Search

Detailed analyses of the bacterial and yeast cell cycles are now possible with flow cytometry. We will focus here on fundamental aspects of cell cycle analysis of the bacterium Escherichia coli, and demonstrate how DNA and cell size distributions may be used to calculate key parameters of the bacterial cell cycle. Also when flow cytometry is merely used to monitor the state of a microbial culture, knowledge of basic DNA replication and cell division patterns is useful, since the DNA and size distributions are very sensitive to changes in the growth conditions. [For reviews on basic cell cycle analysis and advances of flow cytometry in applied microbiology the reader is referred to Ref. 1 and 2, respectively].

Summary In archaea, nothing is known about the β-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 β-alanine aminotransferase. Deletion of barR resulted in a reduced exponential growth rate in the presence of β-alanine. Furthermore, qRT-PCR and promoter activity assays demonstrated that BarR activates the expression of the adjacent aminotransferase gene, but only upon β-alanine supplementation. In contrast, auto-activation proved to be β-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 β-alanine and site-mutant analyses indicated that β-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.

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.

Summary 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.

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.

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.

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.

Archaea is one of the three domains of life and studies of archaeal biology are important for understanding of life in extreme environments, fundamental biogeochemical processes, the origin of life, the eukaryotic cell and their own, unique biology. This thesis presents four studies of the archaeal cell, using the extremophilic Sulfolobus and ocean living Nitrosopumilus as model systems. Cell division in crenarchaea is shown to be carried out by a previously unknown system named Cdv (cell division). The system shares homology with the eukaryotic ESCRT-III system which is used for membrane reorganization during vesicle formation, viral release and cytokinesis. Organisms of the phylum Thaumarchaeota also use the Cdv system, despite also carrying genes for the euryarchaeal and bacterial cell division system FtsZ. The thaumarchaeal cell cycle is demonstrated to be dominated by the prereplicative and replicative stage, in contrasts to the crenarchaeal cell cycle where the cell at the majority of the time resides in the postreplicative stage. The replication rate is remarkably low and closer to what is measured for eukaryotes than other archaea. The gene organization of Sulfolobus is significantly associated with the three origins of replication. The surrounding regions are dense with genes of high importance for the organisms such as highly transcribed genes, genes with known function in fundamental cellular processes and conserved archaeal genes. The overall gene density is elevated and transposons are underrepresented. The archaeal virus SIRV2 displays a lytic life style where the host cell at the final stage of infection is disrupted for release of new virus particles. The remarkable pyramid-like structure VAP (virus associated pyramids), that is formed independently of the virus particle, is used for cell lysis. The research presented in this thesis describes unique features of the archaeal cell and influences our understanding of the entire tree of life.

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.

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.

Much of the current information about the archaeal cell cycle has been generated through studies of the genus Sulfolobus. The overall organization of the cell cycle in these species is well understood, and information about the regulatory principles that govern cell cycle progression is rapidly accumulating. Exciting progress regarding the control and molecular details of the chromosome replication process is evident, and the first insights into the elusive crenarchaeal mitosis and cytokinesis machineries are within reach.

Relative RNA abundance was measured at different cell-cycle stages in synchronized cultures of the hyperthermophilic archaeon Sulfolobus acidocaldarius . Cyclic induction was observed for >160 genes, demonstrating central roles for transcriptional regulation and cell-cycle-specific gene expression in archaeal cell-cycle progression. Many replication genes were induced in a cell-cycle-specific manner, and novel replisome components are likely to be among the genes of unknown function with similar induction patterns. Candidate genes for the unknown genome segregation and cell division machineries were also identified, as well as seven transcription factors likely to be involved in cell-cycle control. Two serine-threonine protein kinases showed distinct cell-cycle-specific induction, suggesting regulation of the archaeal cell cycle also through protein modification. Two candidate recognition elements, CCR boxes, for transcription factors in control of cell-cycle regulons were identified among gene sets with similar induction kinetics. The results allow detailed characterization of the genome segregation, division, and replication processes and may, because of the extensive homologies between the archaeal and eukaryotic information machineries, also be applicable to core features of the eukaryotic cell cycle.

