scholarly journals Proteomic Analysis of a Syntrophic Coculture of Syntrophobacter fumaroxidans MPOBT and Geobacter sulfurreducens PCAT

2021 ◽  
Vol 12 ◽  
Author(s):  
Monir Mollaei ◽  
Maria Suarez-Diez ◽  
Vicente T. Sedano-Nunez ◽  
Sjef Boeren ◽  
Alfons J. M. Stams ◽  
...  
Keyword(s):  
Pure Culture ◽  
Formate Dehydrogenase ◽  
Geobacter Sulfurreducens ◽  
Acetate Metabolism ◽  
Differential Abundance ◽  
Methanospirillum Hungatei ◽  
Interspecies Electron Transfer ◽  
Lower Abundance ◽  
Associated Proteins ◽  
Propionate Degradation

We established a syntrophic coculture of Syntrophobacter fumaroxidans MPOBT (SF) and Geobacter sulfurreducens PCAT (GS) growing on propionate and Fe(III). Neither of the bacteria was capable of growth on propionate and Fe(III) in pure culture. Propionate degradation by SF provides acetate, hydrogen, and/or formate that can be used as electron donors by GS with Fe(III) citrate as electron acceptor. Proteomic analyses of the SF-GS coculture revealed propionate conversion via the methylmalonyl-CoA (MMC) pathway by SF. The possibility of interspecies electron transfer (IET) via direct (DIET) and/or hydrogen/formate transfer (HFIT) was investigated by comparing the differential abundance of associated proteins in SF-GS coculture against (i) SF coculture with Methanospirillum hungatei (SF-MH), which relies on HFIT, (ii) GS pure culture growing on acetate, formate, hydrogen as propionate products, and Fe(III). We noted some evidence for DIET in the SF-GS coculture, i.e., GS in the coculture showed significantly lower abundance of uptake hydrogenase (43-fold) and formate dehydrogenase (45-fold) and significantly higher abundance of proteins related to acetate metabolism (i.e., GltA; 62-fold) compared to GS pure culture. Moreover, SF in the SF-GS coculture showed significantly lower abundance of IET-related formate dehydrogenases, Fdh3 (51-fold) and Fdh5 (29-fold), and the rate of propionate conversion in SF-GS was 8-fold lower than in the SF-MH coculture. In contrast, compared to GS pure culture, we found lower abundance of pilus-associated cytochrome OmcS (2-fold) and piliA (5-fold) in the SF-GS coculture that is suggested to be necessary for DIET. Furthermore, neither visible aggregates formed in the SF-GS coculture, nor the pili-E of SF (suggested as e-pili) were detected. These findings suggest that the IET mechanism is complex in the SF-GS coculture and can be mediated by several mechanisms rather than one discrete pathway. Our study can be further useful in understanding syntrophic propionate degradation in bioelectrochemical and anaerobic digestion systems.

scholarly journals Interspecies Electron Transfer via Hydrogen and Formate Rather than Direct Electrical Connections in Cocultures of Pelobacter carbinolicus and Geobacter sulfurreducens

2012 ◽  
Vol 78 (21) ◽  
pp. 7645-7651 ◽  
Author(s):  
Amelia-Elena Rotaru ◽  
Pravin M. Shrestha ◽  
Fanghua Liu ◽  
Toshiyuki Ueki ◽  
Kelly Nevin ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Formate Dehydrogenase ◽  
Geobacter Sulfurreducens ◽  
Close Relative ◽  
Wild Type ◽  
Geobacter Metallireducens ◽  
Content Type ◽  
Interspecies Electron Transfer ◽  
Essential Components ◽  
Electrical Connections

ABSTRACTDirect interspecies electron transfer (DIET) is an alternative to interspecies H2/formate transfer as a mechanism for microbial species to cooperatively exchange electrons during syntrophic metabolism. To understand what specific properties contribute to DIET, studies were conducted withPelobacter carbinolicus, a close relative ofGeobacter metallireducens, which is capable of DIET.P. carbinolicusgrew in coculture withGeobacter sulfurreducenswith ethanol as the electron donor and fumarate as the electron acceptor, conditions under whichG. sulfurreducensformed direct electrical connections withG. metallireducens. In contrast to the cell aggregation associated with DIET,P. carbinolicusandG. sulfurreducensdid not aggregate. Attempts to initiate cocultures with a genetically modified strain ofG. sulfurreducensincapable of both H2and formate utilization were unsuccessful, whereas cocultures readily grew with mutant strains capable of formate but not H2uptake or vice versa. The hydrogenase mutant ofG. sulfurreducenscompensated, in cocultures, with significantly increased formate dehydrogenase gene expression. In contrast, the transcript abundance of a hydrogenase gene was comparable in cocultures with that for the formate dehydrogenase mutant ofG. sulfurreducensor the wild type, suggesting that H2was the primary electron carrier in the wild-type cocultures. Cocultures were also initiated with strains ofG. sulfurreducensthat could not produce pili or OmcS, two essential components for DIET. The finding thatP. carbinolicusexchanged electrons withG. sulfurreducensvia interspecies transfer of H2/formate rather than DIET demonstrates that not all microorganisms that can grow syntrophically are capable of DIET and that closely related microorganisms may use significantly different strategies for interspecies electron exchange.

