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The caecal supernatants from germ-free, antibiotic-treated and control mice were compared with respect to their content of low-molecular-weight substances (less than 3500 mol. wt.). The supernatants contained about the same amount of free amino acids. After acid hydrolysis, the caecal supernatants of germ-free and antibiotic-treated mice showed a 2.9-fold increase in free amino acids, whereas a similar treatment of the supernatant from control mice resulted in a 2.6-fold increase. By gel filtration on Sephadex G-25, and high-voltage paper electrophoresis at pH 3.5 of the fractions eluted after the void volume, it was found that the caecal supernatants of germ-free and antibiotic-treated mice contained a substance more acidic than aspartic acid. Preparative high-voltage electrophoresis, dansylation, amino acid analysis and a specific colour reaction showed the substance to be beta-aspartylglycine. After a minimal 36 h of treatment with neomycin and bacitracin, a high concentration of beta-aspartylglycine was found, and no enterococci and aerobic Gram-negative rods could be cultured from the caecal contents. The possibility that in one mouse the appearance of beta-aspartylglycine was related to a decrease in Gram-negative rods was ruled out by selective elimination of aerobic Gram-negative rods by using polymyxin B. This suggests that other bacteria concomitantly eliminated with the enterococci and aerobic Gram-negative rods, directly or indirectly, could play a role in the accumulation of beta-aspartylglycine.

β-Aspartylglycine, a substance unique to caecal contents of germ-free and antibiotic-treated mice