Complete WO Phage Sequences Reveal Their Dynamic Evolutionary Trajectories and Putative Functional Elements Required for Integration into the Wolbachia Genome

Wolbachia endosymbionts are ubiquitously found in diverse insects including many medical and hygienic pests, causing a variety of reproductive phenotypes, such as cytoplasmic incompatibility, and thereby efficiently spreading in host insect populations. Recently, Wolbachia-mediated approaches to pest control and management have been proposed, but the application of these approaches has been hindered by the lack of genetic transformation techniques for symbiotic bacteria. Here, we report the genome and structure of active bacteriophages from a Wolbachia endosymbiont. From the Wolbachia strain wCauB infecting the moth Ephestia kuehniella two closely related WO prophages, WOcauB2 of 43,016 bp with 47 open reading frames (ORFs) and WOcauB3 of 45,078 bp with 46 ORFs, were characterized. In each of the prophage genomes, an integrase gene and an attachment site core sequence were identified, which are putatively involved in integration and excision of the mobile genetic elements. The 3′ region of the prophages encoded genes with sequence motifs related to bacterial virulence and protein-protein interactions, which might represent effector molecules that affect cellular processes and functions of their host bacterium and/or insect. Database searches and phylogenetic analyses revealed that the prophage genes have experienced dynamic evolutionary trajectories. Genes similar to the prophage genes were found across divergent bacterial phyla, highlighting the active and mobile nature of the genetic elements. We suggest that the active WO prophage genomes and their constituent sequence elements would provide a clue to development of a genetic transformation vector for Wolbachia endosymbionts.Members of the genus Wolbachia are endosymbiotic bacteria belonging to the Alphaproteobacteria and infecting a wide range of arthropods, including over 60% of insect species, and some filarial nematodes. They are vertically transmitted through the maternal germ line of their host and are known to distort host reproduction by causing cytoplasmic incompatibility (CI), parthenogenesis, male killing, or feminization. The ability of Wolbachia to cause these reproductive phenotypes is thought to be responsible for their efficient and rapid spread into host populations (5, 21, 35, 51).Recently, Wolbachia-mediated pest control approaches have been proposed. A number of insect pests that have important medical and hygienic consequences, such as tsetse flies and mosquitoes that vector devastating human pathogens including African sleeping disease trypanosomes, malaria plasmodia, dengue viruses, Japanese encephalitis viruses, and others, often also carry Wolbachia infections (8, 24, 25, 34). In theory, if maternally transmitted genetic elements coinherited with a CI-inducing Wolbachia, such as mitochondria, the Wolbachia itself, or other coinfecting endosymbionts, are transformed with a gene of interest (like a gene that confers resistance of the vector insect against the pathogen infection), the genetic trait is expected to be spread and fixed in the host insect population, driven by the symbiont-induced reproductive phenotype (1, 2, 10, 11, 13, 32, 43, 44). The paratransgenesis and Wolbachia-driven population replacement approaches are, although potentially promising in controlling such insect-borne diseases, still at a conceptual stage mainly because no technique has been available for Wolbachia transformation.For genetic transformation of bacteria, mobile genetic elements such as plasmids, bacteriophages, and transposons have been used successfully. For example, pUC plasmids, λ phages, and transposons have been widely utilized for transforming Escherichia coli and other model bacterial species (38). While few plasmids and transposons have been reported from Wolbachia, a family of bacteriophages, called WO phages, has been detected from a diverse array of Wolbachia strains (3, 6, 7, 12, 17, 18, 31, 39, 49). For example, in the genomes of the Wolbachia strains wMel from the fruit fly Drosophila melanogaster and wPip from the mosquito Culex quinquefasciatus, three and five WO prophages are present, respectively (26, 52). Many of the prophages are pseudogenized and inactive while some are active and capable of producing phage particles (4, 7, 15, 17, 30, 40). Such active WO phage elements may provide tools for genetic transformation of Wolbachia endosymbionts.λ phage and many other temperate bacteriophages alternate between lytic phase and lysogenic phase in their life cycles. In the lytic phase, phage particles are produced and released via host cell lysis for infection to new host cells. In the lysogenic phase, the phage genome is integrated into the host genome via a site-specific recombination process, and the integrated phage genome, called prophage, is maintained in the host genome and multiplies together with the host DNA replication (38). Upon infection and lysogenic integration of λ phage, both ends of the linear phage genomic DNA are connected by DNA ligase, and the resultant circular phage genome is inserted into the E. coli genome by site-specific recombination at a region containing a core sequence of an attachment (att) site (28). att sites on the phage genome and the bacterial genome are called attP (phage att site) and attB (bacterial att site), respectively. After integration, attP and attB are located on both ends of the prophage, called attL (left prophage att site) and attR (right prophage att site), respectively. The integration and excision processes are mediated by a site-specific recombinase, called λ integrase, encoded in the phage genome (see Fig. S1 in the supplemental material) (27, 50). Hence, the att site and the integrase are the pivotal functional elements that mediate site-specific integration and excision of λ phage. Considering the structural similarity between λ phage and WO phage (31), identification of the att site and integrase from WO phage is of interest in that these elements could be utilized for delivering foreign genes into the Wolbachia genome.In order to identify a functional att site and integrase of WO phage, the complete genome sequences of active prophage elements producing phage particles should be determined. Here, the Wolbachia strain wCauB derived from the almond moth Cadra cautella was investigated because wCauB was reported to actively produce phage particles, and a partial genome sequence of its WO phage has been determined (15). In the original host insect, C. cautella, wCauB coexists with another Wolbachia strain wCauA, and both cause CI phenotypes and produce phage particles (15, 41). Not to be confounded by the coinfecting Wolbachia strains, we used a transfected line of the Mediterranean flour moth Ephestia kuehniella infected with wCauB only, which was generated by interspecific ooplasm transfer (42). It should be noted that a mass preparation procedure for WO phage particles by centrifugation has been established for the wCauB-infected E. kuehniella (15).In this study, we determined the complete genome sequences of two active WO prophages, named WOcauB2 and WOcauB3, that are capable of producing phage particles and that are located on the genome of the Wolbachia strain wCauB. Furthermore, we identified core sequences of att sites and integrase genes of these WO phages that are putatively involved in integration of the genetic elements into the Wolbachia genome.     

