Biology subjects

Deutschbauer, A.

Publications and source records attributed to Deutschbauer, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Tracking gene flow in the gut microbiome reveals phage activation and host-range expansion in response to diet

Horizontal gene transfer (HGT) drives microbial evolution, but what determines when genes move between species within complex communities remains unclear. We developed a junction-resolved metagenomic framework using short-read sequencing to distinguish mobile genetic element activation from de novo transfer and track both longitudinally. In a defined human gut community colonizing mice, mobile element activation was widespread but successful transfer was rare. A prophage carried by invading Phocaeicola vulgatus transferred reproducibly to resident species when the community resisted invasion, but not when dietary perturbation allowed the invader to expand. Transfer required prophage activation and persistence of a permissive recipient and coincided with diversification of a phage receptor-binding protein. Dietary polysaccharides reproduced prophage-associated fitness costs in vitro, and rhamnogalacturonan I induced cell lysis consistent with productive prophage activation. The framework also detected interspecies transfer retrospectively in longitudinal human metagenomes. Thus, diet and community ecology can redirect gene flow through the gut microbiome.

microbiology↗

bioRxiv2026

Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways

Single stranded RNA (ssRNA) and single stranded DNA (ssDNA) bacteriophages represent a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here, we present a comprehensive, genome-wide mapping of the genetic landscape regulating infection cycles for F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Genetic screens across ssRNA phages spanning all four genogroups of the Leviviricetes revealed a highly conserved network of host dependencies, with the notable exception of the F plasmid gene traD. While primary structural receptor components and dsbA mediated disulfide bond formation are universally required across all lineages to ensure F pilus integrity, traD exhibits a strict genogroup-specific requirement during entry, showing variable essentiality across different viral groups despite sharing an identical primary receptor. Our gene dosage screens revealed that an elevated copy number of the hslU protease or the RNA chaperone stpA restricts infection, identifying clear genetic barriers that can perturb the viral life cycle. Parallel assays with filamentous ssDNA phages produced host factor profiles consistent with published literature, while revealing additional variations in host dependency. These screens confirmed that ssDNA phages strictly rely on the host TolQRA complex for entry downstream of pilus engagement. The assays tracked prominent negative fitness signatures across homeostatic clusters, highlighting how the physiological burden of continuous, non-lytic virion extrusion strains the host envelope. Finally, this comparative approach traced the selectivity of our isolation host (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella. This surface shield excludes a large panel of double stranded DNA phages isolated on diverse E. coli strains, while allowing virions from ssDNA and ssRNA phages to engage the extended F pilus and bypass the barrier via native pilus retraction. Together, this work provides a systematic, class-wide map of single stranded phage-host interactions, bridging classical genetics with modern viral discovery while establishing a robust host platform to access uncultured viral diversity and a functional blueprint to design next generation diagnostics, protein antibiotics, and biocontrol tools to halt horizontal gene transfer.

microbiology↗

bioRxiv2026

Surface architecture of the bacterial envelope determines phage adsorption route in pathogenic Escherichia coli O157:H7

The outermost surface layers of Gram-negative bacteria determine phage access to terminal receptors, yet their genetic basis has been mapped almost exclusively in laboratory strains that lack them. Here we apply genome-wide RB-TnSeq fitness profiling to four Escherichia coli O157:H7 strains from distinct phylogenetic clades sharing the O157 O-antigen, using 38 phages with terminal receptors previously mapped in E. coli K-12 strain. RB-TnSeq fitness landscapes across all four pathogenic backgrounds were mostly similar, and dominated by surface-associated loci, including the gfc-etk group 4 capsule operon, O-antigen biosynthesis genes, LPS core assembly genes and outer membrane proteins. Disruption of gfc-etk abolished infection in 11 genetically diverse myoviruses, establishing the O-antigen capsule as a widespread required primary recognition substrate. O-antigen loci generated two classes of fitness score patterns. For 10 phages, disruption increased infectivity, indicating it is a barrier to receptor access; for 3 others, disruption abolished infectivity, demonstrating it can also be a primary recognition substrate. Outer membrane protein receptor identity was conserved across laboratory and pathogenic backgrounds, with the same proteins recognized in both K-12 and O157:H7, while glycan layer state determines whether these receptors are reached. These results demonstrate that outer surface glycan layers can act as primary and optional recognition substrates for phage infection, or as physical barriers preventing terminal receptor access. Extending the ability to probe phage-targeted receptors beyond outer membrane proteins provides a framework for incorporating glycan layer state into predictive models of phage-host interactions.

