A Comparative Metagenome Survey of the Fecal Microbiota of a Breast- and a Plant-Fed Asian Elephant Reveals an Unexpectedly High Diversity of Glycoside Hydrolase Family Enzymes

<div><p>A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant) was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs) were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH) genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs), which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.</p></div

Esterification reactions between 1-propanol and lauric acid (20 mmol each) as well as 1-tetradecanol and myristic acid (15 mmol each).

<p>Synthesis reactions were catalyzed by LipS and CalB (purchased from Sigma-Aldrich, Buchs, Switzerland) under solvent-free conditions at 70°C. Specific activities of LipS and CalB refer to the dry-weights of the lyophilisates. Data are mean values of at least three independent measurements and bars indicate the standard deviation.</p

Protein structure of LipS.

<p><b>A)</b> Ribbon representation of the LipS monomer colored according to secondary structure elements. The inserted lid-domain is indicated in red. The catalytic triad residues Ser126, His257 and Asp227 are shown as stick representation. <b>B)</b> Surface representation of the LipS monomer with the lid-domain (β6, β7, αD₁′) shown as a cartoon representation in red. The active site S126 (in yellow) is completely occluded from the bulk solvent and only accessible through a narrow tunnel. The active site pocket identified by CASTp server is colored in green. Amino acids building a pocket as part of the inserted domain are shown in orange. <b>C)</b> The catalytic triad residues of LipS are properly placed to establish hydrogen bonds.</p

Overall coverage of selected cellulolytic GH family genes in the feces samples in relation to their phylogenetic affiliation.

<p><b>A</b>) three-weeks-old elephant, <b>B</b>) six-years-old elephant.</p

Overall numbers of sequences and contigs generated for the two elephant feces samples.

<p>Overall numbers of sequences and contigs generated for the two elephant feces samples.</p

Phylogenetic analysis of the elephant feces in comparison with other fecal and intestinal metagenome data sets.

<p>Data indicate the phylogenetic relation based on gene similarities in the metagenome sequences. The percent of sequences assigned to each phylum according to IMG/M ER is shown based on the total number of obtained sequences of each data set. Sequence data for the metagenomes were extracted from the IMG/M ER web page of the US Department of Energy Joint Genome Institute and the respective bioprojects (IMG Genome IDs: Six-years-old Elephant (this study): 3300001598; three-weeks-old Elephant (this study): 3300001919; Green Cockroach: 2228664000; Termite: 3300001544; Dog: 2019105001; Reindeer: 2088090000; Neotropical Beetle: 3300000114; Asian Long-Horned Beetle: 2084038013; Bovine Rumen: 2061766007; Northwest Shipworm: 2189573029; Human stool: 7000000038).</p

Selected and recent metagenome studies published from insect or mammalian fecal and gut samples.

<p>n.d., not determined; a), in 2 samples; b), 286 Gb resulted in approx. 2 Gb of assembled DNAs >1 kb scaffolds; c),unassembled; d), in unassembled reads; e), per 503 Mbp unassembled DNA; f), total gene count with respect to the GH1, GH3, GH 5, GH6, GH8, GH9, GH44, GH45, GH48, GH51, GH74, GH94 family enzymes. Total gene counts, size of assembled DNAs, OTUs and numbers of carbohydrate active enzymes were extracted from the indicated references.</p><p>Selected and recent metagenome studies published from insect or mammalian fecal and gut samples.</p

Relative abundances of different phyla and classes in the two elephant feces samples.

<p><b>A:</b> Relative abundance of phyla in the feces of the three-weeks-old and the six-years-old Asian elephant based on 16S rRNA gene sequences. For the three-weeks-old elephant no Archaea were observed. <b>B:</b> Phylogenetic comparison on class level between both elephants. Heat map colors indicate the abundances of the respective 16S rRNA genes.</p

Rarefaction curves calculated for the feces sample of the three-weeks-old and the six-years-old Asian elephant at 3% genetic distance of 16S rRNA genes.

<p>The curve for the six-years-old elephant comprises 8,014 archaeal sequences which were clustered to 54 OTUs. The sequences were denoised employing Acacia. Chimeric sequences were removed using UCHIME in reference mode with the most recent SILVA SSU database as reference dataset (SSURef 115 NR).</p

Biochemical parameters of recombinant LipT and LipS determined using 4-nitrophenol-decanoate (C10) for LipT and –octanoate (C8) for LipS.

<p>The measurements were performed at 75 and 70°C, respectively, in 0.1 M PB pH 8.0.</p><p>Data are mean values of three independent measurements.</p