Increase in streptomycin production caused by tunicamycin.

We reported previously that D-glucosamine or a close derivative was involved in the synthesis of the N-methyl-L-glucosamine moiety of streptomycin'). Since D-glucosamine is a component of the cell wall of streptomycetes2,3), this sugar or its derivatives may be a common precursor of streptomycin and the cell wall. We studied also whether UDP-N-methyl D glucosaminephosphate was involved in the synthesis of the Nmethyl-L-glucosamine moiety of streptomycin and whether UDP-N-acetylmuramyl pentapeptide was present in Streptomyces griseus4). The latter compound should be a precursor of the peptidoglycan in streptomycetes as it is in bacteria. Using specific inhibitors of bacterial cell wall synthesis, some groups5,6) have demonstrated that inhibition of cell wall synthesis in S. griseus affects the synthesis of streptomycin. However, a connection between the intermediates of cell wall synthesis and those of streptomycin biosynthesis has not been reported. We have used tunicamycin, a specific inhibitor of cell wall synthesis in bacteria, to investigate this question. S. griseus ME936-B3 from the Institute of Microbial Chemistry, Tokyo, was used for streptomycin production. Bacillus subtilis IAM 1069, a streptomycin-sensitive strain, was obtained from the Institute of Applied Microbiology, Tokyo University. D-[1-14C]Glucosamine (60.8 mCi/rnmol) and D[6-3H]glucosamine (38 Ci/mmol) were purchased from Radiochemical Centre, Amersham, U.K. S. griseus was grown in the medium previously described4) with or without tunicamycin. In the isotopic experiments, D-[1-14C]glucosamine (10 uCi, 0.16 ismol) was administered to tunicamycin-supplemented medium and D-[6-3H]glucosamine (10 uCi, 0.26 nmol) was administered to the control medium at 24 hours. Streptomycin was determined by the agardiffusion method with B. subtilis as the test organism. Streptomycin hydrochloride was isolated from culture media by the method of HUNTER and HOCKENHULL7). N-Methyl-Lglucosamine hydrochloride was then prepared from it by the method of SILVERMAN and RIEDER8). Nucleotides were isolated from the mycelia by the method of BLUMSON and BADDILEY9). UDPN-acetylmuramyl pentapeptide and UDP-Nmethyl-D-glucosaminephosphate were prepared and identified as described in a previous paper). Cell wall mucopeptide was prepared by the method Of PARK and HANCOCK10 ). When tunicamycin was added to culture media at inoculation, growth of S. griseus was not inhibited (Fig. 1), but the cell shape changed to a spherical form (Fig. 2). Production of streptomycin increased (Fig. 3). However, no increase was observed when tunicamycin was added at 24 or 48 hours after inoculation (data not shown). The incorporation of radioactive D-glucosamine into streptomycin and its N-methyl-Lglucosamine moiety was somewhat increased by addition of tunicamycin to the culture media