Kinetics of derepression of the tryptophan operon of Escherichia coli and Salmonella typhimurium under different culture conditions

Abstract

Studies of the kinetics of derepression of the tryptophan ( trp) operon in cultures of Escherichia coli and Salmonella typhimurium show that the times of appearance (lag times) of newly synthesized t rp † † Abbreviations used: his, histidine biosynthetic operon; lac, lactose operon; tna, gene specifying tryptophanase; ASase, anthranilate synthetase complex; ASase CoI, component I of ASase; TSase α, tryptophan synthetase a polypeptide; trp, tryptophan biosynthetic operon. operon polypeptides following derepression are nearly invariant under many different culture conditions at a fixed temperature and that, following the lag period, specific activities of trp operon polypeptides increase in a smoothly continuous manner. During derepression, newly-synthesized trp operon polypeptides appear sequentially with lag times of approximately 6, 3 and 2 minutes for anthranilate synthetase complex or phosphoribosyl anthranilate transferase at 25, 30 and 37 °C, respectively, and approximately 10, 7 and 4 minutes for tryptophan synthetase α polypeptide at the same temperatures. These lag times are about 45 to 50% longer than transcription times reported for the corresponding regions of the trp operon at the same temperatures. Trimethoprim inhibition, which increases the increment between lag times for the histidine and lactose operons, causes only slight delays in appearance of trp polypeptides or in synthesis of specific regions of trp operon messenger RNA following derepression in cultures of E. coli. Under these conditions, the level of N-formylmethionyl-tRNA in the culture is unchanged compared to an uninhibited control. Concentrations of trimethoprim necessary to lower the level of N-formyl-methionyl-tRNA prevent derepression of trp operon. Under most conditions, the differential rates of synthesis of trp operon polypeptides following derepression of cultures of E. coli are about 250, 490 and 880 molecules/cell/minute for anthranilate synthetase at 25, 30 and 37 °C, respectively, and 180, 450 and 660 molecules/cell/minute for tryptophan synthetase α polypeptide at the same temperatures. These rates are lower when bacterial growth is restricted by histidine limitation, inhibition by 2-thiazolealanine (a histidine analog), or trimethoprim inhibition. Bacterial strains and culture conditions were used which mimic as closely as possible conditions which give simultaneous derepression of the his operon in S. typhimurium, nearly simultaneous induction of the lac operon in S. typhimurium/F′ lac merodiploids, or simultaneous induction of the arabinose operon in E. coli B/r. These conditions include addition of one-carbon compounds ( l-serine, l-methionine, adenine and thymine), use of the specific histidine auxotrophs, and addition of the histidine analog, 2-thiazolealanine. Under all conditions, the trp operon derepresses sequentially, although under some conditions in cultures of E. coli (histidine limitation, trimethoprim inhibition) lag times are increased and under some conditions in cultures of S. typhimurium (histidine limitation, 2-thiazolealanine inhibition) derepression of the trp operon is prevented.