Biology subjects

Ducat, D. C.

Publications and source records attributed to Ducat, D. C..

10 recordsLinked to original sources

bioRxiv2026

A novel class of conserved sucrose-phosphate phosphatases highlights the diversity of cyanobacterial sucrose metabolism

Sucrose metabolism is an important feature of the physiology of the green lineage of photosynthetic organisms and has therefore been the subject of considerable research on plants, algae, and cyanobacteria. Canonical sucrose biosynthesis pathways often involve the condensation of NDP-glucose and fructose-6-phosphate through the action of sucrose-phosphate synthase, then the dephosphorylation of sucrose 6-phosphate into sucrose via sucrose-phosphate phosphatase (SPP). However, many cyanobacterial genomes encode multiple homologs of SPP proteins (SPP-like), including variants that appear to lack key residues reported to be important for sucrose 6-phosphate binding. Herein, we examine these SPP-like proteins, focusing on the biochemical and physiological characterization of the SPP-like protein encoded by the cyanobacterial model, Synechococcus elongatus PCC 7942. Bioinformatic analysis suggests that the SPP-like family of proteins is highly conserved across cyanobacterial species and forms distinct phylogenetic clades that are more widely distributed than the canonical SPP proteins themselves. Biochemical analysis of the purified S. elongatus PCC 7942 SPP-like protein reveals that it not only retains the capacity to dephosphorylate sucrose 6-phosphate, but it also may have physiologically relevant phosphatase activity on 3-phosphoglycerate (3-PGA). We provide evidence that the SPP-like family of proteins represents a well-conserved group of phosphatases across cyanobacteria and suggest some enzymes in this family may have evolved a broader substrate specificity relative to the well-characterized SPP family. Our results have broader implications for cyanobacterial regulation of sucrose biosynthesis and other key steps of central carbon metabolism.

biochemistry↗

bioRxiv2026

Identifying Novel Targets of the Stringent Response in Plants and Cyanobacteria using chemoproteomics

Survival in dynamic environments requires photosynthetic organisms to rapidly sense and respond to stress. The stringent response, mediated by the signaling molecule guanosine-3,5-bisdiphosphate (ppGpp), is crucial for acclimation to environmental changes such as darkness and nitrogen limitation. While it has been extensively characterized in heterotrophic bacteria such as Escherichia coli, the molecular mechanisms and regulatory targets of ppGpp in photosynthetic organisms remain less understood. Here, we report large-scale chemoproteomic identification of ppGpp-binding proteins across plant chloroplasts and cyanobacteria, revealing both conserved and novel targets compared to E. coli. In plants, we found that ppGpp regulates pyrimidine metabolism by inhibiting the chloroplastic enzyme aspartate transcarbamoylase (PyrB). In cyanobacteria, we found that ppGpp activates glucose-1-phosphate adenylyltransferase (GlgC) involved in glycogen synthesis, activates citrate synthase (GltA), and induces carboxysome aggregation. These findings expand the known ppGpp regulatory network in photosynthetic organisms and provide a foundation for understanding how ppGpp coordinates adaptation to nutrient and environmental stresses.

systems biology↗

bioRxiv2025

Plastoquinone redox status influences carboxysome integrity via a RpaA- and ROS-dependent regulatory network

Carboxysomes are bacterial microcompartments that encapsulate Rubisco and are a core component of the cyanobacterial carbon concentration mechanism (CCM). While carboxysome number, size and spatial organization are observed to vary in different environmental conditions (CO2, light, temperature, light quality), molecular mechanisms underlying this potentially adaptive process remain elusive. Herein, we observed that mutants of the circadian rhythm/metabolism factor, Regulator of Phycobilisome Associated A (RpaA), exhibit a striking breakdown of carboxysomes under certain environmental conditions. We find that growth conditions leading to overreduction of the plastoquinone (PQ) pool (mixotrophic growth, high irradiance, or chemical inhibition of electron transfer from PQ to the cytochrome b6f complex) are accompanied by elevated generation of reactive oxygen species (ROS), and correlate with carboxysome breakdown. Carboxysome breakdown is reversed by environmental conditions or chemical inhibitors that prevent PQ overreduction and accompanying ROS generation. Taken together, our data supports a novel link between the redox status of the PQ pool and carboxysome status and/or integrity. Our results have implications for fundamental understanding of cyanobacterial energy balancing pathways and may indicate new research directions for understanding how the carboxysome is remodeled in response to changing environments.

