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The 2,272,351–base pair genome ofNeisseria meningitidisstrain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior ofN. meningitidiscan be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally,N. meningitidiscontains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria , the Cyanobacteria , and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N 2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus , we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron , the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40 T . Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans , consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.

Dichelobacter nodosus causes ovine footrot, a disease that leads to severe economic losses in the wool and meat industries. We sequenced its 1.4-Mb genome, the smallest known genome of an anaerobe. It differs markedly from small genomes of intracellular bacteria, retaining greater biosynthetic capabilities and lacking any evidence of extensive ongoing genome reduction. Comparative genomic microarray studies and bioinformatic analysis suggested that, despite its small size, almost 20% of the genome is derived from lateral gene transfer. Most of these regions seem to be associated with virulence. Metabolic reconstruction indicated unsuspected capabilities, including carbohydrate utilization, electron transfer and several aerobic pathways. Global transcriptional profiling and bioinformatic analysis enabled the prediction of virulence factors and cell surface proteins. Screening of these proteins against ovine antisera identified eight immunogenic proteins that are candidate antigens for a cross-protective vaccine. © 2007 Nature Publishing Group.

Here, we present the complete genome sequence of Thermodesulfobacterium commune DSM 2178 T of the phylum Thermodesulfobacteria .

The completion of the 5,373,180-bp genome sequence of the marine psychrophilic bacterium Colwellia psychrerythraea 34H, a model for the study of life in permanently cold environments, reveals capabilities important to carbon and nutrient cycling, bioremediation, production of secondary metabolites, and cold-adapted enzymes. From a genomic perspective, cold adaptation is suggested in several broad categories involving changes to the cell membrane fluidity, uptake and synthesis of compounds conferring cryotolerance, and strategies to overcome temperature-dependent barriers to carbon uptake. Modeling of three-dimensional protein homology from bacteria representing a range of optimal growth temperatures suggests changes to proteome composition that may enhance enzyme effectiveness at low temperatures. Comparative genome analyses suggest that the psychrophilic lifestyle is most likely conferred not by a unique set of genes but by a collection of synergistic changes in overall genome content and amino acid composition.

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5. Supplementary information The online version of this article (doi:10.1038/nbt1110) contains supplementary material, which is available to authorized users.

Dehalococcoides ethenogenes is the only bacterium known to reductively dechlorinate the groundwater pollutants, tetrachloroethene (PCE) and trichloroethene, to ethene. Its 1,469,720–base pair chromosome contains large dynamic duplicated regions and integrated elements. Genes encoding 17 putative reductive dehalogenases, nearly all of which were adjacent to genes for transcription regulators, and five hydrogenase complexes were identified. These findings, plus a limited repertoire of other metabolic modes, indicate that D. ethenogenes is highly evolved to utilize halogenated organic compounds and H 2 . Diversification of reductive dehalogenase functions appears to have been mediated by recent genetic exchange and amplification. Genome analysis provides insights into the organism's complex nutrient requirements and suggests that an ancestor was a nitrogen-fixing autotroph.

The complete genome sequence of Burkholderia mallei ATCC 23344 provides insight into this highly infectious bacterium's pathogenicity and evolutionary history. B. mallei , the etiologic agent of glanders, has come under renewed scientific investigation as a result of recent concerns about its past and potential future use as a biological weapon. Genome analysis identified a number of putative virulence factors whose function was supported by comparative genome hybridization and expression profiling of the bacterium in hamster liver in vivo . The genome contains numerous insertion sequence elements that have mediated extensive deletions and rearrangements of the genome relative to Burkholderia pseudomallei . The genome also contains a vast number (>12,000) of simple sequence repeats. Variation in simple sequence repeats in key genes can provide a mechanism for generating antigenic variation that may account for the mammalian host's inability to mount a durable adaptive immune response to a B. mallei infection.

The genomes of three strains of Listeria monocytogenes that have been associated with food-borne illness in the USA were subjected to whole genome comparative analysis. A total of 51, 97 and 69 strain-specific genes were identified in L.monocytogenes strains F2365 (serotype 4b, cheese isolate), F6854 (serotype 1/2a, frankfurter isolate) and H7858 (serotype 4b, meat isolate), respectively. Eighty-three genes were restricted to serotype 1/2a and 51 to serotype 4b strains. These strain- and serotype-specific genes probably contribute to observed differences in pathogenicity, and the ability of the organisms to survive and grow in their respective environmental niches. The serotype 1/2a-specific genes include an operon that encodes the rhamnose biosynthetic pathway that is associated with teichoic acid biosynthesis, as well as operons for five glycosyl transferases and an adenine-specific DNA methyltransferase. A total of 8603 and 105 050 high quality single nucleotide polymorphisms (SNPs) were found on the draft genome sequences of strain H7858 and strain F6854, respectively, when compared with strain F2365. Whole genome comparative analyses revealed that the L.monocytogenes genomes are essentially syntenic, with the majority of genomic differences consisting of phage insertions, transposable elements and SNPs.

