Mariprofundus ferrooxydans PV-1 the first genome of a marine Fe(II) oxidizing Zetaproteobacterium.

Mariprofundus ferrooxydans PV-1 has provided the first genome of the recently discovered Zetaproteobacteria subdivision. Genome analysis reveals a complete TCA cycle, the ability to fix CO(2), carbon-storage proteins and a sugar phosphotransferase system (PTS). The latter could facilitate the transport of carbohydrates across the cell membrane and possibly aid in stalk formation, a matrix composed of exopolymers and/or exopolysaccharides, which is used to store oxidized iron minerals outside the cell. Two-component signal transduction system genes, including histidine kinases, GGDEF domain genes, and response regulators containing CheY-like receivers, are abundant and widely distributed across the genome. Most of these are located in close proximity to genes required for cell division, phosphate uptake and transport, exopolymer and heavy metal secretion, flagellar biosynthesis and pilus assembly suggesting that these functions are highly regulated. Similar to many other motile, microaerophilic bacteria, genes encoding aerotaxis as well as antioxidant functionality (e.g., superoxide dismutases and peroxidases) are predicted to sense and respond to oxygen gradients, as would be required to maintain cellular redox balance in the specialized habitat where M. ferrooxydans resides. Comparative genomics with other Fe(II) oxidizing bacteria residing in freshwater and marine environments revealed similar content, synteny, and amino acid similarity of coding sequences potentially involved in Fe(II) oxidation, signal transduction and response regulation, oxygen sensation and detoxification, and heavy metal resistance. This study has provided novel insights into the molecular nature of Zetaproteobacteria.

Draft genome sequence of the chemolithoheterotrophic, halophilic methylotroph Methylophaga thiooxydans DMS010.

Methylophaga thiooxydans is a mesophilic, obligately halophilic bacterium that is capable of methylotrophic growth on a range of one-carbon compounds as well as chemolithoheterotrophic growth at the expense of thiosulfate. Here we present the draft genome sequence of Methylophaga thiooxydans DMS010 (DSM 22068(T), VKM B2586(T)), the type strain of the species, which has allowed prediction of the genes involved in one-carbon metabolism, nitrogen metabolism, and other aspects of central metabolism.

Genome sequence of the Marine Janibacter Sp. Strain HTCC2649.

Janibacter sp. strain HTCC2649 is a novel marine member of the Actinobacteria, family Intrasporangiaceae, and is closely related to Janibacter melonis CM2104(T) and Knoellia sinensis HKI 0119(T). The organism was isolated from a sample collected at Hydrostation S south of Bermuda by using high-throughput culturing techniques. Here we present the genome sequence of Janibacter sp. strain HTCC2649.

Genomic and functional adaptation in surface ocean planktonic prokaryotes.

The understanding of marine microbial ecology and metabolism has been hampered by the paucity of sequenced reference genomes. To this end, we report the sequencing of 137 diverse marine isolates collected from around the world. We analysed these sequences, along with previously published marine prokaryotic genomes, in the context of marine metagenomic data, to gain insights into the ecology of the surface ocean prokaryotic picoplankton (0.1-3.0 µm size range). The results suggest that the sequenced genomes define two microbial groups: one composed of only a few taxa that are nearly always abundant in picoplanktonic communities, and the other consisting of many microbial taxa that are rarely abundant. The genomic content of the second group suggests that these microbes are capable of slow growth and survival in energy-limited environments, and rapid growth in energy-rich environments. By contrast, the abundant and cosmopolitan picoplanktonic prokaryotes for which there is genomic representation have smaller genomes, are probably capable of only slow growth and seem to be relatively unable to sense or rapidly acclimate to energy-rich conditions. Their genomic features also lead us to propose that one method used to avoid predation by viruses and/or bacterivores is by means of slow growth and the maintenance of low biomass.

Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study.

BACKGROUND: We are developing a cross-species comparison strategy to distinguish between cancer driver- and passenger gene alteration candidates, by utilizing the difference in genomic location of orthologous genes between the human and other mammals. As an initial test of this strategy, we conducted a pilot study with human colorectal cancer (CRC) and its mouse model C57BL/6J ApcMin/+, focusing on human 5q22.2 and 18q21.1-q21.2. METHODS: We first performed bioinformatics analysis on the evolution of 5q22.2 and 18q21.1-q21.2 regions. Then, we performed exon-targeted sequencing, real time quantitative polymerase chain reaction (qPCR), and real time quantitative reverse transcriptase PCR (qRT-PCR) analyses on a number of genes of both regions with both human and mouse colon tumors. RESULTS: These two regions (5q22.2 and 18q21.1-q21.2) are frequently deleted in human CRCs and encode genuine colorectal tumor suppressors APC and SMAD4. They also encode genes such as MCC (mutated in colorectal cancer) with their role in CRC etiology unknown. We have discovered that both regions are evolutionarily unstable, resulting in genes that are clustered in each human region being found scattered at several distinct loci in the genome of many other species. For instance, APC and MCC are within 200 kb apart in human 5q22.2 but are 10 Mb apart in the mouse genome. Importantly, our analyses revealed that, while known CRC driver genes APC and SMAD4 were disrupted in both human colorectal tumors and tumors from ApcMin/+ mice, the questionable MCC gene was disrupted in human tumors but appeared to be intact in mouse tumors. CONCLUSIONS: These results indicate that MCC may not actually play any causative role in early colorectal tumorigenesis. We also hypothesize that its disruption in human CRCs is likely a mere result of its close proximity to APC in the human genome. Expanding this pilot study to the entire genome may identify more questionable genes like MCC, facilitating the discovery of new CRC driver gene candidates.

