Bacterial Circadian Rhythms: From Lakes to the Gut

May 20, 2021

Circadian rhythms, or changes in an organism’s behavior and physiology that follow a 24 hour, cyclical pattern, are found throughout the tree of life—plants, fungi, insects and mammals all have them. But what about bacteria? Do these ubiquitous microbes also have circadian rhythms? 

Circadian rhythms are regulated by intracellular clocks composed of interacting genes and proteins that facilitate daily oscillations in gene expression. For many years, bacteria were thought to be clockless. This assumption was rooted in the circadian infradian rule, which suggests that selection for circadian clocks in bacteria is unlikely given that their reproductive cycle is often shorter than 24 hours. In other words, what use is a clock that ticks longer than your lifetime? 

However, with the 1980s came discoveries in cyanobacteria, a group of photosynthesizing microbes found in soil and water, that turned this long-held assumption on its head. New research shows that non-photosynthesizing bacteria also possess circadian rhythms. Even the gut microbiota, a dense and diverse microbial community, exhibits daily oscillations in its composition and function. Learning more about bacterial circadian rhythms could help researchers understand the microbes that play a pivotal role in human health.

Circadian Rhythms in Cyanobacteria

Cyanobacteria are ubiquitous in fresh, brackish and marine water. Not only are they the oldest photosynthetic organisms on Earth, they were the first prokaryotes in which a circadian clock was discovered, and the only bacteria in which such mechanisms have been robustly characterized.
 
Early studies used luciferase reporter assays to identify rhythmicity in cyanobacterial gene expression. In these experiments, Synechococcus elongatus, a species widely recognized as the premier model for cyanobacterial circadian research, was transformed with a plasmid expressing luciferase genes driven by the promoter of a S. elongatus photosynthesis gene. By tracking the bioluminescence of S. elongatus cells under periods of constant light or during light-dark cycles, scientists found that the rhythm of bioluminescence (and thus activity of the photosynthetic gene promoter) was consistent with all criteria of circadian rhythms:                                                         
  1. Free-running and following a 24-hour period. ‘Free-running’ means a rhythm occurs approximately every 24 hours, even under constant environmental conditions. The S. elongatus circadian rhythm continues regardless of whether cells are incubated under constant light or cycles of light and dark.
  2. Able to entrain to environmental cues. Although circadian clocks are endogenous, they are inextricably linked to the external world. They can reset, or entrain, to align with various environmental inputs, called Zeitgebers (German for “time-givers”), including light and food. People experience jet lag, for instance, when their clocks are in the midst of entraining their sleep cycle to a new time zone. In the case of cyanobacteria, photosynthetic gene expression resets in response to different light-dark patterns.
  3. Temperature compensated. A hallmark of circadian rhythms is that they continue over a range of physiological temperatures. This ensures that the timing of physiological and behavioral processes is not thrown out of whack if the environment is a bit cold one minute and a bit hot the next. Scientists found that the cyanobacterial rhythm is maintained regardless of whether bacteria are incubated at 25, 30 or 36℃.
Given photosynthesis is linked to light, the observation that this process exhibits circadian rhythmicity in cyanobacteria makes sense. However, it has since been demonstrated that a large chunk of the cyanobacterial genome, perhaps up to 30%, is under circadian control.
 
With so much genomic rhythmicity, one has to wonder: is having a circadian rhythm useful for these bacteria? By competing S. elongatus mutants with different circadian periods in various light-dark cycles (Zeitgebers), researchers found that strains whose endogenous rhythm most closely aligned with the Zeitgeber cycle survived better than their competitors, suggesting that the ability to biologically track time imparts a fitness advantage to cyanobacteria. 
 
In the 30 years since these initial discoveries, scientists have also characterized the molecular clock regulating cyanobacterial circadian rhythms. The clock consists of 3 proteins: KaiA, KaiB and KaiC. Under periods of light, KaiC is autophosphorylated at 2 residues, a process promoted by interactions with KaiA.  As darkness falls, KaiC is sequentially dephosphorylated, which is facilitated by KaiB-mediated displacement of KaiA. In both its phosphorylated and dephosphorylated states, KaiC indirectly modulates gene expression via interactions with other protein  regulators. The 24-hour cycle of KaiC phosphorylation and dephosphorylation is how the cyanobacterial circadian clock ticks. In fact, the clock will still tick (i.e., undergo its phosphorylation cycles) in vitro when the Kai proteins are mixed with adenosine triphosphate (ATP) in a test tube. 
Phosphorylation and dephosphorylation of KaiC, with the help of KaiA and KaiB, is how the cyanobacterial clock “ticks."
Phosphorylation and dephosphorylation of KaiC, with the help of KaiA and KaiB, is how the cyanobacterial clock “ticks."
Source: Madeline Barron.