microbiology↗

bioRxiv2026

Comprehensive interaction profiling and machine learning prediction of bacteriophage infectivity across clinically diverse Pseudomonas aeruginosa

The rise of antibiotic-resistant bacterial infections has driven renewed interest in bacteriophage therapy, where viruses that specifically kill bacteria are used as targeted antimicrobials. Pseudomonas aeruginosa, a WHO critical-priority pathogen that causes severe infections in hospitalized and immunocompromised patients, presents a major challenge for phage therapy because of its extraordinary genetic diversity. Phages effective against one bacterial strain often fail against others, and existing cross-resistance-profiling approaches require iterative empirical testing of each new patient isolate. To establish a genome-based framework for rapid phage-isolate matching, we assembled a collection of 95 genomically diverse P. aeruginosa phages representing 20 genera and tested each against 99 genetically diverse clinical isolates, generating 9,405 infection outcome measurements. Bacterial O-antigen serotype emerged as the dominant determinant of strain susceptibility, while defense systems, anti-defense systems, and prophage burden contributed smaller strain-specific effects. The full curated multivariate model explained 47% of strain-susceptibility variance. Machine-learning models integrating these features and pangenome-derived gene clusters reached a per-strain AUROC of 0.86. In an in vivo proof-of-concept test against a single held-out strain, the ML-designed cocktail produced a [~]12-fold greater median CFU reduction than the expert-designed cocktail (q = 0.045), with both cocktails substantially reducing burden relative to the untreated control ([~]113-fold for ML, [~]9-fold for CG; both q < 10{square}3). SHAP analysis of the model identified bacterial surface-architecture genes (LPS biosynthesis, outer membrane proteins, type IV pili) as the dominant predictors, with defense-system content modulating which specific phages succeed against a strain rather than uniformly damping susceptibility. Together, these results establish a genome-based framework for predicting phage susceptibility in genetically diverse clinical isolates.

microbiology↗

bioRxiv2026

Metagenome-guided substrate selection enriches Terriglobus, reveals co-occurring taxa, and enables isolation of a novel species

Microbes that remain uncultivated occupy nearly every ecosystem on the planet; this is particularly true in soils, where despite their prevalence, the roles of rarely cultivated microbes in driving biogeochemical cycles and ecosystem function remain poorly explored. We combine metagenome-informed substrate selection with enrichment sub-communities to generate reduced-complexity communities that preserve co-occurrence and expand experimental access to underrepresented soil lineages without requiring prior isolation of each member. Carbohydrate-active enzyme (CAZyme) profiles from soil-derived genomes were used to select carbon compounds predicted to enrich difficult to culture taxa, including members of the phylum Acidobacteriota. Based on 16S rRNA amplicon sequencing, we reproducibly enriched Terriglobus (Acidobacteriota) on multiple metagenome-guided substrates. Select communities with consistent presence and varying abundance of Terriglobus were passaged in a longitudinal design to generate 89 metagenomes; genus-level profiling revealed that community composition varied between biological replicates but remained consistent within replicates over time, providing diverse Acidobacteriota-containing configurations for downstream analysis. Association network inference identified a core set of co-occurring taxa that positively tracked with Terriglobus across the longitudinal series. In parallel, the substrate-guided approach led to isolation of a novel Terriglobus species, the first cultured representative of its GTDB species cluster. Together, these results establish a generalizable strategy for generating communities enriched with rarely cultivated taxa, yielding tractable systems for studying microbial interactions and community assembly in soil.

