Ilka M. Axmann

Genome sequence of the cyanobacterium Prochlorococcus marinus SS120, a nearly minimal oxyphototrophic genome

Prochlorococcus marinus, the dominant photosynthetic organism in the ocean, is found in two main ecological forms: high-light-adapted genotypes in the upper part of the water column and low-light-adapted genotypes at the bottom of the illuminated layer. P. marinus SS120, the complete genome sequence reported here, is an extremely low-light-adapted form. The genome of P. marinus SS120 is composed of a single circular chromosome of 1,751,080 bp with an average G+C content of 36.4%. It contains 1,884 predicted protein-coding genes with an average size of 825 bp, a single rRNA operon, and 40 tRNA genes. Together with the 1.66-Mbp genome of P. marinus MED4, the genome of P. marinus SS120 is one of the two smallest genomes of a photosynthetic organism known to date. It lacks many genes that are involved in photosynthesis, DNA repair, solute uptake, intermediary metabolism, motility, phototaxis, and other functions that are conserved among other cyanobacteria. Systems of signal transduction and environmental stress response show a particularly drastic reduction in the number of components, even taking into account the small size of the SS120 genome. In contrast, housekeeping genes, which encode enzymes of amino acid, nucleotide, cofactor, and cell wall biosynthesis, are all present. Because of its remarkable compactness, the genome of P. marinus SS120 might approximate the minimal gene complement of a photosynthetic organism.

Identification of cyanobacterial non-coding RNAs by comparative genome analysis

BackgroundWhole 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.ResultsBased 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.ConclusionThis 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.

Functioning and robustness of a bacterial circadian clock

Cyanobacteria are the simplest known cellular systems that regulate their biological activities in daily cycles. For the cyanobacterium Synechococcus elongatus, it has been shown by in vitro and in vivo experiments that the basic circadian timing process is based on rhythmic phosphorylation of KaiC hexamers. Despite the excellent experimental work, a full systems level understanding of the in vitro clock is still lacking. In this work, we provide a mathematical approach to scan different hypothetical mechanisms for the primary circadian oscillator, starting from experimentally established molecular properties of the clock proteins. Although optimised for highest performance, only one of the in silico‐generated reaction networks was able to reproduce the experimentally found high amplitude and robustness against perturbations. In this reaction network, a negative feedback synchronises the phosphorylation level of the individual hexamers and has indeed been realised in S. elongatus by KaiA sequestration as confirmed by experiments.

The diversity of cyanobacterial metabolism: genome analysis of multiple phototrophic microorganisms

BackgroundCyanobacteria 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 suns 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.ResultsHere, 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.ConclusionsOur 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.

Small RNAs Establish Delays and Temporal Thresholds in Gene Expression

Noncoding RNAs are crucial regulators of gene expression in prokaryotes and eukaryotes, but how they affect the dynamics of transcriptional networks remains poorly understood. We analyzed the temporal characteristics of the cyanobacterial iron stress response by mathematical modeling and quantitative experimental analyses and focused on the role of a recently discovered small noncoding RNA, IsrR. We found that IsrR is responsible for a pronounced delay in the accumulation of isiA mRNA encoding the late-phase stress protein, IsiA, and that it ensures a rapid decline in isiA levels once external stress triggers are removed. These kinetic properties allow the system to selectively respond to sustained (as opposed to transient) stimuli and thus establish a temporal threshold, which prevents energetically costly IsiA accumulation under short-term stress conditions. Biological information is frequently encoded in the quantitative aspects of intracellular signals (e.g., amplitude and duration). Our simulations reveal that competitive inhibition and regulated degradation allow intracellular regulatory networks to efficiently discriminate between transient and sustained inputs.

A sequestration feedback determines dynamics and temperature entrainment of the KaiABC circadian clock.

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.

