Heatmap display of the curve parameter A (maximum height) comparing them from R. baltica and P. limnophilus based on Phenotype MicroArrays (PM) kinetics of 95 carbon sources (microtiter plate PM02). Visualization of the experimental data was done by employing the R-package “opm”. Substrate utilization patterns of P. limnophilus and R. baltica respectively cluster according to the similarities between their metabolic activities. Substrates mentioned in the text are highlighted with yellow boxes. A dark green color indicates high metabolic activity for the corresponding substrate. Laminarin
Laminarin is a β-1,3-linked D-glucan with occasional β-1,6-linked branches that is used for carbon storage in brown algae (Read et al. 1996). In contrast to P. limnophilus, R. baltica can utilize laminarin (Figure 5: red and blue curve respectively). This finding reflects the ecology of both organisms since R. baltica was shown to dwell on marine brown algae such as kelp, while P. limnophilus is adapted to freshwater habitats (Bengtsson and Øvreås 2010, Lage and Bondoso 2011).
Figure 5: Phenotype MicroArray results for selected ecomimetic compounds
Phenotypic MicroArray respiration kinetics for R. baltica (red) and P. limnophilus (blue) of nine putative ecomimetic carbon substrates. Most of the substrates Planctomycetes might encounter in their native habitats and might serve as a trigger for interorganismic interactions with algae or plants. The curves show the accumulation of reduced tetrazolium dye (y-axis) over the course of time (x-axis).
Mannitol
Like laminarin, mannitol is produced by brown algae and can count for up to 30% of their dry weight (Reed et al. 1985, Zubia et al. 2008). While R. baltica degrades mannitol,
P. limnophilus does not (Figure 5 B). Within marine habitats brown algae use mannitol to
maintain their turgor pressure (Reed et al. 1985) or as storage a compound
(Michel et al. 2010), while brown algae are rather uncommon in limnic habitats. Some
Ectocarpus freshwater species contain only about 10% of the mannitol concentration of their marine counterparts (Dittami et al. 2012). Thus for P. limnophilus mannitol might not serve as a trigger for algae interaction as it might be absent or underrepresented within its habitat. Mannan
The polysaccharide mannan primarily consists of D-mannose and can be found in cell walls of green- or red algae, as well as in higher plants (Popper et al. 2011). While R. baltica could
moderately degrade mannan and its monomer D-mannose, P. limnophilus could not
degrade the polymer mannan, but the monomer D-mannose (Figure 5, Table S2). However, a weak signal does not mean that mannan is less likely to be a trigger for secondary metabolite production as we measure the capability to degrade a certain carbon source for energy consumption. Weak degradation capacity could thus still mean a strong trigger signal. Pectin
Pectin is an herbal polysaccharide that consists predominantly of α-D-galacturonic acid (Mohnen 2008) which is often methylesterified (Pelloux et al. 2007). Besides in higher plants, pectin can also be found in charophyceen green algae (Popper et al. 2011) and it is only degraded by R. baltica. Please note that in Table S3 the utilization of pectin by P. limnophilus
is nevertheless classified by k-means partitioning to be ‘weak’, which can be explained by the constantly large (80-100) but not increasing (indicating a biologically negative reaction) OmniLog values (Figure 5). However, surprisingly, the monomer D-galacturonic acid was not utilized by R. baltica, while close relatives such as D-galacose, methygalactosid, methygalactoside und N-acetyl-D-galactosamin were metabolized.
