The vhuU gene encoding a small subunit of a selenium-containing [NiFe]-hydrogenase in Methanococcus voltae appears to be essential for the cell.
We developed a general method for the site-specific deletion of gene sequences to obtain new selectable markers in the archaeon Methanococcus voltae. Using a deletion in the hisA gene, a vector was integrated into the chromosome by homologous recombination, thereby reconstituting histidine prototrophy. The vector contained the beta-glucuronidase gene uidA of Escherichia coli as a reporter under the control of an M. voltae promoter that normally drives the expression of a selenium-free [NiFe]-hydrogenase after selenium deprivation. This construct has allowed us to check whether the selenium supply was sufficiently low to induce the transcription of the genes encoding the selenium-free hydrogenases. We tried to introduce a chromosomal deletion of the vhuU gene of the archaeon M. voltae by gene replacement and by keeping the cells under selenium deprivation. The gene vhuU encodes the very small, selenocysteine-containing subunit that is part of the primary reaction center of the Vhu hydrogenase. All transformants bearing the deletion also contained the vhuU wild-type gene. Therefore, the vhuU gene appears to be essential for the cell even under conditions that lead to the induction of the selenium-free homologue Vhc of the Vhu hydrogenase.
Pfeiffer
M
M
Genetik, Fachbereich Biologie, Philipps-Universität, D-35032 Marburg, Germany.
Bestgen
H
H
Bürger
A
A
Klein
A
A
D019747:Aldose-Ketose Isomerases / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D004251:DNA Transposable Elements / Q000235:genetics; D019641:DNA, Archaeal / Q000032:analysis; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D019766:Genes, Archaeal / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017017:Methanococcus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012643:Selenium / Q000378:metabolism*
D004251:DNA Transposable Elements; D019641:DNA, Archaeal; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D019747:Aldose-Ketose Isomerases; C079656:Phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxiamide isomerase; D012643:Selenium