Biology subjects

Weisberg, A. J.

Publications and source records attributed to Weisberg, A. J..

13 recordsLinked to original sources

bioRxiv2026

Chromosome-scale nuclear and mitochondrial genome assemblies reveal extreme genome compartmentalization in the hazelnut pathogen Anisogramma anomala

Anisogramma anomala is an obligate biotrophic ascomycete and the causal agent of Eastern filbert blight, the most economically important disease of hazelnut in the United States. Here, we generated a chromosome-scale reference genome for A. anomala isolate OR-Ennis-d and characterized its nuclear and mitochondrial genome architecture. The 344-Mb nuclear genome was resolved into 9 predicted chromosomes and is repeat-rich, with transposable elements comprising 88.8% of the assembly. Although transposable elements are abundant, their divergence landscape and low retention of conserved protein domains support a history of past extensive transposon proliferation followed by widespread degeneration. Repeat accumulation was highly uneven and generated pronounced genome compartmentalization. Approximately half of predicted genes occur in compact, TE-poor regions with short flanking intergenic regions that are enriched for conserved BUSCO orthologs. In contrast, genes in TE-rich, gene-sparse regions with long flanking intergenic regions are enriched for carbohydrate-active enzymes, secreted proteins, and candidate secreted effector proteins, consistent with a two-speed-like genome architecture. The 462-kb mitochondrial genome is among the largest reported for ascomycetes and contains 117 introns, including an intron adjacent to cob codon 143 that may prevent acquisition of the p.G143A substitution associated with resistance to quinone outside inhibitor fungicides. Finally, we identified mitochondrial DNA insertions at 20 nuclear loci totaling at least 616.9 kb, an unusually extensive amount for a fungal genome. This chromosome-scale genome resource reveals the extreme nuclear and mitochondrial genome architecture in A. anomala and provides a foundation for investigating genome evolution and adaptation to hazelnut resistance.

genomics↗

bioRxiv2026

Improved genome assemblies of plant-associated Streptomyces spp. as a resource for understanding plant pathogenicity in the genus

Common scab disease on potato is caused by members of more than 10 pathogenic Streptomyces species. Genome-enabled methods are being increasingly deployed to characterize Streptomyces that cause common scab disease of potato and other tuber and root crops. However, the study of phytopathogenic Streptomyces is constrained by the limited availability of high-quality genome sequences. Here we report improvements to the quality and completeness of genome assemblies for 12 pathogenic type strains of Streptomyces and six closely related non-pathogenic type strains. These assemblies have an average N50 of 7.4 Mbp and with BUSCO scores all greater than 98.5%. Analyses showed that the genomes of phytopathogenic Streptomyces are consistently among the largest Streptomyces genomes sequenced and, relative to those of non-pathogenic strains, are more enriched in genes involved in carbohydrate and amino acid metabolism. Plasmids were not consistently detected across assemblies, suggesting that they are not conserved across species and are not necessary for pathogenicity. Furthermore, comparisons of genome assemblies among both closely and distantly related strains revealed multiple rearrangements within linear chromosomes and reduced synteny near telomeric regions. These improved genome assemblies, many of which correspond to type strains, provide valuable resources for advancing our understanding of the pathogenicity in the genus.

genomics↗

bioRxiv2025

PathogenSurveillance: an automated pipeline for population genomic analyses and pathogen identification

Whole genome sequencing (WGS) offers a comprehensive, organism-agnostic method that effectively meets the need for efficient, reliable, and standardized responses to emerging threats from pathogens and pests. Here, we present PathogenSurveillance, an open-source and automated Nextflow pipeline for population genomic analyses of WGS data. It is designed with features tailored for biosurveillance and is suitable for in-field or point-of-care diagnostics. PathogenSurveillance is flexible, accommodating short- and long-read datasets and mixed samples of prokaryotes and/or eukaryotes. It automates all steps, including reference identification and retrieval from the NCBI Assembly database, and produces customizable interactive reports with summaries, phylogenetic trees, and minimum spanning networks that enable species and subspecies level identification. It also outputs quality control metrics and carefully names and organizes output files to facilitate downstream analyses. The pipeline runs on any Linux-based system and minimizes the need for advanced computational expertise. Source code is available on GitHub under the open-source MIT license. The pipeline expands the toolkit for real-time biosurveillance, enabling rapid detection and monitoring of pathogens and pests for rapid response to novel variants.

bioinformatics↗

bioRxiv2025

Linear dicentric chromosomes in bacterial natural isolates reveal common constraints for replicon fusion

