阐明控制梭菌和相关生物体特有的生长和毒素产生、发病机制和发育过程的生理因素，包括内生孢子形成、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.