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Asset Tag: University of Delaware

September 21, 2019  |  

Chromulinavorax destructans, a pathogenic TM6 bacterium with an unusual replication strategy targeting protist mitochondrion

Most of the diversity of microbial life is not available in culture, and as such we lack even a fundamental understanding of the biological diversity of several branches on the tree of life. One branch that is highly underrepresented is the candidate phylum TM6, also known as the Dependentiae. Their biology is known only from reduced genomes recovered from metagenomes around the world and two isolates infecting amoebae, all suggest that they live highly host-associated lifestyles as parasites or symbionts. Chromulinavorax destructans is an isolate from the TM6/Dependentiae that infects and lyses the abundant heterotrophic flagellate, Spumella elongata. Chromulinavorax destructans is characterized by a high degree of reduction and specialization for infection, so much so it was discovered in a screen for giant viruses. Its 1.2 Mb genome shows no metabolic potential and C. destructans instead relies on extensive transporter system to import nutrients, and even energy in the form of ATP from the host. Accordingly, it replicates in a viral-like fashion, while extensively reorganizing and expanding the host mitochondrion. 44% of proteins contain signal sequences for secretion, which includes many proteins of unknown function as well as 98 copies of ankyrin-repeat domain proteins, known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus.

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September 21, 2019  |  

From the inside out: An epibiotic Bdellovibrio predator with an expanded genomic complement

Bdellovibrio and like organisms are abundant environmental predators of prokaryotes that show a diversity of predation strategies, ranging from intra-periplasmic to epibiotic predation. The novel epibiotic predator Bdellovibrio qaytius was isolated from a eutrophic freshwater pond in British Columbia, where it was a continual part of the microbial community. Bdellovibrio qaytius was found to preferentially prey on the beta-proteobacterium Paraburkholderia fungorum. Despite its epibiotic replication strategy, B. qaytius encodes a complex genomic complement more similar to periplasmic predators as well as several biosynthesis pathways not previously found in epibiotic predators. Bdellovibrio qaytius is representative of a widely distributed basal cluster within the genus Bdellovibrio, suggesting that epibiotic predation might be a common predation type in nature and ancestral to the genus.

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July 19, 2019  |  

Conformation dependent epitopes recognized by prion protein antibodies probed using mutational scanning and deep sequencing.

Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrP(C), into a disease-associated conformation, PrP(Sc), which may be distinguished from one another using conformation specific antibodies. We used mutational scanning by cell-surface display to screen 1,341 PrP single point mutants for attenuated interaction with four anti-PrP antibodies, including several with conformational specificity. Single molecule real time gene sequencing was used to quantify enrichment of mutants, returning on average 26,000 high quality full-length reads for each screened population. Relative enrichment of mutants correlated to the magnitude of the change in binding affinity. Mutations that diminished binding of the antibody ICSM18 represented the core of contact residues in the published crystal structure of its complex. A similarly located binding site was identified for D18, comprising discontinuous residues in helix 1 of PrP, brought into close proximity to one another only when the alpha helix is intact. The specificity of these antibodies for the normal form of PrP likely arises from loss of this conformational feature after conversion to the disease-associated form. Intriguingly, 6H4 binding was found to depend on interaction with the same residues, among others, suggesting that its ability to recognize both forms of PrP depends on a structural rearrangement of the antigen. The application of mutational scanning and deep sequencing provides residue-level resolution of positions in the protein-protein interaction interface that are critical for binding, as well as a quantitative measure of the impact of mutations on binding affinity. Copyright © 2014. Published by Elsevier Ltd.

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July 7, 2019  |  

Complete genome sequence of Sphingobacterium sp. strain ML3W, isolated from wings of Myotis lucifugus infected with white nose syndrome.

Sphingobacterium sp. strain ML3W was isolated from the wing of a bat infected with white nose syndrome. We report the complete 5.33-Mb genome sequence of Sphingobacterium sp. strain ML3W, obtained using Pacific Biosciences technology. Being the second complete Sphingobacterium sequence, this will increase knowledge of the genus. Copyright © 2015 Smith et al.

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July 7, 2019  |  

Draft genome sequence of Erwinia tracheiphila, an economically important bacterial pathogen of cucurbits.

Erwinia tracheiphila is one of the most economically important pathogens of cucumbers, melons, squashes, pumpkins, and gourds in the northeastern and midwestern United States, yet its molecular pathology remains uninvestigated. Here, we report the first draft genome sequence of an E. tracheiphila strain isolated from an infected wild gourd (Cucurbita pepo subsp. texana) plant. The genome assembly consists of 7 contigs and includes a putative plasmid and at least 20 phage and prophage elements. Copyright © 2015 Shapiro et al.

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July 7, 2019  |  

Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium.

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72°C, Geobacillus LC300 has a growth rate of 2.15h(-1) on glucose and 1.52h(-1) on xylose (doubling time less than 30min). The corresponding specific glucose and xylose utilization rates are 5.55g/g/h and 5.24g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio?s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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July 7, 2019  |  

CHOgenome.org 2.0: Genome resources and website updates.

Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019  |  

Complete genome sequence of Vibrio parahaemolyticus environmental strain UCM-V493.

Vibrio parahaemolyticus is the leading bacterial cause of seafood-related gastroenteritis in the world. Here, we report the complete genome sequence and annotation of an environmental strain of V. parahaemolyticus, UCM-V493, with the aim of understanding the differences between the clinical and environmental isolates of the bacteria. We also make some preliminary sequence comparisons with the clinical strain RIMD2210633.

