Biology subjects

Mayer, T.

Publications and source records attributed to Mayer, T..

7 recordsLinked to original sources

bioRxiv2025

Operating CRISPR/Cas12a in a complex nucleic acid sequence background

Since their discovery, CRISPR-Cas systems have been widely applied in areas ranging from genome editing to biosensing, owing to their specific, RNA-guided target recognition. Their performance in complex biological environments has been extensively studied, particularly to optimize guide RNA design and minimize off-target cleavage. Here, we focus on the kinetic inhibition of the interaction between Cas12a - a Class 2, Type V effector - and its target, caused by interference from non-cognate background nucleic acids. This effect is particularly relevant for sensing applications in complex mixtures or cellular contexts, where genome- and transcriptome-derived sequences may impede CRISPR-Cas activity. Using in vitro assays under defined conditions, we systematically examine the influence of background single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) on reaction kinetics. We find that both the purine-to-pyrimidine ratio and the GC content of the guide RNA seed region significantly affect kinetic inhibition by background polynucleotides. Guide RNAs with low GC content and a high purine fraction in the seed region were least affected by background sequences. Experiments with dCas12a-based gene activation in living cells indicate that our in vitro findings may also be relevant for in vivo applications.

biophysics↗

bioRxiv2025

Systematic non-natural base-pairs for strand displacement circuits in complex environments

Nucleic acid strand displacement (SD) circuits have enabled impressive realizations of molecular computing in vitro, but operating them in contention with competing reaction pathways can cause signal leak and circuit failure. Further, operating SD circuits in complex environments, ranging from pools of nucleic acids to living organisms, can exacerbate these challenges. To address these issues, we explore the incorporation of isoC and isoG non-natural bases in single- and multi-step SD cascades and started by studying their promiscuity. We combined the non-natural bases with a mismatch strategy, showing them to be fully compatible. The synthesis we show between strategic placement of non-natural bases and systematic mismatch optimization of SD circuits offers a design space for future circuits that are fast, robust, and biologically compatible. Comparing circuit slowdowns by random pool backgrounds in the presence and absence of non-natural base pairs reveals a significant difference. Using isoC:isoG base pairs in the toehold region shows the greatest effect, with some further positive effects caused by additional non-natural bases in the branch migration domain. Such insights will pave the way for developing SD circuits that operate in complex environments, expanding the applications of molecular computing in a wider range of scenarios.

bioengineering↗

bioRxiv2024

Deterministic colonization arises early during the transition of soil bacteria to the phyllosphere and is shaped by plant-microbe interactions.

BackgroundUpon seed germination, soil bacteria are activated to transition to the plant and eventually colonize mature tissues like leaves. These bacteria are poised to significantly influence plant health, but we know little about their colonization routes. We studied the mechanisms of the transition of soil bacteria to germinating plants and leaves by experimentally manipulating inoculation times and using in-planta isolation to understand bacteria that can make the complex soil-to-leaf transition. Using a trackable, labeled Pseudomonas viridiflava (Pv3D9) amended to soil, we tested how plant-microbe-microbe interactions shape assembly mechanisms in natural soil communites. ResultsWe found that the stages of the transition of bacteria from soil to leaves before true leaf emergence were important in establishing uniquely diverse leaf bacteriomes. A wide diversity of leaf-associated taxa can individually make this transition, but most are still shaped by stochastic processes. Interestingly, deterministic processes drove some important taxa only when they transitioned from soil to leaves, but not when they were inoculated later. The opportunistic pathogen Pv3D9 promoted plant growth in a natural soil, likely by activating plant immunity. These effects in turn strongly affected the soil-to-leaf transition of almost strictly taxa that colonized deterministically, demonstrating the important role of plant-microbe-microbe interactions in controlling deterministic processes. ConclusionsDiverse, well-adapted bacterial taxa make the soil-to-leaf transition during natural colonization resulting in characteristic diversity in healthy leaf microbiomes. The domination of stochastic mechanisms during this colonization indicates that many taxa must strongly compete to establish their niche. During this complex transition, however, specific important taxa emerge that are driven by deterministic processes, suggesting they occupy unique niches. The malleability of these processes suggests that they may be controlled, for example by targeted soil manipulations. This finding is significant given the important roles of these bacteria in plant health and offers directions forward for engineering beneficial plant microbiomes.

microbiology↗

bioRxiv2023

Beyond defense: Glucosinolate structural diversity shapes recruitment of a metabolic network of leaf-associated bacteria