Desulfovibrio vulgaris Hildenborough is a model organism for studying the energy metabolism of sulfate-reducing bacteria (SRB) and for understanding the economic impacts of SRB, including biocorrosion of metal infrastructure and bioremediation of toxic metal ions. The 3,570,858 base pair (bp) genome sequence reveals a network of novel c-type cytochromes, connecting multiple periplasmic hydrogenases and formate dehydrogenases, as a key feature of its energy metabolism. The relative arrangement of genes encoding enzymes for energy transduction, together with inferred cellular location of the enzymes, provides a basis for proposing an expansion to the 'hydrogen-cycling' model for increasing energy efficiency in this bacterium. Plasmid-encoded functions include modification of cell surface components, nitrogen fixation and a type-III protein secretion system. This genome sequence represents a substantial step toward the elucidation of pathways for reduction (and bioremediation) of pollutants such as uranium and chromium and offers a new starting point for defining this organism's complex anaerobic respiration.

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana . The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa , yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.

Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax. Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref. 2) and pXO2 (ref. 3). To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B. anthracis Ames (about 5.23 megabases). We found several chromosomally encoded proteins that may contribute to pathogenicity--including haemolysins, phospholipases and iron acquisition functions--and identified numerous surface proteins that might be important targets for vaccines and drugs. Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B. anthracis to near-neighbours that are not associated with anthrax. By performing a comparative genome hybridization of 19 B. cereus and Bacillus thuringiensis strains against a B. anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives. However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group. The complete sequence of B. anthracis is a step towards a better understanding of anthrax pathogenesis.

The 1,995,275-bp genome ofCoxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method. This bacterium is an obligate intracellular acidophile that is highly adapted for life within the eukaryotic phagolysosome. Genome analysis revealed many genes with potential roles in adhesion, invasion, intracellular trafficking, host-cell modulation, and detoxification. A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis of this organism. Although the lifestyle and parasitic strategies ofC. burnetiiresemble that ofRickettsiaeandChlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles. The presence of 83 pseudogenes displays an ongoing process of gene degradation. Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in theC. burnetiigenome. These analyses suggest that the obligate intracellular lifestyle ofC. burnetiimay be a relatively recent innovation.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

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.

The complete genome of the green-sulfur eubacterium Chlorobium tepidum TLS was determined to be a single circular chromosome of 2,154,946 bp. This represents the first genome sequence from the phylum Chlorobia , whose members perform anoxygenic photosynthesis by the reductive tricarboxylic acid cycle. Genome comparisons have identified genes in C. tepidum that are highly conserved among photosynthetic species. Many of these have no assigned function and may play novel roles in photosynthesis or photobiology. Phylogenomic analysis reveals likely duplications of genes involved in biosynthetic pathways for photosynthesis and the metabolism of sulfur and nitrogen as well as strong similarities between metabolic processes in C. tepidum and many Archaeal species.

Here we present the draft genome of Synergistes jonesii 78-1, ATCC 49833, a member of the Synergistes phylum. This organism was isolated from the rumen of a Hawaiian goat and ferments pyridinediols. The assembly contains 2,747,397 bp in 61 contigs.

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

Background: Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general. Methodology/Principal Findings: We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb). Conclusions/Significance: The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles., Author Summary Extreme environments, such as deep-sea hydrothermal vents, found 2,500 meters below the ocean surface, support large macrofaunal communities via microbially mediated carbon fixation processes using chemicals (chemoautotrophy) rather than light (photoautotrophy). The genome of one such model chemoautotrophic microbe, N. profundicola, was sequenced and described in this work. N. profundicola, distantly related to the pathogenic Helicobacter and Campylobacter species, contains a number of genes and pathways predicted to be important in DNA repair, environmental sensing, and metabolism, which are novel to either its subdivision or to all microbes. The genes and deduced metabolic pathways include several hydrogen uptake and release systems as well as a novel predicted nitrogen assimilation pathway. One gene involved in DNA repair, reverse gyrase, was thought to be a hallmark protein in hyperthermophiles, which are microbes that grow above 80°C. We found this gene to be highly expressed at 65°C, over 20°C above the optimal growth temperature of this organism. Therefore, the genome of this model deep-sea hydrothermal vent chemoautotroph may reflect what is required for life in an extreme environment, hypothesized to be similar to early earth conditions.

