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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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4.
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   

5.
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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6.
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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7.
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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8.
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   

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10.
Molecular analysis of black band disease of corals revealed that samples frozen immediately after collection yielded more proteobacterial 16S rRNA sequences, while unfrozen samples produced more cyanobacterial and sulfur-oxidizing bacterial sequences. These results suggest the need to use multiple approaches for preparation of samples to characterize this complex polymicrobial disease.Black band disease (BBD) is a polymicrobial disease that affects corals on reefs worldwide. It consists of a migrating microbial mat dominated by cyanobacteria that lyses coral tissue, leading to coral colony death, and is one of the most destructive of coral diseases. Microscopic examination of BBD samples consistently reveals an abundance of nonheterocystous, filamentous cyanobacteria and colorless gliding bacteria with internal elemental sulfur granules characteristic of the genus Beggiatoa (6, 17, 18). It is thought that these are key players in the etiology of BBD. However, with one exception (2), previous molecular studies of BBD consistently detected very low proportions of cyanobacteria (4, 8, 9, 19, 20) and only one study has detected Beggiatoa (19). Instead, all molecular BBD studies indicate a highly variable and diverse composition of heterotrophic bacteria, mostly members of the Alphaproteobacteria.It is unknown why the dominant cyanobacteria and filamentous sulfur-oxidizing bacteria observable microscopically in BBD samples are poorly or not at all detected by molecular methods. It is possible that freezing of the samples in these studies is the cause for low detection of BBD cyanobacteria and sulfur oxidizers. Freezing is the common method of sample processing to extract DNA for microbial community analysis of BBD and has been used in all previous molecular studies. However, this approach may impart a bias to detection of specific BBD bacteria. Suomalainen et al. (22) reported that freezing of samples targeting the fish pathogen Flavobacterium columnare destroyed DNA, suggested to be due to the release of DNase and other enzymes from the cell, leaving most of the F. columnare DNA undetectable by PCR. They noted that DNA from bacteria such as Escherichia coli was not affected (22). Bissett et al. (3) speculated that freezing sediments prior to DNA extraction lysed Beggiatoa filaments and caused their DNA to be lost (3). A recent report showed that algae and cyanobacteria with large cell sizes, including filamentous strains, could not be sufficiently cryopreserved (5). While the above-described studies showed or speculated that freezing of samples affects the detection of some microorganisms in environmental samples, none of these studies included detailed investigation of the mechanism responsible for the effect of freezing or of the effect of freezing on the assessment of microbial community composition.In the present study, we investigated the effect of freezing on molecular analysis of the BBD microbial community by using DNA extracts of frozen and unfrozen BBD samples from two coral hosts (Siderastrea siderea and Diploria clivosa), using both universal and cyanobacterium-specific primers targeting the 16S rRNA gene. BBD samples (i.e., the BBD microbial mat) were collected by suctioning the mat off the coral surface using individual sterile syringes while scuba diving. Samples were transferred to 2-ml cryovials (after decanting seawater) upon return to shore and either immediately frozen and stored at −20°C until DNA extraction or maintained at ambient temperature with DNA extracted within 1 h of collection. Eleven samples were collected from reefs of the Florida Keys (United States), Lee Stocking Island (Bahamas), and St. Croix (United States Virgin Islands).Genomic DNA was extracted by the bead-beating method as previously described (12, 19, 20). Frozen samples were first thawed at room temperature, and 500-μl aliquots were directly transferred into multimix lysing matrix tubes by using trimmed pipette tips, excluding any water. Unfrozen samples were transferred to multimix lysing matrix tubes in the same way. The extracted DNA was verified by gel electrophoresis, and DNA extracts from frozen samples were stored at −20°C, whereas DNA extracts from unfrozen samples were kept at 4°C until used for PCR amplification.DNA extracted from both frozen and unfrozen samples was amplified by PCR using universal bacterial