Multipartite bacterial genome organization can confer advantages including coordinated gene regulation and faster genome replication but is challenging to maintain. Agrobacterium tumefaciens lineages often contain a circular chromosome (Ch1), a linear chromosome (Ch2), and multiple plasmids. We previously observed that in some stocks of the lab model strain C58, Ch1 and Ch2 were fused into a linear dicentric chromosome. Here we analyzed Agrobacterium natural isolates from the French Collection for Plant-Associated Bacteria (CFBP) and identified two strains with fused chromosomes. Chromosome conformation capture identified integration junctions that were different from the C58 fusion strain. Genome-wide DNA replication profiling showed both replication origins remain active. Transposon sequencing revealed that partitioning systems of both chromosome centromeres are essential. Importantly, the site-specific recombinases XerCD are required for the survival of the strains containing the fusion chromosome. Our findings show that replicon fusion occurs in natural environments and that balanced replication arm sizes and proper resolution systems enable the survival of such strains. ImportanceMost bacterial genomes are monopartite with a single, circular chromosome. But some species, like Agrobacterium tumefaciens, carry multiple chromosomes. Emergence of multipartite genomes is often related to adaptation to specific niches including pathogenesis or symbiosis. Multipartite genomes confer certain advantages, however, maintaining this complex structure can present significant challenges. We previously reported a laboratory-propagated lineage of A. tumefaciens strain C58 in which the circular and linear chromosomes fused to form a single dicentric chromosome. Here we discovered two environmental isolates of A. tumefaciens containing fused chromosomes derived from a different route, revealing the constraints and diversification of this process. We found that balanced replication arm sizes and the repurposing of multimer resolution systems enable the survival and stable maintenance of dicentric chromosomes. These findings help us better understand how multipartite genomes function across different bacterial species and the role of genomic plasticity in bacterial genetic diversification.

microbiology↗

bioRxiv2025

A comparison of short- and long-read whole genome sequencing for microbial pathogen epidemiology

Whole genome sequencing provides the highest resolution for characterizing pathogen evolution, epidemiology, and diagnostics. Genome assemblies contain information on the identity and potential phenotypes of a pathogen. Likewise, variant calling can inform on transmission patterns and evolutionary relationships. Recent improvements in Oxford Nanopore long-read sequencing have made its use attractive for genomic epidemiology. However, the accuracy and optimal strategy for analysis of Nanopore reads remains to be determined. We compared the use of Illumina short reads and Oxford Nanopore long reads for genome assembly and variant calling of phytopathogenic bacteria. We generated short- and long-read datasets for diverse phytopathogenic Agrobacterium strains. We then analyzed these data using multiple pipelines designed for either short or long reads and compared the results. We found that assemblies made from long reads were more complete than those made from short-read data and contained few sequence errors. Variant calling pipelines differed in their ability to accurately call variants and infer genotypes from long reads. Results suggest that computationally fragmenting long reads can improve the accuracy of variant calling in population-level studies. Using fragmented long reads, pipelines designed for short reads were more accurate at recovering genotypes than pipelines designed for long reads. Further, short- and long-read datasets can be analyzed together with the same pipelines. These findings show that Oxford Nanopore sequencing is accurate and can be sufficient for microbial pathogen genomics and epidemiology. Ultimately, this enhances the ability of researchers and clinicians to understand and mitigate the spread of pathogens. ImportanceGenome assembly and variant calling are important steps in microbial population studies and epidemiology. Most variant calling and genotyping pipelines are designed for Illumina short sequencing reads. Oxford Nanopore Technology long-read sequencing results in more complete genome assemblies but has historically been of lower quality. Here, we show that Nanopore long reads are now of sufficient quality for bacterial whole genome assembly and epidemiology. We benchmarked the accuracy of multiple variant-calling pipelines with short and long reads. Using an optimized variant calling approach, variant calls and genotypes inferred from long reads are as accurate as those inferred from short reads. Importantly, we found that gold-standard variant calling pipelines designed for short reads are also accurate with long reads when long reads are first fragmented into shorter sequences. This finding allows researchers to incorporate the advantages of Nanopore sequencing for genome assembly, while maintaining high accuracy for epidemiology and population analysis.

