Biology subjects

Schubert, O. T.

Publications and source records attributed to Schubert, O. T..

17 recordsLinked to original sources

bioRxiv2026

Amino acid auxotrophy is a feature of a distinct bacterial lifestyle across environments

Amino acid auxotrophy, the loss of biosynthetic potential for an amino acid, is a highly prevalent feature of microbial life, yet the evolutionary processes driving its distribution in nature remain unclear. Here, we sought to determine whether auxotrophy emerges as an independent response to nutrient availability or as part of a broader transition in genomic lifestyle. By first predicting auxotrophies through genome-scale metabolic models and flux-balance analysis, and subsequently applying phylogenetically informed statistical frameworks, we tested the predictions of the Black Queen Hypothesis, which posits that auxotrophy evolves as a dynamic response to metabolite availability, and disentangled the signal of convergent environmental selection from the background of shared evolutionary history. This approach reveals that phylogeny is the dominant predictor of auxotrophy, vastly outweighing environmental context. Beyond correcting for ancestry, our analysis uncovered a previously obscured signal of consistent, co-occurring genomic changes: auxotrophies do not occur as independent losses, but rather as part of a broader reorganization of the genome. We show that auxotrophies co-occur significantly more often than predicted by chance and that the probability of loss scales with the number of other auxotrophies. These findings reveal auxotrophy as a feature of a conserved, phylogenetically entrenched lifestyle rather than a transient response to nutrient availability -- inconsistent with the Black Queen Hypothesis and instead aligning with genome streamlining as the overarching driver of metabolic dependency in natural microbial systems.

evolutionary biology↗

bioRxiv2026

Opposing range-dependent interactions create complex spatial patterns of antibiotic tolerance in multispecies biofilms

Many microbial communities form multispecies biofilms where cells interact through diffusible molecules. In these biofilms, multiple interactions, often with opposing effects, occur simultaneously, yet we lack quantitative frameworks to predict how they combine to shape community functions. Here, we hypothesized that complex spatial patterns can emerge when opposing interactions have distinct spatial ranges. To test this, we studied how two Pseudomonas aeruginosa exoproducts, HQNO and rhamnolipids, jointly modulate Staphylococcus aureus antibiotic tolerance by respectively increasing and decreasing it. Using microfluidics-based imaging, we quantified spatial-tolerance patterns at single-cell resolution and found that tolerance indeed shows a complex spatial pattern: S. aureus cells survived treatment only at intermediate distances from P. aeruginosa, while cells closer or farther away did not. Combining experiments and modelling, we showed that this remarkable pattern emerges because rhamnolipids have a stronger but short-ranged effect, while HQNO has a weaker but longer-ranged effect. We found that spatial arrangement affects overall tolerance by shifting the balance between the two opposing interactions. Finally, using bioprinting, we confirmed that HQNO and rhamnolipids modulate tolerance in highly mixed biofilms. In more segregated biofilms, spatial arrangement still strongly modulated tolerance, but independently of these compounds, suggesting additional interactions. Together, our results show that spatial-tolerance patterns emerge from the combined effect of opposing range-dependent interactions and cannot be predicted from either alone. By predicting how opposing interactions jointly determine community properties, our framework provides a foundation for understanding and ultimately engineering microbiome functions.

systems biology↗

bioRxiv2025

A critical assessment of aptamer and CRISPR-Cas12a-based biosensors for small molecule detection

Analyte detection through aptamer-induced signal generation by CRISPR-Cas enzymes has rapidly emerged as a popular biosensing approach. Here, we investigated the implementability and analytical performance of this setup for the detection of diverse small molecule analytes. We selected nine aptamers from the literature targeting seven analytes and tested a commonly used assay design whereby analyte binding by the aptamer liberates a short complementary DNA strand, which in turn activates Cas12a to generate a fluorescence signal. After extensive optimization, the assay functioned for only two of the seven analytes, and several previously reported results could not be reproduced. While Cas12a fluorescence detection was robust, the low success rate is likely due to aptamers not functioning reliably, underscoring the need for careful aptamer validation. Overall, our study provides a critical assessment of aptamer-Cas12a assay performances and discusses potential strengths, limitations, and pitfalls of this biosensing strategy.

