BACKGROUND
the ability to effectively transfer electrons directly onto insoluble, extracellular substances is one of the hallmark characteristics of geobacter species.
in the natural environment, geobacter species are commonly the most abundant microorganisms in anaerobic soils and sediments where microbial reduction of insoluble fe oxides is important  <cit> .
in addition, geobacter species can be used to make microbial fuel cells - devices in which electrical current is harvested from bacteria that grow by transferring electrons from their food source to the anode of the device  <cit> .
pure cultures of geobacter sulfurreducens produce current densities that are among the highest known  <cit> , and g. sulfurreducens has been enriched from a number of complex communities growing in biofilms on high-efficiency microbial fuel cells  <cit> .
however, the mechanisms for extracellular electron transfer in geobacter species are poorly understood  <cit> .
recently, a new strain of g. sulfurreducens, designated strain kn <dig>  was isolated that is much more effective than wildtype both in electron transfer to electrodes  <cit>  and in fe oxide reduction .
strain kn <dig> was isolated from a culture that grew by transferring electrons to an anode poised at a very low potential   <cit> .
compared to the wildtype strain, when growing in microbial fuel cells kn <dig> forms biofilms on the anode more rapidly; it produces much higher-density current  and power   <cit> ; and it has higher conductivity   <cit> .
because it was recently isolated, little is known about how kn <dig> differs from the wildtype strain.
here we present the analysis of the genome of kn <dig>  <cit>  and compare it to the genome of wildtype g. sulfurreducens pca  <cit>  in order to provide insight into both the phenotype of enhanced extracellular electron transfer and the evolutionary history of the species.
we identify the genes that are unique to each strain and identify polymorphisms between the genomes.
we analyze functional enrichment in the set of genes with sequence changes, and focus on differences in genes involved in electron transfer pathways from intracellular oxidation to electron transfer onto extracellular acceptors.
we use these analyses to suggest how the metabolic network of the species has evolved and how these differences caused the divergent phenotypes.
RESULTS
reannotation of the g. sulfurreducens strains kn <dig> and pca genomes
as published, the g. sulfurreducens strain pca genome was  <dig>  mb with  <dig> open reading frames   <cit>  and the g. sulfurreducens strain kn <dig> genome was  <dig>  mb with  <dig> orfs  <cit> .
because the two genomes were sequenced and annotated several years apart using different methods, we re-annotated both genomes, making several types of corrections in order to assure that comparisons between the two were valid.
orf predictions, start and stop sites of the genes, and functional predictions were compared and reconciled using identical methods on both genomes .
after the re-annotations, the strain kn <dig> genome had  <dig> orfs , and the strain pca genome had  <dig> orfs .
these re-annotations were deposited in the national center for biotechnology information genome database under the accession numbers of ae <dig>  for pca and nc_ <dig> for kn <dig>  a summary of the basic characteristics of the genomes is given in table  <dig>
whole-genome comparison of strain kn <dig> to strain pca
orthologs, proteins predicted to have the same function in both genomes, were identified using a combination of whole-genome alignment and all-versus-all protein sequence alignments.
a whole-genome nucleotide alignment of the pca and kn <dig> genomes showed that they were syntenic, with no large-scale rearrangements or inversions .
orthologs were identified for  <dig> of the  <dig> protein-coding genes in the kn <dig> genome .
a whole-proteome comparison identified orthologs for  <dig> genes .
the two methods agreed for  <dig>  of the pairs, with the genome method identifying  <dig> orthologs with identical sequences: eight transposases, two cytochromes, and a translation elongation factor, as well as three orthologs under  <dig> amino acids in length .the proteome method identified  <dig> orthologs with low sequence similarity or alignment over a short region of the sequence .
for the analysis of polymorphisms between orthologs presented below, we included orthologs identified by only one method if they aligned over at least 50% of the length of the longer protein, and had a sequence identity of at least 30%.
in total,  <dig> of the  <dig> orfs in the kn <dig> strain genome  have orthologs in the pca strain genome .
the relative locations of the conserved genes are largely preserved between the genomes .
genes unique to the kn <dig> strain
there were no orthologs in the pca strain for  <dig> of the orfs  in the kn <dig> strain genome .
in order to better to describe these genes and their evolutionary history, their locations and characteristics were mapped and their sequences were compared to proteins from the  <dig>  organisms in the reference sequence database from the national center for biotechnology information  <cit> .
seventeen of these  <dig> orfs had no significant similarity to any gene in another organism, six were most similar to proteins in the pca strain, and  <dig> had highest similarity to proteins in geobacteraceae species other than pca .
the remaining orfs were most similar to genes from a variety of phylogenetically diverse organisms including desulfovibrio, syntrophobacter, pseudomonas, and vibrio species , suggesting that there are diverse evolutionary histories for these genes.
the  <dig> genes are spread throughout the kn <dig> genome, the majority in clusters together .
in total, there are  <dig> regions in the kn <dig> genome where at least two consecutive genes have no ortholog in the pca genome .
eight are bordered by or contain transposase or integrase genes, three are bordered by trna genes, and one is bordered by a conserved repeated nucleic acid sequence  – indicating that these regions may be or once were mobile genetic elements  <cit> .
one strain-specific region, found at 466250– <dig> in the kn <dig> genome , is inserted relative to pca just downstream of a trna gene, between the orthologs to gsu <dig> and gsu <dig> .
this region encodes a phage integrase and replisome organizer as well as a restriction/modification system.
the two genes for the restriction/modification system  are similar to genes from meiothermus ruber, and are not found in any other geobacteraceae, suggesting that this region may have been acquired by lateral gene transfer.
another kn <dig> strain-specific region is found between 3479100– <dig>  here, kn400_3199-kn400_ <dig> are found between the orthologs to gsu <dig> and gsu <dig> .
the genes include five conserved hypothetical proteins and three proteins involved in metal transport and usage: a metal-transporting atpase, a metal-dependent phosphohydrolase, and a hybrid iron transport/cell signaling protein.
all of these genes are most similar to those found in other geobacteraceae and deltaproteobacteria , suggesting that they might have been lost in the pca strain genome.
