Biology subjects

Fiebig, A.

Publications and source records attributed to Fiebig, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A Caulobacter hub taxon in the soybean root endosphere has a distinctive biofilm and engages in riboflavin-mediated mutualism with Bradyrhizobium

While it is well established that communities of microbes colonize the surfaces of plants as epiphytes and the interior plant tissues as endophytes, their ecological roles and colonization mechanisms remain under characterized. Members of the genus Caulobacter have emerged as hub taxa in epiphytic and endophytic microbiomes of diverse plant hosts, but the factors that support their colonization and community interactions have not been defined. Here, we characterize Caulobacter sp. RL271, a non-canonical member of the genus recently identified as a hub taxon in the soybean root endosphere. We demonstrate that the surface attachment strategies and biofilm architecture of Caulobacter sp. RL271 are determined by a matrix of capsular polysaccharide and cellulose rather than a polar holdfast adhesin, a classical defining feature of the genus. Both polysaccharide components influence the kinetics of plant root colonization in the model plant Arabidopsis thaliana. We further demonstrate that the ecology of Caulobacter sp. RL271 is shaped by its nutritional dependence on exogenous riboflavin and by interactions with Bradyrhizobium diazoefficiens, the nitrogen-fixing symbiont of soybean roots. This work advances understanding of Caulobacter biology and establishes RL271 as a tractable model for dissecting the functional role of hub taxa in root endophyte communities.

microbiology↗

bioRxiv2026

Programmed chromosome elimination correlates with the overexpression of cohesin and additional B chromosome-encoded genes in Aegilops speltoides

Programmed chromosome elimination is a highly controlled developmental process in which specific chromosomes are selectively lost from defined cell types during development. Despite its broad occurrence across plants and animals, the molecular mechanisms driving the tissue-specific elimination of chromosomes remain largely unresolved. Here, we exploit the root-specific elimination of supernumerary B chromosomes in Aegilops speltoides as a tractable model to identify the genetic basis of programmed chromosome loss. A high-quality, chromosome-scale genome assembly was generated, assigning 398 Mb of sequence to the Ae. speltoides B chromosome. Transcriptome profiling across seven tissue types representing chromosome elimination-active, elimination-negative, and B chromosome nondisjunction conditions identified 3,262 genes consistently upregulated in elimination-associated tissues, including 1,035 B genes. Stepwise subtraction of genes expressed in post-elimination and B chromosome-retaining reference tissues, followed by intersection with genes expressed during B nondisjunction in anthers, identified a candidate gene set enriched for chromosome segregation functions. From this set, we prioritized SYN2-B, a B chromosome-encoded cohesin -kleisin subunit whose Arabidopsis thaliana ortholog AtSYN2 induces chromosome bridges and micronucleus formation when misregulated. CENH3-B, an -type centromeric histone variant identified through GO enrichment analysis of B genes expressed in elimination-associated tissues, was shown to be incorporated in centromeres of both A and B chromosomes by transient gene expression assays using protoplasts and 3D structured illumination microscopy. These findings support a model in which B chromosome-encoded perturbations of cohesin activity and centromere composition contribute to selective B chromosome nondisjunction and their elimination in root tissues. Moreover, the SYN2-B promoter is enriched for ethylene response factor-binding sites compared to its A-encoded paralog, suggesting that ethylene is implicated in the root identity pathway driving root-specific B chromosome elimination.

genetics↗

bioRxiv2026

BR-bodies link RNA homeostasis to stress tolerance and intracellular fitness in Brucella

