Retroelement-guided protein diversification abounds in vast lineages of Bacteria and Archaea

Transcription

1 In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 2 ARTICLE NUMBER: Retroelement-guided protein diversification abounds in vast lineages of Bacteria and Archaea Blair G. Paul, David Burstein, Cindy J. Castelle, Sumit Handa, Diego Arambula, Elizabeth Czornyj, Brian C. Thomas, Partho Ghosh, Jeff F. Miller, Jillian F. Banfield and David L. Valentine NATURE MICROBIOLOGY DOI: /nmicrobiol

2 Parcubacteria rifoxyd1_full_scaffold_2802 TR (nt) TR (aa) Observed VR variants (nt & aa) Supplementary Figure 1. Adenine-specific substitutions compared with a TR sequence. An example of the VR variants (nt, nucleotide; aa, amino acid) observed by read-mapping analysis of a Parcubacteria genome DGR. RT (putative)! TR-RNA VP tmrna 1000 id= Length (nt) Supplementary Figure 2. Stringent transcript mapping to DGR-containing features. Showing an example sequence based on high coverage for a DGR feature, where transcripts are mapped to the TR-RNA coding region (purple boxes), as well as to tmrna encoding sequence (pink boxes). The variable protein gene is labeled VP. NATURE MICROBIOLOGY DOI: /nmicrobiol

3 Genome Bin RIFCSPLOWO2_01_FULL_OD1_Yanofskybacteria_49_17 RIFCSPLOWO2_02_FULL_OD1_Yanofskybacteria_47_9b RIFCSPHIGHO2_02_FULL_OD1_Yanofskybacteria_50_12 RIFCSPHIGHO2_01_FULL_OD1_Yanofskybacteria_41_21 RIFCSPHIGHO2_01_FULL_OD1_Yanofskybacteria_48_25b GWA1_OD1_39_13_partial RIFCSPHIGHO2_01_FULL_OD1_Yanofskybacteria_43_32 RIFCSPLOWO2_01_FULLYanofskybacteria_41_34 GWC2_OD1_41_9 RIFCSPHIGHO2_01_FULL_OD1_Yanofskybacteria_44_22 RIFCSPHIGHO2_02_FULL_OD1_Yanofskybacteria_39_10 RIFCSPHIGHO2_01_FULL_OD1_Yanofskybacteria_39_8b RIFCSPHIGHO2_02_FULL_OD1_Yanofskybacteria_38_22b RIFCSPHIGHO2_02_FUL Yanofskybacteria_41_29 RIFCSPHIGHO2_01_FULL_OD1_Yanofskybacteria_44_24 RIFCSPHIGHO2_02_FULL_OD1_Yanofskybacteria_41_11 RIFCSPHIGHO2_02_FULL_OD1_Yanofskybacteria_43_22 RIFCSPHIGHO2_01_FULL_OD1_Yanofskybacteria_43_42 RIFCSPLOWO2_01_FULL_OD1_Yanofskybacteria_42_49 GWD1_OD1_39_16_partial GWF1_OD1_44_227 RIFCSPLOWO2_01_FULL_OD1_Yanofskybacteria_44_88 RIFCSPLOWO2_02_FULL_OD1_Yanofskybacteria_45_18 RIFCSPLOWO2_02_FULL_OD1_Yanofskybacteria_43_10b RIFCSPHIGHO2_12_FULL_OD1_Yanofskybacteria_45_19b RIFCSPHIGHO2_01_FULL_OD1_Yanofskybacteria_44_17 RIFCSPLOWO2_01_FULL_OD1_Yanofskybacteria_49_25 VP TP Cluster RT Cluster Cluster 1 Cluster 5 R Cluster 23 Cluster 0 R Cluster 11, Cluster 00, 36 R 33, 64 Cluster 11 Cluster 0 R Cluster 0, 4 Cluster 1, 20 R Cluster 7 Cluster 0 R Cluster 34 Cluster 0 R Cluster 11 Cluster 30 R Cluster 2, 4 Cluster 5, 20 R Cluster 13 Cluster 69 R Cluster 2 Cluster 5 R _10b 4 Supplementary Figure 3. Phylogenomic distribution of DGR-containing Yanofskybacteria genomes. A concatenated ribosomal tree from Hug et al was used to overlay DGR occurrence (red; black text) versus absence (grey text). Variable protein and reverse transcriptase cluster affiliations are indicated to the right of genomes that contain a DGR. The scale shows substitutions per site. NATURE MICROBIOLOGY DOI: /nmicrobiol