genomics↗

bioRxiv2024

Beav: A bacterial genome and mobile element annotation pipeline

Comprehensive and accurate genome annotation is crucial for inferring the predicted functions of an organism. Numerous tools exist to annotate genes, gene clusters, mobile genetic elements, and other diverse features. However, these tools and pipelines can be difficult to install and run, be specialized for a particular element or feature, or lack annotations for larger elements that provide important genomic context. Integrating results across analyses is also important for understanding gene function. To address these challenges, we present the Beav annotation pipeline. Beav is a command-line tool that automates the annotation of bacterial genome sequences, mobile genetic elements, molecular systems and gene clusters, key regulatory features, and other elements. Beav uses existing tools in addition to custom models, scripts, and databases to annotate diverse elements, systems, and sequence features. Custom databases for plant-associated microbes are incorporated to improve annotation of key virulence and symbiosis genes in agriculturally important pathogens and mutualists. Beav includes an optional Agrobacterium-specific pipeline that identifies and classifies oncogenic plasmids and annotates plasmid-specific features. Following the completion of all analyses, annotations are consolidated to produce a single comprehensive output. Finally, Beav generates publication-quality genome and plasmid maps. Beav is on Bioconda and is available for download at https://github.com/weisberglab/beav.

bioinformatics↗

bioRxiv2023

Transporter-mediated depletion of extracellular proline directly contributes to plant pattern-triggered immunity against a bacterial pathogen

Plants possess cell surface-localized immune receptors that detect pathogen-associated molecular patterns (PAMPs) and initiate defenses that provide effective resistance against microbial pathogens. Many PAMP-induced signaling pathways and cellular responses are known, yet how pattern-triggered immunity (PTI) limits pathogen growth in plants is poorly understood. Through a combined metabolomics and genetics approach, we discovered that plant-exuded proline is a virulence-inducing signal and nutrient for the bacterial pathogen Pseudomonas syringae, and that PAMP-induced depletion of proline from the extracellular spaces of Arabidopsis leaves directly contributes to PTI against P. syringae. We further show that PAMP-induced depletion of extracellular proline requires the amino acid transporter Lysine Histidine Transporter 1 (LHT1). This study demonstrates that depletion of a single extracellular metabolite is an effective component of plant innate immunity. Given the important role for amino acids as nutrients for microbial growth, their depletion at sites of infection may be a broadly effective means for defense against many pathogens.

plant biology↗

bioRxiv2023

Genetically refactored Agrobacterium-mediated transformation

Members of Agrobacterium are costly plant pathogens while also essential tools for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and its complex transcriptional regulation make identifying the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here we use a bottom-up synthetic approach to systematically refactor the tumor-inducing plasmid, wherein the majority of AMT machine components are encoded, into a minimal set of genes capable of plant and fungal transformation that is both controllable and orthogonal to its environment. We demonstrate that engineered vectors can be transferred to new heterologous bacteria, enabling them to transform plants. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, and may lead to the development of strains of bacteria more capable of transforming recalcitrant plant species of societal importance.

microbiology↗

bioRxiv2023

Evolutionary dynamics of proteinaceous MAMPs reveals intrabacterial antagonism of plant immune perception

Plants and animals detect biomolecules termed Microbe-Associated Molecular Patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multi-copy MAMPs on immune induction is unknown. Here we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple Cold Shock Proteins and 46% carry a non-immunogenic form. We uncovered a new mechanism for immune evasion, intrabacterial antagonism, where a non-immunogenic Cold Shock Protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation. Significance StatementPlants recognize pathogens as non-self using innate immune receptors. Receptors on the cell surface can recognize amino acid epitopes present in pathogen proteins. Despite many papers investigating receptor signaling, the vast majority use a single epitope. Here, we analyzed the natural variation across five different epitopes and experimentally characterized their perception in plants. We highlight the importance of analyzing all epitope copies within a pathogen genome. Through genetic and biochemical analyses, we revealed a mechanism for immune evasion, intrabacterial antagonism, where a non-immunogenic epitope blocks perception of immunogenic forms encoded in a single genome. These data can directly inform disease control strategies by enabling prediction of receptor utility and deployment for current and emerging pathogens.

microbiology↗

bioRxiv2021

Species boundaries in the Agrobacterium tumefaciens complex and multi-level modular evolution of their antibacterial type VI secretion system and tumor-inducing plasmids

BackgroundMany bacterial taxa are species complexes and uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized Agrobacterium tumefaciens, a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. ResultsWe utilized 35 genome assemblies to achieve a comprehensive and balanced sampling of A. tumefaciens. Our confident inference of gene content and core-genome phylogeny supported a quantitative guideline for delineating 12 species and allowed for robust investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. ConclusionsWe demonstrated that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. Our strategies for multi-level investigations at scales that range from whole-genomes to intragenic domains and phylogenetic depths of between- and within-species are applicable to other bacteria. Finally, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

genomics↗

bioRxiv2021

Dual adhesive unipolar polysaccharides synthesized by overlapping biosynthetic pathways in Agrobacterium tumefaciens

