Archaellum Research Articles

Structural Basis of Conformational Transitions Involved in Pseudopilus Assembly and Stability

Type II secretion systems (T2SS), type IV pili and archaeal flagella use a conserved plasma membrane machinery to assemble helical filaments promoting macromolecule transport or motility. Using electron microscopy, we observed structural heterogeneity, predominantly twist-angle variations, in T2SS pili. Based on the measured twist-angles we generated 2500 pseudopilus structural models, recapitulating this conformational heterogeneity and twist-angle continuum, by an automated modeling procedure. We analyzed the ensemble of pilus models by making use of self-organizing maps, which allowed us to define a transition path between major conformational basins, leading from low to high twist-angles. We further characterized the free energy landscape of the pilus by performing short molecular dynamics calculations starting from each individual model combined with GBSA analysis. Experimental functional analysis based on predictions derived from our models showed that specific contacts at P-P+3 and P-P+4 interfaces determine fiber stability. Their disruption led to loss of surface pili but did not affect pseudopilus assembly and protein secretion. The results support the one-start assembly model for pseudopili and related fibers, and challenge the piston model of the type II protein secretion.

Haloferax volcanii cells lacking the flagellin FlgA2 are hypermotile

Motility driven by rotational movement of flagella allows bacteria and archaea to seek favourable conditions and escape toxic ones. However, archaeal flagella share structural similarities with bacterial type IV pili rather than bacterial flagella. The Haloferax volcanii genome contains two flagellin genes, flgA1 and flgA2. While FlgA1 has been shown to be a major flagellin, the function of FlgA2 is elusive. In this study, it was determined that although FlgA2 by itself does not confer motility to non-motile ΔflgA1 Hfx. volcanii, a subset of these mutant cells contains a flagellum. Consistent with FlgA2 being assembled into functional flagella, FlgA1 expressed from a plasmid can only complement a ΔflgA1 strain when co-expressed with chromosomal or plasmid-encoded FlgA2. Surprisingly, a mutant strain lacking FlgA2, but expressing chromosomally encoded FlgA1, is hypermotile, a phenotype that is accompanied by an increased number of flagella per cell, as well as an increased flagellum length. Site-directed mutagenesis resulting in early translational termination of flgA2 suggests that the hypermotility of the ΔflgA2 strain is not due to transcriptional regulation. This, and the fact that plasmid-encoded FlgA2 expression in a ΔflgA2 strain does not reduce its hypermotility, suggests a possible regulatory role for FlgA2 that depends on the relative abundance of FlgA1. Taken together, our results indicate that FlgA2 plays both structural and regulatory roles in Hfx. volcanii flagella-dependent motility. Future studies will build upon the data presented here to elucidate the significance of the hypermotility of this ΔflgA2 mutant, and will illuminate the regulation and function of archaeal flagella.

Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park

BackgroundA single cultured marine organism, Nanoarchaeum equitans, represents the Nanoarchaeota branch of symbiotic Archaea, with a highly reduced genome and unusual features such as multiple split genes.ResultsThe first terrestrial hyperthermophilic member of the Nanoarchaeota was collected from Obsidian Pool, a thermal feature in Yellowstone National Park, separated by single cell isolation, and sequenced together with its putative host, a Sulfolobales archaeon. Both the new Nanoarchaeota (Nst1) and N. equitans lack most biosynthetic capabilities, and phylogenetic analysis of ribosomal RNA and protein sequences indicates that the two form a deep-branching archaeal lineage. However, the Nst1 genome is more than 20% larger, and encodes a complete gluconeogenesis pathway as well as the full complement of archaeal flagellum proteins. With a larger genome, a smaller repertoire of split protein encoding genes and no split non-contiguous tRNAs, Nst1 appears to have experienced less severe genome reduction than N. equitans. These findings imply that, rather than representing ancestral characters, the extremely compact genomes and multiple split genes of Nanoarchaeota are derived characters associated with their symbiotic or parasitic lifestyle. The inferred host of Nst1 is potentially autotrophic, with a streamlined genome and simplified central and energetic metabolism as compared to other Sulfolobales.ConclusionsComparison of the N. equitans and Nst1 genomes suggests that the marine and terrestrial lineages of Nanoarchaeota share a common ancestor that was already a symbiont of another archaeon. The two distinct Nanoarchaeota-host genomic data sets offer novel insights into the evolution of archaeal symbiosis and parasitism, enabling further studies of the cellular and molecular mechanisms of these relationships.ReviewersThis article was reviewed by Patrick Forterre, Bettina Siebers (nominated by Michael Galperin) and Purification Lopez-Garcia