biochemistry↗

bioRxiv2025

Phage-mediated lysis increases growth rate of surviving bacterial cells

Bacterial phage infection and subsequent lysis are traditionally considered mechanisms of bacterial mortality and viral propagation; additionally, emerging evidence indicates that they may also contribute to nutrient recycling in broader ecological systems. However, it remains unclear how the nutrients released during cell lysis affect the growth dynamics of the remaining bacterial population. Addressing this gap, we built a controlled system consisting of two Escherichia coli lysogenic strains: one carrying a wild-type{lambda} prophage and the other a temperature-inducible variant that can be induced to lyse at 38 {degrees}C. Using this system, we selectively induced phage lysis in a defined fraction of the population and quantified both total biomass and the biomass of surviving, non-lysed cells. We observed that the biomass loss was consistently smaller than expected based on the fraction of lysed cells, supporting the idea that some of the released biomass is rapidly recycled by the non-lysed population. To formalize the observed dynamics and obtain quantitative insight, we developed a mathematical model showing that nutrients released during lysis can transiently enhance the growth rate of the surviving, non-lysed cells. This effect emerges on a short timescale of minutes, consistent with the rapid onset of biomass compensation observed experimentally. The growth rate increase was confirmed in single-cell experiments using microfluidics and time-lapse microscopy, where we cultured wild-type lysogens in lysate-containing culture supernatants. In summary, the results suggest that nutrients released through lysis are rapidly consumed, leading to an acceleration in the growth rate of non-lysed cells. The consequent partial compensation for cell loss can substantially influence the population dynamics, highlighting phage lysis as a direct modulator of bacterial growth. Overall, our findings provide quantitative insights into how phage-mediated lysis affects the physiology of non-lysed bacterial cells, extending beyond its well-established role in biomass recycling.

microbiology↗

bioRxiv2025

When less is not more: Limits to the evolution of metabolic dependence in spatially structured microbial communities

In microbial communities, evolutionary processes can lead to loss of biosynthetic pathways, creating metabolic dependencies. The Black Queen Hypothesis suggests that such gene loss can confer a fitness advantage by reducing metabolic burden. However, how these dependencies evolve at the level of individual cells in spatially structured communities remains poorly understood. We used a combination of microfluidic single-cell imaging and mathematical modeling to examine the early fate of auxotrophic mutants within E. coli populations. We found that without external amino acids, auxotroph growth is strongly constrained by low amino acid leakage from wildtype neighbors, and further reduced when they form local clusters that drain this limited amino acid pool. A growth advantage was only observed when amino acids were added or when leakage from wildtypes exceeded a threshold. Together, our results reveal insights into determinants of mutant invasion fitness and the trade-offs between reducing metabolic costs and maintaining metabolic autonomy. HighlightsO_LISingle-cell imaging reveals how auxotrophs fare in spatially structured populations C_LIO_LIAuxotrophs grow slowly due to low amino acid leakage from wildtype neighbors C_LIO_LIMutant clustering intensifies local amino acid depletion and reduces auxotroph growth C_LIO_LIModel identifies leakage threshold required for auxotroph invasion from rare C_LI

evolutionary biology↗

bioRxiv2024

Antagonism as a foraging strategy in microbial communities

In natural habitats, nutrient availability limits bacterial growth. We discovered that bacteria can overcome this limitation by acquiring nutrients through lysing neighboring cells via contact-dependent antagonism. Using single-cell live imaging and isotopic markers, we found that during starvation the type VI secretion system, which transfers toxins into neighboring cells, increases growth by enabling the uptake of nutrients from lysing cells. In a spatially structured environment, the gradual lysis of just a few target cells allows for the growth of a new antagonist cell. Genomic adaptations in antagonists, characterized by a reduced metabolic gene repertoire, and the prevalence of the type VI secretion system in natural environments, such as global oceans and soils, suggest that bacterial antagonism has the potential to substantially contribute to nutrient transfer within microbial communities across various ecosystems.

microbiology↗

bioRxiv2023

Polysaccharide breakdown products drive degradation-dispersal cycles of foraging bacteria through changes in metabolism and motility

