Extremophiles – Deepest Darkest Crush

Some microbes can live in unimaginably harsh places — such as in the boiling water of Yellowstone’s hot springs or under intense pressures at the bottom of the sea.

Douglas Bartlett is a marine microbiologist at the Scripps Institution of Oceanography in La Jolla, California. He’s studying a type of bacteria — called Photobacterium profundum — that thrives on the dark, chilly ocean floor — where the water pressure is more than 200 times the pressure of air at sea level.

Douglas Bartlett: The bulk of the volume of where life is found on Earth is found in low temperature, high pressure environments. So we’ve talked about this environment as extreme and yet one might very well consider the surface of our planet, that thin skin on the outside, as being the true extreme.

Low temperatures and high pressures are found elsewhere in our solar system — for example, the possible ocean on Jupiter’s moon Europa. Bartlett says that scientists looking for signs of life on Europa need to understand life in similar places on Earth.

Bartlett is searching for the genes that allow Photobacterium profundum to survive at high pressures. He’s creating thousands of variations of the bacteria, each with a different mutated gene. He puts the mutant bacteria into high-pressure vessels. The ones that die might be missing important genes for surviving in that type of environment.

We’re going to have to know how life can exist in the deep ocean of our planet. Because down below that deep ice layer that exists on Europa is a high pressure environment that extends to … twice the pressure of our deepest ocean trenches here on Earth.

Douglas Bartlett is a marine microbiologist at the Scripps Institution of Oceanography in La Jolla, California. He’s studying a type of bacteria — called Photobacterium profundum — that thrives on the dark, chilly sea floor where the water pressure is about 15,000 pounds per square inch — or about 500 times the pressure found in a car tire — or about one thousand times the pressure of air at sea level.

Transcript of an interview:

I’d love to put in a plug for a fantastic collaborator we have overseas in Padua, Italy and that’s Professor Georgio Vali. Georigio and his team have been doing yeoman’s work determining the genome sequence of this particular deep-sea microorganism and helping use to figure out what that genetic complement means.

Extremophiles is really an anthropomorphic term and it has to do with microorganisms that we humans consider to be extreme and these are a very significant group of microbes because they can be used for a variety of biotechnological purposes and they really highlight the metabolic and global diversity of microorganisms on this planet. So we work with some microorganisms that like to live at refrigeration temperatures, and they like to live at very high pressures, perhaps extending up to 15,000 pounds per square inch and they also live in perpetual darkness and they live with just sporadic sources of food, mostly those that come from surface waters falling plant debris and animal life.

So these are the kinds of extremophiles that we’re interested in, but others work with those that live at very high temperatures or very salty conditions and other kinds of extreme conditions.

We’ve been focusing a lot of our efforts these days on one particular species of microorganism, it’s photobacterium profundum. It’s a species that has been isolated from a variety of deep sea environments by people all over the pacific and we are focusing our attention on this organism because it’s genome sequence is being determined and we have developed genetic techniques for knocking out genes of interest and studying their regulation. So we know find ourselves in this really exciting position of having an exotic organism from the deep ocean and being able to ask questions.

Such as what is the expression pattern life for every single gene in that organism’s genome in response to changes in conditions that are important to life in the deep ocean, like changes in high pressure or temperature or other conditions.

Oddly enough a lot of the organisms that we see in the deep ocean are closely related to organisms that are found in surface waters and the organism that we’re looking at is a cousin of the human pathogen vibrio cholerae. So in addition to gleaning information about life under very unusual conditions, we’re also getting comparative information within this family of bacterium that includes vibrio cholerae. So this will tell us a lot about how this family of bacteria has evolved in some cases to make fish sick, in other cases to make humans sick, and in other cases to live in an exotic deep sea environment.

[in response to query about the extremes the organism is exposed to] In terms of thinking about the pressure that we’re talking about, these are enormous pressures, the Mariana trench extends to a pressure of 15,000 pounds per square inch. So if you think about that in terms of the air in your tires, it’s… five hundred times the pressure that exists in your tires. So its an enormous pressure. And I think that’s probably the best example I can give about what the magnitude of that is really is.

