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Conjugative plasmids inhibit extracellular electron transfer in Geobacter sulfurreducens.

ABSTRACT: Geobacter sulfurreducens is part of a specialized group of microbes with the unique ability to exchange electrons with insoluble materials, such as iron oxides and electrodes. Therefore, G. sulfurreducens plays an essential role in the biogeochemical iron cycle and microbial electrochemical systems. In G. sulfurreducens this ability is primarily dependent on electrically conductive nanowires that link internal electron flow from metabolism to solid electron acceptors in the extracellular environment. Here we show that when carrying conjugative plasmids, which are self-transmissible plasmids that are ubiquitous in environmental bacteria, G. sulfurreducens reduces insoluble iron oxides at much slower rates. This was the case for all three conjugative plasmids tested (pKJK5, RP4 and pB10). Growth with electron acceptors that do not require expression of nanowires was, on the other hand, unaffected. Furthermore, iron oxide reduction was also inhibited in Geobacter chapellei, but not in Shewanella oneidensis where electron export is nanowire-independent. As determined by transcriptomics, presence of pKJK5 reduces transcription of several genes that have been shown to be implicated in extracellular electron transfer in G. sulfurreducens, including pilA and omcE. These results suggest that conjugative plasmids can in fact be very disadvantageous for the bacterial host by imposing specific phenotypic changes, and that these plasmids may contribute to shaping the microbial composition in electrode-respiring biofilms in microbial electrochemical reactors.

SUBMITTER: Fessler M

PROVIDER: S-EPMC10063792 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Conjugative plasmids inhibit extracellular electron transfer in Geobacter sulfurreducens.

Fessler Mathias M Madsen Jonas Stenløkke JS Zhang Yifeng Y

Frontiers in microbiology 20230317

Geobacter sulfurreducens is part of a specialized group of microbes with the unique ability to exchange electrons with insoluble materials, such as iron oxides and electrodes. Therefore, G. sulfurreducens plays an essential role in the biogeochemical iron cycle and microbial electrochemical systems. In G. sulfurreducens this ability is primarily dependent on electrically conductive nanowires that link internal electron flow from metabolism to solid electron acceptors in the ...[more]

PMID: 37007462

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Project description:Multiheme cytochromes have been implicated in Geobacter sulfurreducens extracellular electron transfer (EET). These proteins are potential targets to improve EET and enhance bioremediation and electrical current production by G. sulfurreducens. However, the functional characterization of multiheme cytochromes is particularly complex due to the co-existence of several microstates in solution, connecting the fully reduced and fully oxidized states. Over the last decade, new strategies have been developed to characterize multiheme redox proteins functionally and structurally. These strategies were used to reveal the functional mechanism of G. sulfurreducens multiheme cytochromes and also to identify key residues in these proteins for EET. In previous studies, we set the foundations for enhancement of the EET abilities of G. sulfurreducens by characterizing a family of five triheme cytochromes (PpcA-E). These periplasmic cytochromes are implicated in electron transfer between the oxidative reactions of metabolism in the cytoplasm and the reduction of extracellular terminal electron acceptors at the cell's outer surface. The results obtained suggested that PpcA can couple e(-)/H(+) transfer, a property that might contribute to the proton electrochemical gradient across the cytoplasmic membrane for metabolic energy production. The structural and functional properties of PpcA were characterized in detail and used for rational design of a family of 23 single site PpcA mutants. In this review, we summarize the functional characterization of the native and mutant proteins. Mutants that retain the mechanistic features of PpcA and adopt preferential e(-)/H(+) transfer pathways at lower reduction potential values compared to the wild-type protein were selected for in vivo studies as the best candidates to increase the electron transfer rate of G. sulfurreducens. For the first time G. sulfurreducens strains have been manipulated by the introduction of mutant forms of essential proteins with the aim to develop and improve bioelectrochemical technologies.

Project description:BackgroundA new strain of Geobacter sulfurreducens, strain KN400, produces more electrical current in microbial fuel cells and reduces insoluble Fe(III) oxides much faster than the wildtype strain, PCA. The genome of KN400 was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved.ResultsBoth genomes were re-annotated, resulting in 14 fewer genes (net) in the PCA genome; 28 fewer (net) in the KN400 genome; and ca. 400 gene start and stop sites moved. 96% of genes in KN400 had clear orthologs with conserved synteny in PCA. Most of the remaining genes were in regions of genomic mobility and were strain-specific or conserved in other Geobacteraceae, indicating that the changes occurred post-divergence. There were 27,270 single nucleotide polymorphisms (SNP) between the genomes. There was significant enrichment for SNP locations in non-coding or synonymous amino acid sites, indicating significant selective pressure since the divergence. 25% of orthologs had sequence differences, and this set was enriched in phosphorylation and ATP-dependent enzymes. Substantial sequence differences (at least 12 non-synonymous SNP/kb) were found in 3.6% of the orthologs, and this set was enriched in cytochromes and integral membrane proteins. Genes known to be involved in electron transport, those used in the metabolic cell model, and those that exhibit changes in expression during growth in microbial fuel cells were examined in detail.ConclusionsThe improvement in external electron transfer in the KN400 strain does not appear to be due to novel gene acquisition, but rather to changes in the common metabolic network. The increase in electron transfer rate and yield in KN400 may be due to changes in carbon flux towards oxidation pathways and to changes in ATP metabolism, both of which indicate that the overall energy state of the cell may be different. The electrically conductive pili appear to be unchanged, but cytochrome folding, localization, and redox potentials may all be affected, which would alter the electrical connection between the cell and the substrate.

