[Show abstract][Hide abstract] ABSTRACT: Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the gammaproteobacteria class sharing 96% 16S rRNA-gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl-CoA and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbons-impacted hypersaline environments.
[Show abstract][Hide abstract] ABSTRACT: Corals represent symbiotic meta-organisms that require harmonization among the coral animal, photosynthetic zooxanthellae and associated microbes to survive environmental stresses. We investigated integrated-responses among coral and zooxanthellae in the scleractinian coral Acropora formosa in response to an emerging marine pollutant, the munitions constituent, 1,3,5-trinitro-1,3,5 triazine (RDX; 5 day exposures to 0 (control), 0.5, 0.9, 1.8, 3.7, and 7.2 mg/L, measured in seawater).
[Show abstract][Hide abstract] ABSTRACT: The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome - fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (= pachytene chromosomes), we showed that 45 scaffolds, representing a third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH, while often disagreeing with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.
[Show abstract][Hide abstract] ABSTRACT: RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a U. pugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for D. melanogaster EcR/USP heterodimers. In swapping experiments. UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities.
General and Comparative Endocrinology 01/2014; · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-throughput pyrosequencing of SSU rDNA genes was used to obtain monthly snapshots of eukaryotic and bacterial diversity and community structure at two locations in Lake Texoma, a low salinity lake in the south central United States, over 1 year. The lake experienced two disturbance events (i) a localized bloom of Prymnesium parvum restricted to one of the locations that lasted from January to April, and (ii) a large (17 cm), global rain event in the beginning of May, overlaid onto seasonal environmental change. Eukaryotic species richness as well as both eukaryotic and bacterial community similarity exhibited seasonal patterns, including distinct responses to the rain event. The P. parvum bloom created a natural experiment in which to directly explore the effects of an Ecosystem Disruptive Algal Bloom (EDAB) on the microbial community separated from seasonal changes. Microbial species richness was unaffected by the bloom, however, the eukaryotic community structure (evenness) and the patterns of both eukaryotic and bacterial community similarity at bloom and non-bloom sites were statistically distinct during the 4 months of the bloom. These results indicate that physical and biological disturbances as well as seasonal environmental forces contribute to the structure of both the eukaryotic and bacterial communities.
[Show abstract][Hide abstract] ABSTRACT: The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade ( and species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi ( species), a morning-glory symbiont (), and a bamboo pathogen (), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.
[Show abstract][Hide abstract] ABSTRACT: Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.
PLoS ONE 01/2013; 8(10):e76757. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera1 and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium2, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.
[Show abstract][Hide abstract] ABSTRACT: Viruses are most frequently discovered because they cause disease. To expand knowledge of plant-associated viruses beyond these narrow constraints, non-cultivated plants of the Tallgrass Prairie of the United States were systematically surveyed for evidence of viruses. This report discusses putative viruses of the family Secoviridae identified by the survey. Sequence analysis suggests the presence of at least six viruses in the study site, including Bean pod mottle virus, Maize chlorotic dwarf virus, three previously undescribed viruses within the subfamily Comovirinae and one unclassifiable virus.
Virus Research 04/2012; 167(1):34-42. · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.
[Show abstract][Hide abstract] ABSTRACT: Pyrosequencing analysis of 16S rRNA genes was used to examine impacts of elevated CO(2) (eCO(2)) on soil microbial communities from 12 replicates each from ambient CO(2) (aCO(2)) and eCO(2) settings. The results suggest that the soil microbial community composition and structure significantly altered under conditions of eCO(2), which was closely associated with soil and plant properties.
Applied and Environmental Microbiology 02/2012; 78(8):2991-5. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Native virus-plant interactions require more understanding and their study will provide a basis from which to identify potential sources of emerging destructive viruses in crops. A novel tymovirus sequence was detected in Asclepias viridis (green milkweed), a perennial growing in a natural setting in the Tallgrass Prairie Preserve (TGPP) of Oklahoma. It was abundant within and frequent among A. viridis plants and, to varying extents, within other dicotyledonous and one grass (Panicum virgatum) species obtained from the TGPP. Extracts from A. viridis containing the sequence were infectious to a limited number of species. The virus genome was cloned and determined to be closely related to Kennedya yellow mosaic virus. The persistence of the virus within the Oklahoma A. viridis population was monitored for five successive years. Virus was present in a high percentage of plants within representative areas of the TGPP in all years and was spreading to additional plants. Virus was present in regions adjacent to the TGPP but not in plants sampled from central and south-central Oklahoma. Virus was present in the underground caudex of the plant during the winter, suggesting overwintering in this tissue. The RNA sequence encoding the virus coat protein varied considerably between individual plants (≈3%), likely due to drift rather than selection. An infectious clone was constructed and the virus was named Asclepias asymptomatic virus (AsAV) due to the absence of obvious symptoms on A. viridis.
[Show abstract][Hide abstract] ABSTRACT: In an effort to better understand the ancestral state of the human distal gut microbiome, we examine feces retrieved from archaeological contexts (coprolites). To accomplish this, we pyrosequenced the 16S rDNA V3 region from duplicate coprolite samples recovered from three archaeological sites, each representing a different depositional environment: Hinds Cave (∼8000 years B.P.) in the southern United States, Caserones (1600 years B.P.) in northern Chile, and Rio Zape in northern Mexico (1400 years B.P.). Clustering algorithms grouped samples from the same site. Phyletic representation was more similar within sites than between them. A Bayesian approach to source-tracking was used to compare the coprolite data to published data from known sources that include, soil, compost, human gut from rural African children, human gut, oral and skin from US cosmopolitan adults and non-human primate gut. The data from the Hinds Cave samples largely represented unknown sources. The Caserones samples, retrieved directly from natural mummies, matched compost in high proportion. A substantial and robust proportion of Rio Zape data was predicted to match the gut microbiome found in traditional rural communities, with more minor matches to other sources. One of the Rio Zape samples had taxonomic representation consistent with a child. To provide an idealized scenario for sample preservation, we also applied source tracking to previously published data for Ötzi the Iceman and a soldier frozen for 93 years on a glacier. Overall these studies reveal that human microbiome data has been preserved in some coprolites, and these preserved human microbiomes match more closely to those from the rural communities than to those from cosmopolitan communities. These results suggest that the modern cosmopolitan lifestyle resulted in a dramatic change to the human gut microbiome.
