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Site-Directed Mutagenesis Identifies the Positively Charged Residue Lysine-46 Essential for the Function of the Immunity Protein PedB

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Abstract

The immunity proteins of pediocin-like bacteriocins possess a positively charged region which is located at the C-terminus in all three subclasses. It has been suggested that this region may be involved in directing the immunity protein to the surface of the bacterial cell membrane. The aim of this study was to determine whether the positively charged residue lysine-46 (K46) around the hydrophobic pocket played a key role for immunity activity of subgroup A immunity protein PedB. At first, heterologous expression of the immune gene pedB from Lactobacillus plantarum BM-1 rendered the sensitive Lactobacillus plantarum WQ0815 resistant to bacteriocin BM-1. Then, using site-directed mutagenesis, the residue K46 was replaced by five different amino-acid residues, including arginine (R), aspartate (D), glutamate (E), glutamine (Q), and threonine (T). Western blot analysis confirmed that all mutated pedB genes were successfully expressed in the host L. plantarum WQ0815. Bacteriocin activity assays subsequently showed that any substitution of the K46 residue significantly reduced its immunity activity. Our present results indicated that the positively charged residue K46 located near the hydrophobic pocket was essential for the functionality of the immunity protein PedB.

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Acknowledgments

We thank Professor Elisabeth Sørvig (Agricultural University of Norway) for kindly providing the plasmid pSIP502. This work was supported by the National Natural Sciences Foundation of China (contract No. 21076223).

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Authors and Affiliations

  1. China Agricultural University, College of Food Science and Nutritional Engineering, Beijing, China

    Chunmei Wang, Wanli Zhou & Yanling Hao

  2. Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland

    François P. Douillard

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  1. Chunmei Wang
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  2. François P. Douillard
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  3. Wanli Zhou
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  4. Yanling Hao
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Correspondence to Yanling Hao.

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Wang, C., Douillard, F.P., Zhou, W. et al. Site-Directed Mutagenesis Identifies the Positively Charged Residue Lysine-46 Essential for the Function of the Immunity Protein PedB. Curr Microbiol 69, 423–428 (2014). https://doi.org/10.1007/s00284-014-0596-6

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  • DOI: https://doi.org/10.1007/s00284-014-0596-6

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