primers 27F and 1492R (14) and cyanobacterium-specific primers CYA359F and CYA781R(B) (15) targeting 16S rRNA genes. The purification of PCR products, cloning, and sequencing of plasmid inserts were done as described previously (20). Primer M13F (11) or CYA359F (15) was used to obtain partial sequences, and an additional primer, 518F (13), M13R (11), or CYA781R(B) (15), was used to obtain full-length sequences. Sequence editing, BLAST (1), and phylogenetic analysis using ARB (10) were done as described previously (19, 20). Sequences that matched at similarity identity values of 97% and above were considered to be of the same operational taxonomic unit. Representative gene sequences that were closely related to cyanobacterial sequences were subjected to maximum-parsimony, neighbor-joining, and maximum-likelihood phylogenetic analyses, and a consensus tree was produced based on maximum-parsimony analysis.The results for universal bacterial primers indicated that all of the frozen BBD samples except one (Fig. ​(Fig.1,1, clone library E) were dominated (44 to 87%) by Alphaproteobacteria (Fig. ​(Fig.1;1; see Tables S1 and S2 in the supplemental material). We previously (19) compared the 16S rRNA gene sequences retrieved from seven of these libraries (Fig. ​(Fig.1,1, libraries A to G), all of which were obtained from frozen BBD samples from the host S. siderea, to investigate the diversity of BBD microorganisms between BBD infections. In the present study, we focus on the differences in results obtained using frozen versus unfrozen BBD samples from S. siderea (Fig. ​(Fig.1,1, libraries G and H) and a second coral host, D. clivosa (Fig. ​(Fig.1,1, libraries I and J). The S. siderea samples (libraries G and H) were taken from different host colonies on the same reef (Butler Bay Reef site), whereas the D. clivosa clone libraries were constructed from subsamples of one BBD sample.Open in a separate windowFIG. 1.Dominant bacterial phylogenetic groups, based on 16S rRNA gene sequence types and universal primers, present in clone libraries produced from frozen and unfrozen BBD samples from the coral hosts Siderastrea siderea and Diploria clivosa. The numbers above the bars represent the numbers of sequences in the respective clone libraries. Libraries A to H, frozen (A to G) and unfrozen (H) BBD from S. siderea. Libraries I and J, frozen (I) and unfrozen (J) BBD from D. clivosa. Sampling locations and sampling dates: libraries A and B, Horseshoe Reef, Lee Stocking Island, Bahamas, 19 July 2004; C, Rainbow Garden Reef, Lee Stocking Island, Bahamas, 16 July 2004; D, Watson''s Reef, Florida Keys, 3 May 2005; E, G, and H, Butler Bay Reef site, St. Croix, U.S. Virgin Islands (USVI), 22 October 2005, 1 June 2005, and 5 June 2006, respectively; F, Frederiksted Reef site, St. Croix, USVI, 1 June 2005; I and J, Frederiksted Reef site, St. Croix, USVI, 7 August 2006. All of the sequences from clone libraries A to G have been previously published by Sekar et al. (19, 20).This approach yielded strikingly different results for the two methods. For example, the clone library produced from one frozen sample (Fig. ​(Fig.1,1, library G) from S. siderea contained only one (of 60) cyanobacterium-related sequence (see EF123584 [GenBank sequence accession no.] in Table S1 in the supplemental material), which was phylogenetically related to a sequence from an uncultured planktonic Synechococcus sp. (GenBank sequence accession no. AY172810; Fig. ​Fig.2).2). In contrast, the clone library from the corresponding unfrozen sample (Fig. ​(Fig.1,1, library H) was dominated by a cyanobacterial ribotype which represented 37% of the clones. This ribotype was closely related to an Oscillatoria ribotype (GenBank sequence accession no. AY038527/AF473936) detected in almost all reported BBD molecular studies (2, 4, 7, 23). The sequence was confirmed as the BBD Oscillatoria sequence by phylogenetic analysis using two representative clone sequences (GenBank sequence accession no. EF123639 and EF123644) (Fig. ​(Fig.2).2). The unfrozen S. siderea clone library additionally produced a dominant epsilonproteobacterial ribotype (14 of 15 clones) (see Table S1 in the supplemental material) that was not detected in the corresponding frozen sample. Phylogenetic analysis of two representative sequences (GenBank sequence accession no. EF123607 and EF123613, not shown in Fig. ​Fig.2)2) determined that the sequence was related to a sequence from the sulfur-oxidizing bacterium “Candidatus Arcobacter sulfidicus” (GenBank sequence accession no. AY035822) (24), a species known to deposit filamentous sulfur (21) and reported previously in BBD (9).Open in a separate windowFIG. 