Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Discovery of a Proto-Circadian System in Bacteria



By: Carol A. Rouzer, VICB Communications
Published: April 1, 2016



The KaiC protein of R. palustris regulates light-dark metabolic cycles that convey a survival advantage to the bacteria.


The circadian clock is a complex biochemical pathway that regulates fundamental processes, such as gene expression and metabolism, with broad implications for complex processes such as sleep and behavior. Found in essentially all eukaryotic organisms, the circadian clock conveys a fitness advantage by timing biological functions to coincide with environmental conditions. Classically, circadian rhythms have come to be defined by three characteristics: (1) a periodicity of 24 h that is sustained, even in constant environmental conditions, (2) an entrainment of the 24 h rhythm to the environmental cycle, and (3) temperature compensation, so that changes in environmental temperature have no effect on the periodicity. Despite their ubiquity in eukaryotes, circadian rhythms that exhibit the three canonical characteristics have been reported in relatively few prokaryotic organisms. An important exception, however, is the cyanobacterial circadian clock, which has become a model system for understanding how these complex biochemical processes are regulated. The cyanobacterial clock comprises three proteins: KaiA, KaiB, and KaiC, which establish a 24 h rhythmic cycle of phosphorylation/dephosphorylation of Kai C that is regulated by its interactions with Kai A and Kai B. Although a canonical circadian rhythm has not been observed in other prokaryotes studied thus far, certain species do possess the kaiB and kaiC genes. This led Vanderbilt Institute of Chemical Biology member Carl Johnson and his laboratory to investigate the function of these genes in the Gram negative purple non-sulfur bacterium Rhodopseudomonas palustris. Their research led to the discovery of a proto-circadian system that conveys a survival advantage to this organism [P. Ma, et al. (2016) PLoS Genetics published online March 16, DOI:10.1371/journal.pgen.1005922].


The KaiC protein of R. palustris is structurally very similar to the KaiC of the cyanobacterium Synechococcus elongatus (Figure 1). Important differences include the sequences that serve as phosphorylation sites (TST in S. elongatus and TSS in R. palustris) and in the length of the C-terminus, which serves as an interaction site for KaiA in S. elongatus but not R. palustris. The similarities between the two proteins suggest that the R. palustris KaiC may play a role in some form of circadian rhythmicity that is similar to its role in S. elongatus.



FIGURE 1. Domain structures of the KaiB proteins from S. elongatus and R. palustris. The two proteins have nearly identical overall structures. Significant differences are the phosphorylation target sequences (TST in S. elongatus and TSS in R. palustris, and the length of the C terminus. Figure reproduced under the Creative Commons Attribution License from P. Ma, et al. (2016) PLoS Genetics published online March 16, DOI:10.1371/journal.pgen.1005922.



Remarkable for its metabolic flexibility, R. palustris can live as a photoheterotroph, a chemoheterotroph, a photoautotroph, or a chemoautotroph, meaning that it can use light or chemical energy for biosynthesis of molecules with either CO2 or organic compounds as a source of carbon. The investigators hypothesized that circadian rhythms in these organisms would most likely be observed during photosynthesis, as an ability to coordinate photosynthetic pathways to the light/dark cycle should convey a survival advantage to the bacteria. As circadian rhythms were first discovered in photosynthesizing cyanobacteria through observations of nitrogen fixation, the researchers initially studied the effects of a 24 h light/dark cycle (12 h light followed by 12 h darkness) on nitrogen fixation in R. palustris cultured under photoheterotrophic conditions. The results revealed a clear rhythmicity in the rate nitrogen fixation, with a peak in the middle of the light period that was highly similar in three separate cultures at two different temperatures (Figure 2A and C). In contrast, the bacteria did not display rhythmic changes in nitrogen fixation when placed under conditions of constant light. These findings suggest that under photoheterotrophic conditions, R. palustris displays a rhythmicity that conforms to two of the three canonical characteristics of a circadian clock - entrainment to the environmental light-dark cycle, and temperature independence. However, the bacteria lack the third characteristic of a true circadian clock - the rhythmicity is not retained under constant environmental conditions.


