Biology subjects

van Gestel, J.

Publications and source records attributed to van Gestel, J..

3 recordsLinked to original sources

bioRxiv2025

Selection-free CRISPR-Cas9 editing protocol for distant Dictyostelid species

Dictyostelids are a species-rich clade of cellular slime molds that are widely found in soils and have been studied for over a century. Most research focusses on Dictyostelium discoideum, which - due to its ease of culturing and genetic tractability - has been adopted as a model species in the fields of developmental biology, cell biology and microbiology. Over decades, genome editing methods in D. discoideum have steadily improved but remain relatively time-consuming and limited in scope, effective in a few species only. Here, we introduce a CRISPR-Cas9 editing protocol that is cloning-free, selection-free, highly-efficient, and effective across Dictyostelid species. After optimizing our protocol in D. discoideum, we obtained knock-out efficiencies of [~]80% and knock-in efficiencies of [~]30% without antibiotic selection. Efficiencies depend on template concentrations, insertion sizes, homology arms and target sites. Since our protocol is selection-free, we can isolate mutants as soon as one day post-transfection, vastly expediting the generation of knock-outs, fusion proteins and expression reporters. Our protocol also makes it possible to generate several knock-in mutations simultaneously in the same cells. Boosted by cell-sorting and fluorescent microscopy, we could readily apply our CRISPR-Cas9 editing protocol to phylogenetically distant Dictyostelid species, which diverged hundreds of millions of years ago and have never been genome edited before. Our protocol therefore opens the door to performing broad-scale genetic interrogations across Dicyostelids.

genetics↗

bioRxiv2024

Comprehensive double-mutant analysis of the Bacillus subtilis envelope using double-CRISPRi

Understanding bacterial gene function remains a major biological challenge. Double-mutant genetic interaction (GI) analysis addresses this challenge by uncovering the functional partners of targeted genes, allowing us to associate genes of unknown function with novel pathways and unravel connections between well-studied pathways, but is difficult to implement at the genome-scale. Here, we develop and use double-CRISPRi to systematically quantify genetic interactions at scale in the Bacillus subtilis envelope, including essential genes. We discover > 1000 known and novel genetic interactions. Our analysis pipeline and experimental follow-ups reveal the distinct roles of paralogous genes such as the mreB and mbl actin homologs, and identify new genes involved in the well-studied process of cell division. Overall, our study provides valuable insights into gene function and demonstrates the utility of double-CRISPRi for high-throughput dissection of bacterial gene networks, providing a blueprint for future studies in diverse bacterial species.

genomics↗

bioRxiv2022

Complex extracellular biology drives surface competition in Bacillus subtilis

In nature, many bacteria grow on surfaces, where they form cell collectives that compete for space. Within these collectives, cells often secrete molecules that benefit surface spreading by, for example, reducing surface tension or promoting filamentous growth. Although we have a detailed understanding of how these molecules are produced, much remains unknown about their role in surface competition. Here, we examine sliding motility in Bacillus subtilis and compare how secreted molecules, essential for sliding, affect cooperation and competition on the surface. We specifically examine (i) the lipopeptide surfactin, (ii) the hydrophobin protein BslA and (iii) exopolysaccharides (EPS). We find that these molecules have a remarkably different effect on competition: whereas surfactin acts like a common good, which is costly to produce and benefits cells throughout the surface, BslA and EPS are cost-free and act locally. Accordingly, surfactin deficient mutants can exploit the wild-type strain in competition for space, while BslA and EPS mutants cannot. Using a mathematical model, we show that three factors are important in predicting the outcome of surface competition: the costs of molecule synthesis, the private benefits of molecule production, and the diffusion rate. Our results underscore the intricate extracellular biology that can drive bacterial surface competition.

microbiology↗