PLoS ONE 01/2012; 7(12):e51146. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.
[Show abstract][Hide abstract] ABSTRACT: Viruses are most frequently discovered because they cause disease in organisms of importance to humans. To expand knowledge of plant-associated viruses beyond these narrow constraints, non-cultivated plants of the Tallgrass Prairie Preserve, Osage County, Oklahoma, USA were systematically surveyed for evidence of the presence of viruses. This report discusses viruses of the family Tombusviridae putatively identified by the survey. Evidence of two carmoviruses, a tombusvirus, a panicovirus and an unclassifiable tombusvirid was found. The complete genome sequence was obtained for putative TGP carmovirus 1 from the legume Lespedeza procumbens, and the virus was detected in several other plant species including the fern Pellaea atropurpurea. Phylogenetic analysis of the sequence and partial sequence of a related virus supported strongly the placement of these viruses in the genus Carmovirus. Polymorphisms in the sequences suggested existence of two populations of TGP carmovirus 1 in the study area and year-to-year variations in infection by TGP carmovirus 3.
Virus Research 07/2011; 160(1-2):256-63. · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although Manduca sexta has significantly contributed to our knowledge on a variety of insect physiological processes, the lack of its genome sequence hampers the large-scale gene discovery, transcript profiling, and proteomic analysis in this biochemical model species. Here we report our implementation of the RNA-Seq cDNA sequencing approach based on massively parallel pyrosequencing, which allows us to categorize transcripts based on their relative abundances and to discover process- or tissue-specifically regulated genes simultaneously. We obtained 1,821,652 reads with an average length of 289 bp per read from fat body and hemocytes of naïve and microbe-injected M. sexta larvae. After almost all (92.1%) of these reads were assembled into 19,020 contigs, we identified 528 contigs whose relative abundances increased at least 5- and 8-fold in fat body and hemocytes, respectively, after the microbial challenge. Polypeptides encoded by these contigs include pathogen recognition receptors, extracellular and intracellular signal mediators and regulators, antimicrobial peptides, and proteins with no known sequence but likely participating in defense in novel ways. We also found 250 and 161 contigs that were preferentially expressed in fat body and hemocytes, respectively. Furthermore, we integrated data from our previous study and generated a sequence database to support future gene annotation and proteomic analysis in M. sexta. In summary, we have successfully established a combined approach for gene discovery and expression profiling in organisms lacking known genome sequences.
[Show abstract][Hide abstract] ABSTRACT: We previously isolated a spontaneous mutant of Escherichia coli K-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, that has colonization traits superior to the wild-type parent strain (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). This intestine-adapted strain (E. coli MG1655*) grew faster on several different carbon sources than the wild type and was nonmotile due to deletion of the flhD gene. We now report the results of several high-throughput genomic analysis approaches to further characterize E. coli MG1655*. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at the flhDC locus, that could explain the colonization advantage of E. coli MG1655*. Microarray analysis revealed modest yet significant induction of catabolic gene systems across the genome in both E. coli MG1655* and an isogenic flhD mutant constructed in the laboratory. Catabolome analysis with Biolog GN2 microplates revealed an enhanced ability of both E. coli MG1655* and the isogenic flhD mutant to oxidize a variety of carbon sources. The results show that intestine-adapted E. coli MG1655* is more fit than the wild type for intestinal colonization, because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, resulting in more efficient carbon source utilization and a higher intestinal population. Hence, mutations that enhance metabolic efficiency confer a colonization advantage.
Infection and immunity 03/2011; 79(6):2430-9. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.
Applied and Environmental Microbiology 11/2010; 76(22):7482-90. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The oral biofilm community consists of >800 microbial species, among which Streptococcus mutans is considered a primary pathogen for dental caries. The genomic island TnSmu2 of S. mutans comprises >2% of the genome. In this study, we demonstrate that TnSmu2 harbors a gene cluster encoding nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and accessory proteins and regulators involved in nonribosomal peptide (NRP) and polyketide (PK) biosynthesis. Interestingly, the sequences of these genes and their genomic organizations and locations are highly divergent among different S. mutans strains, yet each TnSmu2 region encodes NRPS/PKS and accessory proteins. Mutagenesis of the structural genes and putative regulatory genes in strains UA159, UA140, and MT4653 resulted in colonies that were devoid of their yellow pigmentation (for strains UA140 and MT4653). In addition, these mutant strains also displayed retarded growth under aerobic conditions and in the presence of H(2)O(2). High-performance liquid chromatography profiling of cell surface extracts identified unique peaks that were missing in the mutant strains, and partial characterization of the purified product from UA159 demonstrated that it is indeed a hybrid NRP/PK, as predicted. A genomic survey of 94 clinical S. mutans isolates suggests that the TnSmu2 gene cluster may be more prevalent than previously recognized.
Applied and Environmental Microbiology 09/2010; 76(17):5815-26. · 3.95 Impact Factor