We reconstructed the complete 2.4 Mb-long genome of a previously uncultivated epsilonproteobacterium, Candidatus Sulfuricurvum sp. RIFRC-1, via assembly of short-read shotgun metagenomic data using a complexity reduction approach. Genome-based comparisons indicate the bacterium is a novel species within the Sulfuricurvum genus, which contains one cultivated representative, S. kujiense. Divergence between the species appears due in part to extensive genomic rearrangements, gene loss and chromosomal versus plasmid encoding of certain (respiratory) genes by RIFRC-1. Deoxyribonucleic acid for the genome was obtained from terrestrial aquifer sediment, in which RIFRC-1 comprised ∼ 47% of the bacterial community. Genomic evidence suggests RIFRC-1 is a chemolithoautotrophic diazotroph capable of deriving energy for growth by microaerobic or nitrate-/nitric oxide-dependent oxidation of S°, sulfide or sulfite or H₂oxidation. Carbon may be fixed via the reductive tricarboxylic acid cycle. Consistent with these physiological attributes, the local aquifer was microoxic with small concentrations of available nitrate, small but elevated concentrations of reduced sulfur and NH(4)(+) /NH₃-limited. Additionally, various mechanisms for heavy metal and metalloid tolerance and virulence point to a lifestyle well-adapted for metal(loid)-rich environments and a shared evolutionary past with pathogenic Epsilonproteobacteria. Results expand upon recent findings highlighting the potential importance of sulfur and hydrogen metabolism in the terrestrial subsurface.

BACKGROUND:Preterm infants represent a unique patient population that is born functionally immature and must accomplish development under the influence of a hospital environment. Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. The purpose of this study was to evaluate the progression of intestinal microbiota community development between preterm infants who remained healthy compared to preterm infants who developed NEC.RESULTS:Weekly fecal samples from ten preterm infants, five with NEC and five matched healthy controls were obtained. Bacterial DNA from individual fecal samples was subjected to sequencing of 16S rRNA-based inventories using the 454 GS-FLX platform. Fecal samples from control infants demonstrated a temporal pattern in their microbiota, which converged toward that of a healthy full term breast-fed infant. Microbiota development in NEC patients diverged from controls beginning three weeks prior to diagnosis. Shotgun metagenomic sequencing was performed to identify functional differences in the respective microbiota of fecal samples from a set of twins in which one twin developed NEC and one did not. The majority of the differentially abundant genes in the NEC patient were associated with carbohydrate metabolism and mapped to members of the family Enterobacteriaceae. This may indicate an adaptation of the community to an altered profile of substrate availability for specific members as a first step towards the development of NEC. We propose that the microbial communities as a whole may metabolize milk differently, resulting in differential substrate availability for specific microbial groups. Additional differentially represented gene sets of interest were related to antibiotic resistance and vitamin biosynthesis.CONCLUSIONS:Our results suggest that there is a temporal component to microbiome development in healthy preterm infants. Thus, bacteriotherapy for the treatment or prevention of NEC must consider this temporal component of the microbial community in addition to its taxonomic composition and functional content.

The democratized world of sequencing is leading to numerous data analysis challenges; MG-RAST addresses many of these challenges for diverse datasets, including amplicon datasets, shotgun metagenomes, and metatranscriptomes. The changes from version 2 to version 3 include the addition of a dedicated gene calling stage using FragGenescan, clustering of predicted proteins at 90% identity, and the use of BLAT for the computation of similarities. Together with changes in the underlying software infrastructure, this has enabled the dramatic scaling up of pipeline throughput while remaining on a limited hardware budget. The Web-based service allows upload, fully automated analysis, and visualization of results. As a result of the plummeting cost of sequencing and the readily available analytical power of MG-RAST, over 78,000 metagenomic datasets have been analyzed, with over 12,000 of them publicly available in MG-RAST.

