Diffusion, permeation, or enzyme limitation: A probe for the kinetics of enzyme induction

The utilization of an exogenous substrate by enzyme inside a bacterial cell can be limited by diffusion up to the cell, penetration of the cell, diffusion within the cell, and/or attack by internal enzyme. For small molecular weight substrates such as galactosides, and for bacteria such as Escherichia coli the diffusion steps are not rate limiting even with the permeases fully induced and the external concentration of substrate low. In permeaseless organisms with more than about 20 enzyme molecules per cell, permeation of O ‐nitrophenyl‐β‐ D ‐galactoside through the membrane is limiting. Thus, a single initiation of transcription of a lactose message suffices to yield enough enzyme molecules to switch an uninduced cell from enzyme limitation to permeability limitation. Subsequent initiations change the cellular activity very little. This transition can be followed by assaying enzyme activity of both intact and lysed cell suspensions. In this way the induction response amongst cells in growing populations at high inducer concentrations has been found to be uniform. It was found that nearly all of the cells from balanced growing culture are immediately inducible even with doubling times as short as 7.6 hrs. At 24 hrs about 1/3 of the cells are inert at any time, but all cells synthesize enzyme within a 3‐hour period. At low inducer concentration or in the present of catabolite repressor the rate of initiation is greatly decreased; this leads to a non uniform distribution of enzyme within the cells, which is readily detected by the experimental technique. In addition to developing the kinetics for enzyme contained in cells, the distribution of the enzyme among uninduced bacteria is presented.