An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis

[Background] Scientific interest in Enterococcus faecalis has increased greatly over recent decades. Some strains are involved in food fermentation and offer health benefits, whereas others are vancomycin-resistant and cause infections that are difficult to treat. The limited availability of vectors able to express cloned genes efficiently in E. faecalis has hindered biotechnological studies on the bacterium’s regulatory and pathogenicity-related genes. The agmatine deiminase (AGDI) pathway of E. faecalis, involved in the conversion of agmatine into putrescine, is driven by a response inducer gene aguR.[Results] This study describes that the exposure to the induction factor (agmatine) results in the transcription of genes under the control of the aguB promoter, including the aguBDAC operon. A novel E. faecalis expression vector, named pAGEnt, combining the aguR inducer gene and the aguB promoter followed by a cloning site and a stop codon was constructed. pAGEnt was designed for the overexpression and purification of a protein fused to a 10-amino-acid His-tag at the C-terminus. The use of GFP as a reporter of gene expression in E. faecalis revealed that under induction with 60 mM agmatine, fluorescence reached 40 arbitrary units compared to 0 in uninduced cells.[Conclusion] pAGEnt vector can be used for the overexpression of recombinant proteins under the induction of agmatine in E. faecalis, with a close correlation between agmatine concentration and fluorescence when GFP was used as reporter.This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). We are also grateful to Bert Poolman for providing the GFP-derivative vectors. The authors thank Adrian Burton for linguistic assistance. Strain L. lactis NZ9000 and plasmid pNZ8048 were kindly provided by the NIZO Food Research. D.M.L. and B.d.R. were the beneficiaries of JAE DOC contracts (CSIC). M.P. is the recipient of an FPU fellowship from the Spanish Ministry of Education, Culture and Sport.Peer reviewe