A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

Increase in Rhamnolipid Synthesis under Iron-Limiting Conditions Influences Surface Motility and Biofilm Formation in Pseudomonas aeruginosa

Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.The biofilm mode of bacterial growth is a surface-attached state in which cells are closely packed and encased in an extracellular polymeric matrix (10, 27). Biofilms are abundant in nature and are of clinical, environmental, and industrial importance. Biofilm development is known to follow a series of complex but discrete and tightly regulated steps (18, 27), including (i) microbial attachment to the surface, (ii) growth and aggregation of cells into microcolonies, (iii) maturation, and (iv) dissemination of progeny cells that can colonize new niches. Over the last decade, several key processes important for biofilm formation have been identified, including quorum sensing (12) and surface motility (28).One of the best-studied model organisms for biofilm development is the bacterium Pseudomonas aeruginosa (10), a notorious opportunistic pathogen which causes many types of infections, including biofilm-associated chronic lung infections in individuals with cystic fibrosis (10, 24, 41). Like most organisms, P. aeruginosa requires iron for growth, as iron serves as a cofactor for enzymes that are involved in many basic cellular functions and metabolic pathways. Recent work has shown that at iron concentrations that are not limiting for growth, this metal serves as a signal for biofilm development (40). Iron limitation imposed, for example, by the mammalian iron chelator lactoferrin blocks the ability of P. aeruginosa biofilms to mature from thin layers of cells attached to a surface into large multicellular mushroom-like biofilm structures (40). By chelating iron, lactoferrin induces twitching motility (a specialized form of surface motility), which causes the cells to move across the surface instead of settling down to form structured communities (39, 40). In a recent paper, Berlutti et al. (5) provided further support for the role of iron in cell aggregation and biofilm formation. They reported that in the liquid phase, iron limitation induced motility and transition to the free-living (i.e., planktonic) mode of growth, while increased iron concentrations facilitated cell aggregation and biofilm formation. We recently demonstrated that iron limitation-induced twitching motility is regulated by quorum sensing (31). Quorum sensing allows bacteria to sense and respond to their population density via the production of small diffusible signal molecules. In P. aeruginosa and many other Gram-negative bacteria, these signal molecules are N-acyl homoserine lactones (acyl-HSLs), which have specific receptors (R proteins) (16, 30). P. aeruginosa possesses two acyl-HSL quorum-sensing systems, one for production of and response to N-3-oxo-dodecanoyl homoserine lactone (3OC₁₂-HSL) (LasR-LasI) and the other for production of and response to N-butanoyl homoserine lactone (C₄-HSL) (RhlR-RhlI) (35, 37). We have reported that an rhlI mutant unable to synthesize the C₄-HSL signal was impaired in iron limitation-induced twitching motility and formed structured biofilms under iron-limiting conditions (31).The correlation between twitching motility, the RhlR-RhlI quorum-sensing system, and iron-regulated biofilm formation led us to hypothesize that rhamnolipids are involved in mediating this process. Rhamnolipids are surface-active amphipathic molecules composed of a hydrophobic lipid and a hydrophilic sugar moiety and compose the main constituents of the biosurfactant produced by P. aeruginosa (reviewed in reference 42). The biosurfactant is required for a form of surface motility called swarming, where it functions as a wetting agent and reduces surface tension (8, 14). Furthermore, elements constituting the biosurfactant were recently shown to modulate the swarming behavior by acting as chemotactic-like stimuli (43). Rhamnolipids are also important in maintaining biofilm structure and inducing biofilm dispersion (6, 11, 29). Their synthesis requires the expression of the rhlAB operon, which is regulated by the RhlR-RhlI quorum-sensing system (14, 25, 32) and is also induced under iron-limiting conditions (14).In this study, we test this hypothesis and demonstrate that rhamnolipid production is induced under iron-limiting conditions and that this promotes twitching motility. We found that increased expression of rhamnolipid synthesis genes during early biofilm development under iron-limiting conditions induces surface motility and results in formation of a thin flat biofilm. Furthermore, a mutant that is incapable of synthesizing rhamnolipids does not display twitching motility under iron-limiting conditions and thus forms structured biofilms under these conditions. These results highlight the importance of biosurfactant production in determining the architecture of mature P. aeruginosa biofilms under iron-limiting conditions.     

