锰氧化物是Mn(Ⅱ)经生物和化学氧化后形成的矿物成分,在元素生物地球化学循环过程中起着重要作用,而不同种类的细菌对Mn(Ⅱ)的氧化作用是自然界中氧化锰矿物形成的主要成因.从山东崅峪采集的铁锰结核棕壤中分离得到一株具有高锰氧化活性的土壤杆菌,其对Mn(Ⅱ)的氧化作用活性明显高于其它分离菌株,达到65 μmol/L.通过个体形态与培养特征观察、生理生化反应、G+C mol％测定和16S rRNA基因序列比对分析等鉴定,确定该菌株为大肠杆菌(Escherichia coli),命名为MB266.进一步对该菌株参与锰氧化作用的多铜氧化酶基因mco进行了克隆、序列与基因结构分析(GenBank登录号:JF682492).结果表明,该基因编码的MCO蛋白与已报道的大肠杆菌Ⅲ型多铜氧化酶的氨基酸同源性达99％,而与已被深入研究的恶臭假单胞菌(Pseudomonas putida)MnB1菌株锰氧化酶CumA的氨基酸序列的相似性仅有19.2％,但MCO与CumA二者的一级结构中都存在2个保守的铜氧化超家族,而每个保守结构域内又都存在2个保守的铜离子结合位点,且二者在二级结构上都存在多个β-折叠片,并形成2个β-桶结构域,这些结构上的共性可能与它们均具有对Mn(Ⅱ)的氧化活性有关.大肠杆菌野生菌株具有锰氧化活性这一特性目前尚未见于文献报道.%Manganese oxides are a type of high-reactive minerals that formed from biochemical and chemical oxidations of manganese( II), which are capable of influencing significantly the transport and fate of many major and trace elements in the biogeochemical cycles. Increasing evidence is showing that microorganisms, especially a variety of bacteria, play a dominant role in the oxidation of dissolved Mn(II) in natural systems. In this study, a soil-borne bacterial isolate with a maximum manganese-oxidizing activity by 65 μmol/L (it was apparently higher than that of other isolates by using the standard leucoberbelin blue assay procedure), was screened from the Fe/Mn nodule-surrounding brown soil samples that taken in Queyu, Shandong Province, China, and was identified as an Es-cherichia coli strain (named as MB266) according to the morphological, physiological and biochemical characteristics, G+C content of its genomic DNA as well as the 16S rRNA gene sequence alignment analysis. Subsequently, the multicopper oxidase encoding gene (mco) of MB266, which was thought to involve in manganese( II) oxidation, was cloned and characterized (GenBank accession number: JF682492). It showed that the corresponding protein, MCO, was highly similar (by 99.0%) with the previously reported E. coli-harboring type IE multicopper oxidase at their amino acid sequences,whereas the similarity of amino acid sequences was very limited (by only 19.2%) with a currently well-characterized bacterial manganese oxidase, manganese oxidase CumA of Pseudomonas putida MnBl. However, the conserved structural domain analysis using online tool CDART revealed that two conserved copper-oxidizing super family domains in both proteins, together with two conserved Cu( II )-binding sites in each conserved domain. Additionally, it appeared that multiple p-sheets in both proteins that were able to form p-barrel domains in their predicted secondary structures. Thus, the structural intercommunity between MCO and CumA might contribute to their similar biochemical activities in manganese oxidation. The manganese-oxidizing activity of an E. coli wild-type strain is a distinctive feature that has not been reported prior to this study.
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