Search bioRxiv⌕ Search

Biology subjects

Sakkos, J. K.

Publications and source records attributed to Sakkos, J. K..

4 recordsLinked to original sources

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↗

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↗

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↗

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↗