Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706

Holck, Askild Lorentz; Axelsson, Lars; Birkeland, Stein-Erik; Aukrust, Thea; Blom, Henning

doi:10.1099/00221287-138-12-2715

Cited by 195 publications

(147 citation statements)

References 25 publications

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“…This result is in accord with the previous observation that the isolated immunity protein does not bind significantly to the parent bacteriocin CbnB2 in vitro (13). Genes for related immunity proteins have been found in other LAB bacteriocin-producing systems (17,(22)(23)(24)(25).…”

Section: Discussionsupporting

confidence: 82%

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2

Quadri

Yan²,

Stiles

et al. 1997

Journal of Biological Chemistry

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Carnobacteriocin B2, a 48-amino acid antimicrobial peptide containing a YGNGV motif that is produced by the lactic acid bacterium Carnobacterium piscicola LV17B, was overexpressed as fusion with maltose-binding protein in Escherichia coli. This fusion protein was cleaved with Factor Xa to allow isolation of the mature bacteriocin that was identical in all respects to that obtained from C. piscicola. Similar methodology permitted production of the precursor precarnobacteriocin B2 (CbnB2P), which has an 18-amino acid leader, as well as six mutants of the mature peptide: CbnF3 (Tyr 3 3 Phe), CbnS33 (Phe 33 3 Ser), CbnI34 (Val 34 3 Ile), CbnI37 (Val 37 3 Ile), CbnG46 (Arg 46 3 Gly), and Cbn28 (truncated frameshift mutation: (carnobacteriocin B2 1-28) ؉ ELTHL). Examination of these compounds for antimicrobial activity showed that although CbnI34, CbnI37, and CbnG46 were fully active, CbnB2P, CbnF3, CbnS33, Cbn28, and all of the fusion proteins had greatly reduced or no antimicrobial activity. Expression of the immunity protein that protects against the action of the parent carnobacteriocin B2 in a previously sensitive organism also protects against the active mutants. Because carnobacteriocin B2 also acts as an inducer of bacteriocin production in C. piscicola, the ability of the precursor CbnB2P and the mutants to exert this effect was examined. All were able to induce Bac ؊ cultures and reestablish the Bac ؉ phenotype except for the truncated Cbn28. The results demonstrate that very minor changes in the peptide sequence may drastically alter antimicrobial activity but that the induction of bacteriocin production is much more tolerant of structural modification, especially at the N terminus.

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Section: Discussionsupporting

confidence: 82%

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2

Quadri

Yan²,

Stiles

et al. 1997

Journal of Biological Chemistry

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“…The group contains at least 17 bacteriocins, of which pediocin PA-1 (Biswas et al, 1991 ;Henderson et al, 1992 ; Marugg et al, 1992 ;Nieto Lozano et al, 1992), leucocin A UAL-187 (Hastings et al, 1991), mesentericin Y105 (He! chard et al, 1992), sakacin P (Tichaczek et al, 1992) and curvacin A (identical to sakacin A ; Tichaczek et al, 1992 ;Holck et al, 1992) were the first to be identified. All pediocin-like bacteriocins are cationic, contain between 35 and 50 amino acid residues, permeabilize target-cell membranes, and have very similar primary structures, but they differ markedly with respect to their target-cell specificities (Chen et al, 1997a, b ; Chikindas et al, 1993 ;Eijsink et al, 1998 ; Fimland et al, 1996 Fimland et al, , 1998Fimland et al, , 2000Johnsen et al, 2000 ;Nes et al, 2002).…”

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confidence: 99%

Mutational analysis of the role of charged residues in target-cell binding, potency and specificity of the pediocin-like bacteriocin sakacin P