in addition to regions of relative insertion in kn <dig>  there are others where, between regions of excellent conservation between the two genomes, there is a stretch of sequence for which there is little or no similarity.
for example, kn400_ <dig> and gsu <dig> are orthologs, and kn400_ <dig> and gsu <dig> are orthologs, but the region between them has low sequence similarity .
however, in both strains, the genes encode products involved in exopolysaccharide biosynthesis .
this may be a region where mutation accumulation was relatively unconstrained, and these genes are attractive targets for studies of differences in the biofilms formed by strains pca and kn <dig>
a few of the genes found in the kn <dig> genome but not the pca genome are isolated, not in the  <dig> clusters described above.
one of these that may have a substantial effect on metabolism is the catalase.
the kn400_ <dig> gene encodes a kate catalase  <cit> , while the catalase of g. sulfurreducens strain pca, gsu <dig>  encodes a katg-type  <cit>  of no sequence similarity.
while each strain encodes only one of the two catalases, there is evidence that each once encoded the other.
kn <dig> has a region with nucleic acid similarity to the katg gene , but a single-base deletion results in a frameshift halfway through it.
in the pca genome, the region 3089478– <dig> aligns with the 5’ end and the 3’ end of the kate gene in kn <dig>  though 90% of the gene has been deleted.
both kate and katg detoxify reactive oxygen species, but their mechanism and expression are different  <cit> .
response to oxidative stress is especially important in fe-reducing, obligate anaerobes like geobacter sulfurreducens – reactive oxygen species cause dna damage and disable many respiratory enzymes, and the presence of ferrous iron can create more of these compounds  <cit> .
a difference in response to oxidative stress between the strains could affect many aspects of growth.
genes unique to the pca strain
<dig> orfs in the pca genome  did not have orthologs in the kn <dig> genome .
gene ontology analysis showed that three types of genes were very highly enriched in this group relative to the whole genome: those involved in dna transposition, dna integration, and rna-dependent dna replication .
as with strain kn <dig>  these orfs were mostly found in clusters within the pca genome .
there are  <dig> regions in the pca genome in which at least two consecutive genes have no orthologs in the kn <dig> genome, .
twelve of these are bordered by  transposases, three by a trna gene, and two by conserved nucleic acid sequences.
compared to the kn <dig> strain, there are more transposases and more diverse types of transposases associated with these regions.
several of these non-orthologous regions in the pca genome are much larger than any of those found in the kn <dig> genome .
in the kn <dig> genome,  <dig> of the non-orthologous regions have at least ten genes, and the largest has  <dig> genes.
in the pca genome,  <dig> regions have at least ten genes, and the largest has  <dig> genes .
the largest region of non-orthology to kn <dig> is between 2316300– <dig> in the pca genome .
this region of  <dig> orfs  is inserted relative to the kn <dig> genome between kn400_ <dig> and a trna gene at  <dig>  two-thirds of the proteins encoded in this region are proteins of unknown function or transposases.
other proteins encoded include five putative transcriptional regulators and two efflux pumps .
the second largest region unique to the pca genome is between 49500– <dig> .
this region of  <dig> orfs  is inserted relative to strain kn <dig> between a trna gene and kn400_ <dig>  the majority of the proteins encoded here are also transposases and proteins of unknown function.
however, this region also encompasses the crispr <dig> locus, six crispr-associated genes and a toxin/antitoxin pair, genes involved in resistance to exogenous dna and potentially in gene regulation  <cit> .
in total, the kn <dig> genome is ca.
<dig> kb smaller than the pca genome, with the difference due to the ca.
<dig> genes in kn <dig> with no ortholog in pca and ca.
<dig> genes in pca with no ortholog in kn <dig>  these genes have a variety of different apparent histories – some are not found in any other organism, some are syntenic between the genomes but lack sequence homology, some appear to have been lost entirely in one of the strains, and some may have arisen from lateral gene transfer from unrelated species.
strain-specific genes of potential interest include a number for membrane transport and polysaccharide metabolism; a restriction/modification system; catalases; a disulfide bond formation operon unique to kn400; and a crispr locus unique to pca.
the evolutionary history of the mobile genetic elements that are enriched in these regions also warrants further investigation.
single nucleotide polymorphisms  between the pca and kn <dig> genomes
using an alignment of the two whole genomes,  <dig>  single nucleotide polymorphism sites and small insertions or deletions between the two strains were identified .
of these,  <dig>   were intragenic in the pca strain, with a rate of  <dig>  snp/kb in coding regions and  <dig>  snp/kb in non-coding regions.
within the  <dig> genes that had orthologs in both genomes, there were  <dig>  snp and  <dig> small-scale insertions or deletions .
the large majority  of the sequence differences between the kn <dig> and the pca orthologs were silent, meaning that the snp caused no changes to translation, and that the protein sequences of the orthologs were identical.
the remaining  <dig> snp  were non-synonymous – they caused a difference in amino acid sequence during translation .
one-third of genes had a change of sequence between the strains −  <dig> of the  <dig> orthologs had at least one snp, with an average of  <dig>  per gene  .
a rate of  <dig> snp/kb was two standard deviations above average.
one-quarter of proteins had a change of sequence between the strains –  <dig> of the  <dig> orthologs had at least one non-synonymous snp, with an average of  <dig>  per ortholog  .
the set of genes that had at least one non-synonymous snp was significantly enriched in genes for: c-type cytochromes, integral membrane proteins, phosphorus and phosphate metabolism, cofactor biosynthesis, and atp-dependent enzymes with a wide variety of functions including: transporters and exporters, helicases, mismatch repair proteins, excinucleases, proteases, and many sensor histidine kinases .
proteins were considered heavily mutated if the corresponding gene had at least  <dig> non-synonymous snp/kb, which was two standard deviations above average .
