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Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides

Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron...

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Autores principales: Chan, Chi Ho, Levar, Caleb E., Jiménez-Otero, Fernanda, Bond, Daniel R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585712/
https://www.ncbi.nlm.nih.gov/pubmed/28674067
http://dx.doi.org/10.1128/JB.00340-17
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author Chan, Chi Ho
Levar, Caleb E.
Jiménez-Otero, Fernanda
Bond, Daniel R.
author_facet Chan, Chi Ho
Levar, Caleb E.
Jiménez-Otero, Fernanda
Bond, Daniel R.
author_sort Chan, Chi Ho
collection PubMed
description Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron acceptors that require contact, such as insoluble metal oxides. To directly investigate the genetic basis of electrode-based respiration, we constructed Geobacter sulfurreducens transposon-insertion sequencing (Tn-Seq) libraries for growth, with soluble fumarate or an electrode as the electron acceptor. Libraries with >33,000 unique insertions and an average of 9 insertions/kb allowed an assessment of each gene's fitness in a single experiment. Mutations in 1,214 different genomic features impaired growth with fumarate, and the significance of 270 genes unresolved by annotation due to the presence of one or more functional homologs was determined. Tn-Seq analysis of −0.1 V versus standard hydrogen electrode (SHE) electrode-grown cells identified mutations in a subset of genes encoding cytochromes, processing systems for proline-rich proteins, sensory networks, extracellular structures, polysaccharides, and metabolic enzymes that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of select genes identified via Tn-Seq revealed a new putative porin-cytochrome conduit complex (extABCD) crucial for growth with electrodes, which was not required for Fe(III) oxide reduction. In addition, four mutants lacking components of a putative methyl-accepting chemotaxis–cyclic dinucleotide sensing network (esnABCD) were defective in electrode colonization but grew normally with Fe(III) oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to Fe(III) oxides. IMPORTANCE Since metal oxide electron acceptors are insoluble, one hypothesis is that cells sense and reduce metals using the same molecular mechanisms used to form biofilms on electrodes and produce electricity. However, by simultaneously comparing thousands of Geobacter sulfurreducens transposon mutants undergoing electrode-dependent respiration, we discovered new cytochromes and chemosensory proteins supporting growth with electrodes that are not required for metal respiration. This supports an emerging model where G. sulfurreducens recognizes surfaces and forms conductive biofilms using mechanisms distinct from those used for growth with metal oxides. These findings provide a possible explanation for studies that correlate electricity generation with syntrophic interspecies electron transfer by Geobacter and reveal many previously unrecognized targets for engineering this useful capability in other organisms.
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spelling pubmed-55857122017-09-13 Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides Chan, Chi Ho Levar, Caleb E. Jiménez-Otero, Fernanda Bond, Daniel R. J Bacteriol Research Article Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron acceptors that require contact, such as insoluble metal oxides. To directly investigate the genetic basis of electrode-based respiration, we constructed Geobacter sulfurreducens transposon-insertion sequencing (Tn-Seq) libraries for growth, with soluble fumarate or an electrode as the electron acceptor. Libraries with >33,000 unique insertions and an average of 9 insertions/kb allowed an assessment of each gene's fitness in a single experiment. Mutations in 1,214 different genomic features impaired growth with fumarate, and the significance of 270 genes unresolved by annotation due to the presence of one or more functional homologs was determined. Tn-Seq analysis of −0.1 V versus standard hydrogen electrode (SHE) electrode-grown cells identified mutations in a subset of genes encoding cytochromes, processing systems for proline-rich proteins, sensory networks, extracellular structures, polysaccharides, and metabolic enzymes that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of select genes identified via Tn-Seq revealed a new putative porin-cytochrome conduit complex (extABCD) crucial for growth with electrodes, which was not required for Fe(III) oxide reduction. In addition, four mutants lacking components of a putative methyl-accepting chemotaxis–cyclic dinucleotide sensing network (esnABCD) were defective in electrode colonization but grew normally with Fe(III) oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to Fe(III) oxides. IMPORTANCE Since metal oxide electron acceptors are insoluble, one hypothesis is that cells sense and reduce metals using the same molecular mechanisms used to form biofilms on electrodes and produce electricity. However, by simultaneously comparing thousands of Geobacter sulfurreducens transposon mutants undergoing electrode-dependent respiration, we discovered new cytochromes and chemosensory proteins supporting growth with electrodes that are not required for metal respiration. This supports an emerging model where G. sulfurreducens recognizes surfaces and forms conductive biofilms using mechanisms distinct from those used for growth with metal oxides. These findings provide a possible explanation for studies that correlate electricity generation with syntrophic interspecies electron transfer by Geobacter and reveal many previously unrecognized targets for engineering this useful capability in other organisms. American Society for Microbiology 2017-09-05 /pmc/articles/PMC5585712/ /pubmed/28674067 http://dx.doi.org/10.1128/JB.00340-17 Text en Copyright © 2017 Chan et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Chan, Chi Ho
Levar, Caleb E.
Jiménez-Otero, Fernanda
Bond, Daniel R.
Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides
title Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides
title_full Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides
title_fullStr Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides
title_full_unstemmed Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides
title_short Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides
title_sort genome scale mutational analysis of geobacter sulfurreducens reveals distinct molecular mechanisms for respiration and sensing of poised electrodes versus fe(iii) oxides
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585712/
https://www.ncbi.nlm.nih.gov/pubmed/28674067
http://dx.doi.org/10.1128/JB.00340-17
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