Agrobacterium tumefaciens, is a member of the Alphaproteobacteria that pathogenizes plants, and associates with biotic and abiotic surfaces via a single cellular pole. A. tumefaciens produces the unipolar polysaccharide (UPP) at the site of surface contact. UPP production is normally surface-contact inducible, but elevated levels of the second messenger cyclic diguanylate monophosphate (cdGMP) bypass this requirement. Multiple lines of evidence suggest that the UPP has a central polysaccharide component. Using an A. tumefaciens derivative with elevated cdGMP and mutationally disabled for other dispensable polysaccharides, a series of related genetic screens have identified a large number of genes involved in UPP biosynthesis, most of which are Wzx-Wzy-type polysaccharide biosynthetic components. Extensive analyses of UPP production in these mutants have revealed that the UPP is comprised of two genetically, chemically and spatially discrete forms of polysaccharide, and that each requires a specific Wzy-type polymerase. Other important biosynthetic, processing and regulatory functions for UPP production are also revealed, some of which are common to both polysaccharides, and a subset of which are specific to each species. Many of the UPP genes identified are conserved among diverse rhizobia, whereas others are more lineage specific. Plain language summaryBacteria attach to a wide variety of surfaces including host tissues, via externalized structures described as adhesins. We define a large set of genes involved in synthesis of a complex unipolar adhesin comprised of two distinct polysaccharides, that is required for surface attachment by the plant-associated pathogen Agrobacterium tumefaciens.

microbiology↗

bioRxiv2019

Re-evaluation of a Tn5::gacA mutant of Pseudomonas syringae pv. tomato DC3000 uncovers roles for uvrC and anmK in promoting virulence

Pseudomonas syringae is a taxon of plant pathogenic bacteria that can colonize and proliferate within the interior space of leaf tissue. This process requires P. syringae to rapidly upregulate the production of virulence factors including a type III secretion system (T3SS) that suppress host defenses. GacS/A is a two-component system that regulates virulence of many plant and animal pathogenic bacteria including P. syringae. We recently investigated the virulence defect of strain AC811, a Tn5::gacA mutant of P. syringae pv. tomato DC3000 that is less virulent on Arabidopsis. We discovered that decreased virulence of AC811 is not caused by loss of GacA function. Here, we report the molecular basis of the virulence defect of AC811. We show that AC811 possesses a nonsense mutation in anmK, a gene predicted to encode a 1,6-anhydromuramic acid kinase involved in cell wall recycling. Expression of a wild-type allele of anmK partially increased growth of AC811 in Arabidopsis leaves. In addition to the defective anmK allele, we also show that the Tn5 insertion in gacA exerts a polar effect on uvrC, a downstream gene encoding a regulator of DNA damage repair. Expression of the wild-type anmK allele together with increased expression of uvrC fully restored the virulence of AC811 during infection of Arabidopsis. These results demonstrate that defects in anmK and uvrC are together sufficient to account for the decreased virulence of AC811, and suggest caution is warranted in assigning phenotypes to GacA function based on insertional mutagenesis of the gacA-uvrC locus.

microbiology↗

bioRxiv2017

Morphological And Transcriptomic Evidence For Ammonium Induction Of Sexual Reproduction In Thalassiosira pseudonana And Other Centric Diatoms

The reproductive strategy of diatoms includes asexual and sexual phases, but in many species, including the model centric diatom Thalassiosira pseudonana, sexual reproduction has never been observed. Furthermore, the environmental factors that trigger sexual reproduction in diatoms are not understood. Although genome sequences of a few diatoms are available, little is known about the molecular basis for sexual reproduction. Here we show that ammonium reliably induces the key sexual morphologies, including oogonia, auxospores, and spermatogonia, in two strains of T. pseudonana, T. weissflogii, and Cyclotella cryptica. RNA sequencing revealed 1,274 genes whose expression patterns changed when T. pseudonana was induced into sexual reproduction by ammonium. Some of the induced genes are linked to meiosis or encode flagellar structures of heterokont and cryptophyte algae. The identification of ammonium as an environmental trigger suggests an unexpected link between diatom bloom dynamics and strategies for enhancing population genetic diversity.

microbiology↗