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Title: Single-genotype syntrophy by Rhodopseudomonas palustris is not a strategy to aid redox balance during anaerobic degradation of lignin monomers

Abstract

Rhodopseudomonas palustris has emerged as a model microbe for the anaerobic metabolism of p-coumarate, which is an aromatic compound and a primary component of lignin. However, under an aerobic conditions, R.palustris must actively eliminate excess reducing equivalents through a number of known strategies (e.g., CO 2 fixation, H 2 evolution) to avoid lethal redox imbalance. Others had hypothesized that to ease the burden of this redox imbalance, a clonal population of R.palustris could functionally differentiate into a pseudo-consortium. Within this pseudo-consortium, one sub-population would perform the aromatic moiety degradation into acetate, while the other sub-population would oxidize acetate, resulting in a single-genotype syntrophy through acetate sharing. Here, the objective was to test this hypothesis by utilizing microbial lelectrochemistry as a research tool with the extrac ellular-electron-transferring bacterium Geobacter sulfurreducens as a reporter strain replacing the hypothesized acetate-oxidizing sub-population. We used a 2×4 experimental design with pure cultures of R. palustris in serum bottles and co-cultures of R. palustris and G.sulfurreducens in bioelectrochemical systems.This experimental design included growth medium with and without bicarbonate to induce non-lethal and lethal redox imbalance conditions, respectively, in R. palustris. Finally, the design also included a mutant strain (NifA*) of R. palustris, which constitutively produces Hmore » 2, to serve both as a positive control for metabolite secretion (H 2) to G. sulfurreducens, and as a non-lethal redox control for without bicarbonate conditions. Our results demonstrate that acetate sharing between different sub-populations of R. palustris does not occur while degrading p-coumarate under either non-lethal or lethal redox imbalance conditions. Furthermore, this work highlights the strength of microbial electrochemistry as a tool for studying microbial syntrophy.« less

Authors:
 [1];  [2]
  1. Cornell Univ., Ithaca, NY (United States); The DOE Joint Genome Institute, Walnut Creek, CA (United States)
  2. Cornell Univ., Ithaca, NY (United States)
Publication Date:
Research Org.:
Cornell Univ., Ithaca, NY (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1312683
Grant/Contract Number:
AR0000312
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Frontiers in Microbiology
Additional Journal Information:
Journal Volume: 7; Journal ID: ISSN 1664-302X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; single-genotype syntrophy; Rhodopseudomonas palustris; microbial electrochemistry; lignin degradation; redox balance

Citation Formats

Doud, Devin F. R., and Angenent, Largus T. Single-genotype syntrophy by Rhodopseudomonas palustris is not a strategy to aid redox balance during anaerobic degradation of lignin monomers. United States: N. p., 2016. Web. doi:10.3389/fmicb.2016.01082.
Doud, Devin F. R., & Angenent, Largus T. Single-genotype syntrophy by Rhodopseudomonas palustris is not a strategy to aid redox balance during anaerobic degradation of lignin monomers. United States. doi:10.3389/fmicb.2016.01082.
Doud, Devin F. R., and Angenent, Largus T. Thu . "Single-genotype syntrophy by Rhodopseudomonas palustris is not a strategy to aid redox balance during anaerobic degradation of lignin monomers". United States. doi:10.3389/fmicb.2016.01082. https://www.osti.gov/servlets/purl/1312683.
@article{osti_1312683,
title = {Single-genotype syntrophy by Rhodopseudomonas palustris is not a strategy to aid redox balance during anaerobic degradation of lignin monomers},
author = {Doud, Devin F. R. and Angenent, Largus T.},
abstractNote = {Rhodopseudomonas palustris has emerged as a model microbe for the anaerobic metabolism of p-coumarate, which is an aromatic compound and a primary component of lignin. However, under an aerobic conditions, R.palustris must actively eliminate excess reducing equivalents through a number of known strategies (e.g., CO2 fixation, H2 evolution) to avoid lethal redox imbalance. Others had hypothesized that to ease the burden of this redox imbalance, a clonal population of R.palustris could functionally differentiate into a pseudo-consortium. Within this pseudo-consortium, one sub-population would perform the aromatic moiety degradation into acetate, while the other sub-population would oxidize acetate, resulting in a single-genotype syntrophy through acetate sharing. Here, the objective was to test this hypothesis by utilizing microbial lelectrochemistry as a research tool with the extrac ellular-electron-transferring bacterium Geobacter sulfurreducens as a reporter strain replacing the hypothesized acetate-oxidizing sub-population. We used a 2×4 experimental design with pure cultures of R. palustris in serum bottles and co-cultures of R. palustris and G.sulfurreducens in bioelectrochemical systems.This experimental design included growth medium with and without bicarbonate to induce non-lethal and lethal redox imbalance conditions, respectively, in R. palustris. Finally, the design also included a mutant strain (NifA*) of R. palustris, which constitutively produces H2, to serve both as a positive control for metabolite secretion (H2) to G. sulfurreducens, and as a non-lethal redox control for without bicarbonate conditions. Our results demonstrate that acetate sharing between different sub-populations of R. palustris does not occur while degrading p-coumarate under either non-lethal or lethal redox imbalance conditions. Furthermore, this work highlights the strength of microbial electrochemistry as a tool for studying microbial syntrophy.},
doi = {10.3389/fmicb.2016.01082},
journal = {Frontiers in Microbiology},
number = ,
volume = 7,
place = {United States},
year = {Thu Jul 14 00:00:00 EDT 2016},
month = {Thu Jul 14 00:00:00 EDT 2016}
}

