The genome-scale DNA-binding profile of BarR, a β-alanine responsive transcription factor in the archaeon Sulfolobus acidocaldarius

The DNA-binding domains of S. acidocaldarius BarR and S. solfataricus Ss-LrpB might share a common ancestor. (PDF 198 kb

The proteasome controls ESCRT-III–mediated cell division in an archaeon

INTRODUCTION: Eukaryotes likely arose from a symbiotic partnership between an archaeal host and an alpha-proteobacterium, giving rise to the cell body and the mitochondria, respectively. Because of this, a number of proteins controlling key events in the eukaryotic cell division cycle have their origins in archaea. These include ESCRT-III proteins, which catalyze the final step of cytokinesis in many eukaryotes and in the archaeon Sulfolobus acidocaldarius. However, to date, no archaeon has been found that harbors homologs of cell cycle regulators, like cyclin-dependent kinases and cyclins, which order events in the cell cycle across all eukaryotes. Thus, it remains uncertain how key events in the archaeal cell cycle, including division, are regulated. RATIONALE: An exception to this is the 20S proteasome, which is conserved between archaea and eukaryotes and which regulates the eukaryotic cell cycle through the degradation of cyclins. To explore the function of the 20S proteasome in the archaeon S. acidocaldarius, we determined its structure by crystallography and carried out in vitro biochemical analyses of its activity with and without inhibition. The impact of proteasome inhibition on cell division and cell cycle progression was examined in vivo by flow cytometry and super-resolution microscopy. Following up with mass spectrometry, we identified proteins degraded by the proteasome during division. Finally, we used molecular dynamics simulations to model the mechanics of this process. RESULTS: Here, we present a structure of the 20S proteasome of S. acidocaldarius to a resolution of 3.7 Å, which we used to model its sensitivity to the eukaryotic inhibitor bortezomib. When this inhibitor was added to synchronous cultures, it was found to arrest cells mid-division, with a stable ESCRT-III division ring positioned at the cell center between the two separated and prereplicative nucleoids. Proteomics was then used to identify a single archaeal ESCRT-III homolog, CdvB, as a key target of the proteasome that must be degraded to enable division to proceed. Examining the localization patterns of CdvB and two other archaeal ESCRT-III homologs, CdvB1 and CdvB2, by flow cytometry and super-resolution microscopy revealed the sequence of events that leads to division. First, a CdvB ring is assembled. This CdvB ring then templates the assembly of the contractile ESCRT-III homologs, CdvB1 and CdvB2, to form a composite division ring. Cell division is then triggered by proteasome-mediated degradation of CdvB, which allows the CdvB1:CdvB2 copolymer to constrict, pulling the membrane with it. During constriction, the CdvB1:CdvB2 copolymer is disassembled, thus vacating the membrane neck to drive abscission, yielding two daughter cells with diffuse CdvB1 and CdvB2. CONCLUSION: This study reveals a role for the proteasome in driving structural changes in a composite ESCRT-III copolymer, enabling the stepwise assembly, disassembly, and contraction of an ESCRT-III–based division ring. Although it is not yet clear how proteasomal inhibition prevents S. acidocaldarius cells from resetting the cell cycle to initiate the next S phase, these data strengthen the case for the eukaryotic cell cycle regulation having its origins in archaea

Tripeptidyl-Peptidase II : Structure, Function and Gene Regulation

The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using Pichia pastoris was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the KMapp is more than 102 higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species

The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius

An actin-based cytoskeleton in archaea

In eukaryotic and bacterial cells, spatial organization is dependent upon cytoskeletal filaments. Actin is a main eukaryotic cytoskeletal element, involved in key processes such as cell shape determination, mechanical force generation and cytokinesis. We describe an archaeal cytoskeleton which forms helical structures within Pyrobaculum calidifontis cells, as shown by in situ immunostaining. The core components include an archaeal actin homologue, Crenactin, closely related to the eukaryotic counterpart. The crenactin gene belongs to a conserved gene cluster denoted Arcade (actin-related cytoskeleton in Archaea involved in shape determination). The phylogenetic distribution of arcade genes is restricted to the crenarchaeal Thermoproteales lineage, and to Korarchaeota, and correlates with rod-shaped and filamentous cell morphologies. Whereas Arcadin-1, -3 and -4 form helical structures, suggesting cytoskeleton-associated functions, Arcadin-2 was found to be localized between segregated nucleoids in a cell subpopulation, in agreement with possible involvement in cytokinesis. The results support a crenarchaeal origin of the eukaryotic actin cytoskeleton and, as such, have implications for theories concerning the origin of the eukaryotic cell.</p