2.Phylogenetic tree derived from the 16S rRNA gene sequences closely related to Synechococcus spp., Xenococcus spp., and Oscillatoria spp. sequences detected in BBD and their neighbors. The tree topology is based on the maximum-parsimony analysis. The bar represents 10% estimated sequence divergence. Boldface type indicates sequences from this study, designated as follows. FRSSBA, UFSSBA, FRSSCY, and UFSSCY indicate sequences retrieved from frozen (FR) and unfrozen (UF) samples of S. siderea (SS) using universal bacterial primers (BA) and cyanobacterium-specific (CY) primers for 16S rRNA gene amplification. FRDCBA, UFDCBA, and UFDCCY indicate sequences retrieved from frozen and unfrozen samples of Diploria clivosa (DC), and the same primer designations are used as for S. siderea sequences. GenBank sequence accession numbers are listed for all sequences. Asterisks designate sequences corresponding to the sequence from the BBD Oscillatoria discussed in the text.Again in clone library I, from the frozen subsample of D. clivosa (see Table S2 in the supplemental material), the Alphaproteobacteria were dominant (44%) and cyanobacteria represented in low percentages (4%). These cyanobacterial sequences were phylogenetically related to sequences of Leptolyngbya spp. (not shown in Fig. ​Fig.2)2) and a planktonic cyanobacterium Xenococcus sp. (GenBank sequence accession no. AF132783) (Fig. ​(Fig.2;2; see Table S2 in the supplemental material). The library from the unfrozen BBD subsample of D. clivosa (see Table S2 in the supplemental material) was dominated by Gammaproteobacteria (35%), followed by cyanobacteria (24%) which had the same cyanobacterial sequence type (BBD Oscillatoria) observed in the unfrozen S. siderea sample (see Table S2 in the supplemental material). For corroboration of these results, we constructed an additional clone library, using universal primers, from an unfrozen BBD sample from S. siderea collected during June 2007; in this sample, 47% of the sequences were also related to the sequence from BBD Oscillatoria.The use of cyanobacterium-specific primers produced results similar to the overall pattern we detected using universal primers. Frozen BBD from S. siderea produced 27 sequences, of which 24 were closely related to sequences from planktonic Synechococcus spp. and Xenococcus sp. (see Table S3 in the supplemental material). This was confirmed by phylogenetic analysis (Fig. ​(Fig.2)2) using representative sequences (GenBank sequence accession no. EU019432, EU019435, EU019439, EU019442, EU019449, and EU019455). In contrast, all of the sequences (n = 37) obtained from unfrozen S. siderea samples were closely related to the sequence from the BBD Oscillatoria (see Table S3 in the supplemental material). Representative sequences (GenBank sequence accession no. EU019460 and EU019467) confirmed this phylogenetic affiliation (Fig. ​(Fig.2).2). Similarly, each of 38 sequences obtained from the unfrozen subsample of D. clivosa with cyanobacterium-specific primers was closely related to the sequence from the BBD Oscillatoria (see Table S3 in the supplemental material), again confirmed by phylogenetic analysis using two representative sequences (GenBank sequence accession no. EU019508 and EU019515) (Fig. ​(Fig.22).There was very little overlap (6 to 10%) between sequences obtained from frozen versus unfrozen BBD samples collected from both coral hosts when considering all of the BBD bacterial sequences detected (see Tables S1 and S2 in the supplemental material). Only four sequences were common to both frozen and unfrozen clone libraries (6% of 62 sequences detected within 136 clones) for S. siderea and seven sequences (10% of 69 sequences detected within 108 clones) for D. clivosa. Statistical analysis (ANOSIM) showed that the sequence types differed significantly between frozen and unfrozen clone libraries (R = 0.987; P = 0.022). Overall, all frozen libraries (libraries A to G and I) were 69% similar to each other, while the two unfrozen libraries (libraries H and J) were 58% similar.The results of our study are significant for the ongoing investigations into the etiology of BBD. While it is well known that the BBD microbial community consists of photoautotrophs (cyanobacteria), sulfate-reducing bacteria, sulfur-oxidizing bacteria, and heterotrophs (16), we are just beginning to understand the roles of these functional groups in the disease process. A first step in this understanding is the valid and repeatable detection of specific members of the BBD consortium. In summary, we show here that unfrozen samples produce better results for detection of BBD cyanobacteria and sulfur-oxidizing bacteria, while frozen samples are best for detection of heterotrophic proteobacterial sequences. The latter is particularly important because of the consistent finding of Proteobacteria associated with toxic dinoflagellates (19, 20), as well as other marine invertebrate pathogens (4), in BBD. We have not studied the mechanism behind the freezing effect (e.g., release of DNase), which is outside the scope of this study. Though the current study was done with BBD samples, the effect of freezing on other microbial mats or biofilms cannot be ignored. Based on the results of this study, we suggest using multiple sample-processing approaches to characterize the microbial communities associated with BBD and other microbial mats.  相似文献   

11.
