Biology subjects

Bergmiller, T.

Publications and source records attributed to Bergmiller, T..

5 recordsLinked to original sources

bioRxiv2026

Biocontainment of phages inhibits bacterial clearance in micro niches

Phage therapy offers a promising solution to the antimicrobial resistance crisis. However, a major concern preventing the adoption of phage therapy is the potential for unintended consequences of phage release; both in regard to preventing the spread of phage resistance, and the proliferation of a non-endemic virus into the microbial ecosystem. Conditional replication (biocontainment) of phages through bioengineering may address these concerns, but the impact on bactericidal efficacy is unknown. Here, we created a biocontained T7 phage (T7{Delta}capsid) lacking the major structural capsid gene, gp10AB, that can only replicate on Escherichia coli strains expressing gp10AB in trans, and assessed its bactericidal efficacy compared with wild-type T7. Congruent with model predictions, T7{Delta}capsid was only able to clear a well-mixed culture of E. coli at a multiplicity of infection (MOI) of 10 or higher, whereas wild-type T7 prohibited growth at an MOI of 0.1. The reduction in efficacy was more evident in a complex structured environment within a microfluidic device, where phage success depends on its ability to penetrate a microbial niche via propagation. In this environment, T7{Delta}capsid was unable to propagate into the bacterial population and unlike wild-type T7, had no impact on the populations growth. This study shows that whilst biocontainment of phages may improve the biosafety of phage therapy, it comes at the cost of its propagation efficacy and niche penetration in relevant environments.

microbiology↗

bioRxiv2024

Programming CRISPRi-dCas9 to control the lifecycle of bacteriophage T7

CRISPR interference (CRISPRi) based on catalytically dead Cas9 nuclease of Streptococcus pyogenes is a programmable and highly flexible tool to interrogate gene function and essentiality in bacteria due to its ability to block transcription elongation at nearly any desired DNA target. Here, I assess how CRISPRi-dCas9 can be programmed to control the life cycle and infectivity of Escherichia coli bacteriophage T7, a highly virulent and obligatory lytic phage, by blocking critical host-dependent promoters that are required for host cell entry and life cycle execution. Using fluorescent reporters demonstrates that the efficacy of CRISPRi-dCas9 towards E. coli RNAP promoters depends on target promoter strength, and that the phages own T7 RNAP can be downregulated very efficiently. Effects on the time to lysis were partially dictated by the chronological order of phage DNA and dCas9 target appearance in the cell, suggesting an accessory role of E. coli RNAP during the early stages of T7 genome ejection. The stringency of the CRISPRi-dCas9 approach was greatly improved when using multiplex sgRNAs to target multiple phage regions simultaneously, resulting in a 25% increase in the time to lysis and up to 8-fold reduction in plaque size. Overall, this work expands CRISPRi-dCas9 as a flexible tool to non-invasively manipulate and probe the lifecycle and infectivity of otherwise native T7 phage.

synthetic biology↗

bioRxiv2023

Bacterial toxin-antitoxin system MazEF as a native defense mechanism against RNA phages in Escherichia coli

Bacteria have evolved a wide range of defense strategies to protect themselves against bacterial viruses (phages). However, the known mechanisms almost exclusively target phages with DNA genomes. While several bacterial toxin-antitoxin systems have been considered to cleave single-stranded bacterial RNA in response to stressful conditions, their role in protecting bacteria against phages with single-stranded RNA genomes has not been studied. Here we investigate the role of a representative toxin-antitoxin system, MazEF, in protecting Escherichia coli against two RNA phages - MS2 and Q{beta}. Our population-level experiments revealed that a mazEF deletion strain is more susceptible to RNA phage infection than the wild-type. At the single-cell level, deletion of the mazEF locus significantly shortened the time to lysis of individual bacteria challenged with RNA phage. At the genomic level, we found that the adenine-cytosine-adenine sequence, directly recognized and cleaved by the MazF toxin, is systematically underrepresented in the genomes of RNA phages that are known to infect E. coli, indicating selection for decreased probability of cleavage. These results suggest that in addition to other physiological roles, RNA-degrading toxin-antitoxin modules can function as a primitive immune system against RNA phages.

microbiology↗

bioRxiv2022

Real-time dynamics of individual chemoreceptor mRNA molecules reveals translation hotspots at the inner membrane of Escherichia coli

Since bacteria lack a nucleus, the location of mRNA molecules is determined by the different characteristics of the encoded proteins, and the transcriptome is spatially arranged into cytosolic and membrane-associated mRNA. While translation of membrane protein-encoding mRNA has been studied in great mechanistic detail using biochemical methods, the spatiotemporal dynamics of this process remains poorly understood at the subcellular level. Here, we investigate the dynamics of individual fluorescently labelled mRNA molecules encoding the transmembrane serine chemoreceptor Tsr, to probe the mechanism of membrane protein translation. Analysis of tsr mRNA diffusion in the proximity of the plasma membrane revealed distinct diffusive modes that reflect the state of the mRNA molecule and its involvement in the process of active translation into the Sec secretion system. We find that the composition, and hence the fluidity of the membrane affects diffusion of membrane targeted mRNAs. Moreover, Tsr translation occurs in localized membrane regions, similar to eukaryotic hotspots. The hotspot localization coincides with the physical location of the transcribed gene, which itself is displaced towards the inner membrane. These findings suggest that inner membrane protein translation is a spatially defined process that occurs in hotspots, indicative of long-lived transertion sites. Our results show an additional layer of spatio-temporal structuring within the bacterial cell, thus revealing a qualitatively different understanding of the basic process of transcription and translation in bacteria. Significance StatementA large fraction of the bacterial proteome is directly synthesized into the inner membrane, and this process shapes the overall distribution of mRNA transcripts within the cell. Although highly dynamic in their nature, bacterial transcriptomes have mostly been studied in fixed cells. Here, we track individual mRNA molecules encoding the serine chemoreceptor in living bacterial cells and find that translation occurs in membrane hotspots that were previously exclusive to eukaryotes. Our results indicate an additional layer of spatio-temporal structuring within the bacterial cell that impacts our understanding of transcription and translation in bacteria.

microbiology↗

bioRxiv2019

Fluorescence photography of patterns and waves of bacterial adaptation at high antibiotic doses

Fisher suggested advantageous genes would spread through populations as a wave so we sought genetic waves in evolving populations, as follows. By fusing a fluorescent marker to a drug efflux protein (AcrB) whose expression provides Escherichia coli with resistance to some antibiotics, we quantified the evolution and spread of drug-resistant E. coli through spacetime using image analysis and quantitative PCR. As is done in hospitals routinely, we exposed the bacterium to a gradient of antibiotic in a disk diffusion drug susceptibility test that we videoed. The videos show complex spatio-genomic patterns redolent of, yet more complex than, Fishers predictions whereby a decelerating wave front of advantageous genes colonises towards the antibiotic source, forming bullseye patterns en route and leaving a wave back of bacterial sub-populations expressing AcrB at decreasing levels away from the drug source. qPCR data show that E. coli sited at rapidly-adapting spatial hotspots gain 2 additional copies of acr, the operon that encodes AcrB, within 24h and imaging data show resistant sub-populations thrive most near the antibiotic source due to non-monotone relationships between inhibition due to antibiotic and distance from the source. In the spirit of Fisher, we provide an explicitly spatial nonlinear diffusion equation that exhibits these properties too. Finally, linear diffusion theory quantifies how the spatial extent of bacterial killing scales with increases in antibiotic dosage, predicting that microbes can survive chemotherapies that have been escalated to 250x the clinical dosage if the antibiotic is diffusion-limited.

evolutionary biology↗