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Putative open reading frames (ORFs) encoding laminin-like proteins are found in all members of the genus Megalocytivirus, family Iridoviridae. This is the first study that identified the VP23R protein encoded by ORF23R of the infectious spleen and kidney necrosis virus (ISKNV), a member of these genes of megalocytiviruses. The VP23R mRNA covering the ISKNV genomic coordinates 19547 to 22273 was transcribed ahead of the major capsid protein. Immunofluorescence analysis demonstrated that VP23R was expressed on the plasma membrane of the ISKNV-infected cells and could not be a viral envelope protein. Residues 292 to 576 of VP23R are homologous to the laminin γ1III2-6 fragment, which covers the nidogen-binding site. An immunoprecipitation assay showed that VP23R could interact with nidogen-1, and immunohistochemistry showed that nidogen-1 was localized on the outer membrane of the infected cells. Electron microscopy showed that a virus-mock basement membrane (VMBM) was formed on the surface of the infected cells and a layer of endothelial cells (ECs) was attached to the VMBM. The VMBM contained VP23R and nidogen-1 but not collagen IV. The attached ECs were identified as lymphatic endothelial cells (LECs), which have unique feature of overlapping intercellular junctions and can be stained by immunohistochemistry using an antibody against a specific lymphatic marker, Prox-1. Such infection signs have never been described in viruses. Elucidating the functions of LECs attached to the surface of the infected cells may be useful for studies on the pathogenic mechanisms of megalocytiviruses and may also be important for studies on lymphangiogenesis and basement membrane functions.Basement membrane (BM), a dense and sheetlike structure that is always associated with cells, is a very important specialized form of extracellular matrix (31, 67). BMs mediate tissue compartmentalization and provide structural support to the epithelium, endothelium, peripheral nerve axons, fat cells, and muscle cells, as well as structural and functional foundations of the vasculature (25, 31, 52). BM is also an important regulator of cell behaviors, such as adhesion, migration, proliferation, and differentiation. BMs are highly cross-linked and insoluble materials. They are highly complex and are made up of more than 50 known components (31, 54). Although the molecular composition of BMs is unique in each tissue, their basic structures are similar. Even if many more isoforms exist in different species, the major BM proteins and their receptors are conserved from Caenorhabditis elegans to mammals. BM consists of a layer of laminin polymer, a layer of type IV collagen network, and the nidogen protein, which acts as a cross-linker of these two networks. Other BM components, such as perlecan and fibulin, interact with the laminin polymer and the type IV collagen network to organize a functional BM on the basolateral aspect of the cells (31, 45, 52).The components of BM are able to self-assemble and form a sheetlike structure, and laminin is the key molecule in this process (50). Laminin protein consists of three different chains (α, β, and γ), which comprise a cross-shaped molecular structure with three short amino-terminal arms and a long carboxyl-terminal triple-helical arm (58, 68). The three short arms of this cross-shaped structure can interact with each other in the presence of calcium. Through the binding of globular G domain at the carboxyl-terminal end of the α chain to the cell receptors (e.g., integrins and dystroglycans), laminin self-assembles into polygonal lattices on cell surfaces. This process initiates BM self-assembly (15, 21, 25, 38, 65, 66). To date, 17 laminin isoforms have been observed in different tissues (51). Among them, laminin-1, the crux of early embryonic BM assembly, has been well studied. Laminin-1 consists of α1, β1, and γ1 chains and can interact with nidogen-1 with high affinity through a laminin-type epidermal growth factor-like (LE) module, γ1III4, within the domain III of the γ1 chain (1, 42). The heptapeptide “NIDPNAV” within the γ1III4 motif of laminin-1 is essential for the interaction between laminin-1 and nidogen-1 (41, 46). Blocking the interactions between laminin-1 and nidogen-1 leads to the disruption of BMs. This indicates that the formation of laminin/nidogen complex is essential for BM assembly and stability (30, 61). Nidogen-1, also called entactin-1, is a dumbbell-shaped sulfated 150-kDa glycoprotein consisted of three domains (G1, G2, and G3) (12). By interacting with collagen IV through its G2 domain and binding with laminin γ1 chain through its G3 domain, nidogen-1 bridges the layers of the laminin network and the collagen IV network to construct the fundamental structure of BMs (48). Collagen IV is a triple-helical trimer composed of three α chains. Through the hexamer formation of the carboxyl-terminal globular non-collagenous-1 (NC1) domain of each α chain, two collagen IV proteins assemble into a dimer. Dimers of collagen IV connect with each other via their amino-terminal 7S domains and self-assemble into a network (24, 27, 31, 32). Six kinds of α chains of collagen IV have been identified in mammals. Among them, α1 and α2 chains are the most abundant forms of collagen IV found in all BMs (19, 23). They commonly form a collagen IV molecule with a α1 and α2 ratio of 2:1 (31, 35).Iridoviruses infect invertebrates and poikilothermic vertebrates, including insects, fish, amphibians, and reptiles. These viruses are a group of icosahedral cytoplasmic DNA viruses with circularly permuted and terminally redundant DNA genomes (6, 8, 9, 10, 57, 62). The family Iridoviridae has been subdivided into five genera: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystisvirus, and Megalocystivirus (7). The genus Megalocystivirus, characterized by the ability to cause swelling of the infected cells, is one group of the most harmful viruses to cultured fish (7, 26, 29). Infectious spleen and kidney necrosis virus (ISKNV), the causative agent of a disease that causes high mortality rates in farmed mandarin fish, Siniperca chuatsi, and large-mouth bass, Micropterus salmoides, is regarded as the type species of Megalocystivirus (7). Similar to infection caused by other members of the Megalocystivirus, fish ISKNV infection is characterized by cell hypertrophy in the spleen, kidney, cranial connective tissue, and endocardium (16, 17). Aside from mandarin fish and large-mouth bass, ISKNV-like virus can also be detected in the tissues of more than 60 marine and freshwater fishes (14, 28, 59, 64). The entire genome of ISKNV has been sequenced, and the organization of open reading frames (ORFs) of ISKNV was analyzed by using DNASTAR Omiga 2.0 and Genescan (18). The ISKNV genome is about 110 kbp and contains 125 putative ORFs (GenBank accession no. AF371960).Putative ORFs, encoding viral proteins containing a fragment homologous to laminin and a putative transmembrane fragment, were found in all of the sequenced genomes of the members of Megalocystivirus. These ORFs include ORF23R of ISKNV (GenBank accession no. AAL98747), laminin-like protein gene of olive flounder iridovirus (GenBank accession no. AAT76907), ORF2 of sea perch iridovirus (GenBank accession no. AAV51313), predicted laminin-type epidermal growth factor-like protein of large yellow croaker iridovirus (GenBank accession no. ABI32391), an unknown gene of red sea bream iridovirus (GenBank accession no. AAQ07956), ORF2 of rock bream iridovirus (GenBank accession no. AAN86692), and laminin-type epidermal growth factor-like protein of orange-spotted grouper iridovirus (GenBank accession no. AAX82335). These putative proteins are highly homologous to each other in amino acid sequence (65 to 99% identity). However, the functions of these proteins have never been identified. This is the first study to identify that the VP23R protein encoded by ORF23R of ISKNV is a plasma membrane-localized viral protein. In addition, we discovered a new function of VP23R in a unique pathological phenomenon of virus infection: the attachment of lymphatic endothelial cells (LECs) to the infected cells. Nidogen-1 assisted VP23R in the construction of a BM-like structure, providing an attachment site for LECs. This unique pathological phenomenon has never been found in viruses and is an attractive direction for studies of pathogenic mechanisms of megalocystiviruses. Moreover, studies on the unique profiles of the virus-mock BM can help us learn more about the functions of BM components and the mechanisms of lymphangiogenesis.  相似文献   