scholarly journals Transcriptomic and Genetic Analysis of Direct Interspecies Electron Transfer

2013 ◽  
Vol 79 (7) ◽  
pp. 2397-2404 ◽  
Author(s):  
Pravin Malla Shrestha ◽  
Amelia-Elena Rotaru ◽  
Zarath M. Summers ◽  
Minita Shrestha ◽  
Fanghua Liu ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Expression Patterns ◽  
Transcript Abundance ◽  
Geobacter Sulfurreducens ◽  
Acetate Metabolism ◽  
Gene Transcript ◽  
Content Type ◽  
Direct Interspecies Electron Transfer ◽  
Interspecies Electron Transfer ◽  
Pilin Gene

ABSTRACTThe possibility that metatranscriptomic analysis could distinguish between direct interspecies electron transfer (DIET) and H2interspecies transfer (HIT) in anaerobic communities was investigated by comparing gene transcript abundance in cocultures in whichGeobacter sulfurreducenswas the electron-accepting partner for eitherGeobacter metallireducens, which performs DIET, orPelobacter carbinolicus, which relies on HIT. Transcript abundance forG. sulfurreducensuptake hydrogenase genes was 7-fold lower in cocultures withG. metallireducensthan in cocultures withP. carbinolicus, consistent with DIET and HIT, respectively, in the two cocultures. Transcript abundance for the pilus-associated cytochrome OmcS, which is essential for DIET but not for HIT, was 240-fold higher in the cocultures withG. metallireducensthan in cocultures withP. carbinolicus. The pilin genepilAwas moderately expressed despite a mutation that might be expected to represspilAexpression. Lower transcript abundance forG. sulfurreducensgenes associated with acetate metabolism in the cocultures withP. carbinolicuswas consistent with the repression of these genes by H2during HIT. Genes for the biogenesis of pili and flagella and severalc-type cytochrome genes were among the most highly expressed inG. metallireducens. Mutant strains that lacked the ability to produce pili, flagella, or the outer surfacec-type cytochrome encoded by Gmet_2896 were not able to form cocultures withG. sulfurreducens. These results demonstrate that there are unique gene expression patterns that distinguish DIET from HIT and suggest that metatranscriptomics may be a promising route to investigate interspecies electron transfer pathways in more-complex environments.

scholarly journals Direct Interspecies Electron Transfer between Geobacter metallireducens and Methanosarcina barkeri

2014 ◽  
Vol 80 (15) ◽  
pp. 4599-4605 ◽  
Author(s):  
Amelia-Elena Rotaru ◽  
Pravin Malla Shrestha ◽  
Fanghua Liu ◽  
Beatrice Markovaite ◽  
Shanshan Chen ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Model Organism ◽  
Methanosarcina Barkeri ◽  
Physical Contact ◽  
Acetate Metabolism ◽  
Geobacter Metallireducens ◽  
Content Type ◽  
Direct Interspecies Electron Transfer ◽  
Interspecies Electron Transfer ◽  
Effective Form

ABSTRACTDirect interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability ofMethanosarcina barkerito participate in DIET was evaluated in coculture withGeobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficientG. metallireducensstrain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficientG. metallireducensisolates to share electrons withM. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. WhenM. barkeriwas grown in coculture with the H2-producingPelobacter carbinolicus, incapable of DIET,M. barkeriutilized H2as an electron donor but metabolized little of the acetate thatP. carbinolicusproduced. This suggested that H2, but not electrons derived from DIET, inhibited acetate metabolism.P. carbinolicus-M. barkericocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H2transfer.M. barkeriis the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H2or electrons derived from DIET for CO2reduction. Furthermore,M. barkeriis genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.

