Do you ever sleep through your alarm? Forget to set it? Set 10 alarms in five minute intervals? Snooze repeatedly until you’re about to be late? Now, we’re not calling out one of the co-authors, Gillian (hey!). However, we’d like to introduce you to your new best friend; someone who’s reliable, on-time, and oh so punctual!
Anabaena spp. is a genus of filamentous cyanobacteria that exist as plankton. They are known for their nitrogen-fixing abilities as well as the symbiotic relationships they can form with plants, fungi, protists, and sponges, as one of the earliest examples of symbiosis on earth! Anabaena is a multicellular organism in which the cells are arranged in a one-dimensional strand filament with local nearest-neighbor cell-cell coupling through septal junctions. Anabaena has shown evidence of coupling metabolic pathways along the bacterial filament through cell-cell communication to preserve the energy efficiency of metabolic reactions. Additionally, Anabaena contains Kai, a post-transcriptional oscillator that regulates the circadian rhythm in Anabaena cultures. This means Anabaena has a robust, coherent, and synchonrized circadian clock built right into its genome. It’s never late for any plans, and will always oscillate on time!
This article discusses the circadian rhythm exhibited by Anabaena. The circadian clock originates from three proteins, KaiA, KaiB, and KaiC. The word “Kai” actually stems from a Japanese character which means “cycle,” deeming it more than appropriate for the characteristics of these fascinating proteins. We’ll start with introducing “kaiC,” a gene belonging to the “kaiABC” gene cluster that regulates bacterial circadian rhythms, specifically in cyanobacteria. The kaiC gene encodes for the KaiC protein, which interacts with the KaiA and KaiB proteins as a “post-translational oscillator.” Further details on the chemical breakdown of this circadian aligned occilitory process can be found here and here in two short, helpful videos that discuss the autophosphorylation processes of our favorite ABC’s. Essentially, at dawn, monomers in a loop of KaiC start in an unphosphorylated state with loosely connected “C rings.” A filamentous loop on KaiC remains exposed, which further binds to KaiA throughout the day, and keeps the KaiC erect. Not only this, but it also hides the binding site for KaiA when the sun goes down and allows the two proteins to stiffen together. When KaiA can no longer bind to the disappeared KaiC binding site, it binds with KaiB which undergoes a rare state change, and turns into a monomer. Authophosphatease activity starts up as autokinase activity ceases. Important for the process of signal transduction, the circadian oscillations can be seen in vitro and the mechanism is quite simple. Impressively, Anabaena exhibits extreme temporal precision. The clock has been shown to be autonomous. In a similar species, Synechococcus, the clock runs a 12-hour light-dark cycle with a high amplitude of oscillation. In contrast, Anabaena undergoes a significantly lower amplitude of oscillation.
One may ask how temporal precision may be achieved in a noisy multicellular environment. There is ample evidence to support that sensing interactions between proteins may result in this precision and synchronization. Within the Anabaena bacterium, protein regulator factors KaiA, KaiB, and KaiC are internally regulated by localized sensing of the other oscillating proteins, as is the known master circadian regulator rpaA. A particularly interesting figure in the research paper, Figure 2, demonstrates the additional 12-hour cycle of oscillation that all four of these proteins undergo, in direct conjunction with controlled exposure to consistent periods of lightness and darkness.
The article presented both an experimental and theoretical study of circadian clocks in multicellular Anabaena. The one-dimensional nature of Anabaena allowed the researchers to follow the “clocks” in each cell along the filament and show the interactions between demographic noise and cell-cell communication. Using DNA microarray analysis, a tool used to determine whether there is a gene mutation in the cell’s DNA, the research team was able to identify high-amplitude circadian oscillations. In the experiment, the researchers followed the output of these clocks in individual cells by monitoring the expression from the promoter of pecB, a clock-controlled gene of high-amplitude oscillations. This pecB gene is part of the pecBACEF operon which codes for the beta subunits of phycoerythrocyanin, a structural component of the phycobilisome rod. This structure plays a major role in light harvesting for photosynthesis. We thought it was an interesting, and somewhat obvious, observation that the same components that contribute to light-harvesting for photosynthesis also contribute to the cell’s natural circadian rhythm, something directly relating to sunlight.
Figure 1. Circadian oscillation in Anabaena. (A) GFP fluorescence in a filament of an Anabaena strain bearing a PpecB–gfp promoter fusion, growing under nitrogen-replete conditions. The snapshots were chosen near maxima and minima of the circadian oscillations. (B) Autofluorescence as a function of time in Anabaena. Snapshots correspond to those in (A), and time 0 corresponds to the time at which filaments were placed in a device for microscope observation (for details, see Materials and methods). For a time-lapse movie, see Video 1 (taken over 6 days).
