In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.Microbial utilization and degradation of alkanes was discovered almost a century ago (27). Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates (30). The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenase (BMO) are specialized on gaseous small-chain alkanes (C₁ to C₄), while medium-chain (C₅ to C₁₆) alkane hydroxylation seems to be the domain of the CYP153 and AlkB enzyme families.Conversion of C₁ to C₄ alkanes to alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression (30) and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth (12). We wished to determine whether it is possible to engineer a medium-chain alkane monooxygenase to hydroxylate small alkanes, thereby circumventing difficulties associated with engineering MMO-like enzymes as well as investigating the fundamental question of whether enzymes unrelated to MMO can support growth on small alkanes.The most intensively studied medium-chain alkane hydroxylases are the AlkB enzymes (2, 20, 29), especially AlkB from Pseudomonas putida GPo1 (13, 28, 32, 35). While most members of the AlkB family act on C₁₀ or longer alkanes, some accept alkanes as small as C₅ (30). A recent study (12) indicated that AlkB from P. putida GPo1 may also be involved in propane and butane assimilation. AlkB selectively oxidizes at the terminal carbon to produce the 1-alkanols. No systematic protein engineering studies have been conducted on this di-iron integral membrane enzyme, although selection and site-directed mutagenesis efforts identified one amino acid residue that sterically determines long-chain alkane degradation (35).The most recent addition to the known biological alkane-hydroxylating repertoire is the CYP153 family of heme-containing cytochrome P450 monooxygenases. Although their activity was detected as early as 1981 (1), the first CYP153 was characterized only in 2001 (16). Additional CYP153 enzymes were identified and studied more recently (9, 10, 31). These soluble class II-type three-component P450 enzymes and the AlkB enzymes are the main actors in medium-chain-length alkane hydroxylation by the cultivated bacteria analyzed to date (31). CYP153 monooxygenases have been the subject of biochemical studies (9, 16, 19), and their substrate range has been explored (10, 14). Known substrates include C₅ to C₁₁ alkanes. The best-characterized member, CYP153A6, hydroxylates its preferred substrate octane predominantly (>95%) at the terminal position (9).Recent studies have shown that high activities on small alkanes can be obtained by engineering bacterial P450 enzymes such as P450cam (CYP101; camphor hydroxylase) and P450 BM3 (CYP102A; a fatty acid hydroxylase) (8, 36). The resulting enzymes, however, hydroxylate propane and higher alkanes primarily at the more energetically favorable subterminal positions; highly selective terminal hydroxylation is difficult to achieve by engineering a subterminal hydroxylase (22). We wished to determine whether a small-alkane terminal hydroxylase could be obtained instead by directed evolution of a longer-chain alkane hydroxylase that exhibits this desirable regioselectivity. For this study, we chose to engineer AlkB from P. putida GPo1 and CYP153A6 from Mycobacterium sp. strain HXN-1500 (9, 33) to enhance activity on butane. Because terminal alkane hydroxylation is the first step of alkane catabolism in P. putida GPo1, we reasoned that it should be possible to establish an in vivo evolution system that uses growth on small alkanes to select for enzyme variants exhibiting the desired activities.The recombinant host Pseudomonas putida GPo12(pGEc47ΔB) was engineered specifically for complementation studies with terminal alkane hydroxylases and was used previously to characterize members of the AlkB and CYP153 families (26, 31). This strain is a derivative of the natural isolate P. putida GPo1 lacking its endogenous OCT plasmid (octane assimilation) (5) but containing cosmid pGEc47ΔB, which carries all genes comprising the alk machinery necessary for alkane utilization, with the exception of a deleted alkB gene (34). We show that this host can be complemented by a plasmid-encoded library of alkane hydroxylases and that growth of the mixed culture on butane leads to enrichment of novel butane-oxidizing terminal hydroxylases.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Ferredoxin-NADP+ Reductase from Pseudomonas putida Functions as a Ferric Reductase