The temperatures are also very low, about two degrees centigrade, a temperature that is lower in the temperature in your refrigerator, and you combine that low temperature and that high pressure and you have a very harsh environment.

It’s one thing to be working with these organisms in stainless steel or titanium pressure vessels, it’s another thing to go down in a deep sea diving submersible and look out a port hole window and get a glimpse of what the deep sea environment can be like and in many ways it is such a strikingly different environment than what all of us are used to, including those of us who live along the coastline. The darkness is really enveloping and the sea floor just is so unique. Sea life may be greatly, may be very diverse in the benthos, but the biomass is also not very great except for the hydrothermal vent environment.

So it’s just really an extraordinary view to be down in the submersible and looking out with the benefit of the submersible lights and see that landscape, and see occasion invertebrates here and there and see a fish swimming by and so on. But it just looks like a very alien world when you’re actually down there.

We know that the deep ocean is that the deep ocean is enriched for a lot of the more recalcitrant organic molecules. Because the food items that are more easily digested are digested on the way down to the deep ocean. So what’s left behind are these humic acids and these other organic molecules that can be very difficult to break down. And so the thinking is that the microorganisms in the deep ocean must certainly have evolved a special transport and hydrolytic processes for breaking down some of these more exotic kinds of food sources. And that may be one of the values that we’ll find in exploring the genomes of deep-sea organisms. That is we’ll find genes that encode enzymes that encode novel degradative abilities.

Yes, so we’re working with a microorganism that we’re able to maintain frozen stocks of. So every time we make an interesting mutant we can save it in a minus 80 degree centigrade freezer. But we need to maintain lots of different mutants. We try to generate mutants in most all of the genes within the genome and those are maintained in microtiter dishes. So we have stacks and stacks of microtiter dishes each well of which contains a different mutant in our ultra low freezer.

So we can maintain this microbe in the refrigerator and these stainless steel and titanium pressure vessels that with hydraulic pumps we can pump up to very high temperatures and we can maintain the cultures that way but we can also maintain them in a state of suspended animation in an ultra-low freezer.

We can maintain those pressure vessels at whatever pressure weâd like, within limits and so often times we’re going down to pressures that are deepest ocean trench or maybe even exceeding that pressure, but the advantage of the microorganism that we do most of our work with is that we can maintain it outside of pressure vessels as long as long as the temperature is cold enough. But some of the other deep sea microorganisms that we’ve done work on have to be maintained within these pressure vessels. In fact, some of these that’s the only way we know how to keep them. So we have to have them in the pressure vessels, pumped up at thousands of pounds per square inch of pressure.

One of the characteristics of many microorganisms is they have relatively small genomes. That is compared to a fish for example. And because of that we donât need millions and millions and a team of 20 different laboratories to go through and obtain the genome sequence information on this organism that we’re studying. So we’re doing it on a relatively modest amount of funding and a relatively small number of people because we’re just talking about a few thousands genes rather than upwards of a million genes that is the case in the human genome for example.

So it doesn’t take as much manpower or resources for us to get this kind of ultimate information on what makes this organism tick, what is the complete assortment of genes that it has.

The most important conditions that we test our mutants for a growth defect in has to do with their ability to grow at very low temperatures or very high pressures. So those are the two parameters that we spend most of our time working with, temperature and pressure.

It turns out that one of the molecules that are turned up within the bacterium are molecules in the production of polyunsaturated fatty acids. The kind of polyunsaturated fatty acids that are useful for us humans to have in our diets. These are the heart smart fatty acids. They’ve also been described as a class of nutrasuticels. So this is just one example of a potential biotechnological spin-off of looking at the genes involved in high pressure and low temperature adaptation.