Mutagenesis and Functional Characterization of the RNA and Protein Components of the toxIN Abortive Infection and Toxin-Antitoxin Locus of Erwinia

Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote “altruistic suicide” of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.Interactions between bacteria and their natural parasites, bacteriophages (phage), have global-scale effects (42). Although the vast majority of the phage infections, which occur at a rate of 10²⁵ infections per s (26), are overlooked by humans, en masse they affect environmental nutrient cycling (18) and have long been known to be vital to the spread and continued diversity of microbial genes (11). A tiny proportion of this activity can directly affect our everyday activities; the lysis of bacteria following phage infection has potential medical benefits, such as use in phage therapy (30), or can be economically damaging, as it is in cases of bacterial fermentation failure (for instance, in the dairy industry [31]).Gram-positive lactococcal strains used in dairy fermentation have been shown to naturally harbor multiple phage resistance mechanisms (16). These mechanisms can be broadly classed as systems which (i) prevent phage adsorption, (ii) interfere with phage DNA injection, (iii) restrict unmodified DNA, and (iv) induce abortive infection. There is also an increasing amount of research that focuses on new systems that use clustered regularly interspaced short palindromic repeats to mediate phage resistance (3). Clustered regularly interspaced short palindromic repeats and associated proteins, although widespread in archaea and bacteria (39), have not been identified yet in lactococcal strains (23).The abortive infection (Abi) systems induce cell death upon phage infection and often rely on a toxic protein to cause “altruistic cell suicide” in the infected host (16). Although Abi systems have been studied predominantly using lactococcal systems, because of their potential economic importance (8) they have been identified in some gram-negative species, such as Escherichia coli, Vibrio cholerae, Shigella dysenteriae, and Erwinia carotovora (9, 14, 36, 38). The prr and lit systems of E. coli have been studied at the molecular level, and their mode of action and mode of activation by incoming phage have been identified (2, 37, 38). In contrast, lactococcal Abi systems have been characterized mainly by the range of phages actively aborted and the scale of these effects, and the Abi systems have been grouped based on general modes of action (8, 12). More recently, research has begun to identify more specific lactococcal Abi activities at the molecular level (12, 17) and has revealed phage activation of two such Abi systems (6, 21).An Abi system was identified on plasmid pECA1039, which was isolated from a strain of the phytopathogen E. carotovora subsp. atroseptica (14). Designated ToxIN, this two-component Abi system operates as a novel protein-RNA toxin-antitoxin (TA) system to abort phage infection in multiple gram-negative bacteria. The toxic activity of the ToxN protein was inhibited by ToxI RNA, which consists of 5.5 direct repeats of 36 nucleotides. It is now recognized that TA loci, which were originally characterized as “plasmid addiction” modules (43), are widely distributed in the chromosomes of archaea and bacteria (19) and in phage genomes, such as that of the extrachromosomal prophage P1 (27). As a result, the precise biological role of TA systems is under debate (29). It is clear, however, that they can be effective phage resistance systems, as is the case for toxIN in E. carotovora subsp. atroseptica (14) and hok/sok and mazEF in E. coli (22, 33). Previously characterized TA systems operate with both components interacting as either RNAs (e.g., hok/sok) (type I) or proteins (e.g., MazE and MazF) (type II). In this study, a mutagenesis approach was used to further characterize the ToxI and ToxN components of the new (type III) protein-RNA TA Abi system. The regulation of the operon and the mode of phage activation were also examined.     

