| Manganese oxide is the highly reactive metallic minerals which rich in earth’s crust, and is considered to be the main causes of the formation of microbial manganese oxide in the environment, in which bacterial manganese oxidation catches the most attention. It is known that manganese-oxidizing bacterias are widely distributed in a variety of environments:sea water, marine sediments, soil and other freshwater bodies, and in which the Firmicutes, Actinobacteria and the Alpha-, Beta- and Gamma-proteobacteria is the major bacterial species of manganese oxide. Despite the current domestic and international study of mechanisms for marine bacterial manganese oxidation is in a deep exploration, but about soil manganese oxide, lack of research of the manganese oxide mechanism and bacterial species, but the. In this study, make a soil-inhabiting E.coli MB266 isolated as the research object, in basis of determining the strain has a high manganese oxidation activity, through Real time-qPCR analysis of manganese oxidation activity of specific genes, knock-out function gene and other means to study the manganese oxidation mechanism of the bacteria.According to various bacteria manganese oxidegenes the literature and the analysis of sequences of the E.coli genome, identified 38 genes possible manganese oxide gene, to design specific primers to analyze these genes of strain MB266 different transcriptional activity changes with Mn (Ⅱ)-induced and non-Mn (Ⅱ)-induced under growth stages by using Real-time-qPCR techniques. The results showed that there are 15 genes in the cell transcriptional activity of different growth stages changed significantly, and in the late logarithmic phase, adding Mn (Ⅱ)-induced the transcriptional activity gets lower. Based on the Mn (Ⅱ)-induced transcriptional activity gets lower and which encodes a large product, and is intracellular oxido reductase, in this study the gens which the transcriptional activity change in a high lever are more than one. We choose the gene mco266 which the activity increases and catalase katE gene which the activity induces as a target gene to knock with the Red recombination system, and analysis the manganese oxidation activity of wild-type strain and mutant strain.The co-expression and in vitro activity of manganese oxide on the cell walls of electron transport cytochrome C gene (ccmF, ccmFGH) and mco266 haven done.Making wild strain MB266 as the starting strain, respectively, mco266 oxidase gene get strain MB267 and knockout catalase gene katE, get mutant strain MB269. Measuring the manganese oxidation activity of mutant strains and wild strain MB266, found that manganese oxidation activity of mco266 deficient strains (MB267) decreased 29.82%, compared with the wild strain, while the manganese oxidation activity of katE deficient strains MB269 with no significant change; further, making MB267 as the starting strain to knockout catalase gene katE, found that manganese oxidation activity of mco266 and katE double deficient-mutant strain did not change significantly, compared with MB267, compensation katE gene expression experiments also found that manganese oxidation activity of the expression compensation recombinant strain is unchanged. These results show that the multi-copper oxidase gene mco266 is one of manganese oxide gene of strain MB266, but obviously not the only one gene of manganese oxide.Mco266 gene and Cytochrome C gene ccmF, ccmFGH gene were amplified from MB266 strain, respectively, together with mco266 gene were connected to E. coli expression vector pTrcHis-C, to obtain the co-expression plasmid, then import to E.coli JM109 which without manganese oxidation activity as body strain, recombinant strains were named MB275 and MB271.The results that the recombinant strain have the activity of manganese oxidation, It was determined that the concentrations of manganese oxide of the recombinant strain MB257 and MB271 were 3.90μM and 4.13μM. show that multicopper oxidase MCO266 and cytochrome C protein complete manganese oxidation together. |
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