synthetic biology↗

bioRxiv2024

Orthogonality of shell proteins across BMC subclasses in cyanobacteria

Bacterial microcompartments (BMC) are protein-based organelles broadly distributed across all bacterial phyla and subclassified into [≥]60 functional variants. Despite their evolutionary and metabolic diversity, shell proteins that structurally compose the BMC surface are closely related across BMC classes. Herein, we sought to identify molecular and physiological features that could promote independent operation of more than one BMC type within the same cell by reducing inter-organelle cross-talk of shell proteins. We heterologously expressed shell proteins from the structurally well-defined BMC of Haliangium ochraceum (HO) within Synechococcus elongatus PCC 7942, a model cyanobacterium containing the {beta}-carboxysome. We find considerable cross-reactivity of the HO hexameric shell protein (HO BMC-H) with components of the {beta}-carboxysome; HO BMC-H can integrate into carboxysomes, disrupt its ultrastructural organization, and impair its associated CO2 fixation reactions. S. elongatus is unable to maintain the integrity of the {beta}-carboxysome over time when HO BMC-H is expressed in the absence of one or more of three broad strategies that act to increase the orthogonality between HO and carboxysome BMC shell proteins: i) reduced expression of promiscuous shell proteins; ii) sequestration of free HO BMC-H proteins via co-expression of other members of the same HO shell protein class, or; iii) heterologous expression of BMC positional system proteins McdAB (Maintenance of carboxysome distribution AB), revealing a putative moonlighting function of the McdAB protein family. Our results have implications for bacteria that encode more than one BMC within their genome and may have translational implications for the use of engineered BMCs for biotechnological applications.

synthetic biology↗

bioRxiv2022

Predicting partner fitness based on spatial structuring in a light-driven microbial community

Microbial communities have vital roles in systems essential to human health and agriculture, such as gut and soil microbiomes, and there is growing interest in engineering designer consortia for applications in biotechnology (e.g., personalized probiotics, bioproduction of high-value products, biosensing). The capacity to monitor and model metabolite exchange in dynamic microbial consortia can provide foundational information important to understand the community level behaviors that emerge, a requirement for building novel consortia. Where experimental approaches for monitoring metabolic exchange are technologically challenging, computational tools can enable greater access to the fate of both chemicals and microbes within a consortium. In this study, we developed an in-silico model of a synthetic microbial consortia of sucrose-secreting Synechococcus elongatus PCC 7942 and Escherichia coli W. Our model was built on the NUFEB framework for Individual-based Modeling (IbM) and optimized for biological accuracy using experimental data. We showed that the relative level of sucrose secretion regulates not only the steady-state support for heterotrophic biomass, but also the temporal dynamics of consortia growth. In order to determine the importance of spatial organization within the consortium, we fit a regression model to spatial data and used it to accurately predict colony fitness. We found that some of the critical parameters for fitness prediction were inter-colony distance, initial biomass, induction level, and distance from the center of the simulation volume. We anticipate that the synergy between experimental and computational approaches will improve our ability to design consortia with novel function. Author SummaryMicrobial communities, play important, yet poorly understood roles in health and agriculture. As we develop a better understanding of how these communities interact together, as well as with their host organisms, there is growing interest in engineering communities with specific functions, such as for treating disease, personalized probiotics, or aiding plants with nutrient uptake. To better understand how these microbes interact with each other, we want to monitor the exchange of metabolites and the locations of the microbes, tasks which at present are technically challenging, if not impossible. Where experimental approaches for monitoring metabolites are limited, computational tools can enable greater access to the fate of both chemicals and microbes within a community. In this study, we developed a computerized model of a synthetic microbial community of two bacteria, one which performs photosynthesis and supplies sugar and another which consumes the sugar for growth. We showed that the relative level of sugar secretion regulates not only the steady-state support for the consumer partners growth, but also how the community changes with time. To determine the importance of spatial organization within the community, we fit a model and used it to predict colony growth. We anticipate that the synergy between experimental and computational approaches will improve our ability to design microbial communities with new functions.

