Archaeal Systems Research Articles

Two new families of the FtsZ-tubulin protein superfamily implicated in membrane remodeling in diverse bacteria and archaea

Several recent discoveries reveal unexpected versatility of the bacterial and archaeal cytoskeleton systems that are involved in cell division and other processes based on membrane remodeling. Here we apply methods for distant protein sequence similarity detection, phylogenetic approaches, and genome context analysis to described two previously unnoticed families of the FtsZ-tubulin superfamily. One of these families is limited in its spread to Proteobacteria whereas the other is represented in diverse bacteria and archaea, and might be the key component of a novel, multicomponent membrane remodeling system that also includes a Von Willebrand A domain-containing protein, a distinct GTPase and membrane transport proteins of the OmpA family.This article was reviewed by Purificación López-García and Gáspár Jékely; for complete reviews, see the Reviewers Reports section.

Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus

The function of initiation factors in and the sequence of events during translation initiation have been intensively studied in Bacteria and Eukaryotes, whereas in Archaea knowledge on these functions/processes is limited. By employing chemical probing, we show that translation initiation factor aIF1 of the model crenarchaeon Sulfolobus solfataricus binds to the same area on the ribosome as the bacterial and eukaryal orthologs. Fluorescence energy transfer assays (FRET) showed that aIF1, like its eukaryotic and bacterial orthologs, has a fidelity function in translation initiation complex formation, and that both aIF1 and aIF1A exert a synergistic effect in stimulating ribosomal association of the Met-tRNAi(Met) binding factor a/eIF2. However, as in Eukaryotes their effect on a/eIF2 binding appears to be indirect. Moreover, FRET was used to analyze for the first time the sequence of events toward translation initiation complex formation in an archaeal model system. These studies suggested that a/eIF2-GTP binds first to the ribosome and then recruits Met-tRNAi(Met), which appears to comply with the operational mode of bacterial IF2, and deviates from the shuttle function of the eukaryotic counterpart eIF2. Thus, despite the resemblance of eIF2 and a/eIF2, recruitment of initiator tRNA to the ribosome is mechanistically different in Pro- and Eukaryotes.

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases

The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure-function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the alpha subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the DeltaH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.

Functional characterization of the DnaK chaperone system from the archaeon Methanothermobacter thermautotrophicusΔH

We characterized the biochemical and functional properties of the DnaK system from the archaeon Methanothermobacter thermautotrophicus ΔH. In contrast to the eubacterial chaperone components the archaeal Hsp70 system shows thermal transitions only slightly above the optimal environmental temperature (65 °C). Nevertheless, it prevents aggregation of luciferase in the physiological temperature range of the organism, but is also fully functional at 30 °C in luciferase refolding. Additionally, GrpE M.th. and DnaJ M.th. substitute their eubacterial counterparts whereas DnaK M.th. is only functional with its native cochaperones which could be attributed to a functional specialization of the eubacterial chaperones during evolution.

SulfoSYS (Sulfolobus Systems Biology): towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. In Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bacteria or Eukarya, e.g. the unusual branched ED (Entner-Doudoroff) pathway, which is utilized for glucose degradation in S. solfataricus. This archaeal model organism of choice is a thermoacidophilic crenarchaeon that optimally grows at 80 degrees C (60-92 degrees C) and pH 2-4. In general, life at high temperature requires very efficient adaptation to temperature changes, which is most difficult to deal with for organisms, and it is unclear how biological networks can withstand and respond to such changes. This integrative project combines genomic, transcriptomic, proteomic and metabolomic, as well as kinetic and biochemical information. The final goal of SulfoSYS is the construction of a silicon cell model for this part of the living cell that will enable computation of the CCM network. In the present paper, we report on one of the first archaeal systems biology projects.

