Genetics of Starvation-Induced Resistance in Escherichia coli

饥饿细菌在生态学上、在生物技术中以及作为生物分化的模型都很重要。饥饿时，未形成孢子的大肠杆菌通过在饥饿早期表达约50个基因而分化为具有明显抵抗力的细胞。Katf蛋白被认为是一种新的sigma因子，是合成这些蛋白中的几种所必需的，因为Katf基因的缺陷基本上阻止了饥饿蛋白的诱导和抗性的形成。这一发现表明，但并不能证明Katf调节的饥饿蛋白参与了细胞抵抗状态的发展。将为这一角色寻找直接证据。将构建一株Katf缺失株。将一个抗药性标记插入克隆的Katf基因，该基因将利用recB recc sbcB菌株与大肠杆菌染色体杂交，然后将突变基因转移到野生型背景中。在可滴定启动子的控制下，Katf结构基因将被引入缺失菌株中。在饥饿过程中会产生不同数量的Katf，并将确定其与对饥饿、氧化、高温和渗透胁迫的抗性程度的相关性。在选定的情况下，将确定双向聚丙烯酰胺凝胶电泳图。这可能有助于揭示哪些蛋白质斑点在饥饿和其他抗性的形成过程中特别重要。类似的实验将在生长的指数阶段进行，以确定过量生产的Katf是否也会在生长过程中产生抗性。有证据表明，Katf的合成可能在转录后水平上受到调控。为了探索这一点，将使用聚合酶链式反应技术构建Katf到LacZ的转录和翻译融合。自然界中的细菌主要处于饥饿状态。因此，在饥饿条件下表达什么生化特征以及这种表达是如何调控的，对于理解自然界中的细菌活动非常重要，这些活动除了疾病原因外，还包括元素的循环和环境解毒。饥饿细菌在以微生物为基础的工业发酵中也很重要，因为使用它们可以使用特殊的生物反应器进行快速和廉价的生物转化。当细菌饥饿时，大肠杆菌表达了大约50个新基因。其中一个子集可能与蛋白质的合成有关，这些蛋白质使细胞对许多不同种类的压力更具抵抗力。这些蛋白质如何使细胞对压力更具抵抗力是一个重要的生物学问题，可以在这个模型系统中方便地进行探索。在这项提案中，目的是明确确定饥饿蛋白的特定子集是否参与抗逆性。这个子集由一种名为Katf的新型遗传元件控制。利用分子生物学技术，将精确调控细胞中的Katf的数量，将其调控的蛋白质的浓度量化，并确定其浓度是否与细胞获得的抗性程度有关。识别决定细胞抵抗状态的蛋白质可以使操纵细胞抵抗成为可能；深入了解它们的生化作用可以提供有关细胞基本功能的信息。

Starving bacteria are important ecologically, in biotechnology, and as models of biological differentiation. Upon starvation, the non- sporeforming bacterium Escherichia coli differentiates into markedly resistant cells by expressing some 50 genes in early starvation. The KatF protein, which is believed to be a novel sigma factor, is required for the synthesis of several of these proteins, since defect in the katF gene substantially prevents starvation protein induction and resistance development. This finding suggests but does not prove the involvement of KatF- regulated starvation proteins in the development of cellular resistant state. Direct evidence will be sought for this role. A katF deletion strain will be constructed. A drug resistance marker will be inserted into the cloned katF gene which will be crossed into the E. coli chromosome, using a recB recC sbcB strain, followed by transfer of the mutated gene into the wild type background. The katF structural gene under the control of a titratable promoter will be introduced into the deletion strain. Different amounts of KatF will be produced during starvation, and correlation to the degree of resistance to starvation, oxidative, heat and osmotic stresses will be determined. Two-dimensional polyacrylamide gel electrophoresis patterns will be determined in selected instances. This may shed light on which protein spots are especially important in the development of starvation and other resistances. Similiar experiments will be done during exponential phase of growth to see if overproduction of KatF can confer resistance during growth as well. There is evidence that KatF synthesis may be regulated at the postranscriptional level. To explore this, transcriptional and tranlational fusions of katF to lacZ will be constructed using the polymerase chain reaction technology. %%% Bacteria in nature exist primarily in a starving state. What biochemical characters are expressed under starvation conditions and how this expression is regulated are therefore important in the understanding of bacterial activities in nature which include besides disease causation, recycling of elements and environmental detoxification. Starving bacteria are also important in microbial based industrial fermentations because their use permits rapid and cheap biotransformations using special bioreactors. Some fifty new genes are expressed by Escherichia coli when this bacterium is starved. A subset of these may be concerned with synthesis of proteins that make the cells more resistant to many different kinds of stresses. How might these proteins make cells more resistant to stresses is an important biological question which can be conveniently explored in this model system. In this proposal, the aim is to clearly determine if a specific subset of starvation proteins is involved in stress resistance. This subset is controlled by a novel genetic element called KatF. By using techniques of molecular biology, the amount of KatF in the cells will be precisely regulated, the concentration of the proteins it regulates will be qunatified, and a determination will be made of whether their concentration correlates with the degree of resistance acquired by the cells. Identification of proteins that determine the cellular resistant state can make it possible to manipulate cellular resistance; and an insight into their biochemical role can provide information about fundamental cellular functions.