<dig> of the orthologs  were heavily mutated .
the set of heavily mutated proteins was significantly enriched in c-type cytochromes and in membrane-bound transport proteins .
some genes had only silent snp, or sequence changes that did not affect the resulting protein sequence, indicating that changes to their sequence may be under negative selective pressure.
solely silent changes were present in  <dig> of the orthologs  .
this set of genes was significantly enriched in genes involved in flagellum biogenesis and amino acid biosynthesis .
the kn <dig> strain, unlike the pca strain, produces flagella and is motile  <cit> .
the most notable silent-mutation-rich region of the genome was cluster gsu1095-gsu <dig>  which encodes a phosphate transporter and phosphate-dependent regulatory genes .
in order to better understand the phenotypic effects of the nucleotide polymorphisms between the strains, several metabolic networks involved in electron transfer out of the cell were examined in more detail.
sequence differences in central energy metabolism
g. sulfurreducens primarily grows by coupling the oxidation of acetate to the reduction of extracellular electron acceptors.
acetate is oxidized by the tca cycle, with the products used to generate a proton gradient for atp synthesis  <cit> .
the electrons that remain from this process are transferred out of the cell to fe or to electrodes.
in total,  <dig> genes in strain pca encode the proteins involved in acetate oxidation, and the kn <dig> strain has orthologs to each of these .
ten of the  <dig> contain a protein sequence change, with fewer per gene than the genome as a whole  .
none are highly mutated.
separately from energy generation, a portion of acetate is shunted to a different pathway and used as the sole source for biomass synthesis via the gluconeogenic and fatty acid pathways  <cit> .
<dig> genes are involved in this other fate of acetate and the kn <dig> strain has orthologs to each of these .
however, two reactions from this pathway show higher sequence changes between the strains – phosphate acetyltransferase and acetate kinase .
in kn <dig> the acetyltransferase has  <dig> snp  and the kinase has  <dig> snp , with a 27-fold higher non-synonymous snp/kb ratio than the other enzymes of central energy metabolism .
the sequence differences in pat and ak stand out as the most pronounced in the otherwise very well-conserved enzymes of central energy metabolism.
it has previously been shown that strain pca cannot grow on acetate alone if either pat or ak is knocked out  <cit> .
this phenotype arises because the coa-transferase that activates acetate for oxidation in the tca cycle requires coa derived from the tca cycle itself.
therefore, additional pathways are needed to produce acetyl-coa for biosynthetic pathways.
with acetate as the sole growth substrate, this anaplerotic function is performed by ak and pat.
differences in these two enzymes could have a substantial effect on the flux between acetate oxidation and reduction, and thereby on electron transport rates out of the cell and on biomass production.
a lower flux through ak/pat relative to the tca cycle would translate to a decreased biomass yield per molecule of acetate oxidized and a higher ratio of electrons transferred to the electrode per cell – the phenotype seen in the kn <dig> strain  <cit> .
sequence differences in extra-cytoplasmic electron transfer proteins
g. sulfurreducens grows by using fe oxide or energy-harvesting electrodes as the terminal electron acceptor  <cit> .
cytochromes, pili, and exopolysaccharides have all been shown to be important for growth using these extracellular electron acceptors  <cit> .
for both the pca and kn <dig> strains, the whole proteomes were scanned for the heme-binding motif of c-type cytochromes  <cit>  .
in each strain,  <dig> genes encode proteins with this c-x-x-c-h motif .
since this definition of cytochrome is minimal, proteins were excluded as cytochromes if they had a predicted function or cellular localization that indicated that they were not involved in electron transport outside the cell; most were proteins with iron-sulfur-binding domains .
in total,  <dig> genes in the pca and  <dig> in the kn <dig> strain were predicted to encode c-type cytochromes .
only one of the pca cytochromes lacked an ortholog in kn400: gsu <dig>  a monoheme cytochrome that has not been shown to be involved in electron transport.
fifty-four of the  <dig> c-type cytochromes contain at least one snp in kn <dig> relative to pca , compared to 36% of genes in the whole genome .
there were more than two-fold more snp per cytochrome compared to the genome as a whole:  <dig>  vs  <dig>  snp/gene, and almost twice as many of the encoded proteins were heavily mutated:  <dig> % vs  <dig> % .
however, all  <dig> of the orthologous cytochromes had the same number of predicted heme-binding sites, with up to  <dig> motifs in a single protein .
so, while the cytochromes have more than the average number of changes to nucleotide and protein sequences, these changes do not result in a change to the number of hemes predicted to be bound to each cytochrome.
this indicates that the proteins’ electron carrying capacity may still function, though perhaps with a changed redox potential due to structural differences  <cit> .
most of the cytochromes previously shown to be required for growth with fe or for optimal current production  <cit>  are identical in kn <dig> and pca, including ppca, maca, omcb, omcc, omcf, and omcz .
however, two cytochromes are among the most heavily mutated genes in the genomes and are required for extracellular electron transfer – omce and omcs  .
the omcs gene is found in a region  that has one of the highest snp rates in the genome:  <dig> snp/kb.
of the  <dig> genes encoded,  <dig> are heavily mutated, one is frameshifted, and three lack orthologs in the kn <dig> strain .
this region also encodes six other c-type cytochromes: gsu <dig>  gsu <dig>  gsu <dig>  gsu <dig> , gsu <dig> and gsu <dig>  finding significant changes to omcs in particular is especially relevant in the kn <dig> strain because this cytochrome has been shown to be localized outside of the cell along the length of the pili  <cit>  where it may facilitate electron transfer from pili to the terminal electron acceptor  <cit> .
changes to the electron-carrying protein at this key position for electrical contact could have a substantial effect on phenotype.
pili are also required for the reduction of extracellular electron acceptors by g. sulfurreducens <cit> .
they are believed to provide a conduit for electron transfer through biofilms and to the cytochromes responsible for fe oxide reduction  <cit> .
there are  <dig> genes involved in biogenesis and assembly of pili .
all  <dig> genes have orthologs in the kn <dig> strain , and they are all well conserved –  <dig> of the genes are identical at the nucleotide level between the strains, and only one has a single non-synonymous mutation, a pilt homolog .
extracellular polysaccharides are also required for electrode reduction; they are involved in anchoring the c-type cytochromes that provide the electrical conduit between the cell and the electrode surface  <cit> .