Having isolating all of these genes, weâll be in a position to transplant them into other kinds of organisms to create transgenetic plants, fishes. In the production of foods like yogurts, perhaps we can enrich those foods, add value to those foods by having greater concentrations of omega three polyunsaturated fatty acids incorporated into them.

The folks in Italy are doing the actual genome sequencing and they’re trying to put the pieces of the sequence information together along with some help from someone in my group. What we’re trying to addition is what is called functional genomics, so it’s one thing to look at that sequence of bases and discern the kinds of proteins that may be encoded for. It’s another to actually do the experiments that show the function of those genes. And so what we’re doing by making mutants in specific genes and looking at the properties of those mutants is ascribing function to the genes.

So this is part of the process of going beyond the genome sequence to try and really figure out what makes the microorganism tick. What’s its physiology, what are its unique properties and we do that by knocking out genes and looking at what the effect is. And that helps us to build back in our mind at least the machinery of the microorganism and understand better how it functions.

The biosphere and astrobiologists

Much of the benefit of what we’re doing has to do with thinking about life globally and one has to keep in mind that the bulk of the volume of where life is found on Earth is found in low temperature, high temperature environments. So we’ve talked about this environment as extreme and yet one might very well consider the surface of our planet, that thin skin on the outside as being the true extreme environment. So we’re looking at the adaptations of life to the largest part of our biosphere.

We’re also doing this with a mindset that other places in the solar system could potentially at least harbor life and one of those areas that astrobiologists love to think about these days is the Jovian moon Europa, this is a moon that believed to have a watery environment that’s at low temperatures and high temperatures. If we’re ever going to understand how life could exist in such a world, we’re going to have to know how life can exist in the deep ocean of our planet. Because down below that deep ice layer that exists on Europa is a high-pressure environment that extends to perhaps 30,000 pounds per square inch. So twice the pressure of our deepest ocean trenches here on Earth.

So that’s important. And then I guess the third reason that I would bring up for studying these deep-sea microbes is the biotechnological potential is really untapped. We donât know what kinds of enzymes we’re going to study that will be useful for food processing, for various industrial processes. And that will come out from identifying the genes and looking at the properties of those gene products. Are they super stable? Do they function under interesting conditions and have interesting catalytic properties that folks in industry, and in particular food industry perhaps would want to know about.

One genome

By developing the technology for screening mutants for defects at low temperature or high pressure, we’ve come up with this micro titer dish based assay that allows us to screens of hundreds of mutants at a time. We’ve now gone through more then 10,000 mutants for defects for growth at high pressure and we hope in a few months to have completed this aspect of the project and have gone through 20,000 mutants.

I think what’s really exciting now a days is we’re in a whole new world of biology because of the advances that have been made in genome sequences and the characterization of genome sequences. So, in the past we looked at the genetics of exotic microorganisms at a time. But genomics changes everything. Now we look at genes throughout the genome, all at once. So we can study the expression of every single gene of this deep-sea microorganism all in the same experiment.

That’s incredibly powerful because it allows us to uncover genetic circuits that we never really would have been able to uncover any other way. So I think really what the future holds is a much greater molecular capability for looking at what enables a microorganism to live in the deep ocean the connections that different genes make to enable that organism to adapt to this extreme environment. And we just get a kind of a resolution of life that was unheard of before the genomics revolution.

Obviously in the future we’re going to want to look at a variety of different species, those that are more extreme, those that come from different environments, but I think you’ve got the highlights.

The organism that we’re working with comes from the western Pacific Ocean, the Sulu Sea. Bu [skip in disk?] we also have plans to do some genome analysis on some even more high pressure adapted microorganism. Such as those that come from the Mariana trench that goes down almost eleven kilometers, so it’s the deepest ocean trench on earth and we would love to look at the microorganisms that come from the bottom of the Mariana trench. These are microorganisms that have to live in pressure vessels in our laboratories. We can not allow them to exist for long periods outside of a very very high pressure. And so it would be a real thrill to look at those organisms in genetic and molecular organisms.

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