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Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.The movement of water across cell membranes has long been thought to occur by free diffusion through the lipid bilayer. However, the discovery of the membrane protein CHIP28 in red blood cells has suggested the involvement of protein channels (29), and it is now well established that transmembrane water permeability is facilitated by aquaporins (AQPs), water channel proteins that are found in bacteria, fungi, plants, and animals (1, 7, 13, 24). AQPs contain six transmembrane α-helices and five connecting loops, and both the N and C termini are located in the cytosol. The monomers assemble into tetrameric complexes, with each monomer forming an individual water channel (11, 14, 24, 33). Apart from the exceptions of AQP11 and AQP12 from mice, as described by K. Ishibashi (15), AQPs have two signature Asn-Pro-Ala motifs, which are located in the second intracellular and the fifth extracellular loops, B and E.While 13 different AQPs have been identified in mammals (16), more than 33 AQP homologues have been discovered in plants (6, 17, 30). Plant AQPs fall into four subclasses: (i) the plasma membrane (PM) intrinsic proteins (PIPs), which are localized in the PM; (ii) the tonoplast intrinsic proteins (TIPs), which are localized in the vacuolar membranes; (iii) the nodulin-26-like intrinsic proteins; and (iv) the small basic intrinsic proteins (24). In Arabidopsis and maize, there are 13 PIPs, which can be divided further into two subfamilies, PIP1 and PIP2 (6, 17).The functions and mechanisms of regulation of plant AQPs have been extensively investigated (7, 13, 18, 24). There have been several reports on the water channel activity (WCA) of specific AQPs and their regulation by protein phosphorylation (3, 4, 8, 12, 18, 25, 32, 33). It has been shown that the WCA of the PIP2 member SoPIP2;1 from spinach is regulated by phosphorylation at two Ser residues (19, 33).The physiologically interesting temperature-dependent opening and closing of tulip (Tulipa gesneriana) petals occur concomitantly with water transport and are regulated by reversible phosphorylation of an undefined PIP (4, 5). Recently, four PIP homologues were isolated from tulip petals, and their WCAs have been analyzed by heterologous expression in Xenopus laevis oocytes (3). It has been shown that the tulip PIP TgPIP2;2 (DDBJ/EMBL/GenBank accession no. AB305617) is ubiquitously expressed in all organs of the tulip and that TgPIP2;2 is the most likely of the TgPIP homologues to be modulated by the reversible phosphorylation that regulates transcellular water transport and mediates petal opening and closing (3, 4). However, while the members of the PIP2 subfamily are characterized as water channels (6), TgPIP2;1 (DDBJ/EMBL/GenBank accession no. AB305616) shows no significant WCA in the oocyte expression system (3). There is growing interest in research on AQPs due to their crucial roles in the physiology of plants and animals (1, 16, 21-24, 26-28, 36). The assay of AQP channel activity is usually performed using either a X. laevis oocyte expression system (29) or a stopped-flow light-scattering spectrophotometer (35), both of which are not widely available. Furthermore, the complexity of these methods and requirement of expertise limit their high-throughput applications. In contrast, a Pichia pastoris expression system is simple to use, inexpensive, and feasible and can be used in high-throughput applications. Although a P. pastoris expression system has been shown to assay the WCA of a TIP (9), extensive research is necessary with other AQPs such as PIPs or AQPs present in intragranular membranes to establish whether this assay system can be used to characterize a water channel and study its regulation mechanisms. With this in view, in the study reported herein, TgPIP2;1 and TgPIP2;2 have been heterologously expressed in P. pastoris, and their WCAs have been assayed. The effects of several factors, such as osmolarity, pH, and inhibitors of protein kinases (PKs) and protein phosphatases (PPs), on the WCA of the recombinant P. pastoris have been investigated. Based on the results, we demonstrate that the P. pastoris heterologous expression system can be used to rapidly characterize PIP channels, to monitor the effects of mutations, and to score the effects of inhibitors and abiotic factors.  相似文献   

