In vivorequirement of selenophosphate for selenoprotein synthesis in archaea

Abstract

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.

Genome Copy Numbers and Gene Conversion in Methanogenic Archaea

“…Therefore, an experiment was designed to clarify whether gene conversion exists in methanogenic archaea and how efficient it is in genome equalization. We made use of a heterozygous mutant that was recently constructed (44). The attempt to replace the selenophosphate synthetase-encoding gene (selD) of M. maripaludis S2 with a puromycin resistance cassette (pacN) revealed that the selD gene is essential and cannot be completely removed from the cell.…”

“…The attempt to replace the selenophosphate synthetase-encoding gene (selD) of M. maripaludis S2 with a puromycin resistance cassette (pacN) revealed that the selD gene is essential and cannot be completely removed from the cell. Therefore, selection in the presence of puromycin yielded a strain (designated SkoD4) that contained two different genomes; the majority of genome copies carried the pacN gene at the selD locus, while a few genome copies retained the native selD gene at this site (44). The strain was used to inoculate four different cultures, which were incubated under four different conditions: (i) in the presence of the "normal" puromycin concentration (2.5 g ml Ϫ1 ), which is totally inhibitory for the wild-type (45); (ii and iii) in the presence of two lower puromycin concentrations (0.6 and 0.15 g ml Ϫ1 , respectively); and (iv) in the absence of puromycin.…”

“…The average increase of wild-type genomes after 14 generations is much higher in the absence of selection than in the presence of the full selection pressure, where it is negligible ( Table 2). The selD gene was ideally suited for the approach because (i) it is an essential gene that cannot be deleted in M. maripaludis S2 during growth on formate and (ii) it has a very low expression level, allowing the cells to grow with only one or two selDcontaining genomes with the same growth rate as the wild-type that is homozygous for selD (44). The experiment shows that genome redundancy can under specific selection conditions allow heterozygoty at a locus within one cell, enabling its survival and growth.…”

“…For the analysis of gene conversion the recently constructed M. maripaludis strain SkoD4 was used, which harbors different genomes that contain either the selD gene or the pacN gene at the selD locus (44). In the presence of selection using puromycin, the copy number of genomes with the puromycin resistance gene pacN is very high, whereas only very few copies of the selD-containing genomes remain in the cell.…”

“…The ploidy levels were determined at different growth rates throughout culture growth. In addition, we made use of a heterozygous mutant strain that was recently constructed (44) to analyze the influence of different selection pressures on the velocity of gene conversion.…”