FIGURE 2. Changes in the rate of nitrogen fixation by wild-type (A and C) and RCKO (B and D) R. palustris during a 24 h light/dark cycle. Data are provided for bacteria cultured at 30 oC (A and B) and 23 oC (C and D). Figure reproduced under the Creative Commons Attribution License from P. Ma, et al. (2016) PLoS Genetics published online March 16, DOI:10.1371/journal.pgen.1005922.


To determine if KaiC is required for the changes in nitrogen fixation observed during a light/dark cycle, the investigators created a strain of R. palustris in which the kaiC gene had been knocked out (RCKO). These bacteria exhibited a rhythmicity in nitrogen fixation when cultured under a 24 h light/dark cycle, but the peak time varied considerably between separate cultures, and the amplitude was substantially reduced by decreasing temperature (Figure 2B and D). RCKO cells expressing the kaiC gene from an ectopic site (RCKO+kaiC), however, exhibited the same rhythmic pattern of nitrogen fixation observed in the wild-type cells. These findings demonstrated that KaiC is required for the coordination of nitrogen fixation to the environmental light/dark cycle.


In cyanobacteria, KaiC exhibits rhythmic changes in both phosphorylation state and abundance. To see if this was also true in R. palustris, the investigators used an RCKO+kaiC strain that expressed a His-tagged KaiC protein. This enabled them to easily detect the presence of KaiC with an antibody directed against the His tag. Using this approach, they were able to discern three distinct phosphorylated states of R. palustris KaiC, in addition to the unphosphorylated protein. Rhythmic changes in the various phosphorylated states occurred in bacteria grown under 24 h light/dark conditions, but not under constant light. No changes in KaiC abundance were detected under either set of culture conditions.


The KaiC of S. elongatus exhibits a very slow, temperature-compensated ATPase activity that is believed to play a key role in establishing the circadian rhythm in the cyanobacterium. The investigators confirmed that the KaiC from R. palustris can also act as an ATPase; however, its activity is much faster than that of the S. elongatus protein, and it is not temperature-compensated. It is unclear how the differences between the two proteins translate into the distinct responses of the two organisms to environmental light/dark cycles.


Despite the fact that the light/dark-driven rhythms observed in R. palustris do not fulfill all of the canonical criteria of a circadian clock, the Johnson lab investigators hypothesized that they should, nonetheless, convey a survival advantage to the bacteria. They tested this hypothesis by observing the growth rate of the wild-type, RCKO, and RCKO+kaiC strains of R. palustris under conditions of a light/dark cycle and constant light at two different temperatures. Consistent with their hypothesis, cells expressing KaiC (wild-type and RCKO+kaiC) exhibited more rapid rates of growth at both temperatures, but only under conditions of an alternating cycle of light and dark (Figure 3). This survival advantage is totally consistent with conditions under which a KaiC-dependent rhythmic change in nitrogen fixation was observed. The investigators conclude that R. palustris exhibits a “proto-circadian clock”, and they argue that evaluation of circadian rhythms in prokaryotes should emphasize biochemical pathways that provide a survival advantage to the organisms rather than a requirement that those pathways satisfy the canonical definition of a circadian clock as observed in eukaryotes.


FIGURE 3. Growth rates of wild-type, RCKO, and RCKO+kaiC strains of R. palustris grown under constant light (A and C), under a 24 h light/dark cycle (B and D) at 30 oC (A and B) and 23 oC (C and D). Figure reproduced under the Creative Commons Attribution License from P. Ma, et al. (2016) PLoS Genetics published online March 16, DOI:10.1371/journal.pgen.1005922.






View PLoS Genetics article: "Evolution of KaiC-Dependent Timekeepers: A Proto-circadian Timing Mechanism Confers Adaptive Fitness in the Purple Bacterium Rhodopseudomonas palustris"










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