Most of Earths biomass is composed of polysaccharides. During biomass decomposition, polysaccharides are degraded by heterotrophic bacteria as a nutrient and energy source and are thereby partly remineralized into CO2. As polysaccharides are heterogeneously distributed in nature, following the colonization and degradation of a polysaccharide hotspot the cells need to reach new polysaccharide hotspots. Even though many studies indicate that these degradation-dispersal cycles contribute to the carbon flow in marine systems, we know little about how cells alternate between polysaccharide degradation and motility, and which environmental factors trigger this behavioral switch. Here, we studied the growth of the marine bacterium Vibrio cyclitrophicus ZF270 on the abundant marine polysaccharide alginate, both in its soluble polymeric form as well as on its breakdown products. We used microfluidics coupled to time-lapse microscopy to analyze motility and growth of individual cells, and RNA sequencing to study associated changes in gene expression. We found that single cells grow at reduced rate on alginate until they form large groups that cooperatively break down the polymer. Exposing cell groups to digested alginate accelerates cell growth and changes the expression of genes involved in alginate degradation and catabolism, central metabolism, ribosomal biosynthesis, and transport. However, exposure to digested alginate also triggers cells to become motile and disperse from cell groups, proportionally increasing with the group size before the nutrient switch, and this is accompanied by high expression of genes involved in flagellar assembly, chemotaxis, and quorum sensing. The motile cells chemotax toward polymeric but not digested alginate, likely enabling them to find new polysaccharide hotspots. Overall, our findings reveal cellular mechanisms that might also underlie bacterial degradation-dispersal cycles, which influence the remineralization of biomass in marine environments. ImportancePolysaccharides, also known as glycans, are the most abundant form of biomass on Earth and understanding how they are degraded by microorganisms is essential for our understanding of the global carbon cycle and the storage and release of CO2 by natural systems. Although group formation is a common strategy used by bacterial cells to degrade ubiquitous polymeric growth substrates in nature, where nutrient hotspots are heterogeneously distributed, little is known about how cells prepare for dispersal from an exhausted nutrient source and re-initiate degradation of new nutrient patches. By quantifying growth, motility and chemotaxis of individual cells and comparing gene expression changes when populations were exposed to either polysaccharides or their degradation products in the form of digested polysaccharides, we show that bacterial cells alter their behavior when they experience a shift from polymeric to digested polysaccharides: After cells form groups during growth on polymers, the exposure to degradation products triggered cells to become motile, enabling dispersal from sessile cell groups. The chemotactic attraction of motile cells to polymeric alginate likely allows cells to move towards new polysaccharide hotspots. Our study sheds light on the cellular processes that drive bacterial growth and behavior during carbon remineralization, an important process resulting in CO2 release from natural systems.

microbiology↗

bioRxiv2022

Effects of interspecies interactions on marine community ecosystem function

Microbial communities perform key ecosystem processes collectively. One such process is the degradation of carbohydrate polymers, which are the dominant pool of organic carbon in natural environments. Carbohydrate polymers are often degraded in a stepwise manner. Individual steps are performed by different microbial species, which form trophic cascades with carbon polymers at the bottom and fully oxidised carbon at the top. It is widely believed that these trophic cascades are hierarchically organised, where organisms at each level rely on organisms at the levels below. However, whether and how the higher-level organisms can also affect processes at the lower levels is not well understood. Here we studied how carbohydrate polymer degradation mediated by secreted enzymes is affected by species at higher trophic levels, i.e., species that cannot produce the enzymes for polymer degradation but can grow in presence of the polymer degraders. We used growth and enzyme assays in combination with transcriptomics to study how chitin degradation by a number of Vibrio strains is affected by the presence of different cross-feeders that consume metabolic by-products. We found that interactions between the degraders and cross-feeders influence the rate of chitin degradation by the community. Furthermore, we show that this is a result of changes in chitinase expression by degraders. Overall, our results demonstrate that interactions between species can influence key ecosystem functions performed by individuals within microbial communities. These results challenge the perspective that trophic cascades based on metabolically coupled microbial communities are unidirectional and provide mechanistic insights into these downstream interactions.

microbiology↗

bioRxiv2022

Towards Synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression

BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient concurrent extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter mhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

microbiology↗

bioRxiv2022

Genome-wide base editor screen identifies regulators of protein abundance in yeast

