Direct cell-to-cell exchange of matter in a synthetic Clostridium syntrophy enables CO2 fixation, superior metabolite yields, and an expanded metabolic space

Charubin, Kamil; Papoutsakis, Eleftherios T.

doi:10.1016/j.ymben.2018.10.006

Cited by 76 publications

(111 citation statements)

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“…6), which display a strong red C. ljungdahlii population at well past 20 h. In other words, there is no loss of red fluorescent proteins in C. ljungdahlii due to its slow growth. This shows that C. ljungdahlii transfers the red-labeled proteins in the coculture to C. acetobutylicum, which grows faster than C. ljungdahlii (13). This results in dilution and replacement of red-labeled proteins within C. ljungdahlii cells with green ZapA-FAST acquired from C. acetobutylicum, which over time will be detected as pure green cells, instead of double-positive hybrid cells.…”

Section: Resultsmentioning

confidence: 96%

“…Syntrophy could then be viewed as an alternate evolutionary trajectory for microbes, similar in principle to that of phage or virus evolutionary trajectory. were grown in Turbo CGM medium as previously described (13). Turbo CGM used for C. ljungdahlii monocultures was supplemented with 5 g/liter fructose, and these cultures were grown in serum bottles with 20 lb/in 2 g of H 2 /CO 2 gas mixture (80/20%).…”

Section: Downloaded Frommentioning

confidence: 99%

“…Furthermore, the large concentrations of 2-propanol produced in coculture combined with electron balances for the individual species' metabolism suggested direct electron transfer (13). These phenotypes were abolished when the two organisms were separated by a permeable membrane (13). Here, we examine these interactions and the hypothesized direct transfer of materials between the two microbes.…”

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confidence: 93%

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Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture

Charubin

Caplan

Papoutsakis

2020

mBio

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Microbial syntrophy is universal in nature, profoundly affecting the composition and function of microbiomes. We have recently reported data suggesting direct cell-to-cell interactions leading to electron and material exchange between the two microbes in the syntrophy between Clostridium ljungdahlii and C. acetobutylicum. Here, transmission electron microscopy and electron tomography demonstrated cell wall and membrane fusions between the two organisms, whereby C. ljungdahlii appears to invade C. acetobutylicum pole to pole. Correlative fluorescence transmission electron microscopy demonstrated large-scale exchange of proteins. Flow cytometry analysis captured the extent and dynamic persistence of these interactions. Dividing hybrid cells were identified containing stained proteins from both organisms, thus demonstrating persistence of cells with exchanged cellular components. Fluorescence microscopy and flow cytometry of one species with stained RNA and the other tagged with a fluorescent protein demonstrated extensive RNA exchange and identified hybrid cells, some of which continued to divide, while some were in an advanced C. acetobutylicum sporulation form. These data demonstrate that cell fusion enables large-scale cellular material exchange between the two organisms. Although unanticipated and never previously reported, these phenomena are likely widely distributed in nature, have profound implications for species evolution and the function of microbial communities, and could find utility in biotechnology. They may shed new light onto little-understood phenomena, such as antibiotic heteroresistance of pathogens, pathogen invasion of human tissues, and the evolutionary trajectory and persistence of unculturable bacteria. IMPORTANCE We report that two different bacterial organisms engage in heterologous cell fusion that leads to massive exchange of cellular material, including proteins and RNA, and the formation of persistent hybrid cells. The interspecies cell fusion observed here involves a syntrophic microbial system, but these heterologous cell fusions were observed even under nonstrict syntrophic conditions, leaving open the possibility that strict syntrophy may not be necessary for interspecies cell fusion and cellular material exchange. Formation of hybrid cells that contain proteins and RNA from both organisms is unexpected and unprecedented. Such fusion events are likely widely distributed in nature, but have gone undetected. The implications are profound and may shed light onto many unexplained phenomena in human health, natural environments, evolutionary biology, and biotechnology.

