Genomic diversity and biosynthetic capabilities of sponge-associated chlamydiae

Abstract

Sponge microbiomes contribute to host health, nutrition, and defense through the production of secondary metabolites. Chlamydiae, a phylum of obligate intracellular bacteria ranging from animal pathogens to endosymbionts of microbial eukaryotes, are frequently found associated with sponges. However, sponge-associated chlamydial diversity has not yet been investigated at the genomic level and host interactions thus far remain unexplored. Here, we sequenced the microbiomes of three sponge species and found high, though variable, Chlamydiae relative abundances of up to 18.7% of bacteria. Using genome-resolved metagenomics 18 high-quality sponge-associated chlamydial genomes were reconstructed, covering four chlamydial families. Among these, Candidatus Sororchlamydiaceae shares a common ancestor with Chlamydiaceae animal pathogens, suggesting long-term co-evolution with animals. Based on gene content, sponge-associated chlamydiae resemble members from the same family more than sponge-associated chlamydiae of other families, and have greater metabolic versatility than known chlamydial animal pathogens. Sponge-associated chlamydiae are also enriched in genes for degrading diverse compounds found in sponges. Unexpectedly, we identified widespread genetic potential for secondary metabolite biosynthesis across Chlamydiae, which may represent an unexplored source of novel natural products. This finding suggests that Chlamydiae members may partake in defensive symbioses and that secondary metabolites play a wider role in mediating intracellular interactions. Furthermore, sponge-associated chlamydiae relatives were found in other marine invertebrates, pointing towards wider impacts of the Chlamydiae phylum on marine ecosystems.

Fig. 1. Bacterial SSU rRNA gene amplicon sequencing and metagenomes reveal variably high relative abundances of Chlamydiae across sponge specimens.

a Relative abundances of phyla from SSU rRNA gene amplicon sequence data with ≥1% relative abundance in a sample. b Heatmap of OTUs with ≥1% relative abundance in a sponge sample from SSU rRNA gene amplicons. Both OTUs and sponges are hierarchically clustered based on presence patterns. c Concatenated maximum likelihood phylogenetic tree of contigs encoding ribosomal proteins (≥5) from across sponge metagenomic assemblies in the context of bacterial reference taxa. The tree is rooted by an archaeal outgroup. The metagenomic origin of each sequence is indicated by the dot plot, with lines corresponding to samples in the following order: P_S1, P_S2, P_S3, O_S4.1, O_S4.2, O_S5, and X_S6. Bars indicate the relative coverage of each ribosomal protein-encoding contig in each metagenome. Phylum affiliation is indicated for each clade according to the colour legend in panel (a). In addition, classes are indicated for Proteobacteria: Alphaproteobacteria (α), Betaproteobacteria (β), Deltaproteobacteria (δ), Epsilonproteobacteria (ε), and Gammaproteobacteria (γ). See legend in panel (a) for phylum colour assignment and patterns corresponding to each sponge species. See Data S1 for sample information, Data S2 for amplicon OTUs, Data S3 for relative abundances and read counts, Data S4 for metagenomic ribosomal protein contigs and their corresponding coverage, and Data S5 for the ribosomal protein phylogenetic tree with sequence accessions.

Fig. 2. Chlamydiae-affiliated metagenome-assembled genomes (MAGs) retrieved from sponges are phylogenetically diverse.

a Concatenated Bayesian phylogeny of Chlamydiae species relationships inferred using 15 marker gene NOGs under the CAT + GTR + Γ4 model of evolution. Branch support is indicated by coloured circles and includes posterior probability (PP) from the Bayesian inference and non-parametric bootstrap support (BP) from a maximum-likelihood reconstruction of the same dataset inferred with the PMSF approximation of the LG + C60 + F + R4 model of evolution (Data S5). Family names are indicated and those not including sponge-associated chlamydiae MAGs collapsed. Sponge-associated chlamydiae MAGs retrieved in this study are highlighted in blue, while chlamydial species groups with ≥95% average nucleotide identity (ANI) are indicated by grey boxes numbered 1–4. The tree is rooted by a PVC outgroup (not shown). See Fig. S3 and Data S5 for additional species trees and uncollapsed phylogenies. b Genome characteristics of sponge MAGs (blue) in the context of species representatives from across Chlamydiae (grey). Boxplots indicate the distribution of GC content across different chlamydial families, with the area of circles indicating genome size. See Data S6 for genome characteristics and ANI.

