Effects of various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, trypsin inhibitors, on the growth of Bacillus subtilis.

Various aromatic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA), trypsin inhibitors, strongly inhibited the growth of Bacillus subtilis 558 and their effects were markedly affected by the species of substitution on the phenyl nucleus of the GMCHA phenyl esters. 4-tert-Butylphenyl ester of GMCHA (GMCHA-OPhtBu), a representative of various GMCHA esters, dose-dependently inhibited the growth of B. subtilis and DNA, RNA and protein syntheses in the cells. The growth inhibition was preceded by suppressive effect of GMCHA-OPhtBu on DNA synthesis. These results suggested the possible involvement of a trypsin-like proteinase in DNA synthesis. A trypsin-like proteinase was partially purified from B. subtilis 558 by DEAE-cellulose column chromatography, ammonium sulfate fractionation and successive chromatographies on Sephadex G-200, phenyl-Sepharose CL-4B, L-arginine-Sepharose 4B and Sephadex G-200 columns. The properties were compared with those of proteinase In, which momentarily appears just before the onset of DNA synthesis and seems to participate in the initiation of DNA replication, and which was purified from E. coli K-12 IAM 1264. The properties of the proteinase from B. subtilis 558 were similar to proteinase In, however, the molecular mass (110000) was different from that of the latter (66000). Various GMCHA esters strongly inhibited the proteinase activity and the order of the effects was closely correlated with that on the cell growth. The proteinase was tentatively called subtilis proteinase In.