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Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.Bacteria use type IV secretion (T4S) to deliver macromolecules to prokaryotes and eukaryotes (12). Animal and human pathogens deliver proteins to their eukaryotic hosts to affect cellular processes causing disease. The plant-pathogenic bacterium Agrobacterium tumefaciens delivers both proteins and DNA to plants and other eukaryotes. DNA delivered by Agrobacterium directs constitutive synthesis of phytohormones in a transformed plant cell, promoting cancerous growth (56). The Ptl toxin of Bordetella pertussis modifies G proteins by ADP-ribosylation, affecting intracellular cell signaling, and CagA of Helicobacter pylori disrupts epithelial cell polarity by inhibiting PAR1 kinase activity (37, 44, 47). T4S is ancestrally related to bacterial conjugation, a mechanism used by bacteria for interbacterial plasmid transfer, enabling them to acquire novel genes for antibiotic resistance, degradation of organic molecules, toxin production, and other virulence traits (29).The VirD4/VirB family of proteins, found conserved in many alphaproteobacteria, mediates T4S (12). The Ti plasmid-encoded Agrobacterium T4S system requires VirD4 and 11 VirB proteins, VirB1 to VirB11, for efficient DNA transfer (7, 54). The membrane and membrane-associated VirB proteins assemble a macromolecular structure at the cell membrane to promote substrate transfer (12). The octopine Ti plasmid pTiA6NC-encoded VirB6 to VirB11 proteins assemble the T4S apparatus at a cell pole (34, 35, 39). The VirD4 coupling protein targets the VirE2 substrate protein to the cell pole (4). A recent study found that the nopaline Ti plasmid pTiC58 T4S system (T4SS) and its substrates form a helical array around the cell circumference (1). Structural studies using Escherichia coli conjugative plasmid pKM101-encoded VirB homologues showed that TraN (VirB7), TraO (VirB9), and TraF (VirB10) form the core complex and that TraF forms a channel at the outer membrane (11, 23). The Agrobacterium VirB proteins assemble a T-pilus, an appendage composed primarily of VirB2, with VirB5 and VirB7 as its minor constituents (38, 40, 41, 48, 50, 55). VirB3, a homolog of the pilin-like TraL protein encoded in E. coli plasmids, is postulated to function in T-pilus assembly (52). Three ATP-utilizing proteins, VirB4, VirB11, and VirD4, supply energy for substrate translocation (3, 9, 34).The membrane topology of all the VirB proteins, except for VirB3, was determined by analyses of random phoA insertion mutants, targeted phoA fusions, and targeted bla fusions (6, 14, 15, 21, 22, 31, 35, 53). phoA and bla, which encode alkaline phosphatase and β-lactamase, respectively, serve as excellent markers for periplasmic proteins, as they are enzymatically active only when targeted to the cell periplasm (8, 30). Green fluorescent protein (GFP) is an ideal cytoplasmic marker because it fluoresces only when located in the cytoplasm (19, 20). When GFP is targeted to the periplasm through fusion with a membrane-spanning domain (MSD), it fails to fold properly and does not fluoresce.The prevailing view, based on in silico analysis, is that VirB3 is a bitopic membrane protein with a periplasmic C terminus. No phoA-positive insertions in virB3, however, were identified in two random mutagenesis studies of the virB operon (6, 15). The small size of VirB3, a polypeptide of 108 amino acids (aa), could be a contributing factor to the negative findings. Yet several PhoA-positive insertions in two smaller VirB proteins, VirB2 (74-aa mature peptide) and VirB7 (41-aa mature peptide), were successfully obtained in both studies. Therefore, the negative findings may also be indicative of the presence of a small periplasmic domain in VirB3. Biochemical studies showed that the nopaline Ti plasmid pTiC58-encoded VirB3 protein (pTiC58 VirB3) associates with the bacterial outer membrane, while VirB2 associates with both the inner and outer membranes (52). The pTiC58 VirB4 protein is required for localization of VirB3 to the outer membrane (33). VirB4 is also required for VirB3 stability (33, 55). A low level of VirB3 accumulated in a nonpolar pTiC58 virB6 deletion mutant; however, addition of virB6 in trans did not restore the level of the protein, even though it restored tumorigenicity (27). VirB3 participates in the formation of protein complexes with the T-pilus proteins VirB2 and VirB5 (55).Homologues of VirB3 are found in many alphaproteobacteria with a T4SS. While most VirB3 homologues are small proteins, several recently identified homologues are fusions of VirB3 and the immediate downstream protein VirB4 (5, 10, 24). These fusion homologs, which include Actinobacillus MagB03 (GenBank accession no. AAG24434), Campylobacter CmgB3/4 (EAQ71805), Yersinia pseudotuberculosis TriC (CAF25448), Citrobacter koseri PilX3-4 (ABV12046), and Klebsiella pneumoniae PilX3-4 (BAF49490), have VirB3 at the N terminus and VirB4 at the C terminus. Agrobacterium VirB4 is an integral membrane protein with a cytoplasmic N terminus (14). Its homologues are expected to have a similar topology. The prevailing view that pTi VirB3 has a periplasmic C terminus is inconsistent with the cytoplasmic location of the N terminus of VirB4 in the VirB3-VirB4 fusion protein homologues.In this study, we report the membrane topology of Agrobacterium VirB3 and demonstrate that the C terminus of the protein resides in the cytoplasm. We also demonstrate that VirB3 is an inner membrane protein, not an outer membrane protein as previously reported (52). The octopine Ti plasmid pTiA6NC VirB4 protein does not affect membrane localization of VirB3 but does stabilize VirB3. VirB4, however, is not sufficient for pTiA6NC VirB3 stabilization. Two additional proteins, VirB7 and VirB8, are required for the stabilization of pTiA6NC VirB3.  相似文献   