microbiology↗

bioRxiv2026

The WalRK two-component system in Streptococcus pneumoniae ensures robustness of secondary wall polymer attachment

Capsular polysaccharide (CPS) is essential for Streptococcus pneumoniae virulence. Yet, the mechanism linking CPS to peptidoglycan (PG) remains unclear. Here, we identified a strong negative genetic interaction between the genes encoding the putative capsule ligase CpsA and the WalK histidine kinase, a component of the WalRK two-component system regulating cell wall homeostasis. In the absence of cpsA, capsule polymers compete with wall teichoic acids for ligase activity to PG. This induces cell wall stress and is sensed by the WalRK system. Overexpression of the PG hydrolase pcsB or disruption of the PG-modifying enzymes pgdA and oatA restored growth of strains lacking cpsA and walK. Furthermore, CpsA overproduction compensates for the loss of other LytR-Cps2A-Psr (LCP) ligases, suggesting it can support capsule and wall teichoic acid syntheses. These findings support the model that LCP ligases are semi-redundant, although they may install secondary polymers on a different residue of PG. This work also suggests that WalRK signaling compensates for reduced capsule and WTA attachment by positively regulating PG hydrolases. SIGNIFICANT STATEMENTStreptococcus pneumoniae causes approximately half a million deaths annually. A powerful public health tool for controlling pneumococcal infections is vaccination against the protective capsule. Yet, the mechanisms by which the capsule layer attaches to the underlying cell wall remain poorly defined. This study shows that the conserved capsule gene CpsA is not strictly required for capsule attachment but instead works together with other LytRIZCpsAIZPsr (LCP) ligases. However, it requires the essential WalRK signaling system to maintain cell envelope integrity. Defects in LCP activity are alleviated by WalRKIZdriven upregulation of peptidoglycan hydrolases, overexpression of PcsB, or inactivating peptidoglycan modifications that limit hydrolysis. These findings reveal coordination among flux to capsule synthesis, secondary wall polymer attachment, and cell wall remodeling. TEASERThe WalRK two-component system responds to cell envelope stress caused by reduced LCP ligase activity.

microbiology↗

bioRxiv2026

Evolutionary and functional genomics reveal that Ralstonia wilt pathogens actively deploy antimicrobial warfare while leveraging physiological adaptations during plant infection

The xylem pathogens in the Ralstonia solanacearum species complex (RSSC) cause wilt diseases that threaten global food security. These diverse pathogens are highly adapted to the in planta environment where they proliferate and cause rapid, aggressive diseases. To understand the genetic underpinnings of these pathogens in planta fitness, we performed forward genetic screens in three RSSC species, using high-throughput random barcode transposon sequencing (RB-TnSeq). We quantified the competitive fitness of hundreds of thousands of mutants during stem colonization of susceptible tomato plants. We compared this in planta forward genetic screen with a prior forward genetic screen performed in a reductionist, plant-like environment: ex vivo xylem sap harvest from healthy tomato plants. The comparative genetic screens revealed both conserved and lineage-specific orthologs that enable RSSCs pathogenic success. Consistently, these diverse pathogens navigated the opportunities and stressors in planta by maintaining a resilient cell envelope barrier, complementing the nutritional availability in xylem sap, fine-tuning their utilization of the host-manipulating type III secretion system, and dynamically regulating their gene expression through a suite of environment sensing and transcriptional regulatory proteins. Surprisingly, the strain-specific fitness factors shed light on ecological interactions beyond pathogen dominance of their susceptible host, even during the single-strain infections, the pathogens wielded arsenals of anti-microbial weapons. Specifically during growth in planta, each RSSC strain required a unique repertoire of type VI secretion system immunity proteins that provide self-protection to their own toxins. We contextualized the in planta fitness factors through evolutionary genomic comparisons of the RSSC wilt pathogens to their non-pathogenic neighbors. Together, these analyses reveal how conserved and lineage-specific fitness determinants have evolved to support pathogenic success in the plant vascular niche.