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12.
Zymomonas mobilis is an ethanol-producing alphaproteobacterium currently considered a major candidate organism for bioethanol production. Here we report the finished and annotated genome sequence of Z. mobilis subsp. mobilis strain NCIMB 11163, a British ale-infecting isolate. This is the first Z. mobilis strain whose genome, chromosomal and plasmid, is presented in its entirety.Zymomonas mobilis is a bacterium vigorously studied as a platform organism for bioethanol production in North America and other parts of the world. Z. mobilis converts sugars such as glucose or sucrose into ethanol and carbon dioxide to almost theoretical yields and to rates higher than those of yeasts (17). Genetically engineered strains that ferment pentoses in addition to naturally utilized hexoses also hold great promise for use in lignocellulosic biomass degradations (5, 22). Besides ethanol, Z. mobilis can produce other high-value chemicals such as sorbitol, levan, or phenylacetylcarbinol and has attracted interest for its unusual membrane steroid content (11). Lastly, Zymomonas is regarded as a safe organism and is even used for medicinal purposes (12, 20), which further facilitates its employment in large-scale biotechnological endeavors.The chromosomal sequence of the Z. mobilis subsp. mobilis industrial strain ATCC 31821 (ZM4) was recently published (19). Here we announce the first entire genome sequence of a Z. mobilis subsp. mobilis strain, that of the United Kingdom-originating strain NCIMB 11163 (B70) (20). Total DNA from NCIMB 11163 (16) was used for whole-genome shotgun sequencing at the U.S. DOE Joint Genome Institute. For this, an 8.7-kb DNA library and 454 and Solexa reads were used (http://www.jgi.doe.gov). Draft assemblies were based on 8,551 Sanger reads and 454 pyrosequencing to 20× coverage, whereas the Phred/Phrap/Consed software package was used for sequence assembly and quality assessment (6, 7, 9; http://www.phrap.com). After the shotgun stage, reads were assembled with parallel Phrap (High Performance Software, LLC), and misassemblies were corrected with Dupfinisher (10) or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). A total of 144 primer walk reactions, five transposon bomb libraries, 53 PCR end reads, and two PCR shatter libraries were necessary to close gaps, resolve repetitive regions, and raise the quality of the finished sequence. The completed genome sequence of NCIMB 11163 was based on 11,048 reads, with an error rate of less than 6 bp out of 100,000 bp.Open reading frame prediction and annotation were performed using Prodigal (http://compbio.ornl.gov/prodigal/) and BLAST (1); tRNAscan-SE and RNAmmer (14, 15) were used for tRNA and rRNA recognition, respectively. Functional assignment of genes was performed by searching translated open reading frames against sequences in the SPTR (TrEMBL) (2), Pfam (8), TIGRFAMs (18), COG (21), and KEGG (13) databases.Z. mobilis NCIMB 11163 contains a single, circular chromosome of 2,124,771 bp and three plasmids, p11163_1, p11163_2, and p11163_3 of 53,380 bp, 40,818 bp, and 4,551 bp, respectively. The overall GC content of the chromosome is 46.83%, whereas those of the plasmids are 42.32%, 43.80%, and 36.37%, respectively. The entire genome of NCIMB 11163 contains 1,884 protein-encoding genes and 51 tRNA and nine rRNA genes, which are chromosomally located.The chromosome of NCIMB 11163 is 68,355 bp larger than that of ZM4 (GenBank accession number NC_006526) (19) and colinear at its largest part with that of ZM4 (genome structure comparisons were performed using ACT) (3). It bears several unique regions, among which are two genomic islands of ca. 25 and 79 kb, with no detectable nucleotide homology to same-species sequences and high regional similarity to chromosomal stretches of Paracoccus denitrificans PD1222 (GenBank accession number CP000489.1), Xanthobacter autotrophicus Py2 (GenBank accession number CP000781.1), and Gluconacetobacter diazotrophicus PAl 5 (GenBank accession number CP001189.1). Genome plasticity in NCIMB 11163 is further indicated by the presence of a type IV secretion system on the 79-kb island, syntenous to the Agrobacterium tumefaciens Ti (IncRh1) conjugal trb system (4), and also by multiple transposase and phage-related genes.In plasmids, housekeeping genes implicated in replication, active partitioning, and plasmid addiction are recognized, as well as genes involved in metabolism, transport, regulation, transposition, and DNA modification. Most notably, p11163_1 bears an arsenical resistance operon inserted in a type II secretion locus, whereas p11163_2, otherwise homologous to the 41-kb ZM4 plasmid (GenBank accession number AY057845), harbors a unique ca. 12-kb CRISPR insertion that interrupts nucleotide colinearity with the aforementioned replicon.  相似文献   