Background: The development of next generation sequencing technology is rapidly changing the face of the genome annotation and analysis field. One of the primary uses for genome sequence data is to improve our understanding and prediction of phenotypes for microbes and microbial communities, but the technologies for predicting phenotypes must keep pace with the new sequences emerging.Scope of review: This review presents an integrated view of the methods and technologies used in the inference of phenotypes for microbes and microbial communities based on genomic and metagenomic data. Given the breadth of this topic, we place special focus on the resources available within the SEED Project. We discuss the two steps involved in connecting genotype to phenotype: sequence annotation, and phenotype inference, and we highlight the challenges in each of these steps when dealing with both single genome and metagenome data.Major conclusions: This integrated view of the genotype-to-phenotype problem highlights the importance of a controlled ontology in the annotation of genomic data, as this benefits subsequent phenotype inference and metagenome annotation. We also note the importance of expanding the set of reference genomes to improve the annotation of all sequence data, and we highlight metagenome assembly as a potential new source for complete genomes. Finally, we find that phenotype inference, particularly from metabolic models, generates predictions that can be validated and reconciled to improve annotations.General significance: This review presents the first look at the challenges and opportunities associated with the inference of phenotype from genotype during the next generation sequencing revolution. This article is part of a Special Issue entitled: Systems Biology of Microorganisms. Published by Elsevier BM.

Sulfate-reducing bacteria (SRB) belonging to the metabolically versatile Desulfobacteriaceae are abundant in marine sediments and contribute to the global carbon cycle by complete oxidation of organic compounds. Desulfobacterium autotrophicum HRM2 is the first member of this ecophysiologically important group with a now available genome sequence. With 5.6 megabasepairs (Mbp) the genome of Db. autotrophicum HRM2 is about 2 Mbp larger than the sequenced genomes of other sulfate reducers (SRB). A high number of genome plasticity elements (> 100 transposon-related genes), several regions of GC discontinuity and a high number of repetitive elements (132 paralogous genes Mbp(-1)) point to a different genome evolution when comparing with Desulfovibrio spp. The metabolic versatility of Db. autotrophicum HRM2 is reflected in the presence of genes for the degradation of a variety of organic compounds including long-chain fatty acids and for the Wood-Ljungdahl pathway, which enables the organism to completely oxidize acetyl-CoA to CO2 but also to grow chemolithoautotrophically. The presence of more than 250 proteins of the sensory/regulatory protein families should enable Db. autotrophicum HRM2 to efficiently adapt to changing environmental conditions. Genes encoding periplasmic or cytoplasmic hydrogenases and formate dehydrogenases have been detected as well as genes for the transmembrane TpII-c(3), Hme and Rnf complexes. Genes for subunits A, B, C and D as well as for the proposed novel subunits L and F of the heterodisulfide reductases are present. This enzyme is involved in energy conservation in methanoarchaea and it is speculated that it exhibits a similar function in the process of dissimilatory sulfate reduction in Db. autotrophicum HRM2.

BACKGROUND:Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described.RESULTS:In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae.CONCLUSION:The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches.

BACKGROUND:The number of prokaryotic genome sequences becoming available is growing steadily and is growing faster than our ability to accurately annotate them.DESCRIPTION:We describe a fully automated service for annotating bacterial and archaeal genomes. The service identifies protein-encoding, rRNA and tRNA genes, assigns functions to the genes, predicts which subsystems are represented in the genome, uses this information to reconstruct the metabolic network and makes the output easily downloadable for the user. In addition, the annotated genome can be browsed in an environment that supports comparative analysis with the annotated genomes maintained in the SEED environment. The service normally makes the annotated genome available within 12-24 hours of submission, but ultimately the quality of such a service will be judged in terms of accuracy, consistency, and completeness of the produced annotations. We summarize our attempts to address these issues and discuss plans for incrementally enhancing the service.CONCLUSION:By providing accurate, rapid annotation freely to the community we have created an important community resource. The service has now been utilized by over 120 external users annotating over 350 distinct genomes.

