Additional file 3 of Functional metagenomics reveals differential chitin degradation and utilization features across free-living and host-associated marine microbiomes

Additional file 2: Detailed Methodology, Extended Results and Discussion. Supplementary Figure S1. RAST annotation of chitin and chitin-derivative degradation and utilization genes in cultivated bacterial symbionts of sponges and octocorals. For details on strains and phylogenetic tree, see legend to Fig. 1. The table on the right side shows chitin degradation (including both hydrolysis and deacetylation processes) and N-acetylglucosamine transport and utilization encoding genes detected on each bacterial genome using RAST-based classification, in contrast with Pfam annotations show in Fig. 1. Values in each cell correspond to the respective coding sequence (CDS) numbers present in each genome, whereby higher CDS numbers are highlighted in dark-gray shading. Entries highlighted in bold represent chitin processing functions examined across the sponge and octocoral metagenome datasets (Fig. 3), while the phylogeny, diversity and taxonomic composition of endo-chitinase encoding genes (EC 3.2.14) are examined in Figs. 2 and 4. For each functional entry, enzyme commission (EC) numbers and specific terminology are given in brackets, when appropriate. 1Chitinases that hydrolyse chitin oligosaccharides - (GlcNAc)4 to (GlcNAc)2 and (GlcNAc)5,6 to (GlcNAc)2 and (GlcNAc)3 but are inactive toward chitin (UniProtKB P96156). 2Corresponds to InterPro database entry IPR002509 (see also Fig. 3) which describes the metal-dependent deacetylation of O- and N- acetylated polysaccharides such as chitin, peptidoglycan and acetylxylan. Supplementary Figure S2. Class-level prokaryotic community profiles of healthy (EG_H) and diseased (EG_N) Eunicella gazella tissue, healthy Eunicella verrucosa (EV01-EV04) and Leptogorgia sarmentosa (LS06-LS08) specimens as well as seawater (SW01-SW04) and sediment samples (SD01-SD03). Taxonomic assignments are based on 16S rRNA gene reads retrieved from unassembled metagenomes using the MGnify metagenomics pipeline version 2.0 (EMBL-EBI) for the octocoral metagenome dataset (project PRJEB13222). Relative abundances are displayed for taxa representing more than 1% of the total dataset reads. Taxa with abundances below 1% across the data are collectively labelled as “rare classes”. Supplementary Figure S3. Maximum Likelihood phylogenetic tree of chiA gene sequences amplified from bacterial isolates. Sequences were obtained for eight marine sponge and 11 octocoral-derived bacterial isolates through PCR amplification from their respective genomic DNA. The evolutionary history was inferred using the General Time Reversible model. The tree with the highest log likelihood (-892.58) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches (1,000 bootstrap replicates). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 3.1129)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 19.83% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 19 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There was a total of 164 positions in the final dataset.