The archaeal Ced system imports DNA.

The intercellular transfer of DNA is a phenomenon that occurs in all domains of life and is a major driving force of evolution. Upon UV-light treatment, cells of the crenarchaeal genus Sulfolobus express Ups pili, which initiate cell aggregate formation. Within these aggregates, chromosomal DNA, which is used for the repair of DNA double-strand breaks, is exchanged. Because so far no clear homologs of bacterial DNA transporters have been identified among the genomes of Archaea, the mechanisms of archaeal DNA transport have remained a puzzling and underinvestigated topic. Here we identify saci_0568 and saci_0748, two genes from Sulfolobus acidocaldarius that are highly induced upon UV treatment, encoding a transmembrane protein and a membrane-bound VirB4/HerA homolog, respectively. DNA transfer assays showed that both proteins are essential for DNA transfer between Sulfolobus cells and act downstream of the Ups pili system. Our results moreover revealed that the system is involved in the import of DNA rather than the export. We therefore propose that both Saci_0568 and Saci_0748 are part of a previously unidentified DNA importer. Given the fact that we found this transporter system to be widely spread among the Crenarchaeota, we propose to name it the Crenarchaeal system for exchange of DNA (Ced). In this study we have for the first time to our knowledge described an archaeal DNA transporter.

DNA Processing Proteins Involved in the UV-Induced Stress Response of Sulfolobales.

UNLABELLED: The ups operon of Sulfolobus species is highly induced upon UV stress. Previous studies showed that the pili encoded by this operon are involved in cellular aggregation, which is essential for subsequent DNA exchange between cells, resulting in homologous recombination. The presence of this pilus system increases the fitness of Sulfolobus cells under UV light-induced stress conditions, as the transfer of DNA takes place in order to repair UV-induced DNA lesions via homologous recombination. Four conserved genes (saci_1497 to saci_1500) which encode proteins with putative DNA processing functions are present downstream of the ups operon. In this study, we show that after UV treatment the cellular aggregation of strains with saci_1497, saci_1498, and saci_1500 deletions is similar to that of wild-type strains; their survival rates, however, were reduced and similar to or lower than those of the pilus deletion strains, which could not aggregate anymore. DNA recombination assays indicated that saci_1498, encoding a ParB-like protein, plays an important role in DNA transfer. Moreover, biochemical analysis showed that the endonuclease III encoded by saci_1497 nicks UV-damaged DNA. In addition, RecQ-like helicase Saci_1500 is able to unwind homologous recombination intermediates, such as Holliday junctions. Interestingly, a saci_1500 deletion mutant was more sensitive to UV light but not to the replication-stalling agents hydroxyurea and methyl methanesulfonate, suggesting that Saci_1500 functions specifically in the UV damage pathway. Together these results suggest a role of Saci_1497 to Saci_1500 in the repair or transfer of DNA that takes place after UV-induced damage to the genomic DNA of Sulfolobus acidocaldarius. IMPORTANCE: Sulfolobales species increase their fitness after UV stress by a UV-inducible pilus system that enables high rates of DNA exchange between cells. Downstream of the pilus operon, three genes that seem to play a role in the repair or transfer of the DNA between Sulfolobus cells were identified, and their possible functions are discussed. Next to the previously described role of UV-inducible pili in the exchange of DNA, we have thereby increased our knowledge of DNA transfer at the level of DNA processing. This paper therefore contributes to the overall understanding of the DNA exchange mechanism among Sulfolobales cells.

BarR, an Lrp-type transcription factor in Sulfolobus acidocaldarius, regulates an aminotransferase gene in a ß-alanine responsive manner.