Organoselenium Coating on Cellulose Inhibits the Formation of Biofilms by Pseudomonas aeruginosa and Staphylococcus aureus

Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims (10), patients with traumatic wounds (33), people with diabetes (27), and patients with surgical wounds (29, 31). Two of the more common causative agents of wound infections are Staphylococcus aureus and Pseudomonas aeruginosa (10, 27, 29, 31, 33). Such infections often lead to fatality; the mortality rate among patients infected with P. aeruginosa ranges from 26% to 55% (9, 49), while mortality from S. aureus infection ranges from 19% to 38% (28, 46, 50). As opportunistic pathogens, S. aureus and P. aeruginosa cause few infections in healthy individuals but readily cause infection once host defenses are compromised, such as with the removal of skin from burns (10). S. aureus infection originates from the normal flora of either the patient or health care workers (48), while P. aeruginosa is acquired from the environment surrounding the patient (41). Once established on the skin, S. aureus and P. aeruginosa are then able to colonize the wound. Infection results if the organisms proliferate in the wound environment.Both P. aeruginosa and S. aureus often exist within burn wounds as biofilms (43, 47). A biofilm is presently defined as a sessile microbial community characterized by cells that are irreversibly attached either to a substratum or to each other (16). Biofilms, which can attain over 100 μm in thickness, are made up of multiple layers of bacteria in an exopolysaccharide matrix (12, 16, 42). Sauer et al. showed that P. aeruginosa biofilms form in distinct developmental stages: reversible attachment, irreversible attachment, two stages of maturation, and a dispersion phase (42). Clinically, biofilms present serious medical management problems through their association with different chronic infections (37). During vascular catheter-related infections and sepsis, biofilms serve as a reservoir of bacteria from which planktonic cells detach and spread throughout the tissue and/or enter the circulatory system, resulting in bacteremia or septicemia (15). Factors specific to the bacterium may influence the formation of bacterial biofilms at different infection sites or surfaces. For example, during the initial attachment stage the flagellum, lipopolysaccharide, and possibly outer membrane proteins play a major role in bringing P. aeruginosa into proximity with the surface as well as mediating the interaction with the substratum (12). Using the murine model of thermal injury, we recently showed that P. aeruginosa forms a biofilm within the thermally injured tissues (43). Clinically, the surgeons debride the infected or dead tissues; however, a few microorganisms may remain on the tissue surface and reinitiate biofilm formation.Antibiotics, silver, or chitosan, attached to or embedded in gauze, have been shown to be efficacious in preventing wound infection (21, 24, 26, 36). However, the resistance of P. aeruginosa and S. aureus to available antibiotics severely limits the choices for antibiotic treatment (13, 40). Additionally, silver compounds, such as silver nitrate and silver sulfadiazine, leaching from dressings are toxic to human fibroblasts even at low concentrations (20, 25). Thus, effective alternative antimicrobial agents that contact the thermally injured/infected tissues and prevent the development of bacterial biofilms are required. Previous studies have shown that selenium (Se) can be covalently bound to a solid matrix and retain its ability to catalyze the formation of superoxide radicals (O₂^·−) (30). These superoxide radicals inhibit bacterial attachment to the solid surface (30). In this study, we examined the ability of a newly synthesized organoselenium-methacrylate polymer (Se-MAP) to block biofilm formation by both S. aureus and P. aeruginosa. These bacteria were chosen since they cause a major share of wound infections and because drug-resistant forms of these bacteria have become a serious problem in the treatment and management of these wound infections (6, 13, 17, 18, 38). Results of the study show that 0.2% (wt/wt) Se in Se-MAP covalently attached to cellulose discs inhibited P. aeruginosa and S. aureus biofilm formation. This could lead to the development of a selenium-based antimicrobial coating for cotton materials that will prevent the bacterial attachment and colonization that can ultimately lead to bacterial biofilm formation during chronic infections.     