Circadian Rhythms in Non-Photosynthesizing Bacteria

Support for circadian rhythms in cyanobacteria is rich, but what about non-photosynthesizing bacteria? Here, the evidence is less clear.  Some bacteria exhibit rhythmic, 24-hour growth, but the cycles fail to meet all criteria for a circadian rhythm, such as temperature compensation. Homologs of kai genes are present in diverse bacterial species, though whether they function as timekeepers is not well understood.
 
Still, there are intriguing reports. One study found that a clinical isolate of the gut bacterium, Klebsiella aerogenes (referred to as Enterobacter aerogenes by the authors), exhibited 24 hour, free-running and temperature-compensated rhythms in the expression of the motility gene, motA. The rhythms of multiple biological replicates synchronized when cells were incubated in vitro with melatonin, a hormone under host circadian control. These findings suggest that the circadian clock of K. aerogenes may entrain to host cues in vivo. Indeed, in the absence of melatonin, there was greater variability in the circadian phases of different cultures. Of note, only 31-44% of cultures exhibited circadian patterns in motA expression—why those cultures demonstrated rhythmicity while others did not is not clear. Ultimately, more research is required to better understand the nuances of the K. aerogenes rhythm.
 
Another recent study illustrated that Bacillus subtilis, a bacterium found in soil and the mammalian gut, exhibits circadian rhythmicity in the expression of genes involved in light detection and biofilm formation. Using a luciferase bioluminescence assay similar to that employed in cyanobacteria, the authors showed that the promoters of these genes controlled expression in 24-hour cycles that entrain to light-dark cycles and compensate for temperature. These rhythms were only observed when B. subilitis formed biofilms, suggesting that circadian rhythms may confer an adaptive advantage in bacterial communities where cells coordinate their behavior to thrive within a given environment.

Circadian Rhythms and the Gut Microbiota

Gut microbiota composition changes over the course of the day.
Gut microbiota composition changes over the course of the day.
Source: Madeline Barron, gut and bacteria icons from the Noun Project.
The identification of circadian rhythmicity in gut-associated bacteria is intriguing in light of research showing that the gut microbiota exhibits daily oscillations in its composition. That is, the abundance of certain taxa peaks during one part of the day (morning) then crashes in another (night). These changes are influenced by Zeitgebers associated with host circadian processes, as illustrated by melatonin-mediated synchronization of K. aerogenes rhythmicity. This underscores the close relationship between host and microbiota, in which processes in one (e.g., circadian rhythms) regulate and are regulated by the other.
 
Studies in mice have shown that loss of host clock genes, high-fat diet, changes in feeding time and jet lag disrupt the rhythmicity of the microbiota. Disruptions in host and microbiota rhythms have important health consequences. In both mice and humans, jet lag dysregulates microbiota rhythm and alters community composition to promote obesity and glucose intolerance, a precursor for type 2 diabetes. Another study found that the rhythmicity of 13 microbial taxa is disrupted in people with type 2 diabetes, which could potentially serve as a biomarker of disease.
 
The negative health outcomes associated with dysregulated microbiota rhythmicity may be tied to altered metabolite production by gut bacteria. For instance, mice on a regular diet exhibit daily oscillations in butyrate levels, a compound produced by the microbiota and a key modulator in host intestinal homeostasis. Mice on a high-fat diet, however, lose this rhythmicity and are prone to obesity. Thus, rhythmic changes in microbiota metabolic function may be one of the many ways in which gut bacteria influence health and susceptibility to disease.
 
Whether gut microbiota rhythmicity is regulated by endogenous bacterial clocks, or is simply a reflection of the community responding to host circadian processes is unclear. In other words, daily fluctuations in microbiota structure and function are not evidence of bona fide bacterial circadian rhythms. Still, given the findings in K. aerogenes and B. subtilis, it is not entirely unlikely that gut bacteria harbor circadian clocks. This raises a number of questions: Which species have circadian rhythms and which ones don’t? What, if anything, can this tell us about the physiology and adaptation of diverse bacteria within the gut? Are there species that drive the rhythm of the gut microbial community at large by coordinating with other bacteria, as hinted by the biofilm-dependent rhythmicity of B. subtilis?
 
Answers to these questions would bolster our understanding of circadian rhythms within the bacterial world. Moreover, they could teach us how to exploit bacterial circadian clocks, and harmonize their ticking with our own, to promote and maintain our health and wellbeing.

Author: Madeline Barron

Madeline Barron
Madeline Barron is a fourth year Ph.D. student at the University of Michigan in the Department of Microbiology and Immunology.