The missing enzymatic link in syntrophic methane formation from fatty acids.

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H2, CO2, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO2 to methane by electrons derived from ß-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO2 by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of ß-oxidation in (methyl)menaquinone-containing organisms.

Motile ghosts of the halophilic archaeon, Haloferax volcanii.

Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5'-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. However, direct evidence is still lacking due to the lack of a versatile model system. Here, we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella and gliding Mycoplasma We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis, and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.

Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerization and interaction with ArlI, the motor ATPase of the archaellum.

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.

DNA processing by the MOBH family relaxase TraI encoded within the gonococcal genetic island.

Relaxases of the MOBH family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOBH family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn2+ or Co2+ dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3' of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site.

Structure and interactions of the archaeal motility repression module ArnA-ArnB that modulates archaellum gene expression in Sulfolobus acidocaldarius.

Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life. Forkhead-associated (FHA) and von Willebrand type A (vWA) domains are involved in several phosphorylation-dependent processes of multiprotein complex assemblies. Although well-studied in eukaryotes and bacteria, the structural and functional contexts of these domains are not yet understood in Archaea. Here, we report the structural base for such an interacting pair of FHA and vWA domain-containing proteins, ArnA and ArnB, in the thermoacidophilic archaeon Sulfolobus acidocaldarius, where they act synergistically and negatively modulate motility. The structure of the FHA domain of ArnA at 1.75 Å resolution revealed that it belongs to the subclass of FHA domains, which recognizes double-pSer/pThr motifs. We also solved the 1.5 Å resolution crystal structure of the ArnB paralog vWA2, disclosing a complex topology comprising the vWA domain, a ß-sandwich fold, and a C-terminal helix bundle. We further show that ArnA binds to the C terminus of ArnB, which harbors all the phosphorylation sites identified to date and is important for the function of ArnB in archaellum regulation. We also observed that expression levels of the archaellum components in response to changes in nutrient conditions are independent of changes in ArnA and ArnB levels and that a strong interaction between ArnA and ArnB observed during growth on rich medium sequentially diminishes after nutrient limitation. In summary, our findings unravel the structural features in ArnA and ArnB important for their interaction and functional archaellum expression and reveal how nutrient conditions affect this interaction.

Effect of UV irradiation on Sulfolobus acidocaldarius and involvement of the general transcription factor TFB3 in the early UV response.

Exposure to UV light can result in severe DNA damage. The alternative general transcription factor (GTF) TFB3 has been proposed to play a key role in the UV stress response in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Reporter gene assays confirmed that tfb3 is upregulated 90-180 min after UV treatment. In vivo tagging and immunodetection of TFB3 confirmed the induced expression at 90 min. Analysis of a tfb3 insertion mutant showed that genes encoding proteins of the Ups pili and the Ced DNA importer are no longer induced in a tfb3 insertion mutant after UV treatment, which was confirmed by aggregation assays. Thus, TFB3 plays a crucial role in the activation of these genes. Genome wide transcriptome analysis allowed a differentiation between a TFB3-dependent and a TFB3-independent early UV response. The TFB3-dependent UV response is characterized by the early induction of TFB3, followed by TFB3-dependent expression of genes involved in e.g. Ups pili formation and the Ced DNA importer. Many genes were downregulated in the tfb3 insertion mutant confirming the hypothesis that TFB3 acts as an activator of transcription. The TFB3-independent UV response includes the repression of nucleotide metabolism, replication and cell cycle progression in order to allow DNA repair.

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.

Secretion of Chromosomal DNA by the Neisseria gonorrhoeae Type IV Secretion System.

Approximately 80% of Neisseria gonorrhoeae and 17.5% of Neisseria meningitidis clinical isolates carry a ~59 kb genomic island known as the gonococcal genetic island (GGI). About half of the GGI consists of genes encoding a type IV secretion system (T4SS), and most of these genes are clustered in a ~28 kb region at one end of the GGI. Two additional genes (parA and parB) are found at the other end of the island. The remainder of the GGI consists mostly of hypothetical proteins, with several being identified as DNA-binding or DNA-processing proteins. The T4SS genes show similarity to those of the F-plasmid family of conjugation systems, with similarity in gene order and a low but significant level of sequence identity for the encoded proteins. However, several GGI-encoded proteins are unique from the F-plasmid system, such as AtlA, Yag, and TraA. Interestingly, the gonococcal T4SS does not act as a conjugation system. Instead, this T4SS secretes ssDNA into the extracellular milieu, where it can serve to transform highly competent Neisseria species, thereby increasing the transfer of genetic information. Although many of the T4SS proteins are expressed at low levels, this system has been implicated in several cellular processes. The secreted ssDNA is involved in the initial stages of biofilm formation, and the presence of the T4SS enables TonB-independent intracellular survival of N. gonorrhoeae strains during infection of cervical cells. Other GGI-like T4SSs have been identified in several other α-, ß-, and γ-proteobacteria, but the function of these GGI-like T4SSs is unknown. Remarkably, the presence of the GGI is related to resistance to several antibiotics. Here, we describe our current knowledge about the GGI and its unique ssDNA-secreting T4SS.

The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM.

Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery ofMyxococcus xanthus PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 ofPseudomonas aeruginosaformed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex.

The nucleotide-dependent interaction of FlaH and FlaI is essential for assembly and function of the archaellum motor.

