15 results on '"Read TD"'
Search Results
2. Direct amplification, sequencing and profiling of Chlamydia trachomatis strains in single and mixed infection clinical samples.
- Author
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Joseph SJ, Li B, Ghonasgi T, Haase CP, Qin ZS, Dean D, and Read TD
- Subjects
- Chlamydia trachomatis genetics, Humans, Chlamydia Infections microbiology, Chlamydia trachomatis isolation & purification, Polymerase Chain Reaction methods, Sequence Analysis, DNA methods
- Abstract
Sequencing bacterial genomes from DNA isolated directly from clinical samples offers the promise of rapid and precise acquisition of informative genetic information. In the case of Chlamydia trachomatis, direct sequencing is particularly desirable because it obviates the requirement for culture in mammalian cells, saving time, cost and the possibility of missing low abundance strains. In this proof of concept study, we developed methodology that would allow genome-scale direct sequencing, using a multiplexed microdroplet PCR enrichment technology to amplify a 100 kb region of the C. trachomatis genome with 500 1.1-1.3 kb overlapping amplicons (5-fold amplicon redundancy). We integrated comparative genomic data into a pipeline to preferentially select conserved sites for amplicon design. The 100 kb target region could be amplified from clinical samples, including remnants from diagnostics tests, originating from the cervix, urethra and urine, For rapid analysis of these data, we developed a framework for whole-genome based genotyping called binstrain. We used binstrain to estimate the proportion of SNPs originating from 14 C. trachomatis reference serotype genomes in each sample. Direct DNA sequencing methods such as the one described here may have an important role in understanding the biology of C. trachomatis mixed infections and the natural genetic variation of the species within clinically relevant ecological niches.
- Published
- 2014
- Full Text
- View/download PDF
3. The complete mitochondrial genome sequence of the world's largest fish, the whale shark (Rhincodon typus), and its comparison with those of related shark species.
- Author
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Alam MT, Petit RA 3rd, Read TD, and Dove AD
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- Animals, Base Sequence, Chromosome Mapping, DNA, Mitochondrial genetics, Endangered Species, Fish Proteins genetics, High-Throughput Nucleotide Sequencing, Molecular Sequence Data, RNA, Ribosomal genetics, RNA, Transfer genetics, Elasmobranchii genetics, Genome, Mitochondrial genetics, Mitochondria genetics, Sequence Analysis, DNA veterinary
- Abstract
The whale shark (Rhincodon typus) is the largest extant species of fish, belonging to the order Orectolobiformes. It is listed as a "vulnerable" species on the International Union for Conservation of Nature (IUCN)'s Red List of Threatened Species, which makes it an important species for conservation efforts. We report here the first complete sequence of the mitochondrial genome (mitogenome) of the whale shark obtained by next-generation sequencing methods. The assembled mitogenome is a 16,875 bp circle, comprising of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a control region. We also performed comparative analysis of the whale shark mitogenome to the available mitogenome sequences of 17 other shark species, four from the order Orectolobiformes, five from Lamniformes and eight from Carcharhiniformes. The nucleotide composition, number and arrangement of the genes in whale shark mitogenome are the same as found in the mitogenomes of the other members of the order Orectolobiformes and its closest orders Lamniformes and Carcharhiniformes, although the whale shark mitogenome had a slightly longer control region. The availability of mitogenome sequence of whale shark will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this species., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
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4. Rapid identification of genetic modifications in Bacillus anthracis using whole genome draft sequences generated by 454 pyrosequencing.
