Abstract
Characterizing the genetic diversity of microbial copper (Cu) resistance at the community level remains challenging, mainly due to the polymorphism of the core functional gene copA. In this study, a local BLASTN method using a copA database built in this study was developed to recover full-length putative copA sequences from an assembled tailings metagenome; these sequences were then screened for potentially functioning CopA using conserved metal-binding motifs, inferred by evolutionary trace analysis of CopA sequences from known Cu resistant microorganisms. In total, 99 putative copA sequences were recovered from the tailings metagenome, out of which 70 were found with high potential to be functioning in Cu resistance. Phylogenetic analysis of selected copA sequences detected in the tailings metagenome showed that topology of the copA phylogeny is largely congruent with that of the 16S-based phylogeny of the tailings microbial community obtained in our previous study, indicating that the development of copA diversity in the tailings might be mainly through vertical descent with few lateral gene transfer events. The method established here can be used to explore copA (and potentially other metal resistance genes) diversity in any metagenome and has the potential to exhaust the full-length gene sequences for downstream analyses.
Highlights
Characterizing the genetic diversity of microbial copper (Cu) resistance at the community level remains challenging, mainly due to the polymorphism of the core functional gene copA
We report a method to recover full-length copA from a tailings metagenome by combining the methods of metagenome assembly, local BLASTN and evolutionary trace (ET) analysis
By building a local database of antibiotic resistance genes (ARGs), a local BLASTN procedure has been established to screen for the presence of ARGs in bacterial genomes[26]; yet this local-BLASTN method has not been applied to metal resistance genes or to explore metagenomes
Summary
Characterizing the genetic diversity of microbial copper (Cu) resistance at the community level remains challenging, mainly due to the polymorphism of the core functional gene copA. A local BLASTN method using a copA database built in this study was developed to recover full-length putative copA sequences from an assembled tailings metagenome; these sequences were screened for potentially functioning CopA using conserved metal-binding motifs, inferred by evolutionary trace analysis of CopA sequences from known Cu resistant microorganisms. Available copA sequences in the literature are thought to be highly polymorphic and available primers only cover a subset of copA sharing high similarity This hinders the assessment of copA diversity and the discovery of novel copA in the environment. We hypothesize that all copA named based on sequence similarity in the literature are not homologs and can be divided into two groups encoding for multicopper oxidase and P-type ATPase, which are both highly conserved. By building a local database of antibiotic resistance genes (ARGs), a local BLASTN procedure has been established to screen for the presence of ARGs in bacterial genomes[26]; yet this local-BLASTN method has not been applied to metal resistance genes or to explore metagenomes
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