genetics↗

bioRxiv2026

RB-TnSeq analysis reveals alcohol and salt tolerance systems in a plant root colonizer Paraburkholderia graminis OAS925

The role of microbial strains in regulating natural stresses and their impact on plant health is well-established. However, the role of microbial tolerance mechanisms in plant response to unnatural or anthropogenic stresses is less understood. Examination of these interactions impact our deeper understanding of plant-microbe interactions and our ability to enhance beneficial functions. In this study we use the model plant Brachypodium distachyon and its prominent root colonizer Paraburkholderia graminis OAS925 to investigate mechanisms of tolerance to alcohol and salt stress. We examined the ability of OAS925 to reduce root growth inhibition during exposure to short chain alcohols and salt. We also examined the tolerance mechanism for OAS925 towards these stresses using RB-TnSeq fitness assays. The most prominent tolerance systems in OAS925 are genes specifically involved in membrane transport (such as the Mla operon), efflux systems (e.g., RND efflux systems), signaling and regulation (PrtR/PrtI, NtrY/NtrX, and EnvZ/OmpR), and oxidative stress response (GshB). Our findings provide a model where bacterial membrane integrity, active solvent efflux, and stress signaling are crucial not only for bacterial survival but also for maintaining the root colonization and biofilm formation that confer protection to the host plant.

microbiology↗

bioRxiv2025

Unraveling Fitness Landscapes in Plant-Associated Bacteroidota

Members of the bacterial phylum Bacteroidota inhabit a wide range of ecosystems. Although significant progress has been made over the past decade in understanding the metabolic functions of clinically relevant Bacteroidota, far less is known about the environmental species. Despite being one of the dominant phyla linked to crops, there is limited information on the mechanisms that enable Bacteroidota to thrive in plant and soil environments. A key obstacle in understanding gene fitness in Bacteroidota is the lack of genome-wide functional studies, largely due to their inherent resistance to antibiotics, which complicates genetic manipulation. In this study, we used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) to measure gene fitness in a plant-associated Bacteroidota, Mucilaginibacter yixingensis YX-36. Our data sheds light on pathways involved in rhizosphere colonization, gliding motility, stress tolerance, and carbon metabolism. Notably, we found that phylum-specific genes such as Polysaccharide Utilization Loci (PULs) and Carbohydrate-Active enzymes (CAZymes) are needed for fitness in the plant niche. Overall, this work advances our understanding of gene functions in environmental Bacteroidota species and provides a foundation for future research on their roles in plant-microbe interactions.

microbiology↗

bioRxiv2025

BarTn7: Optimizing Bacterial Lineage Tracking at Sub-Species Resolution for Population Dynamics in Ecological and Evolutionary Studies

Communities of bacteria undergo population bottlenecks which are crucial to their population, ecological, and evolutionary dynamics. However, conventional amplicon sequencing cannot distinguish such demographic shifts between very closely related lineages. Here we describe BarTn7, an optimized method for bacterial lineage tracking through chromosomal integration of phenotypically neutral DNA barcodes with transposon Tn7. By combining conventional conjugative plasmids into a single vector and leveraging a parts-based strategy to optimize delivery to different recipients, BarTn7 increases barcoding efficiency and enables the systematic application of this tool to diverse bacteria. We tested BarTn7 in multiple bacterial species and confirmed a lack of lineage-specific growth effects. We then used BarTn7 to measure the colonization of Panicum virgatum roots under differing phosphate concentrations. Lineage tracking enabled discrimination between unique colonization events and the proliferation of existing bacteria during root colonization, the ratio of which differed between three plant-associated bacterial species. The strategy further allowed the measurement of phosphate-dependent ingress rates of the root endosphere by Paraburkholderia phytofirmans PsJN. We then demonstrated the effectiveness of BarTn7 to detect adaptive mutation(s) and facilitate identification of mutant lineages. Additionally, we demonstrated that BarTn7 is more accurate than conventional 16S rRNA gene sequencing at measuring community composition of a 5-member synthetic bacterial community (SynCom) and compares favorably to shotgun metagenomics. Our results illustrate the utility of BarTn7 as a simple, cost-efficient, and broadly applicable method of measuring bacterial population dynamics at sub-species resolution.

