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Appl. Environ. Microbiol. doi:10.1128/AEM.02718-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Department of Molecular Biosciences, University of Oslo, Pb 1041 Blindern, 0316 Oslo, Norway

^* To whom correspondence should be addressed. Email: jon.nissen-meyer{at}imbv.uio.no.

	Abstract

The two peptides (Lcn- and Lcn-) of the two-peptide bacteriocin lactococcin G (Lcn) were by site-directed mutagenesis stepwise changed into the corresponding peptides (Ent- and Ent-) of the two-peptide bacteriocin enterocin 1071 (Ent), and the potency and specificity of the various hybrid constructs were determined. Both Lcn and, but to a lesser extent, Ent were active against all the tested lactococci strains, but only Ent was active against the tested enteroccoci 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-) was in contrast highly potent, indicating that these two peptides may form a functional antimicrobial unit. This hybrid combination (Ent-+Lcn-) was in fact more potent against lactoccoci strains than wild type Ent (i.e. Ent-+Ent-), but it was inactive against enterococci 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 in positions 1 to 4, 7, 9 and 10 was > 150 fold less active against enterococci strains but 1-4 times more active against lactococci strains than Ent-+Ent-. Moreover, Ent-to-Lcn single-residue mutations in the region spanning residues 1-7 in Ent- had a more detrimental effect on the activity against enterococci than against lactococci 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: the N8Q mutation had no effect on activity against tested enterococci strains but increased the activity 2-4 fold against the tested lactococcus strains; the P12R mutation reduced the activity > 150 fold and only 2 fold against, respectively, enterococcus and lactococcus strains.

Changing residues in the C-terminal half/part of the Lcn peptides (residues 20-39 and 25-35 in Lcn- and Lcn-, respectively) into those found in the corresponding Ent peptides did not have a marked effect on the activity, but there was 10 fold or greater reduction in the activity upon also introducing Lcn-to-Ent mutations in the mid region (residues 8-19 and 9-24 in Lcn- and Lcn-, respectively). Interestingly, the Lcn-to-Ent F19L+G20A mutation in a Lcn-Ent- hybrid peptide was more detrimental when the altered peptide was combined with Lcn- (> 10 fold reduction) then when it was combined with Ent- ( 2 fold reduction), suggesting that residues 19 and 20 (which are part of a GxxxG-motif) in the peptide may be involved in a specific interaction with the cognate peptide. It is also noteworthy that the K2P and A7P mutations in Lcn- reduced the activity by only 2 fold, suggesting that the first 7 residues in the peptides do not form an -helix.