Notably, as shown in the results in Figure 1, Figure 2B, and Figure 2C, there is significant synchrony in cellular oscillations as they progressed in individual cells along bacterial filaments. These interactions, coupled with cell-cell communication, demonstrate a high spatial coherence of the expression of PpecB-gfp. Shown to occupy nearly all of the same phases along their circadian cycle, Figure 1A contains snapshots of Anabaena at the approximate extremes of their circadian oscillations – with autofluorescence being demonstrated as a function of time. This imaging technique allows scientists to observe under the microscope the cellular processes of naturally occurring substances, such as chlorophyll, collagen and fluorite. Most plant and animal tissues show some autofluorescence when excited with ultraviolet light. Though there were undoubtedly outliers in these natural oscillations, it is obvious that variations in gene expression between cells along a filament may limit both synchrony and spatial coherence. Additionally, the circadian clock has been observed to gate cell division in similar lab studies presented in the paper. To verify the singular influence of cell-to-cell interactions in a “noisy” environment for Anabaena, they first characterized the spectral properties of uncoupled clocks in individual cells of Synechococcus. In addition, mutant forms of kaiABC (ΔkaiABC), rpaA, and pecB were isolated (Figure 2B, Figure 2, Figure 2C) in a “takeaway study” which demonstrated that KaiABC is indeed necessary for cellular oscillation regulation.
Figure 2. Transcriptional oscillations in the core clock genes, rpaA and pecB. (A) Relative expression of kaiA (green), kaiB (red), and kaiC (blue) as a function of time measured by RT-qPCR (Materials and methods). A persistence homology analysis of these data is presented in Figure 2—figure supplement 1. (B, C) Relative expression levels of rpaA and pecB, respectively, in wild-type (full circles) and ΔkaiABC strains (empty circles). Curves have been normalized by their temporal mean. Error bars represent the standard error of the mean of three independent experiments (see Materials and methods). Gray shades represent periods of subjective night. For additional information about regulatory sequences of the kaiABC, rpaA, pecB promoter regions and RpaA binding sites in Anabaena, see (C) (Figure 2—figure supplement 2).
The study of cellular oscillations is something that we have come to be familiar with in our microbiology class through discussions of binary fission as it takes place in different bacterial cells. When most rod-shaped bacteria undergo division, they increase in biomass from intake of nutrients, undergo binary fission, and divide right in the middle, forming two identical symmetrical daughter cells. We explored the interferences that could take place in this process, such as external mediation with the protein FtsZ, which regulates where the separatory “Z ring” forms in the process of bacterial fission through something called “The Min System.” This consists of various proteins, such as MinC, which inhibits FtsZ polymerization, and MinD, a membrane-bound anchor and activator for MinC. Both MinC and MinD are localized in the poles and work together to inhibit FtsZ during polymerization. They accomplish this through an oscillation across the cell. This occurs everywhere except the middle, where FtsZ polymerizes in the only place it is not being inhibited. The Min system shows a fellow “cascade-like” reaction to interruption in the function of oscillation regulator proteins with the observation of a purified “mutant type.” In much the same way, KaiABC proteins are directly regulated by their oscillation in a given environment. We can draw obvious parallels between the comparative studies of both of these cascade-oscillating organisms, which demonstrate the continuous relevance of the relationship between environmental conditions to internal cell regulation of fission, even in a highly complex and biodiverse environment. Understanding the processes of binary fission allows us to further discover the natural processes undergone by bacteria to continuously diversify its genetic variation when reproducing at such a high rate. This bacterial diversity lends itself to such interesting bacterial features, such as the wonderful circadian rhythm that we see with Anabaena!
About the Authors:
Hi! I’m JoJo (right), I’m a senior at Mount Holyoke College majoring in anthropology, minoring in biology, and on the pre-health track. After graduation, I am pursuing both a masters and doctorate in Midwifery at Columbia University. In my free time, I enjoy surfing, aerial silks, and collecting houseplants! I’m Gillian (left), I’m a senior at Mount Holyoke College majoring in biology and anthropology. After graduation, I plan to study repetitive and fractal systems in nature via fields such as genetics molecular and chemical biology, as well as how this relates to patterns of human behavior in anthropology. This past summer I worked at the University of Central Florida as a bioengineer, bone-fortifying nanoparticles delivered in targeted vaccines for ultrasonic release. Outside of class, I love to construct moss terrariums with materials I find in nature. I also love to scuba dive, explore any beach or forest I find, and spend time enjoying the outdoors.
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