In archaea, nothing is known about the ß-alanine degradation pathway or its regulation. In this work, we identify and characterize BarR, a novel Lrp-like transcription factor and the first one that has a non-proteinogenic amino acid ligand. BarR is conserved in Sulfolobus acidocaldarius and Sulfolobus tokodaii and is located in a divergent operon with a gene predicted to encode ß-alanine aminotransferase. Deletion of barR resulted in a reduced exponential growth rate in the presence of ß-alanine. Furthermore, qRT-PCR and promoter activity assays demonstrated that BarR activates the expression of the adjacent aminotransferase gene, but only upon ß-alanine supplementation. In contrast, auto-activation proved to be ß-alanine independent. Heterologously produced BarR is an octamer in solution and forms a single complex by interacting with multiple sites in the 170 bp long intergenic region separating the divergently transcribed genes. In vitro, DNA binding is specifically responsive to ß-alanine and site-mutant analyses indicated that ß-alanine directly interacts with the ligand-binding pocket. Altogether, this work contributes to the growing body of evidence that in archaea, Lrp-like transcription factors have physiological roles that go beyond the regulation of α-amino acid metabolism.

Dissection of key determinants of cleavage activity in signal peptidase III (SPaseIII) PibD.

In Archaea, type IV prepilins and prearchaellins are processed by designated signal peptidase III (SPaseIII) prior to their incorporation into pili and the archaellum, respectively. These peptidases belong to the family of integral membrane aspartic acid proteases that contain two essential aspartate residues of which the second aspartate is located in a conserved GxGD motif. To this group also bacterial type IV prepilin peptidases, Alzheimer disease-related secretases, signal peptide peptidases and signal peptide peptidase-like proteases in humans belong. Here we have performed detailed in vivo analyses to understand the cleavage activity of PibD, SPaseIII from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using an already established in vivo heterologous system cleavage assay, we could successfully identify the key amino acid residues essential for catalysis of PibD. Furthermore, in trans complementation of a pibD S. acidocaldarius deletion mutant with PibD variants having substituted key amino acids has consolidated our observations of the importance of these residues in catalysis. Based on our data, we propose to re-define class III peptidases/type IV prepilin/prearchaellin peptidases as GxHyD group (rather than GxGD) of proteases [Hy-hydrophobic amino acid].

Distinct life cycle stages of an ectosymbiotic DPANN archaeon.

DPANN archaea are a diverse group of microorganisms that are thought to rely on an ectosymbiotic lifestyle; however, the cell biology of these cell-cell interactions remains largely unknown. We applied live-cell imaging and cryo-electron tomography to the DPANN archaeon Nanobdella aerobiophila and its host, revealing two distinct life cycle stages. Free cells possess archaella and are motile. Ectobiotic cells are intimately linked with the host through an elaborate attachment organelle. Our data suggest that free cells may actively seek a new host, while the ectobiotic state is adapted to mediate intricate interaction with the host.

Adhesion pilus retraction powers twitching motility in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

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.

How hyperthermophiles adapt to change their lives: DNA exchange in extreme conditions.

Transfer of DNA has been shown to be involved in genome evolution. In particular with respect to the adaptation of bacterial species to high temperatures, DNA transfer between the domains of bacteria and archaea seems to have played a major role. In addition, DNA exchange between similar species likely plays a role in repair of DNA via homologous recombination, a process that is crucial under DNA damaging conditions such as high temperatures. Several mechanisms for the transfer of DNA have been described in prokaryotes, emphasizing its general importance. However, until recently, not much was known about this process in prokaryotes growing in highly thermophilic environments. This review describes the different mechanisms of DNA transfer in hyperthermophiles, and how this may contribute to the survival and adaptation of hyperthermophilic archaea and bacteria to extreme environments.

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°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.

UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili.

Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UV-inducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species-specific recognition process and that UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self-aggregate, but were partners in mating experiments with wild-type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UV-inducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.

Progress and Challenges in Archaeal Cell Biology.

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.

Methods for Markerless Gene Deletion and Plasmid-Based Expression in Sulfolobus acidocaldarius.

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.

The cell biology of archaea.

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.

Methods to Analyze Motility in Eury- and Crenarchaea.

