ISSN:
1432-072X
Keywords:
Key words Archaea
;
Methanococcus voltae
;
Deletion
;
mutagenesis
;
Adaptation
;
Selenium deprivation
;
[NiFe]-hydrogenases
;
Gene replacement
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract 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 β-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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002030050662
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