bioengineering↗

bioRxiv2022

Developing cyanobacterial quorum sensing toolkits: towards interspecies coordination in mixed autotroph/heterotroph communities

1.There has been substantial recent interest in the promise of sustainable, light-driven bioproduction using cyanobacteria, including developing efforts for microbial bioproduction using mixed autotroph/heterotroph communities, which could provide useful properties, such as division of metabolic labor. However, building stable mixed-species communities of sufficient productivity remains a challenge, partly due to the lack of strategies for synchronizing and coordinating biological activities across different species. To address this obstacle, we developed an inter-species communication system using quorum sensing (QS) modules derived from well-studied pathways in heterotrophic microbes. In the model cyanobacterium, Synechococcus elongatus PCC 7942 (S. elongatus), we designed, integrated, and characterized genetic circuits that detect acyl-homoserine lactones (AHLs), diffusible signals utilized in many QS pathways. We showed that these receiver modules sense exogenously supplied AHL molecules and activate gene expression in a dose-dependent manner. We characterized these AHL receiver circuits in parallel in Escherichia coli W (E. coli W) to dissect species-specific properties, finding broad agreement, albeit with increased basal expression in S. elongatus. Our engineered "sender" E. coli strains accumulated biologically synthesized AHLs within the supernatant and activated receiver strains similarly to exogenous AHL activation. Our results will bolster the design of sophisticated genetic circuits in cyanobacterial/heterotroph consortia and the engineering of QS-like behaviors across cyanobacterial populations. HighlightsO_LIDesigned, built, and tested an inter-species quorum sensing-based communication system. C_LIO_LIThese genetic circuits can sense and respond to exogenous and secreted signals. C_LIO_LICircuit function in S. elongatus was comparable to E. coli, albeit with increased basal expression and lower induction ratios C_LIO_LIDemonstrated inter-species communication in direct co-cultivation C_LIO_LIFirst demonstration of inducible promoters and cross-species gene regulation in S. elongatus based on quorum sensing C_LI

synthetic biology↗

bioRxiv2021

Investigating Carboxysome Morphology Dynamics with a Rotationally Invariant Variational Autoencoder

Carboxysomes are a class of bacterial microcompartments that form proteinaceous organelles within the cytoplasm of cyanobacteria and play a central role in photosynthetic metabolism by defining a cellular microenvironment permissive to CO2 fixation. Critical aspects of the assembly of the carboxysomes remain relatively unknown, especially with regard to the dynamics of this microcompartment. We have recently expressed an exogenous protease as a way of gaining control over endogenous protein levels, including carboxysomal components, in the model cyanobacterium Synechococcous elongatus PCC 7942. By utilizing this system, proteins that compose the carboxysome can be tuned in real-time as a method to examine carboxysome dynamics. Yet, analysis of subtle changes in carboxysome morphology with microscopy remains a low-throughput and subjective process. Here we use deep learning techniques, specifically a Rotationally Invariant Variational Autoencoder (rVAE), to analyze the fluorescence microscopy images and quantitatively evaluate how carboxysome shell remodelling impacts trends in the morphology of the microcompartment over time. We find that rVAEs are able to assist in the quantitative evaluation of changes in carboxysome location, shape, and size over time. We propose that rVAEs may be a useful tool to accelerate the analysis of carboxysome assembly and dynamics in response to genetic or environmental perturbation, and may be more generally useful to probe regulatory processes involving a broader array of bacterial microcompartments.

microbiology↗

bioRxiv2021

Multi-layer Regulation of Rubisco in Response to Altered Carbon Status in Synechococcus elongatus PCC 7942