ABSTRACT Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete that causes bacterial wilt and canker of tomato. The nucleotide sequence of the genome of strain NCPPB382 was determined. The chromosome is circular, consists of 3.298 Mb, and has a high G+C content (72.6%). Annotation revealed 3,080 putative protein-encoding sequences; only 26 pseudogenes were detected. Two rrn operons, 45 tRNAs, and three small stable RNA genes were found. The two circular plasmids, pCM1 (27.4 kbp) and pCM2 (70.0 kbp), which carry pathogenicity genes and thus are essential for virulence, have lower G+C contents (66.5 and 67.6%, respectively). In contrast to the genome of the closely related organism Clavibacter michiganensis subsp. sepedonicus, the genome of C. michiganensis subsp. michiganensis lacks complete insertion elements and transposons. The 129-kb chp/tomA region with a low G+C content near the chromosomal origin of replication was shown to be necessary for pathogenicity. This region contains numerous genes encoding proteins involved in uptake and metabolism of sugars and several serine proteases. There is evidence that single genes located in this region, especially genes encoding serine proteases, are required for efficient colonization of the host. Although C. michiganensis subsp. michiganensis grows mainly in the xylem of tomato plants, no evidence for pronounced genome reduction was found. C. michiganensis subsp. michiganensis seems to have as many transporters and regulators as typical soil-inhabiting bacteria. However, the apparent lack of a sulfate reduction pathway, which makes C. michiganensis subsp. michiganensis dependent on reduced sulfur compounds for growth, is probably the reason for the poor survival of C. michiganensis subsp. michiganensis in soil.

ComparativegenomicsofthepIPO2/pSB102familyofenvironmental plasmids: sequence, evolution, and ecologyof pTer331isolated from Collimonas fungivorans Ter331 Francesca Mela, Kathrin Fritsche, Hidde Boersma, Jan D. van Elsas, Daniela Bartels, Folker Meyer, Wietse de Boer, Johannes A. van Veen & Johan H.J. Leveau Centre for Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Heteren, The Netherlands; Department of Microbial Ecology, University of Groningen, Haren, The Netherlands; and Argonne National Laboratory, Mathematics and Computer Science Division, Argonne, IL, USA

Plasmid pTer331 from the bacterium Collimonas fungivorans Ter331 is a new member of the pIPO2/pSB102 family of environmental plasmids. The 40 457-bp sequence of pTer331 codes for 44 putative ORFs, most of which represent genes involved in replication, partitioning and transfer of the plasmid. We confirmed that pTer331 is stably maintained in its native host. Deletion analysis identified a mini-replicon capable of replicating autonomously in Escherichia coli and Pseudomonas putida. Furthermore, plasmid pTer331 was able to mobilize and retromobilize IncQ plasmid pSM1890 at typical rates of 10(-4) and 10(-8), respectively. Analysis of the 91% DNA sequence identity between pTer331 and pIPO2 revealed functional conservation of coding sequences, the deletion of DNA fragments flanked by short direct repeats (DR), and sequence preservation of long DRs. In addition, we experimentally established that pTer331 has no obvious contribution in several of the phenotypes that are characteristic of its host C. fungivorans Ter331, including the ability to efficiently colonize plant roots. Based on our findings, we hypothesize that cryptic plasmids such as pTer331 and pIPO2 might not confer an individual advantage to bacteria, but, due to their broad-host-range and ability to retromobilize, benefit bacterial populations by accelerating the intracommunal dissemination of the mobile gene pool.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

Nature Biotechnology 24, 1385–1391 (2006); published online 22 October 2006; corrected after print 26 March 2007 In the version of this article initially published, the gene name nosRZDYFLX on p. 1388, left column, line 5, is incorrect; the correct name is nosZRDFYL. The error has been corrected in the PDF version of the article.

Nature Biotechnology 24, 1385–1391 (2006); published online 22 October 2006; corrected after print 26 March 2007 In the version of this article initially published, the gene name nosRZDYFLX on p. 1388, left column, line 5, is incorrect; the correct name is nosZRDFYL. The error has been corrected in the PDF version of the article.

ly, annotating a genome amounts to attaching information to support use of the genome. This includes an almost endless variety of types of analysis and attachment of interpretations. In our experience, it has proven useful to prioritize the analyses, and the following items provide at least a reasonable working notion of what is meant: 1. Genes are identified. This effort includes at least protein-encoding genes and some of the RNA-encoding genes (often just tRNAs and rRNAs). 2. The functions of genes are predicted. 3. Metabolic reconstructions are developed and tied to the specific genes. 4. Prophages, insertion sequences, and transposons are labeled. 5. Frameshifts and pseudogenes are predicted. 6. Regulatory sites and operons are identified as a step toward developing an inventory of regulons. In practice, usually only the locations of genes and their predicted functions are provided by the initial annotation effort. Accordingly the first part of this review deals with the status of gene identification in prokaryotes, and the second part deals with the task of predicting the function to be associated with protein-coding genes. 2. Gene Prediction in Bacteria and Archaea Once the sequence of a prokaryotic genome has been determined, the next step is the definition of the functional Figure 1. Growth of available genomes and SwissProt annotations. While the primary sequence repository (GenBank1) doubles in size every 18 months, high-quality annotations (we take SwissProt2 as an example) cannot keep up with this growth. The graph compares the growth on a logarithmic scale. Figure 2. How annotations are done. This diagram is intended to convey the interactions between the different types of activities that make up the annotation process (blue). A key point is that maintenance and improvement of annotations originate in expert analysis and are reflected through protein family curation, since the “curate genomes” activity is seldom done. Annotation of Bacterial and Archaeal Genomes Chemical Reviews, 2007, Vol. 107, No. 8 3433