Abundance of proteins is extensively regulated both transcriptionally and post-transcriptionally. To systematically characterize how regulation of protein abundance is encoded in the genome and identify protein regulators on a genome-wide scale, we developed a genetic screen that uses a CRISPR base editor. We examined the effects of 16,452 genetic perturbations on the abundance of eleven yeast proteins representing a variety of cellular functions. Thereby, we uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Our results provide unprecedented insights into the components, scale and connectedness of the protein regulatory network.

genetics↗

bioRxiv2021

Island-specific evolution of a sex-primed autosome in the planarian Schmidtea mediterranea

The sexual biotype of the planarian Schmidtea mediterranea is a hermaphrodite indigenous to Tunisia and several Mediterranean islands. Here, we isolated individual chromosomes and used sequencing, Hi-C and linkage mapping to assemble a chromosome-scale genome reference. The linkage map revealed an extremely low rate of recombination on chromosome 1. We confirmed suppression of recombination on chromosome 1 by quantifying recombination events in individual sperm and oocytes. We showed that the extensive heterozygous regions, previously designated as J and V haplotypes, comprise essentially all of chromosome 1. Genome sequencing of individuals isolated in the wild indicated that this heterozygosity has evolved specifically in populations from Sardinia and Corsica. We found that chromosome 1 acquired many genes that determine the development of female and male reproductive systems, along with haplotype-specific expression of such sex specific genes. These molecular signatures and restricted recombination in a hermaphrodite led us to propose that chromosome 1 is a sex-primed autosome, providing direct molecular evidence for the traditional model of how sex chromosomes may have evolved from autosomes.

genetics↗

bioRxiv2020

Network analysis identifies regulators of lineage-specific phenotypes in Mycobacterium tuberculosis

The Mycobacterium tuberculosis (Mtb) complex includes ten phylogenetically distinct human-adapted lineages with varying geographical distributions and pathogenicity. Lineage 1 (L1) is associated with low virulence, while Lineage 2 (L2) is linked to hyper-virulence, increased transmission, and drug resistance. We performed multi-layer comparative analyses integrating whole-genome sequencing with quantitative transcriptomic and proteomic profiling of a panel of L1 and L2 clinical strains, each grown under two in vitro conditions. Our data revealed variable correlations between transcript and protein levels across strains and gene categories, indicating lineage-specific post-translational regulation. Transcriptional and translational differences scaled with phylogenetic distance, with one in three SNPs on average leading to gene expression changes. A newly developed genome-scale transcriptional regulatory model identified master transcription factors--closely linked to the sigma factor network--whose targets were differentially expressed between L1 and L2. Notably, DosR proteins showed higher basal levels and a stronger nitric oxide (NO) response in L2. Time-course validation using an Rv1985c induction and wild-type H37Rv under hypoxia and reaeration confirmed the role of Rv1985c in the differentially expressed genes between L1 and L2. These findings suggest that limited genetic variation can translate into significant phenotypic differences via differential regulation of key transcriptional networks. HighlightsO_LIProteomic and transcriptomic characterization of fully sequenced diverse L1 and L2 clinical strains of Mtb. C_LIO_LIPost translational control mechanisms for regulatory and virulence genes are mitigated in Mtb L2. C_LIO_LIA genome-scale transcriptional framework identifies DosR, Rv1985c, Lsr2 and Rv0691c as master transcription factors responsible for differential target gene expression in L2 compared to L1 strains. C_LIO_LIL1 and L2 DosR proteins respond differently to nitric oxide stress, thus determining a relevant phenotype. C_LIO_LITime-course phenotypic and transcriptomic data under hypoxia and reaeration validate Rv1985c as a key transcription factor differentiating L2 and L1 strains. C_LI

systems biology↗

bioRxiv2019

Integration of transcriptome, proteome and phosphoproteome data elucidates the genetic control of molecular networks

