The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the doublestranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.The isolation and characterization of plasmids are prerequisites for the development of genetic studies on new groups of microorganisms. Plasmids also are essential tools for studying in vivo and in vitro mechanisms such as DNA replication, recombination, and repair (28). Bacterial plasmids have been extensively analyzed and used in molecular and genetic work. In contrast, information about plasmids in the domain Archaea (the third domain of life sensu Woese et al. [49]) is much more limited. Several plasmids in extreme halophiles and methanogens have been described, and a few of them were completely sequenced: pHV2 (6.3 kb) from Haloferax volcanii (7), pGRB1 (1.8 kb) from Halobacterium sp. strain GRB and its relatives pGN101 and pHSB1 (18,19,26), and pME2001 (4.4 kb) from Methanobacterium thermoautotrophicum Marburg (4). The minimal replication regions for pHH1 (ϳ150 kb) and pNRC100 (ϳ200 kb) from Halobacterium salinarium (33, 37) and pHK2 (10.5 kb) from Haloferax sp. strain Aa2.2 (24) were defined and sequenced. The plasmid pGRB1 and its relatives, as well as pHK2, encode homologous proteins that exhibit similarities with Rep proteins from rolling-circle (RC) replicons of the X174 group (24, 25). In contrast, pNRC100 and pHV2 encode homologous Rep proteins (encoded by repH) unrelated to those of the RC replicons (33). Some of the haloarchaeal plasmids have been used successfully for the construction of shuttle vectors (23,29,30). The number of plasmids described for thermophilic archaea is restricted compared with those described for halophiles and methanogens. The first extrachromosomal element detected in a member of the order Sulfolobales, pSB12 (15.5 kb) (50), was in fact the genome of a lysogenic virus, Sulfolobus shibatae virus 1 (SSV1) (for a review, see refere...

Malic enzyme is one of at least five enzymes, known to be present in Corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. To date, no information is available concerning the physiological role of the malic enzyme in this bacterium. The malE gene from C. glutamicum has been cloned and sequenced. The protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. Biochemical characteristics indicate a decarboxylation role linked to NADPH generation. Strains of C. glutamicum in which the malE gene had been disrupted or overexpressed showed no detectable phenotype during growth on either acetate or glucose, but showed a significant modification of growth behavior during lactate metabolism. The wild type showed a characteristic brief period of exponential growth on lactate followed by a linear growth period. This growth pattern was further accentuated in a malE-disrupted strain (⌬malE). However, the strain overexpressing malE maintained exponential growth until all lactate had been consumed. This strain accumulated significantly larger amounts of pyruvate in the medium than the other strains.