Response of Salmonella choleraesuis LT2 Spheroplasts and Permeabilized Cells to the Bacteriocin AS-48

The bacteriocin AS-48 was not active against intact cells of Salmonella choleraesuis LT2 at neutral pH, but it was very effective on spheroplasts, suggesting that the outer membrane (OM) acts as a protective barrier. Cells sublethally injured by heat or treated with OM-permeabilizing agents (i.e., EDTA and Tris) became sensitive to AS-48. The combination of two or more treatments decreased the amount of AS-48 required for cell killing. The activity of AS-48 against heat-injured cells did not change significantly in the pH range of 4.0 to 8.0. AS-48 showed bactericidal activity against intact cells of Salmonella at pH 4.0. The potency of AS-48 increased greatly when the bacteriocin was dissolved at pH 9.0.     

Antilisterial Activity of Peptide AS-48 and Study of Changes Induced in the Cell Envelope Properties of an AS-48-Adapted Strain of Listeria monocytogenes

The peptide AS-48 is highly active on all Listeria species. It has a bactericidal and bacteriolytic mode of action on Listeria monocytogenes CECT 4032, causing depletion of the membrane electrical potential and pH gradient. The producer strain Enterococcus faecalis A-48-32, releases sufficient amounts of AS-48 into the growth medium to suppress L. monocytogenes in cocultures at enterococcus-to-listeria ratios above 1 at 37°C or above 10 at 15°C. As the temperature decreases, the bactericidal effects of AS-48 are less pronounced, but at 2.5 μg/ml it still can inhibit the growth of listeria at 6°C. AS-48 is highly active on liquid cultures, although concentrations above 0.2 μg/ml are required to avoid adaptation of listeria. AS-48-adapted cells can be selected at low (but still inhibitory) concentrations, and they can be inhibited completely by AS-48 at 0.5 μg/ml. The adaptation is lost gradually upon repeated subcultivation. AS48^ad cells are cross-resistant to nisin and show an increased resistance to muramidases. Their fatty acid composition is modified: they show a much higher proportion of branched fatty acids as well as a higher C_{15:0 An}-to-C_{17:0 An} ratio. Resistance to AS-48 is also maintained by protoplasts from AS48^ad cells. Electron microscopy observations show that the cell wall of AS48^ad cells is thicker and less dense. The structure of wild-type cells is severely modified after AS-48 treatment: the cell wall and the cytoplasmic membrane are disorganized, and the cytoplasmic content is lost. Intracytoplasmic membrane vesicles are also observed when the wild-type strain is treated with high AS-48 concentrations.     

Purification, characterization, and biological effects of a second bacteriocin from Enterococcus faecalis ssp. liquefaciens S-48 and its mutant strain B-48-28.

Enterococcus faecalis ssp. liquefaciens S-48 (producer of the peptide antibiotic AS-48) and its mutant B-48-28 (AS-48-) secrete the bacteriocin Bc-48. This substance has been purified to homogeneity from culture supernatants of strain B-48-28; it consists of a protein (80 kDa) stable from pH. 5.5 to 9.0 and sensitive to temperatures above 45 degrees C and to proteases. Its inhibitory spectrum is restricted to strains of Enterococcus faecalis. Bc-48 inhibits protein synthesis but does not affect amino acid uptake. A partial reduction of cell viability, together with autolysis, is also observed. Bc-48 differs from peptide AS-48 in both its molecular properties and mode of action.     

Inhibition of bacterial growth, enterotoxin production, and spore outgrowth in strains of Bacillus cereus by bacteriocin AS-48

Bacteriocin AS-48 showed high bactericidal activity for mesophilic and psychrotrophic strains of Bacillus cereus over a broad pH range. AS-48 inhibition of the enterotoxin-producing strain LWL1 was enhanced by sodium nitrite, sodium lactate, and sodium chloride. The latter also enhanced AS-48 activity against strain CECT 131. Bacterial growth and enterotoxin production by strain LWL1 were completely inhibited at bacteriocin concentrations of 7.5 microg/ml. At subinhibitory bacteriocin concentrations, enterotoxin production decreased markedly and sporulation was delayed. Intact spores were resistant to AS-48 but became gradually sensitive to AS-48 during the course of germination.     

Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain

AIMS: Control of the enterotoxigenic Staphylococcus aureus CECT 976 strain by enterocin AS-48 in laboratory cultures, and behaviour of the AS-48 activity in the presence of food preservatives. METHODS AND RESULTS: Enterocin AS-48 shows inhibitory activity on the majority of the Staphylococcus species tested. This enterocin has a bactericidal and bacteriolytic mode of action on S. aureus CECT 976, a strain selected for this study by its enterotoxigenic character (SEA production). The inhibitory effect of AS-48 was pH and temperature dependent, and enterocin activity was higher at pH 5. The minimum bactericidal concentration (MBC) of AS-48, decreased from 15 microg ml(-1) at 37 degrees C to 10 microg ml(-1) at 15 degrees C. Sublethally injured cells showed an increased sensitivity with a MBC of 5 microg ml(-1). In this way, the highest effectiveness of Ent AS-48 against S. aureus CECT 976 was obtained at 4 degrees C in combination with high concentrations of NaCl (6 and 7%). Interestingly, enterotoxin SEA production by strain CECT 976 was markedly inhibited by subinhibitory concentrations of Ent AS-48. These low concentrations also provoked a delay of bacterial growth. CONCLUSION: The results presented indicated that Ent AS-48 has a potential for application as a protective agent against S. aureus in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have established the conditions for an efficient inhibition of growth and enterotoxin production by S. aureus CECT 976 in culture media by a combination of environmental factors and Ent AS-48.     

Structure of bacteriocin AS-48: from soluble state to membrane bound state

The bacteriocin AS-48 is a membrane-interacting peptide, which displays a broad anti-microbial spectrum against Gram-positive and Gram-negative bacteria. The NMR structure of AS-48 at pH 3 has been solved. The analysis of this structure suggests that the mechanism of AS-48 anti-bacterial activity involves the accumulation of positively charged molecules at the membrane surface leading to a disruption of the membrane potential. Here, we report the high-resolution crystal structure of AS-48 and sedimentation equilibrium experiments showing that this bacteriocin is able to adopt different oligomeric structures according to the physicochemical environment. The analysis of these structures suggests a mechanism for molecular function of AS-48 involving a transition from a water-soluble form to a membrane-bound state upon membrane binding.     

A transferable plasmid associated with AS-48 production in Enterococcus faecalis.

Enterococcus faecalis S-48 produces a peptide antibiotic, AS-48, and a bacteriocin, Bc-48. We have isolated mutants that lack these inhibitory characteristics. Further analysis of the mutants indicates that a plasmid of 56 kilobases (pMB2) may harbor the genes for AS-48. In conjugation experiments, pMB2 has been transferred into a plasmid-free OG1X strain of E. faecalis. The OG1X(pMB2) transconjugant produces the antibiotic AS-48 in solid medium, and the MIC of AS-48 for this strain is the same as that of the donor strain.     

Inhibition of Bacterial Growth, Enterotoxin Production, and Spore Outgrowth in Strains of Bacillus cereus by Bacteriocin AS-48

Bacteriocin AS-48 showed high bactericidal activity for mesophilic and psychrotrophic strains of Bacillus cereus over a broad pH range. AS-48 inhibition of the enterotoxin-producing strain LWL1 was enhanced by sodium nitrite, sodium lactate, and sodium chloride. The latter also enhanced AS-48 activity against strain CECT 131. Bacterial growth and enterotoxin production by strain LWL1 were completely inhibited at bacteriocin concentrations of 7.5 μg/ml. At subinhibitory bacteriocin concentrations, enterotoxin production decreased markedly and sporulation was delayed. Intact spores were resistant to AS-48 but became gradually sensitive to AS-48 during the course of germination.     