- Author
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Chen PE, Willner KM, Butani A, Dorsey S, George M, Stewart A, Lentz SM, Cook CE, Akmal A, Price LB, Keim PS, Mateczun A, Brahmbhatt TN, Bishop-Lilly KA, Zwick ME, Read TD, and Sozhamannan S
- Subjects
- Bacillus anthracis drug effects, Bacillus anthracis physiology, Bacillus anthracis virology, Bacteriophages physiology, Ciprofloxacin pharmacology, Computational Biology, Drug Resistance, Bacterial genetics, Erythromycin pharmacology, Laboratories, Mutation, Time Factors, Bacillus anthracis genetics, Genome, Bacterial genetics, Sequence Analysis, DNA methods
- Abstract
Background: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments., Methodology/principal Findings: We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours., Conclusions/significance: Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in the context of whole genome sequencing-based routine clinical diagnostics as well as epidemiological surveillance of natural disease outbreaks caused by bacterial and viral agents.
- Published
- 2010
- Full Text
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5. Rapid multi-locus sequence typing using microfluidic biochips.
- Author
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Read TD, Turingan RS, Cook C, Giese H, Thomann UH, Hogan CC, Tan E, and Selden RF
- Subjects
- Bacillus cereus classification, Bacterial Typing Techniques, Environmental Microbiology, Phylogeny, Polymerase Chain Reaction, Temperature, Bacillus cereus genetics, Genetic Loci genetics, Microfluidic Analytical Techniques methods, Sequence Analysis, DNA methods
- Abstract
Background: Multiple locus sequence typing (MLST) has become a central genotyping strategy for analysis of bacterial populations. The scheme involves de novo sequencing of 6-8 housekeeping loci to assign unique sequence types. In this work we adapted MLST to a rapid microfluidics platform in order to enhance speed and reduce laboratory labor time., Methodology/principal Findings: Using two integrated microfluidic devices, DNA was purified from 100 Bacillus cereus soil isolates, used as a template for multiplex amplification of 7 loci and sequenced on forward and reverse strands. The time on instrument from loading genomic DNA to generation of electropherograms was only 1.5 hours. We obtained full-length sequence of all seven MLST alleles from 84 representing 46 different Sequence Types. At least one allele could be sequenced from a further 15 strains. The nucleotide diversity of B. cereus isolated in this study from one location in Rockville, Maryland (0.04 substitutions per site) was found to be as great as the global collection of isolates., Conclusions/significance: Biogeographical investigation of pathogens is only one of a panoply of possible applications of microfluidics based MLST; others include microbiologic forensics, biothreat identification, and rapid characterization of human clinical samples.
- Published
- 2010
- Full Text
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6. Finishing genomes with limited resources: lessons from an ensemble of microbial genomes.
- Author
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Nagarajan N, Cook C, Di Bonaventura M, Ge H, Richards A, Bishop-Lilly KA, DeSalle R, Read TD, and Pop M
- Subjects
- Humans, Sequence Analysis, DNA economics, Gammaproteobacteria genetics, Genome, Bacterial, Rickettsia prowazekii genetics, Sequence Analysis, DNA methods, Yersinia genetics
- Abstract
While new sequencing technologies have ushered in an era where microbial genomes can be easily sequenced, the goal of routinely producing high-quality draft and finished genomes in a cost-effective fashion has still remained elusive. Due to shorter read lengths and limitations in library construction protocols, shotgun sequencing and assembly based on these technologies often results in fragmented assemblies. Correspondingly, while draft assemblies can be obtained in days, finishing can take many months and hence the time and effort can only be justified for high-priority genomes and in large sequencing centers. In this work, we revisit this issue in light of our own experience in producing finished and nearly-finished genomes for a range of microbial species in a small-lab setting. These genomes were finished with surprisingly little investments in terms of time, computational effort and lab work, suggesting that the increased access to sequencing might also eventually lead to a greater proportion of finished genomes from small labs and genomics cores.
- Published
- 2010
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7. Genomics. Genome project standards in a new era of sequencing.