Bacterial gene expression depends on the coordinated regulation of RNA synthesis, processing, and decay. The conserved RNA degradosome scaffold, Ribonuclease E (RNase E), assembles into phase-separated bacterial ribonucleoprotein bodies (BR-bodies) through its C-terminal intrinsically disordered region (IDR). This IDR also scaffolds recruitment of degradosome client proteins that carry out post-transcriptional gene regulation. Whether BR-bodies regulate virulence programs to support infection, however, is largely undefined. We show that RNase E of the intracellular pathogen Brucella ovis forms RNA-dependent condensates in vivo and phase-separates with RNA in vitro, with the IDR necessary and sufficient for BR-body assembly. Deleting the IDR (rne({Delta}IDR)) did not impair growth but sensitized Brucella to host-relevant oxidative and cell-envelope stressors. Transcriptome-wide profiling that simultaneously resolved mRNA decay and processing revealed that BR-bodies primarily accelerate mRNA turnover while stabilizing a distinct subset of transcripts. Notably, BR-bodies control the processing and levels of virB type IV secretion system (T4SS) mRNA and the turnover of its key activators, linking condensate-based RNA regulation to a core virulence pathway. Consistent with virB dysregulation, the rne({Delta}IDR) mutant was severely attenuated in mammalian macrophages. To test whether phase separation is sufficient for BR-body function, we replaced the B. ovis IDR with the highly divergent Caulobacter crescentus IDR. This chimera assembled BR-bodies but rescued fitness incompletely, fully restoring oxidative-stress resistance but not cell-envelope stress resistance or intracellular fitness. BR-bodies therefore link RNA metabolism to Brucella stress resistance and infection, and their full function requires both phase separation and additional native IDR-specific activities such as degradosome interactions. ImportanceBiomolecular condensates are non-membrane bound organelles that organize biological processes across all domains of life, but their role in bacterial infection is not well characterized. We show that Brucella, an intracellular bacterial pathogen and causative agent of the disease brucellosis, relies on biomolecular condensates called BR-bodies to control RNA stability and regulate gene expression. Without BR-bodies, Brucella is sensitized to host-relevant chemical stresses, cannot properly regulate essential infection machinery, and has severely diminished fitness within mammalian host cells. These results indicate that control of RNA levels by biomolecular condensates is required for Brucella survival within animal hosts, providing evidence that phase separation is a fundamental mechanism underlying Brucella adaptation to the hostile environment encountered during infection.

microbiology↗

bioRxiv2026

A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus burden at 3 h post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support early infection by B. abortus and B. ovis without impairing general phagocytosis. Gene set enrichment analysis further revealed positive regulators of mTORC1 signaling as key host factors; this result was validated through targeted disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Thus, the AKT-Ragulator-mTORC1 signaling axis contributes to the establishment of a permissive intracellular niche during early Brucella infection. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that support early Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.

microbiology↗

bioRxiv2026

Peripheral Nociceptor Activity and Placebo Hypoalgesia: A Proof-of-Concept Study

Placebo effects illustrate clearly how cognitive states can influence physiological processes. While central mechanisms of placebo hypoalgesia, i.e. reduce sensitivity to painful stimuli due to expectations, are well established, it remains unclear whether such expectations modulate also peripheral activity. In this proof-of-principle study, we combined a well-established placebo paradigm with microneurography to directly assess responses of single nerve fibers in awake humans. Six healthy participants (18 fibers) underwent each a placebo and a control condition in a randomized crossover design. Placebo hypoalgesia was induced using verbal suggestion and classical conditioning. Manipulation checks confirmed that participants expected hypoalgesic effects of a placebo gel after conditioning. At the peripheral level, data on the recovery cycle of the nerve fibers indicated changes in the excitability of sleeping nociceptors (CMi) in the placebo compared to control condition, namely with enhanced and longer-lasting early subnormality. While other microneurographic outcome measures showed no consistent placebo effects, rather strong time-dependent reductions in fiber responsiveness independent of the experimental conditions were observed. These findings provide preliminary evidence that modulation of peripheral nociceptor activity by cognitive states, specifically expectations, is possible. Such modulation of peripheral activity appears to be fiber-type specific. Time effects have been neglected in microneurographic protocols. The pronounced time effects found here highlight the need for optimized such protocols.

neuroscience↗

bioRxiv2025

An N4-like Caulobacter phage requires host smooth lipopolysaccharide biosynthesis for infection

