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

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

Chromosome replication origins: do we really need them?

Replication of the main chromosome in the halophilic archaeon Haloferax volcanii was recently reported to continue despite deletion of all active replication origins. Equally surprising, the deletion strain grew faster than the parent strain. It was proposed that origin-less H. volcanii duplicate their chromosomes via recombination-dependent replication. Here, we recall our present knowledge of this mode of chromosome replication in different organisms. We consider the likelihood that it accounts for the viability of H. volcanii deleted for its main specific replication origins, as well as possible alternative interpretations of the results. The selective advantages of having defined chromosome replication origins are discussed from a functional and evolutionary perspective.

Structure of crenactin, an archaeal actin homologue active at 90°C.

The crystal structure of the archaeal actin, crenactin, from the rod-shaped hyperthermophilic (optimal growth at 90°C) crenarchaeon Pyrobaculum calidifontis is reported at 3.35 Šresolution. Despite low amino-acid sequence identity, the three-dimensional structure of the protein monomer is highly similar to those of eukaryotic actin and the bacterial MreB protein. Crenactin-specific features are also evident, as well as elements that are shared between crenactin and eukaryotic actin but are not found in MreB. In the crystal, crenactin monomers form right-handed helices, demonstrating that the protein is capable of forming filament-like structures. Monomer interactions in the helix, as well as interactions between crenactin and ADP in the nucleotide-binding pocket, are resolved at the atomic level and compared with those of actin and MreB. The results provide insights into the structural and functional properties of a heat-stable archaeal actin and contribute to the understanding of the evolution of actin-family proteins in the three domains of life.

Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius.

In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius.

Mapping of active replication origins in vivo in thaum- and euryarchaeal replicons.

We report mapping of active replication origins in thaum- and euryarchaeal replicons using high-throughput sequencing-based marker frequency analysis. The chromosome of the thaumarchaeon Nitrosopumilus maritimus is shown to contain a single origin of replication, whereas the main chromosome in the halophilic euryarchaea Haloferax mediterranei and Haloferax volcanii each contains two origins. All replication origins specified bidirectional replication, and the two origins in the halophiles were initiated in synchrony. The pHM500 plasmid of H. mediterranei is shown to contain a single origin, and the copy numbers of five plasmid replicons in the two halophiles were inferred to be close to that of the main chromosome. Origin recognition boxes (ORBs) that provide binding sites for Orc1/Cdc6 replication initiator proteins are identified at all chromosomal origins, as well as in a range of additional thaumarchaeal species. An annotation update is provided for all three species.

The cell cycle of archaea.

Growth and proliferation of all cell types require intricate regulation and coordination of chromosome replication, genome segregation, cell division and the systems that determine cell shape. Recent findings have provided insight into the cell cycle of archaea, including the multiple-origin mode of DNA replication, the initial characterization of a genome segregation machinery and the discovery of a novel cell division system. The first archaeal cytoskeletal protein, crenactin, was also recently described and shown to function in cell shape determination. Here, we outline the current understanding of the archaeal cell cycle and cytoskeleton, with an emphasis on species in the genus Sulfolobus, and consider the major outstanding questions in the field.

Four chromosome replication origins in the archaeon Pyrobaculum calidifontis.

Replication origins were mapped in hyperthermophilic crenarchaea, using high-throughput sequencing-based marker frequency analysis. We confirm previous origin mapping in Sulfolobus acidocaldarius, and demonstrate that the single chromosome of Pyrobaculum calidifontis contains four replication origins, the highest number detected in a prokaryotic organism. The relative positions of the origins in both organisms coincided with regions enriched in highly conserved (core) archaeal genes. We show that core gene distribution provides a useful tool for origin identification in archaea, and predict multiple replication origins in a range of species. One of the P. calidifontis origins was mapped in detail, and electrophoretic mobility shift assays demonstrated binding of the Cdc6/Orc1 replication initiator protein to a repeated sequence element, denoted Orb-1, within the origin. The high-throughput sequencing approach also allowed for an annotation update of both genomes, resulting in the restoration of open reading frames encoding proteins involved in, e.g., sugar, nitrate and energy metabolism, as well as in glycosylation and DNA repair.

Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus.

Cell division is mediated by different mechanisms in different evolutionary lineages. While bacteria and euryarchaea utilize an FtsZ-based mechanism, most crenarchaea divide using the Cdv system, related to the eukaryotic ESCRT-III machinery. Intriguingly, thaumarchaeal genomes encode both FtsZ and Cdv protein homologues, raising the question of their division mode. Here, we provide evidence indicating that Cdv is the primary division system in the thaumarchaeon Nitrosopumilus maritimus. We also show that the cell cycle is differently organized as compared to hyperthermophilic crenarchaea, with a longer pre-replication phase and a shorter post-replication stage. In particular, the time required for chromosome replication is remarkably extensive, 15-18 h, indicating a low replication rate. Further, replication did not continue to termination in a significant fraction of N. maritimus cell populations following substrate depletion. Both the low replication speed and the propensity for replication arrest are likely to represent adaptations to extremely oligotrophic environments. The results demonstrate that thaumarchaea, crenarchaea and euryarchaea display differences not only regarding phylogenetic affiliations and gene content, but also in fundamental cellular and physiological characteristics. The findings also have implications for evolutionary issues concerning the last archaeal common ancestor and the relationship between archaea and eukaryotes.

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.

An archaeal origin for the actin cytoskeleton: Implications for eukaryogenesis.

A hallmark of the eukaryotic cell is the actin cytoskeleton, involved in a wide array of processes ranging from shape determination and phagocytosis to intracellular transport and cytokinesis. Recently, we reported the discovery of an actin-based cytoskeleton also in Archaea. The archaeal actin ortholog, Crenactin, was shown to belong to a conserved operon, Arcade (actin-related cytoskeleton in Archaea involved in shape determination), encoding an additional set of cytoskeleton-associated proteins. Here, we elaborate on the implications of these findings for the evolutionary relation between archaea and eukaryotes, with particular focus on the possibility that eukaryotic actin and actin-related proteins have evolved from an ancestral archaeal actin gene. Archaeal actin could thus have played an important role in cellular processes essential for the origin and early evolution of the eukaryotic lineage. Further exploration of uncharacterized archaeal lineages is necessary to find additional missing pieces in the evolutionary trajectory that ultimately gave rise to present-day organisms.

FtsZ-less cell division in archaea and bacteria.

A dedicated cell division machinery is needed for efficient proliferation of an organism. The eukaryotic actin-myosin based mechanism and the bacterial FtsZ-dependent machinery have both been characterized in detail, and a third division mechanism, the Cdv system, was recently discovered in archaea from the Crenarchaeota phylum. Despite these findings, division mechanisms remain to be identified in, for example, organisms belonging to the bacterial PVC superphylum, bacteria with extremely reduced genomes, wall-less archaea and bacteria, and in archaea that carry out the division process without cell constriction. Cytokinesis mechanisms in these clades and individual taxa are likely to include adaptation of host functions to division of bacterial symbionts, transfer of bacterial division genes into the host genome, vesicle formation without a dedicated constriction machinery, cross-wall formation without invagination, as well as entirely novel division mechanisms.

Replication-biased genome organisation in the crenarchaeon Sulfolobus.

BACKGROUND: Species of the crenarchaeon Sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication. RESULTS: We demonstrate that global gene expression in two Sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. The bias by far exceeds what would be anticipated by gene dosage effects alone. In addition, early replicating regions are denser in archaeal core genes (enriched in essential functions), display lower intergenic distances, and are devoid of mobile genetic elements. CONCLUSION: The strong replication-biased structuring of the Sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. The higher-level chromosomal organisation could be of importance for minimizing the impact of DNA damage, and may also be linked to transcriptional regulation.

Comparative and functional analysis of the archaeal cell cycle.

