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58-449: See also: bacterial circadian rhythms In the complex, KaiA enhances the phosphorylation status of kaiC. In contrast, the presence of kaiB in the complex decreases the phosphorylation status of kaiC, suggesting that kaiB acts by antagonising the interaction between kaiA and kaiC. The activity of KaiA activates kaiBC expression, while KaiC represses it. Also in the KaiC family is RadA/Sms,

116-803: A in vitro in vivo test battery, for example for pharmaceutical testing. Results obtained from in vitro experiments cannot usually be transposed, as is, to predict the reaction of an entire organism in vivo . Building a consistent and reliable extrapolation procedure from in vitro results to in vivo is therefore extremely important. Solutions include: These two approaches are not incompatible; better in vitro systems provide better data to mathematical models. However, increasingly sophisticated in vitro experiments collect increasingly numerous, complex, and challenging data to integrate. Mathematical models, such as systems biology models, are much needed here. In pharmacology, IVIVE can be used to approximate pharmacokinetics (PK) or pharmacodynamics (PD). Since

174-409: A clear daily cycle. In arrhythmic mice with clock-component dysfunctions, this rhythmicity disappears. Jet-lag and sleep deprivation can lead to the disruptions of the microbiome daily oscillations, but the changes are usually not dramatic. This interaction is bidirectional as the gut microbiota can also act on the hosts. For example, antibiotics can affect the rhythmic adherence of gut bacteria to

232-640: A competitive advantage over other species in the gut. Some bacteria are known to take hints from the host circadian clock in the form of melatonin . The disrupted gut microbiome has been proven to be related to a lot of diseases in humans gut microbiota . Thus, it is critical to our health to maintain a healthy gut microbiota. The host's circadian clock circadian rhythm controls the gut environment's ~24h cycle of many factors such as temperature changes, nutrients, certain hormones, bile acid levels, immune system functions. The relative abundances of some gut bacteria, such as Firmicutes and Bacteroidetes , display

290-480: A highly conserved eubacterial protein that shares sequence similarity with both RecA strand transferase and lon protease . The RadA/Sms family are probable ATP-dependent proteases involved in both DNA repair and degradation of proteins, peptides , glycopeptides . They are classified in as non-peptidase homologues and unassigned peptidases in MEROPS peptidase family S16 (lon protease family, clan SJ). RadA/Sms

348-468: A histidine kinase SasA and a phosphatase CikA that activate/inactivate the globally acting transcription factor RpaA. A contributing factor to the global transcription programs is rhythms of chromosomal topology in which the circadian clock orchestrates dramatic circadian changes in DNA topology that modulates changes in the transcription rates. The cyanobacterial circadian system is so far unique in that it

406-999: A more detailed or more convenient analysis than can be done with whole organisms; however, results obtained from in vitro experiments may not fully or accurately predict the effects on a whole organism. In contrast to in vitro experiments, in vivo studies are those conducted in living organisms, including humans, known as clinical trials, and whole plants. In vitro ( Latin for "in glass"; often not italicized in English usage ) studies are conducted using components of an organism that have been isolated from their usual biological surroundings, such as microorganisms, cells, or biological molecules. For example, microorganisms or cells can be studied in artificial culture media , and proteins can be examined in solutions . Colloquially called "test-tube experiments", these studies in biology, medicine, and their subdisciplines are traditionally done in test tubes, flasks, Petri dishes, etc. They now involve

464-481: A new steady status that is distinct from the original. The genus Turicibacter , proven to modulate the mood-related neurotransmitter serotonin, was found to overly recover. This effect may lower the serotonin level in the gut, connecting the gut microbiome to effects on the host's mental health. There are 4,616 bacterial species recognized in the human gut. Only 2 of them, Klebsiella aerogenes and Bacillus subtilis, are currently reported to have circadian clocks. It

