Biology subjects

Golden, S. S.

Publications and source records attributed to Golden, S. S..

11 recordsLinked to original sources

bioRxiv2026

A KaiA Conformational Switch Links Day-Night Transitions to Clock Robustness

Biological clocks require mechanisms that enable transition between temporal phases and buffer against environmental fluctuations. In the cyanobacterial KaiABC-based circadian oscillator, KaiA has been regarded as a daytime activator that is passively sequestered at night. Here we define KaiA as a conformationally regulated activator and an active catalyst of the night-to-dawn transition. We identify a single linker helix, 5, that acts as a structural switch, mediating a domain-driven "tug-of-war" that sets the open-closed equilibrium in KaiA. Structure-guided mutations that shift this balance in either direction abolish stable rhythmicity both in vitro and in cells. Also, we show that substoichiometric KaiA dismantles nighttime KaiB-KaiC complexes through interactions with the KaiC C-terminal tails and likely initiates an autocatalytic feedback loop that resets the phosphorylation cycle at dawn. Furthermore, we present two independent kinetic models that offer distinct, deterministic explanations for the clock's low-temperature amplitude decline and failure at 20 {degrees} C.

biochemistry↗

bioRxiv2026

Circadian phase alignment promotes survival under predictive oxidative stress

Circadian clocks are endogenous time-keeping mechanisms that continue to oscillate with a period that approximates 24 h in the absence of cyclic environmental cues. This free-running period (FRP) varies from species to species and can vary within species across latitudinal clines. Because natural environments provide daily cues that reset circadian clocks to exactly 24 h, the importance of the FRP length is unclear. Using the cyanobacterium Synechococcus elongatus we show that the FRP length alters the alignment of a biological process with an environmental reference, known as phase angle of entrainment, across seasonal photoperiods, thereby repositioning stress response programs relative to the external environment. This phase relationship affects the overall fitness of the organism: strains that position stress-response programs to peak at the time at which the stressor peaks outcompete their early- or late-peaking counterparts. Our results suggest that the FRP length is selected for its ability to adjust the phase angle of entrainment across seasonal photoperiods: the wild-type FRP length was uniquely plastic in its ability to adjust stress-response programs to coincide with the stressor in all photoperiod conditions.

microbiology↗

bioRxiv2023

Cyanobacterial sigma factor controls biofilm-promoting genes through intra- and intercellular pathways

Cyanobacteria frequently constitute integral components of microbial communities known as phototrophic biofilms. These assemblages are not only widespread in various environmental contexts but also hold significant industrial relevance. Nevertheless, the governing elements responsible for cyanobacterial biofilm development have remained elusive. This study, which employs the model cyanobacterium Synechococcus elongatus PCC 7942, demonstrates that the RNA polymerase sigma factor SigF1, but not its paralog SigF2, is required for a biofilm-suppression mechanism that operates in this organism. Comprehensive transcriptome analyses identified distinct regulons under the control of each of these sigma factors. Additional data indicate that SigF1 regulates biofilm through its involvement in transcriptional induction of genes that include those for the primary pilus subunit: sigF1 inactivation both prevents pilus assembly and abrogates secretion of a biofilm inhibitor. Consequently, expression is significantly upregulated for the ebfG-operon that encodes matrix components and the genes that encode their corresponding secretion system. Thus, this study uncovers a basic regulatory component of cyanobacterial communal behavior. Elevated expression of biofilm-promoting genes in a sigF1 mutant supports an additional layer of regulation by SigF1 that operates via an intracellular mechanism.

microbiology↗

bioRxiv2023

Phenotypically Complex Living Materials Containing Engineered Cyanobacteria

A cyanobacterial photosynthetic biocomposite material was fabricated using 3D-printing and bioengineered to produce multiple functional outputs in response to an external chemical stimulus. Our investigations show the advantages of utilizing additive manufacturing techniques in controlling the design and shape of the fabricated materials, which proved to be important for the support and growth of obligate phototrophic microorganisms within the material. As an initial proof-of-concept, a synthetic theophylline-responsive riboswitch in Synechococcus elongatus PCC 7942 was used for regulating the expression of a yellow fluorescent protein (YFP) reporter. Upon induction with theophylline, the encapsulated cells produced YFP within the hydrogel matrix. Subsequently, a strain of S. elongatus was engineered to produce an oxidative enzyme that is useful for bioremediation, laccase, expressed either constitutively or under the control of the riboswitch. The responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a useful tool in environmental bioremediation. Finally, cells were engineered to have the capacity for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing unintended environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in patterned volumetric 3D-printed designs, we demonstrate the potential of programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.