The type II secretion system – a dynamic fiber assembly nanomachine

Type II secretion systems (T2SSs) share common origins and structure with archaeal flagella (archaella) and pili, bacterial competence systems and type IV pili. All of these systems use a conserved ATP-powered machinery to assemble helical fibers that are anchored in the plasma membrane. The T2SSs assemble pseudopili, periplasmic filaments that promote extracellular secretion of folded periplasmic proteins. Comparative analysis of T2SSs and related fiber assembly nanomachines might provide important clues on their functional specificities and dynamics. This review focuses on recent developments in the study of pseudopilus structure and biogenesis, and discusses mechanistic models of pseudopilus function in protein secretion.

Insights into FlaI Functions in Archaeal Motor Assembly and Motility from Structures, Conformations, and Genetics

Superfamily ATPases in type IV pili, type 2 secretion, and archaella (formerly archaeal flagella) employ similar sequences for distinct biological processes. Here, we structurally and functionally characterize prototypical superfamily ATPase FlaI in Sulfolobus acidocaldarius, showing FlaI activities in archaeal swimming-organelle assembly and movement. X-ray scattering data of FlaI in solution and crystal structures with and without nucleotide reveala hexameric crown assembly with key cross-subunit interactions. Rigid building blocks form between N-terminal domains (points) and neighboring subunit C-terminal domains (crown ring). Upon nucleotide binding, these six cross-subunit blocks move with respect to each other and distinctly from secretion and pilus ATPases. Crown interactions and conformations regulate assembly, motility, and force direction via a basic-clamp switching mechanism driving conformational changes between stable, backbone-interconnected moving blocks. Collective structural and mutational results identify invivo functional components for assembly and motility, phosphate-triggered rearrangements by ATP hydrolysis, and molecular predictors for distinct ATPase superfamily functions.

Surface appendages of archaea: structure, function, genetics and assembly.

Organisms representing diverse subgroupings of the Domain Archaea are known to possess unusual surface structures. These can include ones unique to Archaea such as cannulae and hami as well as archaella (archaeal flagella) and various types of pili that superficially resemble their namesakes in Bacteria, although with significant differences. Major advances have occurred particularly in the study of archaella and pili using model organisms with recently developed advanced genetic tools. There is common use of a type IV pili-model of assembly for several archaeal surface structures including archaella, certain pili and sugar binding structures termed bindosomes. In addition, there are widespread posttranslational modifications of archaellins and pilins with N-linked glycans, with some containing novel sugars. Archaeal surface structures are involved in such diverse functions as swimming, attachment to surfaces, cell to cell contact resulting in genetic transfer, biofilm formation, and possible intercellular communication. Sometimes functions are co-dependent on other surface structures. These structures and the regulation of their assembly are important features that allow various Archaea, including thermoacidophilic, hyperthermophilic, halophilic, and anaerobic ones, to survive and thrive in the extreme environments that are commonly inhabited by members of this domain.

