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author:("ammann, ilk M")
1.  Revealing a Two-Loop Transcriptional Feedback Mechanism in the Cyanobacterial Circadian Clock 
PLoS Computational Biology  2013;9(3):e1002966.
Molecular genetic studies in the circadian model organism Synechococcus have revealed that the KaiC protein, the central component of the circadian clock in cyanobacteria, is involved in activation and repression of its own gene transcription. During 24 hours, KaiC hexamers run through different phospho-states during daytime. So far, it has remained unclear which phospho-state of KaiC promotes kaiBC expression and which opposes transcriptional activation. We systematically analyzed various combinations of positive and negative transcriptional feedback regulation by introducing a combined TTFL/PTO model consisting of our previous post-translational oscillator that considers all four phospho-states of KaiC and a transcriptional/translational feedback loop. Only a particular two-loop feedback mechanism out of 32 we have extensively tested is able to reproduce existing experimental observations, including the effects of knockout or overexpression of kai genes. Here, threonine and double phosphorylated KaiC hexamers activate and unphosphorylated KaiC hexamers suppress kaiBC transcription. Our model simulations suggest that the peak expression ratio of the positive and the negative component of kaiBC expression is the main factor for how the different two-loop feedback models respond to removal or to overexpression of kai genes. We discuss parallels between our proposed TTFL/PTO model and two-loop feedback structures found in the mammalian clock.
Author Summary
Many organisms possess a true circadian clock and coordinate their activities into daily cycles. Among the simplest organisms harboring such a 24 h-clock are cyanobacteria. Interactions among three proteins, KaiA, KaiB, KaiC, and cyclic KaiC phosphorylation govern the daily rhythm from gene expression to metabolism. Thus, the control of the kaiBC gene cluster expression is important for regulating the cyanobacterial clockwork. A picture has emerged in which different KaiC phospho-states activate and inhibit kaiBC expression. However, the mechanism remains to be solved. Here, we investigated the impact of each KaiC phospho-state on kaiBC expression by introducing a model that combines the circadian transcription/translation rhythm with the KaiABC-protein oscillator. We tested 32 combinations of positive and negative transcriptional regulation. It turns out that the kaiBC expression and KaiC phosphorylation dynamics in wild type and kai mutants can only be described by one mechanism: threonine and double phosphorylated KaiC hexamers activate kaiBC expression and the unphosphorylated state suppresses it. Further, we propose that the activator-to-repressor abundance ratio very likely determines the kaiBC expression dynamics in the simulated kai mutants. Our suggested clock model can be extended by further kinetic mechanisms to gain deeper insights into the various underlying processes of circadian gene regulation.
doi:10.1371/journal.pcbi.1002966
PMCID: PMC3597532  PMID: 23516349
2.  The diversity of cyanobacterial metabolism: genome analysis of multiple phototrophic microorganisms 
BMC Genomics  2012;13:56.
Background
Cyanobacteria are among the most abundant organisms on Earth and represent one of the oldest and most widespread clades known in modern phylogenetics. As the only known prokaryotes capable of oxygenic photosynthesis, cyanobacteria are considered to be a promising resource for renewable fuels and natural products. Our efforts to harness the sun's energy using cyanobacteria would greatly benefit from an increased understanding of the genomic diversity across multiple cyanobacterial strains. In this respect, the advent of novel sequencing techniques and the availability of several cyanobacterial genomes offers new opportunities for understanding microbial diversity and metabolic organization and evolution in diverse environments.
Results
Here, we report a whole genome comparison of multiple phototrophic cyanobacteria. We describe genetic diversity found within cyanobacterial genomes, specifically with respect to metabolic functionality. Our results are based on pair-wise comparison of protein sequences and concomitant construction of clusters of likely ortholog genes. We differentiate between core, shared and unique genes and show that the majority of genes are associated with a single genome. In contrast, genes with metabolic function are strongly overrepresented within the core genome that is common to all considered strains. The analysis of metabolic diversity within core carbon metabolism reveals parts of the metabolic networks that are highly conserved, as well as highly fragmented pathways.