microbiology↗

bioRxiv2025

Phylogeny-agnostic strain-level prediction of phage-host interactions from genomes

Bacteriophages offer promising alternatives to antibiotics for treating drug-resistant infections and engineering microbiomes, but applications are limited by inability to select phages infecting specific bacterial strains. Selecting suitable phages requires either one-to-one experimental assays or strain-level predictions of phage-host interactions. Existing computational approaches either predict host taxonomy at broad ranks unsuitable for strain-level targeting or require species-specific mechanistic knowledge limiting generalizability. Here, we present a phylogenyagnostic machine learning framework predicting strain-level phage-host interactions across diverse bacterial genera from genome sequences alone. Systematically optimizing the workflow over 13.2 million training runs across six datasets (115,037 interactions, 949 bacterial strains, 518 phages), we achieved performance matching species-specific methods (AUROC 0.67-0.94) while eliminating phylogenetic constraints. Comprehensive feature engineering identifies biologically interpretable genetic determinants while minimizing overfitting in sparse, imbalanced datasets. Experimental validation through 1,240 novel interactions confirmed generalizability (AUROC 0.84), while genome-wide RB-TnSeq screens verified that 68.6% of experimentally identified infection mediators were captured computationally, including receptors and cell wall biosynthesis pathways. Model-guided cocktail design achieved up to 97.5% bacterial coverage with five phages, and up to a 3.1-fold improvement in single-phage selection over promiscuity-based selection. This platform enables rational phage therapy design and precision microbiome engineering with applications in combating antimicrobial resistance across clinical, agricultural, and industrial contexts.

microbiology↗

bioRxiv2025

Random barcode transposon-site sequencing in Mycobacterium tuberculosis reveals the functions of uncharacterized genes

Mycobacterium tuberculosis (Mtb) is a human bacterial pathogen that establishes chronic infection in the lung. Although the genome of Mtb was sequenced nearly 25 years ago, the genetic basis of Mtbs success as a human pathogen remains to be fully elucidated. Large-scale genetic approaches to understanding gene function are hindered by the limited throughput of traditional transposon sequencing strategies used in mycobacteria. To create a resource for determining the function of genes, we generated a pooled random barcode transposon-site sequencing (RB-TnSeq) library in Mycobacterium tuberculosis (Mtb). A unique twenty-nucleotide barcode in the transposon allows for rapid, high-throughput genetic screening without the laborious protocol of standard bacterial TnSeq screens. We performed 95 RB-TnSeq screens on an array of carbon sources, nitrogen sources, stressors, and antibiotics. Using the resulting dataset, we examined phenotypes of PE/PPE genes, a mycobacterial gene family whose function has long been elusive, uncovering 187 novel phenotypes across 37 genes in this family. We propose a pathway for lactate utilization in which the ESX-5 type VII secretion system exports PPE3, facilitating the import of D- and L-lactate into the bacterial cell. Notably, we identify a candidate D-lactate dehydrogenase that may mediate this metabolic capability. Additionally, we find that the proton-pumping NADH dehydrogenase Nuo is required for utilization of propionate, highlighting the metabolic flexibility of Mtb. Lastly, we characterize a novel mutant that confers resistance to the new tuberculosis antibiotic pretomanid. Results from these genetic screens will facilitate the development of additional new hypotheses about the function of uncharacterized genes and will expand our knowledge of Mtb metabolism and resistance to stress.