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July 7, 2019  |  

Complete genome sequence of the highly transformable Pseudomonas stutzeri strain 28a24.

Here, we report the complete genome sequence for an isolate of Pseudomonas stutzeri that is highly competent for natural transformation. This sequence enables insights into the genetic basis of natural transformation rate variations and provides an additional data point for genomic comparisons across a ubiquitous and highly diverse bacterial species. Copyright © 2014 Smith et al.

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July 7, 2019  |  

Expansion of the genetic toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene disruption of the endogenous CpaAI restriction enzyme.

Clostridium pasteurianum is one of the most promising biofuel producers within the genus Clostridium owing to its unique metabolic ability to ferment glycerol into butanol. Although an efficient means is available for introducing foreign DNA to C. pasteurianum, major genetic tools, such as gene knockout, knockdown, or genome editing, are lacking, preventing metabolic engineering of C. pasteurianum.Here we present a methodology for performing chromosomal gene disruption in C. pasteurianum using the programmable lactococcus Ll.ltrB group II intron. Gene disruption was initially found to be impeded by inefficient electrotransformation of Escherichia coli-C. pasteurianum shuttle vectors, presumably due to host restriction. By assessing the ability of various vector deletion derivatives to electrotransform C. pasteurianum and probing the microorganism’s methylome using next-generation sequence data, we identified a new C. pasteurianum Type I restriction-methylation system, CpaAII, with a predicted recognition sequence of 5′-AAGNNNNNCTCC-3′ (N?=?A, C, G, or T). Following rescue of high-level electrotransformation via mutation of the sole CpaAII site within the shuttle vectors, we retargeted the intron to the cpaAIR gene encoding the CpaAI Type II restriction endonuclease (recognition site of 5′-CGCG-3′). Intron insertion was potentially hindered by low retrohoming efficiency, yet this limitation could be overcome by a procedure for enrichment of the intron insertion. The resulting ?cpaAIR mutant strain was efficiently electrotransformed with M.FnuDII-unmethylated plasmid DNA.The markerless and plasmidless ?cpaAIR mutant strain of C. pasteurianum developed in this study can serve as a general host strain for future genetic and metabolic manipulation. Further, the associated gene disruption protocol should not only serve as a guide for chromosomal gene inactivation studies involving mobile group II introns, but also prove invaluable for applying metabolic engineering strategies to C. pasteurianum.

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July 7, 2019  |  

Pseudomonas syringae CC1557: a highly virulent strain with an unusually small type III effector repertoire that includes a novel effector.

Both type III effector proteins and nonribosomal peptide toxins play important roles for Pseudomonas syringae pathogenicity in host plants, but whether and how these pathways interact to promote infection remains unclear. Genomic evidence from one clade of P. syringae suggests a tradeoff between the total number of type III effector proteins and presence of syringomycin, syringopeptin, and syringolin A toxins. Here, we report the complete genome sequence from P. syringae CC1557, which contains the lowest number of known type III effectors to date and has also acquired genes similar to sequences encoding syringomycin pathways from other strains. We demonstrate that this strain is pathogenic on Nicotiana benthamiana and that both the type III secretion system and a new type III effector, hopBJ1, contribute to pathogenicity. We further demonstrate that activity of HopBJ1 is dependent on residues structurally similar to the catalytic site of Escherichia coli CNF1 toxin. Taken together, our results provide additional support for a negative correlation between type III effector repertoires and the potential to produce syringomycin-like toxins while also highlighting how genomic synteny and bioinformatics can be used to identify and characterize novel virulence proteins.

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July 7, 2019  |  

Whole-genome sequence of Escherichia coli serotype O157:H7 strain PA20.

Escherichia coli serotype O157:H7 strain PA20 is a Pennsylvania Department of Health clinical isolate. It has been used to study biofilm formation in O157:H7 clinical isolates, where the high incidence of prophage insertions in the mlrA transcription factor disrupts traditional csgD biofilm regulation. Here, we report the complete PA20 genome sequence. Copyright © 2017 Uhlich et al.

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July 7, 2019  |  

Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity.

The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production. During the 2015 fall semester at Bowling Green State University, students isolated 77 soil-derived bacteria and genetically characterized strains using the 16S rRNA gene, identified strains exhibiting antagonistic activity, and performed an expanded SWI workflow using transposon mutagenesis to identify a biosynthetic gene cluster involved in toxigenic compound production. We identified one mutant with loss of antagonistic activity and through subsequent whole-genome sequencing and linker-mediated PCR identified a 24.9 kb biosynthetic gene locus likely involved in inhibitory activity in that mutant. Further assessment against human pathogens demonstrated the inhibition of Bacillus cereus, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus in the presence of this compound, thus supporting our molecular strategy as an effective research pipeline for SWI antibiotic discovery and genetic characterization.© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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July 7, 2019  |  

Complete genome sequence of Spiroplasma citri strain R8-A2T, causal agent of stubborn disease in Citrus species.

Spiroplasma citri causes stubborn disease in Citrus spp. and diseases in other plants. Here, we report the nucleotide sequence of the 1,599,709-bp circular chromosome and two plasmids of S. citri strain R8-A2(T) This information will facilitate analyses to understand spiroplasmal pathogenicity and evolutionary adaptations to lifestyles in plants and arthropod hosts. Copyright © 2017 Davis et al.

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