The nucleotide excision repair (NER) pathway removes bulky lesions such as photoproducts from DNA. In both bacteria and eukarya, lesions located in transcribed strands are repaired significantly faster than those located in non-transcribed strands due to damage signalling by stalled RNA polymerase molecules: a phenomenon known as transcription-coupled repair (TCR). TCR requires a mechanism for coupling the detection of stalled RNA polymerase molecules to the NER pathway, provided in bacteria by the Mfd protein. In the third domain of life, archaea, the pathway of NER is not well defined, there are no Mfd homologues and the existence of TCR has not been investigated. In this report we looked at rates of removal of photoproducts in three different operons of the crenarchaeon Sulfolobus solfataricus following UV irradiation. We found no evidence for significantly faster repair in the transcribed strands of these three operons. The rate of global genome repair in S. solfataricus is relatively rapid, and this may obviate the requirement for a specialized TCR pathway. Significantly faster repair kinetics were observed in the presence of visible light, consistent with the presence of a gene for photolyase in the genome of S. solfataricus.

Motivation: Microarray experiments using probes covering a whole transcriptome are expensive to initiate, and a major part of the costs derives from synthesizing gene-specific PCR primers or hybridization probes. The high costs may force researchers to limit their studies to a single organism, although comparing gene expression in different species would yield valuable information. Results: We have developed a method, implemented in the software DualPrime, that reduces the number of primers required to amplify the genes of two different genomes. The software identifies regions of high sequence similarity, and from these regions selects PCR primers shared between the genomes, such that either one or, preferentially, both primers in a given PCR can be used for amplification from both genomes. To assure high microarray probe specificity, the software selects primer pairs that generate products of low sequence similarity to other genes within the same genome. We used the software to design PCR primers for 2182 and 1960 genes from the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. Primer pairs were shared among 705 pairs of genes, and single primers were shared among 1184 pairs of genes, resulting in a saving of 31% compared to using only unique primers. We also present an alternative primer design method, in which each gene shares primers with two different genes of the other genome, enabling further savings. 3. Availability: The software is freely available at http://www.biotech.kth.se/molbio/microarray/ Contact: anders.andersson@biotech.kth.se

Chromosome replication origins were mapped in vivo in the two hyperthermophilic archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus , by using microarray-based marker frequency analysis. Bidirectional replication was found to be initiated in near synchrony from three separate sites in both organisms. Two of the three replication origins in each species were located in the vicinity of a cdc6 / orc1 replication initiation gene, whereas no known replication-associated gene could be identified near the third origin in either organism. In contrast to initiation, replication termination occurred asynchronously, such that certain replication forks continued to progress for >40 min after the others had terminated. In each species, all replication forks advanced at similar DNA polymerization rates; this was found to be an order of magnitude below that displayed by Escherichia coli and thus closer to eukaryotic elongation rates. In S. acidocaldarius , a region containing short regularly spaced repeats was found to hybridize aberrantly, as compared to the rest of the chromosome, raising the possibility of a centromere-like function.

The recently discovered structural similarities between the archaeal Orc1/Cdc6 and bacterial DnaA initiator proteins for chromosome replication have exciting implications for cell cycle regulation. Together with current attempts to identify archaeal chromosome replication origins, the information is likely to yield fundamental insights into replication control in both archaea and eukaryotes within the near future. Several proteins that affect, or are likely to affect, chromatin structure and genome segregation in archaea have been described recently, including Sph1 and 2, ScpA and B, Sir2, Alba and Rio1p. Important insights into the properties of the MinD and FtsZ cell division proteins, and of putative cytoskeletal elements, have recently been gained in bacteria. As these proteins also are present among archaea, it is likely that the new information will also be essential for understanding archaeal genome segregation and cell division. A series of interesting cell cycle issues has been brought to light through the discovery of the novel Nanoarchaeota phylum, and these are outlined briefly. Exciting areas for extended cell cycle investigations of archaea are identified, including termination of chromosome replication, application of in situ cytological techniques for localization of cell cycle proteins and the regulatory roles of GTP-binding proteins and small RNAs.

We analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus(Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea.

On 19 January 2014 Rolf ('Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.

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.