522-690: A nucleus, prokaryotic cells must have a different mechanism of keeping circadian time. In 1998, Ishiura et al. determined that the KaiABC protein complex was responsible for the circadian negative feedback loop in Synechococcus by mapping 19 clock mutants to the genes for these three proteins.(3) An experiment by Nakajima et al., in 2005, was able to demonstrate the circadian oscillation of the Synechococcus KaiABC complex in vitro . They did this by adding KaiA , KaiB , KaiC , and ATP into

580-443: A peptide isomerization, thereby increasing the activation energy of ATP hydrolysis and slowing it to a 24 hour timescale. In the context of bacterial circadian rhythms , specifically in cyanobacteria , circadian advantage refers to the improved competitive advantage of strains of cyanobacteria that "resonate" with the environmental circadian rhythm . For example, consider a strain with a free-running period (FRP) of 24 hours that

638-505: A period that is as slow as 24 hours and yet still be so precise? One model is that the rate-limiting reaction that determines the period is the very slow rate of ATP hydrolysis by KaiC . KaiC hydrolyses ATP at the remarkably slow rate of only 15 ATP molecules per KaiC monomer per 24 hours. The rate of this ATPase activity is temperature compensated, and the activities of wild-type and period-mutant KaiC proteins are directly proportional to their in vivo circadian frequencies, suggesting that

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696-419: A period that is close to, but not exactly, 24 hours in duration, (b) this " free-running " rhythm is temperature compensated, and (c) the rhythm will entrain to an appropriate environmental cycle. Until the mid-1980s, it was thought that only eukaryotic cells had circadian rhythms. It is now known that cyanobacteria (a phylum of photosynthetic eubacteria ) have well-documented circadian rhythms that meet all

754-424: A rhythmic light/dark cycle (e.g., 12 hours of light alternating with 12 hours of darkness), whereas in "constant" environments (e.g., constant illumination) rhythmic and arhythmic strains grow at comparable rates. Among rhythmic strains with different periods, the strains whose endogenous period most closely matches the period of the environmental cycle is able to out-compete strains whose period does not match that of

812-402: A test tube in the approximate ratio recorded in vivo . They then measured the levels of KaiC phosphorylation and found that it demonstrated circadian rhythmicity for three cycles without damping. This cycle was also temperature compensating. They also tested incubating mutant KaiC protein with KaiA, KaiB, and ATP. They found that the period of KaiC phosphorylation matched the intrinsic period of

870-412: A well-documented circadian timekeeping mechanism is the cyanobacteria. Recent studies have suggested that there might be 24-hour timekeeping mechanisms among other prokaryotes. The purple non-sulfur bacterium Rhodopseudomonas palustris is one such example, as it harbors homologs of KaiB and KaiC and exhibits adaptive KaiC-dependent growth enhancement in 24-hour cyclic environments. However, R. palustris

928-644: Is co-cultured with a strain that has a free-running period (FRP) of 30 hours in a light-dark cycle of 12 hours light and 12 hours dark (LD 12:12). The strain that has a 24-hour FRP will out-compete the 30-hour strain over time under these LD 12:12 conditions. On the other hand, in a light-dark cycle of 15 hours light and 15 hours darkness, the 30-hour strain will out-compete the 24-hour strain. Moreover, rhythmic strains of cyanobacteria will out-compete arhythmic strains in 24-h light/dark cycles, but in continuous light, arhythmic strains are able to co-exist with wild-type cells in mixed cultures. The only prokaryotic group with

986-492: Is involved in recombination and recombinational repair, most likely involving the stabilisation or processing of branched DNA molecules or blocked replication forks because of its genetic redundancy with RecG and RuvABC. The overall fold of the KaiA monomer is that of a four- helix bundle, which forms a dimer in the known structure . KaiA functions as a homodimer . Each monomer is composed of three functional domains :