The temporal and spatial organization of the chromosome replication, genome segregation and cell division processes is less well understood in species belonging to the Archaea, than in those from the Bacteria and Eukarya domains. Novel insights into the regulation and key components of the Sulfolobus acidocaldarius cell cycle have been obtained through genome-wide analysis of cell cycle-specific gene expression, followed by cloning and characterization of gene products expressed at different cell cycle stages. Here, the results of the transcript profiling are further explored, and potential key players in archaeal cell cycle progression are highlighted in an evolutionary context, by comparing gene expression patterns and gene conservation between three selected microbial species from different domains of life. We draw attention to novel putative nucleases and helicases implicated in DNA replication, recombination and repair, as well as to potential genome segregation factors. Focus is also placed upon regulatory features, including transcription factors and protein kinases inferred to be involved in the execution of specific cell cycle stages, and regulation through metabolic coupling is discussed.

Cell division and the ESCRT complex: A surprise from the archaea.

The Archaea constitute the third domain of life, a separate evolutionary lineage together with the Bacteria and the Eukarya.1 Species belonging to the Archaea contain a surprising mix of bacterial (metabolism, life style, genomic organization) and eukaryotic (replication, transcription, translation) features.2 The archaeal kingdom comprises two main phyla, the Crenarchaeota and the Euryarchaeota. Regarding the cell division process in archaeal species (reviewed in ref. 3), members of the Euryarchaeota rely on an FtsZ-based cell division mechanism4 whereas, previously, no division genes had been detected in the crenarchaea. However, we recently reported the discovery of the elusive cell division machinery in crenarchaea from the genus Sulfolobus.5 The minimal machinery consists of three genes, which we designated cdvA, B and C (for cell division), organized into an operon that is widely conserved among crenarchaea. The gene products polymerize between segregating nucleoids at the early mitotic stage, forming a complex that remains associated with the leading edge of constriction throughout cytokinesis. Interestingly, CdvB and CdvC were shown to be related to the eukaryotic ESCRT-III protein sorting machinery (reviewed in ref. 6), indicating shared common ancestry and mechanistic similarities to endosomal vesicle formation and viral (HIV) budding in eukaryotes. We also demonstrated that the cdv operon is subject to checkpoint-like regulation, and that the genes display a complementary phylogenetic distribution within the Archaea domain relative to FtsZ-dependent division systems.5 Here, the findings are further explored and discussed, and topics for further investigation are suggested.

A unique virus release mechanism in the Archaea.

Little is known about the infection cycles of viruses infecting cells from Archaea, the third domain of life. Here, we demonstrate that the virions of the archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) are released from the host cell through a mechanism, involving the formation of specific cellular structures. Large pyramidal virus-induced protrusions transect the cell envelope at several positions, rupturing the S-layer; they eventually open out, thus creating large apertures through which virions escape the cell. We also demonstrate that massive degradation of the host chromosomes occurs because of virus infection, and that virion assembly occurs in the cytoplasm. Furthermore, intracellular viral DNA is visualized by flow cytometry. The results show that SIRV2 is a lytic virus, and that the host cell dies as a consequence of elaborated mechanisms orchestrated by the virus. The generation of specific cellular structures for a distinct step of virus life cycle is known in eukaryal virus-host systems but is unprecedented in cells from other domains.

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.

Cell cycle characteristics of crenarchaeota: unity among diversity.

The hyperthermophilic archaea Acidianus hospitalis, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrobaculum calidifontis, and Sulfolobus tokodaii representing three different orders in the phylum Crenarchaeota were analyzed by flow cytometry and combined phase-contrast and epifluorescence microscopy. The overall organization of the cell cycle was found to be similar in all species, with a short prereplicative period and a dominant postreplicative period that accounted for 64 to 77% of the generation time. Thus, in all Crenarchaeota analyzed to date, cell division and initiation of chromosome replication occur in close succession, and a long time interval separates termination of replication from cell division. In Pyrobaculum, chromosome segregation overlapped with or closely followed DNA replication, and further genome separation appeared to occur concomitant with cellular growth. Cell division in P. aerophilum took place without visible constriction.

Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point.

The intestinal parasite Giardia lamblia undergoes cell differentiations that entail entry into and departure from the replicative cell cycle. The pathophysiology of giardiasis depends directly upon the ability of the trophozoite form to replicate in the host upper small intestine. Thus, cell proliferation is tightly linked to disease. However, studies of cell cycle regulation in Giardia have been hampered by the inability to synchronise cultures. Here we report that Giardia isolates of the major human genotypes A and B can be synchronised using aphidicolin, a mycotoxin that reversibly inhibits replicative DNA polymerases in eukaryotic cells. Aphidicolin arrests Giardia trophozoites in the early DNA synthesis (S) phase of the cell cycle. We identified a set of cell cycle orthologues in the Giardia genome using bioinformatic analyses and showed that synchronised parasites express these genes in a cell cycle stage-specific manner. The synchronisation method also showed that during encystation, exit from the ordinary cell cycle occurs preferentially in G(2) and defines a restriction point for differentiation. Synchronisation opens up possibilities for further molecular and cell biological studies of chromosome replication, mitosis and segregation of the complex cytoskeleton in Giardia.

Variation in gene content among geographically diverse Sulfolobus isolates.

The ability of competitive (i.e., comparative) genomic hybridization (CGH) to assess similarity across entire microbial genomes suggests that it should reveal diversification within and between natural populations of free-living prokaryotes. We used CGH to measure relatedness of genomes drawn from Sulfolobus populations that had been shown in a previous study to be diversified along geographical lines. Eight isolates representing a wide range of spatial separation were compared with respect to gene-specific tags based on a closely related reference strain (Sulfolobus solfataricus P2). For the purpose of assessing genetic divergence, 232 loci identified as polymorphic were assigned one of two alleles based on the corresponding fluorescence intensities from the arrays. Clustering of these binary genotypes was stable with respect to changes in the threshold and similarity criteria, and most of the groupings were consistent with an isolation-by-distance model of diversification. These results indicate that increasing spatial separation of geothermal sites correlates not only with minor sequence polymorphisms in conserved genes of Sulfolobus (demonstrated in the previous study), but also with the regions of difference (RDs) that occur between genomes of conspecifics. In view of the abundance of RDs in prokaryotic genomes and the relevance that some RDs may have for ecological adaptation, the results further suggest that CGH on microarrays may have advantages for investigating patterns of diversification in other free-living archaea and bacteria.

Responses of hyperthermophilic crenarchaea to UV irradiation.

BACKGROUND: DNA damage leads to cellular responses that include the increased expression of DNA repair genes, repression of DNA replication and alterations in cellular metabolism. Archaeal information processing pathways resemble those in eukaryotes, but archaeal damage response pathways remain poorly understood. RESULTS: We analyzed the transcriptional response to UV irradiation in two related crenarchaea, Sulfolobus solfataricus and Sulfolobus acidocaldarius. Sulfolobus species encounter high levels of DNA damage in nature, as they inhabit high temperature, aerobic environments and are exposed to sunlight. No increase in expression of DNA repair genes following UV irradiation was observed. There was, however, a clear transcriptional response, including repression of DNA replication and chromatin proteins. Differential effects on the expression of the three transcription factor B (tfb) genes hint at a mechanism for the modulation of transcriptional patterns in response to DNA damage. TFB3, which is strongly induced following UV irradiation, competes with TFB1 for binding to RNA polymerase in vitro, and may act as a repressor of transcription or an alternative transcription factor for certain promoters. CONCLUSION: A clear response to DNA damage was observed, with down-regulation of the DNA replication machinery, changes in transcriptional regulatory proteins, and up-regulation of the biosynthetic enzymes for beta-carotene, which has UV protective properties, and proteins that detoxify reactive oxygen species. However, unlike eukaryotes and bacteria, there was no induction of DNA repair proteins in response to DNA damage, probably because these are expressed constitutively to deal with increased damage arising due to high growth temperatures.