Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
University of Massachusetts, Amherst
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Nanowires, Capacitors, and Other Novel Electron Transfer Mechanisms in Geobacter Species Elucidated from Genome-Scale Investigations
Gemma Reguera1* (greguera@microbio.umass.edu), Teena Mehta1, Dawn E. Holmes1, Abraham Esteve-Núñez1, Jessica Butler1, Barbara Methe2, Kelly Nevin1, Swades K. Chaudhuri1, Richard Glaven1, Tunde Mester1, Raymond DiDonato1, Kevin McCarthy1, Mark T. Tuominen1, and Derek Lovley1
1University of Massachusetts, Amherst, MA and 2The Institute for Genomic Research, Rockville, MD
Molecular ecology studies have demonstrated that Geobacteraceae are the predominant microorganisms involved in in situ bioremediation of uranium-contaminated groundwater and on the surface of electrodes harvesting electricity from waste organic matter. However, there has been little information on how these organisms transfer electrons outside the cell onto insoluble electron acceptors, such as metals and electrodes, or alternative mechanisms for respiration.
Insoluble Fe(III) oxides are the primary electron acceptor supporting the growth of Geobacter species in subsurface environments, including during uranium bioremediation. Previous studies on electron transfer to Fe(III) oxides in metal-reducing microorganisms have primarily focused on outer-membrane c-type cytochromes functioning as the terminal electron carriers that transfer electrons onto Fe(III) oxides. However, analysis of the genomes of two Pelobacter species, which are also members of the Geobacteraceae indicated that these organisms lacked genes for the outer-membrane cytochromes that are prevalent in Geobacter species. Yet, Pelobacter species can reduce Fe(III) oxide. If it is assumed that the same mechanism for extracellular electron transfer to Fe(III) oxide is conserved within the Geobacteraceae, then these results suggested that outer-membrane c-type cytochromes are not the Fe(III) oxide reductase.
Comparison of the available Geobacteraceae genomes indicated that the genes for pili, are highly conserved and expression studies indicated that PilA, the structural pilin protein, was expressed during growth on Fe(III) oxide, but not soluble, chelated Fe(III). A mutant of Geobacter sulfurreducens in which pilA was deleted could reduce soluble electron acceptors, including Fe(III) citrate, as well as wild-type but could not reduce Fe(III) oxide. Complementation with a functional pilA restored the capacity for Fe(III) oxide reduction. Based on the role of pili in other organisms, it was hypothesized that the pili were required for G. sulfurreducens to attach to Fe(III) oxides, but the pilA mutant attached to Fe(III) oxide as well as the wild-type, suggesting a novel role for pili in Fe(III) oxide reduction. Conducting-probe atomic force microscopy revealed that the pili were highly conductive. In contrast, non-pilin proteins had no detectable conductivity and in instances in which the non-pilin proteins covered the pili filaments, they insulated the pili from the conductive tip. No conductivity was detected in the pili of Shewanella oneidensis, which, unlike Geobacter species, does not need to contact Fe(III) oxides in order to reduce them. These results suggest that the pili of G. sulfurreducens serve as biological nanowires, transferring electrons from the cell surface to the surface of Fe(III) oxides. Electron transfer via pili suggests possibilities for other unique cell-surface and cell-cell interactions, and for bioengineering of novel conductive materials.
The finding that pili, rather than c-type cytochromes, are likely to be responsible for the final electron transfer to Fe(III) oxides leads to the question of why c-type cytochromes are so abundant in Geobacter species, both in quantity and number of genes in the genomes. Some c-type cytochromes, such as the small, periplasmic PpcA, which are highly conserved in Geobacter and Pelobacter genomes, may be intermediaries in electron transfer to Fe(III). However, for many outer-membrane cytochromes, there is little similarity in gene sequences in even closely related Geobacter species. Whole genome gene microarray and proteomic studies revealed much higher expression of multiple cytochrome genes under electron-acceptor limiting conditions. Further investigation suggested that the cytochromes behave as a capacitor capable of accepting electrons from energy-generating electron transfer reactions in the inner membrane and storing these electrons until a suitable electron acceptor is available. This explains how Geobacter species are able to thrive in subsurface environments in which insoluble Fe(III) oxides are heterogeneously dispersed because the capacitor cytochromes permit continued electron transfer during the search for new Fe(III) oxides followed by discharge to the Fe(III) oxide once a suitable source is found.
Surprisingly, the pilA mutant produced electricity as well as the wild-type, suggesting that electron transfer to electrodes proceeded via different mechanisms than electron transfer to Fe(III) oxides. Global analysis of gene expression in G. sulfurreducens with a whole-genome DNA microarray indicated that the outer-member c-type cytochrome gene, omcS, and its co-transcribed homolog, omcT, were the only genes coding for likely electron transfer proteins that were consistently up-regulated during growth on electrodes versus growth with Fe(III) as the electron acceptor. Quantitative PCR demonstrated that mRNA levels for these cytochromes increased as the amount of current harvested with the electrode increased. When omcS and omcT were deleted, current production decreased to ca. a third of the wild type and the potential of the anode went from –0.5 V in the wild type to only –0.15 in the mutant. Complementation of the omcS gene in the mutant restored current production and anode potential to values comparable to wild type. These results suggest that OmcS is likely to be the primary protein mediating electrical contact between the cell and the electrode surface. This finding offers several possibilities for engineering electrode surfaces and/or microorganisms to improve the function of microbial fuel cells.
Fumarate is an electron acceptor in some Geobacter species, such as G. sulfurreducens, but not in others, such as G. metallireducens. Yet the genomes of both organisms contained what appeared to be a heterotrimic type of fumarate reductase, frdCAB, homologous to the fumarate reductase of Wolinella succinogenes. Mutation of the putative catalytic subunit in G. sulfurreducens resulted in a strain that lacked fumarate reductase activity and was unable to respire fumarate. Furthermore, the mutant strain could not grow with acetate as the electron donor, regardless of electron acceptor, and lacked succinate dehydrogenase activity. Oxidation of acetate coupled to Fe(III) reduction was possible in the mutant strain if exogenous fumarate was provided, as fumarate could be converted to succinate through TCA cycle reactions and excreted. Highly similar genes were present in Geobacter metallireducens, which cannot respire fumarate. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, Geobacter species use the same enzyme for both fumarate reduction and succinate oxidation in vivo. This also represents the first example of genetic engineering of a Geobacter species for novel respiratory abilities.
Significant progress was also made in genome-enabled studies of oxygen respiration and novel outer-membrane proteins that were first reported at last year’s meeting and updates will be provided.
* Presenting author
