Royzenblat, S. | bioRxiv Science

bioRxiv2026

Mu-seq reveals previously undetected long-range contact patterns in the Escherichia coli nucleoid

The spatial organization of the Escherichia coli chromosome is a key determinant of its functional and regulatory landscape, but widely used chromatin conformation capture methods, such as Hi-C, provide an incomplete view of the underlying three-dimensional genome connectivity. Here, we introduce Mu-seq, a massively multiplexed Mu transposition-based approach that maps genome-wide DNA-DNA proximity in live cells without crosslinking. By distributing engineered, barcoded Mu prophages throughout the chromosome and tracking their replicative transposition events, Mu-seq generates dense, uniform sampling of physical contacts across the genome, enabling a direct, in vivo snapshot of chromosome architecture. Applying this method in E. coli, we constructed a contact dataset spanning over 200,000 unique loci, revealing interaction patterns not accessible to current crosslinking-based techniques. Mu-seq findings can be broken down into three layers. First, it recapitulates polymer-like enrichment of short-range contacts. Second, contacts identified by Mu-seq are strongly shaped by chromatin state: transcriptionally active and accessible regions disproportionately contribute to long-range connectivity, whereas protein-occupied domains, including H-NS-enriched regions, are depleted of insertions and contacts. Third, beyond distance-dependent polymer behavior and chromatin accessibility, Mu-seq reveals specific long-range associations among functionally related loci, such as ribosomal operons. These findings suggest that bacterial chromosome organization can bring functionally related regions into shared three-dimensional neighborhoods even when they are widely separated along the linear genome. Together, these findings support a layered model of nucleoid organization, in which transcriptionally active loops extend outward from a dense, protein-rich axial core. Mu-seq thus provides a powerful, complementary in vivo framework for probing bacterial chromosome architecture, uncovering regulatory and structural features of genome organization that are largely invisible to crosslinking-based approaches.

microbiology↗

bioRxiv2021

An osmotic laxative renders mice susceptible to prolonged Clostridioides difficile colonization and hinders clearance

Antibiotics are a major risk factor for Clostridioides difficile infections (CDIs) because of their impact on the microbiota. However, non-antibiotic medications such as the ubiquitous osmotic laxative polyethylene glycol (PEG) 3350 also alter the microbiota. Clinicians also hypothesize that PEG helps clear C. difficile. But whether PEG impacts CDI susceptibility and clearance is unclear. To examine how PEG impacts susceptibility, we treated C57Bl/6 mice with 5-day and 1-day doses of 15% PEG in the drinking water and then challenged the mice with C. difficile 630. We used clindamycin-treated mice as a control because they consistently clear C. difficile within 10 days post-challenge. PEG treatment alone was sufficient to render mice susceptible and 5-day PEG-treated mice remained colonized for up to 30 days post-challenge. In contrast, 1-day PEG treated mice were transiently colonized, clearing C. difficile within 7 days post-challenge. To examine how PEG treatment impacts clearance, we administered a 1-day PEG treatment to clindamycin-treated, C. difficile-challenged mice. Administering PEG to mice after C. difficile challenge prolonged colonization up to 30 days post-challenge. When we trained a random forest model with community data from 5 days post-challenge, we were able to predict which mice would exhibit prolonged colonization (AUROC = 0.90). Examining the dynamics of these bacterial populations during the post-challenge period revealed patterns in the relative abundances of Bacteroides, Enterobacteriaceae, Porphyromonadaceae, Lachnospiraceae, and Akkermansia that were associated with prolonged C. difficile colonization in PEG-treated mice. Thus, the osmotic laxative, PEG, rendered mice susceptible to C. difficile colonization and hindered clearance. ImportanceDiarrheal samples from patients taking laxatives are typically rejected for Clostridiodes difficile testing. However, there are similarities between the bacterial communities from people with diarrhea or C. difficile infections (CDI) including lower diversity compared to communities from healthy patients. This observation led us to hypothesize that diarrhea may be an indicator of C. difficile susceptibility. We explored how osmotic laxatives disrupt the microbiotas colonization resistance to C. difficile by administering a laxative to mice either before or after C. difficile challenge. Our findings suggest that osmotic laxatives disrupt colonization resistance to C. difficile, and prevent clearance among mice already colonized with C. difficile. Considering that most hospitals recommend not performing C. difficile testing on patients taking laxatives and laxatives are prescribed prior to administering fecal microbiota transplants via colonoscopy to patients with recurrent CDIs, further studies are needed to evaluate if laxatives impact microbiota colonization resistance in humans.

microbiology↗