Pili Expression in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor

Multiple lines of evidence suggest that electrically conductive pili (e-pili) are an important conduit for long-range electron transport in Geobacter sulfurreducens, a common model microbe for the study of extracellular electron transport mechanisms. One strategy to study the function of e-pili has been to delete the gene for PilB, the pilus assembly motor protein, in order to prevent e-pili expression. However, we found that e-pili are still expressed after the gene for PilB is deleted. Conducting probe atomic force microscopy revealed filaments with the same diameter and similar current-voltage response as e-pili harvested from wild-type G. sulfurreducens or when e-pili are heterologously expressed from the G. sulfurreducens pilin gene in E. coli. Immunogold labeling demonstrated that a G. sulfurreducens strain expressing e-pili with a His-tag continued to express His-tag labelled e-pili when the PilB gene was deleted. Strains with the PilB gene deleted produced maximum current densities comparable to wild-type controls. These results demonstrate that deleting the gene for PilB is not an appropriate strategy for constructing strains of G. sulfurreducens without e-pili, necessitating a reinterpretation of the results of previous studies that have employed this approach.

Download Full-text

Direct Observation of Electrically Conductive Pili Emanating from Geobacter sulfurreducens

10.1101/2021.07.06.451359 ◽

2021 ◽

Author(s):

Xinying Liu ◽

David Jeffrey Fraser Walker ◽

Stephen Nonnenmann ◽

Dezhi Sun ◽

Derek R. Lovley

Keyword(s):

Electron Transfer ◽

Electron Transport ◽

Long Range ◽

Geobacter Sulfurreducens ◽

Metal Corrosion ◽

Extracellular Electron Transfer ◽

Wild Type ◽

Electrically Conductive ◽

In The Wild ◽

Pilin Gene

Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens. However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme c-type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90 % of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in E. coli from the G. sulfurreducens pilin gene. The remaining 10% of filaments had a morphology consistent with filaments comprised of the c-type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90 % filaments with a 3 nm diameter, but greatly reduced conductance, further indicating that the 3 nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface c-type cytochromes, including OmcS, was deleted yielded only 3 nm diameter filaments with the same conductance as in the wild-type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens, consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer.

Download Full-text

Aromatic Amino Acids Required for Pili Conductivity and Long-Range Extracellular Electron Transport in Geobacter sulfurreducens

mBio ◽

10.1128/mbio.00105-13 ◽

2013 ◽

Vol 4 (2) ◽

Cited By ~ 68

Author(s):

Madeline Vargas ◽

Nikhil S. Malvankar ◽

Pier-Luc Tremblay ◽

Ching Leang ◽

Jessica A. Smith ◽

...

Keyword(s):

Amino Acids ◽

Electron Transport ◽

Long Range ◽

Microbial Fuel Cells ◽

Aromatic Amino Acids ◽

Geobacter Sulfurreducens ◽

Control Strain ◽

Content Type ◽

Current Production ◽

Extracellular Electron Transport

ABSTRACTIt has been proposed thatGeobacter sulfurreducensrequires 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 ofG. 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 whichG. sulfurreducensPilA 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 multihemec-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 ofG. sulfurreducensmust be conductive in order for the cells to be effective in extracellular long-range electron transport.IMPORTANCEExtracellular electron transfer byGeobacterspecies 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, andGeobacterorganisms are often abundant in such environments.Geobacter sulfurreducensproduces 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 ofG. sulfurreducensthat produces pili with low conductivity is limited in these extracellular electron transport functions provides further insight into these environmentally significant processes.

Download Full-text

Application of electrochemical surface plasmon resonance (ESPR) to the study of electroactive microbial biofilms

Physical Chemistry Chemical Physics ◽

10.1039/c8cp03898h ◽

2018 ◽

Vol 20 (40) ◽

pp. 25648-25656 ◽

Cited By ~ 4

Author(s):

Joel Golden ◽

Matthew D. Yates ◽

Michelle Halsted ◽

Leonard Tender

Keyword(s):

Surface Plasmon Resonance ◽

Electron Transport ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Electrode Surface ◽

Geobacter Sulfurreducens ◽

Microbial Biofilms ◽

Electrode Interface ◽

Extracellular Electron Transport

Results reveal that for an electrode-grown Geobacter sulfurreducens biofilm, as much as 70% of cytochrome hemes residing within hundreds of nanometers from the electrode surface store electrons even as extracellular electron transport is occurring across the biofilm/electrode interface.

Download Full-text

Thermally activated long range electron transport in living biofilms

Physical Chemistry Chemical Physics ◽

10.1039/c5cp05152e ◽

2015 ◽

Vol 17 (48) ◽

pp. 32564-32570 ◽

Cited By ~ 77

Author(s):

Matthew D. Yates ◽

Joel P. Golden ◽

Jared Roy ◽

Sarah M. Strycharz-Glaven ◽

Stanislav Tsoi ◽

...

