The Role of Mg2+ Transport in Bacterial Heat Tolerance

肠道沙门氏菌是一种对食品安全至关重要的病原体，它的耐热性通过突变显著提高，这种突变导致两种蛋白质的高水平合成，这两种蛋白质可以运输镁离子穿过细菌细胞膜。通过确定这些单核苷酸突变导致哪些细胞成分在高温下受到保护，该项目将提供一个独特的机会来确定导致高温下活力丧失的热敏靶标(S)。这项研究的结果将不仅为沙门氏菌热敏感的原因提供见解，也将为其他细菌的热敏感原因提供见解。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来确保微生物食品安全提供见解。NSF的资助还将使PI能够继续对从高中到博士后的所有级别的科学家进行遗传学、分子生物学和基因组学的整合培训，以解决细菌生理学中的重要问题。拟议的研究目标是测试两种镁离子转运体(MGTA和镁B)的过量生产是否会增强除沙门氏菌以外的其他细菌的耐热性，并阐明增加MGTA和镁B的合成从而增强耐热性的机制。该项目有以下具体目标：1)通过在3个革兰氏阴性菌、一个嗜盐细菌和一个革兰氏阳性细菌中进行这种结构，来检验这样的假设，即过量生产镁离子转运蛋白为细菌耐热衍生物的工程提供了一种通用的方法。2)验证CHR(结构性耐热)突变通过保护细胞蛋白质在高温下的稳定性而提高耐热性的假设。这些实验还将探索蛋白质的变性是否可能是高温下细胞死亡的重要原因。3)验证CHR突变通过在高温下保护细胞膜而导致耐热性增强的假设。这些实验将确定ChR突变是减少高温下的膜渗漏，还是通过改变脂质成分来稳定膜。4)测试CHR突变是否通过诱导热保护基因来增强耐热性。RNAseq将被用来探索CHR突变对全球转录本的影响。这些研究的结果将发表在同行评议的期刊上，并在区域和国家科学会议上公布。

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.