Deep-sea hydrothermal vent bacteria related to human pathogenic Vibrio species.

Vibrio species are both ubiquitous and abundant in marine coastal waters, estuaries, ocean sediment, and aquaculture settings worldwide. We report here the isolation, characterization, and genome sequence of a novel Vibrio species, Vibrio antiquarius, isolated from a mesophilic bacterial community associated with hydrothermal vents located along the East Pacific Rise, near the southwest coast of Mexico. Genomic and phenotypic analysis revealed V. antiquarius is closely related to pathogenic Vibrio species, namely Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio vulnificus, but sufficiently divergent to warrant a separate species status. The V. antiquarius genome encodes genes and operons with ecological functions relevant to the environment conditions of the deep sea and also harbors factors known to be involved in human disease caused by freshwater, coastal, and brackish water vibrios. The presence of virulence factors in this deep-sea Vibrio species suggests a far more fundamental role of these factors for their bacterial host. Comparative genomics revealed a variety of genomic events that may have provided an important driving force in V. antiquarius evolution, facilitating response to environmental conditions of the deep sea.

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

MicroRNAs suppress NB domain genes in tomato that confer resistance to Fusarium oxysporum.

MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing.

The cyanobacterial phylum encompasses oxygenic photosynthetic prokaryotes of a great breadth of morphologies and ecologies; they play key roles in global carbon and nitrogen cycles. The chloroplasts of all photosynthetic eukaryotes can trace their ancestry to cyanobacteria. Cyanobacteria also attract considerable interest as platforms for "green" biotechnology and biofuels. To explore the molecular basis of their different phenotypes and biochemical capabilities, we sequenced the genomes of 54 phylogenetically and phenotypically diverse cyanobacterial strains. Comparison of cyanobacterial genomes reveals the molecular basis for many aspects of cyanobacterial ecophysiological diversity, as well as the convergence of complex morphologies without the acquisition of novel proteins. This phylum-wide study highlights the benefits of diversity-driven genome sequencing, identifying more than 21,000 cyanobacterial proteins with no detectable similarity to known proteins, and foregrounds the diversity of light-harvesting proteins and gene clusters for secondary metabolite biosynthesis. Additionally, our results provide insight into the distribution of genes of cyanobacterial origin in eukaryotic nuclear genomes. Moreover, this study doubles both the amount and the phylogenetic diversity of cyanobacterial genome sequence data. Given the exponentially growing number of sequenced genomes, this diversity-driven study demonstrates the perspective gained by comparing disparate yet related genomes in a phylum-wide context and the insights that are gained from it.

Adaptation genomics of a small-colony variant in a Pseudomonas chlororaphis 30-84 biofilm.

The rhizosphere-colonizing bacterium Pseudomonas chlororaphis 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from a P. chlororaphis 30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genome's 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations in yfiR (cyclic-di-GMP production), fusA (elongation factor), and cyoE (heme synthesis) and a 70-kb deletion. Genetic analysis revealed that the yfiR locus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in the fusA gene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching in P. chlororaphis is not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles in P. chlororaphis natural persistence.

Using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes.

BACKGROUND: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. METHODS: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. RESULTS: We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. CONCLUSIONS: The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.

Comparative genomics of clinical and environmental Vibrio mimicus.

Whether Vibrio mimicus is a variant of Vibrio cholerae or a separate species has been the subject of taxonomic controversy. A genomic analysis was undertaken to resolve the issue. The genomes of V. mimicus MB451, a clinical isolate, and VM223, an environmental isolate, comprise ca. 4,347,971 and 4,313,453 bp and encode 3,802 and 3,290 ORFs, respectively. As in other vibrios, chromosome I (C-I) predominantly contains genes necessary for growth and viability, whereas chromosome II (C-II) bears genes for adaptation to environmental change. C-I harbors many virulence genes, including some not previously reported in V. mimicus, such as mannose-sensitive hemagglutinin (MSHA), and enterotoxigenic hemolysin (HlyA); C-II encodes a variant of Vibrio pathogenicity island 2 (VPI-2), and Vibrio seventh pandemic island II (VSP-II) cluster of genes. Extensive genomic rearrangement in C-II indicates it is a hot spot for evolution and genesis of speciation for the genus Vibrio. The number of virulence regions discovered in this study (VSP-II, MSHA, HlyA, type IV pilin, PilE, and integron integrase, IntI4) with no notable difference in potential virulence genes between clinical and environmental strains suggests these genes also may play a role in the environment and that pathogenic strains may arise in the environment. Significant genome synteny with prototypic pre-seventh pandemic strains of V. cholerae was observed, and the results of phylogenetic analysis support the hypothesis that, in the course of evolution, V. mimicus and V. cholerae diverged from a common ancestor with a prototypic sixth pandemic genomic backbone.