13.
Neisseria meningitidis is a human-specific pathogen known for its capability to cause sepsis and meningitis. Here we report the availability of 2 draft genome sequences obtained from patients infected during the same epidemic outbreak. Both bacterial isolates belong to serogroup C, but their genome sequences show local and remarkable differences compared with each other or with the reference genome of strain FAM18.Neisseria meningitidis is found as a commensal organism of the human nasopharynx in 8 to 25% of the adult population (9), but sporadically, it is able to cross the mucosa and reach the bloodstream, causing severe septicemia and meningitis. Even though the reasons triggering these pathogenic outbreaks are not well understood, several factors related either to the host or the bacterium have been proposed 3, 8).So far, complete genome sequences for N. meningitidis serogroups A (strain Z2491 [GenBank accession no. AL157959]) (4), B (strain MC58 [GenBank accession no. AE002098]) (10), and C (strains FAM18, 8013, and 053442 [GenBank accession no. AM421808, FM999788, and CP000381, respectively]) (1, 5, 6) have been reported, together with the unencapsulated strain α14 (GenBank accession no. AM889136) (7). Here we announce the availability of 2 draft genome sequences for N. meningitidis serogroup C, strains K1207 and S0108, isolated from the same epidemic cluster which occurred in the Veneto region in northern Italy during the 2007-2008 winter (2).The genomes were sequenced using 454 pyrosequencing (Roche), combining shotgun and 30-kb paired-end strategies, according to the manufacturer''s recommendations. The coverage was nearly 27×, and assemblies were performed with Newbler. We obtained 223 and 226 contigs for the 2 genomes, which were finally mapped in 17 and 16 scaffolds, respectively. From both samples, we also isolated a 7-kb plasmid, whose sequence was nearly identical to that of pJS-B, already available in GenBank (accession no. NC_004758).The first analysis was performed by comparing sequences of the two isolates with the most similar complete genome available, strain FAM18. This analysis showed that the genome lengths were almost identical (about 2.2 Mb) and GC contents were comparable (51.91% in both isolates versus 51.62% of strain FAM18). Then, to identify potential differences in coding sequence content, the contigs obtained for both isolates were aligned with those for strain FAM18 using MEGABLAST (11) and LASTZ tools, which showed that in the genomes of the two N. meningitidis isolates, several genes were missing or nonfunctional because of the presence of insertions or deletions. For example, a couple of FAM18 outer membrane proteins (NMC0214 and NMC0215) were completely missing in both genomes, due to a 3-kb deletion, and no homologues were present in other genomic regions.Sequences that did not map on the genome of strain FAM18 were investigated by performing a BLAST analysis on a nonredundant database. Interestingly, besides genes or partial genes belonging to the other completely sequenced N. meningitidis serogroup C strain 053442, the genomes of our isolates contained coding sequences from N. meningitidis serogroups A and B, from other Neisseria species, such as N. gonorrhoeae, N. cinerea, and N. mucosa, and even from other bacterial species, such as cobyrinic acid ac-diamide synthase from Shewanella baltica, attesting once more to the great capability of horizontal gene transfer, which is peculiar to this microorganism.A detailed report of our two isolates will be included in a future publication, with the results of a full comparative analysis between the genomes.  相似文献   

14.