bioengineering↗

bioRxiv2022

Cell specialization in cyanobacterial biofilm development revealed by expression of a cell-surface and extracellular matrix protein

Cyanobacterial biofilms are ubiquitous and play important roles in diverse environments, yet, understanding of the processes underlying development of these aggregates is just emerging. Here we report cell specialization in formation of Synechococcus elongatus PCC 7942 biofilms - a hitherto unknown characteristic of cyanobacterial multicellularity. We show that only a quarter of the cell population expresses at high levels the four-gene ebfG-operon that is required for biofilm formation. Almost all cells, however, are assembled in the biofilm. Detailed characterization of EbfG4 encoded by this operon revealed cell-surface localization as well as its presence in the biofilm matrix. Moreover, EbfG1-3 were shown to form amyloid structures such as fibrils and are thus likely to contribute to the matrix structure. These data suggest a beneficial division of labour during biofilm formation where only some of the cells allocate resources to produce matrix proteins - public goods that support robust biofilm development by the majority of the cells. Additionally, previous studies revealed the operation of a self-suppression mechanism that depends on an extracellular inhibitor, which supresses transcription of the ebfG-operon. Here we revealed inhibitor activity at an early growth stage and its gradual accumulation along the exponential growth phase in correlation with cell density. Data, however, do not support a threshold-like phenomenon known for quorum-sensing in heterotrophs. Together, data presented here demonstrate cell specialization and imply density-dependent regulation thereby providing novel insights into cyanobacterial communal behaviour. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/498973v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@7758f8org.highwire.dtl.DTLVardef@172396aorg.highwire.dtl.DTLVardef@176612org.highwire.dtl.DTLVardef@2e320c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Transcriptomic and phenomic investigations reveal elements in biofilm repression and formation in the cyanobacterium Synechococcus elongatus PCC 7942

Biofilm formation by photosynthetic organisms is a complex behavior that serves multiple functions in the environment. Biofilm formation in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 is regulated in part by a set of small secreted proteins that promotes biofilm formation and a self-suppression mechanism that prevents their expression. Little is known about the regulatory and structural components of the biofilms in PCC 7942, or response to the suppressor signal(s). We performed transcriptomics (RNA-Seq) and phenomics (RB-TnSeq) screens that identified four genes involved in biofilm formation and regulation, more than 25 additional candidates that may impact biofilm formation, and revealed the transcriptomic adaptation to the biofilm state. In so doing, we compared the effectiveness of these two approaches for gene discovery.

microbiology↗

bioRxiv2021

Impairment of a cyanobacterial glycosyltransferase that modifies a pilin results in biofilm development

A biofilm inhibiting mechanism operates in the cyanobacterium Synechococcus elongatus. Here, we demonstrate that the glycosyltransferase homolog, Ogt, participates in the inhibitory process - inactivation of ogt results in robust biofilm formation. Furthermore, a mutational approach shows requirement of the glycosyltransferase activity for biofilm inhibition. This enzyme is necessary for glycosylation of the pilus subunit and for adequate pilus formation. In contrast to wild-type culture in which most cells exhibit several pili, only 25% of the mutant cells are piliated, half of which possess a single pilus. In spite of this poor piliation, natural DNA competence was similar to that of wild-type, therefore, we propose that the unglycosylated pili facilitate DNA transformation. Additionally, conditioned medium from wild-type culture, which contains a biofilm inhibiting substance(s), only partially blocks biofilm development by the ogt-mutant. Thus, we suggest that inactivation of ogt affects multiple processes including production or secretion of the inhibitor as well as the ability to sense or respond to it. Originality-Significance StatementThe molecular mechanisms that underlie biofilm development in cyanobacteria are just emerging. Using the cyanobacterium S. elongatus as a model, we demonstrate that glycosylation of the pilus subunit is crucial for the biofilm self-suppression mechanism, however, it is dispensable for DNA competence.

