Role of Mouse Hepatitis Virus (MHV) Receptor Murine CEACAM1 in the Resistance of Mice to MHV Infection: Studies of Mice with Chimeric mCEACAM1a and mCEACAM1b

Although most inbred mouse strains are highly susceptible to mouse hepatitis virus (MHV) infection, the inbred SJL line of mice is highly resistant to its infection. The principal receptor for MHV is murine CEACAM1 (mCEACAM1). Susceptible strains of mice are homozygous for the 1a allele of mCeacam1, while SJL mice are homozygous for the 1b allele. mCEACAM1a (1a) has a 10- to 100-fold-higher receptor activity than does mCEACAM1b (1b). To explore the hypothesis that MHV susceptibility is due to the different MHV receptor activities of 1a and 1b, we established a chimeric C57BL/6 mouse (cB61ba) in which a part of the N-terminal immunoglobulin (Ig)-like domain of the mCeacam1a (1a) gene, which is responsible for MHV receptor function, is replaced by the corresponding region of mCeacam1b (1b). We compared the MHV susceptibility of these chimeric mice to that of SJL and B6 mice. B6 mice that are homozygous for 1a are highly susceptible to MHV-A59 infection, with a 50% lethal dose (LD₅₀) of 10^2.5 PFU, while chimeric cB61ba mice and SJL mice homozygous for 1ba and 1b, respectively, survived following inoculation with 10⁵ PFU. Unexpectedly, cB61ba mice were more resistant to MHV-A59 infection than SJL mice as measured by virus replication in target organs, including liver and brain. No infectious virus or viral RNA was detected in the organs of cB61ba mice, while viral RNA and infectious virus were detected in target organs of SJL mice. Furthermore, SJL mice produced antiviral antibodies after MHV-A59 inoculation with 10⁵ PFU, but cB61ba mice did not. Thus, cB61ba mice are apparently completely resistant to MHV-A59 infection, while SJL mice permit low levels of MHV-A59 virus replication during self-limited, asymptomatic infection. When expressed on cultured BHK cells, the mCEACAM1b and mCEACAM1ba proteins had similar levels of MHV-A59 receptor activity. These results strongly support the hypothesis that although alleles of mCEACAM1 are the principal determinants of mouse susceptibility to MHV-A59, other as-yet-unidentified murine genes may also play a role in susceptibility to MHV.Differences in susceptibility to a number of viral infections have been documented among inbred mouse strains (20). These differences have been studied as models for the various degrees of susceptibility of individual humans to some viral infections. Numerous host factors have been found to be involved in such differences (2, 15). For example, allelic variations in the virus receptor and coreceptor for HIV-1 are important host factors influencing susceptibility to HIV-1 infection (36).A virus receptor is a molecule with which the virus interacts at an initial step of infection. Therefore, receptors are crucial host determinants of virus susceptibility (15, 16). A variety of receptor proteins has been identified for many different viruses, including the murine coronavirus mouse hepatitis virus (MHV) (12, 50). The principal receptor for MHV is murine carcinoembryonic antigen-related cell adhesion molecule 1 (mCEACAM1; previously called Bgp or MHVR [3]), which is in the immunoglobulin (Ig) superfamily (12, 50). Four isoforms of mCEACAM1a (1a) are expressed on the plasma membranes of a variety of murine cells and tissues (14). The two mCEACAM1 isoforms with a molecular mass of 100 to 120 kDa are composed of four Ig-like ectodomains, a transmembrane (TM) domain, and either a long or a short cytoplasmic tail (Cy) (3, 22). Two other isoforms consist of two Ig-like domains, with either long or short Cy (3, 22). The N-terminal (N) domain is responsible for virus binding (10, 24), the induction of conformational changes in the viral spike protein (S), and membrane fusion during virus entry and syncytium formation (13, 24). The replacement of the N-terminal domain of mCEACAM1a with that of the murine homolog of the poliovirus receptor (PVR) yields a functional receptor for MHV (10), and Ceacam1a-knockout mice are completely resistant to infection with the hepatotropic A59 strain of MHV (17, 25).Wild mice have two alleles of the mCeacam1 gene, called mCeacam1a and mCeacam1b. Inbred mouse strains that are homozygous for mCeacam1a, including BALB/c, C57BL/6 (B6), C3H, and A/J mice, etc., are highly susceptible to infection with strains of MHV. In contrast, the SJL line of inbred mice, which is resistant to death from MHV infection, is homozygous for the mCeacam1b allele (5, 11, 50). The most extensive differences in amino acid sequence between mCEACAM1a and mCEACAM1b are found in the N-terminal domain, where the virus-binding region is located (21, 22, 32). It was initially reported by Boyle et al. that mCEACAM1a proteins had MHV-A59 virus-binding activity in a virus overlay protein blot, while mCEACAM1b did not (5). Those authors speculated that the different viral affinities of these mCEACAM1 proteins may account for the various MHV-A59 susceptibilities of BALB/c mice compared to those of SJL mice (49). However, Yokomori and Lai (53) and Dveksler et al. (11) previously showed that when recombinant CEACAM1a and CEACAM1b proteins are expressed at high levels on cultured cells, both proteins have MHV-A59 receptor activity. Yokomori and Lai suggested that the difference in MHV susceptibility between BALB/c and SJL mice does not depend solely upon the interaction of the virus with mCEACAM1 proteins (52, 53). Dveksler et al. suggested that small differences in MHV-A59 receptor activity between mCEACAM1a and mCEACAM1b could result in very large biological differences during multiple cycles of infection in in vivo infection (11). We then quantitatively showed that recombinant mCEACAM1a expressed in BHK cells has 10- to 30-times-higher MHV-binding activity than mCEACAM1b (31). Similar results were observed in other laboratories (7, 32). Because the mCeacam1 gene is located on chromosome 7 (34) and the gene controlling MHV-A59 susceptibility and the resistance of BALB/c mice versus SJL mice is also located on chromosome 7 close to the mCeacam1 gene (40), we speculated that the mCeacam1 gene is identical to the gene that determines the susceptibility and/or resistance of mice to MHV-A59 and MHV-JHM infection.To examine the above-described hypothesis, we used progeny mice produced by crossing BALB/c and SJL mice. F₂ mice and F₁ mice backcrossed to SJL mice were examined for the mCeacam1 genotype and for MHV-JHM susceptibility (30). Mice homozygous for mCeacam1a (1a/1a) and heterozygous mice (1a/1b) were susceptible to lethal MHV-JHM infection, while mice homozygous for mCeacam1b (1b/1b) were not killed by inoculation with MHV-JHM. These data are consistent with the hypothesis that the susceptibility of mice to MHV is determined by the mCeacam1a allele (30). However, this classical genetic analysis could not prove that mCeacam1 alone determines the susceptibility or resistance of mice to MHV-JHM infection, because this methodology cannot rule out the possibility that a different unknown host gene located close to mCeacam1 on chromosome 7 could also affect MHV-JHM susceptibility. Therefore, we used gene replacement in B6 embryonic stem (ES) cells to create a mouse strain in which the exon encoding the N-terminal part of the N-terminal Ig domain of mCeacam1a was replaced with the corresponding region of mCeacam1b from SLJ mice. We bred the chimeric mCeacam1 gene on the B6 background (called B6 chimeric mCeacam1ba, or cB61ba). We compared these mice, wild-type B6 mice, and SJL mice for their susceptibilities to MHV-A59 infection. We confirmed that the expression of mCEACAM1a makes mice susceptible to lethal infection with MHV-A59. However, surprisingly, we found that cB61ba mice were profoundly resistant to MHV-A59 infection, while the virus could replicate at low levels in SJL mice in a self-limited, unapparent infection. Our results suggest that one or more as-yet-unidentified murine genes may also contribute to murine susceptibility and/or resistance to MHV-A59 infection.     

