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

Herbert, R. A.; Deutschbauer, A.

doi:10.1101/2025.11.24.690285

Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.11.24.690285

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

Herbert, R. A.·Deutschbauer, A.

View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Herbert, R. A., Deutschbauer, A.. 2025-11-24. BarTn7: Optimizing Bacterial Lineage Tracking at Sub-Species Resolution for Population Dynamics in Ecological and Evolutionary Studies. https://doi.org/10.1101/2025.11.24.690285

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

The two-component microbial system of the black soldier fly larvae (BSFL) gut: a plastic microbiota in the midgut, but a stable one in the hindgut

Due to their highly polyphagous capacities, black soldier fly (Hermetia illucens) larvae (BSFL) are increasingly valued for their ability to convert organic waste into valuable biomass that can be used for a variety of purposes. These remarkable digestive capabilities are highly dependent on an extremely plastic gut microbiota. However, the distribution and functioning of bacterial communities in the various gut compartments - particularly in the hindgut - remain little understood. In this study, we used a metabarcoding approach based on 16S gene sequencing to investigate the effect of three carbohydrate-rich diets with distinct molecular compositions on the functional diversity of the BSFL gut microbiota. Our results showed that the midgut harbors a highly substrate-sensitive microbiota, with a high abundance of Actinomyces spp., regardless of the substrate. A bacterial diversity oriented toward fatty acid biosynthesis pathways is promoted by starch-rich environment, whereas a lignocellulosic substrate fosters a midgut microbiota dominated by Paenibacillus spp. In contrast, the hindgut exhibits a distinctly stable and homogeneous bacterial composition dominated by Dysgonomonas spp. Overall, our results provide clear evidence of a two-compartment microbial system, in which the midgut primarily serves as a substrate-adaptive primary degradation chamber, while the hindgut functions as a stable terminal compartment for the final processing of residual substrates and the recycling of nutrients. These findings contribute to our understanding of the functional diversity of the bacterial microbiota along the BSFL digestive tract, which is a key factor in explaining this insect's remarkable polyphagous behavior and optimizing its use for industrial purposes.

microbiology↗

bioRxiv2026

Extreme temperature exposure has negative demographic consequences for Sulfolobus acidocaldarius

Microorganisms inhabiting geothermal springs and volcanic systems experience fluctuating temperatures that can periodically exceed their upper thermal limits, but the demographic consequences of such exposure remain poorly understood. Here, we investigated demographic responses of the thermophilic archaeon Sulfolobus acidocaldarius to an extreme temperature (94.1{degrees}C) under two regimes: sustained exposure varying in duration, and episodic exposure interspersed with recovery at a permissive temperature (75{degrees}C). Under sustained exposure, populations showed no detectable loss of viability after 15 min but declined thereafter, decreasing by approximately five orders of magnitude after 120 min. Under episodic exposure, populations remained viable across nine exposure-recovery cycles but declined in density with successive cycles. Similar responses were observed for three strains, including a DNA mismatch repair knockout ({Delta}nucS), indicating that mismatch repair deficiency did not affect viability or recovery. Together, these results demonstrate that S. acidocaldarius can withstand brief and repeated exposure to near-boiling temperatures, with mortality determined primarily by cumulative exposure duration rather than a fixed thermal threshold.

microbiology↗

bioRxiv2026

Bacteriophage and Antibiotic Resistance Are Positively Associated across a Phylogenetically Diverse Set of Clinical Pseudomonas aeruginosa Isolates

Co-administration of phages and antibiotics has been proposed as a therapeutic approach against antibiotic-resistant bacteria. The relationship, however, between antibiotic resistance and phage resistance in clinical isolates is unclear. Here, we examine associations between phage and antibiotic resistance profiles across a panel of Pseudomonas aeruginosa clinical isolates from the Centers for Disease Control (CDC) and Food and Drug Administration (FDA) Antimicrobial Resistance Isolate (ARI) Bank comprising 55 clinical strains with full genome sequences and antibiotic susceptibility testing (AST) data for 11 clinically relevant antibiotics. As phages in this study, we use three well-characterized, morphologically distinct phages, OMKO1, Luz19, and PAML31-1. We screen for phage resistance using a growth suppression assay, then conduct statistical analysis against antibiotic MIC (Minimum Inhibitory Concentration) data provided by the CDC to define association patterns across this dataset. We find multiple significant susceptibility correlations between pairs of antibiotics and phages, and a positive overall association between average phage resistance and antibiotic resistance across the 55 strains, even controlling for phylogenetic associations (=0.358, p<0.005). We conclude that phage and antibiotic resistance are positively associated across this clinical isolate collection, suggesting that the two resistance phenotypes are not independent in P. aeruginosa. These findings have implications for the development of phage-antibiotic cocktails.

microbiology↗