| The work of this dissertation is concerned about the study of Escherichia coli competence development induced by Ca2+ and the capacity of absorbing external-DNA. We obtained the following results by determining the Ca2+ concentration and locating intracellular Ca2+ of E. coli competence and exploring the molecular mechanism of competence development using AMP, nonselective DNA and quantum dots (QDs) as probes.1. Studies showed that, for divalent cation Ca2+, Mn2+ and Mg2+, Ca2+ was more efficient in inducing E. coli development of competence and accomplishing DNA transformation than the two others. Ca2+ promoted the growth of E. coli but the cells grown in culture medium containing high concentration Ca2+ haven't more special capacity absorbing external-DNA than low concentration Ca2+. The simplest requirement for E. coli absorbing external-DNA was that E. coli cells, proper Ca2+ and DNA must be in a same system. The function of Ca2+ is acted on the process transformation of E. coli, but other processes. Heat shock and other operations increased the DNA transformation efficiency but weren't necessary for E. coli absorbing external-DNA.2. We established a new experiment method for determining the Ca2+ concentration of E. coli competent cells using Fura-2/AM as fluorescent probe, and located the cellular Ca2+ using potassium pyroantimonate [K2H2Sb2O7 4H2O]. The results indicated that, after dealing E. coli with 100mmol/L CaCl2, a little quantity of Ca2+ entered into the cells swiftly and a large quantity of Ca2+ gathered on the surface of cells or periplasm.3. E.coli could take in external-DNA under the conditions of resemble to the nature and the DNA absorbed replicated and expressed regularly. Moreover, the transformation frequency was related to Ca2+ concentration and a proper time was required for accomplishing natural genetic transformation.4. AMP promoted the growth of E. coli lightly. But they have no effects on expression of part of gene in plasmid and chromosome. AMP and GMP prevented E. coli development of competence and then held back E. coli cells absorbing external-DNA. Moreover, AMP displayed more remarkable function when its concentration was higher and the time of dealing with was longer. Others of nonselective DNA didn't affected E. coli absorbing external-DNA regardless their molecular weight, molecular structure and their content of G+C.5. La3+ at concentration from 50 to 150ug/mL stimulated the endogenic metabolism and ectogenic metabolism of E.coli, changed the image of transmission electron microscopy, but had few effects on gene expression. Through many chemical properties of La3+ are similar to Ca2+, they didn't induced E. coli development of competence and absorbing of external-DNA. Furthermore, La3+ at lower concentration 0.5 to 30ug/mL decreased intensively the transformation efficiency and inhibited completely E. coli absorbing external-DNA at high concentration (>30ug/mL).6. Exploring the mechanism of competence development in E. coli using quantum dots as fluorescent probes and observing the change of cells by atomic force microscope (AFM), results showed that E. coli competence cells surface suffered many change, appearing rough and presented holes of about 50-300 nm in diameter under AFM, while the surface of non competent cells appeared smooth and continuous. Water-soluble QDs of 3-4nm could go into competent cells by these holes on cell surface, but could not enter noncompetent cells.In conclusion, based on these results, we believed that there wasn't specific receptor on surface of E. coli. Metal cation exhibited speciality in inducing E. coli development of competence. We suggested that, in process of nonphysiological high concentration Ca2+ inducing E. coli into competent cells, not only Ca2+ had enhanced the penetrability of cell membranes but also part of Ca2+ entered into cells and combined PHB and polyPi into compound on membranes which constituted membrane channels for DNA passage. It is inferred that, as far as Ca2+ promoted E. coli absorbing externa...
|
PDF Full Text Request