Caulobacter species are Alphaproteobacteria that commonly inhabit plant-associated and aquatic microbial communities. Although Caulobacter is widespread and has long served as a model for the study of bacterial cell biology, our understanding of the diversity of viruses that infect Caulobacter species is limited. Here, we describe the discovery and characterization of Circe, a freshwater N4-like podophage belonging to the Schitoviridae family that infects C. crescentus. We isolated two variants, CirceC and CirceH, that differ by a single nucleotide resulting in an F91I substitution in Gp063, an uncharacterized protein found in diverse bacteriophages and bacteria. While both Circe variants adsorb to C. crescentus with similar efficiency, they produce morphologically distinct plaques and display different infection dynamics. Through forward genetic selection and genome-wide transposon fitness profiling, we identified C. crescentus genes involved in cell envelope assembly, membrane sphingolipid biosynthesis, and envelope polysaccharide biosynthesis that influence susceptibility to Circe infection. Loss-of-function mutations in a predicted nucleoside diphosphate sugar epimerase and multiple genes required for smooth lipopolysaccharide (S-LPS) biosynthesis and export conferred strong resistance to infection. These results support a model in which S-LPS functions as a receptor for phage Circe. Our study expands the known repertoire of Caulobacter phages and adds to a growing understanding of the role of envelope polysaccharides in bacterial infection by N4-family phages. IMPORTANCEViruses that infect bacteria and archaea, known as phages, shape microbial community structure and function. Phages initiate infection by binding to specific molecules on the surface of host cells. Yet for many microbes, the identities of infecting phages and their corresponding host receptors remain poorly defined. Caulobacter spp. are ecologically important bacteria that can produce a variety of protein surface structures, including pili, a flagellum, and a surface layer (S-layer), all of which have been identified as phage receptors in this genus. We discovered Caulobacter phage Circe and provide evidence that it relies on host smooth lipopolysaccharide to infect C. crescentus. This study broadens understanding of phage-host interactions in Caulobacter and establishes Circe as a new system to investigate the molecular mechanisms by which phages engage with bacterial cells.

microbiology↗

bioRxiv2025

A Hybrid Knowledge- and Data-driven Model for Automatic Assessment of Chemically Induced Spiking Patterns in C-fiber Microneurography

Analyzing temporal spike patterns in C-fibers recorded via microneurography is challenging due to the use of a single recording electrode, waveform variability, and high similarity of spike shapes across neurons, limiting the interpretation of sensory coding, such as pain and itch. We present a computational pipeline combining peak detection and supervised classification for spike sorting to improve the analysis of discharges, identified through activity-dependent conduction velocity changes. In the knowledge-driven step, we extract spike templates from electrically evoked spikes obtained during low-frequency stimulation and focus on the "best" template as the fiber of interest. Spike detection is further restricted to intervals showing activity-dependent latency shifts, substantially reducing the search space compared to unsupervised clustering. In the data-driven steps, we systematically evaluate three feature sets and machine learning models: One-class SVM, SVM, and XGBoost. For the evaluation, we created a specialized stimulation protocol, providing reliable ground truth labels for all electrically evoked spikes, allowing precise spike time-locking. Compared to Spike2 software, our approach achieved higher F1-scores and reduced false positives, indicating improved spike sorting. Although XGBoost achieved the highest median F1-scores, optimal performance was dependent on individual combinations of feature sets and models for each recording. In some recordings with many nerve fibers and a low signal-to-noise ratio, reliable sorting was not feasible. This highlights the necessity to determine sortability and optimal configurations for individual recordings. To illustrate the potential of our approach to sensory spike train analysis, we present a proof-of-concept application of the pipeline to chemically induced C-fiber activity. These findings represent an important step toward reliable analysis of activity associated with pain and itch signaling.