five of the genes in the extracellular anchoring polysaccharide gene cluster  have non-synonymous snp , including one in the an abc transporter atpase subunit required for polysaccharide production  <cit> .
finally, ompb and ompc are putative copper-binding oxidoreductases required for insoluble fe reduction in g. sulfurreducens <cit> .
the ompb gene  is identical between the two strains, and ompc  has a single synonymous mutation.
thus between the kn <dig> and the pca strains there is both conservation and divergence among the extra-cytoplasmic proteins involved in transferring electrons out of the cell.
most previously known to be required in vivo did not have large sequence differences, including six cytochromes, two copper proteins, and the pilin.
however, cytochromes considered as a class were much more likely to contain non-synonymous snp, and two important cytochromes, omcs and omce, have especially large differences.
notably though, not a single heme-binding site was lost due to the changes.
these data support previous analyses showing that a diversity of cytochromes are characteristic of geobacter species, but individual cytochromes tend to be poorly conserved  <cit> .
this kind of cytochrome diversity and adaptability may be especially important for the use of terminal electron acceptors with a wide variety of redox potentials.
sequence differences in the genes with increased expression during growth on an electrode
two previous studies have analyzed changes in gene expression in the pca strain during growth on an energy-harvesting electrode.
given the improvements in electron transfer to electrodes in the kn <dig> strain relative to pca, any sequence changes in these genes are of particular interest.
the first study looked at gene expression in the early stages of anode biofilm formation, and showed that  <dig> genes had an increase in transcript abundance compared to growth by fe reduction  <cit> .
the second looked at expression changes in established, high-current-density biofilms, and showed that  <dig> genes had an increase in transcript abundance compared to growth with fumarate as an electron acceptor  <cit> .
only one of the  <dig> genes with increased expression on the high-current electrode had substantial changes between the kn <dig> and the pca strain.
ompj is an outer-membrane channel protein known to influence the quantity and localization of cytochromes in g. sulfurreducens <cit> , so changes to its sequence could influence electron transfer very broadly.
ompj  has 3-fold higher expression during growth on the electrode, and it was among the most highly mutated genes between the two strains, with  <dig> non-synonymous changes per kb .
twenty-four genes have increased expression during early-stage growth on the electrode and also had either no ortholog or large sequence differences in the kn <dig> strain .
the omcs cytochrome gene is the most highly up-regulated of all genes and it was among the proteins with the largest sequence difference between the kn <dig> and pca strains .
a third copper-binding oxidoreductase has increased expression during growth on the electrode  <cit> , and the cluster encoding this multicopper oxidase, as well as a cytochrome d and two copper chaperone proteins  had large sequence differences .
unlike ompb and ompc , the role of these proteins in electron transfer has not been studied, though interestingly the most similar complex is found in the fe-oxidizing leptothrix species  <cit> .
in addition to these copper proteins, a transcriptional regulator  with homology to cuer, a copper-responsive transcriptional activator  <cit> , has a four-fold increase in expression on the electrode and was heavily mutated in the kn <dig> strain .
the cuer protein is broadly involved in metal homeostasis, and three general metal efflux pumps also have increased expression on the electrode and sequence changes in kn <dig>  the first pump  was one of the few proteins in pca that lacked orthologs in kn <dig> , and two others have non-synonymous snps .
immediately downstream of one of the metal pumps, a fourth important transporter has large differences between the kn <dig> and pca strains: the only sulfate transporter in the genome  was heavily mutated in kn <dig> .
this is upstream of three additional genes for sulfur metabolism: a sulfite reductase, a sulfur carrier protein, and a thiocarboxylate synthase , which are among the few in pca that lack orthologs entirely in kn <dig> .
these changes indicate that kn <dig> may have a significant difference in its usage of sulfur, copper, and perhaps other metals, which could have far-reaching impact on the types of redox proteins active in the kn <dig> strain.
CONCLUSIONS
the kn <dig> strain of g. sulfurreducens was isolated from a fuel cell that was run for several months poised at increasingly lower potentials.
its 16s rrna genes are identical to those in the well-studied pca strain, but the quantity of nucleotide polymorphisms and their strong enrichment at silent sites, as well as the diversity and distribution of genes that lack orthologs between the genomes all suggest the strains have been subject to substantial selective pressure since their divergence.
further study of strain variation in g. sulfurreducens – these two are the only strains that have been sequenced – and the analysis of more closely related genomes would help to reconstruct a detailed evolutionary history, particularly of the genomic mobility seen in this species.
the kn <dig> strain has a number of favorable characteristics that make it the preferred strain to grow in a microbial fuel cell.
the first is speed of electron transfer: kn <dig> oxidizes the donor and transfers the resulting electrons to the electrode faster than pca  <cit> .
analysis of the differences between the two genomes suggests  several explanations for this phenotype.
kn <dig> does not contain additional genes that pca lacks which are predicted to be involved in donor oxidation, nor does it contain any large sequence changes to the enzymes for acetate oxidation via the tca cycle.
however, there are significant changes to two anaplerotic enzymes that could shift the carbon flux in the kn <dig> strain from biomass production towards more rapid oxidation of acetyl-coa.
if these two enzymes have lower activity in kn <dig>  the tca cycle may complete full turns more often, and this would increase the fraction of acetate that is oxidized for respiration.
interestingly, the increased respiration rate does not lead to increased cell growth in the kn <dig> strain  <cit> , indicating that there may be important changes in atp synthesis or availability.
previously, a pca strain engineered with an unproductive atp sink exhibited a similar phenotype  <cit> .
in the kn <dig> genome, a very broad range of atp-dependent enzymes were more likely to have sequence changes, and the phosphate sensor, transporter and several phosphate regulators were all also changed.
thus, the phosphate levels, the atp:adp ratio, and the energy state of the cell may be different in the kn <dig> strain relative to the pca strain, which could result in broad metabolic differences.
the second favorable characteristic of kn <dig> when grown in a microbial fuel cell is its highly conductive biofilms  <cit>  from which low-potential electrons can be harvested.
