Read the paper as well as the referenced chain of papers in which a soil bacterium was engineered to be an efficient producer of ethanol from Xylose. I love these old-school genetic engineering without-a-known-genome methods!
First, a brief geekout. The species modified in the work in the late 80s and early 90s is a facultative anaerobe, meaning it can get by fine without oxygen but prefers to have it. Without oxygen, like everything else it rearranges the sugar atoms into a lower energy state getting a bit less than 1⁄15 as much energy and much less biomass. They were interested in it because it can consume xylose, a sugar found in plant debris that is difficult for many things to digest. Without oxygen the natural bacterium squirts out a bunch of CO2, a little alcohol, some acetic acid, some lactate, some formic acid, and some weird complicated bigger alcohols when it ferments. The modified strain had a normal enzyme involved in both the production of some of those byproducts and other intermediates and the sucking of some of the carbon into biomass production deleted off the chromosome, plus a non-chromosomal plasmid containing a bacterial gene not normally found in that species that can take the substance piling up in the blocked-up pathway and release it into making ethanol. Has a side effect of making sure they don’t acidify their growth media to all hell with acetic acid and formic acid, which makes them grow much faster in controlled pure culture where nothing else is eating the acetate aerobically. In the process of making energy they have to just throw the carbon through their metabolisms and make ethanol at fully 80% maximum theoretical efficiency.
Both this modified bacterium and the parent bacterium, with the deletion, caused a major disruption in the soil ecology of the soil samples they were added (at 10 million cells per gram) to and temporarily arrested plant shoot growth without arresting root growth, possibly having to do with screwing up the nematode and fungal populations in a chain reaction of effects of things feeding on each other. Both dropped down to something like 100 cells per gram within a few weeks which is probably closer to their natural concentration. The extra modified one also killed more plants, possibly from the transient ethanol production by the initial high population density affecting the ecology or the plants directly. I’d be interested in a double-inoculation experiment with the wild bacterium to see if one could outcompete the other. It’s hard to say because the growth experiments were done in pure culture where the fact that the modded one doesn’t acidify its environment made it grow much faster than the parent strain, but i suspect in the wild the original one would do better because it can make more biomass.
I was frankly surprised and flabbergasted the modified bacteria held onto their plasmids for 8 weeks such that they could even be detected via their plasmid-borne antibiotic resistance. When I have E. coli carrying plasmids for me (full of yeast genes, only bacterial gene is the antibiotic resistance), if I take them out of antibiotic selective pressure for a few hours almost all of them lose their plasmids due to it being easy to lose but hard to gain and due to those with the plasmids making more protein and thus growing slower. Course my lab E coli on rich broth have a generation time of 20 minutes while these guys in soil could’ve had a generation time of days. And the ability to unblock the clogged metabolic pathway caused by the chromosomal deletion could’ve been a selective advantage to holding onto the plasmid—a deleted gene is not something you evolve a workaround for in the timeframe of any experiment short of Lenski’s.
Reasons I would not be terribly worried about this sort of thing:
The inserted gene (pyruvate decarboxylase) is not a rare gene in the eubacterial lineage, and bacteria are throwing genes back and forth horizontally over evolutionary time all the time. The bacteria fall in population quite rapidly to fairly normal levels. The faster growth of the modded bacteria is only observed in pure culture where their lack of acifification more than makes up for their lesser ability to make biomass.
My conclusion: they were right to test it and the dregs from this biofuel production process, full of this bacteria, would make a terrible soil amendment that would screw up your garden/farm’s soil ecology for weeks at least. I’m not terribly surprised; I helped run the university community garden for years and its incredible how long it can take to get the bacterial balance back in the compost bin after someone thoughtlessly throws meat into the pile. Makes a good point about actually testing your genetically modified organisms in their ecological reactions with microbes, plants, and animals they are likely to interact with in the field. These systems are hella complicated and effects can compound up and down the food chain in ways you don’t expect.
