Isolation and Characterization of Pediocin AcH Chimeric Protein Mutants with Altered Bactericidal Activity
| Autor: | Bibek Ray, Kurt W. Miller, Ozlem Osmanagaoglu, Robin Schamber |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Signal peptide
Monosaccharide Transport Proteins Listeria Pediocins Recombinant Fusion Proteins Genetics and Molecular Biology Peptide Biology Polymerase Chain Reaction Applied Microbiology and Biotechnology Maltose-Binding Proteins Maltose-binding protein Transformation Genetic Bacteriocins Bacteriocin Lactococcus Enterococcus faecalis Escherichia coli Point Mutation Amino Acid Sequence Pediococcus Cloning Molecular Protein secondary structure Peptide sequence DNA Primers chemistry.chemical_classification Base Sequence Ecology Escherichia coli Proteins Structural gene food and beverages Amino acid chemistry Biochemistry Periplasmic Binding Proteins biology.protein bacteria ATP-Binding Cassette Transporters Carrier Proteins Leuconostoc Plasmids Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 64:1997-2005 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.64.6.1997-2005.1998 |
| Popis: | Pediocin AcH (same sequence as pediocin PA-1) is a ribosomally synthesized bacteriocin that is produced by certain strains of Pediococcus acidilactici (24, 27). Synthesis is conferred by the papABCD operon, which encodes the pediocin AcH structural gene (papA) and ancillary genes used for production (4, 26). The bacteriocin is translated as a 62-amino-acid preprotein and is converted by the PapD protein to a 44-amino-acid mature form by enzymatic processing of an 18-amino-acid leader peptide (4, 33). The PapC and PapD proteins belong to the ABC export system family of proteins (11). Although an ABC export system is used for production in Pediococcus, the mature sequence region of pediocin AcH can be secreted via the Escherichia coli sec machinery when its N terminus is fused to the E. coli secretory protein maltose binding protein (MBP) (25). These results indicate that PapD is necessary for recognition and processing of the leader peptide rather than for accommodating the mature region as it passes through the membrane. Once secreted, pediocin AcH becomes fully active after the formation of two intramolecular disulfide bonds (14, 19). Independent studies performed with pediocins PA-1 and AcH have begun to reveal their mode of action and structural requirements for activity. These bacteriocins kill susceptible bacteria by permeabilizing the cytoplasmic membrane, causing leakage of ions and small molecules (2, 5, 7, 19). The interaction of pediocin PA-1 with membranes is promoted by acidic phospholipids (5), and positively charged amino acids in this cationic peptide appear to be important for membrane binding (6, 19). In fact, both lysines and histidines may mediate membrane binding in the low-pH environment (pH, ≤5.0) in which Pediococcus strains can grow (3, 19, 35). Other pediocin-like bacteriocins, such as sakacin P (32) (same as sakacin 674) (17), leucocin A (13), and curvacin A (31) (same as sakacin A) (16), also are cationic and may rely in part on electrostatic interactions between basic amino acids and negatively charged phospholipid head groups for membrane adsorption. Pediocins PA-1 and AcH contain two structurally distinct sequence regions (5, 12, 19). The N-terminal 20 amino acids are polar and are highly conserved among pediocin-like bacteriocins. Located within this region is a -Y3-G4-N5-G6-V7- sequence that is present in all family members. While the function of this sequence is unknown, its deletion from pediocin AcH completely inactivates the molecule (25). Based on secondary structure analysis of pediocin PA-1, it has been proposed that the first 18 amino acids fold into a two-strand β hairpin that is stabilized by a β turn at position -G4-N5-G6-V7- (5) and the C9—C14 disulfide bond (5, 14). Solution nuclear magnetic resonance analysis of a related bacteriocin, leucocin A, indicated that its conformation indeed is ordered near the C9—C14 disulfide bond, but specific H-bond interactions expected for a β-hairpin structure were not detected (28). Perhaps due to the short length of these molecules, regions of defined secondary structure may not form until after they have adsorbed to a membrane (20). The C-terminal sequence region of pediocins PA-1 and AcH (residues A21 to C44) is much less polar and conserved than the N-terminal sequence region (19). The C-terminal region is proposed to contain a hydrophobic membrane interaction domain (12), and analysis of the properties of hybrid peptides constructed from pediocin-like bacteriocins (12) and MBP-pediocin AcH chimeric proteins (25) supports this hypothesis. It also has been shown that the membrane interaction domain becomes amphipathic if folded into an α helix (12). While it is well established that membrane binding is promoted by amphipathic α helices (9, 20), it should be noted that the C24—C44 disulfide bond which is unique to pediocins PA-1 and AcH (19) bends the C-terminal region into a loop, and it is unknown whether an amphipathic structure can be formed in the presence of the loop. To facilitate analysis of the structure and mode of action of pediocin AcH, we generated a collection of pediocin AcH substitution mutants that display altered bactericidal activity. Mutants were produced and detected with the E. coli MBP-pediocin AcH chimeric protein secretion system and colony overlay screening methods (25). Over one third of the amino acids in pediocin AcH were found to be important for activity. |
| Databáze: | OpenAIRE |
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