analysis of the differences between the two genomes also suggests  factors that may contribute to this phenotype.
though pili are the conductive material in kn <dig> biofilms, there are no differences in the pilin subunit, biogenesis genes, nor the pilr transcriptional regulator between the strains.
however the cell-electrode connection is also mediated by c-type cytochromes, and there are substantial differences between the strains in these proteins.
the sequences of both cytochromes and of the channel proteins that affect their localization have substantial changes, most notably in the omcs cytochrome that is physically associated with the pili.
in addition, there are changes to several clusters of exopolysaccharide biosynthesis genes in kn <dig> which may further affect cytochrome localization and general conductivity.
excellent conservation of heme-binding sites despite sequence changes indicates that cytochrome redox potential changes also warrant further investigation in the kn <dig> strain.
finally, sensing and response to global redox potential and metal homeostasis may have significant differences in kn <dig> due to changes in proteins involved in the transport and metabolism of copper, of sulfur, and of metals generally.
these types of changes would have a broad impact on electron transport in the species.

 the ability to effectively transfer electrons directly onto insoluble, extracellular substances is one of the hallmark characteristics of geobacter species. in the natural environment, geobacter species are commonly the most abundant microorganisms in anaerobic soils and sediments where microbial reduction of insoluble fe oxides is important. in addition, geobacter species can be used to make microbial fuel cells - devices in which electrical current is harvested from bacteria that grow by transferring electrons from their food source to the anode of the device. pure cultures of geobacter sulfurreducens produce current densities that are among the highest known, and g. sulfurreducens has been enriched from a number of complex communities growing in biofilms on high-efficiency microbial fuel cells. however, the mechanisms for extracellular electron transfer in geobacter species are poorly understood. recently, a new strain of g. sulfurreducens, designated strain kn was isolated that is much more effective than wildtype both in electron transfer to electrodes and in fe oxide reduction. strain kn was isolated from a culture that grew by transferring electrons to an anode poised at a very low potential. compared to the wildtype strain, when growing in microbial fuel cells kn forms biofilms on the anode more rapidly; it produces much higher-density current and power; and it has higher conductivity. because it was recently isolated, little is known about how kn differs from the wildtype strain. here we present the analysis of the genome of kn and compare it to the genome of wildtype g. sulfurreducens pca in order to provide insight into both the phenotype of enhanced extracellular electron transfer and the evolutionary history of the species. we identify the genes that are unique to each strain and identify polymorphisms between the genomes. we analyze functional enrichment in the set of genes with sequence changes, and focus on differences in genes involved in electron transfer pathways from intracellular oxidation to electron transfer onto extracellular acceptors. we use these analyses to suggest how the metabolic network of the species has evolved and how these differences caused the divergent phenotypes.  reannotation of the g. sulfurreducens strains kn and pca genomes as published, the g. sulfurreducens strain pca genome was mb with open reading frames and the g. sulfurreducens strain kn genome was mb with orfs. because the two genomes were sequenced and annotated several years apart using different methods, we re-annotated both genomes, making several types of corrections in order to assure that comparisons between the two were valid. orf predictions, start and stop sites of the genes, and functional predictions were compared and reconciled using identical methods on both genomes. after the re-annotations, the strain kn genome had orfs, and the strain pca genome had orfs. these re-annotations were deposited in the national center for biotechnology information genome database under the accession numbers of ae for pca and nc_ for kn a summary of the basic characteristics of the genomes is given in table whole-genome comparison of strain kn to strain pca orthologs, proteins predicted to have the same function in both genomes, were identified using a combination of whole-genome alignment and all-versus-all protein sequence alignments. a whole-genome nucleotide alignment of the pca and kn genomes showed that they were syntenic, with no large-scale rearrangements or inversions. orthologs were identified for of the protein-coding genes in the kn genome. a whole-proteome comparison identified orthologs for genes. the two methods agreed for of the pairs, with the genome method identifying orthologs with identical sequences: eight transposases, two cytochromes, and a translation elongation factor, as well as three orthologs under amino acids in length.the proteome method identified orthologs with low sequence similarity or alignment over a short region of the sequence. for the analysis of polymorphisms between orthologs presented below, we included orthologs identified by only one method if they aligned over at least 50% of the length of the longer protein, and had a sequence identity of at least 30%. in total, of the orfs in the kn strain genome have orthologs in the pca strain genome. the relative locations of the conserved genes are largely preserved between the genomes. genes unique to the kn strain there were no orthologs in the pca strain for of the orfs in the kn strain genome. in order to better to describe these genes and their evolutionary history, their locations and characteristics were mapped and their sequences were compared to proteins from the organisms in the reference sequence database from the national center for biotechnology information. seventeen of these orfs had no significant similarity to any gene in another organism, six were most similar to proteins in the pca strain, and had highest similarity to proteins in geobacteraceae species other than pca. the remaining orfs were most similar to genes from a variety of phylogenetically diverse organisms including desulfovibrio, syntrophobacter, pseudomonas, and vibrio species, suggesting that there are diverse evolutionary histories for these genes. the genes are spread throughout the kn genome, the majority in clusters together. in total, there are regions in the kn genome where at least two consecutive genes have no ortholog in the pca genome. eight are bordered by or contain transposase or integrase genes, three are bordered by trna genes, and one is bordered by a conserved repeated nucleic acid sequence – indicating that these regions may be or once were mobile genetic elements. one strain-specific region, found at 466250– in the kn genome, is inserted relative to pca just downstream of a trna gene, between the orthologs to gsu and gsu. this region encodes a phage integrase and replisome organizer as well as a restriction/modification system. the two genes for the restriction/modification system are similar to genes from meiothermus ruber, and are not found in any other geobacteraceae, suggesting that this region may have been acquired by lateral gene transfer. another kn strain-specific region is found between 3479100– here, kn400_3199-kn400_ are found between the orthologs to gsu and gsu. the genes include five conserved hypothetical proteins and three proteins involved in metal transport and usage: a metal-transporting