Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. ...However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.
Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly ...acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems level.
Microorganisms release a diversity of organic compounds that couple interspecies metabolism, enable communication, or provide benefits to other microbes. Increased knowledge of microbial metabolite ...production will contribute to understanding of the dynamic microbial world and can potentially lead to new developments in drug discovery, biofuel production, and clinical research. Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that enables detailed chemical characterization of molecules from a specific location on a surface without special sample pretreatment. Due to its ambient nature, living bacterial colonies growing on agar plates can be rapidly analyzed without affecting the viability of the colony. In this study we demonstrate for the first time the utility of nano-DESI for spatial profiling of chemical gradients generated by microbial communities on agar plates. We found that despite the high salt content of the agar used in this study (~350 mM), nano-DESI analysis enables detailed characterization of metabolites produced by the Synechococcus sp. PCC 7002 colonies. High resolution mass spectrometry and MS/MS analysis of the living Synechococcus sp. PCC 7002 colonies allowed us to detect metabolites and lipids on the colony and on the surrounding agar, and confirm their identities. High sensitivity of nano-DESI enabled identification of several glycolipids that have not been previously reported by extracting the cells using conventional methods. Spatial profiling demonstrated that a majority of lipids and metabolites were localized on the colony while sucrose and glucosylglycerol, an osmoprotective compound produced by cyanobacteria, were secreted onto agar. Furthermore, we demonstrated that the chemical gradients of sucrose and glucosylglycerol on agar depend on the age of the colony. The methodology presented in this study will facilitate future studies focused on molecular-level characterization of interactions between bacterial colonies.
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•Metabolically coupled algal-heterotroph binary cultures utilize organic C and CO2.•Food processing wastewater served as the sole C source for co-culture.•Exchange in CO2 and O2 ...linked photosynthesis and heterotrophic respiration.•H. pluvialis accumulated up to 0.8% of astaxanthin per g of dry cell weight.•High value of astaxanthin-rich biomass may offset costs for wastewater treatment.
Growth of heterotrophic bacterium Bacillus subtilis was metabolically coupled with the photosynthetic activity of an astaxanthin-producing alga Haematococcus pluvialis for conversion of starch-containing waste stream into carotenoid-enriched biomass. The H. pluvialis accounted for 63% of the produced co-culture biomass of 2.2 g/L. Importantly, the binary system requires neither exogenous supply of gaseous substrates nor application of energy-intensive mass transfer technologies due to in-situ exchange in CO2 and O2. The maximum reduction in COD, total nitrogen and phosphorus reached 65%, 55% and 30%, respectively. Conducted techno-economic assessment suggested that the astaxanthin-rich biomass may potentially offset the costs of waste treatment, and, with specific productivity enhancements (induction of astaxanthin to 2% and increase H. pluvialis fraction to 80%), provide and additional revenue stream. The outcome of this study demonstrates a successful proof-of-principle for conversion of waste carbon and nutrients into value-added products through metabolic coupling of heterotrophic and phototrophic metabolisms.
We used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella ...putrefaciens W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph-heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic-heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.
Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe₂O₃), ...was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.
The solubility of orthophosphate (PO₄³⁻) in iron-rich sediments can be exceedingly low, limiting the bioavailability of this essential nutrient to microbial populations that catalyze critical ...biogeochemical reactions. Here we demonstrate that dissolved extracellular DNA can serve as a sole source of phosphorus, as well as carbon and energy, for metal-reducing bacteria of the genus SHEWANELLA: Shewanella oneidensis MR-1, Shewanella putrefaciens CN32, and Shewanella sp. strain W3-18-1 all grew with DNA but displayed different growth rates. W3-18-1 exhibited the highest growth rate with DNA. While strain W3-18-1 displayed Ca²⁺-independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca²⁺ for growth with DNA. For S. oneidensis MR-1, the utilization of DNA as a sole source of phosphorus is linked to the activities of extracellular phosphatase(s) and a Ca²⁺-dependent nuclease(s), which are regulated by phosphorus availability. Mass spectrometry analysis of the extracellular proteome of MR-1 identified one putative endonuclease (SO1844), a predicted UshA (bifunctional UDP-sugar hydrolase/5' nucleotidase), a predicted PhoX (calcium-activated alkaline phosphatase), and a predicted CpdB (bifunctional 2',3' cyclic nucleotide 2' phosphodiesterase/3' nucleotidase), all of which could play important roles in the extracellular degradation of DNA under phosphorus-limiting conditions. Overall, the results of this study suggest that the ability to use exogenous DNA as the sole source of phosphorus is widespread among the shewanellae, and perhaps among all prokaryotes, and may be especially important for nutrient cycling in metal-reducing environments.
► Feedback-controlled illumination compensates for culture dynamics. ► Auto-adjusting dilution maintains maximal growth at a fixed culture density. ► Defined light profiles improve steady-state ...comparisons. ► High-resolution real-time monitoring shows acclimation and perturbation responses. ► In-situ performance of light-saturation curves avoids sampling bias.
A custom photobioreactor was designed to enable automatic light adjustments using computerized feedback control. The system consisted of a 7.5-L cylindrical vessel and an aluminum enclosure housing quantum sensors and light-emitting diode arrays, which provide 630 or 680nm light to preferentially excite the major cyanobacterial pigments, phycocyanin and/or chlorophyll a, respectively. Custom-developed software rapidly measures light transmission and subsequently adjusts the irradiance to maintain a defined light profile to compensate for culture dynamics, biomass accumulation, and pigment adaptations during physiological transitions, thus ensuring appropriate illumination across batch and continuous growth modes. In addition to chemostat cultivation, the photobioreactor may also operate as a turbidostat, continuously adjusting the media dilution to achieve maximal growth at a fixed culture density. The cultivation system doubles as an analytical device, using real-time monitoring to avoid sampling bias (e.g., in-situ light-saturation response), determine conditions for optimal growth, and observe perturbation responses at high time-resolution.
A model-based analysis is conducted to investigate metabolism of Shewanella oneidensis MR-1 strain in aerobic batch culture, which exhibits an intriguing growth pattern by sequentially consuming ...substrate (i.e., lactate) and by-products (i.e., pyruvate and acetate). A general protocol is presented for developing a detailed network-based dynamic model for S. oneidensis based on the Lumped Hybrid Cybernetic Model (L-HCM) framework. The L-HCM, although developed from only limited data, is shown to accurately reproduce exacting dynamic metabolic shifts, and provide reasonable estimates of energy requirement for growth. Flux distributions in S. oneidensis predicted by the L-HCM compare very favorably with 13C-metabolic flux analysis results reported in the literature. Predictive accuracy is enhanced by incorporating measurements of only a few intracellular fluxes, in addition to extracellular metabolites. The L-HCM developed here for S. oneidensis is consequently a promising tool for the analysis of intracellular flux distribution and metabolic engineering.
► A cybernetic model is developed for S. oneidensis based on limited dynamic growth data. ► The model accurately predicts dynamic metabolic switches exhibiting triauxic growth pattern. ► Predictions made of intracellular flux distributions correlate well with 13C-MFA results. ► Energy requirement for growth is also reasonably estimated from limited data only. ► With these features, the developed model emerges as a promising tool for metabolic engineering.
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