scholarly journals Comparative proteome analysis of propionate degradation bySyntrophobacter fumaroxidansin pure culture and in coculture with methanogens

2018 ◽  
Vol 20 (5) ◽  
pp. 1842-1856 ◽  
Author(s):  
Vicente T. Sedano-Núñez ◽  
Sjef Boeren ◽  
Alfons J. M. Stams ◽  
Caroline M. Plugge
Keyword(s):  
Pure Culture ◽  
Proteome Analysis ◽  
Propionate Degradation

scholarly journals Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

2001 ◽  
Vol 183 (15) ◽  
pp. 4468-4476 ◽  
Author(s):  
Franz Kaufmann ◽  
Derek R. Lovley
Keyword(s):  
Amino Acid ◽  
Electron Donor ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Geobacter Sulfurreducens ◽  
Oxidoreductase Activity ◽  
Isolation And Characterization ◽  
Apparent Homogeneity

ABSTRACT NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.

scholarly journals Biochemical Evidence for Formate Transfer in Syntrophic Propionate-Oxidizing Cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

2002 ◽  
Vol 68 (9) ◽  
pp. 4247-4252 ◽  
Author(s):  
Frank A. M. de Bok ◽  
Maurice L. G. C. Luijten ◽  
Alfons J. M. Stams
Keyword(s):  
Growth Rates ◽  
Formate Dehydrogenase ◽  
Gradient Centrifugation ◽  
Percoll Gradient ◽  
Electron Carrier ◽  
Biochemical Evidence ◽  
Methanospirillum Hungatei ◽  
Axenic Cultures ◽  
In Cells

ABSTRACT The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H2 lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H2 lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.

scholarly journals Engineering cooperation in an anaerobic co-culture

2021 ◽  
Author(s):  
Aunica L. Kane ◽  
Rachel E. Szabo ◽  
Jeffrey A. Gralnick
Keyword(s):  
Pure Culture ◽  
Culture System ◽  
Shewanella Oneidensis ◽  
Geobacter Sulfurreducens ◽  
The Other ◽  
Evolution Of Cooperation ◽  
Emergence Of Cooperation ◽  
Obligate Mutualism ◽  
Culture Growth ◽  
Malate Metabolism

Over the past century, microbiologists have studied organisms in pure culture, yet it is becoming increasingly apparent that the majority of biological processes rely on multispecies cooperation and interaction. While little is known about how such interactions permit cooperation, even less is known about how cooperation arises. To study the emergence of cooperation in the laboratory, we constructed both a commensal community and an obligate mutualism using the previously non-interacting bacteria Shewanella oneidensis and Geobacter sulfurreducens. Incorporation of a glycerol utilization plasmid (pGUT2) enabled S. oneidensis to metabolize glycerol and produce acetate as a carbon source for G. sulfurreducens establishing a cross-feeding, commensal co-culture. In the commensal co-culture, both species coupled oxidative metabolism to the respiration of fumarate as the terminal electron acceptor. Deletion of the gene encoding fumarate reductase in the S. oneidensis pGUT2 strain shifted the co-culture with G. sulfurreducens to an obligate mutualism where neither species could grow in absence of the other. A shift in metabolic strategy from glycerol catabolism to malate metabolism was associated with obligate co-culture growth. Further targeted deletions in malate uptake and acetate generation pathways in S. oneidensis significantly inhibited co-culture growth with G. sulfurreducens. The engineered co-culture between S. oneidensis and G. sulfurreducens provides a model laboratory system to study the emergence of cooperation in bacterial communities, and the shift in metabolic strategy observed in the obligate co-culture highlights the importance of genetic change in shaping microbial interactions in the environment. Importance Microbes seldom live alone in the environment, yet this scenario is approximated in the vast majority of pure-culture laboratory experiments. Here we develop an anaerobic co-culture system to begin understanding microbial physiology in a more complex setting, but also to determine how anaerobic microbial communities can form. Using synthetic biology, we generated a co-culture system where the facultative anaerobe Shewanella oneidensis consumes glycerol and provides acetate to the strict anaerobe Geobacter sulfurreducens. In the commensal system, growth of G. sulfurreducens is dependent on the presence of S. oneidensis. To generate an obligate co-culture, where each organism requires the other, we eliminated the ability of S. oneidensis to respire fumarate. An unexpected shift in metabolic strategy from glycerol catabolism to malate metabolism was observed in the obligate co-culture. Our work highlights how metabolic landscapes can be expanded in multi-species communities and provides a system to evaluate the evolution of cooperation under anaerobic conditions.

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