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9.
Development of broadly cross-reactive neutralizing antibodies (NAbs) remains a major goal of HIV-1 vaccine development, but most candidate envelope immunogens have had limited ability to cross-neutralize heterologous strains. To evaluate the immunogenicity of subtype A variants of HIV-1, rabbits were immunized with pairs of closely related subtype A envelopes from the same individual. In each immunogen pair, one variant was readily neutralized by a variety of monoclonal antibodies and plasma antibodies, while the other was neutralization resistant, suggesting differences in the exposures of key epitopes. The breadth of the antibody response was evaluated against subtype A, B, C, and D variants of HIV-1. The specificity of the immunogen-derived neutralizing antibody response was also compared to that of the infected individuals from whom these variants were cloned. None of the immunogens produced broad neutralizing antibodies in immunized animals, and most of the neutralizing antibodies were directed to the variable loops, particularly the V3 loop. No detectable antibodies to either of the potentially exposed conserved epitopes, the membrane proximal external region, or the CD4 binding site were found with immunized rabbits. In contrast, relatively little of the neutralizing activity within the plasma samples of the infected individuals was directed to linear epitopes within the variable loops. These data indicate that immunogens designed to expose conserved regions did not enhance generation of broadly neutralizing antibodies in comparison with the immunogens that failed to expose those regions using this immunization approach.The ability to elicit broadly cross-reactive neutralizing antibodies (NAbs) is likely to be an important component of an effective vaccine to human immunodeficiency virus type 1 (HIV-1). Unfortunately, the HIV-1 envelope (Env)-based vaccines developed to date do not elicit such antibodies. Initial vaccines based on soluble, monomeric gp120 generated antibodies capable of only weakly neutralizing the homologous virus, with a very narrow breadth of cross-reactivity (13, 30, 53). Subsequent modifications to the Env immunogens, including variable loop deletions (15, 20, 31, 34, 35, 61, 64-66), alterations in the glycosylation pattern (4, 10, 11, 14, 30, 43, 55, 56), epitope repositioning (39, 46), the use of consensus Envs (22, 36, 37, 47), and the use of soluble trimeric gp140 molecules as immunogens (1-3, 5, 14, 16, 20, 21, 24, 25) have led to only modest enhancements in NAb breadth or potency. These modified Env immunogens have failed to redirect NAbs from the variable loops to more conserved regions of Env (reviewed in reference 33).Differences in Env structure between HIV-1 subtypes may further hinder efforts to elicit broadly cross-reactive antibodies capable of protecting against transmitted strains worldwide. Most immunogens tested to date have been derived from subtype B Envs. However, there are clear antigenic differences between subtype B strains and the subtype A and C strains that account for most infections worldwide (6, 8, 27, 28, 40, 42). For instance, most transmitted subtype A Envs are resistant to the monoclonal antibodies 2G12, b12, 2F5, and 4E10, either because of alterations in the epitopes for these monoclonal antibodies (MAbs) or because the epitopes are shielded in these Envs (6, 8). It is therefore possible that even NAbs specific for a conserved region of subtype B Envs, such as the CD4 binding site, would not be able to access and neutralize a similar epitope on a subtype A Env.In order to evaluate the immunogenicity of subtype A Envs, which account for ∼25% of global HIV-1 infections (12), we previously investigated the types of antibody responses elicited following gp160 priming and gp140 boosting with immunogens derived from four subtype A Envs in comparison to the subtype B Env SF162 (38). These experiments were also designed to explore whether deriving immunogens from HIV-1 Envs isolated from early in infection would better target NAbs to transmitted strains. Although all of the subtype A-based immunogens and the SF162 immunogen elicited anti-V3 NAbs capable of neutralizing the easy-to-neutralize SF162 pseudovirus, only one of the four immunogens generated homologous NAbs (38). Even immunogens with shorter variable loops or fewer potential N-linked glycosylation sites (PNGS) did not lead to enhanced breadth of neutralization against heterologous subtype A or B Envs (38). However, the four subtype A Envs used in these immunizations were generally neutralization resistant to both plasma samples from HIV-1-infected individuals and to monoclonal antibodies (6), raising the possibility that the poor breadth observed could be related to the shielding of conserved epitopes within these Envs.In order to determine whether using subtype A Env immunogens that do not shield conserved epitopes could improve neutralization breadth, here we performed immunizations with pairs of Env immunogens derived from two individuals acutely infected with subtype A HIV-1. The Envs in each pair were very similar in their amino acid sequences yet differed dramatically in their neutralization phenotype (6, 9) (Fig. ​(Fig.1A).1A). The pair from subject Q461 had a neutralization-resistant Env, Q461e2 (termed Q461e2R to indicate neutralization resistance), and a neutralization-sensitive Env, Q461d1 (termed Q461d1S to indicate neutralization sensitivity), which was sensitive to neutralization by plasma, 2F5, 4E10, b12, and soluble CD4 (sCD4). We previously demonstrated that the neutralization sensitivity of the Q461d1S Env is mediated entirely by two amino acid substitutions in gp41, one in the first heptad repeat and one in the membrane proximal external region (MPER) (9). These mutations led to enhanced exposure of both the CD4 binding site and the MPER (9). From subject Q168, the Env Q168b23S was sensitive to autologous and heterologous plasma and to the MPER antibodies 2F5 and 4E10 but resistant to b12 and sCD4, while Q168a2R was weakly neutralized by the MPER antibodies, less sensitive to neutralization by autologous plasma, and resistant to heterologous plasma (6). The Q168a2R and Q168b23S Envs contain identical sequences in the MPER region yet have >500-fold differences in neutralization sensitivity to 2F5 and 4E10, indicating that the exposure of the MPER region, rather than the sequence, likely accounts for the enhanced neutralization of the Q168b23S Env.Open in a separate windowFIG. 1.Analysis of Q461d1S gp140 used for immunizations. (A) SDS-PAGE analysis of final preparation of Q461d1S gp140 from the GNA capture and DEAE and CHAP columns. Lane 1 contains molecular weight standards, lane 2 the concentrated DEAE flowthrough, and lane 3 the final concentrated protein. The purified Q461d1S gp140 protein is indicated by an arrow. The sizes of the molecular weight markers (in thousands) are indicated on the left. (B) Binding of purified gp140 subtype A to CD4 as determined by a high-pressure liquid chromatography (HPLC)-based assay. The bottom line represents the protein obtained after the GNA column, and the top line represents purified protein after all three steps. The trimer and monomer peaks are marked. (C) Summary of neutralization characteristics of all four HIV-1 subtype A Env variants used in the immunizations, adapted from reference 6. The pseudovirus is shown in the far left column. IC50 values for plasma sample (left) and monoclonal antibodies (right) are displayed. The autologous plasma samples were taken 3.7 ypi for subject Q461 and 2.6 ypi for subject Q168. The Kenya pool was derived by pooling plasma from 30 HIV-1-infected individuals in Kenya and has been described previously (6).Thus, to directly test whether using Env immunogens that expose conserved epitopes could enhance neutralization breadth immunization, here we immunized with these pairs of related Envs, in which one variant exposes conserved regions, while the other does not. We also compared the specificity of the NAb responses following immunization with these Envs with the specificities of the NAbs that developed during natural infection in the individuals from whom these variants were cloned.  相似文献   

10.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.

