Article pubs.acs.org/JAFC
Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity Xingrong Ju,† Xinquan Chen,† Lihui Du,*,† Xueyou Wu,† Fang Liu,‡ and Jian Yuan† †
College of Food Science and Engineering, Collaborative Innovation Center for Modern Grain Circulation and Safety, Nanjing University of Finance and Economics, Nanjing 210023, China ‡ Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China S Supporting Information *
ABSTRACT: Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency. KEYWORDS: durancin GL, alanine-scanning mutation, antilisteria, Enterococcus
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domain (which is followed by a hinge) and a flexible, diverse, and hydrophobic/amphiphilic α-helix C-terminal domain.8 The cationic N-terminal domain is structurally stabilized by a conserved disulfide bridge and mediates binding to the target cell surface through electrostatic interaction; the C-terminal domain penetrates into the hydrophobic part of target cell membranes and causes membrane leakage, disruption of membrane integrity, and eventually cell death.9,10 To elucidate the structure−function relationship and mode of action of class IIa bacteriocins, many studies performed site-directed mutagenesis. Mutations are often harmful for bactericidal activity, but some mutations may be similar or better than their wildtype.10−12 Amino acid substitution reveals that dispensable residues are generally distributed across the entire sequence of most bacteriocins.10,12−15 Combinatorial alanine-scanning mutagenesis is a powerful tool for investigating relationship between protein structure and function. This technique is used to systematically examine the behavior of each residue in a peptide and elucidate tolerance mechanisms to bacteriocin modification. Alanine substitution further reveals the contribution of an individual amino acid to protein functionality. Durancin GL is a novel class IIa bacteriocin produced by Enterococcus durans 41D, which was isolated from Hispanicstyle cheese samples. The gene of durancin GL was identified on the 8347 bp plasmid pDGL1.16 Durancin GL consists of 43 amino acids and exhibits high specificity to antilisterial activity, including that of nisin-resistant L. monocytogenes NR30.17 Durancin GL is a promising food-grade preservative in food
INTRODUCTION Bacteriocins are natural peptides secreted by various bacteria to kill other closely related bacteria;1,2 these peptides are synthesized with a cognate immunity protein, which immunizes the bacteriocin-producing bacteria against their own bacteriocins.3 Bacteriocins produced by “food grade” lactic acid bacteria (LAB) have been extensively studied because of their potential applications as nontoxic food preservatives and therapeutic agents for gastrointestinal infections. LAB bacteriocins, such as nisin and pediocin PA-1, are also used as biopreservatives;4,5 the potential of LAB bacteriocins in medical applications is further exemplified by targeting Listeria monocytogenes.6 Bacteriocins from Gram-positive bacteria are generally classified into two groups according to their primary structure, size, mode of action, genetic properties, and modification.5,7 Class I bacteriocins are the lantibiotics, which are highly posttranslationally modified peptides containing lanthionine and methyllanthionine residues. Class II consists of small peptides that do not contain modified residues. Cotter et al. suggested dividing class II bacteriocins into several subclasses: class IIa (pediocin-like bacteriocins), class IIb (two-peptide bacteriocins), and class IIc (circular bacteriocins).5 Class IIa subclass bacteriocins, also known as pediocin-like bacteriocins, are the most studied because of their antilisterial activity; L. monocytogenes is a pathogen responsible for outbreaks of foodborne listeriosis. Class IIa bacteriocins are small (
