September 27, 2013
Bacterium Found In Termite Guts Helps Make Wood A Nutritious Meal
April Flowers for redOrbit.com - Your Universe Online
As termites eat wood, the small chunks they chew off are delivered to feed a community of unique microbes living in their guts. In a complex multi-step process, these microbes turn the hard, fibrous material of the wood into a nutritious meal for the termite host. A key step in this process uses acetogenesis -- where hydrogen converts carbon dioxide into organic carbon. It is unknown, however, which gut bacteria play specific roles in the process.
A research team from the California Institute of Technology (CalTech) used a variety of experimental techniques to discover a previously unidentified bacterium that lives on the surface of a larger microorganism in the termite gut. This new bacterium may be responsible for most gut acetogenesis. The findings of this study were published online in the Proceedings of the National Academy of Sciences.
"In the termite gut, you have several hundred different species of microbes that live within a millimeter of one another. We know certain microbes are present in the gut, and we know microbes are responsible for certain functions, but until now, we didn't have a good way of knowing which microbes are doing what," says Jared Leadbetter, professor of environmental microbiology at Caltech, in whose laboratory much of the research was performed.
Acetogenesis is the process that creates acetate -- a nutritional source for termites -- from the carbon dioxide and hydrogen generated by gut protozoa as they break down decaying wood. Leadbetter and his colleagues identified the specific genes from organisms responsible for acetogensis by examining the entire pool of termite gut microbes.
The research team started by sifting through the microbes' RNA -- genetic information that can provide a snapshot of the genes active at a certain point in time. The team used RNA from the total pool of termite gut microbes to search for actively transcribed formate dehydrogenase (FDH) genes. FDH genes are known to encode a protein necessary for acetogenesis.
The experiment continued by using a method called multiplex microfluidic digital polymerase chain reaction (digital PCR), which allowed the scientists to sequester the previously unstudied individual microbes into tiny compartments to identify the actual microbial species carrying each of the FDH genes. A type of bacteria called spirochetes were found to have FDH genes, but it appears that these microbes alone could not account for all of the acetate produced in the termite gut.
At first, the scientists were unable to identify the microorganism expressing the most active FDH gene in the gut; however, team members Adam Rosenthal, a postdoctoral scholar in biology at Caltech, and Xinning Zhang (PhD '10, Environmental Science and Engineering), noticed that this gene was more abundant in the portion of the gut extract containing wood chunks and larger microbes, like protozoans. Analyzing the chunkier gut extract led to the discovery that the single most active FDH gene was encoded by a previously unstudied species from a group of microbes known as the deltaproteobacteria -- the first evidence that a substantial amount of acetate in the gut may be produced by a non-spirochete.
The research team thought that perhaps the newly identified microbe attaches to the surface of the chunks because the genes from this deltaproteobacterium were found in the chunky particulate matter of the termite gut. A color-coded visualization method called hybridization chain reaction-fluorescent in situ hybridization, or HCR-FISH, was used to test their hypothesis.
Niles Pierce, professor of applied and computational mathematics and bioengineering at Caltech, developed the HCR-FISH technique, which allowed the researchers to simultaneously "paint" cells expressing both the active FDH gene and a gene identifying the deltaproteobacterium with different fluorescent colors simultaneously.
"The microfluidics experiment suggested that the two colors should be expressed in the same location and in the same tiny cell," Leadbetter says.
It turns out they were correct.
"Through this approach, we were able to actually see where the new deltaproteobacterium resided. As it turns out, the cells live on the surface of a very particular hydrogen-producing protozoan," adds Leadbetter.
Based on what is known about the complex food web of the termite gut, Leadbetter says the association between the two organisms makes sense.
"Here you have a large eukaryotic single cell—a protozoan—which is making hydrogen as it degrades wood, and you have these much smaller hydrogen-consuming deltaproteobacteria attached to its surface," he says. "So, this new acetogenic bacterium is snuggled up to its source of hydrogen just as close as it can get."
Leadbetter said that the intimate relationship might never have been discovered relying on phylogenetic inference, which is the standard method for matching a function to a specific organism.
"Using phylogenetic inference, we say, 'We know a lot about this hypothetical organism's relatives, so without ever seeing the organism, we're going to make guesses about who it is related to," he says. "But with the techniques in this study, we found that our initial prediction was wrong. Importantly, we have been able to determine the specific organism responsible and a location of the mystery organism, both of which appear to be extremely important in the consumption of hydrogen and turning it into a product the insect can use."
In addition to identifying a new source of acetogenesis in the termite gut, the results reveal the limitations of making predictions based exclusively on phylogenetic relationships.