Metagenomics-Guided Discovery of Potential Bacterial Metallothionein Genes from the Soil Microbiome That Confer Cu and/or Cd Resistance.

Metallothionein (MT) genes are valuable genetic materials for developing metal bioremediation tools. Currently, a limited number of prokaryotic MTs have been experimentally identified, which necessitates the expansion of bacterial MT diversity. In this study, we conducted a metagenomics-guided analysis for the discovery of potential bacterial MT genes from the soil microbiome. More specifically, we combined resistance gene enrichment through diversity loss, metagenomic mining with a dedicated MT database, evolutionary trace analysis, DNA chemical synthesis, and functional genomic validation to identify novel MTs. Results showed that Cu stress induced a compositional change in the soil microbiome, with an enrichment of metal-resistant bacteria in soils with higher Cu concentrations. Shotgun metagenomic sequencing was performed to obtain the gene pool of environmental DNA (eDNA), which was subjected to a local BLAST search against an MT database for detecting putative MT genes. Evolutional trace analysis led to the identification of 27 potential MTs with conserved cysteine/histidine motifs different from those of known prokaryotic MTs. Following chemical synthesis of these 27 potential MT genes and heterologous expression in Escherichia coli, six of them were found to improve the hosts' growth substantially and enhanced the hosts' sorption of Cu, Cd, and Zn, among which MT5 led to a 13.7-fold increase in Cd accumulation. Furthermore, four of them restored Cu and/or Cd resistance in two metal-sensitive E. coli strains.IMPORTANCE The metagenomics-guided procedure developed here bypasses the difficulties encountered in classic PCR-based approaches and led to the discovery of novel MT genes, which may be useful in developing bioremediation tools. The procedure used here expands our knowledge on the diversity of bacterial MTs in the environment and may also be applicable to identify other functional genes from eDNA.