Isolation and characterization of anaerobic bacteria for symbiotic recycling of uric acid nitrogen in the gut of various termites

Abstract

Recycling of the nitrogenous waste uric acid (UA) of wood-feeding termites by their gut bacteria is one of the significant aspects of symbiosis for the conservation of nitrogen sources. Diverse anaerobic UA-degrading bacteria comprising 16 species were isolated from the gut of eight termite species, and were assigned to Clostridia, Enterobacteriaceae, and low G+C Gram-positive cocci. UA-degrading Clostridia had never been isolated from termite guts. UA-degrading ability was sporadically distributed among phylogenetically various culturable anaerobic bacteria from termite guts. A strain of Clostridium sp., which was commonly isolated from three termite species and represented a probable new species in cluster XIVa of clostridia, utilized UA as a nitrogen source but not as a sole carbon and energy source. This feature is in clear contrast to that of well-studied purinolytic clostridia or previously isolated UA degraders from termite guts, which also utilize UA as a sole carbon and energy source. Ammonia is the major nitrogenous product of UA degradation. Various purines stimulated the growth of this strain when added to an otherwise growth-limiting, nitrogen poor medium. The bacterial species involved the recycling of UA nitrogen in the gut microbial community of termites are more diverse in terms of both taxonomy and nutritional physiology than previously recognized.

Fig. 1

A maximum likelihood tree showing the phylogenetic position of strain NkU-1 in the clostridia cluster XIVa. The tree was reconstructed with 1,345 unambiguously aligned nucleotide sequences of 16S rRNA gene using C. symbiosum as an outgroup. In addition to type strains and isolate LIP5 (17), the analysis included sequences of four clones from termite guts: RsStar407 from R. santonensis(16), BCf1–20 from C. formosanus(51), HsH-8 from H. sjoestedti(30), and AspS5-3 from Archotermopsis sp. (30). The accession number for each sequence is shown. Percent bootstrap value in 1,000 replicates when above 50% is shown at each node. Scale bar represents 0.10 substitutions per position.

Fig. 2

Time course (A) and dose effect (B) of utilization of uric acid as a nitrogen source by strain NkU-1. (A) GY medium supplemented with 0.1% UA was used, and cell growth (OD and protein), UA in the culture, and produced NH₃ were measured at 24-hour intervals. (B) GY medium supplemented with the defined concentration of UA was used and OD, protein content (data not shown), and produced NH₃ were measured after three days culture. Linear correlations were obtained for OD (R²=0.96) and protein (R²=0.90).