Many archaea display swimming motility in liquid medium, which is empowered by the archaellum. Directional movement requires a functional archaellum and a sensing system, such as the chemotaxis system that is used by Euryarchaea. Two well-studied models are the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius. In this chapter we describe two methods to analyze their swimming behavior and directional movement: (a) time-lapse microscopy under native temperatures and (b) spotting on semi-solid agar or gelrite plates. Whereas the first method allows for deep analysis of swimming behavior, the second method is suited for high throughput comparison of multiple strains.

The Role of Polyphosphate in Motility, Adhesion, and Biofilm Formation in Sulfolobales.

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.

"Hot standards" for the thermoacidophilic archaeon Sulfolobus solfataricus.

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology ("SulfoSYS")-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the "-omics" approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics.

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 to repair DNA double-strand breaks via homologous recombination. Substitution of the Sulfolobus 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.IMPORTANCE Type IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The UV-inducible pili system of Sulfolobales (Ups) pili from the crenarchaeal Sulfolobales species are essential in establishing species-specific mating partners, thereby assisting in genome stability. With this work, we show that different Sulfolobus species have specific regions in their Ups pili subunits, which allow them to interact only with cells from the same species. Additionally, different Sulfolobus species have unique surface-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.

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division.

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. Although similar techniques have been applied to the study of halophilic archaea [1-5], our ability to explore the cell biology of thermophilic archaea has been limited by the technical challenges of imaging at high temperatures. Sulfolobus are the most intensively studied members of TACK archaea and have well-established molecular genetics [6-9]. Additionally, studies using Sulfolobus were among the first to reveal striking similarities between the cell biology of eukaryotes and archaea [10-15]. However, to date, it has not been possible to image Sulfolobus cells as they grow and divide. Here, we report the construction of the Sulfoscope, a heated chamber on an inverted fluorescent microscope that enables live-cell imaging of thermophiles. By using thermostable fluorescent probes together with this system, we were able to image Sulfolobus acidocaldarius cells live to reveal tight coupling between changes in DNA condensation, segregation, and cell division. Furthermore, by imaging deletion mutants, we observed functional differences between the two ESCRT-III proteins implicated in cytokinesis, CdvB1 and CdvB2. The deletion of cdvB1 compromised cell division, causing occasional division failures, whereas the ΔcdvB2 exhibited a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRT-III polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division.

Salt-dependent regulation of archaellins in Haloarcula marismortui.

Microorganisms require a motility structure to move towards optimal growth conditions. The motility structure from archaea, the archaellum, is fundamentally different from its bacterial counterpart, the flagellum, and is assembled in a similar fashion as type IV pili. The archaellum filament consists of thousands of copies of N-terminally processed archaellin proteins. Several archaea, such as the euryarchaeon Haloarcula marismortui, encode multiple archaellins. Two archaellins of H. marismortui display differential stability under various ionic strengths. This suggests that these proteins behave as ecoparalogs and perform the same function under different environmental conditions. Here, we explored this intriguing system to study the differential regulation of these ecoparalogous archaellins by monitoring their transcription, translation, and assembly into filaments. The salt concentration of the growth medium induced differential expression of the two archaellins. In addition, this analysis indicated that archaellation in H. marismortui is majorly regulated on the level of secretion, by a still unknown mechanism. These findings indicate that in archaea, multiple encoded archaellins are not completely redundant, but in fact can display subtle functional differences, which enable cells to cope with varying environmental conditions.

Archaeal biofilm formation.

Biofilms are structured and organized communities of microorganisms that represent one of the most successful forms of life on Earth. Bacterial biofilms have been studied in great detail, and many molecular details are known about the processes that govern bacterial biofilm formation, however, archaea are ubiquitous in almost all habitats on Earth and can also form biofilms. In recent years, insights have been gained into the development of archaeal biofilms, how archaea communicate to form biofilms and how the switch from a free-living lifestyle to a sessile lifestyle is regulated. In this Review, we explore the different stages of archaeal biofilm development and highlight similarities and differences between archaea and bacteria on a molecular level. We also consider the role of archaeal biofilms in industry and their use in different industrial processes.