Nucleoid structure and partition in Methanococcus jannaschii: an archaeon with multiple copies of the chromosome. (1/63)

We measured different cellular parameters in the methanogenic archaeon Methanococcus jannaschii. In exponential growth phase, the cells contained multiple chromosomes and displayed a broad variation in size and DNA content. In most cells, the nucleoids were organized into a thread-like network, although less complex structures also were observed. During entry into stationary phase, chromosome replication continued to termination while no new rounds were initiated: the cells ended up with one to five chromosomes per cell with no apparent preference for any given DNA content. Most cells in stationary phase contained more than one genome equivalent. Asymmetric divisions were detected in stationary phase, and the nucleoids were found to be significantly more compact than in exponential phase.  (+info)

Changes in cell size and DNA content in Sulfolobus cultures during dilution and temperature shift experiments. (2/63)

Stationary-phase cultures of different hyperthermophilic species of the archaeal genus Sulfolobus were diluted into fresh growth medium and analyzed by flow cytometry and phase-fluorescence microscopy. After dilution, cellular growth started rapidly but no nucleoid partition, cell division, or chromosome replication took place until the cells had been increasing in size for several hours. Initiation of chromosome replication required that the cells first go through partition and cell division, revealing a strong interdependence between these key cell cycle events. The time points at which nucleoid partition, division, and replication occurred after the dilution were used to estimate the relative lengths of the cell cycle periods. When exponentially growing cultures were diluted into fresh growth medium, there was an unexpected transient inhibition of growth and cell division, showing that the cultures did not maintain balanced growth. Furthermore, when cultures growing at 79 degrees C were shifted to room temperature or to ice-water baths, the cells were found to "freeze" in mid-growth. After a shift back to 79 degrees C, growth, replication, and division rapidly resumed and the mode and kinetics of the resumption differed depending upon the nature and length of the shifts. Dilution of stationary-phase cultures provides a simple protocol for the generation of partially synchronized populations that may be used to study cell cycle-specific events.  (+info)

Halophilic 20S proteasomes of the archaeon Haloferax volcanii: purification, characterization, and gene sequence analysis. (3/63)

A 20S proteasome, composed of alpha(1) and beta subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon Haloferax volcanii. The predominant peptide-hydrolyzing activity of the 600-kDa alpha(1)beta-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75 degrees C. The alpha(1)beta-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the alpha(1)beta-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active alpha(1)beta-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the beta-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and N-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the H. volcanii proteasome (K(i) = 0.2 and 8 microM, respectively). In addition to the genes encoding the alpha(1) and beta subunits, a gene encoding a second alpha-type proteasome protein (alpha(2)) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the alpha(1)beta-proteasome resulted in the copurification of the alpha(2) protein with the alpha(1) and beta subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the alpha(1)beta-proteasome, suggesting that at least two separate 20S proteasomes are synthesized. This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different alpha subunits in this halophilic archaeon.  (+info)

The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum DeltaH contains DNA helicase activity. (4/63)

Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997) J. Biol. Chem. 272, 24508-24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum DeltaH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric ( approximately 10(3) kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3'-to-5' ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.  (+info)

Generation of dominant selectable markers for resistance to pseudomonic acid by cloning and mutagenesis of the ileS gene from the archaeon Methanosarcina barkeri fusaro. (5/63)

Currently, only one selectable marker is available for genetic studies in the archaeal genus Methanosarcina. Here we report the generation of selectable markers that encode resistance to pseudomonic acid (PA(r)) in Methanosarcina species by mutagenesis of the isoleucyl-tRNA synthetase gene (ileS) from Methanosarcina barkeri Fusaro. The M. barkeri ileS gene was obtained by screening of a genomic library for hybridization to a PCR fragment. The complete 3,787-bp DNA sequence surrounding and including the ileS gene was determined. As expected, M. barkeri IleS is phylogenetically related to other archaeal IleS proteins. The ileS gene was cloned into a Methanosarcina-Escherichia coli shuttle vector and mutagenized with hydroxylamine. Nine independent PA(r) clones were isolated after transformation of Methanosarcina acetivorans C2A with the mutagenized plasmids. Seven of these clones carry multiple changes from the wild-type sequence. Most mutations that confer PA(r) were shown to alter amino acid residues near the KMSKS consensus sequence of class I aminoacyl-tRNA synthetases. One particular mutation (G594E) was present in all but one of the PA(r) clones. The MIC of pseudomonic acid for M. acetivorans transformed with a plasmid carrying this single mutation is 70 microgram/ml of medium (for the wild type, the MIC is 12 microgram/ml). The highest MICs (560 microgram/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PA(r) based on the mutant ileS alleles are described. Finally, the implications of the specific mutations we isolated with respect to binding of pseudomonic acid by IleS are discussed.  (+info)

Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon. (6/63)

Despite a rapid increase in the amount of available archaeal sequence information, little is known about the duplication of genetic material in the third domain of life. We identified a single origin of bidirectional replication in Pyrococcus abyssi by means of in silico analyses of cumulative oligomer skew and the identification of an early replicating chromosomal segment. The replication origin in three Pyrococcus species was found to be highly conserved, and several eukaryotic-like DNA replication genes were clustered around it. As in Bacteria, the chromosomal region containing the replication terminus was a hot spot of genome shuffling. Thus, although bacterial and archaeal replication proteins differ profoundly, they are used to replicate chromosomes in a similar manner in both prokaryotic domains.  (+info)

Genome evolution at the genus level: comparison of three complete genomes of hyperthermophilic archaea. (7/63)

We have compared three complete genomes of closely related hyperthermophilic species of Archaea belonging to the Pyrococcus genus: Pyrococcus abyssi, Pyrococcus horikoshii, and Pyrococcus furiosus. At the genomic level, the comparison reveals a differential conservation among four regions of the Pyrococcus chromosomes correlated with the location of genetic elements mediating DNA reorganization. This discloses the relative contribution of the major mechanisms that promote genomic plasticity in these Archaea, namely rearrangements linked to the replication terminus, insertion sequence-mediated recombinations, and DNA integration within tRNA genes. The combination of these mechanisms leads to a high level of genomic plasticity in these hyperthermophilic Archaea, at least comparable to the plasticity observed between closely related bacteria. At the proteomic level, the comparison of the three Pyrococcus species sheds light on specific selection pressures acting both on their coding capacities and evolutionary rates. Indeed, thanks to two independent methods, the "reciprocal best hits" approach and a new distance ratio analysis, we detect the false orthology relationships within the Pyrococcus lineage. This reveals a high amount of differential gains and losses of genes since the divergence of the three closely related species. The resulting polymorphism is probably linked to an adaptation of these free-living organisms to differential environmental constraints. As a corollary, we delineate the set of orthologous genes shared by the three species, that is, the genes that may characterize the Pyrococcus genus. In this conserved core, the amino acid substitution rate is equal between P. abyssi and P. horikoshii for most of their shared proteins, even for fast-evolving ones. In contrast, strong discrepancies exist among the substitution rates observed in P. furiosus relative to the two other species, which is in disagreement with the molecular clock hypothesis.  (+info)

In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin. (8/63)

Although genome analyses have suggested parallels between archaeal and eukaryotic replication systems, little is known about the DNA replication mechanism in Archaea. By two-dimensional gel electrophoreses we positioned a replication origin (oriC) within 1 kb in the chromosomal DNA of Pyrococcus abyssi, an anaerobic hyperthermophile, and demonstrated that the oriC is physically linked to the cdc6 gene. Our chromatin immunoprecipitation assays indicated that P. abyssi Cdc6 and minichromosome maintenance (MCM) proteins bind preferentially to the oriC region in the exponentially growing cells. Whereas the oriC association of MCM was specifically inhibited by stopping DNA replication with puromycin treatment, Cdc6 protein stayed bound to the replication origin after de novo protein synthesis was inhibited. Our data suggest that archaeal and eukaryotic Cdc6 and MCM proteins function similarly in replication initiation and imply that an oriC association of MCM could be regulated by an unknown mechanism in Archaea.  (+info)

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