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

ABSTRACT We present the complete genome sequence of Listeria welshimeri, a nonpathogenic member of the genus Listeria. Listeria welshimeri harbors a circular chromosome of 2,814,130 bp with 2,780 open reading frames. Comparative genomic analysis of chromosomal regions between L. welshimeri, Listeria innocua, and Listeria monocytogenes shows strong overall conservation of synteny, with the exception of the translocation of an FoF1 ATP synthase. The smaller size of the L. welshimeri genome is the result of deletions in all of the genes involved in virulence and of “fitness” genes required for intracellular survival, transcription factors, and LPXTG- and LRR-containing proteins as well as 55 genes involved in carbohydrate transport and metabolism. In total, 482 genes are absent from L. welshimeri relative to L. monocytogenes. Of these, 249 deletions are commonly absent in both L. welshimeri and L. innocua, suggesting similar genome evolutionary paths from an ancestor. We also identified 311 genes specific to L. welshimeri that are absent in the other two species, indicating gene expansion in L. welshimeri, including horizontal gene transfer. The species L. welshimeri appears to have been derived from early evolutionary events and an ancestor more compact than L. monocytogenes that led to the emergence of nonpathogenic Listeria spp.

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones–based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.

The National Microbial Pathogen Data Resource (NMPDR) (http://www.nmpdr.org) is a National Institute of Allergy and Infections Disease (NIAID)-funded Bioinformatics Resource Center that supports research in selected Category B pathogens. NMPDR contains the complete genomes of approximately 50 strains of pathogenic bacteria that are the focus of our curators, as well as >400 other genomes that provide a broad context for comparative analysis across the three phylogenetic Domains. NMPDR integrates complete, public genomes with expertly curated biological subsystems to provide the most consistent genome annotations. Subsystems are sets of functional roles related by a biologically meaningful organizing principle, which are built over large collections of genomes; they provide researchers with consistent functional assignments in a biologically structured context. Investigators can browse subsystems and reactions to develop accurate reconstructions of the metabolic networks of any sequenced organism. NMPDR provides a comprehensive bioinformatics platform, with tools and viewers for genome analysis. Results of precomputed gene clustering analyses can be retrieved in tabular or graphic format with one-click tools. NMPDR tools include Signature Genes, which finds the set of genes in common or that differentiates two groups of organisms. Essentiality data collated from genome-wide studies have been curated. Drug target identification and high-throughput, in silico, compound screening are in development.

MOTIVATION:Novel sequencing techniques can give access to organisms that are difficult to cultivate using conventional methods. When applied to environmental samples, the data generated has some drawbacks, e.g. short length of assembled contigs, in-frame stop codons and frame shifts. Unfortunately, current gene finders cannot circumvent these difficulties. At the same time, the automated prediction of genes is a prerequisite for the increasing amount of genomic sequences to ensure progress in metagenomics.RESULTS:We introduce a novel gene finding algorithm that incorporates features overcoming the short length of the assembled contigs from environmental data, in-frame stop codons as well as frame shifts contained in bacterial sequences. The results show that by searching for sequence similarities in an environmental sample our algorithm is capable of detecting a high fraction of its gene content, depending on the species composition and the overall size of the sample. The method is valuable for hunting novel unknown genes that may be specific for the habitat where the sample is taken. Finally, we show that our algorithm can even exploit the limited information contained in the short reads generated by 454 technology for the prediction of protein coding genes.AVAILABILITY:The program is freely available upon request.