microbiology↗

bioRxiv2024

Improving the annotation of amino acid biosynthesis pathways: GapMind 2024

We previously described GapMind, an automated web-based tool for annotating amino acid biosynthesis pathways in bacterial and archaeal genomes. We used GapMind to identify gaps in biosynthetic pathways and systematically used comparative genomics and high-throughput genetics to identify candidate genes to fill these gaps. We confirmed the activity of ten of the proposed enzymes by using cross-species complementation assays. Highlights include a novel route to glycine, two families that can replace phosphoserine phosphatase, an alternative N-succinyl-L,L-diaminopimelate desuccinylase, an alternative N-acetylornithine deacetylase, and a bifunctional MetB/MetC. We updated GapMind to include these additional enzymes. Across 208 prokaryotes that have high-quality genomes and can grow in minimal media, the average number of unexplained missing steps or gaps in amino acid biosynthesis dropped from 1.4 per genome to 0.7 per genome. The majority of remaining gaps involve the gain or loss of phosphate groups.

microbiology↗

bioRxiv2024

High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly-barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we provide the first evidence that M. maripaludis can utilize the nitrogen sources L-glutamine, D-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP, group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale dataset that can be used as a resource to further understand gene function and metabolism in the archaeal domain. Significance StatementArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly-barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This dataset encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy; thus, providing an extensive dataset for the archaeal community to mine for characterizing additional genes of unknown function.

microbiology↗

bioRxiv2023

A novel phenylpropanoid methyl esterase enables catabolism of aromatic compounds that inhibit biological nitrification

Agriculture is the largest source of greenhouse gases (GHG) production. Conversion of nitrogen fertilizers into more reduced forms by microbes through a process known as biological nitrification drives GHG production, enhances proliferation of toxic algal blooms, and increases cost of crop production. Some plants reduce biological nitrification in soils by exuding a diverse array of biological nitrification inhibitors (BNIs) that inhibit the ammonium oxidizing microbes responsible for nitrification. Applying synthetic biology to enhance and transfer BNI production into food and bioenergy crops is a promising approach to reduce nitrification but the success of this strategy is contingent upon improving our limited understanding of BNIs mechanisms-of-action and degradation in the soil. We addressed this gap by investigating the previously unknown metabolic route through which a prominent class of aromatic BNIs known as phenylpropanoid methyl esters (PPMEs) are catabolized. While neither transcriptomics (RNAseq) or high-throughput functional genomics (RB-TnSeq) methods reduced the genetic search space for pathway discovery into a tenable number of genes, the combination of narrowed the search space to a collection of 4 proteins of unknown function. Using genetic and biochemical analyses we found that two previously uncharacterized enzymes, including a novel phenylpropanoid methyl esterase, funnel PPMEs into an established phenylpropanoid catabolism pathway. Transfer of these two enzymes into bacteria capable of using other phenylpropanoids use of PPMEs as carbon sources. This work both provided insight into BNI catabolism and is the first step towards development of model in vivo plant-microbe systems for studying BNI mechanisms under well controlled conditions.

microbiology↗

bioRxiv2023

Sorgoleone degradation by sorghum-associated bacteria; an opportunity for enforcing plant growth promotion