In archaea, nothing is known about the β-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 β-alanine aminotransferase. Deletion of barR resulted in a reduced exponential growth rate in the presence of β-alanine. Furthermore, qRT-PCR and promoter activity assays demonstrated that BarR activates the expression of the adjacent aminotransferase gene, but only upon β-alanine supplementation. In contrast, auto-activation proved to be β-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 β-alanine and site-mutant analyses indicated that β-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.

The regulation and co-ordination of the cell cycle of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius was investigated with antibiotics. We provide evidence for a core regulation involving alternating rounds of chromosome replication and genome segregation. In contrast, multiple rounds of replication of the chromosome could occur in the absence of an intervening cell division event. Inhibition of the elongation stage of chromosome replication resulted in cell division arrest, indicating that pathways similar to checkpoint mechanisms in eukaryotes, and the SOS system of bacteria, also exist in archaea. Several antibiotics induced cell cycle arrest in the G2 stage. Analysis of the run-out kinetics of chromosome replication during the treatments allowed estimation of the minimal rate of replication fork movement in vivo to 250 bp s-1. An efficient method for the production of synchronized Sulfolobus populations by transient daunomycin treatment is presented, providing opportunities for studies of cell cycle-specific events. Possible targets for the antibiotics are discussed, including topoisomerases and protein glycosylation.

As a basis for studing the essential cellular processes of hyperthermophilic archaea, thermosensitive mutants of Sulfolobus acidocaldarius were isolated and characterized. Exponential-phase liquid cultures were shifted to the non-permissive temperature and growth, viability, and distributions of cell mass and DNA content were measured as a function of time after the shift. The observed phenotypes demonstrate that chromosome replication, nucleoid organization, nucleoid partition and cell division, which normally are tightly co-ordinated during cellular growth, can be inhibited or uncoupled by mutation in this hyperthermophilic archaeon.

We sequenced the ftsZ gene region of the halophilic archaeon Haloferax mediterranei and mapped the transcription start sites for the ftsZ gene. The gene encoded a 363-amino-acid long FtsZ protein with a predicted molecular mass of 38 kDa and an isoelectric point of 4.2. A high level of similarity to the FtsZ protein of Haloferax volcanii was apparent, with 97 and 90% identity at the amino acid and nucleotide levels, respectively. Structural conservation at the protein level was shown by visualization of the FtsZ ring structure in H. mediterranei cells using an antiserum raised against FtsZ of H. volcanii. FtsZ rings were observed in cells in different stages of division, including cells with pleomorphic shapes and cells that appeared to be undergoing asymmetric division. Cells were also observed that displayed constriction-like invaginations in the absence of an FtsZ ring, indicating that morphological data are not sufficient to determine whether pleomorphic Haloferax cells are undergoing cell division. Both the upstream and downstream gene order in the ftsZ region was found to be conserved within the genus Haloferax . Furthermore, the downstream gene order, which includes the secE and nusG genes, is conserved in almost all euryarchaea sequenced to date. The secE and nusG genes are likely to be transcriptionally and translationally coupled in Haloferax , and this co-expression may have been a selective force that has contributed to keeping the gene cluster intact.

The DNA content and light scatter from individual Escherichia coli cells were measured in two flow cytometers with different configurations. The DNA content could be measured with similar resolution either in an Argus flow cytometer equipped with a mercury lamp, or in a FACStar flow cytometer with two argon lasers as light sources. In contrast, light scatter measurements appeared to be a good measure of cell mass only in the Argus instrument. Three DNA stains were compared:, DAPI, Hoechst 33258, and mithramycin A together with ethidium bromide. All three stains yielded DNA histograms of similar quality in both flow cytometers. Optimal results required that the stain and cell concentrations were kept similar, that a fixed rate of sample introduction was used, and that a period of equilibration was allowed during running of each sample. The results demonstrate that conventional, laser-based flow cytometers may be used for high-resolution measurements of bacterial DNA content, thereby making flow cytometry available to an increased number of research groups working with prokaryotes.