Non-Bacterial genomes

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11.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.

Phylum Crenarchaeota

Phylum Deinococcus-Thermus

Phylum Proteobacteria

Phylum Tenericutes

Phylum Firmicutes

Phylum Actinobacteria

Phylum Spirochaetes

Non-Bacterial genomes

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12.
The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.Cytomegalovirus (CMV) is a highly prevalent betaherpesvirus that can cause severe multiorgan disease in immunocompromised individuals (45). The ability of this virus to infect an exceptionally wide variety of different cell types substantially contributes to pathogenesis (5, 68). CMV tropism is largely determined by a finely tuned interplay between cellular and viral factors, many of which act at the earliest stages of infection (30, 68). We recently showed that the cellular protein vimentin is required for efficient onset of infection in primary human foreskin fibroblasts (HF). Interestingly, the degree of reliance on the presence and integrity of vimentin intermediate filaments is dependent on the virus strain, with the broadly tropic strain TB40/E being more negatively affected than the HF-adapted, attenuated strain AD169 (44).Serial passage of clinical isolates in HF or in endothelial cells (EC) has produced strains with different tropisms. The attenuated strains AD169 and Towne were developed as vaccine candidates by propagation in HF for more than 50 (AD169) and 125 (Towne) serial passages (19, 53, 61). During this process, both strains, compared to clinical isolates, accumulated multiple mutations and genomic deletions, resulting in the loss of more than 19 open reading frames (ORFs) (8). The number of serial passages in HF of another commonly used strain, Toledo, has been more moderate (19, 54, 58). This, however, did not prevent the emergence of numerous genomic mutations, including the inversion of an ∼15-kb fragment (8, 16, 56). As a consequence of these changes, productive infections by AD169, Towne, and Toledo are largely restricted to HF. In contrast, propagation of clinical isolates in EC has yielded a series of strains with more-intact genomes and broader tropisms, such as TB40/E, VHL/E, and FIX (VR1814) (25, 60, 71). These strains retain the ability to grow in a wider variety of cell types, including EC, epithelial cells, and dendritic cells (DC), in addition to HF (23, 28, 59, 60, 68).The UL128, UL130, and UL131A gene products were recently identified as essential mediators of CMV infection of EC and epithelial cells (26, 72, 73) and of virus transfer from infected EC to monocyte-derived DC (23). Each of these proteins is independently required for the broader tropisms of EC-propagated CMV isolates (63, 64), and the presence of mutations affecting their functionality has been directly linked to the inability of AD169, Towne, and Toledo to initiate productive infections in EC and epithelial cells (26, 72, 73).We have shown that mature Langerhans-type DC differentiated in vitro from CD34+ hematopoietic progenitor cells are highly permissive to direct infection with TB40/E or VHL/E, with 48 to 72% of cells in culture expressing the viral immediate-early genes IE1 and IE2 at 48 h postinfection (hpi) (28). In contrast, only 2 to 5% and 0% of mature Langerhans cells were IE1/IE2 positive after exposure to Towne and Toledo, respectively. However, productive infection was detected in 12 to 17% of cells infected with AD169, despite the fact that this strain lacks expression of the UL131A gene as a consequence of a frameshift mutation (26). These results suggested the existence of additional viral genes with products involved in mediating tropisms for specific cell types, such as DC. To identify possible candidates, we compared the amino acid sequence of each ORF found in the genome of TB40-BAC4, a sequenced clone of the TB40/E strain in a bacterial artificial chromosome (BAC) (GenBank accession number EF999921) (69), to the sequence of each ORF found in AD169 and AD169-BAC (accession numbers X17403 and AC146999) (10, 49), Towne and Towne-BAC (accession numbers FJ616285, AC146851, and AY315197) (17, 18, 49), and Toledo-BAC (accession number AC146905) (49). The product of a putative ORF, originally identified by Murphy et al. and named conserved ORF 29 (c-ORF29) (49), was considered of particular interest because the amino acid sequence of the putative protein encoded by Toledo and Towne was extended by 79 residues compared to the putative protein encoded by TB40/E and AD169. This led to our speculation that that the extended version might result in a nonfunctional version of the c-ORF29-encoded protein. We thus focused our studies on the products of this ORF.Here, we show for the first time that CMV c-ORF29 encodes a protein expressed at early to late times postinfection (p.i.) and localizes to the nuclear rim in peculiar invaginations of the nuclear lamina and in cytoplasmic vesicular structures at late times p.i. Based on this localization pattern, we named this gene product nuclear rim-associated cytomegaloviral protein, or RASCAL. Surprisingly, no difference was observed in the distributions of RASCAL during infection of HF with TB40/E or Towne, suggesting that the intracellular trafficking of this protein is not affected by the presence of the additional residues at the C terminus of RASCAL from strain Towne (RASCALTowne). Ectopic expression of RASCAL in human embryo kidney 293T (HEK293T) cells further revealed that this protein requires the presence of the nuclear egress complex (NEC) member UL50 to reach the nuclear rim, while coimmunoprecipitation (co-IP) assays provided evidence for the existence of an interaction between RASCAL and UL50. These findings suggest that RASCAL may be a new component of the NEC with possible roles in remodeling the nuclear lamina during nucleocapsid egress from the nucleus.  相似文献   

13.
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14.
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).  相似文献   

