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To date, the US Department of Energy's Joint Genome Institute has produced shotgun libraries for more than 150 microbial whole genome sequencing projects. In an effort to better span repeats and link contigs we now utilize a 3 library approach for all whole genome shotgun projects. Here, we describe the construction, sequencing, and analysis of our 3 library cloning approach with the goal of producing high quality draft assemblies with greater genome coverage and fewer gaps. The libraries were constructed from randomly sheared whole genomic DNA that was size selected to approximately 3 Kb, 8 Kb or 40 Kb and ligated into the appropriate high, low, or single copy cloning vector. The libraries were sequenced to an approximate depth of 4X for the 3 Kb and 8 Kb libraries, and approximately 15X clone coverage for the 40 Kb fosmid library. Analysis to date of a wide array of microbial projects indicates our cloning strategy has been successful in spanning repeat regions and producing longer-range contiguity. These assemblies reduce the amount of finishing required to complete the genome sequence.

Wine grapes present a unique biogeography model, wherein microbial biodiversity patterns across viticultural zones not only answer questions of dispersal and community maintenance, they are also an inherent component of the quality, consumer acceptance, and economic appreciation of a culturally important food product. On their journey from the vineyard to the wine bottle, grapes are transformed to wine through microbial activity, with indisputable consequences for wine quality parameters. Wine grapes harbor a wide range of microbes originating from the surrounding environment, many of which are recognized for their role in grapevine health and wine quality. However, determinants of regional wine characteristics have not been identified, but are frequently assumed to stem from viticultural or geological factors alone. This study used a high-throughput, short-amplicon sequencing approach to demonstrate that regional, site-specific, and grape-variety factors shape the fungal and bacterial consortia inhabiting wine-grape surfaces. Furthermore, these microbial assemblages are correlated to specific climatic features, suggesting a link between vineyard environmental conditions and microbial inhabitation patterns. Taken together, these factors shape the unique microbial inputs to regional wine fermentations, posing the existence of nonrandom "microbial terroir" as a determining factor in regional variation among wine grapes.

During the transformation of grapes to wine, wine fermentations are exposed to a large area of specialized equipment surfaces within wineries, which may serve as important reservoirs for two-way transfer of microbes between fermentations. However, the role of winery environments in shaping the microbiota of wine fermentations and vectoring wine spoilage organisms is poorly understood at the systems level. Microbial communities inhabiting all major equipment and surfaces in a pilot-scale winery were surveyed over the course of a single harvest to track the appearance of equipment microbiota before, during, and after grape harvest. Results demonstrate that under normal cleaning conditions winery surfaces harbor seasonally fluctuating populations of bacteria and fungi. Surface microbial communities were dependent on the production context at each site, shaped by technological practices, processing stage, and season. During harvest, grape- and fermentation-associated organisms populated most winery surfaces, acting as potential reservoirs for microbial transfer between fermentations. These surfaces harbored large populations of Saccharomyces cerevisiae and other yeasts prior to harvest, potentially serving as an important vector of these yeasts in wine fermentations. However, the majority of the surface communities before and after harvest comprised organisms with no known link to wine fermentations and a near-absence of spoilage-related organisms, suggesting that winery surfaces do not overtly vector wine spoilage microbes under normal operating conditions.

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Mollusca) is a member of the Vesicomyidae. Species within this family form symbioses with chemosynthetic Gammaproteobacteria. They exist in environments such as hydrothermal vents and cold seeps and have a rudimentary gut and feeding groove, indicating a large dependence on their endosymbionts for nutrition. The C. magnifica symbiont, Candidatus Ruthia magnifica, was the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced (Newton et al. 2007). Here we expand upon the original report and provide additional details complying with the emerging MIGS/MIMS standards. The complete genome exposed the genetic blueprint of the metabolic capabilities of the symbiont. Genes which were predicted to encode the proteins required for all the metabolic pathways typical of free-living chemoautotrophs were detected in the symbiont genome. These include major pathways including carbon fixation, sulfur oxidation, nitrogen assimilation, as well as amino acid and cofactor/vitamin biosynthesis. This genome sequence is invaluable in the study of these enigmatic associations and provides insights into the origin and evolution of autotrophic endosymbiosis.

Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents, thus not only methanotrophs, but also non-methanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer, Methylobacillus flagellatus (strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mb pairs, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol- and methylamine dehydrogenases, the tetrahydromethanopterin-linked formaldehyde oxidation pathway, the assimilatory and dissimilatory branches of the ribulose monophosphate cycle, and by formate dehydrogenases. Some of the methylotrophy genes are present in more than one (identical or non-identical) copy. The obligate dependence on single carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha ketoglutarate, malate or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared, in terms of methylotrophy functions, to the previously sequenced genomes of three methylotrophs: Methylobacterium extorquens (Alphaproteobacterium, 7 Mbp), Methylibium petroleophilum (Betaproteobacterium, 4 Mbp), and Methylococcus capsulatus (Gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, methylotrophy functions in M. flagellatus were more similar to the ones in M. capsulatus and M. extorquens than to the ones in the more closely related M. petroleophilum, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.

