High‑Yield DNA Extraction of Arsenic‑Resistant Microbes

High‑Yield DNA Extraction of Arsenic‑Resistant Microbes

Chile’s porphyry copper belt—home to giants like Chuquicamata and the Ventanas smelter—provides nearly a third of the world’s copper, but its sulfide ores also concentrate arsenic in minerals such as enargite and tennantite. Decades of mining and smelting have left the surrounding soils contaminated with tens to hundreds of milligrams of arsenic per kilogram.

In these toxic environments, specialized microbes offer insights into natural heavy-metal resistance. A recent study by Soto et al. (2021) isolated one such organism, Brevundimonas sp. B10, from the arsenic-rich rhizosphere near Ventanas, then moved it into the lab for genomic analysis.

You can read the article here: https://www.mdpi.com/1424-2818/13/8/344 

Stepped terraces cutting into a vast porphyry deposit in Chile’s copper belt—where copper and arsenic-bearing minerals like enargite and tennantite are mined side by side.

From Sampling to High‑Yield DNA Prep

They first enriched for arsenic‑tolerant microbes by plating rhizosphere dilutions on LB agar with 50 mg/L As(V) and selecting Brevundimonas sp. B10 colonies. Next, they grew it in liquid culture, pelleted the cells at 20,000×g, and subjected them to mechanical lysis via bead‑beating for 40 seconds. Following RNase A treatment, the lysate was bound to a silica spin column—multiple high‑salt washes removed residual metals and humics, and pure DNA was eluted. Quality checks (NanoDrop ratios of ~1.8/1.7 and a clean agarose‑gel band) confirmed they’d achieved high‑yield DNA prep free of inhibitors.

  • Spectrophotometric purity:
    DNA concentrations were measured on a NanoDrop 2000, and the preparations exhibited clean absorbance profiles (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios consistent with pure DNA).
  • Integrity on agarose gel:
    A single, sharp high–molecular‑weight band with no detectable smearing confirmed that the DNA was intact and free of degradation or co‑extracted inhibitors

A line of heavy haul trucks under the Andes sun. These transport copper ore, rich in both prized metal and arsenic-bearing sulfides, to smelters for processing.

From Clean DNA to a Draft Genome

Once the team confirmed their DNA was pure (sharp, high‑molecular‑weight band on a gel and NanoDrop ratios of about 1.8/1.7), they loaded it onto an Illumina MiSeq machine, which “reads” short stretches of DNA about 251 letters long from both ends, generating roughly 942,000 of these little reads. They then treated the reads like puzzle pieces: SPAdes put most of them together into longer stretches, while MEGAHIT and ABySS each built their own draft assemblies, and GFinisher polished and merged everything into a final draft. 

The result was a 3.34‑million‑letter genome split into seven big scaffolds (66.5% of the letters are G or C), with an N50 of 1.54 Mb—which means half the genome is contained in scaffolds at least that long—and BUSCO confirmed it was 99.5% complete. Finally, the RAST annotation labeled 3,275 genes, including 82 dedicated to defense against toxic metals.

Discoveries: Nature’s Dual + Redundant ars System

All that meticulous DNA extraction and assembly paid off when the team peered into Brevundimonas sp. B10’s genome and uncovered arsenic‑resistance genes. First, they found two complete ars operons. The first operon (arsR‑B‑C‑H) that reduces arsenate As(V) to arsenite As(III) and pumps it out.

  • arsR: a repressor protein that senses arsenite (As III) and turns the operon on or off.
  • arsB (or ACR3): an efflux pump that exports arsenite out of the cell.
  • arsC: an arsenate reductase that converts the more toxic arsenate (As V) into arsenite (As III).
  • arsH: a flavin‑dependent oxidoreductase that broadens the range of arsenic compounds a cell can detoxify.

The second operon (arsR₂‑C₂‑H₂ + ACR3) provides an alternate detox route, ensuring fail‑safe protection under fluctuating arsenic levels.

Next, two extra homologs—arsC3 and arsH3—sit outside those operons, bolstering flexibility so B10 can rapidly ramp up or diversify its arsenate reductases and detox enzymes as environmental pressures shift. 

  • arsC3: a third copy of the arsenate reductase (ArsC) enzyme (outside the ars operon), which converts As(V) to As(III). Having an additional arsC3 means the cell can maintain reduction activity even if one arsC variant is overwhelmed or differently regulated.
  • arsH3: a third flavin‑dependent detox enzyme (outside the ars operon), related to arsH, that likely broadens the range of arsenic‑derived substrates (including organic arsenicals) the bacterium can process.

The first cluster (Contig 8) contains the classic ars operon—ArsH (resistance protein), ArsC (arsenate reductase), ArsR (regulator), and ArsB (efflux pump). The second cluster (Contig 9) features another ArsH, a nearby tyrosine‐phosphatase–like gene, the ACR3 efflux protein, a second ArsC, and its own ArsR regulator. Gene positions (in kb) are shown above each arrow. Image from Soto et al. (2021).

Note: Putting these genes next to each other in an operon means the bacterium can co‑regulate them—turning on the whole detox pathway in one go when arsenic shows up. In addition, these homologs add redundancy and regulatory flexibility, letting B10 fine‑tune its arsenic detox response under varying environmental conditions 

But Brevundimonas sp. B10 doesn’t stop at arsenic: its genome encodes a broad metal‑efflux arsenal, including CzcABC and CusA RND pumps for cobalt, zinc, and cadmium; a P‑type ATPase that exports lead, cadmium, zinc, and mercury; multiple CopB/C/D copper‑binding proteins; and RND (another type of bacterial efflux pump) systems to pump a spectrum of toxic metals.

Why It Matters

Brevundimonas sp. B10’s multi‑operon arsenic systems and multi‑metal efflux pumps make it a prime candidate for mine‑site bioremediation of metal‑contaminated soils. And the DNA extraction approach to bioinformatics provides a blueprint for researchers grappling with the toughest environmental samples.

Further Reading

For the complete methodology and genomic insights, read Soto et al. (2021) in MDPI Biodiversity:

High-Yield Soil DNA Extraction for Arsenic- and Metal-Contaminated Sites ➔

Keywords: soil DNA extraction kit, metal‑contaminated soil, arsenic resistance, Brevundimonas sp. B10, mine bioremediation, heavy‑metal efflux, high‑yield DNA prep

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