Strains MS455 and MS389, endophytic bacteria, were isolated from healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. The complete genomes of both strains were sequenced and identified as Burkholderia species Strain MS455 exhibits broad-spectrum antifungal activities against economically important pathogens, including Aspergillus flavus. Random and site-specific mutations were employed in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA-seq analysis demonstrated ORF1, a homolog to the ambR1 LuxR-type regulatory gene, not only regulates occidiofungin biosynthesis in MS455, but also involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate and corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Strain MS389 shows significant antifungal and antibacterial activities as well. Mutagenesis study identified that the TatC gene, an important unit of twin-arginine translocase (Tat) secretion system, and the LysR-type transcriptional regulatory gene were essential for the antifungal activity of strain MS389. RNA-seq analysis implied that the pyrrolnitrin biosynthesis gene cluster and an uncharacterized NRPS / PKS gene cluster were involved in antifungal activity. By comparing several endophytic bacteria of Burkholderia, including MS455 and MS389, to pathogenic Burkholderia species, endophytic bacteria were observed to harbor multiple antimicrobial biosynthesis genes but lack certain pathogenic or virulence genes. The potential endophytic behavior related genes and characteristics related to antibiotic resistance, secretion system, and CRISPR-Cas profiles were well established. The research findings on strains MS455 and MS389 have provided important genetic clues for understanding their molecular mechanism of antimicrobial activities and exhibited their potential possibility as biocontrol agents.

付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(理学) / 甲第24471号 / 理博第4970号 / 新制||理||1709(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 伊谷 原一, 教授 平田 聡 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Great efforts have been devoted to decipher the sequence composition of the genomes and transcriptomes of diverse organisms. Continuing advances in high-throughput sequencing technologies have led to a decline in associated costs, facilitating a rapid increase in the amount of available genetic data. In particular genome studies have undergone a fundamental paradigm shift where genome projects are no longer limited by sequencing costs, but rather by computational problems associated with assembly. There is an urgent demand for more efficient and more accurate methods. Most recently, “hybrid” methods that integrate short- and long-read data have been devised to address this need. LazyB is a new, low-cost hybrid genome assembler. It starts from a bipartite overlap graph between long reads and restrictively filtered short-read unitigs. This graph is translated into a long-read overlap graph. By design, unitigs are both unique and almost free of assembly errors. As a consequence, only few spurious overlaps are introduced into the graph. Instead of the more conventional approach of removing tips, bubbles, and other local features, LazyB extracts subgraphs whose global properties approach a disjoint union of paths in multiple steps, utilizing properties of proper interval graphs. A prototype implementation of LazyB, entirely written in Python, not only yields significantly more accurate assemblies of the yeast, fruit fly, and human genomes compared to state-of-the-art pipelines, but also requires much less computational effort. An optimized C++ implementation dubbed MuCHSALSA further significantly reduces resource demands. Advances in RNA-seq have facilitated tremendous insights into the role of both coding and non-coding transcripts. Yet, the complete and accurate annotation of the transciptomes of even model organisms has remained elusive. RNA-seq produces reads significantly shorter than the average distance between related splice events and presents high noise levels and other biases The computational reconstruction remains a critical bottleneck. Ryūtō implements an extension of common splice graphs facilitating the integration of reads spanning multiple splice sites and paired-end reads bridging distant transcript parts. The decomposition of read coverage patterns is modeled as a minimum-cost flow problem. Using phasing information from multi-splice and paired-end reads, nodes with uncertain connections are decomposed step-wise via Linear Programming. Ryūtōs performance compares favorably with state-of-the-art methods on both simulated and real-life datasets. Despite ongoing research and our own contributions, progress on traditional single sample assembly has brought no major breakthrough. Multi-sample RNA-Seq experiments provide more information which, however, is challenging to utilize due to the large amount of accumulating errors. An extension to Ryūtō enables the reconstruction of consensus transcriptomes from multiple RNA-seq data sets, incorporating consensus calling at low level features. Benchmarks show stable improvements already at 3 replicates. Ryūtō outperforms competing approaches, providing a better and user-adjustable sensitivity-precision trade-off. Ryūtō consistently improves assembly on replicates, demonstrable also when mixing conditions or time series and for differential expression analysis. Ryūtōs approach towards guided assembly is equally unique. It allows users to adjust results based on the quality of the guide, even for multi-sample assembly.:1 Preface 1.1 Assembly: A vast and fast evolving field 1.2 Structure of this Work 1.3 Available 2 Introduction 2.1 Mathematical Background 2.2 High-Throughput Sequencing 2.3 Assembly 2.4 Transcriptome Expression 3 From LazyB to MuCHSALSA - Fast and Cheap Genome Assembly 3.1 Background 3.2 Strategy 3.3 Data preprocessing 3.4 Processing of the overlap graph 3.5 Post Processing of the Path Decomposition 3.6 Benchmarking 3.7 MuCHSALSA – Moving towards the future 4 Ryūtō - Versatile, Fast, and Effective Transcript Assembly 4.1 Background 4.2 Strategy 4.3 The Ryūtō core algorithm 4.4 Improved Multi-sample transcript assembly with Ryūtō 5 Conclusion & Future Work 5.1 Discussion and Outlook 5.2 Summary and Conclusion

