Biology subjects

Stadler, L.

Publications and source records attributed to Stadler, L..

6 recordsLinked to original sources

bioRxiv2026

Rational engineering enhances the signal and modularity of an RNA barcoding technology to track gene transfer in microbiomes

Horizontal gene transfer drives microbial evolution and offers a powerful strategy for precision microbiome engineering. To track gene transfer within complex communities, we previously developed RNA-Addressable Modification (RAM), an RNA-barcoding technology where a mobile catalytic RNA barcodes host 16S ribosomal RNA (rRNA) upon gene transfer. However, the first-generation RAM suffered from low barcoding efficiency and lacked modularity, limiting its sensitivity and versatility. Here, we present RAM v2, a re-engineered system with significantly enhanced performance and modularity. By incorporating natural ribozyme structural motifs and improved barcode stability, we achieved a [~]200-fold increase in barcoded rRNA signal. To enhance modularity, we integrated CRISPRi-based repression and ribozyme insulators, facilitating easy promoter swapping. We validated RAM v2 on a mobilisable plasmid delivered to a complex wastewater microbial community, demonstrating a substantial increase in signal over the original system while barcoding similar taxa. These improvements enable higher-resolution, more sensitive monitoring of horizontal gene transfer, providing a robust toolkit for accelerating the study of gene transfer in microbial communities and advancing targeted microbiome engineering.

synthetic biology↗

bioRxiv2025

Controlling the taxonomic composition of biological information storage in 16S ribosomal RNA

Microbes can be programmed to record participation in gene transfer by coding biological-recording devices into mobile DNA. Upon DNA uptake, these devices transcribe a catalytic RNA (cat-RNA) that binds to conserved sequences within ribosomal RNA (rRNA) and perform a trans-splicing reaction that adds a barcode to the rRNA. Existing cat-RNA designs were manually generated to be broad-host range, providing no control over the organisms that were barcoded. To achieve control over the organisms barcoded by cat-RNA, we created a program called Ribodesigner that uses input sets of rRNA sequences to create designs with varying specificities. We show how this algorithm can be used to identify designs that enable kingdom-wide barcoding, or selective barcoding of specific taxonomic groups within a kingdom. We use Ribodesigner to create cat-RNA designs that target Pseudomonadales while avoiding Enterobacterales, and we compare the performance of one design to a cat-RNA that was previously found to be broad host range. When conjugated into a mixture of Escherichia coli and Pseudomonas putida, the new design presents increased selectivity compared to a broad host range cat-RNA. Ribodesigner is expected to aid in developing cat-RNA that store information within user-defined sets of microbes in environmental communities for gene transfer studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/651329v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@bcc65borg.highwire.dtl.DTLVardef@18288f2org.highwire.dtl.DTLVardef@ecf5c7org.highwire.dtl.DTLVardef@fbe064_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Comparative analysis of culture- and ddPCR-based wastewater surveillance for carbapenem-resistant bacteria

With the widespread use of last-resort antibiotics, carbapenems, clinical reports of infections associated with carbapenem-resistant Enterobacterales (CRE) have increased. Clinical surveillance for CRE involves susceptibility testing and/or whole genome sequencing of resistant isolates, which is laborious, resource intensive, and requires expertise. Wastewater surveillance can potentially complement clinical surveillance of CRE, and population-level antibiotic resistance (AR) surveillance more broadly. In this study, we quantitatively and qualitatively compared two widely used methods for AR wastewater surveillance: (1) a culture-based approach for quantifying carbapenem-resistant bacteria and (2) a digital droplet PCR (ddPCR) assay targeting five major carbapenemase genes. We developed a multiplexed ddPCR assay to detect five carbapenemase genes and applied it to wastewater samples from three sites over 12 weeks. In parallel, we quantified carbapenem resistant bacteria and carbapenemase-producing bacteria using culture-based methods. We assessed associations between the concentrations of carbapenemase genes and resistant bacteria. Although both approaches showed similar trends in the overall abundance of dominant carbapenem-resistant bacteria and genes, there were weak correlations between the quantitative levels of resistance. Nanopore sequencing of the resistome of the carbapenem-resistant bacteria revealed that discrepancies arose from differences in the sensitivity and specificity of the methods. This study enhances our understanding of the application of wastewater surveillance in tracking carbapenem resistance and highlights how method choice impacts the results from AR wastewater surveillance.

microbiology↗

bioRxiv2020

On a reef far, far away: Offshore transport of floodwaters following extreme storms impacts sponge health and associated microbial communities

