Our inner selves: The microbes that call us home

What do you think of when you think of bacteria? Up until a few years ago I took a decidedly unfriendly view: bacteria (and viruses forthat matter) were something to avoid as much as possible and they didlittle for us besides make us sick.

Bacteria: The Silent Majority

It turns out that the role that microbes play in our lives is much more complex than that of simply a pathogen. All plants and animals arehome to a large and diverse number of microbes.

The numbers are truly staggering. If you do a census of the cells in your body you’ll find 10 times morebacterial cells in you than human cells (Ref. 1). Additionally, there is about 150 times moremicrobial DNA than human DNA in your body. Admittedly prokaryotic cells are much smaller than eukaryotic cells-so by mass there is only "10" pounds or so of bacteria living on and in you, most of which is in your gut.

These bacteria are not just passive residents of our bodies.Rather, it's becoming increasingly apparent that the makeup and composition ofour very own bacterial community, or microbiome, has important consequences forour health. Many disorders, such as type II diabetes, obesity, irritable bowelsyndrome, and asthma have been correlated with a change in the makeup of ourgut microbiome (Ref. 1). In other words, people who have type II Diabetes have, on average,different phyla of bacteria present in their gut than the average individual.This suggests that studying the microbiome is rewarding for two reasons. First,it might lead to new and innovative ways of treating diseases in humans.Second, if you want to understand the basic biology of animals (in particularhumans) it's necessary to understand what our tenants are up to.

In the last decade the study of the microbiome has exploded,driven largely by the rise of cheap high-throughput gene sequencing. Bacteriacan be classified by doing 16s sequencing (Ref. 2). The 16s sequence is ahighly conserved RNA sequence that is used to construct the prokaryoticribosome and because it's believed that that the function of this gene hasn'tchanged over time variations in the 16s sequence can be used to classifybacteria into different phyla. For classifying bacteria this is particularlyuseful because it's often hard to classify bacteria by phenotypes (mostbacteria just look like rods to me) and most bacteria can’t even be cultured ina lab.

The Human Microbiome. Relative abundances of different phyla are shown for different body parts (Ref. 3)

This technique is enormously powerful and it allowsresearchers to get a complete picture of all the bacterial residents of anorganism. Researchers are starting to use this technique to catalogue all thebacteria that lives on humans and the variations in those species, as part ofthe Human Microbiome Project (Ref. 4). The results coming out of this project are really remarkable. The distribution of bacteria on different parts of the bodies varieswildly (see figure above). There’salso evidence that the distribution of bacteria that live in our guts belong toone of three communities, each of which contains hundreds of different species,but that are distinct from each other (Ref. 5). The bacteria that inhabit us are not just a hodgepodge of bacteria taken from the earth around us. Instead our microbiome consists of bacteria that have found their particular environmental niche: us.

While a genetic approach is powerful, it also has itslimitations. There are two ways that gut samples are collected for genesequencing: taking stool samples or dissecting and sequencing the entire gut(obviously impossible with humans). Data taken using the first technique onlylets you get a sense of the bacteria that are leaving the gut and may notaccurately reflect the percentage of different bacteria that reside in the gut.For instance, it may be the case that a subset of bacteria are particularlyeffective at adhering to the wall of the gut and can’t be easily removed. Theother approach, dissecting the gut, only gives you information about the gut asa whole and doesn’t tell you anything about any spatial variation that might bepresent, for instance if different bacteria establish themselves in differentecological niches. The other problem is timing: you can't constantly bemonitoring the types of bacteria in the gut (especially if you’ve dissectedit!) and it may be the case that there are important temporal variations indistributions of bacteria.

With these issues with sequencing in mind there might be alot to be gained by watching the colonization of the gut by bacteria. Our grouphas recently been working with the Guilleman Lab, also right here at theUniversity of Oregon, to do just this, by imaging the dynamics of gut bacteriain zebrafish (a species of minnow).

Now we're not studying zebrafish for the sake ofunderstanding the biology a certain type of fish, but because zebrafish aremodel organism for a variety of human conditions. Model organisms are non-humanspecies that are used to test and understand biological systems.Because many biological features are heavily conserved over the course ofevolution this approach is often well justified.

A: Brightfield image of a zebrafish. Scale bar: 500 microns.

B: Sketch of a zebrafish, with the gut highlighted in green.

Zebrafish are a relatively new model organism (moreestablished ones that you may be familiar with are mice, e. coli, and fruitflies) that was pioneered right here in Eugene (Ref. 6)!

