Huseby, D. L. | bioRxiv Science

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↗

bioRxiv2018

D,D-muramyl endopeptidase Spr contributes to intrinsic vancomycin resistance in Salmonella enterica serovar Typhimurium

The impermeability barrier provided by the outer membrane of enteric bacteria, a feature lacking in Gram-positive bacteria, plays a major role in maintaining resistance to numerous antimicrobial compounds and antibiotics. Here we demonstrate that mutational inactivation of spr, coding for a muramyl endopeptidase, significantly sensitizes Salmonella enterica serovar Typhimurium to vancomycin without any accompanying apparent growth defect or outer membrane destabilization. A similar phenotype was not achieved by deleting the mepA, pbpG, nlpC, yebA or yhdO genes coding for functional homologues to Spr. The spr mutant revealed increased autolytic behavior in response to not only vancomycin, but also to penicillin G, an antibiotic for which the mutant displayed a wild-type MIC. A screen for suppressor mutations of the spr mutant phenotype revealed that deletion of tsp (prc), encoding a periplasmic carboxypeptidase involved in processing Spr and PBP3, restored intrinsic resistance to vancomycin and reversed the autolytic phenotype of an spr mutant. Our data suggest that Spr contributes to intrinsic antibiotic resistance in S. Typhimurium without directly affecting the outer membrane permeability barrier. Furthermore, our data suggests that compounds that target specific cell wall endopeptidases might have the potential to expand the activity spectrum of traditional Gram-positive antibiotics.

microbiology↗