1044-426: Is mechanistically linked to the circadian clock appears to be due to clock triggering of a transcriptional cascade coupled to rhythmic changes in the topology of the entire cyanobacterial chromosome. The S. elongatus luciferase reporter system was used to screen for clock gene mutants, of which many were isolated. The figure shows a few of the many mutants that were discovered. These mutants were used to identify

1102-421: Is much simpler than models for eukaryotic circadian rhythm generators, the principles are largely the same. In both systems the circadian period is dependent on the interactions between proteins within the cell, and when the genes for those proteins are mutated, the expressed period changes. (1)(2) This model of circadian rhythm generation also has implications for the study of circadian “evolutionary biology”. Given

1160-487: Is some evidence of a circadian clock in Bacillus subtilis . Luciferase promoter assays showed gene expression patterns of ytvA, a gene encoding a blue light photoreceptor, that satisfied the criteria of a circadian clock. However, there has yet to be a robust demonstration of a clock in B. subtilis and the potential mechanisms of circadian gene regulation within B. subtilis remain unknown. Another interesting example

1218-592: Is suspected that other gut bacteria may have circadian clocks, too. In vitro In vitro (meaning in glass , or in the glass ) studies are performed with microorganisms , cells , or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology and its subdisciplines are traditionally done in labware such as test tubes, flasks, Petri dishes , and microtiter plates . Studies conducted using components of an organism that have been isolated from their usual biological surroundings permit

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1276-441: Is the case of the microbiome. It is possible that circadian clocks play a role in the gut microbiota behavior. These microorganisms experience daily changes because their hosts eat on a daily routine (consumption in the day for diurnal animals and in the night for nocturnal hosts). The presence of a daily timekeeper might allow gut bacteria to anticipate resources coming from the host temporally, thereby giving those species of bacteria

1334-598: Is the only circadian system in which the structures of full-length clock proteins have been solved. In fact, the structures of all three of the Kai proteins have been determined. KaiC forms a hexamer that resembles a double doughnut with a central pore that is partially sealed at one end. There are twelve ATP-binding sites in KaiC and the residues that are phosphorylated during the in vitro phosphorylation rhythm have been identified. KaiA has two major domains and forms dimers in which

1392-412: Is used in the clinic, it must progress through a series of in vivo trials to determine if it is safe and effective in intact organisms (typically small animals, primates, and humans in succession). Typically, most candidate drugs that are effective in vitro prove to be ineffective in vivo because of issues associated with delivery of the drug to the affected tissues, toxicity towards essential parts of

1450-475: The KaiA / KaiB / KaiC complexes to be visualized as a function of time, which enabled sophisticated mathematical modeling of the in vitro phosphorylation rhythm. Therefore, the cyanobacterial clock components and their interactions can be visualized in four dimensions (three in space, one in time). The temporal formation patterns of the KaiA / KaiB / KaiC complex have been elucidated, along with an interpretation of

1508-547: The N-terminal amplitude-amplifier domain, the central period-adjuster domain and the C-terminal clock-oscillator domain. The N-terminal domain of KaiA, from cyanobacteria, acts as a pseudo-receiver domain, but lacks the conserved aspartyl residue required for phosphotransfer in response regulators. The C-terminal domain is responsible for dimer formation, binding to KaiC, enhancing KaiC phosphorylation and generating

1566-545: The circadian oscillations . The KaiA protein from Anabaena sp. (strain PCC 7120) lacks the N-terminal CheY-like domain. KaiB adopts an alpha-beta meander motif and is found to be a dimer or a tetramer . KaiC belongs to a larger family of proteins ; it performs autophosphorylation and acts as its own transcriptional repressor . It binds ATP . Due to the lack of a nucleus in these organisms, there

1624-410: The fitness of organisms growing under natural conditions? Circadian clocks are assumed to enhance the fitness of organisms by improving their ability to predict and anticipate daily cycles in environmental factors. However, there have been few rigorous tests of this proposition in any organism. Cyanobacteria are one of the few organisms in which such a test has been performed. The adaptive fitness test

