Journal Article

Identification of <i>yrrU</i> as the Methylthioadenosine Nucleosidase Gene in <i>Bacillus subtilis</i>

Agnieszka Sekowska and Antoine Danchin

in DNA Research

Published on behalf of Kazusa DNA Research Institute

Volume 6, issue 5, pages 255-264
Published in print January 1999 | ISSN: 1340-2838
Published online January 1999 | e-ISSN: 1756-1663 | DOI: http://dx.doi.org/10.1093/dnares/6.5.255

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Taking trimethoprim as the selective agent in the presence of thymine, we adapted to Bacillus subtilis a selection procedure depending on the peculiar organisation of the one-carbon metabolism. The corresponding pathways couple synthesis of thymine to tetrahydrofolate consumption as a substrate of the reaction mediated by thymidylate synthase, instead of being a co-enzyme as in the other reactions transferring one-carbon groups. Mutants obtained are thymidylate synthase deficient, and therefore auxotrophic for thymine. This provides positive selection in a first step for gene replacement by a thymidylate synthase cassette, and subsequently against its presence. For systematic recombination of mutations constructed in vitro, we used the property of B. subtilis to grow at high temperature, noting that the thyB gene product is inactive at 46°C, while the product of thyA remains active at this temperature. As the first step, we built up a recipient thyA background, deleting the gene by in situ recombination. This method was used to investigate the function of the yrrU gene, which is presumably involved in a sulfur recycling pathway associated with polyamine biosynthesis. We showed that yrrU codes for a protein recycling methylthioadenosine, probably a nucleosidase. In addition we observed that B. subtilis can use methylthioribose as a sulfur source, and that it is an efficient sulfur scavenger.

Keywords: sulfur salvage; S-adenosyl methionine; spermidine; putrescine; sequential gene disruption

Journal Article.  0 words. 

Subjects: Genetics and Genomics

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