Genome sequences of Pelagibaca bermudensis HTCC2601T and Maritimibacter alkaliphilus HTCC2654T, the type strains of two marine Roseobacter genera.

Pelagibaca bermudensis HTCC2601(T) and Maritimibacter alkaliphilus HTCC2654(T) represent two marine genera in the globally significant Roseobacter clade of the Alphaproteobacteria. Here, we present the genome sequences of these organisms, isolated from the Sargasso Sea using dilution-to-extinction culturing, which offer insight into the genetic basis for the metabolic and ecological diversity of this important group.

Genome sequence of Fulvimarina pelagi HTCC2506T, a Mn(II)-oxidizing alphaproteobacterium possessing an aerobic anoxygenic photosynthetic gene cluster and Xanthorhodopsin.

Fulvimarina pelagi is a Mn(II)-oxidizing marine heterotrophic bacterium in the order Rhizobiales. Here we announce the draft genome sequence of F. pelagi HTCC2506(T), which was isolated from the Sargasso Sea by using dilution-to-extinction culturing. The genome sequence contained a xanthorhodopsin gene as well as a photosynthetic gene cluster, which suggests the coexistence of two different phototrophic mechanisms in a single microorganism.

Complete genome sequence of Croceibacter atlanticus HTCC2559T.

Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559(T), which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559(T) contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559(T), which identified it as an obligate chemoheterotroph.

Genome sequence of the oligotrophic marine Gammaproteobacterium HTCC2143, isolated from the Oregon Coast.

Strain HTCC2143 was isolated from Oregon Coast surface waters using dilution-to-extinction culturing. Here we present the genome of strain HTCC2143 from the BD1-7 clade of the oligotrophic marine Gammaproteobacteria group. The genome of HTCC2143 contains genes for carotenoid biosynthesis and proteorhodopsin and for proteins that have potential biotechnological significance: epoxide hydrolases, Baeyer-Villiger monooxygenases, and polyketide synthases.

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire.

BACKGROUND: Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. RESULTS: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. CONCLUSIONS: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

Genome sequence of the novel marine member of the Gammaproteobacteria strain HTCC5015.

HTCC5015 is a novel, highly divergent marine member of the Gammaproteobacteria, currently without a cultured representative with greater than 89% 16S rRNA gene identity to itself. The organism was isolated from water collected from Hydrostation S south of Bermuda using high-throughput dilution-to-extinction culturing techniques. Here we present the genome sequence of the unique Gammaproteobacterium strain HTCC5015.

Genome sequences of strains HTCC2148 and HTCC2080, belonging to the OM60/NOR5 clade of the Gammaproteobacteria.

Organisms in the OM60/NOR5 clade of the Gammaproteobacteria are ubiquitous in the world's oceans and can make up as much as 11% of bacterial cells in certain areas. Isolated from coastal Oregon water, Gammaproteobacteria HTCC2148 and HTCC2080 are two members of this important clade. Here we present the genome sequences of the OM60 Gammaproteobacteria HTCC2148 and HTCC2080.

Complete genome sequence of Robiginitalea biformata HTCC2501.

Robiginitalea biformata HTCC2501, isolated from the Sargasso Sea by dilution-to-extinction culturing, has been known as an aerobic chemoheterotroph with carotenoid pigments and dimorphic growth phases. Here, we announce the complete sequence of the R. biformata HTCC2501 genome, which contains genes for carotenoid biosynthesis and several macromolecule-degrading enzymes.

Complete genome sequence of Erythrobacter litoralis HTCC2594.

Erythrobacter litoralis has been known as a bacteriochlorophyll a-containing, aerobic, anoxygenic, phototrophic bacterium. Here we announce the complete genome sequence of E. litoralis HTCC2594, which is devoid of phototrophic potential. E. litoralis HTCC2594, isolated by dilution-to-extinction culturing from seawater, could not carry out aerobic anoxygenic phototrophy and lacked genes for bacteriochlorophyll a biosynthesis and photosynthetic reaction center proteins.

Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface-associated life style in the marine environment.

BACKGROUND: Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions. METHODOLOGY/PRINCIPAL FINDINGS: To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicata's oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions. CONCLUSIONS/SIGNIFICANCE: The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community. We have also identified properties that could mediate interactions with surfaces other than its currently recognised hosts. This together with the detection of known virulence genes leads to the hypothesis that P. tunicata maintains a carefully regulated balance between beneficial and detrimental interactions with a range of host surfaces.