A unique cell division machinery in the Archaea

In contrast to the cell division machineries of bacteria, euryarchaea, and eukaryotes, no division components have been identified in the second main archaeal phylum, Crenarchaeota. Here, we demonstrate that a three-gene operon, cdv, in the crenarchaeon Sulfolobus acidocaldarius, forms part of a unique cell division machinery. The operon is induced at the onset of genome segregation and division, and the Cdv proteins then polymerize between segregating nucleoids and persist throughout cell division, forming a successively smaller structure during constriction. The cdv operon is dramatically down-regulated after UV irradiation, indicating division inhibition in response to DNA damage, reminiscent of eukaryotic checkpoint systems. The cdv genes exhibit a complementary phylogenetic range relative to FtsZ-based archaeal division systems such that, in most archaeal lineages, either one or the other system is present. Two of the Cdv proteins, CdvB and CdvC, display homology to components of the eukaryotic ESCRT-III sorting complex involved in budding of luminal vesicles and HIV-1 virion release, suggesting mechanistic similarities and a common evolutionary origin.

Biochemical Characterization of the Minichromosome Maintenance (MCM) Protein of the Crenarchaeote Aeropyrum pernix and Its Interactions with the Origin Recognition Complex (ORC) Proteins

Replication in archaea is carried out by proteins that are homologues of eukaryotic counterparts. However, the archaeal systems tend to be much simpler with fewer different genes encoding the core functions than in eukaryotic counterparts. In many archaea, there is a single minichromosome maintenance (MCM) homologue, presumed to be the replicative helicase and between one and three origin recognition complex (ORC) homologues involved in binding to the replication origins. Here we describe the cloning and characterization of the MCM protein from the crenarchaeote Aeropyrum pernix. Like other eukaryotic and archaeal MCM proteins, it is found to be an ATP-dependent DNA helicase, and the putative active site residues involved in ATP binding and hydrolysis are confirmed by mutation. Deletion of the N-terminal 256 amino acids yielded a protein with higher ATPase activity in the absence of DNA and retained robust helicase activity. Interactions with the ORC proteins of A. pernix were examined, and it was found that both ORC homologues could inhibit the helicase activity of MCM. Further it was found that ORC2 could autophosphorylate in the presence of ATP and more remarkably could phosphorylate MCM in a species-specific manner.

A Role for the ESCRT System in Cell Division in Archaea

Archaea are prokaryotic organisms that lack endomembrane structures. However, a number of hyperthermophilic members of the Kingdom Crenarchaea, including members of the Sulfolobus genus, encode homologs of the eukaryotic endosomal sorting system components Vps4 and ESCRT-III (endosomal sorting complex required for transport-III). We found that Sulfolobus ESCRT-III and Vps4 homologs underwent regulation of their expression during the cell cycle. The proteins interacted and we established the structural basis of this interaction. Furthermore, these proteins specifically localized to the mid-cell during cell division. Overexpression of a catalytically inactive mutant Vps4 in Sulfolobus resulted in the accumulation of enlarged cells, indicative of failed cell division. Thus, the archaeal ESCRT system plays a key role in cell division.

Crystal structure of Methanococcus jannaschii TATA box‐binding protein

As the archaeal transcription system consists of a eukaryotic-type transcription apparatus and bacterial-type regulatory transcription factors, analyses of the molecular interface between the transcription apparatus and regulatory transcription factors are critical to reveal the evolutionary change of the transcription system. TATA box-binding protein (TBP), the central components of the transcription apparatus are classified into three groups: eukaryotic, archaeal-I and archaeal-II TBPs. Thus, comparative functional analysis of these three groups of TBP is important for the study of the evolution of the transcription system. Here, we present the first crystal structure of an archaeal-II TBP from Methanococcus jannaschii. The highly conserved and group-specific conserved surfaces of TBP bind to DNA and TFIIB/TFB, respectively. The phylogenetic trees of TBP and TFIIB/TFB revealed that they evolved in a coupled manner. The diversified surface of TBP is negatively charged in the archaeal-II TBP, which is completely different from the case of eukaryotic and archaeal-I TBPs, which are positively charged and biphasic, respectively. This difference is responsible for the diversification of the regulatory functions of TBP during evolution.

Insight into initiator–DNA interactions: a lesson from the archaeal ORC

Although initiation of DNA replication is considered to be highly coordinated through multiple protein-DNA and protein-protein interactions, it is poorly understood how particular locations within the eukaryotic chromosome are selected as origins of DNA replication. Here, we discuss recent reports that present structural information on the interaction characteristics of the archaeal orthologues of the eukaryotic origin recognition complex with their cognate binding sequences. Since the archaeal replication system is postulated as a simplified version of the one in eukaryotes, by analogy, these works provide insights into the functions of the eukaryotic initiator proteins.