Activation of ExoU Phospholipase Activity Requires Specific C-Terminal Regions

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that utilizes a type III secretion system to subvert host innate immunity. Of the 4 known effector proteins injected into eukaryotic cells, ExoS and ExoU are cytotoxic. The cytotoxic phenotype of ExoU depends on the enzymatic activity of the patatin-like phospholipase A₂ domain localized to the N-terminal half of the protein. Amino acid residues located within the C-terminal region of ExoU are postulated to be required for trafficking or localization to the plasma membrane of eukaryotic cells. This report describes the characterization of a transposon-based linker insertion library in ExoU. Utilizing an unbiased screening approach and sensitive methods for measuring enzymatic activity, we identified regions of ExoU that are critical for activation of the phospholipase activity by the only known cofactor, SOD1. Insertions at D572 and L618 reduced the rate of substrate cleavage. Enzymatic activity could be restored to almost parental levels when SOD1 concentrations were increased, suggesting that the linker insertion disrupted the interaction between ExoU and SOD1. An enzyme-linked immunosorbent assay (ELISA)-based binding test was developed to measure ExoU-SOD1 binding. These experiments suggest that ExoU activation by SOD1 is hampered by linker insertion. ExoU derivatives harboring minimal phospholipase activity retained biological activity in tissue culture assays. These proteins affected primarily cellular architecture in a manner similar to that of ExoT. Our studies suggest that conformational changes in ExoU are facilitated by SOD1. Importantly, the level of phospholipase activity influences the biological outcome of ExoU intoxication.Pseudomonas aeruginosa is a Gram-negative bacterium responsible for severe and potentially fatal opportunistic infections. As a contributor to nosocomial infections, P. aeruginosa is a leading cause of hospital-acquired and ventilator-associated pneumonias (40). Furthermore, P. aeruginosa is responsible for ulcerative keratitis and ocular disease found in conjunction with the use of soft contact lenses (2, 10, 54). Infections with this pathogen are of critical concern for individuals admitted with severe burns, due to the bacterium''s ability to colonize and persist in damaged tissues (35). Patients suffering from cystic fibrosis often succumb to severe lung infections and inflammation due to colonization with antibi otic-resistant, mucoid strains of P. aeruginosa (3). The expression of multiple efflux pumps and the ability to inactivate and modify antibiotics make P. aeruginosa dangerous and difficult to treat (27). Several investigators are exploring ways, as adjuncts or alternatives to antibiotic treatment, to neutralize virulence factors that contribute to the ability of P. aeruginosa to suppress host innate and adaptive immune responses (17, 21, 22, 52).Many Gram-negative bacteria, including P. aeruginosa, encode one or more type III secretion systems (T3SS), which are thought to aid in pathogenesis and increase disease severity (19, 32, 39). Four effectors are translocated by the T3SS of P. aeruginosa and include ExoS, ExoT, ExoU, and ExoY (8, 23, 56, 57). The activity of each effector is dependent upon interaction with a cofactor present in eukaryotic but not prokaryotic cells. ExoS and ExoT are bifunctional enzymes that possess both Rho GTPase-activating protein and ADP-ribosyltransferase activities (23, 25, 51). The ADP ribosylation of eukaryotic proteins by ExoS and ExoT requires activation by members of the 14-3-3 family of scaffolding proteins (13). ExoY is an adenylyl cyclase that causes the accumulation of cyclic AMP (cAMP) in intoxicated cells. The eukaryotic cofactor required for ExoY activity has not been identified (57). ExoU, a potent A₂ phospholipase responsible for membrane disruption and cellular lysis, requires superoxide dismutase 1 (SOD1) for the detection of enzymatic activity (43, 46).ExoU is an important virulence factor of P. aeruginosa, as it causes rapid cell death during in vitro infections and is associated with poor clinical outcomes (19, 39, 44). Several studies have used truncation analyses, linker mutagenesis, and site-specific amino acid substitutions to define regions of ExoU important for various functions (7, 36). ExoU is a 74-kDa, hydrophilic, and slightly acidic protein with a pI of 5.9 (8). The first 52 amino acids are required for interaction with the chaperone SpcU and may be important for translocation through the T3SS (7, 9). Enzymatic activity is attributed to the patatin-like phospholipase domain located between residues 107 and 357 (34, 46). Two catalytic residues, S142 and D344, and a sequence encoding an oxyanion hole (₁₁₂GGAK₁₁₅) are located within this domain (34, 46). The oxyanion hole is thought to stabilize the negative charge of the intermediate structure during substrate cleavage (5). C-terminal residues of ExoU, specifically the last 137 amino acids, have been implicated in membrane localization after translocation into mammalian cells (37). The domain or region(s) required for the activation of ExoU by SOD1 have not been identified.In this study, linker-scanning mutagenesis (the insertion of 15 nucleotides randomly throughout the coding sequence) was used to identify regions of exoU that impair activation of phospholipase activity by SOD1. Our data support the model that SOD1 may be facilitating the activation of ExoU by altering the conformational properties of the enzyme. Understanding the molecular mechanisms mediating SOD1 and ExoU interaction may contribute to the design of therapeutics for the treatment of acute P. aeruginosa infections.     