1682-530: The ATPase activity defines the circadian period. Therefore, some authors have proposed that the KaiC ATPase activity constitutes the most fundamental reaction underlying circadian periodicity in cyanobacteria. Structural analyses of the KaiC ATPase suggested that the slowness of this ATP hydrolysis arises from sequestration of a lytic water molecule in an unfavorable position and coupling of ATP hydrolysis to

1740-572: The N-terminal domains are "swapped" with the C-terminal domains. KaiB has been successfully crystallized from three different species of cyanobacteria and forms dimers or tetramers. The three-dimensional structures have been helpful in elucidating the cyanobacterial clock mechanism by providing concrete models for the ways in which the three Kai proteins interact and influence each other. The structural approaches have also allowed

1798-496: The case of multicellular organisms, organ systems. These myriad components interact with each other and with their environment in a way that processes food, removes waste, moves components to the correct location, and is responsive to signalling molecules, other organisms, light, sound, heat, taste, touch, and balance. This complexity makes it difficult to identify the interactions between individual components and to explore their basic biological functions. In vitro work simplifies

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1856-590: The circadian oscillation by modulating the magnesium binding in KaiC . While the KaiABC phosphorylation/complex cycle can explain key features of this biochemical circadian oscillator, especially how it can link to the output pathways that regulate global gene expression patterns, it does not provide an explanation for why the oscillator has a period of approximately 24 hours, nor how it can be "temperature compensated." Phosphorylation/dephosphorylation reactions and protein complex associations/dissassociations can be very rapid, so why does this biochemical oscillator have

1914-431: The commercial production of antibiotics and other pharmaceutical products. Viruses, which only replicate in living cells, are studied in the laboratory in cell or tissue culture, and many animal virologists refer to such work as being in vitro to distinguish it from in vivo work in whole animals. In vitro studies permit a species-specific, simpler, more convenient, and more detailed analysis than can be done with

1972-405: The core KaiA , KaiB , KaiC clock genes. At first, the cyanobacterial clockwork appeared to be a transcription and translation feedback loop in which clock proteins autoregulate the activity of their own promoters by a process that was similar in concept to the circadian clock loops of eukaryotes. Subsequently, however, several lines of evidence indicated that transcription and translation

2030-419: The core mechanism based on the cycle of KaiC phosphorylation patterns and the dynamics of the KaiA / KaiB / KaiC complex. (See the animation of the phsophorylation/complex cycle.) In addition, single-molecule methods (high-speed atomic force microscopy) have been applied to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on sub-second timescales. These interactions regulate

2088-585: The criteria of bona fide circadian rhythms. In these bacteria , three key proteins whose structures have been determined, KaiA , KaiB , and KaiC can form a molecular clockwork that orchestrates global gene expression. This system enhances the fitness of cyanobacteria in rhythmic environments. Before the mid-1980s, it was believed that only eukaryotes had circadian systems. In 1985–6, several research groups discovered that cyanobacteria display daily rhythms of nitrogen fixation in both light/dark (LD) cycles and in constant light. The group of Huang and co-workers

2146-411: The cyanobacterium with the corresponding mutant genome. These results led them to conclude that KaiC phosphorylation is the basis for circadian rhythm generation in Synechococcus. (2) Cyanobacteria are the simplest organisms that have been observed demonstrating circadian rhythms.(2)(3) The primitiveness and simplicity make the KaiC phosphorylation model invaluable to circadian rhythm research. While it

2204-451: The daily oscillations in luminescence of many individual cyanobacterial colonies on a petri dish; note the synchrony of rhythmicity among the various colonies. Despite predictions that circadian clocks would not be expressed by cells that are doubling faster than once per 24 hours, the cyanobacterial rhythms continue in cultures that are growing with doubling times as rapid as one division every 5–6 hours. Do circadian timekeepers enhance