Complete genome sequence of Rahnella aquatilis CIP 78.65.

Rahnella aquatilis CIP 78.65 is a gammaproteobacterium isolated from a drinking water source in Lille, France. Here we report the complete genome sequence of Rahnella aquatilis CIP 78.65, the type strain of R. aquatilis.

Complete genome sequence of Rahnella sp. strain Y9602, a gammaproteobacterium isolate from metal- and radionuclide-contaminated soil.

Rahnella sp. strain Y9602 is a gammaproteobacterium isolated from contaminated subsurface soils that is capable of promoting uranium phosphate mineralization as a result of constitutive phosphatase activity. Here we report the first complete genome sequence of an isolate belonging to the genus Rahnella.

High-quality draft genome sequence of the Opitutaceae bacterium strain TAV1, a symbiont of the wood-feeding termite Reticulitermes flavipes.

Microbial communities in the termite hindgut are essential for degrading plant material. We present the high-quality draft genome sequence of the Opitutaceae bacterium strain TAV1, the first member of the phylum Verrucomicrobia to be isolated from wood-feeding termites. The genomic analysis reveals genes coding for lignocellulosic degradation and nitrogen fixation.

Complete genome sequences of Desulfosporosinus orientis DSM765T, Desulfosporosinus youngiae DSM17734T, Desulfosporosinus meridiei DSM13257T, and Desulfosporosinus acidiphilus DSM22704T.

Desulfosporosinus species are sulfate-reducing bacteria belonging to the Firmicutes. Their genomes will give insights into the genetic repertoire and evolution of sulfate reducers typically thriving in terrestrial environments and able to degrade toluene (Desulfosporosinus youngiae), to reduce Fe(III) (Desulfosporosinus meridiei, Desulfosporosinus orientis), and to grow under acidic conditions (Desulfosporosinus acidiphilus).

Genome sequencing and mapping reveal loss of heterozygosity as a mechanism for rapid adaptation in the vegetable pathogen Phytophthora capsici.

The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic or genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) in diverse isolates. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30% of the genome. LOH altered genotypes for more than 11,000 single-nucleotide variant sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.

The genome of Pelobacter carbinolicus reveals surprising metabolic capabilities and physiological features.

BACKGROUND: The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing Geobacter species. RESULTS: Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATP synthase and [FeFe]-hydrogenase were differentiated and assigned roles according to their structural properties and genomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way. Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can function catabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that function in 2,3-butanediol production. Pyruvate:ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneck in both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis to production of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages, including three proteins with similarity to the geopilin of electroconductive nanowires. CONCLUSIONS: Several surprising metabolic capabilities and physiological features were predicted from the genome of P. carbinolicus, suggesting that it is more versatile than anticipated.

Epitope-specific CD8+ T lymphocytes cross-recognize mutant simian immunodeficiency virus (SIV) sequences but fail to contain very early evolution and eventual fixation of epitope escape mutations during SIV infection.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) evade containment by CD8(+) T lymphocytes through focused epitope mutations. However, because of limitations in the numbers of viral sequences that can be sampled, traditional sequencing technologies have not provided a true representation of the plasticity of these viruses or the intensity of CD8(+) T lymphocyte-mediated selection pressure. Moreover, the strategy by which CD8(+) T lymphocytes contain evolving viral quasispecies has not been characterized fully. In the present study we have employed ultradeep 454 pyrosequencing of virus and simultaneous staining of CD8(+) T lymphocytes with multiple tetramers in the SIV/rhesus monkey model to explore the coevolution of virus and the cellular immune response during primary infection. We demonstrated that cytotoxic T lymphocyte (CTL)-mediated selection pressure on the infecting virus was manifested by epitope mutations as early as 21 days following infection. We also showed that CD8(+) T lymphocytes cross-recognized wild-type and mutant epitopes and that these cross-reactive cell populations were present at a time when mutant forms of virus were present at frequencies of as low as 1 in 22,000 sequenced clones. Surprisingly, these cross-reactive cells became enriched in the epitope-specific CD8(+) T lymphocyte population as viruses with mutant epitope sequences largely replaced those with epitope sequences of the transmitted virus. These studies demonstrate that mutant epitope-specific CD8(+) T lymphocytes that are present at a time when viral mutant epitope sequences are detected at extremely low frequencies fail to contain the later accumulation and fixation of the mutant epitope sequences in the viral quasispecies.