Comparative genomics of two ecotypes of the marine planktonic copiotroph Alteromonas macleodii suggests alternative lifestyles associated with different kinds of particulate organic matter.

Alteromonas macleodii is a common marine heterotrophic gamma-proteobacterium. Isolates from this microbe cluster by molecular analysis into two major genotypic groups or ecotypes, one found in temperate latitudes in the upper water column and another that is for the most part found in the deep water column of the Mediterranean. Here, we describe the genome of one strain of the 'deep ecotype' (AltDE) isolated from 1000 m in the Eastern Mediterranean and compare this genome with that of the type strain ATCC 27126, a representative of the global 'surface' ecotype. The genomes are substantially different with DNA sequence similarity values that are borderline for microbes belonging to the same species, and a large differential gene content, mainly found in islands larger than 20 kbp, that also recruit poorly to the Global Ocean Sampling project (GOS). These genomic differences indicate that AltDE is probably better suited to microaerophilic conditions and for the degradation of recalcitrant compounds such as urea. These, together with other features, and the distribution of this genotypic group, indicate that this microbe colonizes relatively large particles that sink rapidly to meso and bathypelagic depths. The genome of ATCC 27126 on the other hand has more potential for regulation (two component systems) and degrades more sugars and amino acids, which is consistent with a more transient particle attachment, as would be expected for lineages specialized in colonizing smaller particulate organic matter with much slower sinking rates. The genomic data are also consistent with a picture of incipient speciation driven by niche specialization.

Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria.

BACKGROUND: The picocyanobacterial genus Synechococcus occurs over wide oceanic expanses, having colonized most available niches in the photic zone. Large scale distribution patterns of the different Synechococcus clades (based on 16S rRNA gene markers) suggest the occurrence of two major lifestyles ('opportunists'/'specialists'), corresponding to two distinct broad habitats ('coastal'/'open ocean'). Yet, the genetic basis of niche partitioning is still poorly understood in this ecologically important group. RESULTS: Here, we compare the genomes of 11 marine Synechococcus isolates, representing 10 distinct lineages. Phylogenies inferred from the core genome allowed us to refine the taxonomic relationships between clades by revealing a clear dichotomy within the main subcluster, reminiscent of the two aforementioned lifestyles. Genome size is strongly correlated with the cumulative lengths of hypervariable regions (or 'islands'). One of these, encompassing most genes encoding the light-harvesting phycobilisome rod complexes, is involved in adaptation to changes in light quality and has clearly been transferred between members of different Synechococcus lineages. Furthermore, we observed that two strains (RS9917 and WH5701) that have similar pigmentation and physiology have an unusually high number of genes in common, given their phylogenetic distance. CONCLUSION: We propose that while members of a given marine Synechococcus lineage may have the same broad geographical distribution, local niche occupancy is facilitated by lateral gene transfers, a process in which genomic islands play a key role as a repository for transferred genes. Our work also highlights the need for developing picocyanobacterial systematics based on genome-derived parameters combined with ecological and physiological data.

Novel computational methods for increasing PCR primer design effectiveness in directed sequencing.

BACKGROUND: Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment. RESULTS: We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research. CONCLUSION: The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.

Characterization of a marine gammaproteobacterium capable of aerobic anoxygenic photosynthesis.

Members of the gammaproteobacterial clade NOR5/OM60 regularly form an abundant part, up to 11%, of the bacterioplankton community in coastal systems during the summer months. Here, we report the nearly complete genome sequence of one cultured representative, Congregibacter litoralis strain KT71, isolated from North Sea surface water. Unexpectedly, a complete photosynthesis superoperon, including genes for accessory pigments, was discovered. It has a high sequence similarity to BAC clones from Monterey Bay [Beja O, Suzuki MT, Heidelberg JF, Nelson WC, Preston CM, et al. (2002) Nature 415:630-633], which also share a nearly identical gene arrangement. Although cultures of KT71 show no obvious pigmentation, bacteriochlorophyll a and spirilloxanthin-like carotenoids could be detected by HPLC analysis in cell extracts. The presence of two potential BLUF (blue light using flavin adenine dinucleotide sensors), one of which was found adjacent to the photosynthesis operon in the genome, indicates a light- and redox-dependent regulation of gene expression. Like other aerobic anoxygenic phototrophs (AAnPs), KT71 is able to grow neither anaerobically nor photoautotrophically. Cultivation experiments and genomic evidence show that KT71 needs organic substrates like carboxylic acids, oligopeptides, or fatty acids for growth. The strain grows optimally under microaerobic conditions and actively places itself in a zone of approximately 10% oxygen saturation. The genome analysis of C. litoralis strain KT71 identifies the gammaproteobacterial marine AAnPs, postulated based on BAC sequences, as members of the NOR5/OM60 clade. KT71 enables future experiments investigating the importance of this group of gammaproteobacterial AAnPs in coastal environments.

A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes.

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 3730xl Sanger data and 454 data to generate higher-quality lower-cost assemblies of microbial genomes compared to current Sanger sequencing strategies alone.