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Asset Tag: Plant and Animal Sciences,

July 7, 2019

Vibrio anguillarum is genetically and phenotypically unaffected by long-term continuous exposure to the antibacterial compound tropodithietic acid.

Minimizing the use of antibiotics in the food production chain is essential for limiting the development and spread of antibiotic-resistant bacteria. One alternative intervention strategy is the use of probiotic bacteria, and bacteria of the marine Roseobacter clade are capable of antagonizing fish-pathogenic vibrios in fish larvae and live feed cultures for fish larvae. The antibacterial compound tropodithietic acid (TDA), an antiporter that disrupts the proton motive force, is key in the antibacterial activity of several roseobacters. Introducing probiotics on a larger scale requires understanding of any potential side effects of long-term exposure of the pathogen to the probionts or any compounds they produce. Here we exposed the fish pathogen Vibrio anguillarum to TDA for several hundred generations in an adaptive evolution experiment. No tolerance or resistance arose during the 90 days of exposure, and whole-genome sequencing of TDA-exposed lineages and clones revealed few mutational changes, compared to lineages grown without TDA. Amino acid-changing mutations were found in two to six different genes per clone; however, no mutations appeared unique to the TDA-exposed lineages or clones. None of the virulence genes of V. anguillarum was affected, and infectivity assays using fish cell lines indicated that the TDA-exposed lineages and clones were less invasive than the wild-type strain. Thus, long-term TDA exposure does not appear to result in TDA resistance and the physiology of V. anguillarum appears unaffected, supporting the application of TDA-producing roseobacters as probiotics in aquaculture.It is important to limit the use of antibiotics in our food production, to reduce the risk of bacteria developing antibiotic resistance. We showed previously that marine bacteria of the Roseobacter clade can prevent or reduce bacterial diseases in fish larvae, acting as probiotics. Roseobacters produce the antimicrobial compound tropodithietic acid (TDA), and we were concerned regarding whether long-term exposure to this compound could induce resistance or affect the disease-causing ability of the fish pathogen. Therefore, we exposed the fish pathogen Vibrio anguillarum to increasing TDA concentrations over 3 months. We did not see the development of any resistance to TDA, and subsequent infection assays revealed that none of the TDA-exposed clones had increased virulence toward fish cells. Hence, this study supports the use of roseobacters as a non-risk-based disease control measure in aquaculture. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Chromosome and linear plasmid sequences of a 2015 human isolate of the tick-borne relapsing fever spirochete, Borrelia turicatae.

The sequences of the complete linear chromosome and 7 linear plasmids of the relapsing fever spirochete Borrelia turicatae are presented in this report. The 925,547 bp of chromosome and 380,211 bp of plasmid sequence were predicted to contain a total of 1,131 open reading frames, with an average G+C content of 29.7%. Copyright © 2016 Kingry et al.

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July 7, 2019

Recombination rate heterogeneity within Arabidopsis disease resistance genes.

Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

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July 7, 2019

Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

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July 7, 2019

Complete genome sequence of Bacillus velezensis CBMB205, a phosphate-solubilizing bacterium isolated from the rhizoplane of rice in the Republic of Korea.

Bacillus velezensis CBMB205 (= KACC 13105(T) = NCCB 100236(T)) was isolated from the rhizoplane of rice (Oryza sativa L. cv. O-dae). According to previous studies, this bacterium has several genes that can promote plant growth, such as the phosphorus-solubilizing protein-coding gene. Here, we present the first complete genome of B. velezensis CBMB205. Copyright © 2016 Hwangbo et al.

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July 7, 2019

Complete genome of Nitrosospira briensis C-128, an ammonia-oxidizing bacterium from agricultural soil.

