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

环境条件调节细菌中基因的表达。这些复杂的调控过程为微生物提供了在各种条件下生存的能力。这些信号的范围从温度到PH值和营养供应。在蜡状芽孢杆菌组的成员中，铁的可获得性已经被证明调节与许多途径相关的基因，包括铁的获得。蜡状芽孢杆菌菌群中的铁摄取已成为鉴定毒力因子的重要领域。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病原体炭疽芽孢杆菌。铁铁和血红素铁源已被确定为对许多这些微生物的生长和毒力至关重要。蜡样芽孢杆菌类微生物的一个重要的铁摄取机制是铁载体Petrobactin。作为一种独特的化合物，这种铁载体只在蜡状芽孢杆菌组微生物和海洋水生细菌物种中分离出来。这种小的铁络合剂的水平可以通过生长温度和铁的有效性来改变。虽然大多数铁载体受铁摄取调节器的调节，但在Petrobactin的生产中没有明确定义的调节机制，因为它缺乏Petrobactin操纵子中的铁调节器序列。这项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Tougaloo学院的本科生，这是密西西比州的一所历史悠久的黑人大学。拟议的项目将与Tougaloo学院自然科学部通过加强本科生研究经验和改进课程来增加非裔美国人STEM管道的计划相一致。微生物中铁的有效性已被证明可以调节基因表达。在蜡状芽孢杆菌中，已经检测到几个铁获取系统，包括铁载体介导的运输系统和血红素吸收系统。蜡状芽孢杆菌类微生物产生两种含儿茶酚的铁载体，即杆状杆菌蛋白和石油蛋白。虽然杆状杆菌蛋白通过铁摄取调节来调节铁的可获得性，但在Petrobactin操纵子中没有发现这种调节机制。这项拟议的研究将试图阐明控制Petrobactin产生的机制，以响应包括铁和温度在内的环境信号。Aim One将专注于识别Petrobactin调控基因。转座子突变将被用来鉴定蜡状芽孢杆菌和苏云金芽孢杆菌突变株，这些突变株不能调节Petrobactin的生产。携带微型Tn10转座子元件的温度敏感质粒pIC333将用于转化蜡样芽孢杆菌和苏云金芽孢杆菌。抗生素抗性突变株将在含有转铁蛋白的培养基中培养12小时，然后在链球蛋白存在的情况下培养过夜。能够利用转铁蛋白铁的微生物将被链球蛋白杀死，而转铁蛋白铁预计是由Petrobactin介导的。活细胞将在复杂的含有抗生素的培养液中分离。用铬天青S法、Arnow法和薄层层析法对分离到的突变株的铁载体和儿茶酚的产生进行了研究。基因组测序和互补将被用于识别和确认序列参与Petrobactin的生产。目的二将重点研究蜡样芽孢杆菌在铁和温度条件下的群体成员表型。我们观察到，当细胞在不同的铁源和不同的温度下培养时，Petrobactin生物合成基因的调控是不同的。环境和ATCC蜡样芽胞杆菌组的微生物将在铁充足和缺铁的条件下培养24小时。将在特定的时间点去除等量，以测量不同生长阶段的Petrobactin产量。在每个时间点，细胞活力和孢子浓度也将被测量。转录图谱将被用来识别与蜡样芽孢杆菌和苏云金芽孢杆菌中的铁和生长温度相关的独特信号。这项研究将为蜡样芽孢杆菌群微生物对环境线索的反应提供复杂的调控机制。

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.