Biology subjects

Stadler, L. B.

Publications and source records attributed to Stadler, L. B..

8 recordsLinked to original sources

bioRxiv2026

Microfluidic Capture-Enrichment of individualized microbes from urban wastewater reveals a hidden reservoir of potential pre-emergent pathogens

Current environmental biosurveillance infrastructure excels at tracking known biological threats but is limited in its ability to proactively identify potential pre-emergent pathogens (PEPs). To address this gap, we developed a microfluidics-based Capture-Enrichment pipeline that isolates individual environmental cells via microdroplet encapsulation, preserving community biodiversity while enabling iterative selection against human sera. When applied to urban wastewater, this approach successfully recovered pathogens, commensals, and PEPs that fell below the detection limits of conventional amplicon sequencing. Genomic analysis revealed that wastewater-derived ESKAPE pathogens cluster closely with clinical isolates, showing that the pipeline captures clinically relevant threats. Furthermore, isolated PEPs exhibited known and emerging phenotypes associated with early-stage pathogenesis and antimicrobial resistance. These findings demonstrate that targeting PEPs can fundamentally expand the scope and quality of biosurveillance by providing a scalable strategy to identify, characterize, and close important gaps in our understanding of emerging microbial pathogens.

microbiology↗

bioRxiv2026

Storing >1 byte of information in 16S ribosomal RNA using orthogonal trans-splicing ribozymes

TEXT ABSTRACTCatalytic-RNA (cat-RNA) expressed from mobile DNA can record cellular events, such as the uptake of plasmids via horizontal gene transfer, by splicing a barcode onto 16S ribosomal RNA (rRNA) - a system termed RNA addressable modification (RAM). However, scaling RAM to record multiple simultaneous biological events requires large numbers of orthogonal cat-RNA whose signals reflect the biological features under investigation rather than variability arising from the barcode sequence. Here, we explore how to design orthogonal cat-RNA to record information about multiple plasmid-encoded traits in parallel. We show that cat-RNA having tRNA-derived barcodes with sequence variation in the anticodon stem-loop present greater signal consistency within Escherichia coli than mRNA-derived barcodes. When orthogonal cat-RNA designs harboring tRNA-derived barcodes were evaluated in Vibrio natriegens and Pseudomonas putida, increased variance was observed compared with Escherichia coli. Nevertheless, the signal consistency was sufficient to use these orthogonal cat-RNAs to report on the relative activities of four promoters and two origins of replication by sequencing barcoded-rRNA derived from the three organisms. These results show how RAM can be multiplexed to report on mobile DNA features in microbial communities and illustrate the importance of accounting for variability in RNA outputs when designing and interpreting multiplexed RNA barcoding data. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/738544v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f6d62aorg.highwire.dtl.DTLVardef@6696c6org.highwire.dtl.DTLVardef@134aeb4org.highwire.dtl.DTLVardef@1b9ceeb_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Biocontainment attenuation of mobile DNA host range in a wastewater microbiome

Biocontainment systems designed to attenuate the spread of mobile DNA are challenging to evaluate within microbiomes of engineered environments. To better understand how toxin-based biocontainment systems affect horizontal gene transfer (HGT) in a microbiome, we evaluated the host range of pairs of plasmids using orthogonal catalytic RNA (cat-RNA) that amend distinct barcodes to 16S rRNA following HGT. We show that mobilizable (5 kb) and self-mobilizable (60 kb) plasmids, which use the same RP4 transfer machinery but different origins of replication, overlap in their host range when conjugated in parallel into a wastewater community, with 127 of the 143 amplicon sequence variants (ASVs) presenting barcoding signals from both plasmids (89%). We also find that mobilizable plasmids with or without the Escherichia coli CcdB toxin overlap in host range in a wastewater community. Among the two most abundant orders, CcdB attenuated the barcoding signal in Aeromonadales more consistently than Enterobacteriales, which have F plasmids containing the CcdB-CcdA toxin-antitoxin system used for biocontainment. Also, CcdB decreased the abundance of the mobilizable plasmid by >100-fold and yielded mutations in 85% of the reads. Together, these findings reveal how pairs of plasmids expressing orthogonal cat-RNA can be used to monitor the effects of plasmid-encoded traits on mobile DNA persistence following HGT. They also highlight challenges when using biocontainment systems containing genes related to those found in the microbiomes targeted for engineering.