Terrestrial runoff can negatively impact marine ecosystems through stressors including excess nutrients, freshwater, sediments, and contaminants. Severe storms, which are increasing with global climate change, generate massive inputs of runoff over short timescales (hours to days); such runoff impacted offshore reefs in the northwest Gulf of Mexico (NW GoM) following severe storms in 2016 and 2017. Several weeks after coastal flooding from these events, NW GoM reef corals, sponges, and other benthic invertebrates -185 km offshore experienced mortality (2016 only) and/or sub-lethal stress (both years). To assess the impact of storm-derived runoff on reef filter feeders, we characterized the bacterial communities of two sponges, Agelas clathrodes and Xestospongia muta, from offshore reefs during periods of sub-lethal stress and no stress over a three-year period (2016-2018). Sponge-associated bacterial communities were altered during both flood years. Additionally, we found evidence of wastewater contamination (based on 16S rRNA gene libraries and quantitative PCR) in offshore sponges following these flood years, but not during the no-flood year. We show that flood events from severe storms have the capacity to reach offshore reef ecosystems and impact resident benthic organisms. Such impacts are most readily detected if baseline data on organismal physiology and associated microbiome composition are available. This highlights the need for molecular and microbial time series of benthic organisms in near- and offshore reef ecosystems, and the continued mitigation of stormwater runoff and climate change impacts.

microbiology↗

bioRxiv2019

Comparative analysis of intracellular and extracellular antibiotic resistance gene abundance in anaerobic membrane bioreactor effluent

The growing practice of wastewater reuse poses a significant risk to further dissemination of antibiotic resistance due to the abundance of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in wastewater effluents. Anaerobic membrane bioreactors (AnMBRs) are an emerging wastewater treatment technology capable of reducing the total ARGs and ARB load discharged to receiving environments compared to conventional aerobic treatment processes. While size exclusion is effective at retaining ARB and its associated intracellular ARGs, the abundance and fate of extracellular ARGs in an AnMBR effluent have not been examined. This study elucidates the effect of combined antibiotics loading (ampicillin, erythromycin, and sulfamethoxazole) on the abundance of intracellular and extracellular ARGs in an AnMBR effluent over a period of five weeks. Quantification of targeted genes revealed an overall enrichment of intracellular ARGs (iARGs) and depletion of extracellular (exARGs) in response to antibiotics addition, which suggests exARG uptake as a significant mode of horizontal gene transfer in AnMBR effluents. Comparison of the iARG and exARG abundance profiles showed a potential bias for exARG uptake located on small plasmids compared to large plasmids.\n\nImportanceAntibiotic resistance dissemination is facilitated through horizontal gene transfer (HGT) of ARGs. Currently, conjugation is considered to be the dominant mechanism during wastewater treatment. However, recent studies have detected high abundances of exARGs, implying that transformation may play a greater role in dissemination. While previous studies quantified iARGs and exARGs in wastewater treatment facilities, they did not evaluate temporal changes between the two forms. Further, almost no research has differentiated between iARGs and exARGs in anaerobic processes, which are being considered to replace aerobic activated sludge processes. This study specifically investigates the abundance of targeted iARGs and exARGs in AnMBRs in response to antibiotic pressure to quantify potential exchange of ARGs between intracellular and extracellular compartments. Our findings suggest that exARGs located on small plasmids are preferentially taken up by cells under antibiotic pressure compared to large plasmids, which implies heterogenous HGT mechanisms among the plasmid community.

ecology↗

bioRxiv2018

Diffusion of exit sites on the endoplasmic reticulum - a random walk on a shivering backbone

Major parts of the endoplasmic reticulum (ER) in eukaryotic cells are organized as a dynamic network of membrane tubules connected by three-way junctions. On this network, self-assembled membrane domains, called ER exit sites (ERES), provide platforms at which nascent cargo proteins are packaged into vesicular carriers for subsequent transport along the secretory pathway. While ERES appear stationary and spatially confined on long time scales, we show here via single-particle tracking that they exhibit a microtubule-dependent anomalous diffusion behavior on short and intermediate time scales. By quantifying key parameters of their random walk, we show that the subdiffusive motion of ERES is distinct from that of ER junctions, i.e. ERES are not tied to junctions but rather are mobile on ER tubules. We complement and corroborate our experimental findings with model simulations that also indicate that ERES are not actively moved by microtubules. Altogether, our study shows that ERES perform a random walk on the shivering ER backbone, indirectly powered by microtubular activity. Similar phenomena can be expected for other domains on subcellular structures, setting a caveat for the interpretation of domain tracking data.

biophysics↗