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability,Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the ₉₆LPLGVA₁₀₁ sequence of eVP40 and the corresponding ₈₄LPLGIM₈₉ sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the ₉₆LPLGVA₁₀₁ motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the ₉₆LPLGVA₁₀₁ motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.Ebola and Marburg viruses are members of the family Filoviridae. Filoviruses are filamentous, negative-sense, single-stranded RNA viruses that cause lethal hemorrhagic fevers in both humans and nonhuman primates (5). Filoviruses encode seven viral proteins including: NP (major nucleoprotein), VP35 (phosphoprotein), VP40 (matrix protein), GP (glycoprotein), VP30 (minor nucleoprotein), VP24 (secondary matrix protein), and L (RNA-dependent RNA polymerase) (2, 5, 10, 12, 45). Numerous studies have shown that expression of Ebola virus VP40 (eVP40) alone in mammalian cells leads to the production of virus-like particles (VLPs) with filamentous morphology which is indistinguishable from infectious Ebola virus particles (12, 17, 18, 25, 26, 27, 30, 31, 34, 49). Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1, 5, 7, 10, 12, 18, 25, 27, 34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/Ser-Ala-Pro (PT/SAP) motif (25, 27), the Pro-Pro-x-Tyr (PPxY) motif (11, 12, 18, 19, 41, 53), the Tyr-x-x-Leu (YxxL) motif (3, 15, 27, 37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25, 27, 48, 49), whereas mVP40 contains only a PPxY motif. L domains are believed to act as docking sites for the recruitment of cellular proteins involved in endocytic trafficking and multivesicular body biogenesis to facilitate virus-cell separation (8, 13, 14, 16, 28, 29, 33, 36, 43, 50, 51).In addition to L domains, oligomerization, and plasma-membrane localization of VP40 are two functions of the protein that are critical for efficient budding of VLPs and virions. Specific sequences involved in self-assembly and membrane localization have yet to be defined precisely. However, recent reports have attempted to identify regions of VP40 that are important for its overall function in assembly and budding. For example, the amino acid region ₂₁₂KLR₂₁₄ located at the C-terminal region was found to be important for efficient release of eVP40 VLPs, with Leu₂₁₃ being the most critical (30). Mutation of the ₂₁₂KLR₂₁₄ region resulted in altered patterns of cellular localization and oligomerization of eVP40 compared to those of the wild-type genotype (30). In addition, the proline at position 53 was also implicated as being essential for eVP40 VLP release and plasma-membrane localization (54).In a more recent study, a YPLGVG motif within the M protein of Nipah virus (NiV) was shown to be important for stability, membrane binding, and budding of NiV VLPs (35). Whether this NiV M motif represents a new class of L domain remains to be determined. However, it is clear that this YPLGVG motif of NiV M is important for budding, perhaps involving a novel mechanism (35). Our rationale for investigating the corresponding, conserved motifs present within the Ebola and Marburg virus VP40 proteins was based primarily on these findings with NiV. In addition, Ebola virus VP40 motif maps close to the hinge region separating the N- and C-terminal domains of VP40 (4). Thus, the ₉₆LPLGVA₁₀₁ motif of eVP40 is predicted to be important for the overall stability and function of VP40 during egress. Findings presented here indicate that disruption of these filovirus VP40 motifs results in a severe defect in VLP budding, due in part to impairment in overall VP40 structure, stability and/or intracellular localization.     