Conclusions
Our results have direct implications for resource allocation and further sequencing projects. It can be extrapolated that the number of newly identified genes still significantly increases with increasing number of new sequenced genomes. Furthermore, genome analysis of multiple phototrophic strains allows us to obtain a detailed picture of metabolic diversity that can serve as a starting point for biotechnological applications and automated metabolic reconstructions.
doi:10.1186/1471-2164-13-56
PMCID: PMC3369817  PMID: 22300633
3.  A sequestration feedback determines dynamics and temperature entrainment of the KaiABC circadian clock 
The circadian KaiABC oscillator is driven by a sequestration feedback, which is biochemically realized by a strong affinity of KaiA to exclusively serine phosphorylated KaiBC complexes.A highly non-linear feedback model explains the time courses of the phosphorylation states and the robustness under concerted changes of all Kai proteins.Native mass spectrometry reveals the existence of two KaiA binding sites on KaiC, confirming the theoretical predictions.Temperature entrainment arises from a temperature-dependent change in the abundance of KaiAC and KaiBC complexes.
The circadian rhythm of the cyanobacterium Synechococcus elongatus is controlled by three proteins, KaiA, KaiB, and KaiC. In a test tube, these proteins form complexes of various stoichiometry and the average phosphorylation level of KaiC exhibits robust circadian oscillations in the presence of ATP (Nakajima et al, 2005). Although the three component oscillator is apparently simple, it is highly precise and shows in-phase oscillations over several days (Mihalcescu et al, 2004). If we assume that cyanobacteria gain an evolutionary advantage from predicting the time of maximal sunlight intensity in a very reliable way, a highly robust but entrainable oscillator is likely the optimal solution. To elucidate the mechanism of the opposing properties of the clockwork—robustness and tenability—a mathematical modeling approach is necessary. The model must account for both the measured invariance of the phosphorylation level under concerted concentration changes of all Kai proteins (Kageyama et al, 2006) and for the experimentally observed temperature entrainment (Yoshida et al, 2009).
Here, we show by mathematical modeling, in combination with the measurements of the KaiABC complex formation dynamics from native mass spectrometry, that oscillations in the Kai system are a consequence of KaiA sequestration by KaiC hexamers and KaiBC complexes. Our in vitro model includes the characteristic KaiC phospho-form cycle, originating from two KaiC residues, serine (431) and threonine (432) (Nishiwaki et al, 2000). We allow for three pools that represent different forms of the KaiC complex. Together with the four phosphorylation states of KaiC, this gives a 3 × 4-dimensional model. We used data from the time course experiments of the O'Shea laboratory (Rust et al, 2007) to determine the unknown parameters of the KaiABC system. A global optimization algorithm is used to scan a large range of parameter values, resulting in a mathematical quantitative model of the KaiABC clockwork, see Figure 5. The KaiB response experiment (Rust et al, 2007) turned out to be crucial for identifying the mechanism by which individual KaiC hexamers can be synchronized in their phosphorylation dynamics. Importantly, the dephosphorylation phase can only be explained by a highly non-linear dependency of the KaiBC complex formation on the actual phosphorylation state. This can be realized by allowing only KaiBC complexes with exclusively serine phosphorylated KaiC, [S–KaiBC]6, to inactivate KaiA with a high efficiency.
This theoretical prediction is confirmed by native mass spectrometry, generating semi-quantitative time courses of the KaiABC complex formation dynamics. Our experiments show the existence of two different KaiC binding sites to KaiA. The constant sequestration of free KaiA by the KaiA2C6 complexes is the molecular realization of the dynamic invariance condition because it requires most of the KaiA to be inactive at every instant of time, regardless of the phosphorylation state. The second binding site KaiA4C6 reflects the KaiA-binding domain at the catalytic active center of the KaiC hexamer. This hypothesis is confirmed by comparison of the mass spectrometry signal for KaiA4C6 with predictions from the mathematical model (Figure 7A and B). In the late phosphorylation phase, KaiBC complexes rapidly build up and sequestrate KaiA (Figure 7C), which represent an additional binding site. The relative amount of KaiA6B6C6 confirms the sequestration hypotheses and corresponds to the theoretically estimated amount of sequestrated KaiA. The time of maximum sequestration—as defined by the appearance of the largest observed sequestration complex KaiA10B6C6 (Figure 7E)—agrees with the theoretical expected sequestration maximum (Figure 7F) where [S-KaiBC]6 is maximal.
