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

Abstract

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.

Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis

“…An agmatine induction system combining AguR and P aguB of Enterococcus faecalis was earlier used to develop an expression vector suitable for the latter species [ 47 ]. However, the proposed lactococcal ACE system shows some differences to the E. faecalis system: (1) the aguR gene is in the same orientation as P aguB (reflecting the corresponding organization of the AGDI cluster in each system), (2) the highest expression rate is reached at ≥0.5 mM agmatine in the ACE system but at ≥60 mM in the E. faecalis system, and (3) the level of induction (as determined by fluorescence) is less than that achieved with the E. faecalis system.…”

Fluorescent Lactic Acid Bacteria and Bifidobacteria as Vehicles of DNA Microbial Biosensors

“…A close correlation between agmatine concentration and fluorescence was observed when GFP was used as reporter in the E. faecalis aguR /P aguB controlled expression system. Then, the induction of agmatine in E. faecalis could be used for the overexpression of recombinant proteins [ 71 ].…”

The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis

“…The promoters of the aguR gene and aguBDAC operon are indicated. Adapted from Linares et al (2014) and Ladero et al (2017) .…”

“…Five genes grouped in the agdi cluster are responsible of its biosynthesis: the regulator gene aguR and the metabolic genes aguB, aguD, aguA , and aguC ( Figure 1 ). aguR is constitutively transcribed as a monoscistronic mRNA from its promoter P aguR in a very low expression level, while the catabolic genes are co-transcribed in a single mRNA from the aguB promoter (P aguB ) in a divergent orientation ( Linares et al, 2014 ). Furthermore, increasing concentrations of the substrate, agmatine, increase the transcription of the catabolic genes through the transmembrane regulator AguR ( Suarez et al, 2013 ; Linares et al, 2014 ).…”

“…aguR is constitutively transcribed as a monoscistronic mRNA from its promoter P aguR in a very low expression level, while the catabolic genes are co-transcribed in a single mRNA from the aguB promoter (P aguB ) in a divergent orientation ( Linares et al, 2014 ). Furthermore, increasing concentrations of the substrate, agmatine, increase the transcription of the catabolic genes through the transmembrane regulator AguR ( Suarez et al, 2013 ; Linares et al, 2014 ). Linares et al (2015) suggest that AguR is a one component transcriptional activator that senses agmatine concentrations in the extracellular environment activating the transcription of the aguBDAC operon through its interaction with P aguB .…”