Supplementary material from "Urea hydrolysis by gut bacteria in a hibernating frog: evidence for urea-nitrogen recycling in Amphibia"

Gut bacteria that produce urease, the enzyme hydrolysing urea, contribute to nitrogen balance in diverse vertebrates, although the presence of this system of urea-nitrogen recycling in Amphibia is as yet unknown. Our studies of the wood frog (Rana sylvatica), a terrestrial species that accrues urea in winter, documented robust urease activity by enteric symbionts and, hence, potential to recoup nitrogen from the urea it produces. Ureolytic capacity in hibernating (non-feeding) frogs, whose guts hosted a approximately 33% smaller bacterial population, exceeded that of active (feeding) frogs, possibly due to an inductive effect of high urea on urease expression and/or remodelling of the microbial community. Furthermore, experimentally augmenting the host's plasma urea increased bacterial urease activity. Bacterial inventories constructed using 16S rRNA sequencing revealed that the assemblages hosted by hibernating and active frogs were equally diverse but markedly differed in community membership and structure. Hibernating frogs hosted a greater relative abundance and richer diversity of genera that possess urease-encoding genes and/or have member taxa that reportedly hydrolyse urea. Bacterial hydrolysis of host-synthesized urea likely permits conservation and repurposing of valuable nitrogen not only in hibernating R. sylvatica but, given urea's universal role in amphibian osmoregulation, also in virtually all Amphibia.