Christensen Geoff A, Wymore Ann M, King Andrew J, Podar Mircea, Hurt Richard A, Santillan Eugenio U, Soren Ally, Brandt Craig C, Brown Steven D, Palumbo Anthony V, Wall Judy D, Gilmour Cynthia C, Elias Dwayne A
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
Smithsonian Environmental Research Center, Edgewater, Maryland, USA.
Appl Environ Microbiol. 2016 Sep 16;82(19):6068-78. doi: 10.1128/AEM.01271-16. Print 2016 Oct 1.
Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent discovery of the Hg-methylating gene pair, hgcA and hgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivation-independent strategy.
hgcA和hgcB这两个基因对于微生物汞（Hg）甲基化至关重要。结合汞浓度、生物可利用性和生物地球化学来检测和估算它们的丰度，对于确定高危环境中甲基汞（MeHg）产生的潜在热点至关重要。我们开发了跨越已知hgcAB基因的广谱简并PCR引物，以确定这两个基因在不同环境中的存在情况。这些引物针对一系列具有已发表基因组的纯培养物进行了测试，包括13种δ-变形菌纲、9种厚壁菌门和9种产甲烷古菌的基因组。通过桑格测序确认，所有测试的hgcAB(+)菌株均产生了预期大小的独特PCR产物。此外，我们针对三个主要的汞甲基化进化枝，分别开发了靶向hgcA的进化枝特异性简并定量PCR（qPCR）引物。进化枝特异性qPCR引物分别从64%、88%和86%的测试δ-变形菌纲、厚壁菌门和古菌纯培养物中扩增出hgcA，并且对每个进化枝具有高度特异性。对每种生物的扩增效率和检测限进行了量化。引物敏感性因物种的序列保守性而异。最后，为了开始评估我们的引物组在自然环境中的实用性，我们测试了从添加到沙子和土壤中的纯培养物中回收hgcA和hgcAB的情况。本研究中设计的这些新型定量分子工具将能够更准确地鉴定和量化环境中单个汞甲基化微生物群体。所得数据对于开发准确且可靠的汞甲基化潜力预测模型至关重要，理想情况下是将汞甲基化的地球化学与hgcAB的微生物学和遗传学相结合。重要性：神经毒素甲基汞（MeHg）对人类健康构成严重风险。自然界中MeHg的产生与厌氧微生物有关。最近发现的汞甲基化基因对hgcA和hgcB，使我们能够针对已知能甲基化汞的微生物基因组DNA中的这些基因设计和优化分子探针。本研究中设计的方案允许对纯培养物或环境样品进行定性和定量评估。有了这些方案，我们可以开始通过不依赖培养的策略研究自然界中汞甲基化生物的分布。