Zebrafish aregreat to work with for several reasons. They're relatively easy to raise andbreed. Also, Zebrafish are fairly amenable to genetic manipulation. Finally,and most important to our work, they're optically transparent when they'reyoung. This somewhat unique feature makes it possible to directly imagebacteria colonization of the gut-something you can't do with other modelorganisms, such as mice, that have guts.

Initial colonization of the gut. Left Panel: 4 hours after inoculation with bacteria. Right Panel: 6 hours after inoculation. Scale bar: 100 microns.

Along the lines of model organisms, we are also looking at a "model" gut. Our collaborators have developed a technique to raise germ-freezebrafish. These fish have, from the point of birth, never been exposed to anybacteria. Not a single one. We then introduce fluorescently labeled bacteria(bacteria that have been engineered to produce fluorescent proteins that make them glow when struck by laser light) into the water the fish are living in. This gut is a “model” in thesense that we’re ignoring all the potential complexity that will arise fromhaving a lot of different bacteria in the gut at once and focusing instead onsimpler interactions. From this foundation we can then (hopefully) build up increasinglayers of complexity.

After a couple of hours the bacteria have found their wayinto the zebrafish and we take them and put them on a microscope. Themicroscope that we’re using is called a light sheet microscope (Ref. 7), arelatively new design of microscope that allows us to quickly image with micronlevel resolution along the entire length of the gut. This type of microscopedesign is gentle enough on the zebrafish that we can image them for hours oreven days at a time.

Bacteria colonizing the length of the gut. Anterior is to the left.

Above I’ve included a picture of what the gut looks like 6hours after bacteria have been introduced to the water the fish was in. A couple things areimmediately obvious. The distribution of bacteria is very heterogeneous withthe majority of the bacteria residing towards the anterior of the gut. There isalso a wide range of size of bacterial clumps. In other words, there is a largeamount of spatial variation within the gut and this suggests that we may seethings by imaging that can’t be explored by genomic-based techniques.

Through microscopy we can begin to peer at the dynamics ofan ecosystems that all of us animals carry around with us every day. There area lot of interesting questions one can ask. For instance, what happens when twotypes of bacteria, either from different species or that have differentphenotypes (e.g. a chemotaxis mutant- a bacteria that can’t move in response tothe gradient of different chemicals in its environment), are both introduced tothe gut? If the bacteria are introduced at different times will the firstspecies establish itself in the best environmental niche and prevent the laterarriving species from successfully colonizing the gut? What about if the laterarrival has a competitive advantage (for instance if the first bacterialspecies lacks chemotaxis)? How will that affect competition between thespecies?

We can do a field study of the main players in an environment that stretches not over the length of a forest, but over a humble couple of millimeters of gut. How can you gain information about the spatial and temporalfeatures of an ecosystem (or any system for that matter)? There are a varietyof powerful mathematical techniques that have been developed to extract thistype of information from signals even when the signal is noisy (which all reallife data certainly is!). I’ll go over two of these techniques in class:Fourier and wavelet analysis and show they can be used to answer ecological questions.

References

(1) Our microbial selves: what ecology can teach us. Gonzalez, et. al., EMBO Reports, 12, 775-784 (2011).

URL (firewalled): http://www.nature.com/embor/journal/v12/n8/pdf/embor2011137a.pdf

(2) 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls. J.M Janda and S.L. Abbott, J. Clin Microbiol, 45, 2761-2764 (2007).

URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045242/

(3) Unravelling the effects of the environment and host genotype on the gut microbiome, A. Spor, et. al., Nature Reviews Microbiology, 9, 279-290, (2011).

URL (firewalled): http://www.nature.com/nrmicro/journal/v9/n4/pdf/nrmicro2540.pdf

(4) The Human Microbiome Project: http://commonfund.nih.gov/hmp/overview.aspx

(5) Enterotypes of the human gut microbiome. Arumugum, et. al., Nature, 473, 174-180 (2011).

URL (firewalled): http://www.nature.com/nature/journal/v473/n7346/abs/nature09944.html

This research got plenty of press coverage that is not (or just nominally in the case of the NY times) firewalled, for instance:

https://www.nytimes.com/2011/04/21/science/21gut.html

(6) http://www.neuro.uoregon.edu/k12/george_streisinger.html

Gives a nice biography of George Streisinger and goes into the early history of zebrafish husbandry (which seemed to include a lot of trips to the Albany, OR pet sore)

(7) http://www.lmg.embl.de/lsfm.html

Link contains a nice review of the microscopy technique from the pioneers of this technology.