It previously has been discovered that visible light irradiation of crystalline substrates can lead to enhancement of subsequent enzymatic reaction rates as sharply peaked oscillatory functions of irradiation time. The particular activating irradiation times can vary with source of a given enzyme and thus, presumably, its molecular structure. The experiments reported here demonstrate that the potential for this anomalous enzyme reaction rate enhancement can be transferred from one bacterial species to another coincident with transfer of the genetic determinant for the relevant enzyme. In particular, the effect of crystal-irradiated chloramphenicol on growth of bacterial strains in which a transferable R-factor DNA plasmid coding for chloramphenicol resistance was or was not present (S. panama R+, E. coli R+, and E. coli R-) was determined. Chloramphenicol samples irradiated 10, 35 and 60 sec produced increased growth rates (diminished inhibition) for the resistant S. panama and E. coli strains, while having no such effect on growth rate of the sensitive E. coli strain. Consistent with past findings, chloramphenicol samples irradiated 5, 30 and 55 sec produced decreased growth rates (increased inhibition) for all three strains.
What has evolved as a possibly universal capability on the part of enzymes to recognize unorthodox radiation signals was first reported in 1968 (Comorosan et al., 1968(Comorosan et al., , 1970a(Comorosan et al., ,b,c, 1971a(Comorosan et al., ,b, 1973(Comorosan et al., , 1975(Comorosan et al., , 1980)). The study presented here examines the genetic origin of the relevant enzyme characteristic by utilizing inter-bacterial transfer of an Rfactor plasmid. The particular plasmid chosen codes for production of the enzyme chloramphenicol acetyl transferase (CAT) which, in turn, is responsible for bacterial resistance to chloramphenicol (Smith, et al., 1972).
Comorosan and co-workers have studied extensively the subject phenomenon. Typically, alteration of in vitro activity of an enzyme (Comorosan, et al., 1972b) or growth rate of a microorganism (Comorosan et al., 1973) is obtained as a consequence of visible light irradiation of the enzyme substrate or microorganism growth factor (or inhibitor) in the crystalline state. That is, irradiation is performed prior to dissolution and introduction of the substrate or growth factor into the reaction mixture or growth medium. Probably the most unexpected feature of these observations is that the activity alteration is a periodic function of the duration of exposure of the crystalline substrate to the electromagnetic radiation employed. Thus, if t m is the shortest irradiation time which produces an alteration, the next alteration will occur with irradiation for an increased length of time, t m + τ . The entire collection of irradiation times which produce activity alteration, t*, can be represented as t* = t m + nτ where n is an integer and τ a constant. The magnitudes of t m and τ are on the order of seconds (5-45 seconds) while the width of the activation peaks are ≤ 0.5 seconds. Irradiations for other times t ≠ t*, whether longer or shorter, produce no activity alteration relative to non-irradiated controls. All t m and τ values have been found to be multiples of 5 seconds. These basic observations have been reproduced or demonstrated successfully in other laboratories (Bass, et al., 1973(Bass, et al., , 1976a(Bass, et al., ,b, 1977;;Etzler and Westbrook, 1986;Goodwin and Vieru, 1975;Sherman, et al., 1973Sherman, et al., , 1974)).
The mechanism by which the altered biological responses are produced remains unestablished. For convenience, the responses and their associated irradiation times, t*, are referred to hereafter as “signals.”
In vitro studies (Comorosan, et al., 1971a,b) with systems of enzymes from the glycolysis, gluconeogenesis, and Krebs cycle pathways have revealed patterns in the t* values for the individual reactions. These patterns have led to the suggestion that this phenomenon reflects an innate ability of enzymes to recognize, or respond to, signals which are partly responsible for, or associated with, cellular metabolic control. Further, the t* values for a particular enzyme isolated from mammalian tissues are found to be numerically different from those for the corresponding enzyme from a microorganism. Thus, there is a species dependence which implies a dependence on composition or substructure of the enzyme molecule. Additional support for this proposition has been obtained with isoenzyme rehybridization studies (Comorosan, et al., 1972a). This strange behavior observed in the kinetics of isolated, purified enzymes is also found, at least qualitatively, in studies on growth rate of microorganisms (Comorosan, et al., 1973(Comorosan, et al., , 1975;;Bass and Crisan, 1975;Sherman, et al., 1974). Thus, t* irradiation times corresponding to growth rate enhancements have been found for introduction of crystalirradiated arginine, histidine, and tryptophan into minimal media for growth of yeast strains auxotrophic for these amino acids. Presumably, these growth rate alternations are a consequence of stimulation of some particular enzyme reaction which utilizes the irradiated amino acid. The studies to be described here speak to this presumption.
Corresponding to microbial growth rate enhancements with irradiated growth factors, increased inhibition of growth is obtained for the action of crystal-irradiated antibiotics on sensitive yeast and bacteria (Comorosan, et al., 1975;Bass and Crisan, 1975;Sherman, et al., 1974). Again, the familiar t* = t m + nτ irradiation time dependence is encountered. Presumably, the irradiation process creates photoproducts which stimulate interaction of the antibiotic with cellular receptors (possibly the ultimate target of the antibiotic such as the ribosomes for tetracycline or chloramphenicol). On the other hand, for antibiotic resistant bacteria, two sets of growth alteration antibiotic-irradiation signals, t* 1 and t* 2 , may be found. (As used here, “resistant” bacteria are those which have the capability to actively and appreciably deactivate given antibiotics enzymatically). One set of signals corresponds to incr
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