The Study On The Role Of RpoS In Escherichia Coli Natural Transformation

As a novel transformation system reported these years, Escherichia coli natural transformation contains these processes:natural competence development, DNA uptake and transformants determination. The finding of E. coli natural transformation was of good biology value, since it breaks the traditional view that E. coli could not take exogenous DNA and transform without artificial treatment. Moreover, because E. coli is also one of the most common used materials in genetic engineering, its biosecurity comes under question for its ability of natural gene transfer. So there is extensive space and requirement in the study of mechanism of this transformation system.The general stress response factor σS, so-called RpoS, is an alternative σ factor of RNA polymerase in Escherichia coli. It can partially replace the "housekeeping" σ factor σ70(RpoD) under many stress conditions. RpoS intacts with the core RNA polymerase (RNAP) and controls the expression of a specific but large set of genes. Directly or indirectly, RpoS regulates10%of the E. coli genome (approximately500genes). With the characteristic of "low-expression in exponential phase and high-expression under stress conditions", RpoS was mentioned as a regulator of E. coli natural transformation.In this study, we investigate the effect of RpoS on E. coli natural transformation in an "open system". Some regulation mechanisms was discussed in detail too.In order to make sure whether RpoS has influence exactly on natural transformation process, we continuously exclude the interference of RpoS regulation on other transformation system and the disturbation of transformants survival. The results expressed that, RpoS affects the natural transformation frequency in E. coli, while has no effect on artificial transformation. The transformant survival was not interrupted by RpoS. So the RpoS regulation was exactly on the process of natural transformation.E. coli natural transformation process contains natural competence development and DNA uptake. The RpoS’s effects on which step determin the transformation frequency were investigated in the following work.DNA uptake carried out on plate is one characteristic in natural transformation different from the classical artificial transformation with Ca2+. In order to determine the relationship between RpoS expression and the DNA uptake, three questions should be answered:whether the RpoS deletion would disturb the time that DNA uptake takes place, whether the RpoS expression was changed by the plating which promotes DNA uptake and if the transformation frequency would be changed by the RpoS expression changing in DNA uptake. We finally demonstrate that, although RpoS positive controls the natural transformation, it is not sufficient for transformation complete. The contact of natural competence with solid substrate was necessary for DNA uptake, but this process has nothing to do with RpoS expression.Another characteristic of open system natural transformation is that the process of natural competence development contains two parts:shaking culture and static cultivation. In which step RpoS plays the major role on natural transformation was discussed in the following work. The results demonstrate that the intracellular RpoS content of cells in the process of natural competence development, especially in the early exponential phase, determines the level of transformation frequency. Although not as important as in early exponential phase, the RpoS content in static cultivation enhances the natural transformation.In order to investigate the natural transformation regulation from various angles, besides the function research of single gene rpoS on the natural transformation, another factor AMP, which has been reported to inhibit the natural transformation of Haemophilus influenza, was discussed both in E. coli artificial and natural transformations. We found that intracellular AMP could changes the flow of energy by breaking the energy charge of the competence. This flow of energy blocks the E. coli artificial transformation. However, it enhances the natural transformation at the early exponential phase just like the RpoS regulation.In conclusion, we demonstrate that the intracellular RpoS content in early exponential phase determines the level of transformation frequency, which has not been studies in previous work. Then one regulatory factor in H. influenza natural transformation AMP was found has different effect on E. coli natural transformation. These findings provide detailed understanding of RpoS effect on natural transformation.