Cellobiose
Cellobiose is a disaccharide consisting of two β-1,4-glycosidic linked glucose molecules which is part of cell walls from higher plants and algae (Popper et al. 2011). Cellobiose is released through cellulose degradation (Lynd et al. 2002) and can be used by both, R. baltica
and P. limnophilus as carbon source (Figure 5). However, R. baltica has been shown to lack the capability to degrade cellulose before (Schlesner et al. 2004). One should consider that different cellulose substrates can lead to different degradation results among bacteria (Lynd et al. 2002), more work is needed to ultimately answer if R. baltica can degrade at least certain types of cellulose. In addition, under certain conditions, cellulolytic and non-cellulolytic bacteria compete against each other for the valuable substrate cellobiose that
is released by the cellulolytic species (Chen and Weimer 2001). One can envision the same situation in biofilms formed by several species on water plants and algae. Interestingly, aerobic cellulolytic bacteria are known to produce various antibiotics (Lynd et al. 2002). Since we identified several putative antibiotic-producing genes in Planctomycetes this might be true for cellobiose degrading organisms such as R. baltica as well. Thus, cellobiose might very well be a promising candidate for triggering antibiotic production in Planctomycetes. Sucrose
Both model organisms, R. baltica and P. limnophilus show weak degradation of the disaccharide sucrose that consists of the monomers glucose and fructose, produced through photosynthetic cyanobacteria, green algae and plants (Salerno and Curatti 2003). In addition, both analyzed organisms degrade the monomers D-glucose and D-fructose.
D-Xylose
Xylose is a main component of hemicellulose that is found in all algae and higher plants (Popper et al. 2011). While R. baltica seems to degrade D-xylose at moderate rates,
P. limnophilus gave a strong signal in our phenotypic microarray experiments. Again, the level of substrate degradation does not necessarily correlate with its function as potential trigger for secondary metabolite production.
Chondroitin Sulfate C
Chondroitin sulfates are unbranched polysaccharide chains consisting of repeating
disaccharide units composed of N-acetylgalactosamine and glucuronic acid
(Kwok et al. 2012), that are for example a major component of the brain matrix of higher animals (Kwok et al. 2012). Besides chondroitin sulfate, R. baltica degrades both monomers
N-acetylgalactosamine and D-glucuronic acid, while P. limnophilus is only capable of
N-acetylgalactosamine degradation (Table S2, Table S3). This again reflects the ecological niche of both organisms. While the marine R. baltica SH1 was shown to encode 110 sulfatases (Glöckner et al. 2003) and to be associated with macroalgae (Bengtsson and Øvreås 2010, Lage and Bondoso 2011), P. limnophilus was isolated from a freshwater habitat (Hirsch and Müller 1985). Sulfated polysaccharides are rare in limnic habitats and thus P. limnophilus
might not encounter sufficient amounts of substrates such as chondroitin sulfate C in its natural surroundings, hence has no need to degrade them (Popper et al. 2011, Dantas-Santos et al. 2012). In contrast, marine algae possess sulfated polysaccharides within their cell wall and R. baltica might feed on those. Consequently, previous studies, which used ‘classical’ growth experiments, revealed λ-carrageenan und chondroitin sulfate degradation by R. baltica (Wegner et al. 2012). Surprisingly, no growth was observed on the sulfated polysaccharide fucoidan (Wegner et al. 2012). However, as only one type of fucoidan was analyzed and the chemical composition varies between different algae further investigations are required to draw a final conclusion on fucoidan utilization by R. baltica
N-Acetyl- D-Glucosamin
Besides macro- and microalgae, Planctomycetes can live associated with cyanobacteria (Cai et al. 2013). From this perspective N-acetyl-D-glucosamine (major component of bacterial peptidoglycan) degradation of both, R. baltica and P. limnophilus is an interesting finding. Planctomycetes might either feed on cyanobacteria or of other biofilm members on algal surfaces by degrading their cell walls. However, this hypothesis requires further investigation.
Other Utilized Di-, Tri- and Tetrasaccharides
Adjacent to the carbon substrate discussed above, both, R. baltica and P. limnophilus can degrade several di-, tri- and tetrasaccharides (see Table S2 for details). While we hypothesize that the more complex polysaccharides that are mostly more species specific might provide a better-defined clue on which surface a cell happens to attach, such compounds might serve as triggers for secondary metabolite production as well.