Site-directed mutagenesis identifies the positively charged residue lysine-46 essential for the function of the immunity protein PedB.
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.
China Agricultural University, College of Food Science and Nutritional Engineering, Beijing, China.
Chunmei
C
Wang
François P
FP
Douillard
Wanli
W
Zhou
Yanling
Y
Hao
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D001430:Bacteriocins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D048191:Lactiplantibacillus plantarum / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008239:Lysine / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D001430:Bacteriocins; D008239:Lysine