Published: 15.09.09
Microbiology

Free-for-all on the leaf surface

For the first time, ETH Zurich scientists have examined the genes and proteins of bacteria that live on leaves to clarify which unicellular organisms are found on leaf surfaces and what they are doing there.

Klaus Wilhelm
Electron microscope image of a leaf surface. Up to ten million unicellular organisms live on every square centimeter. (Image: Gerd Innerebner and Roger Wepf/ETH Zurich)
Electron microscope image of a leaf surface. Up to ten million unicellular organisms live on every square centimeter. (Image: Gerd Innerebner and Roger Wepf/ETH Zurich) (large view)

Bacteria are everywhere: in the ground, on the seabed, in boiling hot sources, in the gut. They are even on the surface of plants, and lots of them at that: one to ten million of the unicellular microorganisms live on every square centimeter of stem and foliage. At one billion square kilometers, the entire plant surface is estimated to be four times bigger than that of the earth, thus making the so-called phyllosphere “the largest biological surface inhabited by microorganisms”, as Julia Vorholt, a professor at the Institute of Microbiology at ETH Zurich, puts it. The bacteria in this ecosystem are so numerous that they influence the vital global carbon cycle.

Suitable methods previously lacking

For the layman, it is all the more astonishing that biologists still know so little about this habitat and its inhabitants. However, the scientists have been plagued by one fundamental problem: for decades, the methods simply did not exist to offer a realistic glimpse into the variety of microbial ecosystems. However, in recent years procedures from modern molecular biology have made it possible to gain an increasingly better understanding of the bacteria and their function in complex microbe communities. For instance, Julia Vorholt’s team of researchers has now for the first time analyzed worldwide the metagenome and the metaproteome of a natural bacterial biocoenosis on a grand scale – i.e. the genes and proteins of the bacteria – and thus obtained initial insights into microbial activity on foliage. “Two kinds of bacteria dominate this ecosystem”, explains Vorholt: members of the methylobacterium genus and unicellular organisms from the sphingomonas genus.

The scientists began by collecting leaves from soy and clover plants and mouse-ear cress (Arabidopsis thaliana), then washed the bacteria off and skillfully processed the samples using specifically established methods. The proteins in the complex mixtures were cut into small pieces and analyzed with what Vorholt refers to as “hyper-sensitive mass spectrometers from the Functional Genomics Center Zurich”. The scientists then compared the structure of the fragments with known protein structures in international databases. “This enables us to identify the proteins and get an indication of which proteins the bacteria need under the given environmental conditions”, stresses the microbiologist from ETH Zurich.

Genes and proteins unmask identity

In order to obtain clues about proteins not yet recorded in the databases, the scientists also analyzed the genomes of the bacteria community. The genes indicate which proteins might be produced in a cell – “might” because cells only convert part of their genes into proteins. Together, all the genome and proteome data reveal the identity of the microorganisms, their potential capabilities and activity on the leaf surface.

Result: whatever the plant, bacteria from the sphingomonas and methylobacterium genera and their proteins always dominated the scenery. In all, the researchers found over 20 bacteria genera with about 100 different species. In doing so, the team from ETH Zurich also discovered previously unknown proteins, “which appear to be important for most bacteria on the leaves of all three plants studied”, says Julia Vorholt.

The Zurich team also came across an equally abundant protein among the methylobacteria that is similar to a known protein and important for the unicellular organisms’ metabolism. Methylobacteria convert the methanol produced by the plants into carbon dioxide for energy and nourishment. Bacteria from the sphingomonas genus, however, are less specialized and utilize different food and energy sources, such as sugar for instance. At least, this is what the many transporter proteins discovered by the ETH-Zurich biologists suggest.

Give and take?

The most interesting of the proteins now serve as a spring board for new experiments. The questions: what is the relationship between the bacteria and the plants? Do they solely use them as a source of nourishment and energy? Or do they give the plants something in return? This is easily conceivable; after all, plants are exposed to harmful attacks from microorganisms on a daily basis – transferred by insects or in the air. It is perfectly feasible that the colonization by microbes like methylobacteria or sphingomonas could protect the plants from such attacks. “Maybe the bacteria even produce antibiotics to keep the plants healthy”, speculates Julia Vorholt.

Reference

Delmotte N, Knief C, Chaffron S, Innerebner G, Roschitzki B, Schlapbach R, von Mering C, Vorholt JA. Community proteogenomics reveals insights into the physiology of phyllosphere bacteria. PNAS published online before print September 4, 2009. doi:10.1073/pnas.0905240106

 
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