15.
In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.Current dogma suggests the Gram-negative motile bacterium Escherichia coli colonizes the infant gut within hours of birth and establishes itself as the predominant facultative anaerobe of the colon for the remainder of life (3, 59). While the majority of E. coli strains maintain this harmless existence, some strains have adopted a pathogenic lifestyle. Contemporary tenets suggest that pathogenic strains of E. coli have acquired genetic elements that encode virulence factors and enable the organism to cause disease (12). The large repertoire of virulence factors enables E. coli to cause a variety of clinical manifestations, including intestinal infections mediating diarrhea and extraintestinal infections, such as urinary tract infections, septicemia, and meningitis. Based on clinical manifestation of disease, the repertoire of virulence factors, epidemiology, and phylogenetic profiles, the strains causing intestinal infections can be divided into six separate pathotypes, viz., enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), diffuse adhering E. coli (DAEC), and enterotoxigenic E. coli (ETEC) (33, 35, 39).ETEC is responsible for the majority of E. coli-mediated cases of human diarrhea worldwide. It is particularly prevalent among children in developing countries, where sanitation and clean supplies of drinking water are inadequate, and in travelers to such regions. It is estimated that there are 200 million incidences of ETEC infection annually, resulting in hundreds of thousands of deaths in children under the age of 5 (55, 64). The essential determinants of ETEC virulence are traditionally considered to be colonization of the host small-intestinal epithelium via plasmid-encoded colonization factors (CFs) and subsequent release of plasmid-encoded heat-stable (ST) and/or heat-labile (LT) enterotoxins that induce a net secretory state leading to profuse watery diarrhea (20, 62). More recently, additional plasmid-encoded factors have been implicated in the pathogenesis of ETEC, namely, the EatA serine protease autotransporter (SPATE) and the EtpA protein, which acts as an intermediate in the adhesion between bacterial flagella and host cells (23, 32, 42, 46). Furthermore, a number of chromosomal factors are thought to be involved in virulence, e.g., the invasin Tia; the TibA adhesin/invasin; and LeoA, a GTPase with unknown function (14, 21, 22). E. coli H10407 is considered a prototypical ETEC strain; it expresses colonization factor antigen 1 (CFA/I) and the heat-stable and heat labile toxins. Loss of a 94.8-kb plasmid encoding CFA/I and a gene for ST enterotoxin from E. coli strain H10407 leads to reduced ability to cause diarrhea (17).Here, we report the complete genome sequence and virulence factor repertoire of the prototypical ETEC strain H10407 and the nucleotide sequence and gene repertoire of the plasmids from ETEC strain E1392/75, and we describe a novel conserved secretion system associated with the sequenced ETEC strains.  相似文献   

16.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to this subsequent versions of this list are invited to provide the bibliometric data for such references to the SIGS editorial office.

Non-Bacterial genomes

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17.
Pantoea agglomerans is an ecologically diverse taxon that includes commercially important plant-beneficial strains and opportunistic clinical isolates. Standard biochemical identification methods in diagnostic laboratories were repeatedly shown to run into false-positive identifications of P. agglomerans, a fact which is also reflected by the high number of 16S rRNA gene sequences in public databases that are incorrectly assigned to this species. More reliable methods for rapid identification are required to ascertain the prevalence of this species in clinical samples and to evaluate the biosafety of beneficial isolates. Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) methods and reference spectra (SuperSpectrum) were developed for accurate identification of P. agglomerans and related bacteria and used to detect differences in the protein profile within variants of the same strain, including a ribosomal point mutation conferring streptomycin resistance. MALDI-TOF MS-based clustering was shown to generally agree with classification based on gyrB sequencing, allowing rapid and reliable identification at the species level.Pantoea agglomerans (20) is a ubiquitous plant-epiphytic bacterium that belongs to the family Enterobacteriaceae. While several strains are commercialized for biological control of plant diseases (23), the species also includes two phytopathogenic pathovars that carry distinctive virulence plasmids (32). P. agglomerans has a Jekyll-Hyde nature, being described also as an opportunistic human pathogen (30), which raises biosafety regulatory issues for the utilization of beneficial isolates (45). Clinical reports predominantly involve septicemia following penetrating trauma (16, 56) or nosocomial infections (14, 55). Clinical pathogenicity of this species has not been confidently confirmed (unfulfilled Koch''s postulates). Infections attributed to P. agglomerans are typically of a polymicrobial nature involving patients affected by other diseases (14) and may represent secondary contamination of wounds. Standard clinical diagnostics and identification rely mainly on biochemical profiling analysis or alternatively on 16S rRNA gene sequencing, despite the inadequacy of these techniques for precise discrimination within the Enterobacter and Pantoea genera (5, 20, 39). Problems with correct identification have been observed for automated systems such as the API 20E (24, 39) and Vitek-2/GNI+ (39, 40) (both from bioMerieux) or the Phoenix (11, 38) and Crystal identification systems (40, 48) (both from BD Diagnostic Systems).P. agglomerans is a composite taxon conglomerating former Enterobacter agglomerans, Erwinia milletiae, and Erwinia herbicola strains. Accurate identification is complicated by the unsettled taxonomy of the “P. agglomerans-E. herbicola-E. agglomerans” complex (45). Recent analyses based on gyrB sequencing, multilocus sequence analysis (MLSA) (4), and fluorescent amplified fragment length polymorphisms (fAFLP) (45) indicate that strains belonging to Enterobacter or Erwinia archived in culture collections are often erroneously assigned to P. agglomerans and are likely also misidentified in clinical diagnostics. False classifications of environmental P. agglomerans strains as related Pantoea species, including human- or plant-pathogenic P. ananatis, are also common (45). Inadequate biochemical identification methods and uncertainty regarding current taxonomy are revealed also by the excessive number of 16S rRNA gene sequences incorrectly assigned to P. agglomerans that can be retrieved from GenBank (Fig. ​(Fig.1).1). Sequencing of housekeeping genes, MLSA, and fAFLP are labor-intensive, time-consuming, and impractical approaches as routine diagnostic tools.Open in a separate windowFIG. 1.Taxonomy of putative P. agglomerans isolates based on 16S rRNA gene sequences retrieved from GenBank under the currently accepted species name or under the old basonyms Enterobacter agglomerans and Erwinia herbicola. Out of a total of 331 complete or partial sequences found, 263 could be aligned over their 1,240-bp central region resulting in a minimum evolution tree. For the analysis, gaps and missing data were eliminated only in pairwise sequence comparisons, resulting in a total of 1,114 positions. Nodal supports were assessed by 1,000 bootstrap replicates. Only bootstrap values greater than 50% are shown. The scale bar represents the number of base substitutions per site. The number of “P. agglomerans” sequences clustering with a given reference strain in shown in parentheses. Reference strains and clades containing reference strains are marked in bold, and the corresponding accession numbers are indicated between brackets. For the genus Erwinia the following reference strains were used: E. persicina HK204 [NR_026049.1], E. rhapontici 2OP2 [FJ595873], E. billingiae Eb661 [AM055711], E. tasmaniensis Et2/99 [AM292080], and E. amylovora FAW 23482 [AY456711].Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (31) is an emerging technology for identification of bacteria (26, 46), fungi (17, 33), viruses (29, 51), insects (41), and helminths (42). MALDI-TOF MS-based identification can accurately resolve bacterial identity at the genus, species, and in some taxa subspecies levels (e.g., Salmonella enterica serovars, Listeria genotypes) (1, 18). Identity is based on unique mass/charge ratio (m/z) fingerprints of proteins, which are ionized using short laser pulses directed to bacterial cells obtained from a single colony embedded in a matrix. After desorption, ions are accelerated in vacuum by a high electric potential and separated on the basis of the time taken to reach a detector, which is directly proportional to the mass-to-charge ratio of an ion. This technique has been shown to deliver reproducible protein mass fingerprints starting from an aliquot of a single bacterial colony within minutes and without any prior separation, purification, or concentration of samples. Whole-cell MALDI-TOF MS is a reliable technique across broad conditions (e.g., different growth media, cell growth states), with limited variability in mass-peak signatures within a selected mass range (2,000 < m/z < 20,000) that does not affect reliability of identification (28, 31). MALDI-TOF MS profiles primarily represent ribosomal proteins, which are the most abundant cellular proteins and are synthesized under all growth conditions (47). MALDI-TOF MS identification profiles derived from several characterized strains for a given species are used to develop reference spectra (e.g., SuperSpectrum; AnagnosTec GmbH, Potsdam, Germany), and they include a subset of characteristic and reproducible markers. MALDI-TOF MS identification databases are currently available for a relatively wide range of clinical bacteria, and this method has become an accepted tool for routine clinical diagnostics due to enhanced simplicity, rapidity, and reliability. However, environmental bacteria, such as Pantoea, have not been widely evaluated using MALDI-TOF MS and are largely absent from identification databases, limiting the practical reach of this new technology.Our objectives were to develop a robust method for rapid identification of P. agglomerans and related bacteria based on MALDI-TOF MS and to compare MALDI-TOF MS results against those obtained from a phylogenetic analysis based on gyrB sequencing as well as against biochemical identification methods.  相似文献   

