Gene Induction in Fungi – Lamarckian?
|May 19, 2006||Posted by Dave S. under Biology, Evolution, Science|
As some of you may recall I wrote that I was experimenting with laboratory propagation of volvariella volvacea (Chinese Straw Mushrooms). Recently, among several other lines of R&D, I was experimenting with hydrogels as a nutrient media. So far I’ve been using them as an agar replacement with mixed results. I think the mixed results are due to uneven moisture distribution in the fine powder form I was using but that’s neither here nor there. Since the hydrogels can be loaded with nutrients at room temperature (the big advantage over agar) I decided to play around with another sterilant that would decompose at temperatures required to melt agar. I’ve been extremely successful using ampicillin at 1mg/20ml to prevent bacterial contamination in agar cultures – haven’t had a single bacterial infection in hundreds of agar plates. Ampicillin however breaks down quickly at temperatures over 60C so it must be added to agar at a critical stage after it’s cooled down (agar melts at 95C) some but before it solidifies (about 40C). This requires pouring fast and keeping a 60C water bath on the bench. However, ampicillin is so inexpensive it can be considered free of cost compared to wide spectrum antibiotics that survive pasteurization and autoclave temperatures. Once poured, ampicillin plates must be refrigerated until use as ampicillin in solution breaks down quickly at room temperature (a matter of days).
Anyhow, the new sterilant I was experimenting with is hydrogen peroxide (H2O2). I added a 3% (drugstore concentration) h2o2 solution to liquid nutrient at various ratios from 0.5ml/100ml up to 5ml/100ml, soaked a bit of hydrogel with the solution in culture plates at room temperature, then innoculated them from a vigorous volvariella colony. I did this all in the open air taking no particular sterile precautions (I usually use disinfectant, rubber gloves, scalpel sterized in alcohol flame, and a HEPA laminar flow hood to conduct sterile transfers) doing it with just an alcohol swab on the scalpel and syringe needles and on my kitchen countertop next to the toaster & blender – clean but definitely nowhere near sterile. The lower h2o2 concentrations allowed vigorous if somewhat reduced growth but about half of them contaminated with penicillium fungus (all the plates contained ampicillin so only fungal contamination was possible) after a few days. At higher concentrations of h2o2 new growth was completely arrested but none contaminated. After about 2 weeks I was ready to draw conclusions then move on.
Then a vicious series of thunderstorms with 60 mph winds rolled through the area and I had to stay on my boat for four days riding them out and making sure it remained securely anchored. When I returned home and checked my incubation chamber I was in for a big surprise. One of the h2o2 cultures (the highest concentration I tried at 5/100) that had been completely arrested for the prior two weeks, had bloomed and taken over the entire plate with an aggressive healthy volvariella colony. The rest were unchanged. Just one took off and when it took off it took off like there was no peroxide.
Curious, I wrote to Dr. Rush Wayne who’s pioneered the use of h2o2 in mushroom culture and whose procedures I purchased for a guideline (he’s never worked with this species) and asked if he’d seen anything like this. He said he hadn’t experimented with concentrations that high but he said it would be what he would expect if volvariella had used gene induction to turn on an h2o2 decomposing enzyme. H202 is a naturally occurring sterilant present in the environment and most living things produce enzymes that decompose it. Those enzymes are what cause the fizzing when you pour it on an open wound. Spores don’t have enough of the enzyme present to survive germination in a low h2o2 concentration but a healthy fungal colony already does and hence its efficacy as a sterilant in mushroom culture. One merely has to insure that the nutrient substrate doesn’t have h2o2 decomposing enzymes in it. Almost all processed nutrients have had those enzymes destroyed by the heat of pasteurization which includes the potato/dextrose broth I was using.
Dr. Wayne said some organisms may have latent genes for enzymes that can decompose h2o2 much more rapidly than normal and can turn on those genes through “gene induction” if they encounter high concentrations. None of the mushrooms he’d experimented with had exhibited gene induction for h202 decomposing enzymes. But volvariella is an odd duck amongst mushrooms in its voracious apetite over a wide range of substrates, inability to survive temperatures below 45F, dying (or at least going dormant with asexually produced chlamydospores if conditions are right) easily when it runs out of food (which drastically limits shelf life of harvested mushrooms), and senescence when recultured for very many generations (it mutates rapidly when stressed like when it runs out of fresh agar surface to colonize).
So anyhow, I now have a volvariella colony that mutated beneficially within a matter of weeks in a most Lamarckian way in response to environmental stress. Its progeny inherit the ability to tolerate high h202 concentrations and show no loss of fitness in the absence of h202 (I recultured the colony back onto h202-free agar and it looks just like the control colony recultured at the same time). We’ve blogged here before about Scripps discovery that e.coli can rapidly adapt to antibiotics by enabling certain genes to mutate at extremely high rates and that the antibiotic resistance is heritable (and horizontally transferrable through plasmids). I speculated from that that eukaryotes probably had the same capacity to adapt since they ostensibly evolved from prokaryotes. Volvariella is a eukaryote and I just witnessed it evolving faster than random mutation and natural selection could possibly account for. Yet another nail in the RM+NS coffin…