Isolation and identification of an Escherichia coli strain with high manganese-oxidizing activity and cloning and characterization of its multicopper oxidase gene

Abstract:

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 Escherichia 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 Ⅲ 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 MnB1. 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 b-sheets in both proteins that were able to form b-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.

Key words:Manganese oxidization, Escherichia coli, Identification, Manganese-oxidizing gene