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Section: Resultsmentioning

confidence: 96%

Section: Downloaded Frommentioning

confidence: 99%

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confidence: 93%

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Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture

Charubin

Caplan

Papoutsakis

2020

mBio

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“…Other enhancements have also struggled to achieve net CO 2 uptake. Co-culture of C. acetobutylicum and C. ljungdahlii showed syntrophic metabolic coupling when fermenting glucose, fructose, and CO 2 /H 2 , but no net CO 2 uptake (Charubin and Papoutsakis, 2019). Addition of nitrate to batch CO 2 /H 2 fermentation by C. ljungdahlii, increased biomass concentration and subsequently volumetric productivity of acetate .…”

Section: Discussionmentioning

confidence: 96%

Enhancing CO2-Valorization Using Clostridium autoethanogenum for Sustainable Fuel and Chemicals Production

Heffernan

Valgepea

Lemgruber

et al. 2020

Front. Bioeng. Biotechnol.

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Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO 2 and H 2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 C-mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO 2 and H 2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO 2 , and enhanced CO 2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO 2 and CO. Furthermore, the addition of CO led to superior CO 2-valorizing characteristics (9.7 ± 0.4 g/L ethanol with a 66 ± 2 C-mol% distribution, and 540 ± 20 mmol CO 2 /gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO 2 and H 2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO 2 gas fermentation, which can contribute to mitigating climate change.

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“…E. coli and C. acetobutylicum strains were stored at Ϫ85°C in 20% glycerol. C. acetobutylicum ATCC 824 was grown anaerobically in either liquid or solid 2xYTG medium (43) or Turbo CGM medium (44). Erythromycin was added at the following concentrations where appropriate: 40 g/ml for plates and 100 g/ml for liquid cultures.…”

Section: Methodsmentioning

confidence: 99%

A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)

Streett

Kalis

Papoutsakis

2019

Appl Environ Microbiol

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Visualizing protein localization and characterizing gene expression activity in live Clostridium cells is limited for lack of a real-time, highly fluorescent, oxygen-independent reporter system. Enzymatic reporter systems have been used successfully for many years with Clostridium spp.; however, these assays do not allow for real-time analysis of gene expression activity with flow cytometry or for visualizing protein localization through fusion proteins. Commonly used fluorescent reporter proteins require oxygen for chromophore maturation and cannot be used for most strictly anaerobic Clostridium organisms. Here we show that the fluorescenceactivating and absorption-shifting tag protein (FAST), when associated with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR; now commercially available) and other commercially available ligands, is highly fluorescent in Clostridium acetobutylicum under anaerobic conditions. Using flow cytometry and a fluorescence microplate reader, we demonstrated FAST as a reporter system by employing the promoters of the C. acetobutylicum thiolase (thl), acetoacetate decarboxylase (adc), and phosphotransbutyrylase (ptb) metabolic genes, as well as a mutant P thl and modified ribosome binding site (RBS) versions of P adc and P ptb . Flow cytometrybased sorting was efficient and fast in sorting FAST-expressing cells, and positively and negatively sorted cells could be effectively recultured. FAST was also used to tag and examine protein localization of the predicted cell division FtsZ partner protein, ZapA, to visualize the divisome localization in live C. acetobutylicum cells. Our findings suggest that FAST can be used to further investigate Clostridium divisomes and more broadly the localization and expression levels of other proteins in Clostridium organisms, thus enabling cell biology studies with these organisms. IMPORTANCE FAST in association with the fluorogenic ligand HMBR is characterized as a successful, highly fluorescent reporter system in C. acetobutylicum. FAST can be used to distinguish between promoters in live cells using flow cytometry or a fluorescence microplate reader and can be used to tag and examine protein localization in live, anaerobically grown cells. Given that FAST is highly fluorescent under anaerobic conditions, it can be used in several applications of this and likely many Clostridium organisms and other strict anaerobes, including studies involving cell sorting, sporulation dynamics, and population characterization in pure as well as mixed cultures, such as those in various native or synthetic microbiomes and syntrophic cultures. Citation Streett HE, Kalis KM, Papoutsakis ET. 2019. A strongly fluorescing anaerobic reporter and protein-tagging system for Clostridium organisms based on the fluorescenceactivating and absorption-shifting tag protein (FAST). Appl Environ Microbiol 85:e00622-19. by guest http://aem.asm.org/ Downloaded from RESULTS Expression of FAST off the native and modified C. acetobutylicum thiolase promoter in E. coli and C...

show abstract

Direct cell-to-cell exchange of matter in a synthetic Clostridium syntrophy enables CO2 fixation, superior metabolite yields, and an expanded metabolic space

Cited by 76 publications

References 55 publications

Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture

Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture

Enhancing CO2-Valorization Using Clostridium autoethanogenum for Sustainable Fuel and Chemicals Production

A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)

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