To test further the predictive power of the mathematical model, we reproduced the observed phase synchronization dynamics on entrainment by temperature cycles. The response to a sudden temperature change results in a phase shift of phospho-KaiC, whereas the circadian period does not show any temperature dependency within a physiologically relevant range (Yoshida et al, 2009). As phosphorylation and dephosphorylation dynamics of KaiC alone and incubated with KaiA do not show significant temperature dependence (Tomita et al, 2005), phase entrainment is likely a consequence of temperature-induced changes in binding constants associated with the various KaiABC complexes. From thermodynamic arguments, we expect that an increasing temperature will enhance dissociation of KaiA and KaiB from KaiC. Indeed, a reduction in the net complex formation rate for S-KaiBC, D-KaiBC, and for KaiAC on temperature increase results in the experimentally observed differences in phase response, which compensate during the circadian cycle to assure temperature invariance of the ciracadian period.
The circadian rhythm of the cyanobacterium Synechococcus elongatus is controlled by three proteins, KaiA, KaiB, and KaiC. In a test tube, these proteins form complexes of various stoichiometry and the average phosphorylation level of KaiC exhibits robust circadian oscillations in the presence of ATP. Using mathematical modeling, we were able to reproduce quantitatively the experimentally observed phosphorylation dynamics of the KaiABC clockwork in vitro. We thereby identified a highly non-linear feedback loop through KaiA inactivation as the key synchronization mechanism of KaiC phosphorylation. By using the novel method of native mass spectrometry, we confirm the theoretically predicted complex formation dynamics and show that inactivation of KaiA is a consequence of sequestration by KaiC hexamers and KaiBC complexes. To test further the predictive power of the mathematical model, we reproduced the observed phase synchronization dynamics on entrainment by temperature cycles. Our model gives strong evidence that the underlying entrainment mechanism arises from a temperature-dependent change in the abundance of KaiAC and KaiBC complexes.
doi:10.1038/msb.2010.44
PMCID: PMC2925524  PMID: 20631683
biological network modeling; circadian clock; cyanobacteria; native mass spectrometry; temperature entrainment
4.  Biochemical Evidence for a Timing Mechanism in Prochlorococcus▿ †  
Journal of Bacteriology  2009;191(17):5342-5347.
Organisms coordinate biological activities into daily cycles using an internal circadian clock. The circadian oscillator proteins KaiA, KaiB, and KaiC are widely believed to underlie 24-h oscillations of gene expression in cyanobacteria. However, a group of very abundant cyanobacteria, namely, marine Prochlorococcus species, lost the third oscillator component, KaiA, during evolution. We demonstrate here that the remaining Kai proteins fulfill their known biochemical functions, although KaiC is hyperphosphorylated by default in this system. These data provide biochemical support for the observed evolutionary reduction of the clock locus in Prochlorococcus and are consistent with a model in which a mechanism that is less robust than the well-characterized KaiABC protein clock of Synechococcus is sufficient for biological timing in the very stable environment that Prochlorococcus inhabits.
doi:10.1128/JB.00419-09
PMCID: PMC2725622  PMID: 19502405
5.  A motif-based search in bacterial genomes identifies the ortholog of the small RNA Yfr1 in all lineages of cyanobacteria 
BMC Genomics  2007;8:375.
Background
Non-coding RNAs (ncRNA) are regulators of gene expression in all domains of life. They control growth and differentiation, virulence, motility and various stress responses. The identification of ncRNAs can be a tedious process due to the heterogeneous nature of this molecule class and the missing sequence similarity of orthologs, even among closely related species. The small ncRNA Yfr1 has previously been found in the Prochlorococcus/Synechococcus group of marine cyanobacteria.
Results
Here we show that screening available genome sequences based on an RNA motif and followed by experimental analysis works successfully in detecting this RNA in all lineages of cyanobacteria. Yfr1 is an abundant ncRNA between 54 and 69 nt in size that is ubiquitous for cyanobacteria except for two low light-adapted strains of Prochlorococcus, MIT 9211 and SS120, in which it must have been lost secondarily. Yfr1 consists of two predicted stem-loop elements separated by an unpaired sequence of 16–20 nucleotides containing the ultraconserved undecanucleotide 5'-ACUCCUCACAC-3'.