Genomic variation affects cellular networks by altering diverse molecular layers such as RNA levels, protein abundance, and post-translational protein modifications. However, it remains unclear how these different layers are affected by genetic polymorphisms and give rise to complex physiological phenotypes. To address these questions, we generated high-quality transcriptome, proteome, and phosphoproteome data for a panel of 112 genetically diverse yeast strains. While genetic effects on transcript abundances were generally transmitted to the protein level, we found a significant uncoupling of the transcript-protein relationship for certain protein classes, such as subunits of protein complexes. The additional phosphoproteomics data suggests that the same genetic locus often affects distinct sets of genes within each of these layers. In particular, QTLs tended to affect upstream regulatory proteins at the phosphorylation layer, whereas downstream pathway targets were typically affected at the transcript and protein abundance layers. Underscoring the importance of regulatory protein phosphorylation in linking genetic to phenotypic variation is the finding that the number of protein phosphosites associated with a given genetic locus was more predictive for its influence on cellular growth traits than the number of transcripts or proteins.\n\nThis study shows how multi-layered molecular networks mediate the effects of genomic variants to more complex physiological traits and highlights the important role of protein phosphorylation in mediating these effects.

systems biology↗

bioRxiv2019

Breast cancer classification based on proteotypes obtained by SWATH mass spectrometry

Accurate breast cancer classification is vital for patient management decisions, and better tumour classification is expected to enable more precise and eventually personalized treatment to improve patient outcomes. Here, we present a novel quantitative proteotyping approach based on SWATH mass spectrometry and establish key proteins for breast tumour classification derived from proteotype data. The study was based on 96 tissue samples representing five breast cancer subtypes according to conventional classification. Correlation of SWATH proteotype patterns indicated groups that largely recapitulate these subtypes. However, the proteotype-based classification also revealed varying degrees of heterogeneity within the conventional subtypes, with triple negative tumours being the most heterogeneous. Proteins that contributed most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2, which are associated with oestrogen receptor status, tumour grade, and HER2 status, respectively. While these three key proteins exhibited high levels of correlation between protein and transcript levels (R>0.67), general correlation did not exceed R=0.29, indicating the value of protein-level measurements of biomarkers and disease-regulated genes. Overall, our data shows how large-scale protein-level measurements by next-generation proteomics can lead to improved patient stratification for precision medicine.

cancer biology↗

bioRxiv2018

The GTPase Nog1 couples polypeptide exit tunnel quality control with ribosomal stalk assembly

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the large subunit precursor (pre-60S), these essential steps include eviction of placeholders Arx1 and Mrt4 that prevent premature loading of the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 recruitment to the pre-60S in order to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity extracts the C-terminal tail of Nog1 from the PET, enabling Rei1 to probe PET integrity, and then catalyze Arx1 release. Subsequently, GTPase hydrolysis stimulates Nog1 removal from the pre-60S, permitting Yvh1 to mediate Mrt4 release, and initiate ribosomal stalk assembly. Thus, Nog1 couples quality control and assembly of spatially distant functional centers during ribosome formation.

biochemistry↗

bioRxiv2018

Two parallel pathways implement robust propionate catabolism and detoxification in mycobacteria

Tuberculosis remains a major global health threat with over 1.5 million deaths each year. Mycobacterium tuberculosis success story is related to a flexible metabolism, allowing growth despite restrictive conditions within the human host.\n\nHost lipids stores are a major carbon source in vivo. Their catabolism yields propionyl-CoA, which is processed by two parallel pathways, the methylmalonyl CoA pathway and the methylcitrate pathway. Both pathways are considered potential drug targets. The methylcitrate pathway is upregulated in the pathological context. However, intermediates of this pathway can be cytotoxic and Mtbs preference for its usage remains unclear.\n\nWe combine thermodynamic kinetic modeling, quantitative proteomics and time-resolved metabolomics to characterize the interplay between the two pathways and to show their functionalities in an efficient and fast propionate catabolism.\n\nWe find that the methylcitrate pathway acts as a transcriptionally regulated, high capacity catabolic pathway due to its favorable thermodynamics and metabolic control distribution. In contrast, the methylmalonyl pathway is constitutively fulfilling biosynthetic tasks and can quickly detoxify propionate pulses, but is thermodynamically restricted to lower capacity.

systems biology↗

bioRxiv2016

Updated reference genome sequence and annotation of Mycobacterium bovis AF2122/97

We report an update to the reference genome of the bovine tuberculosis bacillus Mycobacterium bovis AF2122/97 generated using an integrative multi- omics approach. Updates include 42 new CDS, 14 modified annotations, 26 SNP corrections, and disclosure that the RD900 locus, previously described as absent from the genome, is in fact present.

genomics↗