Diversity, assembly and regulation of archaeal type IV pili-like and non-type-IV pili-like surface structures

Archaea have evolved fascinating surface structures allowing rapid adaptation to changing environments. The archaeal surface appendages display such diverse biological roles as motility, adhesion, biofilm formation, exchange of genetic material and species-specific interactions and, in turn, increase fitness of the cells. Intriguingly, despite sharing the same functions with their bacterial counterparts, the assembly mechanism of many archaeal surface structures is rather related to assembly of bacterial type IV pili. This review summarizes our state-of-the-art knowledge about unique structural and biochemical properties of archaeal surface appendages with a particular focus on archaeal type IV pili-like structures. The latter comprise not only widely distributed archaella (formerly known as archaeal flagella), but also different highly specialized archaeal pili, which are often restricted to certain species. Recent findings regarding assembly mechanisms, structural aspects and physiological roles of these type IV pili-like structures will be discussed in detail. Recently, first regulatory proteins involved in transition from both planktonic to sessile lifestyle and in assembly of archaella were identified. To conclude, we provide novel insights into regulatory mechanisms underlying the assembly of archaeal surface structures.

Structure and function of the adhesive type IV pilus of Sulfolobus acidocaldarius

Archaea display a variety of type IV pili on their surface and employ them in different physiological functions. In the crenarchaeon Sulfolobus acidocaldarius the most abundant surface structure is the aap pilus (archaeal adhesive pilus). The construction of in frame deletions of the aap genes revealed that all the five genes (aapA, aapX, aapE, aapF, aapB) are indispensible for assembly of the pilus and an impact on surface motility and biofilm formation was observed. Our analyses revealed that there exists a regulatory cross-talk between the expression of aap genes and archaella (formerly archaeal flagella) genes during different growth phases. The structure of the aap pilus is entirely different from the known bacterial type IV pili as well as other archaeal type IV pili. An aap pilus displayed 3 stranded helices where there is a rotation per subunit of ∼138° and a rise per subunit of ∼5.7 Å. The filaments have a diameter of ∼110 Å and the resolution was judged to be ∼9 Å. We concluded that small changes in sequence might be amplified by large changes in higher-order packing. Our finding of an extraordinary stability of aap pili possibly represents an adaptation to harsh environments that S. acidocaldarius encounters.

Regulation of archaella expression by the FHA and von Willebrand domain‐containing proteins ArnA and ArnB in Sulfolobus acidocaldarius

The ability of microorganisms to sense and respond to sudden changes in their environment is often based on regulatory systems comprising reversible protein phosphorylation. The archaellum (former: archaeal flagellum) is used for motility in Archaea and therefore functionally analogous to the bacterial flagellum. In contrast with archaellum-mediated movement in certain members of the Euryarchaeota, this process, including its regulation, remains poorly studied in crenarchaeal organisms like Sulfolobus species. Recently, it was shown in Sulfolobus acidocaldarius that tryptone limiting conditions led to the induction of archaella expression and assembly. Here we have identified two proteins, the FHA domain-containing protein ArnA and the vWA domain-containing protein ArnB that are involved in regulating archaella expression in S. acidocaldarius. Both proteins are phosphorylated by protein kinases in vitro and interact strongly in vivo. Phenotypic analyses revealed that these two proteins are repressors of archaella expression. These results represent the first step in understanding the networks that underlie regulation of cellular motility in Crenarchaeota and emphasize the importance of protein phosphorylation in the regulation of cellular processes in the Archaea.

Control of archaellation in Sulfolobus acidocaldarius: unravelling of the regulation of surface structure biosynthesis in Archaea begins

Archaea have a variety of surface appendages including archaella (archaeal flagella), pili, hami and cannulae. While expected to be energetically expensive to express, studies focused on the regulation of such structures are nevertheless lacking. In the current issue of Molecular Microbiology, Reimann et al. (2012) identified a two-partner system called ArnA and ArnB in Sulfolobus acidocaldarius that interact strongly with each other and are repressors of archaella expression while also having an enhancing effect on the appearance of type IV pili. ArnA is a forkhead-associated domain-containing protein while ArnB is a von Willebrand domain-containing protein. Both proteins can be phosphorylated in vitro by S. acidocaldarius protein kinases. The repression of archaella expression is dependent on dephosphorylation of the Arn proteins. Deletions of arnA or arnB resulted in increased levels of archaella operon proteins and cells that were hypermotile due to increased archaellation. Direct effects of ArnA/ArnB on transcription from fla promoters were demonstrated using arnA and arnB deletion strains but only a modest increase in transcription was demonstrated in each mutant suggesting that the repression effect observed may be due to protein-protein interactions. This paper represents a significant step forward in our understanding of archaeal surface structure biogenesis.

