Adaptation to herbivory by the Tammar wallaby includes bacterial and glycoside hydrolase profiles different from other herbivores

Abstract

Metagenomic and bioinformatic approaches were used to characterize plant biomass conversion within the foregut microbiome of Australia's "model" marsupial, the Tammar wallaby (Macropus eugenii). Like the termite hindgut and bovine rumen, key enzymes and modular structures characteristic of the "free enzyme" and "cellulosome" paradigms of cellulose solubilization remain either poorly represented or elusive to capture by shotgun sequencing methods. Instead, multigene polysaccharide utilization loci-like systems coupled with genes encoding beta-1,4-endoglucanases and beta-1,4-endoxylanases--which have not been previously encountered in metagenomic datasets--were identified, as were a diverse set of glycoside hydrolases targeting noncellulosic polysaccharides. Furthermore, both rrs gene and other phylogenetic analyses confirmed that unique clades of the Lachnospiraceae, Bacteroidales, and Gammaproteobacteria are predominant in the Tammar foregut microbiome. Nucleotide composition-based sequence binning facilitated the assemblage of more than two megabase pairs of genomic sequence for one of the novel Lachnospiraceae clades (WG-2). These analyses show that WG-2 possesses numerous glycoside hydrolases targeting noncellulosic polysaccharides. These collective data demonstrate that Australian macropods not only harbor unique bacterial lineages underpinning plant biomass conversion, but their repertoire of glycoside hydrolases is distinct from those of the microbiomes of higher termites and the bovine rumen.

Fig. 1.

OTU network map showing OTU interactions between all rarefied samples from the Tammar wallaby (spring and autumn), rumen, and termite. Lines radiating from samples Rumen _FA_8, Rumen_FA_64, Rumen_FA_71, and Rumen_PL are colored blue (fiber-associated fraction and pooled liquid-associated, respectively, from ref. 13), Termite_PL3 colored red [termite lumen study (12)] and Tammar_Spring (T1) and Tammar_Autumn (T2) colored green (present study) are weighted with respect to contribution to the OTU. OTU size is weighted with respect to sequence counts within the OTU. (Inset) The first two principal coordinate axes (PCoA) for the unweighted UniFrac analysis colored by host animal: Rumen (FA_8, ■; FA_64, •; FA_71, ◆; PL, ) blue; Termite (▲) red and Tammar (Spring, ; Autumn,▼) green. For complete inventory and comparisons between the two Tammar wallaby sample dates at an OTU definition (SONS analysis), see Table S2.

Fig. 2.

Gene arrangement in the Bacteroidales-affiliated fosmid and a hypothetical model of polysaccharide-adhesion and hydrolysis coordinated by this gene cluster. (A) Phylopythia affiliated the fosmid clone from which scaffold 78 is derived to the order Bacteroidales, as described in the text. The putative PUL gene cluster consists of an AraC family transcriptional regulator (geneA), an acetylxylan esterase (geneB), susC and susD gene homologs (genes C and D, respectively), and two genes encoding outer membrane-targeted lipoproteins (genes E and F). Genes G, H, and I encode proteins containing GH26, GH5, and GH43 catalytic modules, respectively. Gene J encodes a putative inner-membrane bound “sugar transporter” followed by genes K and L, which encode proteins containing GH5 and GH94 catalytic modules, respectively. (B) The hypothetical model predicts that polysaccharides are bound by the outer membrane-associated components, principally via the SusD homolog in a complex with the SusC, and the two lipoproteins. The GH5-containing proteins generate oligosaccharides, which are transported across the outer membrane, principally via the protein complex described above. These oligosaccharides may be further hydrolyzed by periplasmic GHs or transported to the cytoplasm via a glycoside sugar transporter (encoded by gene J), before hydrolysis by glycoside hydrolases (gene M and I) or terminal phosphorolytic cleavage by the GH94 glycoside phosphorylase (encoded by gene L).