Daily Rhythms in the Cyanobacterium Synechococcus elongatus Probed by High-resolution Mass Spectrometry–based Proteomics Reveals a Small Defined Set of Cyclic Proteins

Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi, and several bacteria. The central mechanism behind these “pacemakers” and the connection to the circadian regulated pathways are still poorly understood. The circadian rhythm of the cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is highly robust and controlled by only three proteins, KaiA, KaiB, and KaiC. This central clock system has been extensively studied functionally and structurally and can be reconstituted in vitro. These characteristics, together with a relatively small genome (2.7 Mbp), make S. elongatus an ideal model system for the study of circadian rhythms. Different approaches have been used to reveal the influence of the central S. elongatus clock on rhythmic gene expression, rhythmic mRNA abundance, rhythmic DNA topology changes, and cell division. However, a global analysis of its proteome dynamics has not been reported yet. To uncover the variation in protein abundances during 48 h under light and dark cycles (12:12 h), we used quantitative proteomics, with TMT 6-plex isobaric labeling. We queried the S. elongatus proteome at 10 different time points spanning a single 24-h period, leading to 20 time points over the full 48-h period. Employing multidimensional separation and high-resolution mass spectrometry, we were able to find evidence for a total of 82% of the S. elongatus proteome. Of the 1537 proteins quantified over the time course of the experiment, only 77 underwent significant cyclic variations. Interestingly, our data provide evidence for in- and out-of-phase correlation between mRNA and protein levels for a set of specific genes and proteins. As a range of cyclic proteins are functionally not well annotated, this work provides a resource for further studies to explore the role of these proteins in the cyanobacterial circadian rhythm.

Biochemical Evidence for a Timing Mechanism in Prochlorococcus

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.

Insight into cyanobacterial circadian timing from structural details of the KaiB–KaiC interaction

Significance The Kai system is a widely studied model in theoretical biology and systems biology. It is to date the only known circadian clock that can be reconstituted in vitro. Essential to the rhythmicity of the system is the formation of the KaiC–KaiB complex. Many aspects of this interaction, such as the mode of binding of KaiB, the stoichiometry of the interaction, and the exact binding interfaces have long remained ambiguous. We present a mass spectrometry-based structural model of the KaiC–KaiB interaction that answers many of these outstanding questions on the basis of direct experimental evidence. This structural model sheds light on the intricate workings of the in vitro oscillator. Circadian timing in cyanobacteria is determined by the Kai system consisting of KaiA, KaiB, and KaiC. Interactions between Kai proteins change the phosphorylation status of KaiC, defining the phase of circadian timing. The KaiC–KaiB interaction is crucial for the circadian rhythm to enter the dephosphorylation phase but it is not well understood. Using mass spectrometry to characterize Kai complexes, we found that KaiB forms monomers, dimers, and tetramers. The monomer is the unit that interacts with KaiC, with six KaiB monomers binding to one KaiC hexamer. Hydrogen–deuterium exchange MS reveals structural changes in KaiC upon binding of KaiB in both the CI and CII domains, showing allosteric coupling upon KaiB binding. Based on this information we propose a model of the KaiB–KaiC complex and hypothesize that the allosteric changes observed upon complex formation relate to coupling KaiC ATPase activity with KaiB binding and to sequestration of KaiA dimers into KaiCBA complexes.

Diversity of KaiC-based timing systems in marine Cyanobacteria.

The coordination of biological activities into daily cycles provides an important advantage for the fitness of diverse organisms. Most eukaryotes possess an internal clock ticking with a periodicity of about one day to anticipate sunrise and sunset. The 24-hour period of the free-running rhythm is highly robust against many changes in the natural environment. Among prokaryotes, only Cyanobacteria are known to harbor such a circadian clock. Its core oscillator consists of just three proteins, KaiA, KaiB, and KaiC that produce 24-hour oscillations of KaiC phosphorylation, even in vitro. This unique three-protein oscillator is well documented for the freshwater cyanobacterium Synechococcus elongatus PCC 7942. Several physiological studies demonstrate a circadian clock also for other Cyanobacteria including marine species. Genes for the core clock components are present in nearly all marine cyanobacterial species, though there are large differences in the specific composition of these genes. In the first section of this review we summarize data on the model circadian clock from S. elongatus PCC 7942 and compare it to the reduced clock system of the marine cyanobacterium Prochlorococcus marinus MED4. In the second part we discuss the diversity of timing mechanisms in other marine Cyanobacteria with regard to the presence or absence of different components of the clock.