A multilocus sequence typing (MLST) analysis was used to examine the genetic structure and diversity within the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae). The allelic diversity within these replicons was high compared to the reported diversity within the corresponding chromosomes of the same strains (P. van Berkum et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized linkage disequilibrium (LD) between certain pSymA loci: e.g., nodC and nifD. Although both of these observations could be explained by positive (or diversifying) selection by plant hosts, results of tests for positive selection did not provide consistent support for this hypothesis. The strong LD observed between the nodC and nifD genes could also be explained by their close proximity on the pSymA replicon. Evidence was obtained that some nodC alleles had a history of intragenic recombination, while other alleles of this locus had a history of intergenic recombination. Both types of recombination were associated with a decline in symbiotic competence with Medicago sativa as the host plant. The combined observations of LD between the nodC and nifD genes and intragenic recombination within one of these loci indicate that the symbiotic gene region on the pSymA plasmid has evolved as a clonal segment, which has been laterally transferred within the natural populations.Plants of the genus Medicago are legumes that often benefit from a mutualistic symbiosis with rhizobia. The most agriculturally significant species of rhizobia that nodulate these plants are Sinorhizobium meliloti (9) and Sinorhizobium medicae (22). Previously reported population genetic analyses of these bacteria have focused on the study of how allelic variants at multiple loci are distributed within and among natural populations (2, 3, 10, 26, 31, 32). This was also the focus of the present study, but it was extended by examining more loci in many more strains of both species of Sinorhizobium coupled with an analysis having a range of symbiotic genotypes. One goal was to determine if there were any obvious correlations between the megaplasmid genotypes observed and their symbiotic competence. A second goal was to determine if selection by their host plants may have influenced the evolution of their symbiotic relationships.The genes for symbiosis reside on the extrachromosomal replicons pSymA (1,354,226 nucleotides [nt]) and pSMED02 (1,245,408 nt) in the genomes of S. meliloti Rm1021 and S. medicae WSM419, respectively (GenBank accession no. AE006469 and CP000740, respectively). Besides these two plasmids, these two strains each harbor one other large extrachromosomal replicon, pSymB (1,683,333 nt) and pSMED01 (1,570,951 nt), respectively (GenBank accession no. AL591985 and CP000739, respectively).Multilocus sequence typing (MLST) (16) is a form of genomic indexing that is commonly used to study the population genetic structure and phylogenetic relatedness within diverse groups of bacteria. In this method, nucleotide sequences of a fixed set of common loci are obtained from a collection of strains, and polymorphic sites among these sequences are used to derive an allelic profile or sequence type (ST) for each genome. Comparisons of the resulting data can be used to infer phylogenetic relationships among the organisms in the sample population, and they also can be used to infer how evolutionary processes, such as recombination and selection, have shaped the genetic structure of the population. For example, levels of intergenic recombination among chromosomal genes in natural populations of Neisseria meningitidis reportedly are relatively high, while corresponding levels within populations of Staphylococcus aureus were low (28). Depending on the specific pairs of loci examined, the levels of linkage disequilibrium (LD) (a lack of intergenic recombination) among several chromosomally carried core genes of S. meliloti were reported to be generally moderate to high (26).The MLST approach has been used to confirm that the chromosomes of S. meliloti and S. medicae are sexually isolated (2, 3, 31) and to provide evidence that horizontal gene transfer (HGT) does occur between the symbiotic megaplasmids of these species (3, 32). It has also been used to demonstrate that levels of intergenic recombination, as indicated by linkage disequilibrium, differ between the three replicons of S. meliloti (26). Levels of intergenic recombination within the pSymB replicons of these strains are generally high, unlike the chromosomes and pSymA replicons within the same strains (26). Bailly et al. (3) hypothesized that the region of the pSymA plasmid that contains the nodulation (nod) genes is frequently transferred in natural populations. They also suggested that selective pressures from the host plant may have influenced both nod gene diversity and patterns of polymorphism across the entire nod gene region.In the present study, multilocus allelic variation of the two megaplasmids was examined among 231 Medicago-nodulating rhizobia that originated primarily from southwest Asia (10). Previously, 91 different chromosomal sequence types (STs) were identified among the same strains from sequence variation in 10 loci (31). This collection of strains had earlier been divided into two closely related groups based on results of multilocus enzyme electrophoresis (10), and this result was subsequently cited in support of separating the Medicago-nodulating rhizobia into the two species S. meliloti and S. medicae (22).The objectives of this study were (i) to use MLST to examine the genetic relationships within and among the large extrachromosomal replicons in S. meliloti and S. medicae, (ii) to estimate levels of intergenic and intragenic recombination in these replicons, (iii) to evaluate the nitrogen-fixing competence of representative symbiotic genotypes with Medicago sativa, and (iv) to determine whether positive (or diversifying) selection may have influenced the genetic structure of the megaplasmids.  相似文献   

15.