synthetic biology↗

bioRxiv2025

Seqwin: Ultrafast Identification of Signature Sequences in Microbial Genomes

MotivationPolymerase chain reaction (PCR) enables rapid, cost-effective diagnostics but requires prior identification of genomic regions that allow sensitive and specific detection of target microbial groups, herein referred to as microbial signature sequences. We introduce Seqwin, an open-source framework designed to automate microbial genome signature discovery. Tens of thousands of microbial genomes are now available for a single species, limiting the application of existing manual and automated approaches for identifying signatures. Modern approaches that are capable of leveraging all available microbial genomes will ensure sensitive and accurate DNA signature identification and enable robust pathogen detection for clinical, environmental, and public health applications. ResultsSeqwin builds weighted pan-genome minimizer graphs and uses a traversal algorithm to identify signature sequences that occur frequently in target genomes but remain rare in non-targets. Unlike earlier tools that depend on strict presence or absence of sequences, Seqwin accommodates natural sequence variation and scales to very large genome collections. When applied to genomes from C. difficile, M. tuberculosis, and S. enterica, Seqwin recovered more high-quality signatures than alternative methods with lower computational burden. Seqwins analysis of nearly 15,000 S. enterica genomes yielded over 200 candidate signatures in 5 minutes. Seqwin provides an open-source solution for the long-standing need for scalable microbial signature discovery and diagnostic assay design. Availability and ImplementationSeqwin is freely available for academic use (https://github.com/treangenlab/Seqwin) and can be installed via Bioconda. Benchmarking datasets, outputs, and scripts are available on Zenodo https://doi.org/10.5281/zenodo.19176444. Contacttreangen@rice.edu, xw66@rice.edu Supplementary MaterialsProvided as separate PDF and data files.

bioinformatics↗

bioRxiv2025

Cross-order detection of bacteriophage transduction in communities using ribosomal RNA barcoding

Bacteriophages (phages) facilitate gene transfer and microbial evolution in all ecosystems and have applications as tools for engineering microbiomes and as antimicrobials. Historic efforts to map phage hosts, such as plaque assays, are limited to culturable bacteria, are low throughput, and are hard to apply in environmentally-relevant contexts. To overcome these limitations, a synthetic ribozyme that stores information about DNA uptake in 16S ribosomal RNA (rRNA) was used to identify phage-host interactions by integrating the ribozyme into phage-plasmid P1 and performing targeted 16S rRNA sequencing following transduction. Experiments in synthetic and wastewater communities revealed Aeromonadales as a novel P1 host order and transduction of P1 into pathogens. Host range varied across phagemids with different origins of replication and phage particles with different tail fibers. This work shows how autonomous barcoding can be used in phages to identify the molecular controls on their host range in communities.

synthetic biology↗

bioRxiv2023

Long duration environmental biosensing by recording analyte detection in DNA using recombinase memory

Microbial biosensors that convert environmental information into real-time visual outputs are limited in their sensing abilities in complex environments, such as soil and wastewater. Alternative reporter outputs are needed that stably record the presence of analytes. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12- homoserine lactone) over 8 days ([~]70 generations) following analyte exposure. These biosensors use analyte sensing to trigger the expression of a recombinase which flips a segment of DNA, creating a genetic memory, and initiates fluorescent protein expression. The initial designs failed over time due to unintended DNA flipping in the absence of the analyte and loss of the flipped state after exposure to the analyte. Biosensor performance was improved by decreasing recombinase expression, removing the fluorescent protein output, and using qPCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterized Pseudomonas isolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration and in situ investigation of microbial signaling, community shifts, and gene transfer beyond the reach of traditional environmental biosensors. IMPORTANCELiving microbial sensors can monitor chemicals and biomolecules in the environment in real-time, but they remain limited in their ability to function on the week, month, and year timescales. To determine if environmental microbes can be programmed to record the detection of analytes over longer timescales, we evaluated whether the sensing of a microbial signaling molecule could be recorded through a DNA rearrangement. We show that off-the-shelf DNA memory is suboptimal for long-duration information storage, use iterative design to enable robust functioning over more than a week, and demonstrate DNA memory in an uncharacterized wastewater Pseudomonas isolate. Memory biosensors will be useful for monitoring the role of quorum sensing in wastewater biofilm formation, and variations of this design are expected to enable studies of ecological processes in situ that are currently challenging to monitor using real-time biosensors and analytical instruments.

synthetic biology↗

bioRxiv2023

Information storage across a microbial community using universal RNA memory

Biological recorders can code information in DNA, but they remain challenging to apply in complex microbial communities. To program microbiome information storage, a synthetic catalytic RNA (cat-RNA) was used to write information in ribosomal RNA (rRNA) about gene transfer host range. By reading out native and modified rRNA using amplicon sequencing, we find that 140 out of 279 wastewater microbial community members from twenty taxonomic orders participate in conjugation and observe differences in information storage across amplicon sequence variants. Twenty of the variants were only observed in modified rRNA amplicons, illustrating information storage sensitivity. This autonomous and reversible RNA-addressable memory (RAM) will enable biosurveillance and microbiome engineering across diverse ecological settings and studies of environmental controls on gene transfer and cellular uptake of extracellular materials. One-Sentence SummaryRibosomal RNA sequencing detects cellular events recorded across a wastewater microbial community using synthetic biology.

synthetic biology↗

bioRxiv2023

Olivar: fully automated and variant aware primer design for multiplex tiled amplicon sequencing of pathogen genomes

Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 2 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer SNPs overlapping with primers and predicted PCR byproducts. We also compared Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates ([~]90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluated Olivar on real wastewater samples and found that Olivar had up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available as a web application at https://olivar.rice.edu. Olivar can also be installed locally as a command line tool with Bioconda. Source code, installation guide and usage are available at https://github.com/treangenlab/Olivar.

bioinformatics↗