Characterization of a Putative Ancestor of Coxsackievirus B5

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.The group B coxsackieviruses (CVBs) (serotypes 1 to 6) were discovered in the 1950s in a search for new poliovirus-like viruses (33, 61). Infections caused by CVBs are often asymptomatic but may occasionally result in severe diseases of the heart, pancreas, and central nervous system (99). CVBs are small icosahedral RNA viruses belonging to the Human enterovirus B (HEV-B) species within the family Picornaviridae (89). In the positive single-stranded RNA genome, the capsid proteins VP1 to VP4 are encoded within the P1 region, whereas the nonstructural proteins required for virus replication are encoded within the P2 and P3 regions (4). The 30-nm capsid has an icosahedral symmetry and consists of 60 copies of each of the four structural proteins. The VP1, VP2, and VP3 proteins are surface exposed, whereas the VP4 protein lines the interior of the virus capsid (82). The coxsackievirus and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, serves as the major cell surface attachment molecule for all six serotypes of CVB (5, 6, 39, 60, 98). Some strains of CVB1, CVB3 and CVB5 also interact with the decay-accelerating factor (DAF) (CD55), a member of the family of proteins that regulate the complement cascade. However, the attachment of CVBs to DAF alone does not permit the infection of cells (6, 7, 59, 85).Picornaviruses exist as genetically highly diverse populations within their hosts, referred to as quasispecies (20, 57). This genetic plasticity enables these viruses to adapt rapidly to new environments, but at the same time, it may compromise the structural integrity and enzymatic functionality of the virus. The selective constraints imposed on the picornavirus genome are reflected in the different regions used for different types of evolutionary studies. The highly conserved RNA-dependent RNA polymerase (3D^pol) gene is used to establish phylogenetic relationships between more-distantly related viruses (e.g., viruses belonging to different genera) (38), whereas the variable genomic sequence encoding the VP1 protein is used for the classification of serotypes (13, 14, 69, 71, 72).In 1963, Pauling and Zuckerkandl proposed that comparative analyses of contemporary protein sequences can be used to predict the sequences of their ancient predecessors (73). Experimental reconstruction of ancestral character states has been applied to evolutionary studies of several different proteins, e.g., galectins (49), G protein-coupled receptors (52), alcohol dehydrogenases (95), rhodopsins (15), ribonucleases (46, 88, 110), elongation factors (32), steroid receptors (10, 96, 97), and transposons (1, 45, 87). In the field of virology, reconstructed ancestral or consensus protein sequences have been used in attempts to develop vaccine candidates for human immunodeficiency virus type 1 (21, 51, 66, 81) but rarely to examine general phenotypic properties.In this study, a CVB5 virus with a probable ancestral virion (CVB5-P1anc) was constructed and characterized. We first analyzed in detail the evolutionary relationships between structural genes of modern CVB5 isolates and inferred a time scale for their evolutionary history. An ancestral virion sequence was subsequently inferred by using a maximum likelihood (ML) method. This sequence was then synthesized de novo, cloned into a replicative backbone of an infectious CVB5 cDNA clone, and transfected into HeLa cells. The hypothetical CVB5-P1anc assembled into functional virus particles that displayed phenotypic properties similar to those of contemporary clinical isolates. This is the first report describing the reconstruction and characterization of a fully functional picornavirus with a putative ancestral capsid.     

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-α/β responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-α/β production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.Ebola viruses (EBOVs) are zoonotic, enveloped negative-strand RNA viruses belonging to the family Filoviridae which cause lethal viral hemorrhagic fever in humans and nonhuman primates (47). Currently, information regarding EBOV-encoded virulence determinants remains limited. This, coupled with our lack of understanding of biochemical and structural properties of virulence factors, limits efforts to develop novel prophylactic or therapeutic approaches toward these infections.It has been proposed that EBOV-encoded mechanisms to counter innate immune responses, particularly interferon (IFN) responses, are critical to EBOV pathogenesis (7). However, a role for viral immune evasion functions in the pathogenesis of lethal EBOV infection has yet to be demonstrated. Of the eight major EBOV gene products, two viral proteins have been demonstrated to counter host IFN responses. The VP35 protein is a viral polymerase cofactor and structural protein that also inhibits IFN-α/β production by preventing the activation of interferon regulatory factor (IRF)-3 and -7 (3, 4, 8, 24, 27, 34, 41). VP35 also inhibits the activation of PKR, an IFN-induced, double-stranded RNA (dsRNA)-activated kinase with antiviral activity, and inhibits RNA silencing (17, 20, 48). The VP24 protein is a minor structural protein implicated in virus assembly and regulation of viral RNA synthesis, and changes in VP24 coding sequences are also associated with adaptation of EBOVs to mice and guinea pigs (2, 13, 14, 27, 32, 37, 50, 52). Further, VP24 inhibits cellular responses to both IFN-α/β and IFN-γ by preventing the nuclear accumulation of tyrosine-phosphorylated STAT1 (44, 45). The functions of VP35 and VP24 proteins are manifested in EBOV-infected cells by the absence of IRF-3 activation, impaired production of IFN-α/β, and severely reduced expression of IFN-induced genes, even after treatment of infected cells with IFN-α (3, 19, 21, 22, 24, 25, 28).Previous studies proposed that VP35 basic residues 305, 309, and 312 are required for VP35 dsRNA binding activity (26). VP35 residues K309 and R312 were subsequently identified as critical for binding to dsRNA, and mutation of these residues impaired VP35 suppression of IFN-α/β production (8). In vivo, an EBOV engineered to carry a VP35 R312A point mutation exhibited reduced replication in mice (23). However, because the parental recombinant EBOV into which the mutation was built did not cause disease in these animals, the impact of the mutation on viral pathogenesis could not be fully evaluated. Further, the lack of available structural and biochemical data to explain how the R312A mutation affects VP35 function limited avenues for the therapeutic targeting of critical VP35 functions. Recent structural analyses of the VP35 carboxy-terminal interferon inhibitory domain (IID) suggested that additional residues from the central basic patch may contribute to VP35 dsRNA binding activity and IFN-antagonist function (30). However, a direct correlation between dsRNA and IFN inhibitory functions of VP35 with viral pathogenesis is currently lacking.In order to further define the molecular basis for VP35 dsRNA binding and IFN-antagonist function and to define the contribution of these functions to EBOV pathogenesis, an integrated molecular, structural, and virological approach was taken. The data presented below identify two VP35 carboxy-terminal basic amino acids, K319 and R322, as required for its dsRNA binding and IFN-antagonist functions. Interestingly, these residues are outside the region originally identified as being important for dsRNA binding and IFN inhibition (26). However, they lie within the central basic patch identified by prior structural studies (26, 30). Introduction of these mutations (VP35 with these mutations is designated KRA) into recombinant EBOV renders this otherwise fully lethal virus avirulent in guinea pigs. KRA-infected animals also develop EBOV-specific antibodies and become fully resistant to subsequent challenge with wild-type (WT) virus. Our data further reveal that the KRA EBOV is immunogenic and likely replicates to low levels early after infection in vivo. However, the mutant virus is subsequently cleared by host immune responses. These data demonstrate that the VP35 central basic patch is important not only for IFN-antagonist function but also for EBOV immune evasion and pathogenesis in vivo. High-resolution structural analysis, coupled with our in vitro and in vivo analyses of the recombinant Ebola viruses, provides the molecular basis for loss of function by the VP35 mutant and highlights the therapeutic potential of targeting the central basic patch with small-molecule inhibitors and for future vaccine development efforts.     