- Author
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Chain PS, Grafham DV, Fulton RS, Fitzgerald MG, Hostetler J, Muzny D, Ali J, Birren B, Bruce DC, Buhay C, Cole JR, Ding Y, Dugan S, Field D, Garrity GM, Gibbs R, Graves T, Han CS, Harrison SH, Highlander S, Hugenholtz P, Khouri HM, Kodira CD, Kolker E, Kyrpides NC, Lang D, Lapidus A, Malfatti SA, Markowitz V, Metha T, Nelson KE, Parkhill J, Pitluck S, Qin X, Read TD, Schmutz J, Sozhamannan S, Sterk P, Strausberg RL, Sutton G, Thomson NR, Tiedje JM, Weinstock G, Wollam A, and Detter JC
- Subjects
- Computational Biology, Databases, Nucleic Acid standards, Genome, Genomics standards, Sequence Analysis, DNA standards
- Published
- 2009
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8. Genotyping of Bacillus cereus strains by microarray-based resequencing.
- Author
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Zwick ME, Kiley MP, Stewart AC, Mateczun A, and Read TD
- Subjects
- Bacillus cereus genetics, Genetic Variation, Genome, Bacterial, Genotype, Phylogeny, Bacillus cereus classification, Microarray Analysis methods, Sequence Analysis, DNA methods
- Abstract
The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.
- Published
- 2008
- Full Text
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9. Scaffolding and validation of bacterial genome assemblies using optical restriction maps.
- Author
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Nagarajan N, Read TD, and Pop M
- Subjects
- Base Sequence, Molecular Sequence Data, Algorithms, Chromosome Mapping methods, Genome, Bacterial genetics, Optics and Photonics, Restriction Mapping methods, Sequence Alignment methods, Sequence Analysis, DNA methods
- Abstract
Motivation: New, high-throughput sequencing technologies have made it feasible to cheaply generate vast amounts of sequence information from a genome of interest. The computational reconstruction of the complete sequence of a genome is complicated by specific features of these new sequencing technologies, such as the short length of the sequencing reads and absence of mate-pair information. In this article we propose methods to overcome such limitations by incorporating information from optical restriction maps., Results: We demonstrate the robustness of our methods to sequencing and assembly errors using extensive experiments on simulated datasets. We then present the results obtained by applying our algorithms to data generated from two bacterial genomes Yersinia aldovae and Yersinia kristensenii. The resulting assemblies contain a single scaffold covering a large fraction of the respective genomes, suggesting that the careful use of optical maps can provide a cost-effective framework for the assembly of genomes., Availability: The tools described here are available as an open-source package at ftp://ftp.cbcb.umd.edu/pub/software/soma
- Published
- 2008
- Full Text
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10. Microarray-based resequencing of multiple Bacillus anthracis isolates.
- Author
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Zwick ME, Mcafee F, Cutler DJ, Read TD, Ravel J, Bowman GR, Galloway DR, and Mateczun A
- Subjects
- Bacillus anthracis classification, Bacillus anthracis pathogenicity, Chromosomes, Bacterial genetics, Genetic Variation genetics, Genome, Bacterial, Molecular Sequence Data, Recombination, Genetic genetics, Reproducibility of Results, Sequence Analysis, DNA economics, Sequence Analysis, DNA standards, Time Factors, Bacillus anthracis genetics, Bacillus anthracis isolation & purification, Oligonucleotide Array Sequence Analysis, Sequence Analysis, DNA methods
- Abstract
We used custom-designed resequencing arrays to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains. Sequence quality was shown to be very high by replication (discrepancy rate of 7.4 x 10(-7)) and by comparison to independently generated shotgun sequence (discrepancy rate < 2.5 x 10(-6)). Population genomics studies of microbial pathogens using rapid resequencing technologies such as resequencing arrays are critical for recognizing newly emerging or genetically engineered strains.
- Published
- 2005
- Full Text
- View/download PDF
11. Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis.