Wie Archaeen Kontakt mit der Umwelt aufnehmen

Members of the third domain of life, the Archaea, exhibit a number of different cell surface structures which are similar in assembly and function to bacterial type IV pili. These pili have been shown to be involved in processes like attachment, motility, and the exchange of DNA in Archaea. Intriguingly, the archaeal flagellum, called the archaellum, functionally resembles the bacterial flagellum, although it is structurally alike type IV pili.

Molecular analysis of the crenarchaeal flagellum

The ability to move towards favourable conditions provides fundamental advantages to organisms. Interestingly, flagella as motility structures evolved independently in the bacterial and the archaeal kingdom. Whereas bacterial flagella have been intensively studied, our knowledge regarding the archaeal counterpart is mostly restricted to Euryarchaeota rather than crenarchaeal flagella. We therefore investigated the flagellar assembly system of the crenarchaeal model organism Sulfolobus acidocaldarius in vivo. Promoter studies and qRT-PCR analyses of the flagella gene cluster provided evidence that the expression of the fla genes was induced by tryptone starvation. Moreover, we confirmed presence of a secondary fla promoter within the flaB gene that regulates the transcription of downstream genes flaX-J. Markerless in-frame deletions for all fla genes encoded in the fla gene cluster were constructed. Western blot analysis of all fla deletion strains suggested hierarchical protein interactions during the archaeal flagella assembly. Moreover, functional analysis by thermomicroscopy revealed non-motile cells for each of the mutant strains. Electron micrographs demonstrated that lack of motility coincided with the loss of flagellar assembly. Thus we demonstrated that all seven fla genes are essential for crenarchaeal flagellum assembly and function.

Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding

Microbial motility frequently depends on flagella or typeIV pili. Using recently developed archaeal genetic tools, archaeal flagella and its assembly machinery have been identified. Archaeal flagella are functionally similar to bacterial flagella and their assembly systems are homologous with typeIV pili assembly systems of Gram-negative bacteria. Therefore elucidating their biochemistry may result in insights in both archaea and bacteria. FlaI, a critical cytoplasmic component of the archaeal flagella assembly system in Sulfolobus acidocaldarius, is a member of the typeII/IV secretion system ATPase superfamily, and is proposed to be bi-functional in driving flagella assembly and movement. In the present study we show that purified FlaI is a Mn2+-dependent ATPase that binds MANT-ATP [2'-/3'-O-(N'- methylanthraniloyl)adenosine-5'-O-triphosphate] with a high affinity and hydrolyses ATP in a co-operative manner. FlaI has an optimum pH and temperature of 6.5 and 75°C for ATP hydrolysis. Remarkably, archaeal, but not bacterial, lipids stimulated the ATPase activity of FlaI 3-4-fold. Analytical gel filtration indicated that FlaI undergoes nucleotide-dependent oligomerization. Furthermore, SAXS (small-angle X-ray scattering) analysis revealed an ATP-dependent hexamerization of FlaI in solution. The results of the present study report the first detailed biochemical analyses of the motor protein of an archaeal flagellum.