neuroscience↗

bioRxiv2025

The hiuABC Operon Mediates Xenosiderophore Utilization in Caulobacter crescentus

Caulobacter species are common residents of soil and aquatic ecosystems, where bioavailable iron is often extremely limited. Like other diderm bacteria, C. crescentus can acquire Fe(III) via outer-membrane TonB-dependent transporters (TBDTs) that recognize and import ferric siderophore complexes. Although C. crescentus is not known to synthesize siderophores, it encodes multiple TBDTs that are transcriptionally regulated by the ferric uptake repressor (Fur), suggesting it acquires iron by scavenging xenosiderophores produced by neighboring microbes. To identify C. crescentus genes required for xenosiderophore utilization, we developed a barcoded transposon screen using ferrioxamine B (FXB), a hydroxamate-family siderophore produced by soil actinomycetes, as a model substrate. This screen identified hiuABC, a conserved, Fur-regulated operon that supports FXB-dependent iron acquisition. We provide evidence that hiuA encodes the primary TBDT responsible for uptake of ferrioxamines and ferrichrome (FC), structurally distinct members of the hydroxamate siderophore family. hiuB encodes a PepSY-domain protein with structural similarity to Pseudomonas aeruginosa FoxB, a known periplasmic ferri-siderophore reductase. hiuC encodes a small, hypothetical membrane protein predicted to form a functional complex with HiuB in the inner membrane. Both hiuB and hiuC are required for utilization of FXB and ferrioxamine E (FXE), indicating a shared role in iron acquisition from ferrioxamines. Surprisingly, utilization of FC as an iron source required hiuB but not hiuC, suggesting a substrate-specific role for HiuC in ferri-siderophore processing. We conclude that the conserved hiuABC operon encodes a set of proteins that enable bacteria to acquire iron from structurally diverse hydroxamate-family siderophores. ImportanceIron is often a limiting nutrient due to its poor solubility in the presence of oxygen. To overcome this, some microbes produce specialized molecules known as siderophores, which tightly bind and solubilize iron, facilitating its uptake into the cell. Caulobacter species are common in freshwater, marine, and soil environments, and there is emerging evidence they play important roles in plant-associated microbial communities. Here we report the discovery of a three-gene system that allows C. crescentus to acquire iron from a set of siderophores produced by select soil bacteria and fungi. We define functional roles for each protein component of this system, which informs a mechanism by which Caulobacter can pirate iron-scavenging molecules produced by its neighbors.

microbiology↗

bioRxiv2025

Non-redundant cardiolipin synthases support membrane integrity and stress resilience in Bacteroides fragilis

Gut-resident bacteria must tolerate diverse membrane-disrupting agents, including bile acids, to maintain colonization. Cardiolipin is an anionic phospholipid that supports membrane integrity and stress resilience in many bacteria. However, cardiolipin synthases remain poorly characterized in the Bacteroidota, a dominant phylum of the human gut microbiota. The prevalent gut commensal Bacteroides fragilis encodes two predicted cardiolipin synthases, ClsA and ClsB. We previously identified both cls genes as bile-acid fitness factors in B. fragilis P207, but the individual contributions of ClsA and ClsB to cell physiology had remained undefined. Here we combine targeted gene deletion with high-resolution lipidomics, metabolomics, and elemental mass spectrometry to show that the two enzymes have non-redundant functions in the cell. Cardiolipin is a minor lipid in the B. fragilis membrane, and both Cls enzymes contribute to its production. clsA and clsB differ in growth-phase expression, in their effects on cell morphology, and in their associated cardiolipin species. Loss of each enzyme also produces distinct changes in fitness under several membrane-perturbing stresses and in the broader cellular metabolome, including compound classes with documented bioactivity in mammalian hosts. In contrast to the acute ion-gradient disruption caused by the secondary bile acid deoxycholate, deletion of both synthases did not measurably alter steady-state intracellular ion levels under standard growth conditions, indicating that cardiolipin loss does not perturb basal ion homeostasis under these conditions. Together, these results define non-redundant roles for two cardiolipin synthases in a common member of the human gut microbiota. ImportanceInflammatory bowel diseases affect millions of people worldwide. The gut bacterium Bacteroides fragilis is a normal member of the human intestinal microbiota that can also bloom to high abundance in inflamed guts. To survive within the gut, B. fragilis must maintain the integrity and function of its cell membrane. In this study, we characterize the functional role of two B. fragilis genes that contribute to the synthesis of the membrane lipid, cardiolipin. We find that the two cardiolipin synthase genes are not functionally interchangeable; each impacts the cells lipid pool in distinct ways and contributes differently to how B. fragilis responds to membrane stress. Our work provides insight into how a common gut bacterium adapts to conditions encountered in the intestine, and improves understanding of membrane biology in this important group of gut microbes.

