A counter-selectable marker for Bacillus
Microbial Cell Factories volume 5, Article number: P82 (2006)
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.
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.
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Rasmussen, M.D., Martinussen, J., Defoor, E.M.C. et al. A counter-selectable marker for Bacillus. Microb Cell Fact 5 (Suppl 1), P82 (2006). https://doi.org/10.1186/1475-2859-5-S1-P82