Assembly and function of the archaeal flagellum

Motility is a common behaviour in prokaryotes. Both bacteria and archaea use flagella for swimming motility, but it has been well documented that structures of the flagellum from these two domains of life are completely different, although they contribute to a similar function. Interestingly, information available to date has revealed that structurally archaeal flagella are more similar to bacterial typeIV pili rather than to bacterial flagella. With the increasing genome sequence information and advancement in genetic tools for archaea, identification of the components involved in the assembly of the archaeal flagellum is possible. A subset of these components shows similarities to components from typeIV pilus-assembly systems. Whereas the molecular players involved in assembly of the archaeal flagellum are being identified, the mechanics and dynamics of the assembly of the archaeal flagellum have yet to be established. Recent computational analysis in our laboratory has identified conserved highly charged loop regions within one of the core proteins of the flagellum, the membrane integral protein FlaJ, and predicted that these are involved in the interaction with the assembly ATPase FlaI. Interestingly, considerable variation was found among the loops of FlaJ from the two major subkingdoms of archaea, the Euryarchaeota and the Crenarchaeota. Understanding the assembly pathway and creating an interaction map of the molecular players in the archaeal flagellum will shed light on the details of the assembly and also the evolutionary relationship to the bacterial typeIV pili-assembly systems.

Detailed structural and assembly model of the type II secretion pilus from sparse data

Many gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called "pilins." In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to position-specific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure.

Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.

The Iho670 Fibers of Ignicoccus hospitalis : a New Type of Archaeal Cell Surface Appendage

Ignicoccus hospitalis forms many cell surface appendages, the Iho670 fibers (width, 14 nm; length, up to 20 mum), which constitute up to 5% of cellular protein. They are composed mainly of protein Iho670, possessing no homology to archaeal flagellins or fimbrins. Their existence as structures different from archaeal flagella or fimbriae have gone unnoticed up to now because they are very brittle.

Archaeal flagella as matrices for new nanomaterials

Archaeal flagella are among the most perspective biopolymer structures for use in nanotechnology and have certain advantages over both bacterial analogues and viral particles. Such advantages include their resistance to dissociating agents and their adhesion properties to certain types of surfaces. As a rule, archaeal flagella consist of several protein subunit types (flagellins). This allows multifunctional filaments to be designed with each flagellin differently modified. One of the reasons that archaeal flagella have not been used in nanotechnology is the lack of data about the spatial structure of subunits in flagella. We present an experimental approach for determining flagellin molecule sites which are exposed on the flagellum surface and suitable for directed insertion of groups capable of binding certain ligands. It was shown that modified flagella of halophilic archaea Halobacterium salinarum can be used as scaffolds for designing nowel nanomaterials.

UV‐inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis

The archaeal flagellum is a unique motility apparatus in the prokaryotic domain, distinct from the bacterial flagellum. Most of the currently recognized archaeal flagella-associated genes fall into a single fla operon that contains the genes for the flagellin proteins (two or more genes designated as flaA or flaB), some variation of a set of conserved proteins of unknown function (flaC, flaD, flaE, flaF, flaG and flaH), an ATPase (flaI) and a membrane protein (flaJ). In addition, the flaD gene has been demonstrated to encode two proteins: a full-length gene product and a truncated product derived from an alternate, internal start site. A systematic deletion approach was taken using the methanogen Methanococcus maripaludis to investigate the requirement and a possible role for these proposed flagella-associated genes. Markerless in-frame deletion strains were created for most of the genes in the M. maripaludis fla operon. In addition, a strain lacking the truncated FlaD protein [FlaD M(191)I] was also created. DNA sequencing and Southern blot analysis confirmed each mutant strain, and the integrity of the remaining operon was confirmed by immunoblot. With the exception of the DeltaFlaB3 and FlaD M(191)I strains, all mutants were non-motile by light microscopy and non-flagellated by electron microscopy. A detailed examination of the DeltaFlaB3 mutant flagella revealed that these structures had no hook region, while the FlaD M(191)I strain appeared identical to wild type. Each deletion strain was complemented, and motility and flagellation was restored. Collectively, these results demonstrate for first time that these fla operon genes are directly involved and critically required for proper archaeal flagella assembly and function.