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Susan S Golden

Publications and source records attributed to Susan S Golden.

At least 19 recordsLinked to original sources
PubMed2006

Quinone sensing by the circadian input kinase of the cyanobacterial circadian clock.

Circadian rhythms are endogenous cellular programs that time metabolic and behavioral events to occur at optimal times in the daily cycle. Light and dark cycles synchronize the endogenous clock with the external environment through a process called entrainment. Previously, we identified the bacteriophytochrome-like circadian input kinase CikA as a key factor for entraining the clock in the cyanobacterium Synechococcus elongatus PCC 7942. Here, we present evidence that CikA senses not light but rather the redox state of the plastoquinone pool, which, in photosynthetic organisms, varies as a function of the light environment. Furthermore, CikA associates with the Kai proteins of the circadian oscillator, and it influences the phosphorylation state of KaiC during resetting of circadian phase by a dark pulse. The abundance of CikA varies inversely with light intensity, and its stability decreases in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The pseudo-receiver domain of CikA is crucial for sensitivity to DBMIB, and it binds the quinone directly, a demonstration of a previously unrecognized ligand-binding role for the receiver fold. Our results suggest that resetting the clock in S. elongatus is metabolism-dependent and that it is accomplished through the interaction of the circadian oscillator with CikA.

Bacterial Proteins↗
PubMed2006

The pseudo-receiver domain of CikA regulates the cyanobacterial circadian input pathway.

CikA (circadian input kinase) is a component of the cyanobacterial circadian clock that aids in synchronizing the endogenous oscillator with the external environment. cikA mutants of the prokaryotic circadian model organism Synechococcus elongatus PCC 7942 fail to reset the phase of the circadian rhythm of gene expression after an environmental time cue, and also exhibit reduced amplitude and shortened period of circadian oscillation. CikA has histidine protein kinase (HPK) activity that is modulated in vitro by GAF and pseudo-receiver (PsR) domains. Here we show that the PsR domain negatively regulates HPK activity in vivo and also serves as an interaction module to dock CikA at a specific subcellular location. Phenotypes conferred by alleles that encode CikA variants showed that all domains except the featureless N-terminus are required for CikA function. Overexpression of all alleles that encode the PsR domain, whether or not the HPK is functional, caused a dominant arrhythmic phenotype, whereas overexpressed variants that lack PsR did not. Subcellular localization of intact CikA identified a polar focus whereas a variant without PsR showed uniform distribution in the cell, consistent with a model in which PsR mediates interaction with other input pathway components.

Bacterial Proteins↗
PubMed2005

LdpA: a component of the circadian clock senses redox state of the cell.

The endogenous 24-h (circadian) rhythms exhibited by the cyanobacterium Synechococcus elongatus PCC 7942 and other organisms are entrained by a variety of environmental factors. In cyanobacteria, the mechanism that transduces environmental input signals to the central oscillator of the clock is not known. An earlier study identified ldpA as a gene involved in light-dependent modulation of the circadian period, and a candidate member of a clock-entraining input pathway. Here, we report that the LdpA protein is sensitive to the redox state of the cell and exhibits electron paramagnetic resonance spectra consistent with the presence of two Fe4S4 clusters. Moreover, LdpA copurifies with proteins previously shown to be integral parts of the circadian mechanism. We also demonstrate that LdpA affects both the absolute level and light-dependent variation in abundance of CikA, a key input pathway component. The data suggest a novel input pathway to the circadian oscillator in which LdpA is a component of the clock protein complex that senses the redox state of a cell.

Bacterial Proteins↗
PubMed2005

Circadian rhythms from multiple oscillators: lessons from diverse organisms.

The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.

Animals↗
PubMed2005

High-throughput functional analysis of the Synechococcus elongatus PCC 7942 genome.

Synechococcus elongatus PCC 7942 was the first cyanobacterial strain to be reliably transformed by exogenously added DNA and has become the model organism for cyanobacterial circadian rhythms. With a small genome (2.7 Mb) and well-developed genetic tools, PCC 7942 provides an exceptional opportunity to elucidate the circadian mechanism through genetics. We describe a project to create mutations in every locus of the genome, both to assay each locus for its potential contribution to the circadian clock and to archive data for the cyanobacterial community. Cosmid clones that carry inserts of PCC 7942 DNA are saturated with transposon insertions in vitro to provide sequencing templates and substrates for mutagenesis of the PCC 7942 genome via homologous recombination. We have mutagenized 53% of the chromosome from 50 chromosome-bearing cosmids and identified the positions of insertions in 31 of those cosmids and the 46 kb plasmid, pANL. PCC 7942 mutants defective for 490 different genes have been screened for circadian phenotypes. Mutagenesis of three apparently essential loci, including clpPIIclpX, resulted in circadian phenotypes. We developed an effective antisense suppression method to further the analysis of essential genes. When completed, the set of comprehensive mutations will provide the community with a unique resource whose impact will extend beyond circadian research.