Inactivation of Burkholderia cepacia Complex Phage KS9 gp41 Identifies the Phage Repressor and Generates Lytic Virions

The Burkholderia cepacia complex (BCC) is made up of at least 17 species of Gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3′ end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage φE125 and Burkholderia pseudomallei phage φ1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo.The Burkholderia cepacia complex (BCC) is a group of at least 17 Gram-negative species, the first identified strains of which were characterized as onion pathogens by W. H. Burkholder (9). Although these bacteria have a number of beneficial activities, including the promotion of crop growth and the degradation of organic pollutants, they have gained notoriety in the last two decades as serious opportunistic pathogens (19, 21, 25). BCC species, particularly B. multivorans and B. cenocepacia, cause serious respiratory infections in patients with cystic fibrosis and chronic granulomatous disease (42, 7). These infections are especially problematic due to symptom severity, the inherent antibiotic resistance of Bcc species, and the potential for rapid spread through susceptible patient populations (25, 23). Difficulties in treating these infections have led to the unfortunate practice of segregating patients, which has high economic, social, and psychological costs (18).Because of these clinical difficulties, interest in the isolation and characterization of Burkholderia-specific bacteriophages (or phages) has increased in recent years, with the apparent potential for using phages as therapeutic agents. Phage therapy is the clinical application of phages to prevent and/or to treat infections, which offers a promising alternative to antibiotic treatment for resistant bacteria such as those of the BCC (33, 39). A second benefit of these phage studies is that they may provide insight into the possible mechanisms of BCC virulence. For example, BcepMu, a transposable phage that specifically infects strains of B. cenocepacia, was found to carry genes similar to exeA, involved in toxin secretion, and mdmB and oafA, two acyltransferases (44). Finally, as Burkholderia phages tend to be underrepresented in comparative studies with respect to Escherichia coli and lactic acid bacteria phages, BCC-specific phage studies provide novel information about a relatively uncharacterized group of viruses.Although phage therapy using temperate virions can be effective (39), there are several reasons why lytic phages are generally considered the most appropriate candidates for use in phage therapy. One of the concerns is that phage integration can lead to lysogenic conversion and enhanced virulence (8). A second concern is that integration of temperate phages results in superinfection immunity due to expression of the phage repressor from the prophage. This protein binds to the operators of infecting phage DNA and represses gene expression, preventing both the initiation of the lytic cycle and the establishment of lysogeny (14). A third concern is that lysogeny affects the kinetics of infection. When a phage infects a cell and undergoes lysogeny instead of entering the lytic cycle, the cell survives, and no new phage particles are released (27). A final problem is that prophages can lead to specialized transduction after induction. Specialized transduction occurs after inexact excision of a prophage from the bacterial chromosome. Bacterial DNA flanking the prophage is packaged into the capsid, and this sequence, which can potentially encode virulence factors, can subsequently recombine into the chromosome of a new host (14).It has been estimated that more than half of tailed phages have evolved a temperate lifestyle, although some estimates have been greater than 90% (1, 22). This situation makes the isolation of naturally lytic phages extremely difficult, particularly when they must have a specific host range that includes clinically relevant bacterial species, such as B. cenocepacia (24). The use of classical genetics to produce lytic phage variants, for example, by plating temperate phages on lysogens and screening for clear plaque vir mutants, is complicated by the fact that such mutations are undefined.This report describes the characterization of KS9 (vB_BcenS_KS9), a prophage of Burkholderia pyrrocinia LMG 21824 (41), and its conversion to a lytic phage through specific molecular modification of gene 41 encoding its putative lytic phase repressor. Preliminary characterization of short sequences by Seed and Dennis (41) indicated that the genome of KS9, whose host range includes Bcc B. cenocepacia K56-2, shows similarity to the genomes of two non-BCC Burkholderia phages: φE125, a prophage of Burkholderia thailandensis E125 (47), and φ1026b, a prophage of Burkholderia pseudomallei 1026b (17). However, no phages closely related to KS9 have been functionally tested to demonstrate that proteins similar to gp41 function as true phage repressors. In the present study, we have used the BCC infection model of Galleria mellonella (40) to assess both the contribution of the KS9 prophage to BCC host virulence and the ability of a genetically modified KS9 to treat B. cenocepacia infections without stably integrating into the host bacterial chromosome as a prophage.     

