Volume 5 Supplement 1

The 4th Recombinant Protein Production Meeting: a comparative view on host physiology

Open Access

A counter-selectable marker for Bacillus

  • Michael D Rasmussen1,
  • Jan Martinussen2,
  • Els Marie Celine Defoor2 and
  • Gitte Bak Poulsen1
Microbial Cell Factories20065(Suppl 1):P82

DOI: 10.1186/1475-2859-5-S1-P82

Published: 10 October 2006

Background

A putative gene, denoted ysbC, was previously identified by genome sequencing of a Lactococcus lactis strain, but the gene was not annotated in the databases, and no function of the predicted encoded polypeptide was identified. Studies show that the ysbC encodes a membrane associated orotate transporter enabling orotate to be taken up by the cell and used as a pyrimidine precursor. It was further shown that orotate transporters are quite rare in bacteria and that most bacillus species do not possess such a function.

Results

In order to exploit the functional properties of the ysbC gene it was investigated if an orotate analogue fluoro-orotate could be transported by YsbC. Fluoro-orotate is a toxic pyrimidine precursor which will be incorporated in the host DNA and very efficiently stop further DNA synthesis and divisions of the cell. Lactococcus strains with or without the ysbC gene was tested for resistance against fluoro-orotate on minimal plates. Only Lactococcus strains without the ysbC plasmid was able to grow on minimal plates with fluoro-orotate. The conclusion is that orotate (and fluoro-orotate) can only be transported over the cell membrane when the specific orotate transporter encoded by ysbC is present.

The gene was transferred to Bacillus subtilis to investigate if the orotate transporter would be functional in this background. Experiments in Bacillus subtilis using a plasmids with the ysbC gene show that transformants can not grow on minimal plates supplemented with Fluoro-orotate. Further experiments presented on the poster show that the ysbC gene can be of use in general bacillus recombinant technology as an efficient counter selectable marker.

Authors’ Affiliations

(1)
Dept. of Bacterial Gene Technologyy, Novozymes
(2)
Dept of Molecular Physiology and Genetics, Biocentrum-DTU

Copyright

© Rasmussen et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

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