Phytoplankton consortia as a blueprint for mutually beneficial eukaryote-bacteria ecosystems based on the biocoenosis of Botryococcus consortia

Abstract

Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host`s development, growth and health. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described at the phylogenetic level but rarely functionally. Multifaceted analysis of the microbial consortium of the ancient phytoplankton Botryococcus as an attractive model food web revealed that its all abundant bacterial members belong to a niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies seem adversely to affect the algal cell morphology. Synthetic rearrangement of a minimal community consisting of an alga, a mutualistic and a parasitic bacteria underpins the model of a eukaryote that maintains its own mutualistic microbial community to control its surrounding biosphere. This model of coexistence, potentially useful for defense against invaders by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction is an opportunity to unravel the relationships within the consortia and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.

Figure 1

Taxonomy of the B. braunii bacterial community and selected functional categories encoded in reconstructed MAGs and complete genomes. (a) Normalized read-based comparison and taxonomic assignment of different B. braunii metagenome datasets. The circle size represents the amount of classified reads for the respective taxonomic group (minimum 50 reads per taxonomic group). Abbreviations: P, phylum; G, genus; O, order. (b) Abundance of selected functional categories encoded in the high-quality draft and complete bacterial genomes. Numbers in parentheses represent the maximal total number of genes within each pathway. Percentage: per cent of total number of genes per pathway identified within each genome. All annotated genes within the respective pathways are presented on the EMGB platform (https://emgb.cebitec.uni-bielefeld.de/Bbraunii-bacterial-consortium/). The pathways statistics are summarized in the Table S5.

Figure 2

Physiological effect of the individual bacterial isolates on the microalgae during co-cultivation. (a–d) Determination of algal growth and product formation performance of axenic and xenic B. braunii cultures, supplemented with bacterial isolates. Shown are growth analysis of B. braunii in the presence of (a) Mycobacterium sp. Bb-A and (b) Brevundimonas sp. Bb-A. The inserts within the graph show the maximal observed growth difference in comparison to the control. (c) Comparison of the hydrocarbon levels observed in samples cultivated with bacterial isolates in relation to the axenic B. braunii culture (values set to 100%) in the course of the cultivation; (d) Morphological characteristic of algal cells during the cultivation (pictures were taken after 9 days of cultivation) with the bacterial isolates (red arrows indicate the hydrocarbons produced by the microalga). Graphs imbedded in the pictures show the progress of the bacterial growth (cell number on log₁₀ scale). The error bars represent standard error of mean values of three biological and three technical replicates (SE; n = 9). Asterisks represent p values as determined via Student’s t-test (*=  < 0.05, **=  < 0.01, ***=  < 0.001).

Figure 3

Essential cofactors (inter-)dependencies of the bacterial core community and isolates of B. braunii. (a) Heatmap of the reconstructed de novo biosynthesis of B-vitamins encoded in the high-quality draft and complete bacterial genomes. Numbers in parentheses represent the maximal total number of genes within each pathway. Percentage: per cent of total number of genes per pathway identified within each genome. All annotated genes within the respective pathways are presented on the EMGB platform (https://emgb.cebitec.uni-bielefeld.de/Bbraunii-bacterial-consortium/). The pathways statistics are summarized in the Table S5 and S8. (b) Growth assay to confirm B-vitamin auxotrophy and prototrophy in axenic Brevundimonas sp. Bb-A (blue) and Mycobacterium sp. Bb-A (orange) strains, respectively. (c) Biotin concentration levels detected in cell-free supernatants from the cultures of axenic B. braunii as well as in the presence of Mycobacterium sp. Bb-A (orange) and Brevundimonas sp. Bb-A (blue). The error bars represent standard error of mean values of three biological and three technical replicates (SE; n = 9).

Figure 4

Bacterial symbionts fend their eukaryotic host from hydrocarbonoclastic invaders. Physiology of a synthetic alga-bacteria-bacteria community, consisting of Brevundimonas sp. Bb-A (blue) and Mycobacterium sp. Bb-A (orange) in co-culture with B. braunii (green) under photoautrophic conditions with three different inoculum ratios (10:90, 50:50 or 90:10 per cent based on cfu (colony-forming units)). Shown are (a) the progression of bacterial proliferation (cfu) and (b) detected relative hydrocarbon levels (normalized to the axenic control) during the co-cultivation. The error bars represent standard error of mean values of three biological and three technical replicates (SE; n = 9).

Figure 5

Alga-bacteria interactions within the Botryococcus braunii phycosphere. The depiction summarizes the observed generic potential encoded in the (meta)genomes of bacterial associates (Figs. 1 and 3), along with experimental validation within the minimal synthetic community (Figs. 2 and 4). The color code indicates the functional grouping of the community members: supportive B-vitamin-auxotrophic (blue) and hydrocarbonoclastic prototrophic (red). The potential for degradation of hydrocarbons and carbohydrates of the bacterial associates is illustrated via yellow and striped pacmans, respectively. The genetic predisposition to the synthesis of bacteriocins and antibiotics encoded by the secondary metabolite biosynthesis gene clusters, is indicated by the halos and triangles, respectively. Abbreviation: HC, Hydrocarbons; CH, carbohydrates; B₇, Biotin; N, nitrogen; P, phosphorus.