Chromosomes, Bacterial↗
PubMed2005

Stability of the Synechococcus elongatus PCC 7942 circadian clock under directed anti-phase expression of the kai genes.

The kaiA, kaiB and kaiC genes encode the core components of the cyanobacterial circadian clock in Synechococcus elongatus PCC 7942. Rhythmic expression patterns of kaiA and of the kaiBC operon normally peak in synchrony. In some mutants the relative timing of peaks (phase relationship) between these transcription units is altered, but circadian rhythms persist robustly. In this study, the importance of the transcriptional timing of kai genes was examined. Expressing either kaiA or kaiBC from a heterologous promoter whose peak expression occurs 12 h out of phase from the norm, and thus 12 h out of phase from the other kai locus, did not affect the time required for one cycle (period) or phase of the circadian rhythm, as measured by bioluminescence reporters. Furthermore, the data confirm that specific cis elements within the promoters of the kai genes are not necessary to sustain clock function.

Bacterial Proteins↗
PubMed2005

Diel infection of a cyanobacterium by a contractile bacteriophage.

Light was found to strongly influence the infection of a freshwater cyanobacterium (Synechococcus elongatus PCC 7942) by a contractile DNA phage named AS-1. Phage progeny production was correlated with the amount of light in the laboratory and occurred in a diel pattern under natural light. At least one effect of light on AS-1 infection is at the level of adsorption.

Adsorption↗
PubMed2004

Structure of the N-terminal domain of the circadian clock-associated histidine kinase SasA.

Circadian oscillators are endogenous biological systems that generate the approximately 24 hour temporal pattern of biological processes and confer a reproductive fitness advantage to their hosts. The cyanobacterial clock is the simplest known and the only clock system for which structural information for core component proteins, in this case KaiA, KaiB and KaiC, is available. SasA, a clock-associated histidine kinase, is necessary for robustness of the circadian rhythm of gene expression and implicated in clock output. The N-terminal domain of SasA (N-SasA) interacts directly with KaiC and likely functions as the sensory domain controlling the SasA histidine kinase activity. N-SasA and KaiB share significant sequence similarity and, thus, it has been proposed that they would be structurally similar and may even compete for KaiC binding. Here, we report the NMR structure of N-SasA and show it to be different from that of KaiB. The structural comparisons provide no clear details to suggest competition of SasA and KaiB for KaiC binding. N-SasA adopts a canonical thioredoxin fold but lacks the catalytic cysteine residues. A patch of conserved, solvent-exposed residues is found near the canonical thioredoxin active site. We suggest that this surface is used by N-SasA for protein-protein interactions. Our analysis suggests that the structural differences between N-SasA and KaiB are the result of only a few critical amino acid substitutions.

Amino Acid Sequence↗
PubMed2004

NMR structure of the KaiC-interacting C-terminal domain of KaiA, a circadian clock protein: implications for KaiA-KaiC interaction.

KaiA is a two-domain circadian clock protein in cyanobacteria, acting as the positive element in a feedback loop that sustains the oscillation. The structure of the N-terminal domain of KaiA is that of a pseudo-receiver, similar to those of bacterial response regulators, which likely interacts with components of the clock-resetting pathway. The C-terminal domain of KaiA is highly conserved among cyanobacteria and enhances the autokinase activity of KaiC. Here we present the NMR structure of the C-terminal domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. This domain adopts a novel all alpha-helical homodimeric structure. Several mutations known to affect the period of the circadian oscillator are shown to be located at an exposed groove near the dimer interface. This NMR structure and a 21-A-resolution electron microscopy structure of the hexameric KaiC particle allow us to postulate a mode of KaiA-KaiC interaction, in which KaiA binds a linker region connecting two globular KaiC domains.

Bacterial Proteins↗
PubMed2004

PsfR, a factor that stimulates psbAI expression in the cyanobacterium Synechococcus elongatus PCC 7942.

