An integration vector for use in Methanococcus voltae was constructed, based on the Escherichia coli vector pUC18. It carries the structural gene for puromycin transacetylase from Streptomyces alboniger, which is flanked by expression signals of M. voltae structural genes and hisA gene sequences of this bacterium. Transformed M. voltae cells are puromycin resistant. Several types of integration of the vector into the chromosome were found. Only one case was due to nonhomologous recombination. The integrated sequences were stable under selective pressure but were slowly lost in some cases in the absence of the selective drug. The vector could be excised from M. voltae chromosomal DNA, recircularized and transformed back into E. coli.

Two regions of homology to Anabaena nifH (nitrogenase Fe protein) were detected in the total DNA of the thermophilic nitrogen-fixing archaebacterium Methanococcus thermolithotrophicus. A 2.8 kb HindIII fragment carrying one of these regions was previously cloned and shown to contain a nifH gene (Souillard et al., 1988) now referred to as ORFnifH2. A 3.4 kb PstI fragment and an overlapping 3.8 kb BglII fragment, containing the second region of homology, were cloned, and a DNA region of 4073 bp was sequenced. It contained four complete open reading frames (ORFs) (ORF nifH1, ORF105, ORF128, ORFnifD) and two truncated ORFs (ORFnifK and ORF96). Five ORFs were transcribed in the same direction in the order of ORFnifH1-ORF105-ORF128-ORFnifD-ORFnifk. ORFnifH1, ORFnifD and ORFnifK were assigned from their similarity to eubacterial nifH and nifDK (nitrogenase MoFe protein) genes. Transcription studies showed that ORFnifH1 and ORFnifD were expressed only under nitrogen-fixation conditions, whereas no ORFnifH2 mRNA was detected under the same conditions. A DNA probe containing ORFnifH1 hybridized with a 1.8 kb mRNA, as detected by a Northern blotting experiment. A transcriptional start site was localized 87 and 88 bp upstream from the ATG codon of ORFnifH1. This site is preceded, 21 bp upstream, by the sequence 5'-TTTATATA-3' already found at the same position in several archaebacterial promoters. ORFnifH1 mRNA was too small to encode ORFnifDK. This was confirmed by the fact that another transcription start site was localized 85 bp upstream from the ATG codon of ORFnifD.

The action of novobiocin and coumermycin (two coumarins which interact with the gyrB subunit of eubacterial DNA gyrase) and ciprofloxacin (a fluoroquinolone which interacts with the gyrA subunit of DNA gyrase) was tested on several archaebacteria, including five methanogens, two halobacteria, and a thermoacidophile. Most strains were sensitive to doses of coumarins (0.02 to 10 Rg/ml) which specifically inhibit DNA gyrase in eubacteria. Ciprofloxacin inhibited growth of the haloalkaliphilic strain Natronobacterium gregoryi and of the methanogen Methanosarcina barkeri. In addition, ciprofloxacin partly relieved the sensitivity to coumarins (and vice versa). Novobiocin inhibited DNA replication in Halobacterium halobium rapidly and specifically. Topological analysis has shown that the 1.7-kilobase plasmid from Halobacterium sp. strain GRB is negatively supercoiled; this plasmid was relaxed after novobiocin treatment. These results support the existence in archaebacteria of a coumarin and quinolone target related to eubacterial DNA gyrase.The procaryotes are now divided into eubacteria and archaebacteria which constitute two independent lines of evolutionary descent (25,35,36 (13,24). In contrast, eubacteria contain a type II DNA topoisomerase (DNA gyrase) which introduces negative superturns in topologically closed DNA and a type I DNA topoisomerase (protein w) which removes negative supertums (10). Both type I and type II DNA topoisomerases also coexist in eucaryotic cells. These two enzymes only remove superturns; i.e., they have no gyrase or reverse gyrase activity (for reviews on DNA topoisomerases see references 15, 22, and 32).Eucaryotic and eubacterial type II DNA topoisomerases also differ in their antibiotic sensitivities. DNA gyrase is composed of two types of subunits, gyrA and gyrB, which are, respectively, the targets of antibiotics of the quinolone and coumarin families. Coumarins are specific inhibitors of DNA gyrase at low concentrations (<10 ,ug/ml), whereas quinolones have no activity on the eucaryotic enzyme, even at high doses. Preliminary results have been reported on the action of these antibiotics on archaebacteria. The coumarin novobiocin inhibits growth of Thermoplasma acidophilum (9) and halobacteria (4, 14). However, this does not necessarily mean that a coumarin-sensitive DNA gyrase-like enzyme exists in archaebacteria. Indeed, novobiocin has the general property of interacting with arginine-rich proteins at concentrations above 100 ,ug/ml (8) and consequently non-* Corresponding author. specifically inhibits various enzymatic activities (including eucaryotic type II DNA topoisomerase and S. acidocaldarius reverse gyrase). Thus, it was troublesome that novobiocin concentrations reported to inhibit archaebacteria varied from 0.02 to 200 ,ug/ml, depending on the strains used and the authors reporting (3,14). In addition, the archaebacterium Halobacterium halobium is resistant to the quinolones nalidixic and oxolinic acids and to the fluoroquinolones pefloxacin and norfloxacin (14). These t...

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