Role of TonB1 in Pyoverdine-Mediated Signaling in Pseudomonas aeruginosa

Pyoverdines are siderophores secreted by Pseudomonas aeruginosa. Uptake of ferripyoverdine in P. aeruginosa PAO1 occurs via the FpvA receptor protein and requires the energy-transducing protein TonB1. Interaction of (ferri)pyoverdine with FpvA activates pyoverdine gene expression in a signaling process involving the cytoplasmic-membrane-spanning anti-sigma factor FpvR and the sigma factor PvdS. Here, we show that mutation of a region of FpvA that interacts with TonB1 (the TonB box) prevents this signaling process, as well as inhibiting bacterial growth in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid). Signaling via wild-type FpvA was also eliminated in strains lacking TonB1 but was unaffected in strains lacking either (or both) of two other TonB proteins in P. aeruginosa, TonB2 and TonB3. An absence of pyoverdine-mediated signaling corresponded with proteolysis of PvdS. These data show that interactions between FpvA and TonB1 are required for (ferri)pyoverdine signal transduction, as well as for ferripyoverdine transport, consistent with a mechanistic link between the signaling and transport functions of FpvA.Pseudomonas aeruginosa is an opportunistic pathogen that is able to cause severe infections in patients with cystic fibrosis and in immunocompromised individuals, such as burn victims. Under conditions of iron limitation, P. aeruginosa secretes an iron-scavenging compound (siderophore) called pyoverdine. Ferripyoverdine is transported back into the bacteria by an outer membrane (OM) receptor protein, FpvA. The transport of ferripyoverdine via FpvA requires energy provided by a TonB complex (36, 42, 50). TonB is an energy-transducing protein that couples the energy of the cytoplasmic membrane (CM) to a variety of OM receptors required for the import of ferrisiderophores and other molecules. TonB acts in a complex with two CM-associated proteins, ExbB and ExbD, both of which are required for full TonB function (5, 37). The TonB-ExbB-ExbD complex has been identified in many gram-negative bacterial species and is thought to be a conserved mechanism for energy transduction to OM receptor proteins (31). TonB-dependent receptors contain a conserved protein motif known as the TonB box (5). Direct interaction between TonB and the TonB box has been demonstrated for several TonB-dependent receptors (8, 26, 33, 35, 47). Mutations of the TonB box, particularly mutations that are likely to affect the secondary structure, can result in a TonB-uncoupled phenotype characterized by loss of TonB-dependent functions (ferrisiderophore transport) with no loss of TonB-independent functions, such as internalization of bacteriophage (37).The P. aeruginosa PAO1 genome contains three tonB genes, tonB1 (PA5531) (36), tonB2 (PA0197) (55), and tonB3 (PA0406) (20), encoding proteins of 342, 270, and 319 amino acids (aa), respectively. The TonB1 and TonB2 amino acid sequences display 31% identity over a section of 187 aa, but otherwise, the three PAO1 TonB proteins show similarity (30 to 40% aa identity) to each other only over short (<70-aa) regions. TonB1 is considered to be the primary TonB protein involved in iron