Photosynthetic organisms possess a variety of mechanisms to achieve balance between absorbed light (source) and the capacity to metabolically utilize or dissipate this energy (sink). While regulatory processes that detect changes in metabolic status/balance are relatively well-studied in plants, analogous pathways remain poorly characterized in photosynthetic microbes. Herein, we explore systemic changes that result from alterations in carbon availability in the model cyanobacterium Synechococcus elongatus PCC 7942 by taking advantage of an engineered strain where influx/efflux of a central carbon metabolite, sucrose, can be regulated experimentally. We observe that induction of a high-flux sucrose export pathway leads to depletion of internal carbon storage pools (glycogen), and concurrent increases in photosynthetic parameters. Further, a proteome-wide analysis and fluorescence reporter-based analysis revealed that upregulated factors following the activation of the metabolic sink are strongly concentrated on ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and axillary modules involved in Rubisco maturation. Carboxysome number and Rubisco activity also increase following engagement of sucrose secretion. Conversely, reversing the flux of sucrose by feeding exogenous sucrose heterologously results in increased glycogen pools, decreased Rubisco abundance, decreased photosystem II quantum efficiency, and carboxysome reorganization. Our data suggest that Rubisco activity and organization are key outputs connected to regulatory pathways involved in metabolic balancing in cyanobacteria. One sentence summaryRubisco activity and organization are key outputs involve in source-sink balancing in cyanobacteria.

microbiology↗

bioRxiv2018

Biotransformation of 2,4-dinitrotoluene in a phototrophic co-culture of engineered Synechococcus elongatus and Pseudomonas putida

In contrast to the current paradigm of using microbial monocultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed-species consortia to perform functions that are difficult to program into individual strains. Additionally, the division of labor between specialist species found in natural consortia can lead to increased catalytic efficiency and stability relative to a monoculture or a community composed of generalists. In this work, we have designed a synthetic co-culture for phototrophic degradation of xenobiotics, composed of a cyanobacterium, (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). Cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose as the only carbon source and to degrade the environmental pollutant 2,4-dinitrotoluene (2,4-DNT). The synthetic consortium is able to degrade 2,4-DNT with only light and CO2 as inputs for the system, and it was stable over time through repeated backdilutions. Furthermore, cycling this consortium through low nitrogen medium promoted the accumulation of polyhydroxyalkanoate (PHA)-an added-value biopolymer-in P. putida, thus highlighting the versatility of this production platform. Altogether, the synthetic consortium allows for simultaneous bioproduction of PHA and remediation of the industrial pollutant 2,4-DNT, using light and CO2 as inputs.\n\nImportanceIn this study, we have created an artificial consortium composed of two bacterial species that enables the degradation of the industrially-produced environmental pollutant 2,4-DNT while simultaneously producing PHA bioplastic. In these co-cultures, the photosynthetic cyanobacteria fuel an engineered P. putida strain programmed both to use sucrose as a carbon source and to perform the biotransformation of 2,4-DNT. The division of labor in this synthetic co-culture is reminiscent of that commonly observed in microbial communities and represents a proof-of-principle example of how artificial consortia can be employed for bioremediation purposes. Furthermore, this co-culture system enabled the utilization of freshwater sources that could not be utilized in classical agriculture settings, reducing the potential competition of this alternative method of bioproduction with current agricultural practices, as well as remediation of contaminated water streams.

synthetic biology↗

bioRxiv2018

Protein Gradients on the Nucleoid Position the Carbon-fixing Organelles of Cyanobacteria

In BriefWe describe a novel positioning system that utilizes the cyanobacterial nucleoid to segregate, transport and equidistantly position the carbon-fixation machinery (carboxysomes).\n\nSummaryCarboxysomes are protein-based bacterial organelles that encapsulate a key enzyme of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we show that a small protein, termed McdB, localizes to carboxysomes through interactions with carboxysome shell proteins to drive emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly interacts to stimulate McdA ATPase activity, and indicate that carboxysome-dependent McdA depletion zone formation on the nucleoid is required for directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a new class of self-organizing proteins that follow a Brownian-ratchet mechanism, challenging the cytoskeletal model of organelle transport, for equidistant positioning of carboxysomes in cyanobacteria. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria more broadly.

microbiology↗