Xylose is a major constituent of angiosperm lignocellulose, so its fermentation is important for bioconversion to fuels and chemicals. Pichia stipitis is the best-studied native xylose fermenting yeast. Genes from P. stipitis have been used to engineer xylose metabolism in Saccharomycescerevisiae, and the regulation of the P. stipitis genome offers insights into the mechanisms of xylose metabolism in yeasts. We have sequenced, assembled and finished the genome of P.stipitis. As such, it is one of only a handful of completely finished eukaryotic organisms undergoing analysis and manual curation. The sequence has revealed aspects of genome organization, numerous genes for biocoversion, preliminary insights into regulation of central metabolic pathways, numerous examples of co-localized genes with related functions, and evidence of how P. stipitis manages to achieve redox balance while growing on xylose under microaerobic conditions.

The basidiomycete Melampsora larici-populina is responsible for the leaf rust disease in Populus species. Urediniospore germlings of this obligate biotrophic fungus usually penetrate the host plant through stomatal openings, differentiates a series of infection structures in the intercellular space, and exhibits highly localized penetration of the host cell wall to establish a haustorium. Hyphae then proliferate in the leaf parenchyma and produce golden pustules filled with masses of urediniospores on the lower leaf surfaces. M. larici-populina causes severe economic losses in European poplar plantations and has recently been detected in Canada. Selection for resistance to this biotrophic pathogen is thus an important challenge for poplar breeders. Severe damages occur through decreased photosynthesis efficiency, early defoliation and increased susceptibility to other pests and diseases. The PGF is sequencing this organism by the whole genome shotgun (WGS) method. Generally we make genomic libraries containing 3 different insert sizes. For all libraries, the DNA is randomly sheared, fragments end repaired and size selected prior to cloning. After cleanup the 3kb insert is ligated into pUC18 vector and the 8kb and 40kb fragments are ligated into pMCL200 and pcc1Fos vectors. The libraries are sequenced to a depth of approximately 4X, 4X, and 0.5-1X coverage respectively. The JGI has recently sequenced the genome to approximately 4x coverage of this plant pathogen, and is currently sequencing to the 8-9X depth prior to final assembly. The initial size was estimated to be approximately 61 Million bases (Mb), and analysis at the 4X assembly allowed us to revise this estimate to approximately 95 Mb. Library construction, genome assembly and size analysis will be presented.

The US DOE Joint Genome Institute (JGI) is a high-throughput genomics facility involved in sequencing a variety of organisms. A major effort at JGI is the sequencing of genomes and microbial community samples of relevance to the DOE missions of carbon sequestration, bioremediation and energy production. cDNA/EST sequencing is an integral part of genomic sequencing because it provides crucial information for gene models and genome annotation. The 454 sequencing platform is an integrated system of emulsion-based PCR amplification of hundreds of thousands of DNA fragments linked to high throughput parallel pyrosequencing in picoliter-sized wells. Several strategies have been designed and carried out at JGI to use the 454 platform for cDNA/EST sequencing. cDNA libraries constructed by conventional methods were subjected to direct 454 sequencing. In addition, special primers and adaptors were also designed for library construction so the directional sequencing feature of the 454 technology can be used to sequence a particular end of the cDNA/EST fragments. Adaptor sequences used by 454 library construction can be incorporated into polyT primer, cap primer and/or random primer for cDNA/EST library construction. The 454 sequencing platform can deliver 200 to 400 thousand cDNA/EST reads from a single run and does not require cloning step, potentially improving the coverage obtained through traditional Sanger sequencing. The large numbers of short reads generated by the 454 platform can be aligned to genome assemblies to extend and confirm gene models. Results from different strategies of library construction combined with 454 sequencing will be presented. The coverage of the library and the novelty rate are compared with traditional Sanger sequencing. The possible assembly problems caused by short reads with slightly higher error rate from 454 will also be addressed.

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the 28 Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approx. 98.5 percent, have been reported to occur as obligate endosymbionts of 30 ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent 32 strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S34 23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont 36 sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments 38 revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the 40 two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex lifecycle. 42 These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and 44 obligately free-living bacteria ever revealed.

