Sequence-based Functional Metagenomics Reveals Novel Natural Diversity of Functioning CopA in Environmental Microbiomes

Genomics, Proteomics & Bioinformatics

Available online 8 September 2022

Genomics, Proteomics & BioinformaticsAbstract

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 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 are 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.

Keywords

Functional metagenomics

Natural diversity

CopA

Evolutionary trace analysis

Cu resistance

Data availability

The 175 novel sequences can be downloaded from genome warehourse accession no. GWHBISN00000000 from bioproject PRJCA008249 (https://ngdc.cncb.ac.cn/) [55], GeneBank from NCBI (Accession No. ON553002 to ON553176), and Table S3. The procedure developed to mine the natural diversity of novel CopA proteins from eDNA is available in https://github.com/ebg-lab/CopA and BioCode in https://ngdc.cncb.ac.cn/biocode/tools/BT007306.

Competing interests

The authors declare no competing financial interests.

© 2022 The Authors. Published by Elsevier B.V. and Science Press on behalf of Beijing Institute of Genomics, Chinese Academy of Sciences/China National Center for Bioinformation and Genetics Society of China.

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