In early 2020 the World Health Organization (WHO) declared a COVID-19 pandemic outbreak. As of March 2023, the Kingdom of Saudi Arabia has reported over 829 thousand cases and over 9 thousand deaths. Due to the public health emergency, the wild-type SARS-CoV-2 genome was fully sequenced early on the pandemic course. Afterward, different viral variants emerged around the world. The variant prevalence fluctuates over time, with some strains presenting increased transmissibility, changes in disease severity, and decreased efficacy of containment measures. In this respect, genomic surveillance and timely data sharing to public repositories represent a tool to track and detect emerging changes that require addressing with public health measurements. However, the Kingdom's genome contribution to the GISAID database represents merely 0.27% of the reported cases. To address this gap, we incorporated whole genome sequencing and the available metadata, to track the circulating viral lineages and identify the variants of concern (VOC) and variants of interest (VOI) over 14 months, from December 2021 to January 2023. We sequenced 581 genomes using the MinION MK1C platform of Oxford Nanopore Technologies (ONT) and assigned the viral lineages, the data primarily derived from COVID-19-positive patients from the city of Jeddah. The VOC Omicron was the principal circulating variant in the country (~99%), which aligns with the global trend. Our report included the sublineages BA.2.75, BQ.1, CH.1.1, XBB, and XBB.1.5, which are under WHO monitoring. We also reported the VOC Delta and the recombinant strains XF, XZ, and XPB. Finally, to validate the use of ONT for genomic surveillance, we compared the data quality of 93 samples sequenced in both ONT MinION and Illumina NaovaSeq platforms. There was 90% correspondence in the Nextstrain clade assignment (84/93) and 78% correspondence in the Pangolin sublineage assignment (73/93). MinION offered a shorter turnaround time, while Illumina produced a consistently higher breadth of genome coverage. In conclusion, the continued viral evolution patterns are reflected in the Kingdom's variant prevalence, where only Omicron circulated by January 2023. Likewise, current Variants Under Monitoring (VUM) were already identified in KSA, highlighting the need for increased genomic surveillance.

Francisella tularensis is the etiological agent of tularemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated as a potential biothreat agent. Currently there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica, and novicida. In addition, genomic studies have further subdivided type A tularensis into two subclassifications, type A.I and type A.II. These two subclassifications differ in geographic distribution with type A.I appearing mainly in the Eastern United States and type A.II appearing mainly in the Western United States. Because of differences of virulence among the subspecies, it is important to be able to quickly identify each of the subspecies rapidly and accurately. This work describes the development of a multiplex real-time polymerase chain reaction (PCR) assay which was shown to be ~98% successful at identifying the known subspecies of F. tularensis. Furthermore, F. tularensis is thought be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. Eleven arbitrarily chosen virulence genes were screened for positive selection along with 10 arbitrarily chosen housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution, as evidenced by several of its virulence genes which are undergoing strong, positive selection.

