Regulation of hemolysin genes in Bacillus

描述（申请人提供）：炭疽的病原体炭疽芽孢杆菌的毒力因子研究主要集中在质粒编码胶囊以及致死性和水肿毒素。很少有关于成孔溶血素的研究报道。研究有限的一个主要原因是，尽管炭疽杆菌的基因组中存在溶血素基因，但人们普遍认为炭疽杆菌是一种非溶血病原体。然而，最近的研究表明，炭疽杆菌在一定条件下可以产生溶血素。另一种非溶血性芽孢杆菌也携带假定的溶血素基因。本研究计划的长期目标是研究溶血素是否在炭疽杆菌的致病性中发挥作用，如从巨噬细胞成功逃逸到细胞外环境中。拟议的研究将旨在了解毒力因子如溶血素是如何在环境变化的反应中被诱导的，以及作为调控基因单一突变的结果。“潜伏”毒力基因的激活可能对各种细菌感染产生强烈影响。该提案的具体目的是：1)识别激活？枯草芽孢杆菌-溶血素基因的基因组定位和DNA序列分析。2)表征？-溶血素基因通过调控突变和氧限制利用转录lacZ融合溶血素基因的启动子。3)探讨硝酸盐对？-溶血素基因在枯草芽孢杆菌和炭疽芽孢杆菌中的好氧/厌氧表达。硝酸盐依赖诱导需要硝酸还原酶活性的假设将通过使用枯草芽孢杆菌硝酸还原酶突变体和测量好氧炭疽芽孢杆菌培养物的硝酸还原酶活性来检验。将编码crp家族转录调节因子的炭疽芽孢杆菌基因引入枯草芽孢杆菌，以确定该调节因子是否能够在有氧条件下激活呼吸硝酸盐还原酶操纵子的表达，从而导致好氧诱导？-枯草芽孢杆菌中的溶血素。

DESCRIPTION (provided by applicant): Studies of virulence factors of Bacillus anthracis, the causative agent of anthrax, have mainly focused on plasmid-encoded capsule as well as lethal and edema toxins. Very few studies have been reported on pore-forming hemolysins. A primary reason for the limited studies is that B. anthracis is generally accepted as a nonhemolytic pathogen, although hemolysin genes are present in the genome. However, recent studies have shown that B. anthracis can produce hemolysin under certain conditions. Another nonhemolytic sporeformer Bacillus subtilis also carries putative hemolysin genes. The long-term objectives of the research plan are to investigate whether hemolysin plays a role in pathogenicity of B. anthracis such as successful escape from macrophage into extracellular milieu. The proposed studies will aim at understanding how virulence factors such as hemolysins are induced in response to environmental changes and as a result of a single mutation in regulatory genes. The activation of 'latent' virulence genes could have a strong impact on various bacterial infections. The specific aims of the proposal are: 1) To identify regulatory mutation(s) that activate ?-hemolysin gene(s) in B. subtilis by genome mapping and DNA sequence analysis. 2) To characterize the Induction mechanism of the ?-hemolysin gene by the regulatory mutation and by oxygen limitation using a transcriptional lacZ fusion to the promoter of the hemolysin gene. 3) To investigate the stimulatory effect of nitrate on anaerobic expression of ?-hemolysin gene in B. subtilis and on aerobic/anaerobic expression in B. anthracis. The hypothesis that nitrate-dependent induction requires nitrate reductase activity will be examined by using a B. subtilis nitrate reductase mutant and measuring nitrate reductase activity of aerobic B. anthracis cultures. B. anthracis gene(s) encoding putative transcriptional regulator(s) of the CRP-family will be introduced into B. subtilis to determine whether the putative regulator is able to activate expression of the respiratory nitrate reductase operon under aerobic conditions, and thus leading to the aerobic induction of ?-hemolysin in B. subtilis.