A Melatonin-Sensitive Circadian Oscillator in the Human Gut Commensal, Enterobacter Aerogenes

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Biological circadian clocks are ubiquitous among eukaryotic organisms, which share remarkably conserved formal properties and molecular components. Among prokaryotes, circadian clocks have only been definitively demonstrated in the cyanobacterium Synechococcus elongatus, in which rhythmicity derives from the rhythmic phosphorylation of the hexameric protein KaiC, induced by KaiA, and dephosphorylation, induced by KaiB. Although clocks have only been demonstrated definitively in Synechococcus, orthologues of Kai proteins are widespread among cyanobacteria. We have recently observed that the human commensal, enteric proteobacterium Enterobacter aerogenes exhibits increased swarming behavior at 370C in the presence of the pineal and enteric hormone melatonin. Further, we observed that swarming was expressed in a circadian pattern at 370C, but not at 270C or at 400C. To further explore this process, we transformed E. aerogenes to express luciferase (luxCDABE) with a MotA promoter. Circadian rhythms in bioluminescence were observed at 270C, 370C and 400C in a temperature compensated fashion. This is the first demonstration of temperature compensated circadian rhythms in prokaryotes outside Cyanobacteria. It raises the possibility that human circadian clocks synchronize intestinal microbiota through entrainment of bacterial circadian clocks. We propose to further characterize the Enterobacter circadian clock through the identification of molecular components of this clock by candidate gene approaches by knockout of genes identified through bioinformatic analyses pointing to both melatonin sensitivity and sequence similarity to Kai genes and by mutagenesis screen. Mutant or knockout genes that exhibit circadian phenotypes will be complemented to recover WT phenotypes. Secondly, we will further characterize the Enterobacter clock to determine whether circadian rhythms are restricted to bacterial motility or are more widespread through transformation of bacteria with plasmids containing luxCDABE and one of several other promoter regions, including putative Kai orthologues and manganese transporters bearing putative melatonin binding sites. Finally, we will test the hypothesis that circadian patterns of bacterial swarming and their gene expression proxies are entrained to time of day and/or meal-time through the host’s secretion of melatonin as its Zeitgeber. Overall, this project is likely to identify the components of the first prokaryotic circadian clock outside of the cyanobacterial phylum, and the first in an organism that interacts directly with humans. The results are likely to have broad implications for the regulation of the human microbiota and gut physiology.
Effective start/end date9/15/177/31/21


  • National Institute of General Medical Sciences: $567,758.00


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