The genomic basis of trophic strategy in marine bacteria.

Many marine bacteria have evolved to grow optimally at either high (copiotrophic) or low (oligotrophic) nutrient concentrations, enabling different species to colonize distinct trophic habitats in the oceans. Here, we compare the genome sequences of two bacteria, Photobacterium angustum S14 and Sphingopyxis alaskensis RB2256, that serve as useful model organisms for copiotrophic and oligotrophic modes of life and specifically relate the genomic features to trophic strategy for these organisms and define their molecular mechanisms of adaptation. We developed a model for predicting trophic lifestyle from genome sequence data and tested >400,000 proteins representing >500 million nucleotides of sequence data from 126 genome sequences with metagenome data of whole environmental samples. When applied to available oceanic metagenome data (e.g., the Global Ocean Survey data) the model demonstrated that oligotrophs, and not the more readily isolatable copiotrophs, dominate the ocean's free-living microbial populations. Using our model, it is now possible to define the types of bacteria that specific ocean niches are capable of sustaining.

Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, is a bacterium autochthonous to the aquatic environment, and a serious public health threat. V. cholerae serogroup O1 is responsible for the previous two cholera pandemics, in which classical and El Tor biotypes were dominant in the sixth and the current seventh pandemics, respectively. Cholera researchers continually face newly emerging and reemerging pathogenic clones carrying diverse combinations of phenotypic and genotypic properties, which significantly hampered control of the disease. To elucidate evolutionary mechanisms governing genetic diversity of pandemic V. cholerae, we compared the genome sequences of 23 V. cholerae strains isolated from a variety of sources over the past 98 years. The genome-based phylogeny revealed 12 distinct V. cholerae lineages, of which one comprises both O1 classical and El Tor biotypes. All seventh pandemic clones share nearly identical gene content. Using analogy to influenza virology, we define the transition from sixth to seventh pandemic strains as a "shift" between pathogenic clones belonging to the same O1 serogroup, but from significantly different phyletic lineages. In contrast, transition among clones during the present pandemic period is characterized as a "drift" between clones, differentiated mainly by varying composition of laterally transferred genomic islands, resulting in emergence of variants, exemplified by V. cholerae O139 and V. cholerae O1 El Tor hybrid clones. Based on the comparative genomics it is concluded that V. cholerae undergoes extensive genetic recombination via lateral gene transfer, and, therefore, genome assortment, not serogroup, should be used to define pathogenic V. cholerae clones.

Complete genome sequence of the haloalkaliphilic, hydrogen-producing bacterium Halanaerobium hydrogeniformans.

Halanaerobium hydrogenoformans is an alkaliphilic bacterium capable of biohydrogen production at pH 11 and 7% (wt/vol) salt. We present the 2.6-Mb genome sequence to provide insights into its physiology and potential for bioenergy applications.

Genome sequence of the ethanol-producing Zymomonas mobilis subsp. mobilis lectotype strain ATCC 10988.

Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome.

Genome sequence of the ethanol-producing Zymomonas mobilis subsp. pomaceae lectotype strain ATCC 29192.

Zymomonas mobilis is an alphaproteobacterium studied for bioethanol production. Different strains of this organism have been hitherto sequenced; they all belong to the Z. mobilis subsp. mobilis taxon. Here we report the finished and annotated genome sequence of strain ATCC 29192, a cider-spoiling agent isolated in the United Kingdom. ATCC 29192 is the lectotype of the second-best-characterized subspecies of Z. mobilis, Z. mobilis subsp. pomaceae. The nucleotide sequence of ATCC 29192 deviates from that of Z. mobilis subsp. mobilis representatives, which justifies its distinct taxonomic positioning and proves particularly useful for comparative and functional genomic analyses.

Genome sequence of Nitrosomonas sp. strain AL212, an ammonia-oxidizing bacterium sensitive to high levels of ammonia.

Nitrosomonas sp. strain AL212 is an obligate chemolithotrophic ammonia-oxidizing bacterium (AOB) that was originally isolated in 1997 by Yuichi Suwa and colleagues. This organism belongs to Nitrosomonas cluster 6A, which is characterized by sensitivity to high ammonia concentrations, higher substrate affinity (lower K(m)), and lower maximum growth rates than strains in Nitrosomonas cluster 7, which includes Nitrosomonas europaea and Nitrosomonas eutropha. Genome-informed studies of this ammonia-sensitive cohort of AOB are needed, as these bacteria are found in freshwater environments, drinking water supplies, wastewater treatment systems, and soils worldwide.