microbiology↗

bioRxiv2025

Reversion of a RND transporter pseudogene uncovers latent stress resistance in Brucella ovis

Small-molecule screens can advance therapeutic discovery while yielding new insights into pathogen biology. Through a luminescence-based screen, we identified clinically approved dihydropyridines that reduced the fitness of the intracellular pathogen Brucella ovis within mammalian phagocytes. Given the established role of dihydropyridines as inhibitors of mammalian L-type calcium channels and our observation that drug treatment perturbed calcium and manganese levels in host phagocytes, we initially hypothesized a host-directed mechanism of action. However, dose-response assays in axenic medium revealed that these drugs can directly inhibit B. ovis growth. To investigate the genetic basis of B. ovis susceptibility to dihydropyridine treatment, we selected for mutants capable of growing in the presence of cilnidipine. Cilnidipine-resistant isolates carried single-base deletions in the bepE pseudogene that restored an open reading frame encoding an RND-family transporter subunit. B. ovis is an ovine venereal pathogen that has experienced significant pseudogenization in its recent evolutionary history. Frameshift mutations that restored bepE function in B. ovis increased its resistance not only to dihydropyridines but also to a broad range of membrane-disrupting agents, including bile acid. Conversely, deleting bepE in Brucella abortus, a related zoonotic species that retains an intact version of the gene, increased its sensitivity to bile acid in vitro and to cilnidipine in the intracellular niche. We conclude that bepE is a key determinant of chemical stress resistance in Brucella spp., and that its pseudogenization in B. ovis contributes to the documented hypersensitivity of this host-restricted lineage to chemical stressors. Author SummaryBrucella species are intracellular bacterial pathogens that infect diverse wildlife and livestock and can also cause disease in humans. We developed a high-throughput assay to identify compounds that reduce the intracellular fitness of Brucella ovis, a globally distributed pathogen of sheep. This screen identified two dihydropyridine-class hypertension drugs as inhibitors of B. ovis fitness within mammalian cells. While dihydropyridines are established inhibitors of calcium channels in mammals, our results revealed that they also directly inhibited B. ovis growth. The B. ovis genome contains over 300 pseudogenes, and we discovered that resistance to dihydropyridines and other chemical stressors can emerge through frameshift mutations that resurrect the function of bepE, a conserved pseudogene encoding a membrane transporter subunit. In other Brucella species, bepE remains intact, and we show that deleting this gene in the zoonotic pathogen Brucella abortus increases susceptibility to both dihydropyridine treatment and membrane-disrupting stress. Our study provides evidence that pseudogenes like bepE can serve as a latent reservoir of adaptive functions in B. ovis, including drug and stress resistance.

microbiology↗

bioRxiv2025

The Salmonella phage shock protein system is required for defense against host antimicrobial peptides