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  • Understanding of cellular processes involved in the anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of toxic organic compounds. In natural environment the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. Here we show that the metabolically versatile phototrophic bacterium Rhodopseudomonas palustris may form its own syntrophic consortia, when it grows anaerobically on p-coumarate or benzoate as a sole carbon source. In the study we reveal the consortia from a comparison of large-scale measurements of mRNA and protein expressions under p-coumarate andmore » benzoate degrading conditions using a novel computational approach referred as phenotype fingerprinting. In this approach marker genes for known R. palustris phenotypes are employed to calculate their expression from the gene and protein expressions in each studied condition. Subpopulations of the consortia are inferred from the expression of phenotypes and known metabolic modes of the R. palustris growth. We find that p-coumarate degrading condition leads to at least three R. palustris subpopulations utilizing p-coumarate, benzoate, and CO2 and H2. Benzoate degrading condition also produces at least three subpopulations utilizing benzoate, CO2 and H2, and N2 and formate. Communication among syntrophs and inter-syntrophic dynamics in each consortium are indicated by up-regulation of transporters and genes involved in the curli formation and chemotaxis. The photoautotrphic subpopulation found in both consortia is characterized by activation of two cbb operons and the uptake hydrogenase system. A specificity of N2-fixing subpopulation in the benzoate degrading consortium is the preferential activation of the vanadium nitrogenase over the molybdenum nitrogenase. The N2-fixing subpopulation in the consortium is confirmed by consumption of dissolved nitrogen gas under the benzoate degrading conditions.« less
  • To assess the applicability of latex cell coatings as an ‘off-the-shelf’ biocatalyst, the effect of osmoprotectants, temperature, humidity and O 2 on preservation of H 2 production in Rhodopseudomonas palustris coatings was evaluated. Immediately following latex coating coalescence (24 h) and for up to 2 weeks of dry storage, rehydrated coatings containing different osmoprotectants displayed similar rates of H2 production. Beyond 2 weeks of storage, sorbitol-treated coatings lost all H 2 production activity, whereas considerable H 2 production was still detected in sucrose- and trehalose-stabilized coatings. The relative humidity level at which the coatings were stored had a significant impactmore » on the recovery and subsequent rates of H 2 production. After 4 weeks storage under air at 60% humidity, coatings produced only trace amounts of H 2 (0–0.1% headspace accumulation), whereas those stored at < 5% humidity retained 27–53% of their H 2 production activity after 8 weeks of storage. In conWhen stored in argon at < 5% humidity and room temperature, R. palustris coatings retained full H 2 production activity for 3 months, implicating oxidative damage as a key factor limiting coating storage. Overall, the results demonstrate that biocatalytic latex coatings are an attractive cell immobilization platform for preservation of bioactivity in the dry state.« less
  • Rhodopseudomonas palustris grows anaerobically on structurally diverse aromatic compounds and has served as a model organism in studies of anaerobic aromatic compound degradation. The authors examined {Delta}-1-chca degradation by intact cells of R. paulustris and reexamined enzymatic activities proposed to be involved in the conversion of {Delta}-1-chca to a ring cleavage product. The study confirmed the proposed degradation pathway illustrated. Profiles of intracellular metabolites formed during short-term incubations of whole cells indicated that benzoate grown and {Delta}-1-chc grown cells degrade benzoate by the same route. 27 refs., 6 figs., 3 tabs.
  • Alkali-treated extracts of Rhodopseudomonas palustris growing photosynthetically on benzoate were examined by gas chromatography/mass spectrometry for partially reduced benzoate derivatives. Two cyclic dienes, cyclohexa-2,5-diene-1-carboxylate and cyclohexa-1,4-diene-1-carboxylate, were detected. Either compound supported cell growth as effectively as benzoate. These results suggest that these cyclohexadienecarboxylates, probably as their coenzyme A esters, are the initial reduction products formed during anaerobic benzoate metabolism by R. palustris.
  • A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 ..mu..mol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg/sup 2 +/ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent K/sub m/ valuesmore » of 0.6 to 2 ..mu..M for benzoate, 2 to 3 ..mu..M for ATP, and 90 to 120 ..mu..M for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.« less