- Author
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Paulsen IT, Banerjei L, Myers GS, Nelson KE, Seshadri R, Read TD, Fouts DE, Eisen JA, Gill SR, Heidelberg JF, Tettelin H, Dodson RJ, Umayam L, Brinkac L, Beanan M, Daugherty S, DeBoy RT, Durkin S, Kolonay J, Madupu R, Nelson W, Vamathevan J, Tran B, Upton J, Hansen T, Shetty J, Khouri H, Utterback T, Radune D, Ketchum KA, Dougherty BA, and Fraser CM
- Subjects
- Adhesins, Bacterial genetics, Bacterial Adhesion, Bacterial Proteins genetics, Carrier Proteins genetics, Carrier Proteins metabolism, Chromosomes, Bacterial genetics, Conjugation, Genetic, Conserved Sequence, DNA Transposable Elements, Digestive System microbiology, Drug Resistance, Multiple, Bacterial, Enterococcus faecalis drug effects, Enterococcus faecalis pathogenicity, Enterococcus faecalis physiology, Gene Transfer, Horizontal, Gram-Positive Bacterial Infections microbiology, Humans, Lysogeny, Open Reading Frames, Oxidative Stress, Plasmids, Synteny, Virulence genetics, Virulence Factors genetics, Biological Evolution, Enterococcus faecalis genetics, Genome, Bacterial, Interspersed Repetitive Sequences, Sequence Analysis, DNA, Vancomycin Resistance genetics
- Abstract
The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.
- Published
- 2003
- Full Text
- View/download PDF
12. Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis.
- Author
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Heidelberg JF, Paulsen IT, Nelson KE, Gaidos EJ, Nelson WC, Read TD, Eisen JA, Seshadri R, Ward N, Methe B, Clayton RA, Meyer T, Tsapin A, Scott J, Beanan M, Brinkac L, Daugherty S, DeBoy RT, Dodson RJ, Durkin AS, Haft DH, Kolonay JF, Madupu R, Peterson JD, Umayam LA, White O, Wolf AM, Vamathevan J, Weidman J, Impraim M, Lee K, Berry K, Lee C, Mueller J, Khouri H, Gill J, Utterback TR, McDonald LA, Feldblyum TV, Smith HO, Venter JC, Nealson KH, and Fraser CM
- Subjects
- Amino Acid Sequence, Biodegradation, Environmental, Cell Respiration, Electron Transport, Gene Expression, Metals metabolism, Molecular Sequence Data, Open Reading Frames genetics, Organic Chemicals metabolism, Oxidation-Reduction, Plasmids, Proteomics methods, Sequence Alignment methods, Shewanella classification, Shewanella pathogenicity, Species Specificity, Water Pollutants, Chemical metabolism, Water Purification methods, Gene Expression Regulation, Bacterial, Genome, Bacterial, Sequence Analysis, DNA, Sequence Analysis, Protein, Shewanella genetics, Shewanella metabolism
- Abstract
Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities, conferred in part by multicomponent, branched electron transport systems. Here we report the sequencing of the S. oneidensis genome, which consists of a 4,969,803-base pair circular chromosome with 4,758 predicted protein-encoding open reading frames (CDS) and a 161,613-base pair plasmid with 173 CDSs. We identified the first Shewanella lambda-like phage, providing a potential tool for further genome engineering. Genome analysis revealed 39 c-type cytochromes, including 32 previously unidentified in S. oneidensis, and a novel periplasmic [Fe] hydrogenase, which are integral members of the electron transport system. This genome sequence represents a critical step in the elucidation of the pathways for reduction (and bioremediation) of pollutants such as uranium (U) and chromium (Cr), and offers a starting point for defining this organism's complex electron transport systems and metal ion-reducing capabilities.
- Published
- 2002
- Full Text
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13. Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis.