Read the paper as well as the referenced chain of papers in which a soil bacterium was engineered to be an efficient producer of ethanol from Xylose. I love these old-school genetic engineering without-a-known-genome methods!
First, a brief geekout. The species modified in the work in the late 80s and early 90s is a facultative anaerobe, meaning it can get by fine without oxygen but prefers to have it. Without oxygen, like everything else it rearranges the sugar atoms into a lower energy state getting a bit less than 1⁄15 as much energy and much less biomass. They were interested in it because it can consume xylose, a sugar found in plant debris that is difficult for many things to digest. Without oxygen the natural bacterium squirts out a bunch of CO2, a little alcohol, some acetic acid, some lactate, some formic acid, and some weird complicated bigger alcohols when it ferments. The modified strain had a normal enzyme involved in both the production of some of those byproducts and other intermediates and the sucking of some of the carbon into biomass production deleted off the chromosome, plus a non-chromosomal plasmid containing a bacterial gene not normally found in that species that can take the substance piling up in the blocked-up pathway and release it into making ethanol. Has a side effect of making sure they don’t acidify their growth media to all hell with acetic acid and formic acid, which makes them grow much faster in controlled pure culture where nothing else is eating the acetate aerobically. In the process of making energy they have to just throw the carbon through their metabolisms and make ethanol at fully 80% maximum theoretical efficiency.
Both this modified bacterium and the parent bacterium, with the deletion, caused a major disruption in the soil ecology of the soil samples they were added (at 10 million cells per gram) to and temporarily arrested plant shoot growth without arresting root growth, possibly having to do with screwing up the nematode and fungal populations in a chain reaction of effects of things feeding on each other. Both dropped down to something like 100 cells per gram within a few weeks which is probably closer to their natural concentration. The extra modified one also killed more plants, possibly from the transient ethanol production by the initial high population density affecting the ecology or the plants directly. I’d be interested in a double-inoculation experiment with the wild bacterium to see if one could outcompete the other. It’s hard to say because the growth experiments were done in pure culture where the fact that the modded one doesn’t acidify its environment made it grow much faster than the parent strain, but i suspect in the wild the original one would do better because it can make more biomass.
I was frankly surprised and flabbergasted the modified bacteria held onto their plasmids for 8 weeks such that they could even be detected via their plasmid-borne antibiotic resistance. When I have E. coli carrying plasmids for me (full of yeast genes, only bacterial gene is the antibiotic resistance), if I take them out of antibiotic selective pressure for a few hours almost all of them lose their plasmids due to it being easy to lose but hard to gain and due to those with the plasmids making more protein and thus growing slower. Course my lab E coli on rich broth have a generation time of 20 minutes while these guys in soil could’ve had a generation time of days. And the ability to unblock the clogged metabolic pathway caused by the chromosomal deletion could’ve been a selective advantage to holding onto the plasmid—a deleted gene is not something you evolve a workaround for in the timeframe of any experiment short of Lenski’s.
Reasons I would not be terribly worried about this sort of thing:
The inserted gene (pyruvate decarboxylase) is not a rare gene in the eubacterial lineage, and bacteria are throwing genes back and forth horizontally over evolutionary time all the time. The bacteria fall in population quite rapidly to fairly normal levels. The faster growth of the modded bacteria is only observed in pure culture where their lack of acifification more than makes up for their lesser ability to make biomass.
My conclusion: they were right to test it and the dregs from this biofuel production process, full of this bacteria, would make a terrible soil amendment that would screw up your garden/farm’s soil ecology for weeks at least. I’m not terribly surprised; I helped run the university community garden for years and its incredible how long it can take to get the bacterial balance back in the compost bin after someone thoughtlessly throws meat into the pile. Makes a good point about actually testing your genetically modified organisms in their ecological reactions with microbes, plants, and animals they are likely to interact with in the field. These systems are hella complicated and effects can compound up and down the food chain in ways you don’t expect.