atpase, a metal-dependent phosphohydrolase, and a hybrid iron transport/cell signaling protein. all of these genes are most similar to those found in other geobacteraceae and deltaproteobacteria, suggesting that they might have been lost in the pca strain genome. in addition to regions of relative insertion in kn there are others where, between regions of excellent conservation between the two genomes, there is a stretch of sequence for which there is little or no similarity. for example, kn400_ and gsu are orthologs, and kn400_ and gsu are orthologs, but the region between them has low sequence similarity. however, in both strains, the genes encode products involved in exopolysaccharide biosynthesis. this may be a region where mutation accumulation was relatively unconstrained, and these genes are attractive targets for studies of differences in the biofilms formed by strains pca and kn a few of the genes found in the kn genome but not the pca genome are isolated, not in the clusters described above. one of these that may have a substantial effect on metabolism is the catalase. the kn400_ gene encodes a kate catalase, while the catalase of g. sulfurreducens strain pca, gsu encodes a katg-type of no sequence similarity. while each strain encodes only one of the two catalases, there is evidence that each once encoded the other. kn has a region with nucleic acid similarity to the katg gene, but a single-base deletion results in a frameshift halfway through it. in the pca genome, the region 3089478– aligns with the 5’ end and the 3’ end of the kate gene in kn though 90% of the gene has been deleted. both kate and katg detoxify reactive oxygen species, but their mechanism and expression are different. response to oxidative stress is especially important in fe-reducing, obligate anaerobes like geobacter sulfurreducens – reactive oxygen species cause dna damage and disable many respiratory enzymes, and the presence of ferrous iron can create more of these compounds. a difference in response to oxidative stress between the strains could affect many aspects of growth. genes unique to the pca strain orfs in the pca genome did not have orthologs in the kn genome. gene ontology analysis showed that three types of genes were very highly enriched in this group relative to the whole genome: those involved in dna transposition, dna integration, and rna-dependent dna replication. as with strain kn these orfs were mostly found in clusters within the pca genome. there are regions in the pca genome in which at least two consecutive genes have no orthologs in the kn genome,. twelve of these are bordered by transposases, three by a trna gene, and two by conserved nucleic acid sequences. compared to the kn strain, there are more transposases and more diverse types of transposases associated with these regions. several of these non-orthologous regions in the pca genome are much larger than any of those found in the kn genome. in the kn genome, of the non-orthologous regions have at least ten genes, and the largest has genes. in the pca genome, regions have at least ten genes, and the largest has genes. the largest region of non-orthology to kn is between 2316300– in the pca genome. this region of orfs is inserted relative to the kn genome between kn400_ and a trna gene at two-thirds of the proteins encoded in this region are proteins of unknown function or transposases. other proteins encoded include five putative transcriptional regulators and two efflux pumps. the second largest region unique to the pca genome is between 49500–. this region of orfs is inserted relative to strain kn between a trna gene and kn400_ the majority of the proteins encoded here are also transposases and proteins of unknown function. however, this region also encompasses the crispr locus, six crispr-associated genes and a toxin/antitoxin pair, genes involved in resistance to exogenous dna and potentially in gene regulation. in total, the kn genome is ca. kb smaller than the pca genome, with the difference due to the ca. genes in kn with no ortholog in pca and ca. genes in pca with no ortholog in kn these genes have a variety of different apparent histories – some are not found in any other organism, some are syntenic between the genomes but lack sequence homology, some appear to have been lost entirely in one of the strains, and some may have arisen from lateral gene transfer from unrelated species. strain-specific genes of potential interest include a number for membrane transport and polysaccharide metabolism; a restriction/modification system; catalases; a disulfide bond formation operon unique to kn400; and a crispr locus unique to pca. the evolutionary history of the mobile genetic elements that are enriched in these regions also warrants further investigation. single nucleotide polymorphisms between the pca and kn genomes using an alignment of the two whole genomes, single nucleotide polymorphism sites and small insertions or deletions between the two strains were identified. of these, were intragenic in the pca strain, with a rate of snp/kb in coding regions and snp/kb in non-coding regions. within the genes that had orthologs in both genomes, there were snp and small-scale insertions or deletions. the large majority of the sequence differences between the kn and the pca orthologs were silent, meaning that the snp caused no changes to translation, and that the protein sequences of the orthologs were identical. the remaining snp were non-synonymous – they caused a difference in amino acid sequence during translation. one-third of genes had a change of sequence between the strains − of the orthologs had at least one snp, with an average of per gene. a rate of snp/kb was two standard deviations above average. one-quarter of proteins had a change of sequence between the strains – of the orthologs had at least one non-synonymous snp, with an average of per ortholog. the set of genes that had at least one non-synonymous snp was significantly enriched in genes for: c-type cytochromes, integral membrane proteins, phosphorus and phosphate metabolism, cofactor biosynthesis, and atp-dependent enzymes with a wide variety of functions including: transporters and exporters, helicases, mismatch repair proteins, excinucleases, proteases, and many sensor histidine kinases. proteins were considered heavily mutated if the corresponding gene had at least non-synonymous snp/kb, which was two standard deviations above average. of the orthologs were heavily mutated. the set of heavily mutated proteins was significantly enriched in c-type cytochromes and in membrane-bound transport proteins. some genes had only silent snp, or sequence changes that did not affect the resulting protein sequence, indicating that changes to their sequence may be under negative selective pressure. solely silent changes were present in of the orthologs. this set of genes was significantly enriched in genes involved in flagellum biogenesis and amino acid biosynthesis. the kn strain, unlike the pca strain, produces flagella and is motile. the most notable silent-mutation-rich region of the genome was cluster gsu1095-gsu which encodes a phosphate transporter and phosphate-dependent regulatory genes. in order to better understand the phenotypic effects of the nucleotide polymorphisms between the strains, several metabolic networks involved in electron transfer out of the cell were examined in more detail. sequence differences in central energy metabolism g. sulfurreducens primarily grows by coupling the oxidation of acetate to the reduction of extracellular electron acceptors. acetate is oxidized by the tca cycle, with the products used to generate a proton gradient for atp synthesis. the electrons that remain from this process are transferred out of the cell to fe or to electrodes. in