18.
The binding affinities and specificities of six truncated S-layer homology domain (SLH) polypeptides of mosquitocidal Bacillus sphaericus strain C3-41 with the purified cell wall sacculi have been assayed. The results indicated that the SLH polypeptide comprised of amino acids 31 to 210 was responsible for anchoring the S-layer subunits to the rigid cell wall layer via a distinct type of secondary cell wall polymer and that a motif of the recombinant SLH polypeptide comprising amino acids 152 to 210 (rSLH152-210) was essential for the stable binding of the S-layer with the bacterial cell walls. The quantitative assays revealed that the KD (equilibrium dissociation constant) values of rSLH152-210 and rSLH31-210 with purified cell wall sacculi were 1.11 × 10−6 M and 1.40 × 10−6 M, respectively. The qualitative assays demonstrated that the SLH domain of strain C3-41 could bind only to the cell walls or the cells treated with 5 M guanidinium hydrochloride of both toxic and nontoxic B. sphaericus strains but not to those from other bacteria, indicating the species-specific binding of the SLH polypeptide of B. sphaericus with bacterial cell walls.Crystalline bacterial cell surface layers (S-layers) cover the cell surfaces of many bacteria and archaea during all stages of growth and division. S-layers are composed of identical protein or glycoprotein subunits, which can assemble into two-dimensional crystalline arrays and exhibit oblique, square, or hexagonal symmetry (27, 28, 30). S-layers play key roles in the interaction between bacterial cells and environment as protective coats, molecular sieves, ion traps, cell adhesion mediators, and attachment structures (4, 21, 26, 29). Many S-layer proteins possess an N-terminal region with highly conserved amino acid sequences, which is called an S-layer homology (SLH) domain. An SLH domain contains one, two, or three repeating SLH motifs (6, 16). Each SLH motif is composed of about 55 amino acids containing 10 to 15 conserved residues (6, 17). It is suggested that the SLH domain of S-layer proteins is responsible for the binding of the S-layer subunits to the rigid cell wall layer (6, 15, 17, 19, 25), while the middle and C-terminal parts include the domains which are involved in the self-assembly process (27). In the case of Bacillaceae, secondary cell wall polymers (SCWP) are responsible for binding with SLH domains (13, 18, 19), but the SLH domains of some other bacteria have an affinity for peptidoglycan (33).Bacillus sphaericus is a gram-positive soil bacterium that represents a strictly aerobic group of mesophilic endospore-forming bacteria. Due to its specific toxicity to target mosquito larvae and the limited environment impact, some strains of this bacterium have been successfully used worldwide in integrated mosquito control programs. Previous studies revealed that some nontoxic strains of B. sphaericus contained S-layer proteins, and the S-layer proteins of B. sphaericus NCTC 9602, JG-A12, P1, and CCM 2177 have been studied in detail elsewhere (3, 7-9, 12, 22).B. sphaericus C3-41, a highly active strain isolated from a mosquito-breeding site in China in 1987, has different levels of toxicity against Culex spp., Anopheles spp., and Aedes spp. This strain belongs to the flagella serotype H5a5b, like strains 2362 and 1593 (32), and it has been developed as a commercial larvicide (JianBao) for mosquito larva control in China during the last decade (31). The genomic analysis of strain C3-41 revealed that an S-layer protein gene (slpC) (GenBank accession no. EF535606) exists on the chromosomal genome and its sequence is identical to the S-layer protein of B. sphaericus 2362 (1, 10), composed of 3,531 bp encoding a protein of 1,176 amino acids with a molecular size of 125 kDa. Although the binding function of S-layers has been identified in some nontoxic B. sphaericus strains (6, 11), it is not well documented in mosquitocidal B. sphaericus strains, and there are few reports on the binding function of each SLH motif and the binding specificity.In this study, the binding affinities and specificities of each SLH motif of S-layer protein from mosquitocidal B. sphaericus C3-41 alone and in combination with the different cell wall preparations have been investigated, and the species-specific binding of SLH polypeptide with bacterial cell walls has been demonstrated.  相似文献   

19.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.