Metabolite exchange between plant roots and their associated rhizosphere microbiomes underpins plant growth promotion by microbes. Sorghum bicolor is a cereal crop that feeds animals and humans and is used for bioethanol production. Its root tips exude large amounts of a lipophilic benzoquinone called sorgoleone. Sorgoleone is an allelochemical that suppresses the growth of competing plant seedlings and is mineralized by microbes in soil. As an avenue to understand how sorghum and its root microbiome may be connected through root exudates, we identified the molecular determinants of microbial sorgoleone degradation and the distribution of this trait among microbes. We isolated and studied from sorghum-associated soils, three bacterial strains classified as Acinetobacter, Burkholderia, and Pseudomonas species that grow with sorgoleone as a sole carbon and energy source. The genomes of these strains were sequenced and subjected to transcriptomic and gene fitness analyses to identify candidate sorgoleone degradation genes. Follow up mutational analysis showed that sorgoleone catabolism is dependent on four contiguous genes that are conserved among the species we sequenced. Phylogenetic analysis of the sorgoleone degradation gene cluster showed that sorgoleone catabolism is enriched in sorghum-associated Streptomyces strains. The discovery of bacteria that grow on a compound like sorgoleone that is plant specific and not widely distributed in the environment, provides an opportunity to study how a plant exudate can enforce the development of a rhizosphere specific microbiome for the mutual benefit of plant and microbe. SignificanceThe grain crop sorghum exudes an herbicidal compound called sorgoleone from its root tips, which inhibits the growth of other plants. We isolated bacteria that grow on sorogleone and identified a cluster of bacterial genes required for sorogleone degradation that can be used as a biomarker for this trait. An approach to improve the production of crops in stressful conditions such as drought, is to encourage their association with plant growth promoting bacteria. Our discovery of sorgoleone degradation genes opens the door to engineering bacteria that receive benefit from sorghum in the form of a plant-specific growth substrate, and in return promote the growth of this crop.

microbiology↗

bioRxiv2023

An integrated transcriptomics-functional genomics approach reveals a small RNA that modulates Bacteroides thetaiotaomicron sensitivity to tetracyclines

Gene expression plasticity allows bacteria to adapt to diverse environments, tie their metabolism to available nutrients, and cope with stress. This is particularly relevant in a niche as dynamic and hostile as the human intestinal tract, yet transcriptional networks remain largely unknown in gut Bacteroides spp. Here, we map transcriptional units and profile their expression levels in Bacteroides thetaiotaomicron over a suite of 15 defined experimental conditions that are relevant in vivo, such as variation of temperature, pH, and oxygen tension, exposure to antibiotic stress, and growth on simple carbohydrates or on host mucin-derived glycans. Thereby, we infer stress- and carbon source-specific transcriptional regulons, including conditional expression of capsular polysaccharides and polysaccharide utilization loci, and expand the annotation of small regulatory RNAs (sRNAs) in this organism. Integrating this comprehensive expression atlas with transposon mutant fitness data, we identify conditionally important sRNAs. One example is MasB, whose inactivation led to increased bacterial tolerance of tetracyclines. Using MS2 affinity purification coupled with RNA sequencing, we predict targets of this sRNA and discuss their potential role in the context of the MasB-associated phenotype. Together, this transcriptomic compendium in combination with functional sRNA genomics--publicly available through a new iteration of the Theta-Base web browser (www.helmholtz-hiri.de/en/datasets/bacteroides-v2)--constitutes a valuable resource for the microbiome and sRNA research communities alike.

molecular biology↗

bioRxiv2022

Eliminating Genes for a Two Component System Increases PHB Productivity in Cupriavidus basilensis 4G11 Under PHB Suppressing, Non-Stress Conditions

Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly-hydroxybutyrate (PHB) making them attractive candidate bioplastic producers. The native production of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB-TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two component system which, when omitted from the genome, are capable of increased PHB productivity in balanced, non-stress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain, upon deleting each of two genes individually from the genome. The increased PHB phenotype disappears, however, in nitrogen-free unbalanced growth conditions suggesting the phenotype is specific to fast-growing, replete, non-stress growth. Bioproduction modeling suggests this phenotype could be due to a decreased reliance on metabolic stress induced by nitrogen limitation to initiate PHB production in the mutant strains. Such strains may allow for the use of single stage, continuous bioreactor systems, which are far simpler than PHB bioproduction schemes used previously. Bioproductivity modeling suggests that omitting this regulation in the cells may increase PHB productivity up to 24% relative to the wildtype organism when using single stage continuous systems. This work furthermore expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes.

genomics↗