A trypsin inhibitor trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester suppresses the onset of DNA synthesis in Escherichia coli cells synchronized by phosphate starvation.

Abstract

trans-4-Guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPh'Bu), a trypsin inhibitor, dose-dependently inhibited the growth of Escherichia coli K-12 IAM1264. Growth inhibition was preceded by dose- and time-dependent inhibition of DNA synthesis. These results strongly suggested participation of a trypsin-like proteinase in DNA synthesis. To clarify this suggestion, the effects of GMCHA-OPh'Bu on the doubling time and on the uptake of [methyl-3H]thymidine into DNA were examined with E. coli K-12 IAM1264 synchronized by a modified version of phosphate starvation. The synchrony lasted for two or three cycles with a doubling time of 55 min and a cell division period of 15 min. The cell cycle of E. coli was divided into three periods, cell division period (P), the period between cell division and initiation of chromosome replication (Q) and the period between initiation of chromosome replication and cell division (R). The R period was subdivided into two periods, R1 in which the rate of thymidine uptake into DNA was increasing, and R2 in which it was constant. The addition of GMCHA-OPh'Bu at the R1 period did not affect the already-initiated round of cell division, however, it retarded the next round. The addition at P, Q or R2 retarded the cell division in the same round, causing prolongation of the R1 period. A sharp and momentary appearance of trypsin-like proteinase activity peaked at the Q/R1 boundary in one cell cycle, and inhibition of the activity prolonged the R1 period.(ABSTRACT TRUNCATED AT 250 WORDS)