Biology subjects

Staals, R. H.

Publications and source records attributed to Staals, R. H..

3 recordsLinked to original sources

bioRxiv2026

Establishment of titration-based control of DNA replication in Escherichia coli

Escherichia coli couples the initiation of DNA replication with cell size by modulating the activity of the replication initiator protein DnaA. The activity of DnaA is regulated by both its interconversion between an active and inactive form and its titration on binding sites on the chromosome. Whereas its interconversion has been thoroughly studied, the extent to which DnaA titration can control replication initiation is poorly understood. Here, we describe the control of E. coli DNA replication via titration by modulating the expression of an always-active DnaA variant in four growth conditions. While we obtained stable cell cycles during slow growth, faster growth associated with overlapping replication forks led to replicative instability and DNA damage. Overall, our results provide insights into the limits of titration-based systems in the control of genome replication and their potential role in the evolutionary trajectory of E. coli. Finally, this study provides design principles for a simplified, titration-only regulatory mechanism for DNA replication in synthetic cells.

molecular biology↗

bioRxiv2024

Towards sequential conjugation-assisted laboratory evolution (SCALE) of Cas nucleases

DNA endonucleases such as the CRISPR-associated enzymes Cas9 and Cas12a constitute powerful molecular tools with a wide range of applications. To facilitate optimization of these enzymes, we investigated a fully in vivo selection system for laboratory evolution based on plasmid conjugation. In the proposed method, DNA cleavage promotes plasmid transfer to other cells, allowing cleaving variants to spread through the bacterial culture via consecutive conjugations. We have investigated whether the conjugation system of the F plasmid of E. coli is suitable for laboratory evolution of Cas nucleases. Through a series of conjugation experiments, we found (i) that the F plasmid can spread through bacterial cultures, (ii) that the FinOP conjugation inhibition system is a potential nuclease target as it can prevent transfer of non-cleaving variants (counterselection), and (iii) that a trimmed version of the F plasmid allows for consecutive conjugations. Overall, we were able to show that in many regards, conjugation can indeed be used as means of in vivo laboratory evolution. We discuss the potential of the here presented concept for a yet to be developed laboratory evolution platform.

molecular biology↗

bioRxiv2019

Type I-F CRISPR-Cas resistance against virulent phage infection triggers abortive infection and provides population-level immunity

Type I CRISPR-Cas systems are the most abundant and widespread adaptive immune systems of bacteria and can greatly enhance bacterial survival in the face of temperate phage infection. However, it is less clear how these systems protect against virulent phages. Here we experimentally show that type I CRISPR immunity of Pectobacterium atrosepticum leads to rapid suppression of two unrelated virulent phages, {Phi}TE and {Phi}M1. However, unlike the case where bacteria are infected with temperate phages, this is the result of an abortive infection-like phenotype, where infected cells do not survive the infection but instead become metabolically inactive and lose their membrane integrity. Our findings challenge the view of CRISPR-Cas as a system that protects the individual cell and supports growing evidence of an Abi-like function for some types of CRISPR-Cas systems.

microbiology↗