Macrophages are professional phagocytes that play a major role in engulfing and eliminating invading pathogens. Some intracellular pathogens, such as Salmonella enterica serovar Typhimurium, exploit macrophages as niches for their replication, which requires precise and dynamic modulation of bacterial gene expression in order to resist the hostile intracellular environment. Here, we present a comprehensive analysis of the global transcriptome of S. Typhimurium across four stages of infection of primary macrophages. Our results revealed a profound change in early-stage gene expression dominated by pathways linked to metabolic processes required for Salmonella adaptation to the proinflammatory conditions of the macrophage. We identified the phage shock protein (Psp) system to be highly expressed in intracellular S. Typhimurium, with sustained high expression over the course of infection. We determined that the Psp system is regulated by the virulence-associated two-component system SsrA-SsrB, which coordinates its expression with critical bacterial functions required for immune evasion and intracellular survival. Functional assays demonstrated that the Psp system mediates resistance to host antimicrobial peptides, including cathelicidin-related antimicrobial peptide (CRAMP), which we demonstrate supports bacterial persistence in host tissues and survival within macrophages. Our findings establish the Psp system as a new and critical adaptive mechanism for evading host immune defenses and highlight the utility of temporal transcriptomics in unraveling the genetic strategies employed by S. Typhimurium during macrophage infection. Author summarySalmonella enterica is an important global pathogen that infects a wide range of mammalian hosts, requiring it to survive in diverse and hostile environments. A key aspect of Salmonella pathogenesis is its ability to reside within host immune cells like macrophages, where it must rapidly adapt to intracellular conditions. To do so, the bacteria must ensure correct spatiotemporal expression of virulence genes to maximise fitness in each environment. To better understand how Salmonella modulates its gene expression during infection of host cells, we defined its transcriptome at four distinct stages of primary murine macrophage infection. Our findings reveal that the first stage of early infection is dominated by changes in gene expression of metabolic circuits. Furthermore, we identified the phage-shock protein (Psp) system as highly expressed within intracellular Salmonella throughout the course of infection as a result of regulatory evolution that coordinates its expression with virulence genes. We showed that this system is required for bacterial survival within macrophages and host tissues by mediating resistance to host cationic antimicrobial peptides. These findings highlight the dynamic nature of Salmonellas transcriptional response during macrophage infection and uncover a previously unknown function of the Psp system as an adaptive mechanism for evading host immune defenses.

microbiology↗

bioRxiv2024

Supervised Spike Sorting Feasibility of Noisy Single-Electrode Extracellular Recordings: Systematic Study of Human C-Nociceptors recorded via Microneurography

Sorting spikes from noisy single-channel in-vivo extracellular recordings is challenging, particularly due to the lack of ground truth data. Microneurography, an electrophysiological technique for studying peripheral sensory systems, employs experimental protocols that time-lock a subset of spikes. Stable propagation speed of nerve signals enables reliable sorting of these spikes. Leveraging this property, we established ground truth labels for data collected in two European laboratories and designed a proof-of-concept open-source pipeline to process data across diverse hardware and software systems. Using the labels derived from the time-locked spikes, we employed a supervised approach instead of the unsupervised methods typically used in spike sorting. We evaluated multiple low-dimensional representations of spikes and found that raw signal features outperformed more complex approaches, which are effective in brain recordings. However, the choice of the optimal features remained dataset-specific, influenced by the similarity of average spike shapes and the number of fibers contributing to the signal. Based on our findings, we recommend tailoring lightweight algorithms to individual recordings and assessing the "sortability feasibility" based on achieved accuracy and the research question before proceeding with sorting of non-time-locked spikes in future projects.

neuroscience↗

bioRxiv2024

Molecular architecture of human dermal sleeping nociceptors

Human dermal sleeping nociceptors display ongoing activity in neuropathic pain, affecting 10% of the population. Despite advances in rodents, a molecular marker for these mechano-insensitive C-fibers (CMis) in human skin remains elusive, preventing targeted therapy. In this translational Patch-seq study, we combine single-cell transcriptomics following electrophysiological characterization with single-nucleus and spatial transcriptomics from pigs and humans. We functionally identified CMis in pig sensory neurons with patch-clamp using adapted protocols from human microneurography. We identified oncostatin-M-receptor (OSMR) and somatostatin (SST) as marker genes for CMis. Following dermal injection in healthy human volunteers, oncostatin-M, the ligand of OSMR, exclusively modulates CMis. We identified the entire molecular architecture of human dermal sleeping nociceptors, providing new therapeutic targets and the basis for a mechanistic understanding of neuropathic pain. One Sentence SummaryWe identify the molecular architecture and specifically OSMR and SST as molecular markers for human dermal sleeping nociceptors, key players in the generation of neuropathic pain. Short versionIn this Patch-seq study, we identify OSMR and SST as molecular markers for human dermal sleeping nociceptors, key players in the generation of neuropathic pain.