Conclusion
Starting with an ncRNA previously found in a narrow group of cyanobacteria only, we show here the highly specific and sensitive identification of its homologs within all lineages of cyanobacteria, whereas it was not detected within the genome sequences of E. coli and of 7 other eubacteria belonging to the alpha-proteobacteria, chlorobiaceae and spirochaete. The integration of RNA motif prediction into computational pipelines for the detection of ncRNAs in bacteria appears as a promising step to improve the quality of such predictions.
doi:10.1186/1471-2164-8-375
PMCID: PMC2190773  PMID: 17941988
6.  Identification of cyanobacterial non-coding RNAs by comparative genome analysis 
Genome Biology  2005;6(9):R73.
The first genome-wide and systematic screen for non-coding RNAs (ncRNAs) in cyanobacteria. Several ncRNAs were computationally predicted and their presence was biochemically verified. These ncRNAs may have regulatory functions, and each shows a distinct phylogenetic distribution.
Background
Whole genome sequencing of marine cyanobacteria has revealed an unprecedented degree of genomic variation and streamlining. With a size of 1.66 megabase-pairs, Prochlorococcus sp. MED4 has the most compact of these genomes and it is enigmatic how the few identified regulatory proteins efficiently sustain the lifestyle of an ecologically successful marine microorganism. Small non-coding RNAs (ncRNAs) control a plethora of processes in eukaryotes as well as in bacteria; however, systematic searches for ncRNAs are still lacking for most eubacterial phyla outside the enterobacteria.
Results
Based on a computational prediction we show the presence of several ncRNAs (cyanobacterial functional RNA or Yfr) in several different cyanobacteria of the Prochlorococcus-Synechococcus lineage. Some ncRNA genes are present only in two or three of the four strains investigated, whereas the RNAs Yfr2 through Yfr5 are structurally highly related and are encoded by a rapidly evolving gene family as their genes exist in different copy numbers and at different sites in the four investigated genomes. One ncRNA, Yfr7, is present in at least seven other cyanobacteria. In addition, control elements for several ribosomal operons were predicted as well as riboswitches for thiamine pyrophosphate and cobalamin.
Conclusion
This is the first genome-wide and systematic screen for ncRNAs in cyanobacteria. Several ncRNAs were both computationally predicted and their presence was biochemically verified. These RNAs may have regulatory functions and each shows a distinct phylogenetic distribution. Our approach can be applied to any group of microorganisms for which more than one total genome sequence is available for comparative analysis.
doi:10.1186/gb-2005-6-9-r73
PMCID: PMC1242208  PMID: 16168080
7.  Experimental and computational analysis of transcriptional start sites in the cyanobacterium Prochlorococcus MED4 
Nucleic Acids Research  2003;31(11):2890-2899.
In contrast to certain model eubacteria, little is known as to where transcription is initiated in the genomes of cyanobacteria, which are largely distinct from other prokaryotes. In this work, 25 transcription start sites (TSS) of 21 different genes of Prochlorococcus sp. MED4 were determined experimentally. The data suggest more than one TSS for the genes ftsZ, petH, psbD and ntcA. In contrast, the rbcL-rbcS operon encoding ribulose 1,5-bisphosphate carboxylase/oxygenase lacks a detectable promoter and is co-transcribed with the upstream located gene ccmK. The entire set of experimental data was used in a genome-wide scan for putative TSS in Prochlorococcus. A –10 element could be defined, whereas at the –35 position there was no element common to all investigated sequences. However, splitting the data set into sub-classes revealed different types of putative –35 boxes. Only one of them resembled the consensus sequence TTGACA recognized by the vegetative σ factor (σ70) of enterobacteria. Using a scoring matrix of the –10 element, more than 3000 TSS were predicted, about 40% of which were estimated to be functional. This is the first systematic study of transcription initiation sites in a cyanobacterium.
PMCID: PMC156731  PMID: 12771216

Results 1-7 (7)