microbiology↗

bioRxiv2020

Structural mimicry confers robustness in the cyanobacterial circadian clock

The histidine kinase SasA enhances robustness of circadian rhythms in the cyanobacterium S. elongatus by temporally controlling expression of the core clock components, kaiB and kaiC. Here we show that SasA also engages directly with KaiB and KaiC proteins to regulate the period and enhance robustness of the reconstituted circadian oscillator in vitro, particularly under limiting concentrations of KaiB. In contrast to its role regulating gene expression, oscillator function does not require SasA kinase activity; rather, SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex. Cooperativity gives way to competition with increasing concentrations of SasA to define a dynamic window by which SasA directly modulates clock robustness. One Sentence SummarySasA controls the assembly of clock protein complexes through a balance of cooperative and competitive interactions.

biochemistry↗

bioRxiv2020

Reconstitution of an intact clock that generates circadian DNA binding in vitro

Circadian clocks control gene expression in the complex milieu of cells. Here, we reconstituted under defined conditions in vitro the cyanobacterial circadian clock system which includes an oscillator, signal-transduction pathways, transcription factor, and promoter DNA. The system oscillates autonomously with a near 24 h period, remains phase coherent for many days, and allows real-time observation of each component simultaneously without user intervention. This reassembled clock system provides new insights into how a circadian clock exerts control over gene expression and can serve in the area of synthetic biology as a new platform upon which to build even more complexity. One Sentence SummaryAn autonomously oscillating circadian clock-controlled gene regulatory circuit is studied in vitro using a real-time high-throughput assay.

biochemistry↗

bioRxiv2019

The circadian clock and darkness control natural competence in cyanobacteria

Natural genetic competence-based transformation contributed to the evolution of prokaryotes, including the cyanobacterial phylum that established oxygenic photosynthesis. The cyanobacterium Synechococcus elongatus is noted both as a model system for analyzing a prokaryotic circadian clock and for its facile, but poorly understood, natural competence. Here a genome-wide screen aimed at determining the genetic basis of competence in cyanobacteria identified all genes required for natural transformation in S. elongatus, including conserved Type IV pilus, competence-associated, and newly described genes, and revealed that the circadian clock controls the process. The findings uncover a daily program that determines the state of competence in S. elongatus and adapts to seasonal changes of day-length. Pilus biogenesis occurs daily in the morning, but competence is maximal upon the coincidence of circadian dusk and the onset of darkness. As in heterotrophic bacteria, where natural competence is conditionally regulated by nutritional or other stress, cyanobacterial competence is conditional and is tied to the daily cycle set by the cells most critical nutritional source, the Sun.

microbiology↗

bioRxiv2018

Genome-wide fitness assessment during diurnal growth reveals an expanded role of the cyanobacterial circadian clock protein KaiA

The recurrent pattern of light and darkness generated by Earths axial rotation has profoundly influenced the evolution of organisms, selecting for both biological mechanisms that respond acutely to environmental changes and circadian clocks that program physiology in anticipation of daily variations. The necessity to integrate environmental responsiveness and circadian programming is exemplified in photosynthetic organisms such as cyanobacteria, which depend on light-driven photochemical processes. The cyanobacterium Synechococcus elongatus PCC 7942 is an excellent model system for dissecting these entwined mechanisms. Its core circadian oscillator, consisting of three proteins KaiA, KaiB, and KaiC, transmits time-of-day signals to clock-output proteins, which reciprocally regulate global transcription. Research performed under constant light facilitates analysis of intrinsic cycles separately from direct environmental responses, but does not provide insight into how these regulatory systems are integrated during light-dark cycles. Thus, we sought to identify genes that are specifically necessary in a day-night environment. We screened a dense bar-coded transposon library in both continuous light and daily cycling conditions and compared the fitness consequences of loss of each nonessential gene in the genome. Although the clock itself is not essential for viability in light-dark cycles, the most detrimental mutations revealed by the screen were those that disrupt KaiA. The screen broadened our understanding of light-dark survival in photosynthetic organisms, identified unforeseen clock-protein interaction dynamics, and reinforced the role of the clock as a negative regulator of a night-time metabolic program that is essential for S. elongatus to survive in the dark.\n\nSignificanceUnderstanding how photosynthetic bacteria respond to and anticipate natural light-dark cycles is necessary for predictive modeling, bioengineering, and elucidating metabolic strategies for diurnal growth. Here, we identify the genetic components that are important specifically under light-dark cycling conditions and determine how a properly functioning circadian clock prepares metabolism for darkness, a starvation period for photoautotrophs. This study establishes that the core circadian clock protein KaiA is necessary to enable rhythmic de-repression of a night-time circadian program.

microbiology↗