Keyword(s):

Electron Transport ◽

Long Range ◽

Geobacter Sulfurreducens ◽

Electron Hopping ◽

Thermally Activated ◽

Conductivity Electron ◽

Extracellular Electron Transport ◽

Conductive Surfaces

The rate of extracellular electron transport through living, electrode-grown Geobacter sulfurreducens biofilms decreases with decreasing temperature, consistent with incoherent redox conductivity (electron hopping) among hemes of c-type cytochromes to conductive surfaces.

Download Full-text

Improper Localization of the OmcS Cytochrome May Explain the Inability of thexapD-Deficient Mutant ofGeobacter sulfurreducensto Reduce Fe(III) Oxide

10.1101/114900 ◽

2017 ◽

Cited By ~ 8

Author(s):

Kelly A. Flanagan ◽

Ching Leang ◽

Joy E. Ward ◽

Derek R. Lovley

Keyword(s):

Electron Transfer ◽

Electron Transport ◽

Type Strain ◽

Wild Type Strain ◽

Geobacter Sulfurreducens ◽

Extracellular Electron Transfer ◽

Outer Cell ◽

Wild Type ◽

Redox Active ◽

In The Wild

AbstractExtracellular electron transfer through a redox-active exopolysaccharide matrix has been proposed as a strategy for extracellular electron transfer to Fe(III) oxide byGeobacter sulfurreducens,based on the phenotype of axapD-deficient strain. Central to this model was the assertion that thexapD-deficient strain produced pili decorated with the multi-hemec-type cytochrome OmcS in manner similar to the wild-type strain. Further examination of thexapD-deficient strain with immunogold labeling of OmcS and transmission electron microscopy revealed that OmcS was associated with the outer cell surface rather than pili. PilA, the pilus monomer, could not be detected in thexapD-deficient strain under conditions in which it was readily detected in the wild-type strain. Multiple lines of evidence in previous studies have suggested that long-range electron transport to Fe(III) oxides proceeds through electrically conductive pili and that OmcS associated with the pili is necessary for electron transfer from the pili to Fe(III) oxides. Therefore, an alternative explanation for the Fe(III) oxide reduction phenotype of thexapD-deficientstrain is that the pili-OmcS route for extracellular electron transport to Fe(III) oxide has been disrupted in thexapD-deficient strain.

Download Full-text

Adaptation to Disruption of the Electron Transfer Pathway for Fe(III) Reduction in Geobacter sulfurreducens

Journal of Bacteriology ◽

10.1128/jb.187.17.5918-5926.2005 ◽

2005 ◽

Vol 187 (17) ◽

pp. 5918-5926 ◽

Cited By ~ 53

Author(s):

Ching Leang ◽

L. A. Adams ◽

K.-J. Chin ◽

K. P. Nevin ◽

B. A. Methé ◽

...

Keyword(s):

Electron Transfer ◽

Steady State ◽

Electron Transport ◽

Outer Membrane ◽

Geobacter Sulfurreducens ◽

Significant Shift ◽

Deficient Mutant ◽

Wild Type ◽

Transcript Levels ◽

Electron Transfer Pathway

ABSTRACT Previous studies demonstrated that an outer membrane c-type cytochrome, OmcB, was involved in Fe(III) reduction in Geobacter sulfurreducens. An OmcB-deficient mutant was greatly impaired in its ability to reduce both soluble and insoluble Fe(III). Reintroducing omcB restored the capacity for Fe(III) reduction at a level proportional to the level of OmcB production. Here, we report that the OmcB-deficient mutant gradually adapted to grow on soluble Fe(III) but not insoluble Fe(III). The adapted OmcB-deficient mutant reduced soluble Fe(III) at a rate comparable to that of the wild type, but the cell yield of the mutant was only ca. 60% of that of the wild type under steady-state culturing conditions. Analysis of proteins and transcript levels demonstrated that expression of several membrane-associated cytochromes was higher in the adapted mutant than in the wild type. Further comparison of transcript levels during steady-state growth on Fe(III) citrate with a whole-genome DNA microarray revealed a significant shift in gene expression in an apparent attempt to adapt metabolism to the impaired electron transport to Fe(III). These results demonstrate that, although there are many other membrane-bound c-type cytochromes in G. sulfurreducens, increased expression of these cytochromes cannot completely compensate for the loss of OmcB. The concept that outer membrane cytochromes are promiscuous reductases that are interchangeable in function appears to be incorrect. Furthermore, the results indicate that there may be different mechanisms for electron transfer to soluble Fe(III) and insoluble Fe(III) oxides in G. sulfurreducens, which emphasizes the importance of studying electron transport to the environmentally relevant Fe(III) oxides.

