Distribution and diversity of 'Tectomicrobia', a deep-branching uncultivated bacterial lineage harboring rich producers of bioactive metabolites

Abstract

Genomic and functional analyses of bacterial sponge symbionts belonging to the uncultivated candidate genus 'Entotheonella' has revealed them as the prolific producers of bioactive compounds previously identified from their invertebrate hosts. These studies also suggested 'Entotheonella' as the first members of a new candidate phylum, 'Tectomicrobia'. Here we analyzed the phylogenetic structure and environmental distribution of this as-yet sparsely populated phylum-like lineage. The data show that 'Entotheonella' and other 'Tectomicrobia' are not restricted to marine habitats but widely distributed among terrestrial locations. The inferred phylogenetic trees suggest several intra-phylum lineages with diverse lifestyles. Of these, the previously described 'Entotheonella' lineage can be more accurately divided into at least three different candidate genera with the terrestrial 'Candidatus Prasianella', the largely terrestrial 'Candidatus Allonella', the 'Candidatus Thalassonella' comprising sponge-associated members, and the more widely distributed 'Candidatus Entotheonella'. Genomic characterization of 'Thalassonella' members from a range of sponge hosts did not suggest a role as providers of natural products, despite high genomic similarity to 'Entotheonella' regarding primary metabolism and implied lifestyle. In contrast, the analysis revealed a correlation between the revised 'Entotheonella' 16S rRNA gene phylogeny and a specific association with sponges and their natural products. This feature might serve as a discovery method to accelerate the identification of new chemically rich 'Entotheonella' variants, and led to the identification of the first 'Entotheonella' symbiont in a non-tetractinellid sponge, Psammocinia sp., indicating a wide host distribution of 'Entotheonella'-based chemical symbiosis.

Fig. 1. Phylogeny and environmental distribution of the candidate phylum Tectomicrobia based on 16 S rRNA gene sequences.

A Phylogenetic tree inferred from 456 tectomicrobial sequences under the neighbor-joining criterion. Support of individual branches by bootstrap values above 50% is indicated at the respective nodes for the neighbor-joining method (left) and the maximum-likelihood method (right). Scale bar, 0.01 changes per nucleotide position. The number of sequences comprising a specific group is shown inside or next to the corresponding clade. The phylum Nitrospinota was used as an outgroup. The environmental distribution of sequences assigned to specific groups is shown as a pie chart next to the corresponding group. The subgroups ‘t1’ and ‘t2’, present in Clade 2, contained 10 and 11 sequences of mainly terrestrial origin respectively, but further data are needed to resolve their relationship with the rest of the Clade. B Median sequence identities within (main diagonal) and between (off-diagonal) the genus-level clades described in this study. A more detailed version of this figure, showing higher and lower-level divisions, is provided in Fig. S5. C Pie chart diagram illustrating the environmental distribution of ‘Tectomicrobia’ based on 456 16 S rRNA gene sequences. A list of sequences generated in this study can be found in Table S3.

Fig. 2. Phylogeny of ‘Thalassonella’.

Detailed view of ‘Thalassonella subgroup for the tree shown in Fig. 2. Bootstrap values above 50% are given for the neighbor-joining (left) and maximum-likelihood (right) methods. Sequences generated in this study are indicated with a black circle. Scale bar, 0.005 changes per nucleotide position.

Fig. 3. Phylogeny of ‘Entotheonella’.

Detailed view of ‘Entotheonella’ for the tree shown in Fig. 2. Bootstrap values above 50% are given for the neighbor-joining (left) and maximum-likelihood method (right). Sequences generated in this study are indicated with a black circle and ‘Entotheonella’ bacteria previously linked to the production of bioactive natural products are highlighted with a black asterisk. Sequences derived from soil samples are highlighted in red. Scale bar, 0.01 changes per nucleotide position.

Fig. 4. CARD-FISH localization of ‘Ca. Entotheonella consociata’ in Psammocinia sp.

Overlay of a bright-field image of a representative thin slice of Psammocinia sp. A with a fluorescent image obtained from CARD-FISH labeling of ‘Entotheonella’ (B). Scale bar: 20 µm.

Fig. 5. Analysis of putative ‘Tectomicrobia’ members from shotgun metagenomics.

A Reproduction of Fig. 1A showing the placement of 15 putative ‘Tectomicrobia’ bins in red by alignment of recovered 16 S sequences. B Unrooted phylogenomic tree of putative ‘Tectomicrobia’ MAGs containing 16 S sequences based on concatenation of conserved single-copy genes, including for reference three ‘Entotheonella’ draft genomes (‘E. factor’, NCBI ID AZHW01; ‘E. gemina’, AZHX01; ‘E. serta’, PPXO01) and three draft genomes from the adjacent bacterial phylum Nitrospinae (MHDL01, PCWG01, DCWK01). The MAGs Gb4_35 and Gb10_1 showed a genome completeness below 75% and were therefore not included in the analyses of Table S6. Gb4_35 had an inconclusive taxonomic affiliation based on its 16 S rRNA gene but was assigned to the ‘Thalassonella’ sublittoral to upper bathyal Geodia group through phylogenomics.

Fig. 6. BGC analysis of ‘Thalassonella’ MAGs.

A Table of conserved BGCs in 18 ‘Thalassonella’-assigned MAGs, including MAGs assigned on the basis of the phylogenomic tree in Fig. 1B. MAGs containing an ortholog of a cluster are indicated with a ‘+‘, and other, non-conserved clusters are shown in the final column. B Representative architectures for the four conserved BGCs as found in ‘Thalassonella’. Putative transport elements are shown in green and regulatory elements in yellow, with core biosynthetic machinery in orange and tailoring enzymes in blue. For the latter two, predicted functions are assigned based on homology. For multi-domain enzymes, the domain architecture is elaborated, with a black circle representing a thiolation domain. For a conservation analysis of these clusters, along with a comparison to the related ‘Entotheonella’ BGCs, see Figs. S10–13.