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You can find hordes of bacteria on the surfaces of a plant’s leaves, stems, flowers and fruits. These bacteria range from beneficial to benign, with the occasional bad actor. If you examined Arabidopsis thaliana, you likely would find two bacteria called Sphingomonas melonis and Methylobacterium extorquens inhabiting the entire above-ground plant surface.

S. melonis (blue) and M. extorquens (green) share a leafy habitat. DANIEL MÃœLLER/ETH ZURICH

To understand better the interactions and adaptations that allow these bacteria and many others to call the plant home, researchers recently performed proteomic analyses of these two organisms. By applying a technique known as SWATH MS, the researchers identified a set of shared proteins, which indicates common mechanisms that underlie successful leaf colonization. They described their work in a in the journal .

“Historically, people have only looked at the roots,” says at the ETH Zurich’s Institute of Microbiology. This is largely because of the role symbiotic root bacteria play in providing the host with nutrients. How the bacteria interact with the part of the plant that is above the ground has been looked into only recently, says Müller. “It became increasingly obvious that the leaves and the phyllosphere in general — all the above-ground parts of plants — are also colonized, and they have an impact on the host cells too,” he adds. Müller is a postdoctoral researcher in the lab of , the lead author on the MCP paper.

Despite their shared phylogenetic class of Alphaproteobacteria, S. melonis and M. extorquens have evolved to occupy different ecological niches on plants. S. melonis has adapted to a diet of amino acids and hydrocarbon compounds; M. extorquens subsists primarily on methanol, oxalate and alkanesulfonates and also carries out anoxygenic photosynthesis. Additionally, S. melonis has been demonstrated to confer protection against a common leaf pathogen. Researchers believe that the bacteria might provide other symbiotic benefits to the host.

“We are lacking a lot of functional information,” says Müller. “This proteomics approach was one of the first steps towards providing such insights. We know what is present in terms of bacterial taxa, but we need to understand what they are actually doing there and how they might influence each other.”

To examine which of their proteins S. melonis and M. extorquens activate when occupying the phyllosphere, Müller and colleagues inoculated surface-sterilized seeds of A. thaliana with samples of each strain. They collected the bacteria from the growing plants after 28 days and subjected them to an analysis by shotgun proteomics. From the shotgun proteomics data, the investigators constructed a database containing mass-spectrometric information about every protein of interest. The libraries the researchers generated contained information for about 71 percent of the total proteome of both S. melonis and M. extorquens.

Next, to quantify the bacterial proteomes, Müller and colleagues ionized and fragmented the proteins expressed by the bacteria by tandem mass spectrometry. This allowed them to record the mass-to-charge ratios of all fragment ions, along with other characteristics that helped match the fragments to the database.

The researchers then analyzed this quantitative information with special software. They identified 635 candidate proteins for M. extorquens that were regulated significantly on leaf surfaces compared with minimal media and 545 candidate proteins that were regulated significantly on leaf surfaces for S. melonis. Between the two bacteria, there was a shared subset of 17 proteins.

This means that “despite different modes of metabolism, common adaptive strategies seem to exist, such as acquiring limiting macroelements such as sulfur or phosphorus,” says Müller. “Among the shared proteins are some of unknown function, potentially indicating that new functions are essential for leaf colonization.”

Future work for Müller and colleagues will include examining the differences in the protein repertoires of different, co-existing bacteria to understand better how they manage to share a plant between them.