The Role of Mg2+ Transport in Bacterial Heat Tolerance

肠道沙门氏菌是一种对食品安全很重要的病原体，其耐热性因突变而显著增强，突变导致两种蛋白质的高水平合成，这两种蛋白质可跨细菌细胞膜转运镁离子。通过确定哪些细胞组分在高温下受到这些单DNA核苷酸突变的保护，该项目将提供一个独特的机会来鉴定导致高温下活力丧失的热敏靶标。本研究获得的结果将不仅为沙门氏菌而且也为其他细菌的热敏性原因提供见解。成功构建耐热细菌品种将为改良菌株的工程设计提供一种简单的方法，这些菌株可用于合成生物燃料和其他化学品，这些化学品的生产或分离将在更高的温度下得到加强。此外，由于高温处理是灭活食品病原体的最广泛使用和最具成本效益的方法，对耐热性调节的分析可以为开发更有效的方法以确保微生物食品安全提供见解。NSF的资助也将使PI能够继续培训从高中到博士后的各级科学家，将遗传学，分子生物学，和基因组学来解决细菌生理学中的重要问题。拟议研究的目标是测试两种Mg 2+转运蛋白的过度生产是否（MgtA和MgtB）将赋予除了沙门氏菌之外的其他细菌增加的耐热性，并阐明MgtA和MgtB的合成增加赋予增强的耐热性的机制。该项目具有以下具体目的：1）通过在3种革兰氏阴性菌、嗜盐菌和革兰氏阳性菌中进行这种构建，测试Mg2+转运蛋白的过量生产为细菌耐热衍生物的工程化提供了通用方法的假设。2)测试chr（组成性耐热）突变通过保护细胞蛋白在高温下的稳定性而赋予耐热性增加的假设。这些实验还将探索蛋白质的变性是否是高温下细胞死亡的重要原因。3)测试chr突变通过在高温下保护膜而增强耐热性的假设。这些实验将确定在高温下chr突变是否减少膜渗漏或通过改变脂质组成来稳定膜。4)测试chr突变是否通过诱导热保护基因来增强耐热性。RNAseq将用于探测chr突变对全局转录组的影响。这些研究的结果将发表在同行评审的期刊上，并在区域和国家科学会议上发表。

Heat tolerance of Salmonella enterica, a pathogen important to food safety, is dramatically enhanced by mutations that result in high-level synthesis of two proteins that transport magnesium ions across bacterial cell membranes. By determining which cellular components are protected at high temperature as a consequence of these single DNA nucleotide mutations, this project will provide a unique opportunity to identify the heat sensitive target(s) that are responsible for loss of viability at high temperature. Results obtained in this study will provide insights into the causes of thermosensitivity not only in Salmonella but also in other bacteria. Success in the construction of thermotolerant varieties of bacteria would provide a simple approach to the engineering of improved strains that can be used for the synthesis of biofuels and other chemicals whose production or isolation would be enhanced at higher temperatures. Also, because high-temperature treatment is the most widely used and cost-effective means for inactivating food pathogens, analysis of the regulation of thermotolerance could provide insights into the development of more efficient ways to ensure microbiological food safety. Funding by the NSF will also enable the PI to continue the training of scientists at all levels from high school through postdoctoral in the integration of genetics, molecular biology, and genomics to address important problems in bacterial physiology.The goals of the proposed research are to test whether the overproduction of two Mg2+ transporters (MgtA and MgtB) will confer increased thermotolerance on other bacteria besides Salmonella and to elucidate the mechanism by which increased synthesis of MgtA and MgtB confers enhanced thermotolerance. The project has the following Specific Aims: 1) Test the hypothesis that overproduction of Mg2+ transporters provides a general method for the engineering of thermotolerant derivatives of bacteria by carrying out such constructions in 3 Gram negative species, a halophile, and in a Gram positive bacterium. 2) Test the hypothesis that the chr (constitutively heat resistant) mutations impart increased thermotolerance by protecting the stability of cellular proteins at high temperature. These experiments will also probe whether denaturation of proteins could be an important cause of cell death at high temperature. 3) Test the hypothesis that the chr mutations result in enhanced heat resistance by protecting membranes at high temperature. These experiments will determine whether the chr mutations decrease membrane leakage at high temperature or stabilize membranes by altering lipid composition. 4) Test whether the chr mutations confer enhanced heat tolerance by inducing thermoprotective genes. RNAseq will be used to probe the effect of the chr mutations on global transcriptomes. Results of these studies will be published in peer-reviewed journals and presented at regional and national scientific conferences.