4 0.2 RIFCSPHIGHO2_01_FULL_OD1_Giovannonibacteria_45_23 RIFCSPLOWO2_01_FULL_OD1_Giovannonibacteria_45_140 GWA1_OD1_44_29 GWA2_OD1_rel_44_26 RIFCSPLOWO2_01_FULL_OD1_Giovannonibacteria_46_32 RIFCSPLOWO2_01_FULL_OD1_Giovannonibacteria_44_16 RIFCSPLOWO2_12_FULL_OD1_Giovannonibacteria_44_32 RIFCSPHIGHO2_02_FULL_OD1_Giovannonibacteria_44_11 RIFCSPHIGHO2_02_FULL_OD1_Giovannonibacteria_46_20 RIFCSPHIGHO2_02_OD1_Giovannonibacteria_43_16 RIFCSPLOWO2_12_FULL_OD1_Giovannonibacteria_43_11c RIFCSPLOWO2_12_FULL_RIF_OD1_44_15 RIFCSPLOWO2_01_FULL_OD1_Giovannonibacteria_46_13 RIFOXYD1_FULL_OD1_48_21 GWF2_OD1_42_19 GWA2_OD1_53_7_partial DGR in partial genome bin (<75% complete) DGR in near-complete bin near-complete bin without DGR partial bin without DGR Supplementary Figure 4. Phylogenomic distribution of DGRs in near-complete and partial Giovannonibacteria genomes. A concatenated ribosomal tree from Hug et al was used to overlay DGR occurrence (red branches and square/circle symbols). The scale shows substitutions per site. a Cluster Size (# of Sequences) 100 Variable TPs (Full Proteins Sequences) VR Domains Cluster # b Assigned Function Uncharacterized DUF1566 (PF07603) AAA+ ATPase CLec-like domain (FGE-sulfatase) Tyrosine kinase PcfJ-like protein Guanylate cyclase Ubiquitin-activating E1 FCCH Supplementary Figure 5. Variable protein clusters and functional classes for combined binned and unbinned genome fragments. a, Clustering analysis of variable protein sequences (amino acid) with at least 30% intracluster similarity. Distributions of globally aligned variable proteins, and separately, VR domains, are shown on distinct lines as indicated in the legend. b, Relative proportions of characterized versus uncharacterized variable proteins and corresponding putative functional classifications NATURE MICROBIOLOGY DOI: /nmicrobiol

5 Supplementary Figure 6. Length distribution of DGR variable proteins. A non-redundant list of variable proteins was generated using CD-Hit (intracluster global alignment identity >95%). Sequence lengths are indicated in amino acids (aa). Bin: RIFCSPHIGHO2_01_FULL_OD1_Kaiserbacteria_54_36 rifcsphigho2_01_scaffold_730 (DGR) srk ftsfts RT prk VP hp rec TR VR gwa2_scaffold_1695 (no DGR) sps slp cps VP Alignment (amino acid) AAA_5 ATPase Domain 96.4% a.a. id. gtf gtf VP-like hp-like gtf gtf gtf gtf gtf gtf gtf 81.2% a.a. id. 83.4% a.a. id. scaffold 730 TR scaffold 730 VR scaffold 730 annotations: VP - DGR variable protein RT - DGR reverse transcriptase hp - hypothetical protein rec - recombinase srk/perk - kinase-like fts - cell division protein scaffold 1695 annotations: VP-like - similar to DGR variable protein hp-like - similar to hypothetical protein gtf - glycosyltransferase sps - spore coat polysaccharide biosynthesis protein cps - capsular polysaccharide biosynthesis protein slp - S-layer array-like protein Supplementary Figure 7. Paralagous variable proteins in DGR and non-dgr loci of a Kaiserbacteria genome. Sequences with homology are highlighted with a yellow connector and amino acid similarity is shown below each annotation (VP-like and hp-like). Selected neighborhood annotations are indicated by abbreviated gene names. An expanded view depicts the alignment of variable protein and VP-like amino acid sequences. At right, the aligned VR, TR, and VR-like sequences show variable residues. NATURE MICROBIOLOGY DOI: /nmicrobiol

6 Bin: RIFOXYA2_FULL_OD1_Nomurabacteria_42_12 rifoxyd2_full_scaffold_547 (no DGR) rbps1 VP-like 77.4% a.a. id. murc murd mure rifoxyd2_full_scaffold_2276 (DGR) rec VP RT N-Methyl TM VP Alignment (amino acid) TM DUF1566 (CLec-like) VR scaffold 2276 annotations: VP - DGR variable protein RT - DGR reverse transcriptase rec - recombinase scaffold 547 annotations: VP-like - similar to DGR variable protein murc - N-acetylmuramic acid ligase murd - (UDP-Mur-NAC-L-Ala) D-glutamate ligase mure - (UDP-Mur-NAC-L-Ala-D-Glu) diaminopimelate ligase rbps1 - ribosomal binding protein S1 Supplementary Figure 8. Paralagous variable proteins in DGR and non-dgr loci of a Nomurabacteria genome. Sequences with homology are highlighted with a yellow connector and amino acid similarity is shown below the VP-like annotation. Selected neighborhood annotations are indicated by abbreviated gene names. An expanded view depicts the alignment of variable protein and VP-like amino acid sequences. A putative transmembrane region is highlighted in red (TM). DGR-RT consensus: - Bordetella pertussis phage - L. pneumophila str. Corby - ANMV-1 (methane seep virus) - uncultivated nanoarchaea (1) tblastn DGR protein vs groundwater genomes (Geneious v8.1.4) complete and partial genomes from 30 groundwater metagenomes complete and partial genomes with RT-like region (2) (3) extract RT regions +/- 5kb flanking fragment to sliding windows (200bp; 50bp step) (Geneious v8.1.4) (Python v2.7.12) (4) (5) (6) BLAST all vs. all 200bp fragments parse BLASTall output for putative TR-VR pairs AGCCAGTACCA TGCCCGTTCCG map TR-VR onto scaffolds; manually inspect to assign VP (BLAST+ command line) AGCCAGTACCA TGCCCGTTCCG (Python v2.7.12) AGCCAGTACCA TGCCCGTTCCG (Geneious v8.1.4) Supplementary Figure 9. Overview of DGR feature identification. The schematic depicts steps taken to identify RT-like regions on partial or complete genomes reconstructed from groundwater metagenomes. Software/programs used to conduct each step are listed in parentheses. NATURE MICROBIOLOGY DOI: /nmicrobiol