- Author
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Read TD, Salzberg SL, Pop M, Shumway M, Umayam L, Jiang L, Holtzapple E, Busch JD, Smith KL, Schupp JM, Solomon D, Keim P, and Fraser CM
- Subjects
- Animals, Anthrax microbiology, Bacillus anthracis classification, Bacillus anthracis isolation & purification, Bacillus anthracis pathogenicity, Bacterial Typing Techniques, Base Sequence, Bioterrorism, Chromosome Inversion, Computational Biology, Disease Outbreaks, Genetic Markers, Genomics, Humans, Minisatellite Repeats, Molecular Sequence Data, Mutation, Phenotype, Phylogeny, Plasmids, Recombination, Genetic, Repetitive Sequences, Nucleic Acid, Sequence Deletion, Species Specificity, Transposases genetics, Virulence genetics, Bacillus anthracis genetics, Genetic Variation, Genome, Bacterial, Polymorphism, Single Nucleotide, Sequence Analysis, DNA
- Abstract
Comparison of the whole-genome sequence of Bacillus anthracis isolated from a victim of a recent bioterrorist anthrax attack with a reference reveals 60 new markers that include single nucleotide polymorphisms (SNPs), inserted or deleted sequences, and tandem repeats. Genome comparison detected four high-quality SNPs between the two sequenced B. anthracis chromosomes and seven differences among different preparations of the reference genome. These markers have been tested on a collection of anthrax isolates and were found to divide these samples into distinct families. These results demonstrate that genome-based analysis of microbial pathogens will provide a powerful new tool for investigation of infectious disease outbreaks.
- Published
- 2002
- Full Text
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14. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.
- Author
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Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, Heidelberg J, DeBoy RT, Haft DH, Dodson RJ, Durkin AS, Gwinn M, Kolonay JF, Nelson WC, Peterson JD, Umayam LA, White O, Salzberg SL, Lewis MR, Radune D, Holtzapple E, Khouri H, Wolf AM, Utterback TR, Hansen CL, McDonald LA, Feldblyum TV, Angiuoli S, Dickinson T, Hickey EK, Holt IE, Loftus BJ, Yang F, Smith HO, Venter JC, Dougherty BA, Morrison DA, Hollingshead SK, and Fraser CM
- Subjects
- Antigens, Bacterial, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins immunology, Bacterial Proteins metabolism, Bacterial Vaccines, Base Composition, Carbohydrate Metabolism, Carrier Proteins genetics, Carrier Proteins metabolism, Chromosomes, Bacterial genetics, Computational Biology, DNA Transposable Elements, DNA, Bacterial chemistry, DNA, Bacterial genetics, Gene Duplication, Genes, Bacterial, Hexosamines metabolism, Oligonucleotide Array Sequence Analysis, Recombination, Genetic, Repetitive Sequences, Nucleic Acid, Species Specificity, Streptococcus pneumoniae immunology, Streptococcus pneumoniae metabolism, Virulence, rRNA Operon, Genome, Bacterial, Sequence Analysis, DNA, Streptococcus pneumoniae genetics, Streptococcus pneumoniae pathogenicity
- Abstract
The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.
- Published
- 2001
- Full Text
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15. Unity in variety-the pan-genome of the chlamydiae
- Author
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Collingro, A, Tischler, P, Weinmaier, T, Penz, T, Heinz, E, Brunham, RC, Read, TD, Bavoil, PM, Sachse, K, Kahane, S, Friedman, MG, Rattei, T, Myers, GSA, and Horn, M
- Subjects
DNA, Bacterial ,Evolutionary Biology ,Chlamydiales ,Gene Transfer, Horizontal ,Base Sequence ,Cell Membrane ,Molecular Sequence Data ,Genetic Variation ,Sequence Analysis, DNA ,Evolution, Molecular ,Bacterial Proteins ,RNA, Ribosomal, 16S ,Host-Pathogen Interactions ,Chlamydia ,Symbiosis ,Bacterial Secretion Systems ,Phylogeny ,Genome, Bacterial ,Bacterial Outer Membrane Proteins ,Plasmids - Abstract
Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote. © 2011 The Author.
- Published
- 2011
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