A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.Virus infections depend on complex interactions between viral and cellular proteins. Consequently, the nature of these interactions has important implications for viral cell type specificity, tissue tropism, and pathogenesis. Group B coxsackieviruses (CVB1 to CVB6), members of the genus Enterovirus within the family of Picornaviridae, are human pathogens that cause a broad spectrum of diseases, ranging from mild upper respiratory illnesses to more severe infections of the central nervous system, heart, and pancreas (61). These viruses have also been associated with certain chronic muscle diseases and myocardial infarction (2, 3, 12, 13, 22).The positive single-stranded RNA genome (approximately 7,500 nucleotides in length) of CVBs is encapsidated within a small T=1, icosahedral shell (30 nm in diameter) comprised of repeating identical subunits made up of four structural proteins (VP1 to VP4). Parts of VP1, VP2, and VP3 are exposed on the outer surface of the capsid, whereas VP4 is positioned on the interior. The virion morphology is characterized by a star-shaped mesa at each 5-fold icosahedral symmetry axis, surrounded by a narrow depression referred to as the “canyon” (69). All six serotypes of CVB can use the coxsackie and adenovirus receptor (CAR) for cell attachment and entry (9, 55, 82). Some strains of CVB1, -3, and -5 also use decay accelerating factor ([DAF] CD55) for initial attachment to the host cell; however, binding to DAF alone is insufficient to permit entry into the cell (10, 54, 76).Picornaviruses are generally characterized by their cytolytic nature in cell culture. However, several in vivo and in vitro studies have shown that some picornaviruses, e.g., poliovirus, Theiler''s murine encephalomyelitis virus, foot-and-mouth disease virus, CVB3, CVB4, and CVB5, may also establish persistent, noncytolytic infections (4, 29, 35, 39, 62, 74). Recently, it has been shown that the diverse outcomes of picornaviral infections may depend on interactions between the virus and the apoptotic machinery of the infected cell (14, 30, 71). Several picornaviral proteins have been identified as inducers of an apoptotic response, including viral capsid proteins VP1, VP2, and VP3, as well as nonstructural proteins 2A and 3C (7, 20, 32, 33, 42, 50, 63). In addition, antiapoptotic activity has been assigned to the nonstructural proteins 2B and 3A (16, 59).Picornaviruses have the potential to adapt rapidly to new host environments. Virus features affecting adaptability include high mutation rates, short replication times, large populations, and frequent incidences of recombination (25-27, 53). Consequently, picornaviruses exist as genetically heterogenous populations, referred to as viral quasispecies (25, 26).Previously, the CVB2 prototype strain Ohio-1 (CVB2O) was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells (66). Two amino acid changes were identified in the capsid-coding region, and one was identified in the 2C-coding region of the adapted virus. Further characterization of the virus-host interaction showed that the infection was not affected by anti-DAF antibodies, indicating the use of an alternative receptor.In this study, the amino acid substitutions associated with the adaptation of CVB2O to cytolytic infection of RD cells were evaluated. Site-directed mutagenesis studies showed that a single amino acid change in the VP1 capsid protein was responsible for the cytolytic RD phenotype. In addition, as indicated by caspase activation and DNA degradation, the apoptotic pathway was activated in RD cells infected by the cytolytic virus.     