Download Full-text

Use of Interdigitated Microelectrode Arrays (IDAs) to Investigate Extracellular Electron Transport Through Geobacter Sulfurreducens Biofilms

ECS Meeting Abstracts ◽

10.1149/ma2012-01/39/1442 ◽

2012 ◽

Keyword(s):

Electron Transport ◽

Microelectrode Arrays ◽

Geobacter Sulfurreducens ◽

Interdigitated Microelectrode ◽

Extracellular Electron Transport

Download Full-text

GeobacterStrains Expressing Poorly Conductive Pili Reveal Constraints on Direct Interspecies Electron Transfer Mechanisms

mBio ◽

10.1128/mbio.01273-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 26

Author(s):

Toshiyuki Ueki ◽

Kelly P. Nevin ◽

Amelia-Elena Rotaru ◽

Li-Ying Wang ◽

Joy E. Ward ◽

...

Keyword(s):

Electron Transfer ◽

Electron Flow ◽

Geobacter Sulfurreducens ◽

Wild Type ◽

Anaerobic Digesters ◽

Content Type ◽

Direct Interspecies Electron Transfer ◽

Interspecies Electron Transfer ◽

Anaerobic Soils ◽

Pilin Gene

ABSTRACTCytochrome-to-cytochrome electron transfer and electron transfer along conduits of multiple extracellular magnetite grains are often proposed as strategies for direct interspecies electron transfer (DIET) that do not require electrically conductive pili (e-pili). However, physical evidence for these proposed DIET mechanisms has been lacking. To investigate these possibilities further, we constructedGeobacter metallireducensstrain Aro-5, in which the wild-type pilin gene was replaced with thearo-5pilin gene that was previously shown to yield poorly conductive pili inGeobacter sulfurreducensstrain Aro-5.G. metallireducensstrain Aro-5 did not reduce Fe(III) oxide and produced only low current densities, phenotypes consistent with expression of poorly conductive pili. LikeG. sulfurreducensstrain Aro-5,G. metallireducensstrain Aro-5 displayed abundant outer surface cytochromes. Cocultures initiated with wild-typeG. metallireducensas the electron-donating strain andG. sulfurreducensstrain Aro-5 as the electron-accepting strain grew via DIET. However,G. metallireducensAro-5/G. sulfurreducenswild-type cocultures did not. Cocultures initiated with the Aro-5 strains of both species grew only when amended with granular activated carbon (GAC), a conductive material known to be a conduit for DIET. Magnetite could not substitute for GAC. The inability of the two Aro-5 strains to adapt for DIET in the absence of GAC suggests that there are physical constraints on establishing DIET solely through cytochrome-to-cytochrome electron transfer or along chains of magnetite. The finding that DIET is possible with electron-accepting partners that lack highly conductive pili greatly expands the range of potential electron-accepting partners that might participate in DIET.IMPORTANCEDIET is thought to be an important mechanism for interspecies electron exchange in natural anaerobic soils and sediments in which methane is either produced or consumed, as well as in some photosynthetic mats and anaerobic digesters converting organic wastes to methane. Understanding the potential mechanisms for DIET will not only aid in modeling carbon and electron flow in these geochemically significant environments but will also be helpful for interpreting meta-omic data from as-yet-uncultured microbes in DIET-based communities and for designing strategies to promote DIET in anaerobic digesters. The results demonstrate the need to develop a better understanding of the diversity of types of e-pili in the microbial world to identify potential electron-donating partners for DIET. Novel methods for recovering as-yet-uncultivated microorganisms capable of DIET in culture will be needed to further evaluate whether DIET is possible without e-pili in the absence of conductive materials such as GAC.

Download Full-text

Going Wireless: Fe(III) Oxide Reduction without Pili by Geobacter sulfurreducens Strain JS-1

Applied and Environmental Microbiology ◽

10.1128/aem.01122-14 ◽

2014 ◽

Vol 80 (14) ◽

pp. 4331-4340 ◽

Cited By ~ 54

Author(s):

Jessica A. Smith ◽

Pier-Luc Tremblay ◽

Pravin Malla Shrestha ◽

Oona L. Snoeyenbos-West ◽

Ashley E. Franks ◽

...

Keyword(s):

Electron Transfer ◽

Electron Transport ◽

Type Strain ◽

Culture Medium ◽

Wild Type Strain ◽

Geobacter Sulfurreducens ◽

Electron Shuttle ◽

Oxide Reduction ◽

Wild Type ◽

Content Type

ABSTRACTPrevious studies have suggested that the conductive pili ofGeobacter sulfurreducensare essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain ofG. sulfurreducensreduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of thec-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every 3 days, the wild-type strain outcompeted the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA being continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron shuttle-producing Fe(III) reducers in many anaerobic soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current, consistent with the concept that long-range electron transport throughG. sulfurreducensbiofilms is more effective via pili.

Download Full-text