Additional file 2 of A multi-OMIC characterisation of biodegradation and microbial community succession within the PET plastisphere

Additional file 1: Figure S1. DNA yields from all PET succession experiment mesocosms and the inoculum. The DNA concentration in all negative extraction controls was too low to measure aside from day 30 (0.04 ng μL-1). Where DNA yields were significantly (two sample T-test p<0.05) higher than the no carbon control these days are marked with an asterisk. Figure S2. Relative abundances of taxa within all samples, with each bar representing the mean of three biological replicates. Each row shows taxa grouped to a different taxonomic level (shown on y label) and other represents all that were present at below 0.5% relative abundance at that level. ASVs are classified to species level where possible: ASV1 Vibrio, ASV2 Alteromonas, ASV3 Bacillus, ASV4 Vibrio, ASV5 Sediminibacterium salmoneum, ASV6 Pseudoalteromonas, ASV7 Sunxiuqinia, ASV8 Alcanivorax, ASV9 Thiobacimonas profunda, ASV10 Thalassospira lucentensis, ASV11 Methylophaga, ASV12 Pseudoalteromonas, ASV13 Vibrio, ASV14 Thalassospira, ASV15 Alcanivorax, ASV16 Thalassospira, ASV17 Vibrio alginolyticus, ASV18 Halomonas, ASV19 Catenococcus, ASV20 Pseudomonas, ASV21 Pseudoalteromonas, ASV22 Thalassospira, ASV23 Shewanella, ASV26 Exiguobacterium, ASV28 Roseivirga, ASV31 Alteromonas, ASV34 Oricola cellulosilytica, ASV36 Lysobacter maris, ASV39 Catenococcus, ASV40 Exiguobacterium, ASV42 Tistlia, ASV43 Azomonas, ASV45 Rhizobiaceae, ASV46 Maritimibacter, ASV48 Catenococcus, ASV49 Halomonadaceae, ASV53 Vibrionaceae, ASV54 Oricola, ASV55 Catenococcus, ASV56 Sphingomonas, ASV63 Vibrionaceae, ASV64 Parvibaculum, ASV65 Catenococcus, ASV70 Catenococcus, ASV73 Catenococcus, ASV75 Vibrionaceae, ASV76 Aestuariibacter aggregatus, ASV78 Catenococcus, ASV85 Hyphobacterium, ASV145 Tenacibaculum litoreum, ASV153 Mesoflavibacter zeaxanthinifaciens, ASV203 Tenacibaculum. Figure S3. Diversity for all samples across 42 days of incubation. Showing Simpsons index of diversity (top) and species richness (bottom). Figure S4. Fourier transform infrared spectra of PET powder and weathered PET powder before incubation with communities or isolates. All wavelengths are shown in (A) and ratios between the wavenumbers at 1410 and 1711, 1240, 725 and 1090 cm-1 are shown in (B), while (C), (D), (E) and (F) show 1000-600, 1200-1000, 1600-1200 and 1800-1600 cm-1, respectively. Each line shows the mean absorbance for three technical replicates for each of three biological replicates (i.e. nine total measurements) per treatment while bars and error bars represent means and standard deviations for biological replicates. Asterisks denote significant differences (two independent samples T-test, p<0.05) between the wavenumber ratios before and after thermal weathering of PET. Dashed lines indicate the wavenumbers used for ratio calculations. Figure S5. Distribution of Thioclava sp. BHET1 (left) and Bacillus sp. BHET2 (right) in surface (top) or deep-chlorophyll maximum (bottom) waters samples by the Tara oceans expedition [2–4]. Sequences within the Tara oceans mitags dataset that shared above 90, 95 or 97% identity with each of the Thioclava sp. BHET1 and Bacillus sp. BHET2 16S rRNA genes were identified and the relative abundance of all matches were summed to give the abundances shown here. We also calculated the coverage for each of Thioclava sp. BHET1 and Bacillus sp. BHET2 in the assembled Tara oceans metagenomes. These were co-assembled for each ocean and the same values are therefore plotted for all stations within each ocean. For both relative abundance and coverage, purple indicates 0% and green indicates 3% or higher. Figure S6. Growth of the isolates Thioclava sp. BHET1 (A-C) and Bacillus sp. BHET2 (D-F) on a range of common growth substrates across three days of incubation. Panels show biological replicates. Figure S7. Predicted abundance of genes that are potentially involved in PET degradation in PICRUSt2-assembled predicted metagenomes for all communities over time. Genes that are in the standard PET degradation pathway (i.e. shown in Fig. 4) are shown in red in (A), while those that are predicted to be involved in PET degradation based on the proteomics results of isolates (Fig. 3) are shown in blue. The abundance and taxonomic contributions to each KEGG ortholog is shown in (B). Note that K14037 was not found in the PICRUSt2 predicted metagenome and is therefore