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

Abstract

BackgroundScientific 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.ResultsThis 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.ConclusionpAGEnt 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.

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range

“…One specific tool that has eluded enterococcus researchers is an expression vector system that tightly represses expression of the cloned gene while allowing high-level expression when induced. The expression systems available tend toward leakiness under noninducing conditions and/or are inducible by toxic peptides (such as nisin [9]) or nutrients (10) that could alter the metabolism of the strain under investigation. Our previous attempt to study the mode of action of the Fst pAD1 toxin using the nisininducible expression was compromised by synergistic toxic effects of nisin and Fst pAD1 (11).…”

Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities

“…For complementation of the E. faecalis EFT41963 (gmdH) and EFT41964 (mmdH) mutants, the corresponding wild-type genes were cloned into the vector pAGEnt (32). This vector contains the gene aguR, which encodes the activator of the agmatine operon aguBDAC, and the aguB promoter.…”

Efficient engineered probiotics using synthetic biology approaches: A review

“…Also, the NICE system is an inducible system, so it is not appropriate to use when the cells are present in an inaccessible position to add inducer. The agmatine‐controlled expression is a tightly controlled expression system but it is not used as an alternative to the NICE system as it is dose responsive . The P Zn ‐zitR, which regulate the expression of zit operon and Zirex (zinc‐regulated expression system), is employed concurrently to provide tight coexpression in the NICE system under the nontoxic Zn 2+ levels .…”