The highly consistent gene order and axial colinear expression patterns found in vertebrate hox gene clusters are less well conserved across the rest of bilaterians. We report the first deuterostome instance of an intact hox cluster with a unique gene order where the paralog groups are not expressed in a sequential manner. The finished sequence from BAC clones from the genome of the sea urchin, Strongylocentrotus purpuratus, reveals a gene order wherein the anterior genes (Hox1, Hox2 and Hox3) lie nearest the posterior genes in the cluster such that the most 3' gene is Hox5. (The gene order is : 5'-Hox1, 2, 3, 11/13c, 11/13b, '11/13a, 9/10, 8, 7, 6, 5 - 3)'. The finished sequence result is corroborated by restriction mapping evidence and BAC-end scaffold analyses. Comparisons with a putative ancestral deuterostome Hox gene cluster suggest that the rearrangements leading to the sea urchin gene order were many and complex.

In this poster we will provide an overview of the Joint Genome Institute's Microbial Sequencing projects with an emphasis on recent efforts to improve data quality by performing various quality control operations during sequencing and prior to beginning finishing. Some of these methods have been applied tooled projects in an attempt to correct obvious data problems and, therefore, provide a minimum standard of quality satisfied by all projects. We will describe a rapid and automated protocol which removes low quality and likely contamination from our projects. Application of this protocol typically removes between 5 and 30 percent of the reads in a project without negatively affecting the number of major contigs in draft assemblies.

Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents; thus, not only methanotrophs but also nonmethanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer, Methylobacillus flagellatus (strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mbp, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin-linked formaldehyde oxidation pathway and the assimilatory and dissimilatory ribulose monophosphate cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical) copy. The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha-ketoglutarate, malate, or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs, Methylobacterium extorquens (an alphaproteobacterium, 7 Mbp), Methylibium petroleiphilum (a betaproteobacterium, 4 Mbp), and Methylococcus capsulatus (a gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in M. flagellatus were more similar to those in M. capsulatus and M. extorquens than to the ones in the more closely related M. petroleiphilum species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.

Methylibium petroleiphilum PM1 is a methylotroph distinguished by its ability to completely metabolize the fuel oxygenate methyl tert-butyl ether (MTBE). Strain PM1 also degrades aromatic (benzene, toluene, and xylene) and straight-chain ([C.sub.5] to [C.sub.12]) hydrocarbons present in petroleum products. Whole-genome analysis of PM1 revealed an ~4-Mb circular chromosome and an ~600-kb megaplasmid, containing 3,831 and 646 genes, respectively. Aromatic hydrocarbon and alkane degradation, metal resistance, and methylotrophy are encoded on the chromosome. The megaplasmid contains an unusual t-RNA island, numerous insertion sequences, and large repeated elements, including a 40-kb region also present on the chromosome and a 29-kb tandem repeat encoding phosphonate transport and cobalamin biosynthesis. The megaplasmid also codes for alkane degradation and was shown to play an essential role in MTBE degradation through plasmid-curing experiments. Discrepancies between the insertion sequence element distribution patterns, the distributions of best BLASTP hits among major phylogenetic groups, and the G+C contents of the chromosome (69.2%) and plasmid (66%), together with comparative genome hybridization experiments, suggest that the plasmid was recently acquired and apparently carries the genetic information responsible for PMI's ability to degrade MTBE. Comparative genomic hybridization analysis with two PM1-like MTBE-degrading environmental isolates (~99% identical 16S rRNA gene sequences) showed that the plasmid was highly conserved (ca. 99% identical), whereas the chromosomes were too diverse to conduct resequencing analysis. PM1's genome sequence provides a foundation for investigating MTBE biodegradation and exploring the genetic regulation of multiple biodegradation pathways in M. petroleiphilum and other MTBE-degrading beta-proteobacteria.

The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, [beta]-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by [beta]-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.

Author(s): Gene W. Tyson [1]; Jarrod Chapman [3, 4]; Philip Hugenholtz [1]; Eric E. Allen [1]; Rachna J. Ram [1]; Paul M. Richardson [4]; Victor V. Solovyev [4]; Edward M. [...]

A bioinformatic and phenotypic analysis is described that has revealed diverse metabolic capabilities of JS666 that might be useful for bioremediation and biocatalysis. The roles of mobile genetic elements in shaping the genome and metabolic abilities of the bacterium are described.

A high-density polygenitic DNA microarray (16S microarray) containing 500,000 probes is employed to monitor changes in bacterial populations during redemption process to determine the relation between microbial community composition and less-soluble uranium U (IV) reoxidation. Observations suggest that reoxidation of uranium under reducing conditions occur despite elevated microbial activity and consistent presence of metal-reducing bacteria.

Research has been conducted on methane-oxidising archaea. The hypothesis that these bacteria have co-opted the methanogenic pathway has been tested via the isolation of methane-oxidising archaea genes by different approaches including PCR surveys of naturally occurring populations.