Biology subjects

van Wolferen, M.

Publications and source records attributed to van Wolferen, M..

6 recordsLinked to original sources

bioRxiv2026

Regulation of Sulfolobus acidocaldarius surface structures by the PP2A core interaction module

Protein phosphorylation is a central regulatory mechanism that enables organisms to adapt to changing environmental conditions. The hyperthermophilic archaeon Sulfolobus acidocaldar-ius encodes only two phosphatases: the dual-specificity phosphatase PTP and the ser-ine/threonine phosphatase PP2A. PP2A has previously been implicated in archaellum regula-tion, and its deletion results in a hypermotile phenotype. Under starvation conditions, PP2A associates with a stress regulatory module comprising the archaellum repressors ArnA and ArnB, the universal stress protein UspA, and a GPN-loop GTPase. Here, we investigated PP2A-associated proteins under normal growth conditions and following UV-induced DNA damage. Pulldown experiments using a genomically HA-tagged PP2A strain identified a PP2A-associated basal regulatory module consisting of ArnA, ArnB, ArnE, and PTP, distinct from the previously described starvation-associated network. In addition, several proteins involved in the biogenesis and regulation of type IV pili co-purified with PP2A. Functional analyses using thermomicroscopy and electron microscopy revealed that deletion of {triangleup}pp2a, {triangleup}arnA, or {triangleup}arnB abolishes Aap-pilus formation and twitching motility, demonstrating that the PP2A regulatory network controls both swimming and surface-associated motility. In con-trast, the same network exerted only a modulatory effect on UV-induced cell aggregation. Together, our findings establish PP2A as a central regulator coordinating multiple archaeal surface structures through phosphorylation-dependent signaling.

microbiology↗

bioRxiv2025

Archaeal SegAB forms a bipolar structure that promotes chromosome segregation in spherical cells

Archaeal segAB operons are thought to promote chromosome segregation, but their mechanism remains unknown. We employ comparative genomics, structural biology, genetic knockouts, and quantitative cell biology to investigate how SegA and SegB proteins work together to segregate chromosomes in the thermophilic archaeon Sulfolobus acidocaldarius. In vitro, SegB binds a centromeric DNA sequence adjacent to the segAB operon, and in vivo forms a distinct focus on each segregating chromosome. SegA, a ParA-like ATPase, binds DNA non-specifically in vitro and promotes chromosome compaction and segregation in vivo. During division, SegA shifts from chromosome-associated puncta to form a single, elongated figure that runs between separating SegB foci. Late in division, SegA retreats to regions surrounding separated SegB foci. Elongated SegA figures appear in segB knockout cells but no longer lie perpendicular to the division plane. We propose that SegA and SegB interact to form a bipolar, DNA-segregating structure radically different from bacterial ParABS systems.

cell biology↗

bioRxiv2024

New components of the community based DNA-repair mechanism in Sulfolobales

After exposure to UV light, Sulfolobus acidocaldarius cells aggregate in a species-specific manner to exchange DNA and repair double-strand breaks via homologous recombination. The formation of cell-cell interactions is mediated by Ups pili. DNA exchange subsequently occurs through the Ced system, which imports DNA. To identify novel players in these processes, we investigated several genes upregulated after UV exposure by creating in-frame deletion mutants and performing cell aggregation and DNA exchange assays. This led to the identification of two novel components involved in the Ups and Ced systems: UpsC, a minor pilin of the Ups pili, and CedD, a VirD4-like ATPase essential for DNA import. Altogether, these findings provide new insights into the fascinating DNA damage response of Sulfolobales.

microbiology↗

bioRxiv2024

The use of thermostable fluorescent proteins for live imaging in Sulfolobus acidocaldarius

Among hyperthermophilic organisms, in vivo protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon S. acidocaldarius has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in S. acidocaldarius. To achieve this, we expressed various previously published and optimized thermostable fluorescent proteins along with fusion proteins of interest and analyzed the cells using flow cytometry and (thermo-) fluorescent microscopy. Of the tested proteins, Thermal Green Protein (TGP) exhibited the brightest fluorescence when expressed in Sulfolobus cells. By optimizing the linker between TGP and a protein of interest, we could additionally successfully fuse proteins with minimal loss of fluorescence. TGP-CdvB and TGP-PCNA1 fusions displayed localization patterns consistent with previous immunolocalization experiments. These initial results in protein localization in S. acidocaldarius at high temperatures, combined with recent advancements in thermomicroscopy, open new avenues in the field of archaeal cell biology. This progress finally enables localization experiments in thermophilic archaea, which have so far been limited to mesophilic organisms.

microbiology↗

bioRxiv2023

Sulfolobus acidocaldarius adhesion pili power twitching motility in the absence of a dedicated retraction ATPase

Type IV pili are ancient and widespread filamentous organelles found in most bacterial and archaeal phyla where they support a wide range of functions, including substrate adhesion, DNA uptake, self aggregation, and cell motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess homologs of PilT, it was thought that archaeal pili cannot retract. Here, we employ live-cell imaging under native conditions (75{degrees}C and pH 2), together with automated single-cell tracking, high-temperature fluorescence imaging, and genetic manipulation to demonstrate that S. acidocaldarius exhibits bona fide twitching motility, and that this behavior depends specifically on retractable adhesion pili. Our results demonstrate that archaeal adhesion pili are capable of retraction in the absence of a PilT retraction ATPase and suggests that the ancestral type IV pilus machinery in the last universal common ancestor (LUCA) relied on such a bifunctional ATPase for both extension and retraction.

cell biology↗

bioRxiv2019

Species-specific recognition of Sulfolobales mediated by UV inducible pili and S-layer glycosylation patterns

The UV-inducible pili system of Sulfolobales (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA in order to repair DNA double strand breaks via homologous recombination. Substitution of the S. acidocaldarius pilin subunits UpsA and UpsB with their homologs from Sulfolobus tokodaii showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of N-glycosylation in the recognition process. In addition, the N-glycan decorating the S-layer of S. tokodaii is different from the one of S. acidocaldarius. Therefore, each Sulfolobus species seems to have developed a unique UpsA binding pocket and unique N-glycan composition to ensure aggregation and consequently also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination.\n\nImportanceType IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The Ups-pili from the crenarchaeal Sulfolobales species are essential in establishing species-specific mating partners, ensuring genome stability. With this work, we show that different Sulfolobus species have species-specific regions in their Ups-pilin subunits, which allow them to interact only with cells from the same species. Additionally, different Sulfolobus species all have unique S-layer N-glycosylation patterns. We propose that the unique features of each species allow the recognition of specific mating partners. This knowledge for the first time gives insights into the molecular basis of archaeal self-recognition.

microbiology↗