Aims: To exploit the enterocin regulatory system for regulated expression of genes in Enterococcus .
Methods and Results: Production of some pediocin-like bacteriocins such as enterocin A in Enterococcus is regulated by a three-component system comprising a histidine kinase (entK ), a response regulator (entR) and an induction factor (entF ). Exposure to the induction factor results in the transcription of gene(s) under the control of the enterocin A promoter, including entA which encodes the bacteriocin. In an effort to exploit this system for expression of genes in Enterococcus , a number of vectors were constructed which contain the entA promoter followed by convenient cloning sites to introduce gene(s) of interest. These vectors were used in an enterococcal background which does not produce induction factor but does produce both the kinase and regulator proteins. The system was tested using the reporter genes ltnI (lacticin 3147 immunity) and gusA (beta-glucuronidase) under the control of the entA promoter.
Conclusions: Upon addition of the induction factor, the beta -glucuronidase activity increased 20-fold when compared with uninduced cells. In addition, concentrations of as little as 0.2 nm synthetic EntF were sufficient to give maximal expression.
Significance and Impact of the Study: The potential benefit of having an expression system based on EntF is that gene expression can be finely controlled upon addition of low concentrations of a peptide that can easily be artificially synthesized.