Insights into structure-activity relationships in the C-terminal region of divercin V41, a class IIa bacteriocin with high-level antilisterial activity

Abstract

Divercin V41 (DvnV41) is a class IIa bacteriocin with potent antilisterial activity isolated from Carnobacterium divergens V41. Previously, we expressed from a synthetic gene, in Escherichia coli Origami, a recombinant DvnV41 designated DvnRV41, which possesses four additional amino acids (AMDP) in the N-terminal region that result from enzymatic cleavage and retains the initial DvnV41 activity. To unravel the relationship between the structure of DvnRV41 and its particularly elevated activity, we produced by site-directed mutagenesis eight variants in which a single amino acid replacement was specifically introduced into the sequence. The point mutations were designed to change either conserved residues in class IIa bacteriocins or residues specific to DvnV41 located mainly in the C-terminal region. The fusion proteins were purified from the cytosoluble fractions by immobilized affinity chromatography. DvnRV41 and its variants were released from the fusion proteins by enzymatic cleavage, using enterokinase. The purity of DvnRV41 and of the variants was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, and mass spectrometry. The antibacterial activity of DvnRV41 and its variants was assessed using different indicator strains, including Listeria monocytogenes EGDe and Enterococcus faecalis JH2-2. The activity of all of the variants appeared to be less than the activity of DvnRV41. The decrease in activity did not appear to be related to a global conformational change, as determined by circular dichroism. Overall, the variants of DvnRV41 produced in the present study provide interesting insights into structure-activity relationships of class IIa bacteriocins.

FIG. 1.

(A) Sequence of DvnRV41 fusion protein. (B) Overview of the mutations created in the primary structure of DvnRV41 by site-directed mutagenesis. Cysteine residues are underlined. Compared to natural DvnV41, the DvnRV41 recombinant contains four additional amino acids at the N terminus (AMDP) due to specific cleavage by enterokinase. The highlighted residues correspond to amino acids inserted in the sequence in order to replace the original amino acids.

FIG. 2.

Expressed and purified W19F variant. (A) 16.5% Tricine-SDS-PAGE. Lane 1, 3 μl of molecular weight protein markers (Bio-Rad); lane 2, 2 μl of CSF before purification; lane 3, 2 μl of purified fusion protein DvnRV41-fP-W19F; lane 4, 5 μl of DvnRV41-fP-W19F digested with enterokinase EKMax (Invitrogen); lane 5, 5 μl of purified variant W19F. (B) Western blotting with DvnV41 polyclonal antibodies (45). Lane 1, 3 μl of fusion protein DvnRV41-fP-W19F after purification and desalting; lane 2, 3 μl of digested DvnRV41-fP-W19F; lane 3, 3 μl of purified variant W19F.