Determination of Essential and Variable Residues in Pediocin PA-1 by NNK Scanning

Abstract

Pediocin PA-1 is an antimicrobial peptide (called bacteriocin) that shows inhibitory activity against the foodborne pathogen Listeria monocytogenes. To elucidate which residue(s) is responsible for this function, the antimicrobial activities of pediocin PA-1 mutants were evaluated and compared. Each of the 44 native codons was replaced with the NNK triplet oligonucleotide in a technique termed NNK scanning, and 35 mutations at each position were examined for antimicrobial activities using a modified colony overlay screening method. As a consequence, the functional responsibility of each residue was estimated by counting the number of active mutants, allowing us to identify candidate essential/variable residues. Activity was abrogated by many of the mutations at residues Y2, G6, C9, C14, C24, W33, G37, and C44, indicating that these residues may be essential. In contrast, activity was retained by almost all versions harboring mutations at K1, T8, G10, S13, G19, N28, and N41, indicating that these are functionally redundant residues. Sequence analysis revealed that only the wild type was active and 14 and 11 substitutions were inactive at G6 and C14, respectively, while 12 and 11 substitutions were active and 2 and 0 substitutions were inactive at T8 and K1, respectively. These findings suggest that NNK scanning is effective for determining essential and variable residues in pediocin PA-1, leading to an elucidation of structure-function relationships and to improvements in the antimicrobial function efficiently by peptide engineering.Various antimicrobial peptides, called bacteriocins, are produced by lactic acid bacteria to kill or inhibit the growth of closely related species (1). Bacteriocins are thought to have promise for use as safe food preservatives (7). For example, nisin is currently used in more than 40 countries as an antimicrobial additive (41), and pediocin PA-1 is currently being investigated in this context, as it has a strong inhibitory effect on the food-borne pathogen Listeria monocytogenes (2, 38). Pediocin PA-1, composed of 44 residues, is secreted by Pediococcus acidilactici (23,29). More than 20 members of the pediocin-like (class IIa) bacteriocins have been identified to date (16). These peptides contain a consensus YGNGV motif and can be structurally divided into a highly conserved hydrophilic N-terminal domain and a relatively variable hydrophobic C-terminal domain (14). Nuclear magnetic resonance structure (18,42,44) and circular dichroism spectral (17,27,45) analyses have suggested that the N-terminal domain has a threestranded ␤-sheet structure, while the C-terminal domain is folded into an amphiphilic ␣-helical structure. It is thought that the bacteriocins bind to bacteria via electrostatic interactions (4, 28) and then insert into the bacterial membrane via their amphiphilic ␣-helical region, subsequently forming a membrane pore that triggers dissipation of the membrane electrochemical potential (3) and/or nutrient leakage from within the bacterial cell (5,22). A mannose phosphotran...

Target recognition, resistance, immunity and genome mining of class II bacteriocins from Gram-positive bacteria

“…Such techniques are particularly suitable for class II bacteriocins, given their gene-encoded nature. Mutational analysis and gene shuffling of class II bacteriocins has been performed in several studies (Haugen et al, 2011(Haugen et al, , 2008Oppegård et al, 2007Oppegård et al, , 2008Quadri et al, 1997;Tominaga & Hatakeyama, 2006; however, systematic engineering efforts to improve antibacterial potencies have so far mainly been conducted for the lantibiotic bacteriocins (Field et al, 2008). Notably, the specific antimicrobial activity of some bacteriocins has been increased through cloning and expression in other producer hosts.…”

Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins

“…Determination of antimicrobial activity. Colony overlay assays were performed as described previously (45). Briefly, single E. coli JE5505 colonies containing the desired expression vector were picked with toothpicks and touched lightly onto the surface of 2.5% LB agar plates.…”

“…The presently used oligonucleotide and primer sequences are listed in the supplemental material. The enterocin A, divercin V41, and carnobacteriocin BM1-encoding fragment was obtained essentially as described previously (45), using EntF and EntR, DivF and DivR, and CarF and CarR, respectively, as oligonucleotides and EntAExtF and EntACR, DivExtF and DivExtR, and CarExtF and CarExtR, respectively, as primers. The resultant fragment was cloned into the pFLAG-ATS expression vector (Sigma).…”

“…The Nterminal domain was selected for shuffling because this region contains several conserved sequences that could be used as shuffling scaffolds. Within this domain, four regions were selected for shuffling based on multiple alignment and data from a previous study examining which residues were essential or variable for pediocin PA-1 activity (45). The antimicrobial activities of the resulting shuffled peptides against Leuconostoc lactis YKLAB10, which had been isolated from a sour-spoiled prepared food product, were examined.…”

Synthesis of Trypsin-Resistant Variants of the Listeria -Active Bacteriocin Salivaricin P

“…Theoretically, engineering of a bacteriocin to reduce its sensitivity to proteases is also a viable option. Engineering of bacteriocins has been used to generate variants which have helped scientists to better understand the structure-function relationships of these peptides (6,7,10,11,14,24,25,28,31,34), and there have been recent successes in which the antimicrobial activity of bacteriocins has been enhanced through a bioengineering-based approach (1,8,9,15,17,19,35). Despite this, bioengineered peptides with enhanced protease resistance have been generated only in the case of the class I bacteriocin gallidermin.…”