neuroscience↗

bioRxiv2024

Whole-Genome Sequencing of the Wild Barley Diversity Collection: A Resource for Identifying and Exploiting Genetic Variation for Cultivated Barley Improvement

To exploit allelic variation in Hordeum vulgare subsp. spontaneum, the Wild Barley Diversity Collection was evaluated for several agronomic traits and subjected to paired-end Illumina sequencing at [~]9X depth, generating 109.5 million single nucleotide polymorphisms after alignment to the Morex V3 assembly. A genome-wide association study of lemma color identified one marker-trait association (MTA) on chromosome 1HL close to HvBlp, the cloned gene controlling black lemma. Four MTAs were identified for stem rust resistance: one co-locating to the complex RMRL1-RMRL2 locus on 5HL, and three novel loci on 1HS, 1HL, and 5HL. Six MTAs for days to heading (DTH) on vernalized plants were identified on all chromosomes except 1H and 6H. Two MTAs for DTH on non-vernalized plants were identified on chromosomes 1HL and 2HS. All MTAs for DTH were novel. The whole genome sequence data described herein will facilitate the identification and utilization of new alleles for barley improvement.

genomics↗

bioRxiv2024

A pangenome and pantranscriptome of hexaploid oat

Oat grain is a traditional human food rich in dietary fiber that contributes to improved human health. Interest in the crop has surged in recent years owing to its use as the basis for plant-based milk analogs. Oat is an allohexaploid with a large, repeat-rich genome that was shaped by subgenome exchanges over evolutionary timescales. In contrast to many other cereal species, genomic research in oat is still at an early stage, and surveys of structural genome diversity and gene expression variability are scarce. Here, we present annotated chromosome-scale sequence assemblies of 33 wild and domesticated oats along with an atlas of gene expression across six tissues of different developmental stages in 23 accessions. We describe the interplay of gene expression diversity across subgenomes, accessions and tissues. Gene loss in the hexaploid is accompanied by compensatory up-regulation of the remaining homeologs, but this process is constrained by subgenome divergence. Chromosomal rearrangements have significantly impacted recent oat breeding. A large pericentric inversion associated with early flowering explains distorted segregation on chromosome 7D and a homeologous sequence exchange between chromosomes 2A and 2C in a semidwarf mutant has risen to prominence in Australian elite varieties. The oat pangeome will promote the adoption of genomic approaches to understanding the evolution and adaptation of domesticated oats and will accelerate their improvement.

genomics↗

bioRxiv2024

A co-conserved gene pair supports Caulobacter iron homeostasis during chelation stress

Synthetic metal chelators are widely used in industrial, clinical, and agricultural settings, leading to their accumulation in the environment. We measured the growth of Caulobacter crescentus, a soil and aquatic bacterium, in the presence of the ubiquitous chelator ethylenediaminetetraacetic acid (EDTA) and found that it restricts growth by lowering intracellular iron levels. Using barcoded transposon sequencing, we identified an operonic gene pair, cciT-cciO, that is required to maintain iron homeostasis in laboratory media during EDTA challenge. cciT encodes one of four TonB-dependent transporters that are regulated by the ferric uptake repressor (Fur) and stands out among this group of genes in its ability to support Caulobacter growth across diverse media conditions. The function of CciT strictly requires cciO, which encodes a cytoplasmic FeII dioxygenase-family protein. Our results thus define a functional partnership between an outer membrane iron receptor and a cytoplasmic dioxygenase that are broadly co-conserved in Proteobacteria. We expanded our analysis to natural environments by examining the growth of mutant strains in freshwater from two lakes, each with biochemical and geochemical profiles that differ markedly from standard laboratory media. In lake water, Caulobacter growth did not require cciT or cciO and was less affected by EDTA treatment. This result aligns with our observation that EDTA toxicity is influenced by common forms of biologically chelated iron and the spectrum of free cations present in the medium. Our study defines a conserved iron acquisition system in Proteobacteria and bridges laboratory-based physiology studies with real-world conditions. IMPORTANCEMetal-chelating chemicals are widely used across industries, including as preservatives in the food sector, but their full impact on microbial physiology is not well understood. We identified two genes, cciT and cciO, that function together to support Caulobacter crescentus iron balance when cells are exposed to the common synthetic chelator, EDTA. CciT is an outer membrane transporter and CciO is a dioxygenase-family protein that are mutually conserved in many bacteria, including several human pathogens, where mutations in cciT homologs are linked to clinical resistance to the siderophore antibiotic, cefiderocol. This study identifies a conserved genetic system that supports iron homeostasis during chelation stress and illuminates the iron acquisition versatility and stress resilience of Caulobacter in freshwater environments.