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   

16.
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   

17.
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18.
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   

19.
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   

20.
While characterizing modified vaccinia virus recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary recombinant vaccines.Vaccinia virus (VACV), the first recombinant virus shown to induce a protective immune response against an unrelated pathogen (21, 22), is being employed as a vector for veterinary and wildlife vaccines (19). Development of recombinant VACV for human use, however, has been impeded by safety concerns. For this reason, there is interest in modified VACV Ankara (MVA), a highly attenuated smallpox vaccine with an exemplary safety profile even in immunodeficient animals (17, 26, 27). MVA is severely host range restricted and propagates poorly or not at all in most mammalian cells because of a block in virion assembly (29). Initial experiments with recombinant MVA (rMVA) demonstrated its ability to robustly express foreign proteins (29) and induce protective humoral and cell-mediated immunity (30). Currently, rMVA candidate vaccines expressing genes from a wide variety of pathogens are undergoing animal and human testing (13).While developing candidate human immunodeficiency virus (HIV) and other vaccines, we encountered a tendency for mutant rMVA that had lost the ability to express foreign proteins to arise after tissue culture passage (28, 34, 37). This instability may initially go undetected, however, unless individual plaques are isolated and analyzed. Nevertheless, once established in the population, the nonexpressors can rapidly overgrow the original rMVA. These considerations are particularly important for production of large vaccine seed stocks of rMVA. The instability of cloned genes in MVA is surprising, since MVA had already undergone genetic changes during its adaptation through hundreds of passages in chicken embryo fibroblasts (CEFs) and is now quite stable. Indeed, identical 167,000-bp genome sequences have been reported for three independent plaque isolates, accession numbers U94848, AY603355, and DQ983236, and by Antoine et al. (1). Although the cause of the instability of the gene inserts had not been previously investigated, harmful effects of the recombinant protein seem to play a role in the selective advantage of nonexpressing mutants. Thus, reducing the expression level of parainfluenza virus and measles virus transmembrane proteins and deleting part of the cytoplasmic tail of HIV Env improves the stability of rMVAs (28, 34, 37). Reducing expression, however, can also decrease immunogenicity and therefore may be undesirable (36).In view of the importance of understanding and overcoming this pernicious instability problem, we carried out a systematic study of HIV env and gag-pol genes that were unstable in rMVA. We also considered that the analysis would provide basic information regarding the kinds of errors that can occur during replication of the VACV genome, which encodes its own cytoplasmic replication system (20). The most common mutations, which led to loss of recombinant gene expression, were large deletions that extended deep into the MVA flanks and frameshift mutations within consecutive identical nucleotides in the insert. The frequency of viable mutations was minimized by introducing the recombinant gene between two essential, highly conserved MVA genes and by making silent codon alterations to interrupt the homonucleotide runs. In addition, we constructed a panel of recombinant viruses with chimeric and truncated env genes to determine the basis for the selection of nonexpressing mutants and to prevent their expansion during virus propagation. Understanding the causes of the instability and taking preemptive actions should facilitate the development of MVA and other poxviruses as human and veterinary vaccines. In addition, these insights may have application to other DNA expression vectors.  相似文献   

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