Analysis of the Two-Peptide Bacteriocins Lactococcin G and Enterocin 1071 by Site-Directed Mutagenesis

Abstract

The two peptides (Lcn-␣ and Lcn-␤) of the two-peptide bacteriocin lactococcin G (Lcn) were changed by stepwise site-directed mutagenesis into the corresponding peptides (Ent-␣ and Ent-␤) of the two-peptide bacteriocin enterocin 1071 (Ent), and the potencies and specificities of the various hybrid constructs were determined. Both Lcn and, to a lesser extent, Ent were active against all the tested lactococcal strains, but only Ent was active against the tested enterococcal strains. The two bacteriocins thus differed in their relative potencies to various target cells, despite their sequence similarities. The hybrid combination Lcn-␣؉Ent-␤ had low potency against all strains tested, indicating that these two peptides do not interact optimally. The reciprocal hybrid combination (i.e., Ent-␣؉Lcn-␤), in contrast, was highly potent, indicating that these two peptides may form a functional antimicrobial unit. In fact, this hybrid combination (Ent-␣؉Lcn-␤) was more potent against lactococcal strains than wild-type Ent was (i.e., Ent-␣؉Ent-␤), but it was inactive against enterococcal strains (in contrast to Ent but similar to Lcn). The observation that Ent-␣ is more active against lactococci in combination with Lcn-␤ and more active against enterococci in combination with Ent-␤ suggests that the ␤ peptide is an important determinant of target cell specificity. Especially the N-terminal residues of the ␤ peptide seem to be important for specificity, since Ent-␣ combined with an Ent-␤ variant with Ent-to-Lcn mutations at positions 1 to 4, 7, 9, and 10 was >150-fold less active against enterococcal strains but one to four times more active against lactococcal strains than Ent-␣؉Ent-␤. Moreover, Ent-to-Lcn single-residue mutations in the region spanning residues 1 to 7 in Ent-␤ had a more detrimental effect on the activity against enterococci than on that against lactococcal strains. Of the single-residue mutations made in the N-terminal region of the ␣ peptide, the Ent-to-Lcn mutations N8Q and P12R in Ent-␣ influenced specificity, as follows: the N8Q mutation had no effect on activity against tested enterococcal strains but increased the activity 2-to 4-fold against the tested lactococcal strains, and the P12R mutation reduced the activity >150-fold and only ϳ2-fold against enterococcal and lactococcal strains, respectively. Changing residues in the C-terminal half/part of the Lcn peptides (residues 20 to 39 and 25 to 35 in Lcn-␣ and Lcn-␤, respectively) to those found in the corresponding Ent peptides did not have a marked effect on the activity, but there was an ϳ10-fold or greater reduction in the activity upon also introducing Lcn-to-Ent mutations in the mid-region (residues 8 to 19 and 9 to 24 in Lcn-␣ and Lcn-␤, respectively). Interestingly, the Lcn-to-Ent F19L؉G20A mutation in an Lcn-Ent-␤ hybrid peptide was more detrimental when the altered peptide was combined with Lcn-␣ (>10-fold reduction) than when it was combined with Ent-␣ (ϳ2-fold reduction), suggesting that residues 19 and 20 (which are part of a GXXXG motif)...

The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G

“…Peptides were produced by a two-plasmid expression system using L. sakei Lb790/pSAK20 cells transformed with previously constructed plasmids (derived from pLT100) carrying the gene encoding either one of the wild-type peptides (Lcn-␣, Lcn-␤, Ent1071A, or Ent1071B) or one of the hybrid peptides (the Lcn-␣-Ent1071A hybrid designated ␣ [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]/A or the Lcn-␤-Ent1071B hybrid designated ␤[1-13]/B ) (24). The two-plasmid expression system is based on the production and secretion of the class IIa bacteriocin sakacin A (2, 3).…”

“…The two-plasmid expression system is based on the production and secretion of the class IIa bacteriocin sakacin A (2, 3). The peptides were purified using a cation-exchange column, followed by reverse- http://aem.asm.org/ phase chromatography, as described previously (24,30). Depending on the peptide purified, between 100 and 500 g of peptide per liter of culture was obtained.…”

“…Lactococcin G is perhaps the best-characterized two-peptide bacteriocin (12,18,19,21,24,26,27). It consists of the 39-residue ␣ peptide (termed Lcn-␣) and the 35-residue ␤ peptide (termed Lcn-␤) (Fig.…”

The Abi Proteins and Their Involvement in Bacteriocin Self-Immunity

“…This strategy has previously proven successful for heterologous production of both one-and two-peptide bacteriocins (5,11,29,36 Fig. 5A.…”

Fermentation Processes Using Lactic Acid Bacteria Producing Bacteriocins for Preservation and Improving Functional Properties of Food Products

“…Na + , K + , Li + , Cs + , Rb + and choline) leading to the destruction of proton motive force by dissipation of the transmembrane pH gradient and eventually to the cell death (Simova et al, 2009;Oppegård et al, 2007). The bactericidal or bacteriostatic activity possessed by bacteriocins is influenced by the following factors: bacteriocin dose and purification degree, physiological status of the indicator cells (e.g.…”