Project description:UnlabelledIt has been proposed that Geobacter sulfurreducens requires conductive pili for long-range electron transport to Fe(III) oxides and for high-density current production in microbial fuel cells. In order to investigate this further, we constructed a strain of G. sulfurreducens, designated Aro-5, which produced pili with diminished conductivity. This was accomplished by modifying the amino acid sequence of PilA, the structural pilin protein. An alanine was substituted for each of the five aromatic amino acids in the carboxyl terminus of PilA, the region in which G. sulfurreducens PilA differs most significantly from the PilAs of microorganisms incapable of long-range extracellular electron transport. Strain Aro-5 produced pili that were properly decorated with the multiheme c-type cytochrome OmcS, which is essential for Fe(III) oxide reduction. However, pili preparations of the Aro-5 strain had greatly diminished conductivity and Aro-5 cultures were severely limited in their capacity to reduce Fe(III) compared to the control strain. Current production of the Aro-5 strain, with a graphite anode serving as the electron acceptor, was less than 10% of that of the control strain. The conductivity of the Aro-5 biofilms was 10-fold lower than the control strain's. These results demonstrate that the pili of G. sulfurreducens must be conductive in order for the cells to be effective in extracellular long-range electron transport.ImportanceExtracellular electron transfer by Geobacter species plays an important role in the biogeochemistry of soils and sediments and has a number of bioenergy applications. For example, microbial reduction of Fe(III) oxide is one of the most geochemically significant processes in anaerobic soils, aquatic sediments, and aquifers, and Geobacter organisms are often abundant in such environments. Geobacter sulfurreducens produces the highest current densities of any known pure culture, and close relatives are often the most abundant organisms colonizing anodes in microbial fuel cells that harvest electricity from wastewater or aquatic sediments. The finding that a strain of G. sulfurreducens that produces pili with low conductivity is limited in these extracellular electron transport functions provides further insight into these environmentally significant processes.

Project description:CsrA is a post-transcriptional regulator that controls biofilm formation, virulence, carbon metabolism, and motility, among other phenotypes in bacteria. Although CsrA has been extensively studied in γ-proteobacteria and firmicute, it is unknown which cellular processes are targeted for regulation in δ-proteobacteria. In this work, we constructed and characterized the ΔcsrA mutant strain in Geobacter sulfurreducens to determine the involvement of the CsrA protein in the regulation of biofilm and extracellular electron transfer. The ΔcsrA mutant strain grows on acetate-fumarate and reduces soluble Fe(III) similarly to the wild type strain, but has higher rates of insoluble Fe(III) reduction than the wild type. Quantification and characterization of biofilms by violet staining and confocal laser scanning microscopy showed that the ΔcsrA strain produces up to twice as much biofilm as the wild type strain, containing more than 95% viable cells. Transcriptome analysis by RNA-seq shows that in ΔcsrA biofilms developed on an inert support, 244 (103 upregulated and 141 downregulated) genes changed their expression, including those related to extracellular electron transfer, exopolysaccharide synthesis, c-di-GMP synthesis and degradation. To validate the transcriptome data, RT-qPCR confirmed the differential expression of several selected genes in the ΔcsrA strain. Current production in microbial fuel cells was tested and the ΔcsrA strain was found to produce 45-50% more current than the wild type. To verify the genes that changed expression in the graphite electrodes in the ΔcsrA strain, a transcriptome analysis was performed. 181 genes changed their expression in the ΔcsrA biofilms of which 113 genes showed differentially expression only in MFC and 68 genes changed their expression as well as transcriptome from biofilms grown on inert support. In silico analysis of the 5'-UTR regions revealed that 76 genes that changed expression in the RNA-seq analysis have a consensus sequence for CsrA binding. This is the first report describing the involvement of CsrA in the regulation of extracellular electron transfer and biofilm in a member of the γ-proteobacteria.

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Conjugative plasmids inhibit extracellular electron transfer in Geobacter sulfurreducens.

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Conjugative plasmids inhibit extracellular electron transfer in <i>Geobacter sulfurreducens</i>.

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