Characteristics and Probable Etiology of Luminescent Oscillations in Microaerobic Cultures of Photobacterium Phosphoreum

Purpose: Bacterial bioluminescence has been postulated to be an adaptation to physical environments of low oxygen concentration as well as an adaptation to the biotic environment. The in vivo luminescence of cultures of luminescent bacteria grown under microaerobic conditions had apparently not been investigated previously. I conducted experiments in which in vivo luminescence emitted by microaerobic cultures of Photobacterium phosphoreum was monitored non-destructively. I found luminescence oscillated under these conditions. The cause of luminous oscillations was sought for three reasons. First, biochemical and biophysical mechanisms responsible for generating long-period oscillations are controversial, and some models deny the capability for such oscillations to prokaryotes. Second, the hypothesis that luminescence adapts bacteria to low oxygen environments had apparently only been tested on cultures grown with oxygenation. Third, interpretation of temporal changes of luminescence is important for the adaption of sequential luminescent analysis to bioassays based on this easily quantified biochemical product. Methods: In vivo luminescence was measured non-destructively by growing cultures of P. phosphoreum in sterile scintillation vials. The study of other factors relevant to in vivo luminescence was done on parallel cultures. Parallel cultures were derived from the same flask of inoculated media, and were treated treated similarly afterward. This allowed the concomitant study of several variables under undisturbed microaerobiosis. Experiments were of two types. In the first type, coincident temporal structure of luminescence and other parameters was sought by parallel measurements. In the second type of experiment, perturbations were applied to cultures and their effects on subsequent luminous oscillations were examined. Results: Luminous oscillations do not seem to share a common etiology. The first luminous peak was relatively resistant to the effects of nutrition, media conditioned by previous growth of this bacterium, and to the protein synthesis inhibitors chloramphenicol and puromycin. The amplitude, but not the timing of this peak was affected by pargyline. The timing, but not the amplitude of this peak was affected by aldehyde perturbation. Later luminous peaks were labile to factors that the first peak was resistant to, as well as resistant to the aldehyde perturbation that the first luminous peak was labile to. Microaerobiosis apparently increased in vivo luminescence emitted by cultures during the first 24 hours of observation. The first peak of in vivo luminescence occurs after a time span similar to that required for oxygen content to reach a minimum in microaerobic cultures of P. phosphoreum. An abrupt increase in luciferase synthesis also occurs after a similar lag time. Luminescent peaks after the first may be reduced in amplitude and slowed in periodicity by changing the conditions within scintillation vial cultures so that oxygen content is increased. Conclusions: The most likely etiology for the first luminous peak is that pre-existing luciferase is expressed by relief of aldehyde limitation. Subsequent luminous peaks are labile to many factors. One hypothesis consistent with observations in this work is that availability of FMNH₂ from the microbial metabolism exerts control over in vivo luminescence. In low oxygen conditions, considerable control of in vivo luminescence seems to be exercised independently of both luciferase content and the concentration of bacteria in a culture. The temporal similarity between the time required to reach minimum oxygen content, the time before the first luminous peak, and the time before a burst of luciferase synthesis occurs in microaerobic cultures may support the hypothesis that luminescence is functionally significant in low-oxygen conditions. This is based on the assumption that both in vivo luminescence and de novo synthesis of luciferase are linked to environmental conditions having functional significance for luminescence. Automated sequential luminescence monitoring may be a useful tool for bioassays. Luminous oscillations may be minimized by appropriate conditions within scintillation vial cultures. The non-destructive nature of such measurements allows the collection of data on in vivo biochemical events. Lability of luminescence to nutritional factors suggests use as a bioassay for nutrients. In my latest work, I have used the reversion of a dim mutant of this strain of P. phosphoreum to wild-type luminescence as the basis of an assay for mutagenic substances.