Shining a light on internal clocks - the bacterium Bacillus subtilis [Professor Ákos Kovács, Technical University of Denmark]

Research headed by scientists at the Ludwig Maximilians University (LMU), Munich, has shown that—just like humans and other organisms—bacteria have internal clocks that align with the 24-hour day-night cycle. “We’ve found for the first time that nonphotosynthetic bacteria can tell the time,” said LMU research lead Martha Merrow, PhD. “They adapt their molecular workings to the time of day by reading the cycles in the light or in the temperature environment.”

The authors say their findings answer a long-standing biological question, and could have implications for the timing of drug delivery, industrial biotechnology, and even how we develop timely solutions for crop protection. Reporting on their research in Science Advances (“A circadian clock in a non-photosynthetic prokaryote”), Merrow and an international team of colleagues concluded, “Our work opens the field of circadian clocks in the free-living, nonphotosynthetic prokaryotes, bringing considerable potential for impact upon biomedicine, ecology, and industrial processes.”

Biological clocks, or circadian rhythms, are internal timing mechanisms that are widespread across nature, enabling living organisms to cope with the major changes that occur from day to night, even across seasons. “Circadian clocks create a 24-hour temporal structure, which allows organisms to occupy a niche formed by time rather than space,” the authors wrote. Existing inside cells, these molecular rhythms use external cues such as daylight and temperature to synchronize biological clocks to their environment. It is why we experience the jarring effects of jet lag as our internal clocks are temporarily mismatched before aligning to the new cycle of light and dark at our travel destination.

A growing body of research over the past two decades has demonstrated the importance of these molecular metronomes to essential processes, for example, sleep and cognitive functioning in humans, and water regulation and photosynthesis in plants. However, despite the fact that bacteria represent a significant proportion of the planet’s total biomass, and are important for health, ecology, and industrial biotechnology, little is known about their 24-hour biological clocks. In fact, previous studies have shown that photosynthetic bacteria, which require light to make energy, also have biological clocks. But free-living, nonphotosynthetic bacteria have remained a mystery in this regard. So while circadian clocks are “… pervasive throughout nature,” the authors wrote, “… they remain largely unknown in the nonphotosynthetic bacteria, despite bacteria representing about 15% of the living matter on Earth.”

The newly reported work by Merrow and colleagues has now detected free-running circadian rhythms in the nonphotosynthetic soil bacterium Bacillus subtilis. For their research, the team applied a technique called luciferase reporting, which involves exploiting an enzyme that produces bioluminescence, to allow the researchers to visualize how active a gene is inside an organism. They focused on ytvA, a gene that encodes a blue light photoreceptor, and also on an enzyme called KinC, which is involved in inducing formation of biofilms and spores in the bacterium.

The researchers were able to observe levels of the genes in constant dark, in comparison to cycles of 12 hours of light and 12 hours of dark. The results showed that the pattern of ytvA levels was adjusted to the light and dark cycle, with levels increasing during the dark and decreasing in the light. A cycle was also still observed in constant darkness. The researchers also noted how it took several days for a stable pattern to appear, and saw that the pattern could be reversed if the conditions were inverted. These two observations are common features of circadian rhythms and their ability to “entrain” to environmental cues.

The investigators then carried out similar experiments using daily temperature changes; for example, increasing the length or strength of the daily cycle, and found the rhythms of ytvA and kinC adjusted in a way consistent with circadian rhythms, and not just simply switching on and off in response to the temperature. “Here, we identify in Bacillus subtilis circadian rhythms sharing the canonical properties of circadian clocks: free-running period, entrainment, and temperature compensation,” the team noted. “We show that gene expression in B. subtilis can be synchronized in 24-hour light or temperature cycles and exhibit phase-specific characteristics of entrainment.”

The team believes their research could be used to help address questions, for example, is the time of day of bacterial exposure important for infection? Can industrial biotechnological processes be optimized by taking the time of day into account? Is the time of day of anti-bacterial treatment important?

“We suggest that the incorporation of temporal structures into industrial, biomedical, and agricultural applications for bacteria might provide important translational opportunities,” they concluded. “Our discovery of circadian rhythms in the Eubacteria should motivate future insights into the mechanisms and evolution of circadian rhythms across life.”

Co-author Antony Dodd, PhD, from the John Innes Centre, in the U.K., further commented, “Our study opens doors to investigate circadian rhythms across bacteria. Now that we have established that bacteria can tell the time we need to find out the processes that cause these rhythms to occur and understand why having a rhythm provides bacteria with an advantage.” Merrow added, “In addition to medical and ecological questions we wish to use bacteria as a model system to understand circadian clock mechanisms. The lab tools for this bacterium are outstanding and should allow us to make rapid progress.”

Ákos Kovács, PhD, a co-author at the Technical University of Denmark, also pointed out, “Bacillus subtilis is used in various applications from laundry detergent production to crop protection, besides recently exploiting as human and animal probiotics, thus engineering a biological clock in this bacterium will culminate in diverse biotechnological areas.”

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