total, genes in strain pca encode the proteins involved in acetate oxidation, and the kn strain has orthologs to each of these. ten of the contain a protein sequence change, with fewer per gene than the genome as a whole. none are highly mutated. separately from energy generation, a portion of acetate is shunted to a different pathway and used as the sole source for biomass synthesis via the gluconeogenic and fatty acid pathways. genes are involved in this other fate of acetate and the kn strain has orthologs to each of these. however, two reactions from this pathway show higher sequence changes between the strains – phosphate acetyltransferase and acetate kinase. in kn the acetyltransferase has snp and the kinase has snp, with a 27-fold higher non-synonymous snp/kb ratio than the other enzymes of central energy metabolism. the sequence differences in pat and ak stand out as the most pronounced in the otherwise very well-conserved enzymes of central energy metabolism. it has previously been shown that strain pca cannot grow on acetate alone if either pat or ak is knocked out. this phenotype arises because the coa-transferase that activates acetate for oxidation in the tca cycle requires coa derived from the tca cycle itself. therefore, additional pathways are needed to produce acetyl-coa for biosynthetic pathways. with acetate as the sole growth substrate, this anaplerotic function is performed by ak and pat. differences in these two enzymes could have a substantial effect on the flux between acetate oxidation and reduction, and thereby on electron transport rates out of the cell and on biomass production. a lower flux through ak/pat relative to the tca cycle would translate to a decreased biomass yield per molecule of acetate oxidized and a higher ratio of electrons transferred to the electrode per cell – the phenotype seen in the kn strain. sequence differences in extra-cytoplasmic electron transfer proteins g. sulfurreducens grows by using fe oxide or energy-harvesting electrodes as the terminal electron acceptor. cytochromes, pili, and exopolysaccharides have all been shown to be important for growth using these extracellular electron acceptors. for both the pca and kn strains, the whole proteomes were scanned for the heme-binding motif of c-type cytochromes. in each strain, genes encode proteins with this c-x-x-c-h motif. since this definition of cytochrome is minimal, proteins were excluded as cytochromes if they had a predicted function or cellular localization that indicated that they were not involved in electron transport outside the cell; most were proteins with iron-sulfur-binding domains. in total, genes in the pca and in the kn strain were predicted to encode c-type cytochromes. only one of the pca cytochromes lacked an ortholog in kn400: gsu a monoheme cytochrome that has not been shown to be involved in electron transport. fifty-four of the c-type cytochromes contain at least one snp in kn relative to pca, compared to 36% of genes in the whole genome. there were more than two-fold more snp per cytochrome compared to the genome as a whole: vs snp/gene, and almost twice as many of the encoded proteins were heavily mutated: % vs %. however, all of the orthologous cytochromes had the same number of predicted heme-binding sites, with up to motifs in a single protein. so, while the cytochromes have more than the average number of changes to nucleotide and protein sequences, these changes do not result in a change to the number of hemes predicted to be bound to each cytochrome. this indicates that the proteins’ electron carrying capacity may still function, though perhaps with a changed redox potential due to structural differences. most of the cytochromes previously shown to be required for growth with fe or for optimal current production are identical in kn and pca, including ppca, maca, omcb, omcc, omcf, and omcz. however, two cytochromes are among the most heavily mutated genes in the genomes and are required for extracellular electron transfer – omce and omcs. the omcs gene is found in a region that has one of the highest snp rates in the genome: snp/kb. of the genes encoded, are heavily mutated, one is frameshifted, and three lack orthologs in the kn strain. this region also encodes six other c-type cytochromes: gsu gsu gsu gsu, gsu and gsu finding significant changes to omcs in particular is especially relevant in the kn strain because this cytochrome has been shown to be localized outside of the cell along the length of the pili where it may facilitate electron transfer from pili to the terminal electron acceptor. changes to the electron-carrying protein at this key position for electrical contact could have a substantial effect on phenotype. pili are also required for the reduction of extracellular electron acceptors by g. sulfurreducens. they are believed to provide a conduit for electron transfer through biofilms and to the cytochromes responsible for fe oxide reduction. there are genes involved in biogenesis and assembly of pili. all genes have orthologs in the kn strain, and they are all well conserved – of the genes are identical at the nucleotide level between the strains, and only one has a single non-synonymous mutation, a pilt homolog. extracellular polysaccharides are also required for electrode reduction; they are involved in anchoring the c-type cytochromes that provide the electrical conduit between the cell and the electrode surface. five of the genes in the extracellular anchoring polysaccharide gene cluster have non-synonymous snp, including one in the an abc transporter atpase subunit required for polysaccharide production. finally, ompb and ompc are putative copper-binding oxidoreductases required for insoluble fe reduction in g. sulfurreducens. the ompb gene is identical between the two strains, and ompc has a single synonymous mutation. thus between the kn and the pca strains there is both conservation and divergence among the extra-cytoplasmic proteins involved in transferring electrons out of the cell. most previously known to be required in vivo did not have large sequence differences, including six cytochromes, two copper proteins, and the pilin. however, cytochromes considered as a class were much more likely to contain non-synonymous snp, and two important cytochromes, omcs and omce, have especially large differences. notably though, not a single heme-binding site was lost due to the changes. these data support previous analyses showing that a diversity of cytochromes are characteristic of geobacter species, but individual cytochromes tend to be poorly conserved. this kind of cytochrome diversity and adaptability may be especially important for the use of terminal electron acceptors with a wide variety of redox potentials. sequence differences in the genes with increased expression during growth on an electrode two previous studies have analyzed changes in gene expression in the pca strain during growth on an energy-harvesting electrode. given the improvements in electron transfer to electrodes in the kn strain relative to pca, any sequence changes in these genes are of particular interest. the first study looked at gene expression in the early stages of anode biofilm formation, and showed that genes had an increase in transcript abundance compared to growth by fe reduction. the second looked at expression changes in established, high-current-density biofilms, and showed that genes had an increase in transcript abundance compared to growth with fumarate as an electron acceptor. only one of the genes with increased expression on the high-current electrode had substantial changes between the kn and the pca strain. ompj is an outer-membrane channel protein known to influence the quantity and localization of cytochromes in g. sulfurreducens, so changes to its sequence