Fig. 3. Clusters of orthologous group (COG) pathways across Chlamydiae indicate that sponge-associated chlamydiae have similar metabolic profiles to other members of their respective families.

Bars are coloured according to COG category and show the number of COGs identified from the pathway in the given genome. COG categories with similar profiles across Chlamydiae genomes were excluded but can be found in Fig. S5 and Data S9. Sponge-associated chlamydiae MAGs are in blue, with relevant families coloured accordingly. Order and family names are indicated at the bottom with the following short forms: Criblamydiaceae (Crib.), Parilichlamydiaceae (Parili.), and Waddliaceae (Wad.). See Fig. S5 for an overview of relative pathway completeness and Data S9 for an overview of each COG across Chlamydiae genomes.

Fig. 4. Sponge-associated chlamydiae genomes encode genes related to fermentation, degradation, and typical of eukaryotes.

The presence of selected enriched genes across Chlamydiae genomes from families with sponge-associated members is shown, alongside a schematic overview of pyruvate fermentation to acetoin, and genes related to degrading acetoin, scyllo-inositol, taurine, and D-xylose. Protein function and gene orthologs are indicated to the left alongside numbers corresponding to the schematic overview. The number of representative genomes from other chlamydial families encoding the given gene is indicated to the right out of the total number of genomes considered in parentheses. See Data S8 for a full overview of gene presence across Chlamydiae representatives and corresponding gene annotations for sponge-associated chlamydiae.

Fig. 5. Biosynthetic gene clusters (BGCs) are found across Chlamydiae and sponge-associated chlamydiae genomes.

a The number and type of BGCs (found in >1 genome) identified across Chlamydiae. Order and family classification is indicated alongside shortened species names. See Data S10 for a full overview of BGCs. Maximum-likelihood phylogenies of a group of polyketide cyclases (SnoaL-like; PF07366) (b), which are involved in secondary metabolite biosynthesis, and of phosphoenolpyruvate mutase (PepM; PF13714) (c), which performs the first step in phosphonate biosynthesis. Ultrafast bootstrap support (ufBP) is indicated by a black circle (c). Branch and clade colours indicate taxonomy according to the legend (b, c). The presence of Chlamydiae families including sponge-associated chlamydiae genomes is indicated by the coloured circles, with the taxonomy of the sister clade indicated where supported (b). See Data S5 for uncollapsed phylogenies and sequence accessions.

Fig. 6. Relatives of sponge-associated chlamydiae are primarily found in marine habitats, and no other putative eukaryotic hosts were identified in the sponge metagenomes based on SSU rRNA genes.

a Percentage of amplicon samples from various environments with SSU rRNA genes ≥95% identity to the indicated chlamydial sponge MAG. A representative from each chlamydial species group with MAGs that include SSU rRNA genes is shown (Fig. 2a). Only environments with ≥100 samples, and clear labels are shown. See Data S11 for full IMNGS results of SSU rRNA gene searches against NCBI SRA amplicon datasets. b Presence of eukaryotic SSU rRNA genes, and their corresponding taxonomy, across sponge sample metagenome assemblies. See Data S4 for full taxonomy, contig IDs, and contig coverage. c. Sponge-associated chlamydiae are restricted to specific chlamydial groups. Maximum-likelihood phylogeny of small subunit rRNA genes from across Chlamydiae (and outgroup sequences) inferred using the GTR + F + R10 model and shown as a cladogram for clarity (See Data S5 for sequence accessions and branch lengths). Included are reference chlamydial sequences (black), sponge-associated chlamydiae genomes and amplicons from the present study (orange and blue), chlamydial amplicons previously obtained from these sponge species (light blue; Naim et al., 2014) [20], and chlamydial amplicons obtained from a prior study of sponge microbial diversity (green; Thomas et al.) [18]. Sequences from Chlamydiae genomes, and thus representing sequenced genomic diversity, are shown by stars. Chlamydial families are coloured according to the legend and labelled.