New Arsenate Reductase Gene ( arrA ) PCR Primers for Diversity Assessment and Quantification in Environmental Samples

Abstract

The extent of arsenic contamination in drinking water and its potential threat to human health have resulted in considerable research interest in the microbial species responsible for arsenic reduction. The arsenate reductase gene (arrA), an important component of the microbial arsenate reduction system, has been widely used as a biomarker to study arsenate-reducing microorganisms. A new primer pair was designed and evaluated for quantitative PCR (qPCR) and high-throughput sequencing of the arrA gene, because currently available PCR primers are not suitable for these applications. The primers were evaluated in silico and empirically tested for amplification of arrA genes in clones and for amplification and high-throughput sequencing of arrA genes from soil and groundwater samples. In silico, this primer pair matched (Ն90% DNA identity) 86% of arrA gene sequences from GenBank. Empirical evaluation showed successful amplification of arrA gene clones of diverse phylogenetic groups, as well as amplification and high-throughput sequencing of independent soil and groundwater samples without preenrichment, suggesting that these primers are highly specific and can amplify a broad diversity of arrA genes. The arrA gene diversity from soil and groundwater samples from the Cache Valley Basin (CVB) in Utah was greater than anticipated. We observed a significant correlation between arrA gene abundance, quantified through qPCR, and reduced arsenic (As III ) concentrations in the groundwater samples. Furthermore, we demonstrated that these primers can be useful for studying the diversity of arsenate-reducing microbial communities and the ways in which their relative abundance in groundwater may be associated with different groundwater quality parameters. IMPORTANCE Arsenic is a major drinking water contaminant that threatens the health of millions of people worldwide. The extent of arsenic contamination and its potential threat to human health have resulted in considerable interest in the study of microbial species responsible for the reduction of arsenic, i.e., the conversion of As V to As III . In this study, we developed a new primer pair to evaluate the diversity and abundance of arsenate-reducing microorganisms in soil and groundwater samples from the CVB in Utah. We observed significant arrA gene diversity in the CVB soil and groundwater samples, and arrA gene abundance was significantly correlated with the reduced arsenic (As III ) concentrations in the groundwater samples. We think that these primers are useful for studying the ecology of arsenate-reducing microorganisms in different environments.KEYWORDS arsenate reductase gene (arrA), high-throughput sequencing, qPCR of arrA gene, arsenic reduction

Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations

“…3 and SI Appendix, Fig. S6) and is conserved with few exceptions across several hundred partial ArrA sequences analyzed previously (42). In Arx, the motif is XGRGWG ( Fig.…”

“…Regardless of their mechanistic roles, the active-site residues revealed by the Arr structure provide a powerful handle for the annotation of ambiguous sequences as Arr or Arx. The sequence similarity between these enzymes makes it difficult to distinguish them without a phylogenetic analysis (41,42), but doing so is essential to understand the directionality of arsenic redox transformations based on metagenomic data. The (R/K)GRY motif in Arr and the XGRGWG motif in Arx provide a structural basis for this distinction.…”

“…The (R/K)GRY motif in Arr and the XGRGWG motif in Arx provide a structural basis for this distinction. Although PCR amplification with arrA primers has been used to identify Arr-encoding organisms in the environment (27,42), the similarity between ArrA and ArxA raises questions about the specificity of this detection. For example, the "cluster V" group of arrA sequences identified by PCR (42) contains the Arx motif, suggesting this group is Arx rather than Arr, even though the amplified sequences appeared more related to arrA than to arxA (42).…”

Isolation and molecular identification of native As-resistant bacteria: As(III) and As(V) removal capacity and possible mechanism of detoxification

“…This process was repeated for an additional 30 cycles, followed by a final extension at 72 °C for 10 min. Finally, the ampli cation of the genes was veri ed in a 1% agarose gel stained with ethidium bromide (Mirza et al, 2017).…”

New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing

“…Amplicon NGS surveys are now also increasingly applied to target functional genes that are indicative of a particular microbial guild, such as mcrA for methane oxidizers (Cai et al, 2018), amoA for ammonia oxidizers (Roots et al, 2019), pmoA for methanotrophs (Chiri et al, 2020), and dsrAB for sulfate reducers (Zeleke et al, 2013;Pelikan et al, 2016). To date, investigations targeting metal biotransformation using NGS have only involved the arrA gene for dissimilatory arsenate reducers (Mirza et al, 2017) and the arsM gene for arsenate methylated microorganisms (Zhang S. Y. et al, 2015). However, no available PCR primer is suitable for applications in high-throughput sequencing for genes that encode As(III)-oxidases.…”