The motor of the membrane-anchored archaeal motility structure, the archaellum, contains FlaX, FlaI and FlaH. FlaX forms a 30 nm ring structure that acts as a scaffold protein and was shown to interact with the bifunctional ATPase FlaI and FlaH. However, the structure and function of FlaH has been enigmatic. Here we present structural and functional analyses of isolated FlaH and archaellum motor subcomplexes. The FlaH crystal structure reveals a RecA/Rad51 family fold with an ATP bound on a conserved and exposed surface, which presumably forms an oligomerization interface. FlaH does not hydrolyze ATP in vitro, but ATP binding to FlaH is essential for its interaction with FlaI and for archaellum assembly. FlaH interacts with the C-terminus of FlaX, which was earlier shown to be essential for FlaX ring formation and to mediate interaction with FlaI. Electron microscopy reveals that FlaH assembles as a second ring inside the FlaX ring in vitro. Collectively these data reveal central structural mechanisms for FlaH interactions in mediating archaellar assembly: FlaH binding within the FlaX ring and nucleotide-regulated FlaH binding to FlaI form the archaellar basal body core.

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili.

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the ß-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.

Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion.

TraK and TraB are conserved outer membrane proteins of the Neisseria gonorrhoeae Type IV secretion system and are expressed at low levels in wild-type cells.

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.

PomZ, a ParA-like protein, regulates Z-ring formation and cell division in Myxococcus xanthus.

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z-ring at the division site. Here, we show that lack of the ParA-like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome-free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z-rings and incorrect positioning of the few Z-rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z-ring formation and is a spatial regulator of Z-ring formation and cell division. The cell cycle-dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z-ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z-ring formation to this position.

Secreted single-stranded DNA is involved in the initial phase of biofilm formation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an obligate human pathogen that colonizes the genital tract and causes gonorrhoea. Neisseria gonorrhoeae can form biofilms during natural cervical infections, on glass and in continuous flow-chamber systems. These biofilms contain large amounts of extracellular DNA, which plays an important role in biofilm formation. Many clinical isolates contain a gonococcal genetic island that encodes a type IV secretion system (T4SS). The T4SS of N. gonorrhoeae strain MS11 secretes ssDNA directly into the medium. Biofilm formation, studied in continuous flow-chamber systems by confocal laser scanning microscopy (CLSM), was strongly reduced, especially in the initial phases of biofilm formation, in the presence of Exonuclease I, which specifically degrades ssDNA or in a ΔtraB strain that does not secrete ssDNA. To specifically detect ssDNA in biofilms using CLSM, a novel method was developed in which thermostable fluorescently labelled ssDNA- and ss/dsDNA-binding proteins were used to visualize ssDNA and total DNA in biofilms and planktonic cultures. Remarkably, mainly dsDNA was detected in biofilms of the ssDNA secreting strain. We conclude that the secreted ssDNA facilitates initial biofilm formation, but that the secreted ssDNA is not retained in mature biofilms.

Proteins containing photosynthetic reaction centre domains modulate FtsZ-based archaeal cell division.

Cell division in all domains of life requires the orchestration of many proteins, but in Archaea most of the machinery remains poorly characterized. Here we investigate the FtsZ-based cell division mechanism in Haloferax volcanii and find proteins containing photosynthetic reaction centre (PRC) barrel domains that play an essential role in archaeal cell division. We rename these proteins cell division protein B 1 (CdpB1) and CdpB2. Depletions and deletions in their respective genes cause severe cell division defects, generating drastically enlarged cells. Fluorescence microscopy of tagged FtsZ1, FtsZ2 and SepF in CdpB1 and CdpB2 mutant strains revealed an unusually disordered divisome that is not organized into a distinct ring-like structure. Biochemical analysis shows that SepF forms a tripartite complex with CdpB1/2 and crystal structures suggest that these two proteins might form filaments, possibly aligning SepF and the FtsZ2 ring during cell division. Overall our results indicate that PRC-domain proteins play essential roles in FtsZ-based cell division in Archaea.

Putative nucleotide-based second messengers in archaea.

Second messengers transfer signals from changing intra- and extracellular conditions to a cellular response. Over the last few decades, several nucleotide-based second messengers have been identified and characterized in especially bacteria and eukaryotes. Also in archaea, several nucleotide-based second messengers have been identified. This review will summarize our understanding of nucleotide-based second messengers in archaea. For some of the nucleotide-based second messengers, like cyclic di-AMP and cyclic oligoadenylates, their roles in archaea have become clear. Cyclic di-AMP plays a similar role in osmoregulation in euryarchaea as in bacteria, and cyclic oligoadenylates are important in the Type III CRISPR-Cas response to activate CRISPR ancillary proteins involved in antiviral defense. Other putative nucleotide-based second messengers, like 3',5'- and 2',3'-cyclic mononucleotides and adenine dinucleotides, have been identified in archaea, but their synthesis and degradation pathways, as well as their functions as secondary messengers, still remain to be demonstrated. In contrast, 3'-3'-cGAMP has not yet been identified in archaea, but the enzymes required to synthesize 3'-3'-cGAMP have been found in several euryarchaeotes. Finally, the widely distributed bacterial second messengers, cyclic diguanosine monophosphate and guanosine (penta-)/tetraphosphate, do not appear to be present in archaea.

Archaeal GPN-loop GTPases involve a lock-switch-rock mechanism for GTP hydrolysis.

IMPORTANCE: GPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea, the mechanism by which these GTPases mediate their function is unknown. Our study on an archaeal representative from Sulfolobus acidocaldarius showed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement of SaGPN for motility appears to be due to its large footprint on the archaeal proteome.

Processing and maturation of the pilin of the type IV secretion system encoded within the gonococcal genetic island.

The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili.

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

Microbial motility frequently depends on flagella or type IV 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 type IV 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 type II/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.