RUI: Petobactin Mediated Iron-Acquisition in Bacillus cereus Group Microbes
RUI:Petobactin 介导的蜡状芽孢杆菌群微生物中的铁获取
基本信息
- 批准号:1412858
- 负责人:
- 金额:$ 35.43万
- 依托单位:
- 依托单位国家:美国
- 项目类别:Standard Grant
- 财政年份:2014
- 资助国家:美国
- 起止时间:2014-07-01 至 2018-06-30
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
环境条件调节细菌基因的表达。这些复杂的调控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。在蜡样芽孢杆菌群的成员中,铁的可用性已被证明可以调节与许多途径相关的基因,包括铁的获取。蜡样芽孢杆菌群微生物的铁摄取已成为鉴定毒力因素的兴趣领域。这一组包括人类病原体蜡样芽孢杆菌、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。铁和血红素铁来源已被确定为许多这些微生物的生长和毒力的关键。蜡样芽孢杆菌群微生物的一个重要的铁摄取机制是铁载体岩杆菌。这是一种独特的化合物,这种铁载体只在蜡样芽孢杆菌群微生物和海洋海洋杆菌中被分离出来。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。虽然大多数铁载体受铁摄取调节剂的调节,但由于在石油蛋白操纵子中缺乏铁调节序列,石油蛋白的产生没有明确的调节机制。本研究的重点是研究铁和温度对铁吸收的信号机制。这项研究将涉及密西西比州历史悠久的黑人学院图加卢学院的本科生。拟议的项目将与Tougaloo学院自然科学部的计划相协调,该计划通过加强本科生研究经验和改进课程来增加非裔美国人的STEM管道。技术描述。铁在微生物中的可用性已被证明可以调节基因表达。在蜡样芽孢杆菌群微生物中,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。蜡样芽孢杆菌群微生物产生两种含铁载体的儿茶酚,即杆菌杆菌素和石油杆菌素。虽然bacillibactin通过铁摄取调节受铁可用性的调节,但在petrobactin操纵子中没有发现这种调节机制。拟议的研究将寻求阐明控制石油蜡蛋白生产的机制,以响应环境信号,包括铁和温度。第一个目标将集中于确定石油蛋白调控基因。转座子诱变将用于鉴定蜡样芽孢杆菌和苏云金芽孢杆菌突变体不能调节石油杆菌蛋白的产生。携带mini-Tn10转座子元件的温敏质粒pIC333将用于转化蜡样芽孢杆菌和苏云金芽孢杆菌。抗生素抗性突变体将在含转铁蛋白的培养基中培养12小时,然后在链霉素存在下培养过夜。能够利用转铁蛋白铁的微生物,预计是由石油杆菌蛋白介导的,将被链霉素杀死。活细胞将在含抗生素的复杂培养基上分离。铬azurrol S测定法、Arnow测定法和薄层色谱法将用于鉴定分离突变体中铁载体和儿茶酚的产生。基因组测序和互补将被用于鉴定和确认与石油蜡蛋白生产有关的序列。目的二将重点研究蜡样芽孢杆菌群体成员在铁和温度生长条件下的表型特征。我们观察到,在不同铁源和不同温度下培养的细胞中,岩杆菌蛋白生物合成基因受到不同的调控。环境和ATCC蜡样芽孢杆菌群微生物将在补铁和缺铁条件下培养24小时。等分将在特定时间点移除,以测量不同生长阶段的石油胶产量。在每个时间点,还将测量细胞运动和孢子浓度。转录谱分析将用于鉴定蜡样芽孢杆菌和苏云金芽孢杆菌中与铁和生长温度相关的独特特征。本研究将深入了解蜡样芽孢杆菌群微生物对环境信号的复杂调控机制。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Bianca Garner其他文献
Transferrin Impacts Bacillus thuringiensis Biofilm Levels
转铁蛋白影响苏云金芽孢杆菌生物膜水平
- DOI:
- 发表时间:
2016 - 期刊:
- 影响因子:0
- 作者:
Bianca Garner;E. Brown;M. Taplin;Á. García;Baracka Williams - 通讯作者:
Baracka Williams
Bianca Garner的其他文献
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