Marine Sponges as Chloroflexi Hot Spots: Genomic Insights and High-Resolution Visualization of an Abundant and Diverse Symbiotic Clade

Abstract

Members of the widespread bacterial phylum Chloroflexi can dominate high-microbial-abundance (HMA) sponge microbiomes. In the Sponge Microbiome Project, Chloroflexi sequences amounted to 20 to 30% of the total microbiome of certain HMA sponge genera with the classes/clades SAR202, Caldilineae, and Anaerolineae being the most prominent. We performed metagenomic and single-cell genomic analyses to elucidate the functional gene repertoire of Chloroflexi symbionts of Aplysina aerophoba. Eighteen draft genomes were reconstructed and placed into phylogenetic context of which six were investigated in detail. Common genomic features of Chloroflexi sponge symbionts were related to central energy and carbon converting pathways, amino acid and fatty acid metabolism, and respiration. Clade-specific metabolic features included a massively expanded genomic repertoire for carbohydrate degradation in Anaerolineae and Caldilineae genomes, but only amino acid utilization by SAR202. While Anaerolineae and Caldilineae import cofactors and vitamins, SAR202 genomes harbor genes encoding components involved in cofactor biosynthesis. A number of features relevant to symbiosis were further identified, including CRISPR-Cas systems, eukaryote-like repeat proteins, and secondary metabolite gene clusters. Chloroflexi symbionts were visualized in the sponge extracellular matrix at ultrastructural resolution by the fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM) method. Carbohydrate degradation potential was reported previously for "Candidatus Poribacteria" and SAUL, typical symbionts of HMA sponges, and we propose here that HMA sponge symbionts collectively engage in degradation of dissolved organic matter, both labile and recalcitrant. Thus, sponge microbes may not only provide nutrients to the sponge host, but they may also contribute to dissolved organic matter (DOM) recycling and primary productivity in reef ecosystems via a pathway termed the sponge loop. IMPORTANCE Chloroflexi represent a widespread, yet enigmatic bacterial phylum with few cultivated members. We used metagenomic and single-cell genomic approaches to characterize the functional gene repertoire of Chloroflexi symbionts in marine sponges. The results of this study suggest clade-specific metabolic specialization and that Chloroflexi symbionts have the genomic potential for dissolved organic matter (DOM) degradation from seawater. Considering the abundance and dominance of sponges in many benthic environments, we predict that the role of sponge symbionts in biogeochemical cycles is larger than previously thought.

Keywords: Chloroflexi; DOM degradation; FISH-CLEM; metabolism; metagenomic binning; single-cell genomics; sponge symbiosis.

FIG 1

Heatmap showing the relative abundance of Chloroflexi classes/clades in 63 HMA sponges extracted from Earth Microbiome Project (EMP) data (31). The top panel shows the mean relative abundance of Chloroflexi classes/clades in all sponges (means ± standard deviations [error bars]). The right panel displays the mean relative abundance of the phylum Chloroflexi in predicted (light green names and medium green bars) and classified (dark green names and bars) HMA sponges (means ± standard deviations) determined by machine learning (12). Results of cluster analysis based on Bray-Curtis dissimilarities on mean relative abundances of OTUs within the phylum are presented on the left side. Sponges are marked with stars when all species samples came from one of the three major sample locations.

FIG 2

Concatenated protein tree. Maximum likelihood phylogenetic analysis of Chloroflexi metagenome bins and SAGs (in red) from 1,914 positions of 60 sequences using ribosomal proteins. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown. The initial tree for the heuristic search was obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model. Multiple sequences are included in the collapsed branches representing Chloroflexi classes/clades (bold). To root the tree, three representative genomes from the phylum Actinobacteria were used. Reference genomes with accession numbers can be found in Table S1 in the supplemental material.

FIG 3

Distribution of Chloroflexi clades in Aplysina aerophoba mesohyl using fluorescence in situ hybridization (FISH). The image shows the overlay of all probes. SAR202 cells are displayed in green, Caldilineae cells in orange, and Anaerolineae cells in red. The nucleotide stain DAPI (white/gray) served as a reference for the localization of unstained cells. Bar, 10 µm.

FIG 4

Summarized metabolic features which were found only in Anaerolineae and Caldilineae (left side, blue and green arrows, respectively) or in SAR202 genomes (right side, red arrows). The central metabolic pathways (glycolysis, TCA cycle, purine, pyrimidine histidine biosynthesis) located in the middle of the figure are general features found in all genomes. Lines are dashed when pathways or transporter could not be annotated completely (single enzymes of the pathway or single genes from the transporter were missing) or could not be annotated in both genomes of one clade. Gray dashed arrows indicate that those transporters were not identified.

FIG 5

Pathway for synthesis of thiamine (A) and riboflavin (B) in both SAR202 genomes. (A) Members of classes Anaerolineae and Caldilineae carry genes encoding components involved in the import of thiamine (see also Fig. 4). (B) The conversion of riboflavin into the biologically active forms (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) was encoded by genes in the genomes of all three classes (filled pies compared to empty pies). The colors represent the genomes. The numbers are KEGG identifiers. PPP, pentose phosphate pathway. Gray arrows and numbers indicate unidentified enzymes.

FIG 6

Visualization of sponge-associated Chloroflexi in Aplysina aerophoba mesohyl using FISH-CLEM. (A and B) SAR202 cells are displayed in green, Anaerolineae in red (A), and Caldilineae in orange (B). The nucleotide stain DAPI (blue) served as reference for the localization of unstained cells. In both panels, the picture on the right is the overlay of all probes and DAPI. Bars, 1 µm.