Nitrosospira briensis C-128 is an ammonia-oxidizing bacterium isolated from an acid agricultural soil. N. briensis C-128 was sequenced with PacBio RS technologies at the DOE-Joint Genome Institute through their Community Science Program (2010). The high-quality finished genome contains one chromosome of 3.21 Mb and no plasmids. We identified 3073 gene models, 3018 of which are protein coding. The two-way average nucleotide identity between the chromosomes of Nitrosospira multiformis ATCC 25196 and Nitrosospira briensis C-128 was found to be 77.2 %. Multiple copies of modules encoding chemolithotrophic metabolism were identified in their genomic context. The gene inventory supports chemolithotrophic metabolism with implications for function in soil environments.

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July 7, 2019

Microsatellite length scoring by Single Molecule Real Time Sequencing – Effects of sequence structure and PCR regime.

Microsatellites are DNA sequences consisting of repeated, short (1-6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level.

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July 7, 2019

Draft genome sequence of Ustilago trichophora RK089, a promising malic acid producer.

The basidiomycetous smut fungus Ustilago trichophora RK089 produces malate from glycerol. De novo genome sequencing revealed a 20.7-Mbp genome (301 gap-closed contigs, 246 scaffolds). A comparison to the genome of Ustilago maydis 521 revealed all essential genes for malate production from glycerol contributing to metabolic engineering for improving malate production. Copyright © 2016 Zambanini et al.

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July 7, 2019

Structural basis for recombinatorial permissiveness in the generation of Anaplasma marginale Msp2 antigenic variants.

Sequential expression of outer membrane protein antigenic variants is an evolutionarily convergent mechanism used by bacterial pathogens to escape host immune clearance and establish persistent infection. Variants must be sufficiently structurally distinct to escape existing immune effectors yet retain core structural elements required for localization and function within the outer membrane. We examined this balance using Anaplasma marginale, which generates antigenic variants in the outer membrane protein Msp2 using gene conversion. The overwhelming majority of Msp2 variants expressed during long-term persistent infection are mosaics, derived by recombination of oligonucleotide segments from multiple alleles to form unique hypervariable regions (HVR). As a result, the mosaics are not under long-term selective pressure to encode a functional protein; consequently, we hypothesized that the Msp2 HVR is structurally permissive for mosaic expression. Using an integrated approach of predictive modeling with determination of native Msp2 protein structure and function, we demonstrate that structured elements, most notably ß-sheets, are significantly concentrated in the highly conserved N- and C-terminal domains. In contrast the HVR is overwhelmingly random coil with the structured a-helices and ß-sheets confined to the genomically defined “structural tethers” that separate the antigenically variable microdomains. This structure is supported by the surface exposure of the HVR microdomains and the slow diffusion type porin function in native Msp2. Importantly, the predominance of random coil provides plasticity for formation of functional HVR mosaics and realization of the full potential of segmental gene conversion to dramatically expand the variant repertoire. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

The genomic sequence of the oral pathobiont strain NI1060 reveals unique strategies for bacterial competition and pathogenicity.

Strain NI1060 is an oral bacterium responsible for periodontitis in a murine ligature-induced disease model. To better understand its pathogenicity, we have determined the complete sequence of its 2,553,982 bp genome. Although closely related to Pasteurella pneumotropica, a pneumonia-associated rodent commensal based on its 16S rRNA, the NI1060 genomic content suggests that they are different species thriving on different energy sources via alternative metabolic pathways. Genomic and phylogenetic analyses showed that strain NI1060 is distinct from the genera currently described in the family Pasteurellaceae, and is likely to represent a novel species. In addition, we found putative virulence genes involved in lipooligosaccharide synthesis, adhesins and bacteriotoxic proteins. These genes are potentially important for host adaption and for the induction of dysbiosis through bacterial competition and pathogenicity. Importantly, strain NI1060 strongly stimulates Nod1, an innate immune receptor, but is defective in two peptidoglycan recycling genes due to a frameshift mutation. The in-depth analysis of its genome thus provides critical insights for the development of NI1060 as a prime model system for infectious disease.

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