We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels., Author Summary Bacterial endophytes are capable of inhabiting the living tissues of plants without causing them significant harm. Klebsiella pneumoniae 342 (Kp342) is a model for this plant host-bacterial association, in part due to its capacity to colonize in high numbers the interior of plants including wheat and maize, two of the most important crops in the world. Kp342 possesses the ability to capture atmospheric nitrogen gas and turn it into an organic form (a process known as nitrogen fixation), of which part may be used as fertilizer by its plant host. Here, we describe the genome sequence and analysis of this model endophyte. When the Kp342 genome is compared to the genome of a closely related pathogenic relative, we can begin to surmise that its preference to engage in a harmonious relationship with plants is a result of many interacting factors. These include differences in its protein secretion systems, the manner in which its genes are regulated, and its ability to sense and respond to its environment. The study of endophytes is increasing in intensity due to the roles they may play in multiple biotechnological applications, including enhancing crop growth and nutrition, bioremediation, and development of plant-derived products and biofuels.

Arthrobacter sp. strains are among the most frequently isolated, indigenous, aerobic bacterial genera found in soils. Member of the genus are metabolically and ecologically diverse and have the ability to survive in environmentally harsh conditions for extended periods of time. The genome of Arthrobacter aurescens strain TC1, which was originally isolated from soil at an atrazine spill site, is composed of a single 4,597,686 basepair (bp) circular chromosome and two circular plasmids, pTC1 and pTC2, which are 408,237 bp and 300,725 bp, respectively. Over 66% of the 4,702 open reading frames (ORFs) present in the TC1 genome could be assigned a putative function, and 13.2% (623 genes) appear to be unique to this bacterium, suggesting niche specialization. The genome of TC1 is most similar to that of Tropheryma, Leifsonia, Streptomyces, and Corynebacterium glutamicum, and analyses suggest that A. aurescens TC1 has expanded its metabolic abilities by relying on the duplication of catabolic genes and by funneling metabolic intermediates generated by plasmid-borne genes to chromosomally encoded pathways. The data presented here suggest that Arthrobacter's environmental prevalence may be due to its ability to survive under stressful conditions induced by starvation, ionizing radiation, oxygen radicals, and toxic chemicals., Synopsis Soil systems contain the greatest diversity of microorganisms on earth, with 5,000–10,000 species of microorganism per gram of soil. Arthrobacter sp. strains have a primitive life cycle and are among the most frequently isolated, indigenous soil bacteria, found in common and deep subsurface soils, arctic ice, and environments contaminated with industrial chemicals and radioactive materials. To better understand how these bacteria survive in environmentally harsh conditions, the authors used a structural genomics approach to identify genes involved in soil survival of Arthrobacter aurescens strain TC1, a bacterium originally isolated for its ability to degrade the herbicide atrazine. They found that the genome of this bacterium comprises a single circular chromosome and two plasmids that encode for a large number proteins involved in stress responses due to starvation, desiccation, oxygen radicals, and toxic chemicals. A. aurescens' metabolic versatility is in part due to the presence of duplicated catabolic genes and its ability to funnel plasmid-derived intermediates into chromosomally encoded pathways. Arthrobacter's array of genes that allow for survival in stressful conditions and its ability to produce a temperature-tolerant “cyst”-like resting cell render this soil microorganism able to survive and prosper in a variety of environmental conditions.

Coastal aquatic environments are typically more highly productive and dynamic than open ocean ones. Despite these differences, cyanobacteria from the genus Synechococcus are important primary producers in both types of ecosystems. We have found that the genome of a coastal cyanobacterium, Synechococcus sp. strain CC9311, has significant differences from an open ocean strain, Synechococcus sp. strain WH8102, and these are consistent with the differences between their respective environments. CC9311 has a greater capacity to sense and respond to changes in its (coastal) environment. It has a much larger capacity to transport, store, use, or export metals, especially iron and copper. In contrast, phosphate acquisition seems less important, consistent with the higher concentration of phosphate in coastal environments. CC9311 is predicted to have differences in its outer membrane lipopolysaccharide, and this may be characteristic of the speciation of some cyanobacterial groups. In addition, the types of potentially horizontally transferred genes are markedly different between the coastal and open ocean genomes and suggest a more prominent role for phages in horizontal gene transfer in oligotrophic environments.