In this paper a gene (psfR) is reported that regulates psbAI activity in Synechococcus elongatus, a unicellular photoautotrophic cyanobacterium that carries out oxygenic (plant-type) photosynthesis and exhibits global circadian regulation of gene expression. In S. elongatus, a family of three psbA genes encodes the D1 protein of the photosystem II reaction centre. Overexpression of psfR results in increased expression of psbAI, but does not affect the circadian timing of psbAI expression. psfR overexpression affected some, but not all of the genes routinely surveyed for circadian expression. PsfR acts (directly or indirectly) on the psbAI basal promoter region. psfR knockout mutants exhibit wild-type psbAI expression, suggesting that other factors can regulate psbAI expression in the absence of functional PsfR. PsfR contains two receiver-like domains (found in bacterial two-component signal transduction systems), one of which lacks the conserved aspartyl residue required for phosphoryl transfer. PsfR also contains a GGDEF domain. The presence of these domains and the absence of a detectable conserved DNA-binding domain suggest that PsfR may regulate psbAI expression via protein-protein interactions or GGDEF activity (the production of cyclic dinucleotides) rather than direct interaction with the psbAI promoter.

Circadian Rhythm↗
PubMed2004

Phase determination of circadian gene expression in Synechococcus elongatus PCC 7942.

The authors analyzed the upstream regulatory region of purF, a gene that is expressed in a minority phase that peaks at dawn (class 2 circadian phasing) in Synechococcus elongatus, to determine whether specific cis elements are responsible for this characteristic expression pattern. Fusions of various promoter-bearing fragments to luciferase reporter genes showed that normal class 2 phasing of purF expression was correlated with promoter strength. No specific cis element that is separable from the promoter was responsible for determining phase. Very weak promoter activity of unstable phasing was mapped to a 50-bp segment. Inclusion of sequences that flank this minimal promoter either upstream or downstream increased the promoter strength and stabilized the phase in class 2, but neither segment was individually necessary. Because the data suggested a role for the overall promoter context rather than a specific "phase element," the authors proposed that DNA topology is important in the phase determination of circadian gene expression in S. elongatus. To test this hypothesis, they fused the well-characterized DNA topology-dependent Escherichia coli fis promoter to luciferase and showed that it acts as a class 2 promoter in S. elongatus.

Base Sequence↗
PubMed2003

Biochemical properties of CikA, an unusual phytochrome-like histidine protein kinase that resets the circadian clock in Synechococcus elongatus PCC 7942.

We recently described the cikA (circadian input kinase A) gene, whose product supplies environmental information to the circadian oscillator in the cyanobacterium Synechococcus elongatus PCC 7942. CikA possesses three distinct domains: a GAF, a histidine protein kinase (HPK), and a receiver domain similar to those of the response regulator family. To determine how CikA functions in providing circadian input, we constructed modified alleles to tag and truncate the protein, allowing analysis of each domain individually. CikA covalently bound bilin chromophores in vitro, even though it lacks the expected ligand residues, and the GAF domain influenced but did not entirely account for this function. Full-length CikA and truncated variants that carry the HPK domain showed autophosphorylation activity. Deletion of the GAF domain or the N-terminal region adjacent to GAF dramatically reduced autophosphorylation, whereas elimination of the receiver domain increased activity 10-fold. Assays to test phosphorelay from the HPK to the cryptic receiver domain, which lacks the conserved aspartyl residue that serves as a phosphoryl acceptor in response regulators, were negative. We propose that the cryptic receiver is a regulatory domain that interacts with an unknown protein partner to modulate the autokinase activity of CikA but does not work as bona fide receiver domain in a phosphorelay.

Adenosine Triphosphate↗
PubMed2003

Timekeeping in bacteria: the cyanobacterial circadian clock.

The prokaryotes known as cyanobacteria possess an endogenous 24h biological (circadian) clock that provides temporal coordination for physiological processes. Although the cyanobacterial clock has the same fundamental properties as circadian clocks in eukaryotes, its components are non-homologous to those of animals, plants or fungi. Moreover, its mechanism is likely to be very different from that depicted in eukaryotic clock models. The picture that is emerging for the timing mechanism in cyanobacteria is of a multiprotein, multimeric, molecular machine composed of proteins whose domains exhibit twists on common themes. Signal transduction into and out of the clock core appears to occur via histidine protein kinase-based phosphorylation relays.

Biological Clocks↗
PubMed2003

Cyanobacterial circadian clocks--timing is everything.

For more than three billion years, the organisms on this planet have known, like Little Orphan Annie, that "The sun'll come out tomorrow", and many have honed their biochemistry to exploit this knowledge. The cyanobacteria have had ample time to fashion a suitable timepiece, as they are among the oldest inhabitants of the earth. For these organisms, light is food, and it is a nutrient that shows up at the same time every day. Not surprisingly, cyanobacteria have learned to arrange their days around dinnertime.

Bacterial Proteins↗