Abstract
The gene pair hgcAB is essential for microbial mercury methylation. Our understanding of its abundance and diversity in nature is rapidly evolving. In this study we developed a new broad-range primer set for hgcAB, plus an expanded hgcAB reference library, and used these to characterize Hg-methylating communities from diverse environments. We applied this new Hg-methylator database to assign taxonomy to hgcA sequences from clone, amplicon, and metagenomic datasets. We evaluated potential biases introduced in primer design, sequence length, and classification, and suggest best practices for studying Hg-methylator diversity. Our study confirms the emerging picture of an expanded diversity of HgcAB-encoding microbes in many types of ecosystems, with abundant putative mercury methylators Nitrospirae and Chloroflexi in several new environments including salt marsh and peat soils. Other common microbes encoding HgcAB included Phycisphaerae, Aminicenantes, Spirochaetes, and Elusimicrobia. Combined with high-throughput amplicon specific sequencing, the new primer set also indentified novel hgcAB sequences similar to Lentisphaerae, Bacteroidetes, Atribacteria, and candidate phyla WOR-3 and KSB1 bacteria. Gene abundance data also corroborate the important role of two “classic” groups of methylators (Deltaproteobacteria and Methanomicrobia) in many environments, but generally show a scarcity of hgcAB+ Firmicutes. The new primer set was developed to specifically target hgcAB sequences found in nature, reducing degeneracy and providing increased sensitivity while maintaining broad diversity capture. We evaluated mock communities to confirm primer improvements, including culture spikes to environmental samples with variable DNA extraction and PCR amplification efficiencies. For select sites, this new workflow was combined with direct high-throughput hgcAB sequencing. The hgcAB diversity generated by direct amplicon sequencing confirmed the potential for novel Hg-methylators previously identified using metagenomic screens. A new phylogenetic analysis using sequences from freshwater, saline, and terrestrial environments showed Deltaproteobacteria HgcA sequences generally clustered among themselves, while metagenome-resolved HgcA sequences in other phyla tended to cluster by environment, suggesting horizontal gene transfer into many clades. HgcA from marine metagenomes often formed distinct subtrees from those sequenced from freshwater ecosystems. Overall the majority of HgcA sequences branch from a cluster of HgcAB fused proteins related to Thermococci, Atribacteria (candidate division OP9), Aminicenantes (OP8), and Chloroflexi. The improved primer set and library, combined with direct amplicon sequencing, provide a significantly improved assessment of the abundance and diversity of hgcAB+ microbes in nature.
Original language | English |
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Article number | 541554 |
Journal | Frontiers in Microbiology |
Volume | 11 |
DOIs | |
State | Published - Oct 6 2020 |
Funding
This research was sponsored by the Office of Biological and Environmental Research, Office of Science, US Department of Energy (DOE) as part of the Mercury Science Focus Area at Oak Ridge National Laboratory, which is managed by UT-Battelle LLC for the DOE under contract DE-AC05-00OR22725. We wish to thank and acknowledge the staff at the University of Minnesota Genomics Center (UMGC) for advice and assistance with high-throughput sequencing of custom hgcAB amplicon libraries. We also thank Brandy Toner (University of Minnesota), for peat, hollow and hummock samples from SPRUCE, Caroline Pierce for SPRUCE Hg/MeHg data, Helen Hsu-Kim for sediment samples from Sandy Creek, NC, United States, and Baohua Gu for rice paddy soils from Yanwuping, China. This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). Funding. This research was sponsored by the Office of Biological and Environmental Research, Office of Science, US Department of Energy (DOE) as part of the Mercury Science Focus Area at Oak Ridge National Laboratory, which is managed by UT-Battelle LLC for the DOE under contract DE-AC05-00OR22725. We wish to thank and acknowledge the staff at the University of Minnesota Genomics Center (UMGC) for advice and assistance with high-throughput sequencing of custom hgcAB amplicon libraries. We also thank Brandy Toner (University of Minnesota), for peat, hollow and hummock samples from SPRUCE, Caroline Pierce for SPRUCE Hg/MeHg data, Helen Hsu-Kim for sediment samples from Sandy Creek, NC, United States, and Baohua Gu for rice paddy soils from Yanwuping, China. This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).
Keywords
- amplicon sequencing
- environmental microbiology
- hgcAB
- mercury methylation
- microbial diversity
- primer development