not shown here. Also shown is all monooxygenases and all dioxygenases. Taxonomic contributions shown are scaled by the relative abundance of each taxon as well as the number of gene copies possessed by that taxon. All taxa with a total contribution below 0.5% are grouped to Other. See Table S2 for individual ASVs and Table S12 for NSTI values for all treatments at all time points. Figure S7. Predicted abundance of genes that are potentially involved in PET degradation in PICRUSt2-assembled predicted metagenomes for all communities over time. Genes that are in the standard PET degradation pathway (i.e. shown in Fig. 4) are shown in red in (A), while those that are predicted to be involved in PET degradation based on the proteomics results of isolates (Fig. 3) are shown in blue. The abundance and taxonomic contributions to each KEGG ortholog is shown in (B). Note that K14037 was not found in the PICRUSt2 predicted metagenome and is therefore not shown here. Also shown is all monooxygenases and all dioxygenases. Taxonomic contributions shown are scaled by the relative abundance of each taxon as well as the number of gene copies possessed by that taxon. All taxa with a total contribution below 0.5% are grouped to Other. See Table S2 for individual ASVs and Table S12 for NSTI values for all treatments at all time points. Figure S8. nMDS plot showing Bray-Curtis distance between metabolomic analyses of culture supernatants of the community incubations used for MiSeq on day 42. Supernatants from incubations with no inoculum are shown with crosses while supernatants from incubations with the microbial community are shown with circles. Table S1. Results of PERMANOVA and ANOSIM tests for statistical significance (using Bray-Curtis distance) on all succession experiment samples. ANOSIM results are mentioned in the text as these values are more conservative. Table S2. ASVs identified by the PRC analysis. This includes ASV classifications using DADA2 and BLAST, PRC species weights, the closest representative whole genomes (from the NCBI database, where this was >97% similarity), whether these genomes potentially contain PETases and MHETases, PICRUSt2 nearest sequenced taxon indices (NSTI), the KEGG orthologs present in the genomes according to PICRUSt2 and other relevant information. Table S3. Analysis of early, middle or late colonisers, showing the day on which that ASV was most abundant in that treatment. Only ASVs that were above 0.5% in abundance in at least one time point in that treatment were included. Table S4. Genomic analysis of Thioclava sp. BHET1 and Bacillus sp. BHET2 (separate excel file). Table S5. Sequences that were used to construct the Hidden Markov Models (HMMs) for PETase, pcaG, pcaH, tphA2, tphA3 and tphB. Table S6. Potential PETases found in the genomes of Thioclava sp. BHET1 and Bacillus sp. BHET2 using a Hidden Markov Model (HMM) constructed with known PETases. Table S7. Peptides and protein groups for cellular and extracellular proteomics performed on Thioclava sp. BHET1 and Bacillus sp. BHET2 growing with fructose, TPA, BHET and PET (separate excel file). Table S8. Proteomic analysis of cellular and extracellular proteomics performed on Thioclava sp. BHET1 and Bacillus sp. BHET2 growing with fructose, TPA, BHET and PET (separate excel file). Table S9. Metabolomic analysis performed on Thioclava sp. BHET1 and Bacillus sp. BHET2 growing with fructose, TPA, BHET and PET (separate excel file). Table S10. Metabolomic analysis performed on microbial communities after incubation with BHET, amorphous PET, PET powder and weathered PET powder (separate excel file). Table S11. Proteins in the Thioclava sp. BHET1 cellular proteome that are potentially related to PET, BHET and TPA degradation, including relative abundance within the proteome and fold change when compared with the positive control. Table S12. Proteins that are potentially involved in xenobiotics degradation that were upregulated in one or more treatments in the Bacillus sp. BHET2 cellular proteome, including relative abundance within the proteome and fold change when compared with the positive control. Table S13. Details of the PETases found within the PICRUSt2 artificial metagenome and predicted to be in ASVs (separate excel file). Table S14. Nearest Sequenced Taxon Indices (NSTI) for all samples included in the PICRUSt2 analysis. Table S15. Comparison of different kits for DNA extraction from plastic pieces incubated with microbial communities.