Biology subjects

Brandis, G.

Publications and source records attributed to Brandis, G..

5 recordsLinked to original sources

bioRxiv2026

Spaghettification: How extreme replichore imbalance impacts bacterial replication and morphology

Bacterial chromosomes are typically organized into two similarly sized replichores to synchronize bidirectional replication and fusion within a Tus/ter-defined termination zone. Previous studies have suggested that replichore balance in both Salmonella and E. coli is maintained by selection for efficient and properly coordinated replication termination and chromosome segregation. However, there is a lack of understanding of the causal relationship between replichore asymmetry, cellular fitness, and replication-fork convergence, as well as how the termination machinery behaves when the two replichores differ substantially in length. Here we dissect the impact of replichore asymmetry by engineering 41 isogenic Salmonella inversion mutants spanning progressive imbalances, complemented by duplication-based asymmetries. Our constructed strains show that even cells with severely imbalanced chromosomes, where one replichore is 11-fold longer in size than the other (0.38 Mb vs. 4.48 Mb) are viable. However, relative fitness declined linearly with replichore size difference at approximately -0.17 per Mb. Additionally, highly imbalanced strains exhibited substantial division defects with severe cell elongation and lysis. Replication profiling showed that, even under extreme replichore asymmetry, forks consistently converge at the innermost ter sites of the shorter replichore and that two sequential Tus/ter barriers were sufficient for complete replication fork arrest. Together, these results establish a quantitative rule linking replichore asymmetry to fitness, define the termination position under imbalance, and reveal a dual Tus/ter barrier that safeguards termination when one replichore reaches the terminus region ahead of the other. Collectively these results connect chromosome architecture to replication mechanics and cell-division outcomes.

microbiology↗

bioRxiv2026

16S rRNA Modifications Are Dispensable for Viability but Collectively Optimize Ribosome Biogenesis and Translation Initiation in Escherichia coli

Ribosomal RNAs contain numerous conserved nucleotide modifications, yet the functional importance of most of these modifications remains unclear. In Escherichia coli, deletion of individual 16S rRNA modification enzymes generally produces only minor phenotypes, raising questions about their biological significance. Here, we generated a comprehensive collection of deletion mutants lacking individual and combined 16S rRNA modifications, culminating in a strain lacking all known 30S ribosomal subunit modifications. Despite the absence of all known 16S rRNA modifications, cells remained viable, exhibiting a fitness defect of ~30% at 37 {degrees}C that increased to ~50% at 20 {degrees}C, consistent with impaired ribosome biogenesis. We identified strong epistatic interactions between modifications in the 3' major and 3' minor domains of 16S rRNA, resulting in disproportionately large effects on both fitness and antibiotic susceptibility. Live-cell single-molecule tracking revealed a marked increase in the fraction of non-translating ribosomes and a prolonged time required to enter productive translation, whereas translational elongation by actively engaged 70S ribosomes remained largely unaffected. In addition, fluorescence-based measurements showed that unmodified ribosomes exhibited increased stringency during translation initiation, reducing utilization of near-cognate start codons. Together, these findings demonstrate that 16S rRNA modifications are not essential for viability but collectively enhance the efficiency, robustness, and fidelity of ribosome assembly and translational initiation.

microbiology↗

bioRxiv2025

Evolutionary trajectories determine the feasibility of collateral sensitivity based antibiotic treatment strategies in critical bacterial pathogens

The rise of antibiotic resistance among pathogenic bacteria necessitates innovative therapeutic strategies. A promising technique is the use of collateral sensitivity where resistance to one antibiotic increases susceptibility to another. In this study, we explored the clinical relevance of collateral sensitivity through experimental evolution and genetic engineering in six critical bacterial pathogens using 23 distinct antibiotics. Our in-depth analysis of Escherichia coli showed that clinically relevant resistance mutations did not confer collateral sensitivity to the tested antibiotics. We were able to identify at least three new classes of ciprofloxacin-resistance mutations that cause collateral sensitivity to multiple antibiotics. However, these mutations incur significant fitness costs and are absent in ciprofloxacin-resistant clinical isolates. Our further analysis showed that the development of collateral effects differed significantly between the tested species. Most species showed development of collateral sensitivity to gentamicin during ciprofloxacin-resistance evolution but Acinetobacter baumanii developed collateral resistance instead. Overall, Pseudomonas aeruginosa showed the most consistent development of collateral sensitivity among the tested species, highlighting it as a promising candidate for the use of collateral-sensitivity-based treatment strategies. Our findings provide insights into the potential of collateral sensitivity as a therapeutic strategy and contribute to the development of more effective antibiotic treatment regimens.

microbiology↗

bioRxiv2024

Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre

Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally-important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the "critical region" of the peptidyl transferase center (PTC) in 23S rRNA that exhibited only a minor growth defect at 37{degrees}C (although major at 20{degrees}C). However, our combined KO of modification enzymes RluC and RlmE resulted in conditional lethality (at 20{degrees}C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20 and 37{degrees}C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Rates of protein synthesis in vivo, as judged by the kinetics of {beta}-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37{degrees}C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

biochemistry↗

bioRxiv2022

Antibiotic perseverance increases the risk of resistance development

The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for five out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the effective population size and led to an up to 40-fold increase in antibiotic resistance development in Gram-negative and Gram-positive species. We conclude that antibiotic perseverance is a common phenomenon across the bacterial kingdom that has the potential to impact antibiotic resistance development.

microbiology↗