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.     

Different Roles of DosS and DosT in the Hypoxic Adaptation of Mycobacteria

The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.Oxygen sensing is important for facultative anaerobes to adapt to changes in metabolic necessities during the transition between aerobic and anaerobic conditions. Although Mycobacterium tuberculosis (MTB) is an obligate aerobe, a gradual depletion of O₂ from its culture is known to lead to a drastic change in gene expression (8, 21, 24, 28, 34, 37, 39). Approximately 48 genes of M. tuberculosis were reported to be induced under early hypoxic conditions, which is mediated by the DosSR (DevSR) two-component system (16, 24, 34). The induction of the DosR regulon is important for survival of M. tuberculosis under hypoxic conditions and for it to enter the nonreplicating dormant state (2, 19). The DosSR two-component system consists of the DosS histidine kinase (HK) and its cognate DosR response regulator (RR) (24, 26, 29). The DosT HK, which shares high sequence similarity to DosS over the length of their primary structures, was also found to cross talk with DosR (26, 30). The N-terminal domains of DosS and DosT contain two tandem GAF domains (GAF-A and GAF-B from their N termini), and the three-dimensional structure of the GAF-A and GAF-B domains was determined (5, 25). A b-type heme is embedded in the GAF-A domain, composed of one five-stranded antiparallel β-sheet and four α-helices (5, 14, 25, 32). The heme is positioned nearly perpendicular to the β-sheet, and H149 and H147 of the polypeptides serve as the proximal axial ligands for DosS and DosT, respectively (5, 25). The ligand-binding state at the distal axial position of heme and the redox state of the heme iron modulate the autokinase activity of DosS and DosT. The O₂-bound (oxyferrous) and ferric forms of the HKs are inactive, whereas the unliganded ferrous (deoxyferrous) form as well as NO- and CO-bound forms are active (17, 36). The heme iron of DosT is stable against autooxidation of Fe²⁺ to Fe³⁺ in the presence of O₂, indicating that its conversion between deoxyferrous and oxyferrous forms is the mechanism by which DosT recognizes O₂ (17). However, the autooxidation property of oxyferrous DosS remains controversial. Kumar et al. (17) and Cho et al. (5) reported that DosS undergoes autooxidation on exposure to O₂, while other research groups demonstrated that the oxyferrous form of DosS is stable against autooxidation (13, 14, 36). Recently, different roles of DosS and DosT in O₂ sensing by M. tuberculosis were suggested. DosT plays a more important role in the early phase of hypoxic conditions than DosS when the growth of M. tuberculosis is transferred from aerobic to hypoxic conditions (11).Mycobacterium smegmatis possesses a single DevS HK that phosphorylates the DevR RR (20). The DevSR two-component system is also implemented in hypoxic adaptation of this bacterium (20). Like DosT of M. tuberculosis, the autokinase activity of M. smegmatis DevS was shown to be controlled by the ligand-binding state of its heme (18). Regarding the autooxidation property, DevS of M. smegmatis was suggested to be similar to DosT rather than DosS; i.e., the heme iron in DevS is resistant to autooxidation from an oxyferrous to a ferric state in the presence of O₂ (18).In this paper we report several lines of evidence for the functional difference between DosS and DosT in the hypoxic adaptation of mycobacteria and discuss the implications of these findings.     

An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA⁺ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpL_M and ImpK_L, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpL_M and ImpK_L are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.     

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).     

Hitherto Unknown [Fe-Fe]-Hydrogenase Gene Diversity in Anaerobes and Anoxic Enrichments from a Moderately Acidic Fen

Newly designed primers for [Fe-Fe]-hydrogenases indicated that (i) fermenters, acetogens, and undefined species in a fen harbor hitherto unknown hydrogenases and (ii) Clostridium- and Thermosinus-related primary fermenters, as well as secondary fermenters related to sulfate or iron reducers might be responsible for hydrogen production in the fen. Comparative analysis of [Fe-Fe]-hydrogenase and 16S rRNA gene-based phylogenies indicated the presence of homologous multiple hydrogenases per organism and inconsistencies between 16S rRNA gene- and [Fe-Fe]-hydrogenase-based phylogenies, necessitating appropriate qualification of [Fe-Fe]-hydrogenase gene data for diversity analyses.Molecular hydrogen (H₂) is important in intermediary ecosystem metabolism (i.e., processes that link input to output) in wetlands (7, 11, 12, 33) and other anoxic habitats like sewage sludges (34) and the intestinal tracts of animals (9, 37). H₂-producing fermenters have been postulated to form trophic links to H₂-consuming methanogens, acetogens (i.e., organisms capable of using the acetyl-coenzyme A [CoA] pathway for acetate synthesis) (7), Fe(III) reducers (17), and sulfate reducers in a well-studied moderately acidic fen in Germany (11, 12, 16, 18, 22, 33). 16S rRNA gene analysis revealed the presence of Clostridium spp. and Syntrophobacter spp., which represent possible primary and secondary fermenters, as well as H₂ producers in this fen (11, 18, 33). However, H₂-producing bacteria are polyphyletic (30, 31, 29). Thus, a structural marker gene is required to target this functional group by molecular methods. [Fe-Fe]-hydrogenases catalyze H₂ production in fermenters (19, 25, 29, 30, 31), and genes encoding [Fe-Fe]-hydrogenases represent such a marker gene. The objectives of this study were to (i) develop primers specific for highly diverse [Fe-Fe]-hydrogenase genes, (ii) analyze [Fe-Fe]-hydrogenase genes in pure cultures of fermenters, acetogens, and a sulfate reducer, (iii) assess [Fe-Fe]-hydrogenase gene diversity in H₂-producing fen soil enrichments, and (iv) evaluate the limitations of the amplified [Fe-Fe]-hydrogenase fragment as a phylogenetic marker.     