transport in P. aeruginosa. tonB1 mutants are impaired for growth in iron-limited medium and are defective for siderophore-mediated iron transport and heme utilization (36, 50, 55). Moreover, direct interaction between TonB1 and the ferripyoverdine receptor FpvA has been demonstrated in vitro (1). The tonB2 gene is not required for growth in iron-limited medium (55). However, tonB1 tonB2 double mutants grow even less well under iron limitation than tonB1 mutants, indicating that TonB2 may be able to partially complement TonB1 in its role in iron acquisition (55). The tonB3 gene is required for twitching motility and assembly of extracellular pili (20), but it is not known whether TonB3 has a role in iron acquisition. Genes encoding ExbB and ExbD proteins are located directly downstream of tonB2 (55) but are not found in association with tonB1 or tonB3.Besides its role in ferripyoverdine transport, FpvA is part of a signal transduction pathway and thus belongs to a subset of TonB-dependent receptors known as TonB-dependent transducers (reviewed in references 23 and 51). Mutational analysis has shown that the ferripyoverdine transport and signaling roles of FpvA are separate and discrete functions (21, 46). Besides FpvA, the signal transduction pathway involves a CM-spanning anti-sigma factor protein, FpvR, and (ferri)pyoverdine. (It was previously thought that both ferri- and apopyoverdine could bind FpvA (43). However, it was recently reported that only ferripyoverdine is able to form a high-affinity interaction with FpvA (13). The designation (ferri)pyoverdine will be used here to represent the active signaling molecule. FpvA and (ferri)pyoverdine regulate the activity of FpvR, which in turn regulates the activities of two extracytoplasmic function family sigma factors, PvdS and FpvI (3, 25). Upon binding of (ferri)pyoverdine to FpvA, a signal is transmitted to FpvR, resulting in activation of PvdS and FpvI. Activation of PvdS is required for maximal synthesis of pyoverdine itself, as well as two secreted proteins (25). Activation of FpvI leads to increased expression of fpvA (3, 39). In the absence of pyoverdine-mediated signaling, caused by the lack of FpvA or pyoverdine or overexpression of FpvR, suppression of PvdS- and FpvI-dependent gene expression occurs (3, 25), and this is associated with proteolysis of PvdS (49).Analogous siderophore transport and signaling systems involving an OM TonB-dependent transducer, a CM-bound anti-sigma factor, and an extracytoplasmic function family sigma factor have been described in other bacteria, including the ferric citrate (Fec) system in Escherichia coli and the pseudobactin (Pup) system in Pseudomonas putida (reviewed in reference 6). The TonB protein is required for signaling in both the Fec (14, 33) and Pup (24) systems. Similarly, a TonB system is required for hemophore transport and signaling in Serratia marcescens (4). The aim of this study was to investigate whether TonB was required for pyoverdine-mediated signaling in P. aeruginosa, and if so, to identify which of the three TonB proteins was involved.     

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.     

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).