Archaeal actin-family filament systems

Actin represents one of the most abundant and conserved eukaryotic proteins over time, and has an important role in many different cellular processes such as cell shape determination, motility, force generation, cytokinesis, amongst many others. Eukaryotic actin has been studied for decades and was for a long time considered a eukaryote-specific trait. However, in the early 2000s a bacterial actin homolog, MreB, was identified, characterized and found to have a cytoskeletal function and group within the superfamily of actin proteins. More recently, an actin cytoskeleton was also identified in archaea. The genome of the hyperthermophilic crenarchaeon Pyrobaculum calidifontis contains a five-gene cluster named Arcade encoding for an actin homolog, Crenactin, polymerizing into helical filaments spanning the whole length of the cell. Phylogenetic and structural studies place Crenactin closer to the eukaryotic actin than to the bacterial homologues. A significant difference, however, is that Crenactin can form single helical filaments in addition to filaments containing two intertwined proto filaments. The genome of the recently discovered Lokiarchaeota encodes several different actin homologues, termed Lokiactins, which are even more closely related to the eukaryotic actin than Crenactin. A primitive, dynamic actin-based cytoskeleton in archaea could have enabled the engulfment of the alphaproteobacterial progenitor of the mitochondria, a key-event in the evolution of eukaryotes.</p

Archaeal actin from a hyperthermophile forms a single-stranded filament

The prokaryotic origins of the actin cytoskeleton have been firmly established, but it has become clear that the bacterial actins form a wide variety of different filaments, different both from each other and from eukaryotic F-actin. We have used electron cryomicroscopy (cryo-EM) to examine the filaments formed by the protein crenactin (a crenarchaeal actin) from Pyrobaculum calidifontis, an organism that grows optimally at 90 °C. Although this protein only has ∼20% sequence identity with eukaryotic actin, phylogenetic analyses have placed it much closer to eukaryotic actin than any of the bacterial homologs. It has been assumed that the crenactin filament is double-stranded, like F-actin, in part because it would be hard to imagine how a single-stranded filament would be stable at such high temperatures. We show that not only is the crenactin filament single-stranded, but that it is remarkably similar to each of the two strands in F-actin. A large insertion in the crenactin sequence would prevent the formation of an F-actin-like double-stranded filament. Further, analysis of two existing crystal structures reveals six different subunit-subunit interfaces that are filament-like, but each is different from the others in terms of significant rotations. This variability in the subunit-subunit interface, seen at atomic resolution in crystals, can explain the large variability in the crenactin filaments observed by cryo-EM and helps to explain the variability in twist that has been observed for eukaryotic actin filaments

Alpinia speciosa K. Schum.

原著和名: ゲッタウ科名: ショウガ科 = Zingiberaceae採集地: 沖縄県 石垣島 (琉球 石垣島)採集日: 1978/5/15採集者: 萩庭丈壽整理番号: JH015424国立科学博物館整理番号: TNS-VS-96542

DNA-Binding Properties of a Novel Crenarchaeal Chromatin-Organizing Protein in Sulfolobus acidocaldarius

In archaeal microorganisms, the compaction and organization of the chromosome into a dynamic but condensed structure is mediated by diverse chromatin-organizing proteins in a lineage-specific manner. While many archaea employ eukaryotic-type histones for nucleoid organization, this is not the case for the crenarchaeal model species Sulfolobus acidocaldarius and related species in Sulfolobales, in which the organization appears to be mostly reliant on the action of small basic DNA-binding proteins. There is still a lack of a full understanding of the involved proteins and their functioning. Here, a combination of in vitro and in vivo methodologies is used to study the DNA-binding properties of Sul12a, an uncharacterized small basic protein conserved in several Sulfolobales species displaying a winged helix&ndash;turn&ndash;helix structural motif and annotated as a transcription factor. Genome-wide chromatin immunoprecipitation and target-specific electrophoretic mobility shift assays demonstrate that Sul12a of S. acidocaldarius interacts with DNA in a non-sequence specific manner, while atomic force microscopy imaging of Sul12a&ndash;DNA complexes indicate that the protein induces structural effects on the DNA template. Based on these results, and a contrario to its initial annotation, it can be concluded that Sul12a is a novel chromatin-organizing protein