2262-465: The environment. Similar results were later obtained in plants and mice. In eukaryotes, about 10–20% of the genes are rhythmically expressed (as gauged by rhythms of mRNA abundance). However, in cyanobacteria, a much larger percentage of genes are controlled by the circadian clock. For example, one study has shown that the activity of essentially all promoters in the genome are rhythmically regulated. The mechanism by which this global gene regulation

2320-418: The extrapolations. In the case of early effects or those without intercellular communications, the same cellular exposure concentration is assumed to cause the same effects, both qualitatively and quantitatively, in vitro and in vivo . In these conditions, developing a simple PD model of the dose–response relationship observed in vitro , and transposing it without changes to predict in vivo effects

2378-570: The full range of techniques used in molecular biology, such as the omics . In contrast, studies conducted in living beings (microorganisms, animals, humans, or whole plants) are called in vivo . Examples of in vitro studies include: the isolation, growth and identification of cells derived from multicellular organisms (in cell or tissue culture ); subcellular components (e.g. mitochondria or ribosomes ); cellular or subcellular extracts (e.g. wheat germ or reticulocyte extracts); purified molecules (such as proteins , DNA , or RNA ); and

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2436-432: The hosts’ rhythmic and arrhythmic feeding behaviors contributed differently to the recoveries of their gut microbiota from antibiotic treatment. Researchers found that rhythmic behavior after antibiotic ablation facilitates complete recovery of the gut microbiota. On the other hand, arrhythmic behavior after antibiotic ablation hinders the gut microbiota's proper recovery. Instead, this behavior promotes microbiota recovery to

2494-436: The immune system. Another advantage of in vitro methods is that human cells can be studied without "extrapolation" from an experimental animal's cellular response. In vitro methods can be miniaturized and automated, yielding high-throughput screening methods for testing molecules in pharmacology or toxicology. The primary disadvantage of in vitro experimental studies is that it may be challenging to extrapolate from

2552-412: The intestinal epithelium and in turn, rewire the hosts’ chromatin and transcription oscillations in the intestines and in the livers. Some of the current research in this field is focused on whether or not gut bacteria have intrinsic circadian rhythms. If so, researchers speculate that they may use their host's feeding patterns as zeitgebers . A long-term study on mice was conducted to determine whether

2610-673: The organism that were not represented in the initial in vitro studies, or other issues. A method which could help decrease animal testing is the use of in vitro batteries, where several in vitro assays are compiled to cover multiple endpoints. Within developmental neurotoxicity and reproductive toxicity there are hopes for test batteries to become easy screening methods for prioritization for which chemicals to be risk assessed and in which order. Within ecotoxicology in vitro test batteries are already in use for regulatory purpose and for toxicological evaluation of chemicals. In vitro tests can also be combined with in vivo testing to make

2668-519: The research of the aforementioned pioneers, the collaborative group of Takao Kondo , Carl H. Johnson , Susan Golden , and Masahiro Ishiura genetically transformed the cyanobacterium Synechococcus elongatus with a luciferase reporter that allowed rhythmic gene expression to be assayed non-invasively as rhythmically "glowing" cells. This system allowed an exquisitely precise circadian rhythm of luminescence to be measured from cell populations and even from single cyanobacterial cells. The figure shows

2726-514: The results of in vitro work back to the biology of the intact organism. Investigators doing in vitro work must be careful to avoid over-interpretation of their results, which can lead to erroneous conclusions about organismal and systems biology. For example, scientists developing a new viral drug to treat an infection with a pathogenic virus (e.g., HIV-1) may find that a candidate drug functions to prevent viral replication in an in vitro setting (typically cell culture). However, before this drug

2784-519: The simplicity of cyanobacteria and of this circadian system, it may be safe to assume that eukaryotic circadian oscillators are derived from a system similar to that present in cyanobacterium. (1) Bacterial circadian rhythms Bacterial circadian rhythms , like other circadian rhythms , are endogenous "biological clocks" that have the following three characteristics: (a) in constant conditions (i.e. constant temperature and either constant light {LL} or constant darkness {DD}) they oscillate with

