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

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(DcopA).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.

Assembly and variation of root-associated microbiota of rice during their vegetative growth phase with and without lindane pollutant

Soil-derived microbiota associated with plant roots are conducive to plant growth and stress resistance.However,the spatio-temporal dynamics of microbiota in response to organochlorine pollution during the unstable vegetative growth phase of rice is not well understood.In this study,we focused on the rice(Oryza sativa L.)microbiota across the bulk soil,rhizosphere and endosphere compartments during the vegetative growth phase in two different soils with and without lindane pollutant.The results showed that the factors of growth time,soil types and rhizo-compartments had significant influence on the microbial communities of rice,while lindane mostly stimulated the construction of endosphere microbiota at the vegetative phase.Active rice root-soil-microbe interactions induced an inhibition effect on lindane removal at the later vegetative growth phase in rice-growth-dependent anaerobic condition,likely due to the root oxygen loss and microbial mediated co-occurring competitive electron-consuming redox processes in soils.Each rhizocompartment owned distinct microbial communities,and therefore,presented specific ecologically functional categories,while the moderate functional differences were also affected by plants species and residual pollution stress.This work revealed the underground micro-ecological process of microbiota and especially their potential linkage to the natural attenuation of residual organochlorine such as lindane.