Leaf bacteria are critical for plant health, but little is known about how plant traits control their recruitment. Aliphatic glucosinolates (GLSs) are secondary metabolites present in leaves of Brassicaceae plants in genotypically-defined mixtures. Upon damage, they are broken down to products that deter herbivory and inhibit pathogens. Using two A. thaliana genotypes with different aliphatic GLS profiles, we find that structural variants differentially affect commensal leaf bacteria: In the model genotype Col-0, GLS breakdown products (mostly from 4-methylsulfinylbutyl-glucosinolate) are potentially highly toxic to bacteria but have no effect on natural leaf colonization. In contrast, in an A. thaliana genotype from a wild population, GLS (mostly allyl-GLS) enriches Burkholderiales bacteria, an effect also detected in nature. Indeed, in-vitro as a carbon source, intact allyl-GLS specifically enriches a Burkholderiales-containing community in which Burkholderiales depend on other bacteria but in turn increase community growth rates. Metabolism of different GLSs is linked to breakdown product detoxification, helping explain GLS structural control of community recruitment.

microbiology↗

bioRxiv2022

Toehold-Mediated Strand Displacement in Random Sequence Pools

Toehold-mediated strand displacement (TMSD) has been used extensively for molecular sensing and computing in DNA-based molecular circuits. As these circuits grow in complexity, sequence similarity between components can lead to cross-talk causing leak, altered kinetics, or even circuit failure. For small non-biological circuits, such unwanted interactions can be designed against. In environments containing a huge number of sequences, taking all possible interactions into account becomes infeasible. Therefore, a general understanding of the impact of sequence backgrounds on TMSD reactions is of great interest. Here, we investigate the impact of random DNA sequences on TMSD circuits. We begin by studying individual interfering strands and use the obtained data to build machine learning models that estimate kinetics. We then investigate the influence of pools of random strands and find that the kinetics are determined by only a small subpopulation of strongly interacting strands. Consequently, their behavior can be mimicked by a small collection of such strands. The equilibration of the circuit with the background sequences strongly influences this behavior, leading to up to one order of magnitude difference in reaction speed. Finally, we compare two established and a novel technique that speed up TMSD reactions in random sequence pools: a threeletter alphabet, protection of toeholds by intramolecular secondary structure, or by an additional blocking strand. While all of these techniques were useful, only the latter can be used without sequence constraints. We expect that our insights will be useful for the construction of TMSD circuits that are robust to molecular noise.

bioengineering↗

bioRxiv2022

Leaf bacterial community structure and variation in wild ruderal plants are shaped by the interaction of host species and defense chemistry with environment

Variable phenotypes help plants ensure fitness and survival in the face of unpredictable environmental stresses. Leaf bacteria (bacteriomes) can extend plant phenotypes and are well-known to vary from one plant to the next, but little is known about controls on this variation. Here, we find in 9 populations of Arabidopsis thaliana that core leaf bacteriomes are largely, but not completely, shared with other ground-dwelling ruderal plant species. Strength of differentiation between plant species and between A. thaliana populations shifts from year to year, becoming stronger when plants within populations have more similar leaf bacteriomes (less plant-to-plant variation or stochasticity). Interestingly, across different populations, plants with shared leaf aliphatic glucosinolate chemotypes exhibited similar strong year-to-year stochasticity shifts. Therefore, stochasticity of leaf bacteriomes in plant populations changes in specific ways and might be controlled by plant traits, with important implications for how plants adapt to complex and shifting environments.

ecology↗

bioRxiv2020

Obtaining deeper insights into microbiome diversity using a simple method to block host and non-targets in amplicon sequencing

Microbiome profiling is revolutionizing our understanding of biological mechanisms such as metaorganismal (host+microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci is an instrumental tool for characterization of the highly diverse microbiomes of natural systems. Investigations in many study systems are hindered by loss of essential sequencing depth due to amplification of non-target DNA from hosts or overabundant microorganisms. This issue requires urgent attention to address ecologically relevant problems using high throughput, high resolution microbial profiling. Here, we introduce a simple, low cost and highly flexible method using standard oligonucleotides ("blocking oligos") to block amplification of non-targets and an R package to aid in their design. They can be dropped into practically any two-step amplicon sequencing library preparation pipeline. We apply them in leaves, a system presenting exceptional challenges with host and non-target microbial amplification. Blocking oligos designed for use in eight target loci reduce undesirable amplification of host and non-target microbial DNA by up to 90%. In addition, 16S and 18S "universal" plant blocking oligos efficiently block most plant hosts, leading to increased microbial alpha diversity discovery without biasing beta diversity measurements. By blocking only chloroplast 16S amplification, we show that blocking oligos do not compromise quantitative microbial load information inherent to plant-associated amplicon sequencing data. Using these tools, we generated a near-complete survey of the Arabidopsis thaliana leaf microbiome based on diversity data from eight loci and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. The blocking oligo approach has potential to make new questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems-based insights into microbial discovery.

microbiology↗