Polarity in Archaeal Operon Transcription in Thermococcus kodakaraensis

An in vivo archaeal gene reporter system has been established based on TK1761, a gene that encodes a nonessential beta-glycosidase in Thermococcus kodakaraensis. Following the introduction of nonsense codons into promoter-proximal genes, polarity in operon expression in this archaeon has been established by both microarray hybridization assays and a reporter gene expression system.

Relationship of Geographic Distance, Depth, Temperature, and Viruses with Prokaryotic Communities in the Eastern Tropical Atlantic Ocean

The richness and biogeographical distribution pattern of bacterial and archaeal communities was assessed by terminal restriction fragment length polymorphism analysis of polymerase chain reaction-amplified fragments of the 16S rRNA gene at the surface (15-25 m depth), in the deep chlorophyll maximum layer (DCM; 50 m depth), and deep waters (75-1000 m depth) of the eastern tropical Atlantic Ocean. Additionally, prokaryotic and viral abundance and the frequency of infected prokaryotic cells (FIC) were determined along with physico-chemical parameters to identify factors influencing prokaryotic richness and biogeography. Viral abundance was highest in the DCM layer averaging 45.5 x 10(6) ml(-1), whereas in the mixed surface layer and in the waters below the DCM, average viral abundance was 11.3 x 10(6) and 4.3 x 10(6) ml(-1), respectively. The average estimate of FIC was 8.3% in the mixed surface layer and the DCM and 2.4% in deeper waters. FIC was positively related to prokaryotic and viral abundance and negatively to archaeal richness. There was no detectable effect of geographic distance (maximum distance between stations approximately 4600 km) or differences between water masses on bacterial and archaeal community composition. Bacterial communities showed a clear depth zonation, whereas changes in archaeal community composition were related to temperature and FIC. The results indicate that planktonic archaeal virus host systems are a dynamic component of marine ecosystems under natural conditions.

Genetic and Proteomic Analyses of a Proteasome-Activating Nucleotidase A Mutant of the Haloarchaeon Haloferax volcanii

The halophilic archaeon Haloferax volcanii encodes two related proteasome-activating nucleotidase proteins, PanA and PanB, with PanA levels predominant during all phases of growth. In this study, an isogenic panA mutant strain of H. volcanii was generated. The growth rate and cell yield of this mutant strain were lower than those of its parent and plasmid-complemented derivatives. In addition, a consistent and discernible 2.1-fold increase in the number of phosphorylated proteins was detected when the panA gene was disrupted, based on phosphospecific fluorescent staining of proteins separated by 2-dimensional gel electrophoresis. Subsequent enrichment of phosphoproteins by immobilized metal ion and metal oxide affinity chromatography (in parallel and sequentially) followed by tandem mass spectrometry was employed to identify key differences in the proteomes of these strains as well as to add to the restricted numbers of known phosphoproteins within the Archaea. In total, 625 proteins (approximately 15% of the deduced proteome) and 9 phosphosites were identified by these approaches, and 31% (195) of the proteins were identified by multiple phosphoanalytical methods. In agreement with the phosphostaining results, the number of identified proteins that were reproducibly exclusive or notably more abundant in one strain was nearly twofold greater for the panA mutant than for the parental strain. Enriched proteins exclusive to or more abundant in the panA mutant (versus the wild type) included cell division (FtsZ, Cdc48), dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase system (EI, DhaK), and oxidoreductase homologs. Differences in transcriptional regulation and signal transduction proteins were also observed, including those differences (e.g., OsmC and BolA) which suggest that proteasome deficiency caused an up-regulation of stress responses (e.g., OsmC versus BolA). Consistent with this, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems were enriched and identified as unique to the panA mutant. The cumulative proteomic data not only furthered our understanding of the archaeal proteasome system but also facilitated the assembly of the first subproteome map of H. volcanii.