2002

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The significance of charged residues for the target-cell binding, potency and specificity of pediocin-like bacteriocins has been studied by site-directed mutagenesis of sakacin P. Most of the charged residues are located in the N-terminal half, which is thought to mediate the initial binding of these bacteriocins to target cells through electrostatic interaction. All the mutated peptides in which the net positive charge was reduced by one (by replacing a charged residue with threonine) exhibited reduced binding to target cells and a 2-15-fold reduction in potency. The least deleterious of these mutations was the removal of the positive charge in position 8 (H8T). This mutation was, in fact, less deleterious than the conservative His to Lys mutation, indicating that the positive charge in position 8 per se is not of major importance. Somewhat more deleterious was the removal of positive charges at the N-and C-terminal ends (K1T, K43T). Most deleterious was the elimination of the positive charge at positions 11 and (but to a lesser extent) 12, demonstrating the importance of the cationic patch in the middle of the N-terminal half of pediocin-like bacteriocins. Mutated peptides in which the net positive charge was increased by one were also constructed. Some of these exhibited increased cell binding and a potency that was the same as (44K, i.e. an extra positive charge at the Cterminus), or somewhat greater (T20K) than, that of sakacin P, whereas others (0K, i.e. an extra positive charge at the N-terminus) had reduced potency. Sakacin P contains only one negatively charged residue (Asp17). This negative charge and its orientation in space were crucial for activity, since the Asp to Asn mutation and (especially) the conservative Asp to Glu mutation were deleterious. Mutations that made the peptide less cationic had, overall, less effect on the potency toward the Carnobacterium piscicola strain than on the potency toward the three other strains tested, whereas the opposite was the case for mutations that made the peptide more cationic. Thus, charged residues in the N-terminal half may -apparently via the initial electrostatic binding of the bacteriocin to target cells -influence the target-cell specificity.Keywords : antimicrobial peptides, mutagenesis, lactic acid bacteria INTRODUCTIONMany Gram-positive bacteria produce antimicrobial peptides, generally termed bacteriocins. These peptides are usually cationic, less than 50 amino acid residues long, contain an amphiphilic or hydrophobic region, and often kill their target cells by permeabilizing the cell membrane (Nes et al., 1996(Nes et al., , 2002. Antimicrobial peptides with these characteristics are also produced by plants and a wide variety of animals, including humans, and are thus widely distributed in nature M. Kazazic, J. Nissen-Meyer and G. Fimland phospholipid-containing membranes and\or acidic cell walls, whereas their amphiphilic\hydrophobic character enables membrane permeabilization. The pediocin-like bacteriocins constitute a dominant and well-studie...

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“…The family contains at least 15 different bacteriocins, of which pediocin PA-1 (3,19,23,30), leucocin A UAL-187 (17), mesentericin Y105 (18), sakacin P (35) and curvacin A (identical to sakacin A [20,35]) were the first to be identified. All pediocin-like bacteriocins are cationic, contain between 35 and 50 amino acid residues, permeabilize target cell membranes, and have very similar primary structures but differ markedly with respect to their target cell specificity (5-7, 10-14, 22, 24, 29, 32, 37).…”

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confidence: 99%

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

Uteng

Hauge

Brondz

et al. 2002

Appl Environ Microbiol

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A rapid and simple two-step procedure suitable for both small-and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.Gene-encoded, ribosomally synthesized antimicrobial peptides are widely distributed in nature, being produced by bacteria, plants, and a wide variety of animals, including humans (28,29,32,34). The peptides are often cationic and amphiphilic or hydrophobic, and many of them kill bacteria by permeabilizing the target cell membrane. The peptides may be developed into new and useful antimicrobial additives and drugs. An example of this is the antimicrobial peptide nisin, which is produced by lactic acid bacteria (LAB). This peptide is used as a food preservative (9) and has been considered for use for treatment of gastric Heliobacter infections and/or ulcers (16).There has especially been considerable interest in antimicrobial peptides (bacteriocins) produced by LAB because of the "food-grade quality" and industrial importance of these bacteria. LAB are used in food production, are part of the natural microbial flora in food that humans have consumed for centuries, and constitute a significant part of the indigenous flora of mammals, including humans. Thus, LAB and the bacteriocins that they produce may be considered safe agents for preventing growth of pathogenic and/or undesirable microorganisms.Many of the LAB bacteriocins belong to the pediocin-like family; these bacteriocins are of special interest because of their antilisterial activity. The family contains at least 15 different bacteriocins, of which pediocin PA-1 (3,19,23,30), leucocin A UAL-187 (17), mesentericin Y105 (18), sakacin P (35) and curvacin A (identical to sakacin A [20,35]) were the first to be identified. All pediocin-like bacteriocins are cationic, contain between 35 and 50 amino acid residues, permeabilize target cell membranes, and have very similar primary structures but differ markedly with respect to their target cell specificity (5-7, 10-14, 22, 24, 29, 32, 37).The use of pediocin-like bacteriocins and other antimicrobial peptides as additives or drugs requires a simple and rapid method by which large quantities may be purified to homogeneity. Present methods for purification of pediocin-like bacteriocins and other cationic bacteriocins generally include a centrifugati...

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Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706

Cited by 195 publications

References 25 publications

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2

Mutational analysis of the role of charged residues in target-cell binding, potency and specificity of the pediocin-like bacteriocin sakacin P

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

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