Marleen van Wolferen

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 ∆pp2a, ∆arnA, or ∆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.

Since its isolation from a Yellowstone hot spring in 1972, Sulfolobus acidocaldarius has become one of the most important model organisms for archaeal biology. Initially studied for its remarkable adaptation to high temperature and low pH, it has evolved into a genetically tractable system that has contributed substantially to our understanding of archaeal physiology, molecular biology, and evolution. Here, we summarize more than 5 decades of research on S. acidocaldarius, highlighting key developments in genetic and microscopy tools and their impact on our understanding of archaeal cell biology. Studies in this organism have improved our understanding of archaeal metabolism, chromosome organization, DNA replication and segregation, cell division, protein glycosylation, biofilm formation, and the assembly and regulation of archaeal surface structures, including archaella and type IV pili. Beyond fundamental biology, S. acidocaldarius has also served as a valuable source of thermostable enzymes and other biomolecules with biotechnological potential. Today, S. acidocaldarius is still one of the main model organisms for archaeal biology. Ongoing advances in genetics, imaging, and structural biology continue to expand its experimental potential, while its phylogenetic position within the Thermoproteota makes it a powerful system for investigating the evolutionary origins of eukaryotic cellular complexity.

After exposure to ultraviolet (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 Crenarchaeal system for exchange of DNA (Ced), which imports DNA. To identify novel players in these processes, we investigated that 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 DNA damage response mechanisms 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.

Type IV pili are filamentous appendages found in most bacteria and archaea, where they can support functions such as surface adhesion, DNA uptake, aggregation, and 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 PilT homologs, it was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility. Here, we use live-cell imaging, automated cell tracking, fluorescence imaging, and genetic manipulation to show that the hyperthermophilic archaeon Sulfolobus acidocaldarius exhibits twitching motility, driven by retractable adhesion (Aap) pili, under physiologically relevant conditions (75 °C, pH 2). Aap pili are thus capable of retraction in the absence of a PilT homolog, suggesting that the ancestral type IV pili in the last universal common ancestor (LUCA) were capable of retraction.

The past decade has revealed the diversity and ubiquity of archaea in nature, with a growing number of studies highlighting their importance in ecology, biotechnology and even human health. Myriad lineages have been discovered, which expanded the phylogenetic breadth of archaea and revealed their central role in the evolutionary origins of eukaryotes. These discoveries, coupled with advances that enable the culturing and live imaging of archaeal cells under extreme environments, have underpinned a better understanding of their biology. In this Review we focus on the shape, internal organization and surface structures that are characteristic of archaeal cells as well as membrane remodelling, cell growth and division. We also highlight some of the technical challenges faced and discuss how new and improved technologies will help address many of the key unanswered questions.

A well-functioning genetic system, which is important for studying gene functions in vivo, requires a transformation method, a vector system and a selection system. Sulfolobus acidocaldarius is a crenarchaeal model organism that grows optimally at 75 °C and a pH of 3. These extreme growth conditions cause some difficulties in developing a genetic system. With continuous efforts, versatile genetic tools have been developed for different species from the order of Sulfolobales. In this chapter, we describe the methods for the available genetic tools in S. acidocaldarius including a (1) transformation method, (2) pop in/pop out strategy to generate markerless deletion mutants and (3) a plasmid-based expression system. Key words Genetics Archaea Sulfolobus Transformation Gene deletion Complementation Protein expression

Over the past decades there has been a growing interest in the domain of archaea. In this chapter we highlight the recent advances that have been made in studying the cell biology of archaea. We particularly focus on methods for genetic manipulation and imaging of different archaeal species and discuss the technical limitations at the often-extreme growth conditions. Several ongoing developments will help us overcoming these limitations, thereby facilitating future studies in the existing field of archaeal cell biology. Key words Archaea Cell biology Genetics Fluorescence microscopy Localization Thermomicroscopy

Polyphosphates (polyP) are polymers of orthophosphate residues linked by high-energy phosphoanhydride bonds that are important in all domains of life and function in many different processes, including biofilm development. To study the effect of polyP in archaeal biofilm formation, our previously described Sa. solfataricus polyP (−) strain and a new polyP (−) S. acidocaldarius strain generated in this report were used. These two strains lack the polymer due to the overexpression of their respective exopolyphosphatase gene (ppx). Both strains showed a reduction in biofilm formation, decreased motility on semi-solid plates and a diminished adherence to glass surfaces as seen by DAPI (4′,6-diamidino-2-phenylindole) staining using fluorescence microscopy. Even though arlB (encoding the archaellum subunit) was highly upregulated in S. acidocardarius polyP (−), no archaellated cells were observed. These results suggest that polyP might be involved in the regulation of the expression of archaellum components and their assembly, possibly by affecting energy availability, phosphorylation or other phenomena. This is the first evidence indicating polyP affects biofilm formation and other related processes in archaea.