The Single-Stranded DNA Genome of Novel Archaeal Virus Halorubrum Pleomorphic Virus 1 Is Enclosed in the Envelope Decorated with Glycoprotein Spikes

Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses.In recent years there has been growing interest in viruses infecting hosts in the domain Archaea (43). Archaeal viruses were discovered 35 years ago (52), and today about 50 such viruses are known (43). They represent highly diverse virion morphotypes in contrast to the vast majority (96%) of head-tail virions among the over 5,000 described bacterial viruses (1). Although archaea are widespread in both moderate and extreme environments (13), viruses have been isolated only for halophiles and anaerobic methanogenes of the kingdom Euryarchaeota and hyperthermophiles of the kingdom Crenarchaeota (43).In addition to soil and marine environments, high viral abundance has also been detected in hypersaline habitats such as salterns (i.e., a multipond system where seawater is evaporated for the production of salt) (19, 37, 50). Archaea are dominant organisms at extreme salinities (36), and about 20 haloarchaeal viruses have been isolated to date (43). The majority of these are head-tail viruses, whereas electron microscopic (EM) studies of highly saline environments indicate that the two other described morphotypes, spindle-shaped and round particles, are the most abundant ones (19, 37, 43). Thus far, the morphological diversity of the isolated haloarchaeal viruses is restricted compared to viruses infecting hyperthermophilic archaea, which are classified into seven viral families (43).All of the previously described archaeal viruses have a double-stranded DNA (dsDNA) genome (44). However, a newly characterized haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1), has a single-stranded DNA (ssDNA) genome (39). HRPV-1 and its host Halorubrum sp. were isolated from an Italian (Trapani, Sicily) solar saltern. Most of the studied haloarchaeal viruses lyse their host cells, but persistent infections are also typical (40, 44). HRPV-1 is a nonlytic virus that persists in the host cells. In liquid propagation, nonsynchronous infection cycles of HRPV-1 lead to continuous virus production until the growth of the host ceases, resulting in high virus titers in the growth medium (39).The pleomorphic virion of HRPV-1 represents a novel archaeal virus morphotype constituted of lipids and two major structural proteins VP3 (11 kDa) and VP4 (65 kDa). The genome of HRPV-1 is a circular ssDNA molecule (7,048 nucleotides [nt]) containing nine putative open reading frames (ORFs). Three of them are confirmed to encode structural proteins VP3, VP4, and VP8, which is a putative ATPase (39). The ORFs of the HRPV-1 genome show significant similarity, at the amino acid level, to the minimal replicon of plasmid pHK2 of Haloferax sp. (20, 39). Furthermore, an ∼4-kb region, encoding VP4- and VP8-like proteins, is found in the genomes of two haloarchaea, Haloarcula marismortui and Natronomonas pharaonis, and in the linear dsDNA genome (16 kb) of spindle-shaped haloarchaeal virus His2 (39). The possible relationship between ssDNA virus HRPV-1 and dsDNA virus His2 challenges the classification of viruses, which is based on the genome type among other criteria (15, 39).HRPV-1 is proposed to represent a new lineage of pleomorphic enveloped viruses (39). A putative representative of this lineage among bacterial viruses might be L172 of Acholeplasma laidlawii (14). The enveloped virion of L172 is pleomorphic, and the virus has a circular ssDNA genome (14 kb). In addition, the structural protein pattern of L172 with two major structural proteins, of 15 and 53 kDa, resembles that of HRPV-1.The structural approach has made it possible to reveal relationships between viruses where no sequence similarity can be detected. It has been realized that several icosahedral viruses infecting hosts in different domains of life share common virion architectures and folds of their major capsid proteins. These findings have consequences for the concept of the origin of viruses. A viral lineage hypothesis predicts that viruses within the same lineage may have a common ancestor that existed before the separation of the cellular domains of life (3, 5, 8, 26). Currently, limited information is available on the detailed structures of viruses infecting archaea. For example, the virion structures of nontailed icosahedral Sulfolobus turreted icosahedral virus (STIV) and SH1 have been determined (21, 23, 46). However, most archaeal viruses represent unusual, sometimes nonregular, morphotypes (43), which makes it difficult to apply structural methods that are based on averaging techniques.A biochemical approach, i.e., controlled virion dissociation, gives information on the localization and interaction of virion components. In the present study, controlled dissociation was used to address the virion architecture of HRPV-1. A comparative lipid analysis of HRPV-1 and its host was also carried out. Our results show that the unique virion type is composed of a flexible membrane decorated with the glycosylated spikes of VP4 and internal membrane protein VP3. The circular ssDNA genome resides inside the viral membrane vesicle without detected association to any nucleoproteins.     

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).     

