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Applied and Environmental Microbiology, February 2006, p. 1141-1147, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1141-1147.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Saitama Industrial Technology Center North Institute, 2-133 Suehiro, Kumagaya, Saitama 360-0031,¹ Rational Evolutionary Design of Advanced Biomolecules (REDS) Group/JST, Saitama Small Enterprise Promotion Corporation SKIP City, Kawaguchi, Saitama 333-0844, Japan²

Received 24 June 2005/ Accepted 11 November 2005

Pediocin PA-1 is an antimicrobial peptide (called bacteriocin) that shows inhibitory activity against the food-borne 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.

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• Tominaga, T., Hatakeyama, Y. (2007). Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins. Appl. Environ. Microbiol. 73: 5292-5299 [Abstract] [Full Text]