could influence electron transfer very broadly. ompj has 3-fold higher expression during growth on the electrode, and it was among the most highly mutated genes between the two strains, with non-synonymous changes per kb. twenty-four genes have increased expression during early-stage growth on the electrode and also had either no ortholog or large sequence differences in the kn strain. the omcs cytochrome gene is the most highly up-regulated of all genes and it was among the proteins with the largest sequence difference between the kn and pca strains. a third copper-binding oxidoreductase has increased expression during growth on the electrode, and the cluster encoding this multicopper oxidase, as well as a cytochrome d and two copper chaperone proteins had large sequence differences. unlike ompb and ompc, the role of these proteins in electron transfer has not been studied, though interestingly the most similar complex is found in the fe-oxidizing leptothrix species. in addition to these copper proteins, a transcriptional regulator with homology to cuer, a copper-responsive transcriptional activator, has a four-fold increase in expression on the electrode and was heavily mutated in the kn strain. the cuer protein is broadly involved in metal homeostasis, and three general metal efflux pumps also have increased expression on the electrode and sequence changes in kn the first pump was one of the few proteins in pca that lacked orthologs in kn, and two others have non-synonymous snps. immediately downstream of one of the metal pumps, a fourth important transporter has large differences between the kn and pca strains: the only sulfate transporter in the genome was heavily mutated in kn. this is upstream of three additional genes for sulfur metabolism: a sulfite reductase, a sulfur carrier protein, and a thiocarboxylate synthase, which are among the few in pca that lack orthologs entirely in kn. these changes indicate that kn may have a significant difference in its usage of sulfur, copper, and perhaps other metals, which could have far-reaching impact on the types of redox proteins active in the kn strain.  the kn strain of g. sulfurreducens was isolated from a fuel cell that was run for several months poised at increasingly lower potentials. its 16s rrna genes are identical to those in the well-studied pca strain, but the quantity of nucleotide polymorphisms and their strong enrichment at silent sites, as well as the diversity and distribution of genes that lack orthologs between the genomes all suggest the strains have been subject to substantial selective pressure since their divergence. further study of strain variation in g. sulfurreducens – these two are the only strains that have been sequenced – and the analysis of more closely related genomes would help to reconstruct a detailed evolutionary history, particularly of the genomic mobility seen in this species. the kn strain has a number of favorable characteristics that make it the preferred strain to grow in a microbial fuel cell. the first is speed of electron transfer: kn oxidizes the donor and transfers the resulting electrons to the electrode faster than pca. analysis of the differences between the two genomes suggests several explanations for this phenotype. kn does not contain additional genes that pca lacks which are predicted to be involved in donor oxidation, nor does it contain any large sequence changes to the enzymes for acetate oxidation via the tca cycle. however, there are significant changes to two anaplerotic enzymes that could shift the carbon flux in the kn strain from biomass production towards more rapid oxidation of acetyl-coa. if these two enzymes have lower activity in kn the tca cycle may complete full turns more often, and this would increase the fraction of acetate that is oxidized for respiration. interestingly, the increased respiration rate does not lead to increased cell growth in the kn strain, indicating that there may be important changes in atp synthesis or availability. previously, a pca strain engineered with an unproductive atp sink exhibited a similar phenotype. in the kn genome, a very broad range of atp-dependent enzymes were more likely to have sequence changes, and the phosphate sensor, transporter and several phosphate regulators were all also changed. thus, the phosphate levels, the atp:adp ratio, and the energy state of the cell may be different in the kn strain relative to the pca strain, which could result in broad metabolic differences. the second favorable characteristic of kn when grown in a microbial fuel cell is its highly conductive biofilms from which low-potential electrons can be harvested. analysis of the differences between the two genomes also suggests factors that may contribute to this phenotype. though pili are the conductive material in kn biofilms, there are no differences in the pilin subunit, biogenesis genes, nor the pilr transcriptional regulator between the strains. however the cell-electrode connection is also mediated by c-type cytochromes, and there are substantial differences between the strains in these proteins. the sequences of both cytochromes and of the channel proteins that affect their localization have substantial changes, most notably in the omcs cytochrome that is physically associated with the pili. in addition, there are changes to several clusters of exopolysaccharide biosynthesis genes in kn which may further affect cytochrome localization and general conductivity. excellent conservation of heme-binding sites despite sequence changes indicates that cytochrome redox potential changes also warrant further investigation in the kn strain. finally, sensing and response to global redox potential and metal homeostasis may have significant differences in kn due to changes in proteins involved in the transport and metabolism of copper, of sulfur, and of metals generally. these types of changes would have a broad impact on electron transport in the species. 
the genome of kn was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved. geobacter sulfurreducens kn400comparative genomicsmicrobial fuel cellextracellular electron transfer both genomes were re-annotated, resulting in fewer genes in the pca genome; fewer in the kn genome; and ca. a new strain of geobacter sulfurreducens, strain kn produces more electrical current in microbial fuel cells and reduces insoluble fe oxides much faster than the wildtype strain, 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. the increase in electron transfer rate and yield in kn 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. 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. substantial sequence differences were found in % of the orthologs, and this set was enriched in cytochromes and integral membrane proteins. 25% of orthologs had sequence differences, and this set was enriched in phosphorylation and atp-dependent enzymes.
 a new strain of geobacter sulfurreducens, strain kn produces more electrical current in microbial fuel cells and reduces insoluble fe oxides much faster than the wildtype strain, pca. the genome of kn was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved.  both genomes were re-annotated, resulting in fewer genes in the pca genome; fewer in the kn genome; and ca. gene start and stop sites moved. 96% of genes in kn 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 single nucleotide polymorphisms 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 were found in % 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.  the improvement in external electron transfer in the kn 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 kn 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. geobacter sulfurreducens kn400comparative genomicsmicrobial fuel cellextracellular electron transfer 