Previous studies revealed that one species of methanogenic archaea, Methanocaldococcus jannaschii, is polyploid, while a second species, Methanothermobacter thermoautotrophicus, is diploid. To further investigate the distribution of ploidy in methanogenic archaea, species of two additional genera-Methanosarcina acetivorans and Methanococcus maripaludis-were investigated. M. acetivorans was found to be polyploid during fast growth (t D ‫؍‬ 6 h; 17 genome copies) and oligoploid during slow growth (doubling time ‫؍‬ 49 h; 3 genome copies). M. maripaludis has the highest ploidy level found for any archaeal species, with up to 55 genome copies in exponential phase and ca. 30 in stationary phase. A compilation of archaeal species with quantified ploidy levels reveals a clear dichotomy between Euryarchaeota and Crenarchaeota: none of seven euryarchaeal species of six genera is monoploid (haploid), while, in contrast, all six crenarchaeal species of four genera are monoploid, indicating significant genetic differences between these two kingdoms. Polyploidy in asexual species should lead to accumulation of inactivating mutations until the number of intact chromosomes per cell drops to zero (called "Muller's ratchet"). A mechanism to equalize the genome copies, such as gene conversion, would counteract this phenomenon. Making use of a previously constructed heterozygous mutant strain of the polyploid M. maripaludis we could show that in the absence of selection very fast equalization of genomes in M. maripaludis took place probably via a gene conversion mechanism. In addition, it was shown that the velocity of this phenomenon is inversely correlated to the strength of selection.

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SummaryBiosynthesis of selenocysteine, the 21st proteinogenic amino acid, occurs bound to a dedicated tRNA in all three domains of life, Bacteria, Eukarya and Archaea, but differences exist between the mechanism employed by bacteria and eukaryotes/archaea. The role of selenophosphate and the enzyme providing it, selenophosphate synthetase, in archaeal selenoprotein synthesis was addressed by mutational analysis. Surprisingly, MMP0904, encoding a homologue of eukaryal selenophosphate synthetase in Methanococcus maripaludis S2, could not be deleted unless selD, encoding selenophosphate synthetase of Escherichia coli, was present in trans, demonstrating that the factor is essential for the organism. In contrast, the homologous gene of M. maripaludis JJ could be readily deleted, obviating the strain's ability to synthesize selenoproteins. Complementing with selD restored selenoprotein synthesis, demonstrating that the deleted gene encodes selenophosphate synthetase and that selenophosphate is the in vivo selenium donor for selenoprotein synthesis of this organism. We also showed that this enzyme is a selenoprotein itself and that M. maripaludis contains another, HesB-like selenoprotein previously only predicted from genome analyses. The data highlight the use of genetic methods in archaea for a causal analysis of their physiology and, by comparing two closely related strains of the same species, illustrate the evolution of the selenium-utilizing trait.

:selenocysteine synthase with the corresponding genes from M. maripaludis S2 restored the mutant's ability to synthesize selenoproteins. However, only partial restoration of the wild-type selenoproteome was observed as only selenocysteine-containing formate dehydrogenase was synthesized. Quantification of transcripts showed that disrupting the pathway of selenocysteine synthesis leads to downregulation of selenoprotein gene expression, concomitant with upregulation of a selenium-independent backup system, which is not re-adjusted upon complementation. This transcriptional arrest was independent of selenophosphate but depended on the 'history' of the mutants and was inheritable, which suggests that a stable genetic switch may cause the resulting hierarchy of selenoproteins synthesized.

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