The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5′-side of a 2′-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity.

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na ؉ (Li ؉ )/H ؉ antiporter activity on the antiporter-deficient Escherichia coli strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive E. coli clones were obtained during the initial screening of 1,480,000 recombinant E. coli strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li ؉ -resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na ؉ /H ؉ antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of E. coli K-12 containing nhaA, nhaR, and gef. This region is flanked by highly conserved insertion elements. The sequence identity with E. coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E. coli. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E. coli and enabled the antiporter-deficient E. coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na ؉ /H ؉ antiporter activity in everted membrane vesicles that were derived from the E. coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The K m values of both antiporters for Na ؉ were approximately 10-fold higher than the values for Li ؉ .

Flow cytometry and epifluorescence microscopy results for the euryarchaeon Methanothermobacter thermautotrophicus were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.Methanothermobacter thermautotrophicus has become an important model system for biochemical characterization of the archaeal chromosome replication machinery, providing the first functional evidence of helicase activity from an archaeal MCM complex (8,12,19) as well as the first structural information concerning these proteins (9, 17). Information concerning the activity and site-specific binding of the two Cdc6 homologues, which are known to play a key role in the control of eukaryotic DNA replication initiation, is also available for this species (7, 10). Insights into the organization of the cell cycle of M. thermautotrophicus will therefore have significant implications for understanding the mechanisms by which the eukaryote-like replication proteins can interface with the bacterial-type cell division proteins found in the euryarchaea (2) and will allow investigations of the cell cycle control mechanisms of these organisms.M. thermautotrophicus (DSMZ 1053) cultures were grown in a 3-liter bioreactor under chemoautotrophic conditions (16), with H 2 and CO 2 as the sole energy and carbon sources. Growth from an initial estimated optical density at 600 nm (OD 600 ) of 0.002 resulted in at least five doublings before sampling and analysis commenced. The culture showed exponential growth to an OD 600 of about 1 (1,500 min), a transition phase between 1,500 and 2,100 min, and then a stationaryphase plateau (Fig. 1A). Flow cytometry (5) was used to determine the DNA content of M. thermautotrophicus filaments at different growth stages (Fig. 1B and C). A correlation with the positions of peaks from flow cytometry of Escherichia coli MG1655 seqA::Tn10 cells, grown in M9 glucose medium to an OD of 0.1 and treated with 200 g of rifampin/ml for 3 h to induce replication runout (20), indicated that the M. thermautotrophicus peaks shown in Fig. 1C could only correspond to two, four, and eight genomes (Fig. 1D). This result was confirmed by a plot of fluorescence versus the inferred chromosome content (Fig. 1E) (3), which yielded superimposed straight lines for both M. thermautotrophicus (genome size, 1.75 Mb; 49.5% GC) (22) and E. coli (genome size, 4.64 Mb; 50.8% GC) (6). The peaks in the DNA content distribution for M. thermautotrophicus at the 1,225-min time point therefore corresponded to 4 and 8 chromosomes, with a shoulder in the graph around a DNA content of 12 chromosomes (Fig. 1C). The DNA content gradually decreased with increasing ODs, and the eight-chromosome peak could no longer be detected after the 2,125-min time point. The four-chromosome...

To date, methanogens are the only group within the archaea where firing DNA replication origins have not been demonstrated in vivo. In the present study we show that a previously identified cluster of ORB (origin recognition box) sequences do indeed function as an origin of replication in vivo in the archaeon Methanothermobacter thermautotrophicus. Although the consensus sequence of ORBs in M. thermautotrophicus is somewhat conserved when compared with ORB sequences in other archaea, the Cdc6-1 protein from M. thermautotrophicus (termed MthCdc6-1) displays sequence-specific binding that is selective for the MthORB sequence and does not recognize ORBs from other archaeal species. Stabilization of in vitro MthORB DNA binding by MthCdc6-1 requires additional conserved sequences 3' to those originally described for M. thermautotrophicus. By testing synthetic sequences bearing mutations in the MthORB consensus sequence, we show that Cdc6/ORB binding is critically dependent on the presence of an invariant guanine found in all archaeal ORB sequences. Mutation of a universally conserved arginine residue in the recognition helix of the winged helix domain of archaeal Cdc6-1 shows that specific origin sequence recognition is dependent on the interaction of this arginine residue with the invariant guanine. Recognition of a mutated origin sequence can be achieved by mutation of the conserved arginine residue to a lysine or glutamine residue. Thus despite a number of differences in protein and DNA sequences between species, the mechanism of origin recognition and binding appears to be conserved throughout the archaea.