Nucleoid structure and distribution in thermophilic organisms from the Archaea domain were studied. Combined phase-contrast and fluorescence microscopy of DAPI (4',6-diamidino-2-phenylindole)-stained Sulfolobus acidocaldarius and Sulfolobus solfataricus cells revealed that the nucleoids were highly structured. Different nucleoid distribution within the cells, representing different partition stages, was observed. The conformation of the nucleoids differed between exponentially growing and stationary-phase cells. Also, the stationary-phase cells contained two chromosomes, and the nucleoids occupied a larger part of the interior of the cells than in the exponentially growing cells. The part of the cell cycle during which fully separated nucleoids could be detected was short. Since the postreplication period is long in these organisms, there was a considerable time interval between termination of chromosome replication and completion of nucleoid separation, similar to the G2 phase in eukaryotic cells. The length of the visible cell constriction period was found to be in the same range as that of eubacteria. Finally, cell-cell connections were observed under certain conditions. Possible eubacterial, eukaryotic, and unique features of nucleoid processing and cell division in thermophilic archaea are discussed.

Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature. Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point. The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S. Sazer and S. W. Sherwood, J. Cell Sci. 97: 509-516, 1990). Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis. To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion. The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size. With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content. Premature and abnormal mitosis ('cut') could be observed for the orp1 mutant after only 4 h at restrictive temperature.

Two so-called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini-derivative pOU47 despite the presence of a functional partition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2 bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA. Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild-type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling-circle structures with double-stranded tails. Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling-circle replication.

The crystal structure of the archaeal actin, crenactin, from the rod-shaped hyperthermophilic (optimal growth at 90°C) crenarchaeonPyrobaculum calidifontisis reported at 3.35 Å resolution. 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.

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.

We have constructed intP1 and intFs strains of Escherichia coli in which the basic replicons of either plasmid P1 or plasmid F (oriS) were integrated into an inactivated oriC, such that chromosome replication is controlled by the integrated plasmid replicon. In this study, we have further analysed these strains, and density-shift experiments revealed that chromosome replication occurred randomly during the cell cycle. Flow-cytometry analyses of exponentially growing populations supported this conclusion, and also showed that the DNA/mass ratio of the strains decreased with increasing growth rate. Flow cytometry of exponentially growing cultures treated with rifampicin demonstrated that initiation of replication was uncoordinated in cells containing multiple replication origins.

We report the construction of intP1 and intFs strains, in which the basic replicon from either plasmid P1 or plasmid F (oriS) has been integrated in both orientations into the origin of replication, oriC, of the Escherichia coli chromosome. In these strains, oriC is no longer functional and chromosome-replication is instead controlled by the integrated plasmid replicon. The strains were viable, showing that the deviation from normal chromosome-replication control was not large enough to prohibit cell survival. The strains showed a broader cell-size distribution than a wild-type strain and were more filamentous in rich than in minimal media, although cells of wild-type size were also present. Cells which contained aberrantly shaped or aberrantly distributed nucleoids were also observed. Marker-frequency analysis indicated that chromosome replication was predominantly bidirectional in both intFs strains. In the intP1 strains, the degree of bidirectionality depended upon the orientation of the integrated replicon.

Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.

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.

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.

We have previously constructed Escherichia coli strains in which an R1 plasmid is integrated into the origin of chromosome replication, oriC. In such intR1 strains, oriC is inactive and initiation of chromosome replication instead takes place at the integrated R1 origin. Due to the large size of the chromosome, replication intermediates generated at the R1 origin in these strains are considerably more long-lived than those in unintegrated R1 plasmids. We have taken advantage of this and performed primer extensions on total DNA isolated from intR1 strains, and mapped the free 5' DNA ends that were generated as replication intermediates during R1 replication in vivo. The sensitivity of the mapping was considerably improved by the use of a repeated primer extension method (RPE). The free DNA ends were assumed to represent normal in vivo start sites for leading strand DNA synthesis in plasmid R1. The ends were mapped to a short region approximately 380 bp away from the R1 minimal origin, and the positions agreed well with previous in vitro mappings. The same start positions were also utilized in the absence of the DnaA protein, indicating that DnaA is not required for determination of the position at which DNA synthesis starts during initiation of replication at the R1 origin.