Since its introduction a decade ago, whole-genome shotgun sequencing (WGS) has been the main approach for producing cost-effective and high-quality genome sequence data. Until now, the Sanger sequencing technology that has served as a platform for WGS has not been truly challenged by emerging technologies. The recent introduction of the pyrosequencing-based 454 sequencing platform (454 Life Sciences, Branford, CT) offers a very promising sequencing technology alternative for incorporation in WGS. In this study, we evaluated the utility and cost-effectiveness of a hybrid sequencing approach using 3730 xl Sanger data and 454 data to generate higher-quality lower-cost assemblies of microbial genomes compared to current Sanger sequencing strategies alone.

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique “genomic islands” identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens., Synopsis Ehrlichiosis is an acute disease that triggers flu-like symptoms in both humans and animals. It is caused by a range of bacteria transmitted by ticks or flukes. Because these bacteria are difficult to culture, however, the organisms are poorly understood. The genomes of three emerging human pathogens causing ehrlichiosis were sequenced. A database was designed to allow the comparison of these three genomes to sixteen other bacteria with similar lifestyles. Analysis from this database reveals new species-specific and disease-specific genes indicating niche adaptations, pathogenic traits, and other features. In particular, one of the organisms contains more than 100 copies of a single gene involved in interactions with the host(s). These comparisons also enabled a reconstruction of the metabolic potential of five representative genomes from these bacteria and their close relatives. With this work, scientists can study these emerging pathogens in earnest.

The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae , the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for ≈80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.

Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

Virulence and immunity are poorly understood inMycobacterium tuberculosis.We sequenced the complete genome of theM. tuberculosisclinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample ofM. tuberculosisclinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms amongM. tuberculosisstrains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

The 2,160,267 bp genome sequence ofStreptococcusagalactiae, the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the U.S. and Europe, is predicted to encode 2,175 genes. Genome comparisons amongS. agalactiae,Streptococcus pneumoniae,Streptococcus pyogenes, and the other completely sequenced genomes identified genes specific to the streptococci and toS. agalactiae. Thesein silicoanalyses, combined with comparative genome hybridization experiments between the sequenced serotype V strain 2603 V/R and 19S. agalactiaestrains from several serotypes using whole-genome microarrays, revealed the genetic heterogeneity amongS. agalactiaestrains, even of the same serotype, and provided insights into the evolution of virulence mechanisms.

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.

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living α-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii , the plant pathogen Agrobacterium tumefaciens , and the bovine and human pathogen Brucella abortus .

A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.

A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.

Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The ∼150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development., Author Summary Vector-transmitted blood parasites cause some of the most widely distributed, serious, and poorly controlled diseases globally, including the most severe form of human malaria caused by Plasmodium falciparum. In livestock, tick-transmitted blood parasites include the protozoa Theileria parva, the cause of East Coast fever and Babesia bovis, the cause of tick fever, to which well over half of the world's cattle population are at risk. There is a critical need to better understand the mechanisms by which these parasites are transmitted, persist, and cause disease in order to optimize methods for control, including development of vaccines. This manuscript presents the genome sequence of B. bovis, and provides a whole genome comparative analysis with P. falciparum and T. parva. Genome-wide characterization of the B. bovis antigenically variable ves1 family reveals interesting differences in organization and expression from the related P. falciparum var genes. The second largest gene family (smorf) in B. bovis was newly discovered and may itself be involved in persistence, highlighting the utility of this approach in gene discovery. Organization and structure of the B. bovis genome is most similar to that of Theileria, and despite common features in clinical outcome is limited to microregional similarity with P. falciparum. Comparative gene analysis identifies several previously unknown proteins as homologs of vaccine candidates in one or more of these parasites, and candidate genes whose expression might account for unique properties such as the ability of Theileria to reversibly transform leukocytes.

Corrigendum: Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5

Nature 438, 1151–1156 (2005) There are two errors in the author listings for this Letter: the surname of Anne Lafon was misspelt as 'Lafton' and the affiliation of Hiroyuki Horiuchi should have been number 16 (and not 15, as was published).

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis . Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.