Biochemical Studies on Gramicidin S Non-producing Mutants of Bacillus brevis ATCC 9999

Mutants which could not produce gramicidin S were isolated from Bacillus brevis ATCC 9999 by UV irradiation or N-methyl-N'-nitro-N-nitrosoguanidine treatment. They were classified into three groups by means of complementation testsin vitro using purified complementary fractions of gramicidin S synthetase, Fraction I and Fraction II [phenylalanine racemase, EC 5.1.1.11]. The former activates L-proline, L-valine, L-ornithine, and L-leucine and the latter L- and D-phenylalanine. The first group lacked Fraction II activity, the second both Fraction I and Fraction II activities, and the third Fraction I activity. ATP-32PP1 exchange activities depending on the constituent amino acids of gramicidin S were determined after fractionation of mutant extracts by sucrose density gradient centrifugation. It was found that the third group of mutants was possibly classified into several subgroups. One mutant hh had activating activities for three amino acids (L-proline, L-valine, and L-ornithine), but lacked L-leucine activating activity intrinsic to Fraction I. The other mutant n-7 lacked activating activities for all four amino acids which should be present in the region of Fraction I, although L-proline activating activity was detected in the region of Fraction II. D-Phenylalanyl-L-proline diketopiperazine synthesizing activity was not present in the mutant hh extract, but was detected in the mutant n-7 extract. This result indicates that some modifications of Fraction I result in failure to associate with racemase in the case of mutant hh, despite the fact that the L-proline activating subunit of Fraction I alone could associate with racemase to make an active complex for diketopiperazine synthesis in the case of mutant n-7.