Structure and function of archaeal RNA polymerases

RNA polymerases (RNAPs) are essential to all life forms and therefore deserve our special attention. The archaeal RNAP is closely related to eukaryotic RNAPII in terms of subunit composition and architecture, promoter elements and basal transcription factors required for the initiation and elongation phase of transcription. RNAPs of this class are large and sophisticated enzymes that interact in a complex manner with DNA/RNA scaffolds, substrates NTPs and a plethora of transcription factors - interactions that often result in an allosteric regulation of RNAP activity. The 12 subunits of RNAP play distinct roles including RNAP assembly and stability, catalysis and functional contacts with exogenous factors. Due to the availability of structural information of RNAPs at high-resolution and wholly recombinant archaeal transcription systems, we are beginning to understand the molecular mechanisms of archaeal RNAPs and transcription in great detail.

Phylogenomics of the archaeal flagellum: rare horizontal gene transfer in a unique motility structure

BackgroundAs bacteria, motile archaeal species swim by means of rotating flagellum structures driven by a proton gradient force. Interestingly, experimental data have shown that the archaeal flagellum is non-homologous to the bacterial flagellum either in terms of overall structure, components and assembly. The growing number of complete archaeal genomes now permits to investigate the evolution of this unique motility system.ResultsWe report here an exhaustive phylogenomic analysis of the components of the archaeal flagellum. In all complete archaeal genomes, the genes coding for flagellum components are co-localized in one or two well-conserved genomic clusters showing two different types of organizations. Despite their small size, these genes harbor a good phylogenetic signal that allows reconstruction of their evolutionary histories. These support a history of mainly vertical inheritance for the components of this unique motility system, and an interesting possible ancient horizontal gene transfer event (HGT) of a whole flagellum-coding gene cluster between Euryarchaeota and Crenarchaeota.ConclusionOur study is one of the few exhaustive phylogenomics analyses of a non-informational cell machinery from the third domain of life. We propose an evolutionary scenario for the evolution of the components of the archaeal flagellum. Moreover, we show that the components of the archaeal flagellar system have not been frequently transferred among archaeal species, indicating that gene fixation following HGT can also be rare for genes encoding components of large macromolecular complexes with a structural role.

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria.

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

DNA replication initiation: mechanisms and regulation in bacteria

In all organisms, multi-subunit replicases are responsible for the accurate duplication of genetic material during cellular division. Initiator proteins control the onset of DNA replication and direct the assembly of replisomal components through a series of precisely timed protein-DNA and protein-protein interactions. Recent structural studies of the bacterial protein DnaA have helped to clarify the molecular mechanisms underlying initiator function, and suggest that key structural features of cellular initiators are universally conserved. Moreover, it appears that bacteria use a diverse range of regulatory strategies dedicated to tightly controlling replication initiation; in many cases, these mechanisms are intricately connected to the activities of DnaA at the origin of replication. This Review presents an overview of both the mechanism and regulation of bacterial DNA replication initiation, with emphasis on the features that are similar in eukaryotic and archaeal systems.

Crystal structure of an archaeal homologue of multidrug resistance repressor protein, EmrR, from hyperthermophilic archaea Sulfolobus tokodaii strain 7

MarR family proteins, MarR, MexR, and EmrR, are known as bacterial regulators for a phenotype resistant to multiple antibiotic drugs. Genomic data have indicated the presence of bacterial-type transcriptional regulators, including MarR family proteins in archaea, though the archaeal transcription system is close to that of eukaryote. To elucidate the structural basis of the transcriptional regulation mechanism of archaeal MarR family proteins, the crystal structure of the ST1710 protein, which was identified as an archaeal EmrR homologue, StEmrR, from hyperthermophilic archaeon Sulfolobus tokodaii strain 7 was determined at 1.45-A resolution. The protein was composed of two N- and C-terminal dimerization domains, and the DNA-binding domain consisted of a winged helix motif, as in the case of bacterial MarR family proteins. Despite the relatively low overall structural similarity between StEmrR and bacterial MarR family proteins, the structure of the DNA-binding domain displayed high structural similarity. A comparison with the crystal structures of bacterial MarR family proteins revealed that structural variation was mainly due to the different orientation of the two helices at the N- and C-termini. Our results indicated that the distance between the two DNA-binding domains of MarR family proteins would be changed by the rotation of the two terminal helices to interact with the target DNA.

Characterization of the archaeal SRP system

Characterization of the archaeal SRP system