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.     

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

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

Detection,Distribution, and Organohalogen Compound Discovery Implications of the Reduced Flavin Adenine Dinucleotide-Dependent Halogenase Gene in Major Filamentous Actinomycete Taxonomic Groups

Halogenases have been shown to play a significant role in biosynthesis and introducing the bioactivity of many halogenated secondary metabolites. In this study, 54 reduced flavin adenine dinucleotide (FADH₂)-dependent halogenase gene-positive strains were identified after the PCR screening of a large collection of 228 reference strains encompassing all major families and genera of filamentous actinomycetes. The wide distribution of this gene was observed to extend to some rare lineages with higher occurrences and large sequence diversity. Subsequent phylogenetic analyses revealed that strains containing highly homologous halogenases tended to produce halometabolites with similar structures, and halogenase genes are likely to propagate by horizontal gene transfer as well as vertical inheritance within actinomycetes. Higher percentages of halogenase gene-positive strains than those of halogenase gene-negative ones contained polyketide synthase genes and/or nonribosomal peptide synthetase genes or displayed antimicrobial activities in the tests applied, indicating their genetic and physiological potentials for producing secondary metabolites. The robustness of this halogenase gene screening strategy for the discovery of particular biosynthetic gene clusters in rare actinomycetes besides streptomycetes was further supported by genome-walking analysis. The described distribution and phylogenetic implications of the FADH₂-dependent halogenase gene present a guide for strain selection in the search for novel organohalogen compounds from actinomycetes.It is well known that actinomycetes, notably filamentous actinomycetes, have a remarkable capacity to produce bioactive molecules for drug development (4, 6). However, novel technologies are demanded for the discovery of new bioactive secondary metabolites from these microbes to meet the urgent medical need for drug candidates (5, 9, 31).Genome mining recently has been used to search for new drug leads (7, 20, 42, 51). Based on the hypothesis that secondary metabolites with similar structures are biosynthesized by gene clusters that harbor certain homologous genes, such homologous genes could serve as suitable markers for distinct natural-product gene clusters (26, 51). A wide range of structurally diverse bioactive compounds are synthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) systems in actinomycetes, therefore much attention has been given to revealing a previously unrecognized biosynthetic potential of actinomycetes through the genome mining of these genes (2, 3, 22). However, the broad distribution of PKS and NRPS genes and their high numbers even in a single actinomycete complicate their use (2, 3). To rationally exploit the genetic potential of actinomycetes, more and more special genes, such as tailoring enzyme genes, are being utilized for this sequence-guided genetic screening strategy (20, 38).Tailoring enzymes, which are responsible for the introduction and generation of diversity and bioactivity in several structural classes during or after NRPS, PKS, or NRPS/PKS assembly lines, usually include acyltransferases, aminotransferases, cyclases, glycosyltransferases, halogenases, ketoreductases, methyltransferases, and oxygenases (36, 45). Halogenation, an important feature for the bioactivity of a large number of distinct natural products (16, 18, 30), frequently is introduced by one type of halogenase, called reduced flavin adenine dinucleotide (FADH₂)-dependent (or flavin-dependent) halogenase (10, 12, 35). More than 4,000 halometabolites have been discovered (15), including commercially important antibiotics such as chloramphenicol, vancomycin, and teicoplanin (43).Previous investigations of FADH₂-dependent halogenase genes were focused largely on related gene clusters in the genera Amycolatopsis (33, 44, 53) and Streptomyces (8, 10, 21, 27, 32, 34, 47-49) and also on those in the genera Actinoplanes (25), Actinosynnema (50), Micromonospora (1), and Nonomuraea (39); however, none of these studies has led to the rest of the major families and genera of actinomycetes. In addition, there is evidence that FADH₂-dependent halogenase genes of streptomycetes usually exist in halometabolite biosynthetic gene clusters (20), but we lack knowledge of such genes and clusters in other actinomycetes.In the present study, we show that the distribution of the FADH₂-dependent halogenase gene in filamentous actinomycetes does indeed correlate with the potential for halometabolite production based on other genetic or physiological factors. We also showed that genome walking near the halogenase gene locus could be employed to identify closely linked gene clusters that likely encode pathways for organohalogen compound production in actinomycetes other than streptomycetes.