RUI: Petobactin Mediated Iron-Acquisition in Bacillus cereus Group Microbes

环境条件调节细菌中基因的表达。这些复杂的调节过程使微生物能够在各种条件下生存。这些线索可以从温度到pH值和营养可用性。在蜡状芽孢杆菌组的成员中，铁的可用性已被证明可以调节与许多途径相关的基因，包括铁的获得。铁在蜡状芽孢杆菌群微生物中的摄取已成为鉴定毒力因子的感兴趣的领域。这一组包括人类病原体蜡状芽孢杆菌、昆虫病原体苏云金芽孢杆菌和人畜共患病原体炭疽芽孢杆菌。三价铁和血红素铁来源已被确定为对许多这些微生物的生长和毒性至关重要。 B的一种重要的铁吸收机制。蜡状菌群微生物是铁载体岩杆菌素。这种铁载体是一种独特的化合物，仅在蜡状芽孢杆菌群微生物和海洋海洋生物中分离出来。这种小的铁螯合剂的水平可以通过生长温度和铁的可用性来改变。虽然大多数铁载体由铁摄取调节剂调节，但在petrobactin生产中没有明确定义的调节机制，因为它缺乏petrobactin操纵子内的铁调节剂序列。 该研究的重点将解决控制铁吸收的信号机制，以响应铁和温度。这项研究将涉及图格鲁学院的本科生，这是密西西比一所历史悠久的黑人学院。拟议的项目将与Tougaloo学院自然科学部的计划合作，通过加强本科生的研究经验和提高课程，增加非洲裔美国人的STEM管道。技术描述 微生物中的铁可用性已被证明可以调节基因表达。在蜡样芽孢杆菌组微生物中，已经检测到几种铁获取系统，包括铁载体介导的运输和血红素摄取系统。 蜡状芽孢杆菌群微生物产生两种含儿茶酚的铁载体，芽孢杆菌素和石油杆菌素。虽然bacillibactin通过三价铁摄取调节由铁的可用性调节，但在petrobactin操纵子内没有鉴定出这样的调节机制。拟议的研究将寻求阐明控制petrobactin生产的机制，以响应环境信号，包括铁和温度。目的一是鉴定petrobactin的调控基因。 转座子诱变将用于鉴定蜡状芽孢杆菌和苏云金芽孢杆菌突变体不能调节petrobactin的生产。携带mini-Tn 10转座子元件的温度敏感质粒pIC 333将用于转化B。cereus和B. 苏云金杆菌抗生素抗性突变体将在含转铁蛋白的培养基中培养12小时，然后在链黑菌素存在下培养过夜。能够利用转铁蛋白铁的微生物（预计由petrobactin介导）将被链黑菌素杀死。活细胞将在含抗生素的复合培养基上分离。 将使用Chrome天青S测定、Arnow测定和薄层色谱法表征分离的突变体中的铁载体和儿茶酚产生。基因组测序和互补将用于鉴定和确认参与petrobactin生产的序列。目标二将集中于表征B。在铁和温度生长条件下的蜡状菌群成员表型。 我们已经观察到，当细胞在不同的铁源和不同的温度下培养时，petrobactin生物合成基因的差异调节。 环境和ATCC B。蜡状菌群微生物将在充满铁和耗尽铁的条件下培养24小时。将在特定时间点取出等分试样，以测量各个生长阶段期间的petrobactin产量。 在每个时间点，还将测量细胞活力和孢子浓度。转录谱将用于鉴定与B中的铁和生长温度相关的独特特征。cereus和B.苏云金杆菌本研究将为深入了解B的复杂调控机制提供帮助。蜡状菌群微生物对环境线索的反应。

Environmental conditions regulate the expression of genes in bacteria. These complex regulatory processes afford microbes the ability to survive under various conditions. These cues can range from temperature to pH and nutritional availability. In members of the Bacillus cereus group, iron availability has been demonstrated to regulate genes associated with numerous pathways, including iron acquisition. Iron uptake in Bacillus cereus group microbes have emerged as areas of interest in the identification virulence factors. This group includes the human pathogen Bacillus cereus, the insect pathogen Bacillus thuringiensis, and the zoonotic pathogen Bacillus anthracis. Ferric iron and heme iron sources have been identified as critical for the growth and virulence of many of these microbes. An important iron uptake mechanism for the B. cereus group microbes is the siderophore petrobactin. A unique compound, this siderophore has only been isolated within the Bacillus cereus group microbes and the marine Marinobacter species. The level of this small, ferric chelator can be altered by both growth temperature and iron availability. While most siderophore are regulated by the ferric uptake regulator, there are no clearly defined regulatory mechanisms involved in petrobactin production, as it lacks the ferric regulator sequence within the petrobactin operon. The focus of the study will address the signaling mechanisms that govern iron uptake in response to iron and temperature. This research will involve undergraduates from Tougaloo College, a historically black college in Mississippi. The proposed project will work in concert with the Tougaloo College Natural Sciences Division's plan to increase the African-American STEM pipeline by strengthening the undergraduate research experience and enhancing the curriculum.TECHNICAL DESCRIPTION. Iron availability in microbes has been demonstrated to regulate gene expression. In the Bacillus cereus group microbes, several iron acquisition systems have been detected, including siderophore mediated transport and heme uptake systems. Bacillus cereus group microbes produce two catechol containing siderophores, bacillibactin and petrobactin. While bacillibactin is regulated by iron availability via the ferric iron uptake regulation, no such regulatory mechanism is identified within the petrobactin operon. The proposed study will seek to elucidate the mechanisms governing petrobactin production in response to environmental signals, including iron and temperature. Aim one will focus on identifying petrobactin regulatory genes. Transposon mutagenesis will be employed to identify Bacillus cereus and Bacillus thuringiensis mutants not capable of regulating petrobactin production. The temperature sensitive plasmid pIC333, which carries the mini-Tn10 transposon element, will be used to transform B. cereus and B. thuringiensis. Antibiotic resistant mutants will be cultured for 12 hours in transferrin containing medium and then cultured overnight in the presence of streptonigrin. Microbes capable of utilizing transferrin iron, which is predicted to be mediated by petrobactin, will be killed by the streptonigrin. Viable cells will be isolated on complex, antibiotic containing medium. The chrome azurol S assay, the Arnow assay and thin layer chromatography will be used to characterize siderophore and catechol production in isolated mutants. Genome sequencing and complementation will be used to identify and confirm sequence involvement in petrobactin production. Aim two will focus on characterizing B. cereus group member phenotypes under iron and temperature growth conditions. We have observed that petrobactin biosynthesis genes are differentially regulated when cells were cultured in different iron sources and under different temperatures. Environmental and ATCC B. cereus group microbes will be cultured in iron replete and deplete conditions for 24 hours. Aliquots will be removed at specific time points to measure petrobactin production during the various growth phases. At each of the time points, cell motility and spore concentration will also be measured. Transcriptional profiling will be used to identify unique signatures associated with iron and growth temperature in B. cereus and B. thuringiensis. This study will provide insight into the complex regulatory mechanisms of B. cereus group microbes in response to environmental cues.