The Autolysin LytA Contributes to Efficient Bacteriophage Progeny Release in Streptococcus pneumoniae

Most bacteriophages (phages) release their progeny through the action of holins that form lesions in the cytoplasmic membrane and lysins that degrade the bacterial peptidoglycan. Although the function of each protein is well established in phages infecting Streptococcus pneumoniae, the role—if any—of the powerful bacterial autolysin LytA in virion release is currently unknown. In this study, deletions of the bacterial and phage lysins were done in lysogenic S. pneumoniae strains, allowing the evaluation of the contribution of each lytic enzyme to phage release through the monitoring of bacterial-culture lysis and phage plaque assays. In addition, we assessed membrane integrity during phage-mediated lysis using flow cytometry to evaluate the regulatory role of holins over the lytic activities. Our data show that LytA is activated at the end of the lytic cycle and that its triggering results from holin-induced membrane permeabilization. In the absence of phage lysin, LytA is able to mediate bacterial lysis and phage release, although exclusive dependence on the autolysin results in reduced virion egress and altered kinetics that may impair phage fitness. Under normal conditions, activation of bacterial LytA, together with the phage lysin, leads to greater phage progeny release. Our findings demonstrate that S. pneumoniae phages use the ubiquitous host autolysin to accomplish an optimal phage exiting strategy.Streptococcus pneumoniae (pneumococcus), a common and important human pathogen, is characterized by the high incidence of lysogeny in isolates associated with infection (34, 44). Pneumococcal bacteriophages (phages) share with the majority of bacteriophages infecting other bacterial species the “holin-lysin” system to lyse the host cell and release their progeny at the end of the lytic cycle. Genes encoding both holins and lysins (historically termed “endolysins”) are indeed found in the genomes of all known pneumococcal phages (8, 28, 31, 37). Supporting this mechanism, a lytic phenotype in the heterologous Escherichia coli system was achieved only by the simultaneous expression of the Ejh holin and the Ejl endolysin of pneumococcal phage EJ-1 (8). When these proteins were independently expressed, cellular lysis was not perceived. Similar results were shown for pneumococcal phage Cp-1, not only in E. coli, but also in the pneumococcus itself (28).Phage lysins destroy the pneumococcal peptidoglycan network due to their muralytic activity, whereas holins have been shown in S. pneumoniae to form nonspecific lesions (8), most likely upon a process of oligomerization in the cytoplasmic membrane, as observed for the E. coli phage λ (13, 14, 43). It was generally proposed that holin lesions allow access of phage lysins to the cell wall (52, 54), as the majority of phage lysins, including the pneumococcal endolysins, lack a typical N-terminal secretory signal sequence and transmembrane domains (8). However, recent evidence also highlights the possibility for a holin-independent targeting of phage lysins to the cell wall, where holin lesions seem to be crucial for the activation of the already externalized phage lysins (42, 50, 51). Regardless of the mechanism operating in S. pneumoniae to activate phage lysins, holin activity compromises membrane integrity.Pneumococcal cells present their own autolytic activity, mainly due to the presence of a powerful bacterial cell wall hydrolase, LytA (an N-acetylmuramoyl-l-alanine-amidase), responsible for bacterial lysis under certain physiological conditions (47). Although other bacterial species also encode peptidoglycan hydrolases, the extensive lysis shortly after entering stationary phase caused by LytA is a unique feature of S. pneumoniae. Interestingly, LytA is translocated across the cytoplasmic membrane to the cell wall—where it remains inactive—in spite of the absence of a canonical N-terminal sequence signal (7). In the cell wall, autolysin activities are tightly regulated by mechanisms that seem to be related to the energized state of the cell membrane. In fact, depolarizing agents are able to trigger autolysis in Bacillus subtilis (16, 17), and bacteriocin-induced depletion of membrane potential triggers autolysis of some species of the genera Lactococcus and Lactobacillus, closely related to streptococci (29). It is therefore possible that the holin-inflicted perturbations of the S. pneumoniae cytoplasmic membrane upon the induction of the lytic cycle may trigger not only the lytic activity of the phage lysin, but also that of inactive LytA located in the cell wall. Accordingly, LytA could also participate in the release of phage particles at the end of the infectious cycle, especially considering its powerful autolytic activity. Previous studies have suggested a role for the host autolytic enzyme in the release of phage progeny (11, 38), but in fact, the evidence is unclear and dubious, considering that the existence of phage-encoded lysins was unknown or very poorly understood and some of the experimental conditions used to show a role of LytA could have also affected the activity of the phage lysin (38).To clarify the possible role of the bacterial autolysin in host lysis, we used the S. pneumoniae strain SVMC28, lysogenic for the SV1 prophage (34), which contains a typical “holin-lysin” cassette, and a different host strain lysogenized with the same SV1 phage. Our results show that LytA is activated by the holin-induced membrane disruption, just like the phage endolysin. In the absence of the endolysin, LytA is capable of mediating host lysis, releasing functional phage particles able to complete their life cycle. Still, sole dependence on LytA results in an altered pattern of phage release that may reduce phage fitness. Importantly, we also show that, together with the endolysin, the concurrent LytA activation is critical for optimal phage progeny release.     

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.