Diffusion-Controlled Insult of Prokaryotes: We have transformed MG1655 E. coli using a 9 kb insert from Vibrio fischeri that encodes enzymatic luciferase activities for the light reaction (including the synthesis of the substrate luciferin, N-(b-ketocaproyl)homoserine) as well as the regulatory functions necessary for the expression of the genes (including the autoinducer).  Thus, bioluminescence in this mutant, MG1655-lux, an ATP-driven reaction that is localized in the cytosol, is self-sustaining.  We have carried out a preliminary examination of the reaction of MG1655-lux with HOCl under stopped-flow (turbulent mixing) conditions.  Representative data are summarized in Figure 2.  The kinetic traces of Figure 2 are total light emission, as detected by a photomultiplier tube, as a function of time.  There are no other light sources in this experiment other than the organism itself.  Furthermore, that source of light is certainly originating from the cytoplasm of the E. coli cells since there is virtually no ATP in the periplasm and the enzymatic reaction cannot be sustained extracellularly. The mixing event, which takes place in ca. 1 ms, does not influence the light emission, as shown in the control experiment that involves rapid mixing of the cell with PBS that does not contain HOCl.  The time-trace that is observed when the MG1655-lux is mixed with 50-500 mM HOCl exhibits exponential decay of light emission, beginning with the level of emission that is observed without chemical insult (E₀), and ending with no light emission.  We have demonstrated that the HOCl is in large excess because the spectrum of OCl^- remains largely unperturbed after the mixing event.  A careful measurement of the small change in absorbance for OCl^- yields the same pseudo-first-order rate constant that was determined by the change in emission of MG1655-lux under the same conditions.  It is unclear whether the luciferase enzyme or its supply of ATP is affected.  In either case, the salient point is that cytoplasmic processes are influenced on the stopped-flow timescale and the initial chemical insult is rate-limiting (although kinetically more facile processes may occur thereafter).  We note that these stopped-flow experiments are, to our knowledge, the first that involve monitoring a chemical reaction with cells on the millisecond timescale, although there have been several stopped-flow experiments reported that have involved employing light scattering to follow osmotically-induced transport phenomenon in cells. 

Acknowledgement:  We are very grateful to the National Science Foundation (CHE-0503984), the National Institutes of Health (5 P20 RR018741-02), and the Department of Education (GAANN) for their financial support of this project.