Predicting Species-Resolved Macronutrient Acquisition during Succession in a Model Phototrophic Biofilm Using an Integrated 'Omics Approach

Abstract

The principles governing acquisition and interspecies exchange of nutrients in microbial communities and how those exchanges impact community productivity are poorly understood. Here, we examine energy and macronutrient acquisition in unicyanobacterial consortia for which species-resolved genome information exists for all members, allowing us to use multi-omic approaches to predict species' abilities to acquire resources and examine expression of resource-acquisition genes during succession. Metabolic reconstruction indicated that a majority of heterotrophic community members lacked the genes required to directly acquire the inorganic nutrients provided in culture medium, suggesting high metabolic interdependency. The sole primary producer in consortium UCC-O, cyanobacterium Phormidium sp. OSCR, displayed declining expression of energy harvest, carbon fixation, and nitrate and sulfate reduction proteins but sharply increasing phosphate transporter expression over 28 days. Most heterotrophic members likewise exhibited signs of phosphorus starvation during succession. Though similar in their responses to phosphorus limitation, heterotrophs displayed species-specific expression of nitrogen acquisition genes. These results suggest niche partitioning around nitrogen sources may structure the community when organisms directly compete for limited phosphate. Such niche complementarity around nitrogen sources may increase community diversity and productivity in phosphate-limited phototrophic communities.

Keywords: carbon fixation; metagenomics; metaproteomics; metatranscriptomics; nitrate reduction; periphyton; phosphate transport; sulfate reduction.

FIGURE 1

Macronutrient acquisition functions detected in phototrophic consortium member genomes. Isolates belonging to the Cyanobacteria are green, Alphaproteobacteria are red, Gammaproteobacteria are blue, and Bacteroidetes are yellow. ^aGenomic information derived from sequenced isolates. ^bFor description of the types of proteorhodopsins, see Supplementary Notes 2. ^cSimilar to form IV RuBisCo, and thus unlikely to permit autotrophy (Supplementary Notes 3). ^dThese organisms contain multiple complete nitrate reductases; only one gene of each type of multi-subunit nitrate reductase is denoted to conserve space. ^eWhere organisms have multiple phosphate-acquisition systems, pst denotes the presence of pstSABC. ^fThe pstAB genes are contiguous to a contig edge in the Rhodobacteraceae sp. HLUCCA08 reconstruction; pstSC are assumed to be within the gap between contigs.

FIGURE 2

Metatranscriptomic and metaproteomic measures of member activity dynamics. (A) Metatranscriptomic measurement of member activity during succession, represented as the fraction of the total reads per sample attributed to each organism. Values are plotted on a log₁₀ scale, error bars denote the 95% confidence interval of the mean. (B) Metaproteomic evaluation of member activity during succession, represented as the share of peptide spectral counts attributed to each of the members. Values are plotted on a log₁₀ scale. Error bars are omitted for clarity, but variance data are presented in Supplementary Table S3. (C) Ratio of member relative activity as assessed by metatranscriptomics to that observed by metaproteomics during succession. Flat lines (m = 0) indicate a monotonic relationship between metatranscriptomic and metaproteomic estimations. (D) Organism-centric changes in metatranscriptomic and metaproteomic estimates of relative activity across all members between sequential time points. The y-axis indicates the between-time point fold change in the relative abundance of an organism’s peptide spectral counts, and the x-axis indicates the fold change in an organism’s RNA read abundance. Colored dots represent the seven most dominant species according to the key, and black dots indicate fold changes for less-abundant species.

FIGURE 3

Cyanobacterial energy capture and macronutrient acquisition during succession. Expression of Phormidium sp. OSCR proteins involved in light energy capture and carbon fixation (A), nitrogen and phosphorus acquisition (B) and sulfur acquisition (C), expressed as a percentage of total spectral counts per sample attributed to this species. The expression of the large subunit of the cyanobacterial beta’ subunit of RNA polymerase, RpoC1, is provided for reference in all panels. Data are plotted on a log₂ scale; error bars represent the 95% confidence interval of the mean. For energy and carbon capture systems (A), data represent a sum of all the subsystem-specific cyanobacterial gene products notated in Supplementary Table S2.

FIGURE 4

Metatranscriptomic evaluation of phosphate acquisition gene expression in major heterotrophic species during succession. Expression of the substrate-binding subunit pstS is plotted as an indicative gene for pstSABC, as other transporters and phosphates are each encoded by a single gene. In organisms where they are present, the pst genes are operonic and expression of pstABC displayed similar trends as pstS (Supplementary Table S4). Organism-specific RPKM is plotted on a log₂ scale; error bars indicate 95% confidence interval of the mean (where the 95% confidence interval is inclusive of zero or below, negative error bars are not shown). Filled symbols indicate that two or more peptides were observed for a gene’s product at a given time point; open symbols denote no or inadequate proteomic evidence. Expression of rpoC is included as a reference.

FIGURE 5

Metatranscriptomic evaluation of ammonium incorporation gene expression in major heterotrophic species reveals species-specific patterns of nitrogen limitation during succession. Organism-specific RPKM is plotted on a log₂ scale; error bars indicate 95% confidence interval of the mean (where the 95% confidence interval is inclusive of zero or below, negative error bars are not shown). Filled symbols indicate that two or more peptides were observed for a gene’s product at a given time point; open symbols denote no or inadequate proteomic evidence. Expression of rpoC is included as a reference.

FIGURE 6

Localization of A. marincola HL-49 within UCC-O biofilms via fluorescence in situ hybridization. Image represents a maximum-intensity Z-projection of a stack of confocal images, scale bar denotes 20 mm. Fluorescence intensity from FISH probes targeted against A. marincola HL-49 16S ribosomal RNA is depicted in yellow. Cyanobacterial chlorophyll a and phycocyanin auto-fluorescence intensity appear in red within filaments of Phormidium sp. OSCR. Fluorescence images are overlaid upon a differential interference contrast image of consortium biomass to display cell boundaries.