microbiology↗

bioRxiv2024

Adaptive diversification through structural variation in barley

Pangenomes are collections of annotated genome sequences of multiple individuals of a species. The structural variants uncovered by these datasets are a major asset to genetic analysis in crop plants. Here, we report a pangenome of barley comprising long-read sequence assemblies of 76 wild and domesticated genomes and short-read sequence data of 1,315 genotypes. An expanded catalogue of sequence variation in the crop includes structurally complex loci that have become hot spots of gene copy number variation in evolutionarily recent times. To demonstrate the utility of the pangenome, we focus on four loci involved in disease resistance, plant architecture, nutrient release, and trichome development. Novel allelic variation at a powdery mildew resistance locus and population-specific copy number gains in a regulator of vegetative branching were found. Expansion of a family of starch-cleaving enzymes in elite malting barleys was linked to shifts in enzymatic activity in micro-malting trials. Deletion of an enhancer motif is likely to change the developmental trajectory of the hairy appendages on barley grains. Our findings indicate that rapid evolution at structurally complex loci may have helped crop plants adapt to new selective regimes in agricultural ecosystems.

plant biology↗

bioRxiv2023

Untangling the Role of Pathobionts from Bacteroides Species in Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD) arise from a convergence of underlying genetic susceptibility, environmental factors, and shifts in gut microbiota function and membership. Although the latter may trigger and contribute to IBD, there is little consensus on a specific causative pathogen. In this study, we demonstrate that commensal Bacteroides fragilis strains from ulcerative colitis (UC) patients before and during the development of ileal pouchitis engraft and promote colitis in specific pathogen free (SPF) IL-10 deficient (IL-10-/-) mice, but not in wild type SPF mice or when mono-associated in germ free mice. The colitis in IL-10-/- mice was also associated with significant alterations in commensal microbiota potentially important for maintaining intestinal and immune homeostasis. UC pouchitis B. fragilis also engrafts in DSS-induced colitis in WT SPF mice, indicating a fitness advantage under conditions of mucosal inflammation over other commensals in the gut microbiota. These findings show that gut inflammation promotes the expansion and fitness of UC-derived Bacteroides species that is associated with changes in the SPF gut microbiota and may be promote colitis in genetically susceptible hosts. ImportanceThis study supports the notion that human inflammatory bowel diseases arise from the emergence of indigenous pathobionts in genetically-prone subjects. Colitis-promoting pathobionts are well-suited to establish themselves in the host inflammatory environment and outcompete endogenous microbiota. Once engrafted, the pathobiont can further aggravate inflammation in a genetically-susceptible host. Such complex interplay among several factors creates a vicious pro-inflammatory cycle and promotes disease development. These findings are consistent with our previous clinical observation that B. fragilis, an otherwise low-abundance commensal species, expands prior to the development of UC pouchitis. We believe these findings are relevant to the pathogenesis of UC pouchitis and possibly human inflammatory bowel diseases in general, underscoring the role of commensal to pathobiont transitions, rather than classical pathogens, in promoting and exacerbating the onset of human IBD.

microbiology↗