Hot-spring DNA and the contamination problem
A student collects a water sample from a geothermal spring in the Taupō Volcanic Zone. The spring water is 75°C, pH 3.2, with high dissolved arsenic and very low oxygen. The sample contains a microbial mat. Back in the lab, DNA sequencing gives a 16S rRNA sequence that is 99.9% identical to a common soil bacterium, Bacillus subtilis, which grows best at 25–35°C and pH 6–7. The student's hypothesis is that the mat contains a novel thermophilic acidophile. The sampling method used a sterile bottle but no negative field control, and the bottle was stored in a warm car for four hours before processing. The lab's negative extraction control is clean. A second sample from a nearby 40°C, pH 6.5 spring contains the same sequence. The student has no microscopy or culture data yet.
1. Does the sample provide strong evidence for a novel thermophilic acidophile? Explain.
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No. The 99.9% match to B. subtilis, a mesophilic soil bacterium, suggests the sequence is either that organism or a close relative, not a novel thermophile. The field method lacked a negative control, so contamination from soil, dust, hands or the bottle cannot be excluded. Storage in a warm car for four hours could allow soil bacteria to persist or grow and could degrade heat-sensitive DNA. The clean extraction control only shows the lab reagents are clean; it does not show the field sample was uncontaminated. The same sequence in a nearby cooler spring also suggests a widespread soil organism, not a hot-spring specialist. To test the hypothesis, the student needs microscopy, culturing at 75°C, RNA evidence and replicate field controls.
2. If a microbe really lived at 75°C and pH 3.2, explain two adaptations and how you would test them.
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Thermostable proteins: amino acid changes increase ionic bonds and packing so enzymes do not unfold at 75°C. Test by measuring enzyme activity after heating, or compare protein stability. Membrane lipids: saturated or ether-linked lipids reduce membrane fluidity at high temperature; test membrane composition and permeability. Acid stress: proton pumps and a less permeable membrane maintain a near-neutral cytoplasm at pH 3.2; test internal pH with fluorescent dyes or growth at different external pH. DNA repair: high temperature damages DNA; test survival after heat or UV exposure. Each adaptation must be linked to a measurable prediction, not just named.