Exploring functional contexts of symbiotic sustain within lichen-associated bacteria by comparative omics

Abstract

Symbioses represent a frequent and successful lifestyle on earth and lichens are one of their classic examples. Recently, bacterial communities were identified as stable, specific and structurally integrated partners of the lichen symbiosis, but their role has remained largely elusive in comparison to the well-known functions of the fungal and algal partners. We have explored the metabolic potentials of the microbiome using the lung lichen Lobaria pulmonaria as the model. Metagenomic and proteomic data were comparatively assessed and visualized by Voronoi treemaps. The study was complemented with molecular, microscopic and physiological assays. We have found that more than 800 bacterial species have the ability to contribute multiple aspects to the symbiotic system, including essential functions such as (i) nutrient supply, especially nitrogen, phosphorous and sulfur, (ii) resistance against biotic stress factors (that is, pathogen defense), (iii) resistance against abiotic factors, (iv) support of photosynthesis by provision of vitamin B12, (v) fungal and algal growth support by provision of hormones, (vi) detoxification of metabolites, and (vii) degradation of older parts of the lichen thallus. Our findings showed the potential of lichen-associated bacteria to interact with the fungal as well as algal partner to support health, growth and fitness of their hosts. We developed a model of the symbiosis depicting the functional multi-player network of the participants, and argue that the strategy of functional diversification in lichens supports the longevity and persistence of lichens under extreme and changing ecological conditions.

Figure 1

Taxonomic spectrum visualized with Krona (www.krona.sourceforge.net/) of contigs in the metagenome for all domains of life. Circles represent taxonomic classifications in ascending order up to the family level (outermost circle). Less-abundant taxa are listed outside the charts together with their relative abundance.

Figure 2

(a) Leaf-like thallus visualization of bacteria on a cross-section by 3D reconstruction of FISH image stacks. Eubacteria (red) and Alphaproteobacteria (yellow) were found widespread on both, the upper and the lower cortex, while Betaproteobacteria (pink) were less abundant and locally contained. Fungal hyphae (blue) and algae located under the upper cortex (green) were visualized without specific FISH probes, due to the naturally occurring fluorescence of the internal structures. (b) Model of the lichen symbiosis depicting the functional network of the participants. The model includes relevant functions of the colonizing bacteria, which are derived from metagenomic/metaproteomic analysis, as well as cultivation-dependent experiments.

Figure 3

Distribution of particular functions prior and after exclusion of less-abundant taxa. The number of contigs assigned to all organisms within the sample (blue bars) is visualized in contrast to assigned contigs of the eight most-abundant taxa (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes and Firmicutes; red bars) and Alphaproteobacteria (green bars). The full colour version of this figure is available at ISME Journal online.

Figure 4

Principal Coordinates Analysis (PCoA) including 20 publicly available metagenomic datasets from MG-RAST and the Lobaria metagenome (red dot). All datasets were compared with subsytems and calculated using normalized values and the Bray-Curtis distance matrix. Single metagenomes from different biomes are labeled with their MG-RAST accession numbers and grouped in colored ellipses. The full colour version of this figure is available at ISME Journal online.

Figure 5

Voronoi Treemap visualization of the prokaryotic metaproteome on the taxonomic (a) and functional (b) level. The taxonomic and functional distributions were carried out by Prophane 2.0. Taxonomic groups are indicated by different colors (a and b) and the functional classes are separated by black lines (based on TIGRRoles). Each cell represents at least one protein (or protein group) assigned to the respective phylum.

Figure 6

Functional recovery of the bacterial lichen microbiome metagenome on the metaproteomic level using Voronoi treemaps. The protein sequences were compared with metagenomic contigs by BLAST and then functionally characterized using HMMER3 and TIGRFAMs. Upper panel shows all functions covered by the metagenome (third level) and the respective subroles (second level) and main roles (first level). The mainrole labels are shown. On the lower panel, metaproteomic coverage is shown (blue cells: functions present only in the metagenome; grey cells: functions present in both metagenome and metaproteome).