Pseudomonas putida harbors two ferredoxin-NADP⁺ reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k_cat/K_m value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.Commonly, Fprs are ubiquitous, monomeric, reversible flavin enzymes. Fprs evidence a profound preference for NADP(H) over NAD(H) (3). They harbor a prosthetic flavin cofactor (FAD) and catalyze the reversible electron exchange between NADPH and either ferredoxin (Fd) or flavodoxin (Fld) (4, 5). In oxygenic photosynthesis, the Fd is reduced by the photosystem and subsequently passes electrons on to NADP⁺ via the Fpr. This reaction provides the cellular NADPH pool required for CO₂ assimilation and other biosynthetic processes (4, 5). In heterotrophic organisms such as bacteria, reduced ferredoxin, owing to the reverse enzymatic activity of the Fpr, can donate an electron to several Fd-dependent enzymes, such as nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase, allowing ferredoxin to function in a variety of systems, including oxidative stress (1, 4, 5).Iron is the fourth most abundant element in the natural environment and exists primarily as an oxidized form, Fe(III), which has very low solubility under neutral pH conditions (9, 34) and thus presents problems in terms of bioavailability. However, ferrous iron, of Fe(II), is soluble and available at neutral pH in bacterial cytosol (34). Most bacteria secrete siderophores, which are natural chelators of ferric iron. After they bind to ferric iron, that complex enters the bacteria and releases ferric iron into the cytosol in ferric or ferrous form (9). In the bacterial cytosol, ferric iron must be reduced to ferrous form, and thus ferric reductase is essential to bacterial iron utilization.Commonly, prokaryotic ferric reductases are divided into two groups—namely, the bacterial and archaeal types (34). The typical bacterial type ferric reductase is Escherichia coli Fre, which also functions as a flavin reductase. In other words, the ferric reductase can reduce free flavin as flavin reductase, rather than having the flavin cofactor as a prosthetic group in E. coli (38). The archaeal ferric reductase harbors a flavin cofactor in the enzyme and thus does not require a flavin carrier for ferric reduction (26, 34). E. coli Fre includes a Rosmann folding structure at the NAD(P) binding region, whereas the archaeal ferric reductase (FeR) of Archaeoglobus fulgidus does not evidence that folding structure (6, 34). Many bacterial ferric reductases utilize free flavins, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and riboflavin, as electron carrier and, NADH (NAD) or NADP as electron donors to ferric reductase (14, 34). However, reduced ferric iron by reduced free flavin gives rise to the Fenton reaction, which generates the hydroxyl radical within the cell (20, 38). The Fenton reaction is known to generate hydroxyl radicals from ferrous iron and hydrogen peroxide (20). The hydroxyl radical is the most reactive radical and can damage DNA, proteins, and membrane lipids (16, 20, 34, 38). Therefore, the fine-tuning of ferric reduction regulation is required for the survival of bacterial cells.Many Pseudomonas strains, including Pseudomonas putida, a gram-negative soil model bacteria, and Pseudomonas aeruginosa, a human pathogen bacteria, do not harbor annotated ferric reductase within their genome sequences. Commonly, the pathogens compete with the host for available iron, whichis crucial for their survival within the host. Thus, studies of P. aeruginosa regarding iron utilization, siderophores, and ferric reduction are considered to be essential for a better understanding of human infections (9, 19). Studying the physiology and ecology of P. putida also provides us with a new framework for elucidating the basis of the metabolic versatility and environmental stress response of soil microorganisms. Thus, the study of ferric reductase in strains of Pseudomonas at the molecular level is certainly required. From the structural perspective, ferric reductases are generally considered to be contained within the structurally diverse ferredoxin-NADP⁺ reductase (Fprs; EC 1.18.1.2) superfamily, which is frequently involved in the transfer of electrons between Fd/Fld and NADP(H) (2, 15, 34). Thus, we tested the role of the Fpr as a ferric reductase using free flavin (FMN or FAD), NADH, or NADPH as electron donors, and ferric-citrate or ferric-EDTA as terminal electron acceptors (37). We determined that FprA could efficiently utilize NADPH in ferric reduction. Rather, FprB could use NADH as an electron donor and may perform a crucial role as a NADH-dependent ferric reductase under iron stress conditions.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.