2842-547: The system under study, so the investigator can focus on a small number of components. For example, the identity of proteins of the immune system (e.g. antibodies), and the mechanism by which they recognize and bind to foreign antigens would remain very obscure if not for the extensive use of in vitro work to isolate the proteins, identify the cells and genes that produce them, study the physical properties of their interaction with antigens, and identify how those interactions lead to cellular signals that activate other components of

2900-497: The timing and intensity of effects on a given target depend on the concentration time course of candidate drug (parent molecule or metabolites) at that target site, in vivo tissue and organ sensitivities can be completely different or even inverse of those observed on cells cultured and exposed in vitro . That indicates that extrapolating effects observed in vitro needs a quantitative model of in vivo PK. Physiologically based PK ( PBPK ) models are generally accepted to be central to

2958-434: The two groups oscillated with opposite phases. This led them to conclude that the Synechococcus sp. genome was regulated by a circadian clock. (1) The circadian oscillators in eukaryotes that have been studied function using a negative feedback loop in which proteins inhibit their own transcription in a cycle that takes approximately 24 hours. This is known as a transcription-translation-derived oscillator (TTO).(2) Without

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3016-434: The whole organism. Just as studies in whole animals more and more replace human trials, so are in vitro studies replacing studies in whole animals. Living organisms are extremely complex functional systems that are made up of, at a minimum, many tens of thousands of genes, protein molecules, RNA molecules, small organic compounds, inorganic ions, and complexes in an environment that is spatially organized by membranes, and in

3074-466: Was done by mixing cyanobacterial strains that express different circadian properties (i.e., rhythmicity vs. arhythmicity, different periods, etc.) and growing them in competition under different environmental conditions. The idea was to determine if having an appropriately functional clock system enhances fitness under competitive conditions. The result was that strains with a functioning biological clock out-compete arhythmic strains in environments that have

3132-428: Was doubt as to whether or not cyanobacteria would be able to express circadian rhythms. Kondo et al. were the first to definitively demonstrate that cyanobacteria do in fact have circadian rhythms. In a 1993 experiment, they used a luciferase reporter inserted into the genetically tractable Synechococcus sp., which was grown in a 12:12 light-dark cycle to ensure “entrainment”. There were two sets of bacteria so that one

3190-416: Was in light while the other was in darkness during this entrainment period. Once the bacteria entered the stationary phase, they were transferred into test tubes kept in constant light, except for 5-minute recording periods every 30 minutes, in which the tubes were kept in darkness to measure their levels of bioluminescence . They found that the level of bioluminescence cycled at a near 24-hour period, and that

3248-406: Was not necessary for circadian rhythms of Kai proteins, the most spectacular being that the three purified Kai proteins can reconstitute a temperature-compensated circadian oscillation in a test tube. In vivo, the output of this biochemical KaiABC oscillator to rhythms of gene expression appears to be mediated by KaiC phosphorylation status (see below) regulating a biochemical cascade involving

3306-417: Was reported to show a poor intrinsic free-running rhythm of nitrogen fixation under constant conditions. The lack of rhythm in R. palustris in constant conditions has implications for the adaptive value of intrinsic timekeeping mechanism. Therefore, the R. palustris system was proposed as a "proto" circadian timekeeper that exhibit some parts of circadian systems (kaiB and kaiC homologs), but not all. There

3364-413: Was the first to recognize clearly that the cyanobacterium Synechococcus sp. RF-1 was exhibiting circadian rhythms, and in a series of publications beginning in 1986 demonstrated all three of the salient characteristics of circadian rhythms described above in the same organism, the unicellular freshwater Synechococcus sp. RF-1. Another ground-breaking study was that of Sweeney and Borgese. Inspired by

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