Yes / Aeromonas hydrophila has emerged worldwide as a human pathogen. Here, we report the draft whole-genome sequence of a freshwater isolate from Malaysia, A. hydrophila strain M062, and its N-acylhomoserine lactone genes are also reported here. / University of Malaya via High-Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01, no. A-000001- 50001), and aUM-MOHEHIR grant (UM C/625/1/HIR/MOHE/CHAN/ 14/1, no. H-50001-A000027)

Yes / Trypanosomes cause a variety of diseases in man and domestic animals in Africa, Latin America, and Asia. In the Trypanozoon subgenus, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis, whereas Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum are responsible for nagana, surra, and dourine in domestic animals, respectively. The genetic relationships between T. evansi and T. equiperdum and other Trypanozoon species remain unclear because the majority of phylogenetic analyses has been based on only a few genes. In this study, we have conducted a phylogenetic analysis based on genome-wide SNP analysis comprising 56 genomes from the Trypanozoon subgenus. Our data reveal that T. equiperdum has emerged at least once in Eastern Africa and T. evansi at two independent occasions in Western Africa. The genomes within the T. equiperdum and T. evansi monophyletic clusters show extremely little variation, probably due to the clonal spread linked to the independence from tsetse flies for their transmission. / Funding was received from the Research Foundation Flanders (FWO, grants 1501413N and 1101614N) and the European DG Health and Food Safety (SANTE). We thank the Center of Medical Genetics at the University of Antwerp for hosting the NGS facility.

Yes / Background: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. Methods: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005–2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. Findings: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. Interpretation: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting. / ERC grant [INTERRUPTB; no. 311725] to BdJ, FG and CJM; an ERC grant to TS [PhyPD; no. 335529]; an FWO PhD fellowship to PM [grant number 1141217N]; the Leibniz Science Campus EvolLUNG for MM and SN; the German Centre for Infection Research (DZIF) for TAK, MM, CU, PB and SN; a SNF SystemsX grant (TBX) to JP and TS and a Marie Heim-Vögtlin fellowship granted to DK by the Swiss National Science Foundation. The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation - Flanders (FWO) and the Flemish Government – department EWI.

Yes / We investigated whether companion drug resistance was associated with adverse outcome of the shorter MDR-TB regimen in Bangladesh, after adjusting for fluoroquinolone resistance. MDR/RR-TB patients registered for treatment with a standardized gatifloxacin-based shorter MDR-TB regimen were selected for the study. Drug resistance was determined using the proportion method, gatifloxacin and isoniazid minimum inhibitory concentration testing for selected isolates, and whole genome sequencing. Low-level and high-level fluoroquinolone resistance were the most important predictors of adverse outcomes, with pyrazinamide resistance having a significant yet lower impact. In patients with fluoroquinolone-/second-line injectable-susceptible TB, non-eligibility to the shorter MDR-TB regimen (initial resistance to either pyrazinamide, ethionamide, or ethambutol) was not associated with adverse outcome (aOR 1.01; 95%CI 0.4-2.8). Kanamycin resistance was uncommon (1.3%). Increasing levels of resistance to isoniazid predicted treatment failure, also in a subgroup of patients with high-level fluoroquinolone-resistant TB. Our results suggest that resistance to companion drugs of the shorter MDR-TB regimen, except kanamycin resistance, is of no clinical importance as long as fluoroquinolone susceptibility is preserved. Hence, contrary to current WHO guidelines, exclusions to the standard regimen are justified only in the case of fluoroquinolone, and possibly kanamycin resistance. / Damien Foundation Belgium for its financial and logistic support to run the project including its research activities. European Research Council (Starting Grant INTERRUPTB 311725).

yes / BACKGROUND: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis allows rapid, accurate inferences about the sources, location and timing of transmission. However, in an era of heightened concern for personal privacy and science distrust, such inferences could result in unintended harm and undermine the public´s trust. METHODS: We held interdisciplinary stakeholder discussions and performed ethical analyses of real-world illustrative cases to identify principles that optimise benefit and mitigate harm of M. tuberculosis WGS-driven TB source investigations.RESULTS: The speed and precision with which real-time WGS can be used to associate M. tuberculosis strains with sensitive information has raised important concerns. While detailed understanding of transmission events could mitigate harm to vulnerable patients and communities when otherwise unfairly blamed for TB outbreaks, the precision of WGS can also identify transmission events resulting in social blame, fear, discrimination, individual or location stigma, and the use of defaming language by the public, politicians and scientists. Public health programmes should balance the need to safeguard privacy with public health goals, transparency and individual rights, including the right to know who infects whom or where.CONCLUSIONS: Ethical challenges raised by real-time WGS-driven TB source investigation requires public health authorities to move beyond their current legal mandate and embrace transparency, privacy and community engagement.