Summary The direct visualization of plasmid DNA inside Escherichia coli cells is demonstrated using phase-fluorescence microscopy of DAPI (4′,6-diamidino-2-phenylindole)-stained bacteria. Small as well as large plasmids could be detected, both in minicells and in cells of larger size. For large plasmids, even single molecules appeared to be within the detection limit. The fluorescence generated from monomers of small plasmids was probably below this limit, and for these plasmids the observed signals may represent aggregates. The distribution of the fluorescence foci might reflect specific plasmid positioning during partition and/or replication.

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.

In the life cycle of a bacterium there are several key processes: cellular growth, chromosome replication and decatenation, nucleoid partition, septum formation, and cell division. These processes have to be carefully controlled and co-ordinated both with respect to each other and to the growth of the cell, and could be viewed as parts of a single cycle in which each step is dependent upon the previous one. Alternatively, they could be independently controlled and carefully tuned to each other without actually constituting a true cycle. In this review, using Escherichia coli as model system, we discuss these two ways of describing the bacterial life cycle. The evidence supporting independent control of the processes is presented, and some of the key questions in the elucidation of the regulation of the bacterial life cycle are discussed.

The phenotypic effect of the tus::kan mutation in an Escherichia coli strain, where the chromosome is replicated unidirectionally by an integrated R1 miniplasmid, was examined by flow cytometry and phase fluorescence microscopy. The tus+ cells exhibited perturbed cell division, as indicated by the presence of many elongated cells and filaments. Inactivation of the tus gene led to a reduction in the frequency of such elongated cells, presumably by eliminating Tus-mediated polar arrests of replication forks at ter sites, thereby shortening the time required for completion of chromosome replication.

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.

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.

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.

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.

The transcriptional response to UV irradiation was analyzed in two related crenarchaea, Sulfolobus solfataricus and Sulfolobus acidocaldarius, showing a clear response to DNA damage but no increase in the expression of DNA repair genes., 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.

The nucleotide excision repair (NER) pathway removes bulky lesions such as photoproducts from DNA. In both bacteria and eukarya, lesions located in transcribed strands are repaired significantly faster than those located in non-transcribed strands due to damage signalling by stalled RNA polymerase molecules: a phenomenon known as transcription-coupled repair (TCR). TCR requires a mechanism for coupling the detection of stalled RNA polymerase molecules to the NER pathway, provided in bacteria by the Mfd protein. In the third domain of life, archaea, the pathway of NER is not well defined, there are no Mfd homologues and the existence of TCR has not been investigated. In this report we looked at rates of removal of photoproducts in three different operons of the crenarchaeon Sulfolobus solfataricus following UV irradiation. We found no evidence for significantly faster repair in the transcribed strands of these three operons. The rate of global genome repair in S. solfataricus is relatively rapid, and this may obviate the requirement for a specialized TCR pathway. Significantly faster repair kinetics were observed in the presence of visible light, consistent with the presence of a gene for photolyase in the genome of S. solfataricus.

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.

The discovery of multiple chromosome replication origins in Sulfolobus species has added yet another eukaryotic trait to the archaea, and brought new levels of complexity to the cell cycle in terms of initiation of chromosome replication, replication termination and chromosome decatenation. Conserved repeated DNA elements — origin recognition boxes — have been identified in the origins of replication, and shown to bind the Orc1/Cdc6 proteins involved in cell cycle control. The origin recognition boxes aid in the identification and characterization of new origins, and their conservation suggests that most archaea have a similar replication initiation mechanism. Cell-cycle-dependent variation in Orc1/Cdc6 levels has been demonstrated, reminiscent of variations in cyclin levels during the eukaryotic cell cycle. Information about archaeal chromosome segregation is also accumulating, including the identification of a protein that binds to short regularly spaced repeats that might constitute centromer-like elements. In addition, studies of cell-cycle-specific gene expression have potential to reveal, in the near future, missing components in crenarchaeal chromosome replication, genome segregation and cell division. Together with an increased number of physiological and cytological investigations of the overall organization of the cell cycle, rapid progress of the archaeal cell cycle field is evident, and archaea, in particular Sulfolobus species, are emerging as simple and powerful models for the eukaryotic cell cycle.