Phylum Euryarchaeota

Phylum Crenarchaeota

Phylum Deinococcus-Thermus

Phylum Proteobacteria

Phylum Tenericutes

Phylum Firmicutes

Phylum Actinobacteria

Non-Bacterial genomes

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20.
The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand β-sheet holding two α-helices of three and four turns that are oriented antiparallel to the β-strands. Two antiparallel two-strand β-sheets and two 310-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.The coronavirus replication cycle begins with the translation of the 29-kb positive-strand genomic RNA to produce two large polyprotein species (pp1a and pp1ab), which are subsequently cleaved to produce 15 or possibly 16 nonstructural proteins (nsp''s) (11). Among these, nsp3 is the largest nsp and also the largest coronavirus protein. nsp3 is a glycosylated (16, 22), multidomain (36, 51), integral membrane protein (38). All known coronaviruses encode a homologue of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp3, and sequence analysis suggests that at least some functions of nsp3 may be found in all members of the order Nidovirales (11). Hallmarks of the coronavirus nsp3 proteins include one or two papain-like proteinase domains (3, 12, 16, 31, 56, 62), one to three histone H2A-like macrodomains which may bind RNA or RNA-like substrates (5, 9, 48, 54, 55), and a carboxyl-terminal Y domain of unknown function (13). An extensive bioinformatics analysis of the coronavirus replicase proteins by Snijder et al. (51) provided detailed annotations of the then-recently sequenced SARS-CoV genome (35, 47), including the identification of a domain unique to SARS-CoV and the prediction of the ADP-ribose-1″-phosphatase (ADRP) activity of the X domain (since shown to be one of the macrodomains).Only limited information is so far available regarding the ways in which the functions of nsp3 are involved in the coronavirus replication cycle. Some functions of nsp3 appear to be directed toward protein; e.g., the nsp3 proteinase domain cleaves the amino-terminal two or three nsp''s from the polyprotein and has deubiquitinating activity (4, 6, 14, 30, 53, 60). Most homologues of the most conserved macrodomain of nsp3 appear to possess ADRP activity (9, 34, 41-43, 48, 59) and may act on protein-conjugated poly(ADP-ribose); however, this function appears to be dispensable for replication (10, 42) and may not be conserved in all coronaviruses (41). The potential involvement of nsp3 in RNA replication is suggested by the presence of several RNA-binding domains (5, 36, 49, 54, 55). nsp3 has been identified in convoluted membrane structures that are also associated with other replicase proteins and that have been shown to be involved in viral RNA synthesis (16, 24, 52), and nsp3 papain-like proteinase activity is essential for replication (14, 62). Other conserved structural features of nsp3 include two ubiquitin-like domains (UB1 and UB2) (45, 49). We have also recently reported that nsp3 is a structural protein, since it was identified as a minor component of purified SARS-CoV preparations, although it is not known whether nsp3 is directly involved in virogenesis or is incidentally incorporated due to protein-protein or protein-RNA interactions (36).A nucleic acid-binding region (NAB) is located within the polypeptide segment of residues 1035 to 1203 of nsp3. The NAB is expected to be located in the cytoplasm, along with the papain-like protease, ADRP, a region unique to SARS-CoV (the SARS-CoV unique domain [SUD]), and nsp3a, since both the N and C termini of nsp3 were shown previously to be cytoplasmic (38). Two hydrophobic segments are membrane spanning (38), and the NAB is located roughly 200 residues in the N-terminal direction from the first membrane-spanning segment. This paper presents the next step in the structural coverage of nsp3, with the determination of the NAB structure. The structural studies included nuclear magnetic resonance (NMR) characterization of two constructs, an nsp3 construct comprising residues 1035 to 1181 [nsp3(1035-1181)] and nsp3(1066-1203), and complete NMR structure determination for the construct nsp3(1066-1181) (see Fig. ​Fig.8).8). The structural data were then used as a platform from which to investigate the nature of the previously reported single-stranded RNA (ssRNA)-binding activity of the NAB (36). Since no three-dimensional (3D) structures for the corresponding domains in other group II coronaviruses are known and since the SARS-CoV NAB has only very-low-level sequence identity to other proteins, such data could not readily be derived from comparisons with structurally and functionally characterized homologues.Open in a separate windowFIG. 8.Sequence alignment of the polypeptide segment nsp3(1066-1181) that forms the globular domain of the SARS-CoV NAB with homologues from other group II coronaviruses. Protein multiple-sequence alignment was performed using ClustalW2 and included sequences from SARS-CoV Tor2 (accession no. AAP41036) and representatives of three protein clusters corresponding to three group II coronavirus lineages identified by a BLAST search: bat coronavirus HKU5-5 (BtCoV-HKU5-5; accession no. ABN10901), BtCoV-HKU9-1 (accession no. P0C6T6), and human coronavirus HKU1-N16 (HCoV-HKU1-N16; accession no. ABD75496). Above the sequences, the positions in full-length SARS-CoV nsp3, the locations of the regular secondary structures in the presently solved NMR structure of the SARS-CoV NAB globular domain, and the residue numbering in this domain are indicated. Amino acids are colored according to conservation and biochemical properties, following ClustalW conventions. Residues implicated in interactions with ssRNA are marked with inverted black triangles. In the present context, the key features are that there is only one position with conservation of K or R (red) and that there are extended sequences with conservation of hydrophobic residues (blue) (see the text).  相似文献   

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