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Updated: Aug 29, 2025

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans
Published on: September 13, 2022
Sequence-based Functional Metagenomics Reveals Novel Natural Diversity of Functional CopA in Environmental
Wenjun Li1, Likun Wang2, Xiaofang Li2
1Hebei Key Laboratory of Soil Ecology, Centre for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050022, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Abstract:
Exploring the natural diversity of functional genes/proteins from environmental DNA in high throughput remains challenging. In this study, we developed a sequence-based functional metagenomics procedure for mining the diversity of copper (Cu) resistance gene copA in global microbiomes, by combining the metagenomic assembly technology, local BLAST, evolutionary trace analysis (ETA), chemical synthesis, and conventional functional genomics. In total, 87 metagenomes were collected from a public database and subjected to copA detection, resulting in 93,899 hits. Manual curation of 1214 hits of high confidence led to the retrieval of 517 unique CopA candidates, which were further subjected to ETA. Eventually, 175 novel copA sequences of high quality were discovered. Phylogenetic analysis showed that almost all these putative CopA proteins were distantly related to known CopA proteins, with 55 sequences from totally unknown species. Ten novel and three known copA genes were chemically synthesized for further functional genomic tests using the Cu-sensitive Escherichia coli (ΔcopA). The growth test and Cu uptake determination showed that five novel clones had positive effects on host Cu resistance and uptake. One recombinant harboring copA-like 15 (copAL15) successfully restored Cu resistance of the host with a substantially enhanced Cu uptake. Two novel copA genes were fused with the gfp gene and expressed in E. coli for microscopic observation. Imaging results showed that they were successfully expressed and their proteins were localized to the membrane. The results here greatly expand the diversity of known CopA proteins, and the sequence-based procedure developed overcomes biases in length, screening methods, and abundance of conventional functional metagenomics.
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