Inhibition of Bacillus licheniformis LMG 19409 from ropy cider by enterocin AS-48

AIMS: To determine the activity of enterocin AS-48 against ropy-forming Bacillus licheniformis from cider. METHODS AND RESULTS: Enterocin AS-48 was tested on B. licheniformis LMG 19409 from ropy cider in MRS-G broth, fresh-made apple juice and in two commercial apple ciders (A and B). Bacillus licheniformis was rapidly inactivated in MRS-G by 0.5 microg ml(-1)AS-48 and in fresh-made apple juice by 3 microg ml(-1). Concentration-dependent inactivation of this bacterium in two commercial apple ciders (A and B) stored at 4, 15 and 30 degrees C for 15 days was also demonstrated. Counts from heat-activated endospores in cider A plus AS-48 decreased very slowly. Application of combined treatments of heat (95 degrees C) and enterocin AS-48 reduced the time required to achieved complete inactivation of intact spores in cider A to 4 min for 6 microg ml(-1) and to 1 min for 12 microg ml(-1). D and z values also decreased as the bacteriocin concentration increased. CONCLUSION: Enterocin AS-48 can inhibit ropy-forming B. licheniformis in apple cider and increase the heat sensitivity of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study support the potential use of enterocin AS-48 to control B. licheniformis in apple cider.     

Permeation of bacterial cells, permeation of cytoplasmic and artificial membrane vesicles, and channel formation on lipid bilayers by peptide antibiotic AS-48.

Peptide AS-48 induces ion permeation, which is accompanied by the collapse of the cytoplasmic membrane potential, in sensitive bacteria. Active transport by cytoplasmic membrane vesicles is also impaired by AS-48. At low concentrations, this peptide also causes permeability of liposomes to low-molecular-weight compounds without a requirement for a membrane potential. Higher antibiotic concentrations induce severe disorganization, which is visualized under electron microscopy as aggregation and formation of multilamellar structures. Electrical measurements suggest that AS-48 can form channels in lipid bilayers.     

Synergistic effect of enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli O157:H7

AIMS: To determine the effects of outer membrane (OM) permeabilizing agents on the antimicrobial activity of enterocin AS-48 against Escherichia coli O157:H7 CECT 4783 strain in buffer and apple juice. METHODS AND RESULTS: We determined the influence of pH, EDTA, sodium tripolyphosphate (STPP) and heat on E. coli O157:H7 CECT 4783 sensitivity to enterocin AS-48 in buffer and in apple juice. Enterocin AS-48 was not active against intact cells of E. coli O157:H7 CECT 4783 at neutral pH. However, cells sublethally injured by OM permeabilizing agents (EDTA, STPP, pH 5, pH 8.6 and heat) became sensitive to AS-48, decreasing the amount of bacteriocin required for inhibition of E. coli O157:H7 CECT 4783. CONCLUSIONS: The results presented indicate that enterocin AS-48 could potentially be applied with a considerably wider range of protective agents, such as OM permeabilizing agents, with increased efficacy in inhibiting E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study support the potential use of enterocin AS-48 to control E. coli O157:H7 in combination with other hurdles.     

Purification of bacteriocin AS-48 from an Enterococcus faecium strain and analysis of the gene cluster involved in its production

The cyclic bacteriocin AS-48 has previously been shown to be produced by Enterococcus faecalis strains. A bacteriocin has been purified from an E. faecium strain (E. faecium 7C5), and it has been found to possess molecular mass, cyclization and amino acid sequence typical of bacteriocin AS-48. In addition to the structural gene as-48A, the sequence analysis of the AS-48 gene cluster present in E. faecium 7C5 has revealed the presence of several putative coding regions presumably involved in bacteriocin production and immunity. The results of DNA hybridization assays have indicated that the AS-48 gene cluster and the gene pd78 are present on the same plasmid, possibly the pPD1 plasmid, in E. faecium 7C5.     

Influence of Physico-Chemical Factors on the Oligomerization and Biological Activity of Bacteriocin AS-48

Bacteriocin AS-48 forms a mixture of monomers and oligomers in aqueous solutions. Such oligomers can be clearly differentiated by SDS-PAGE after formaldehyde crosslinking, and we have verified that these associates are stable to acid treatment after fixation. In addition, they show antimicrobial activity and are recognized by anti-AS-48 antibodies. AS-48 oligomers can be dissociated by the detergents SDS and Triton X-100. The degree of oligomerization of AS-48 depends on the pH of the solution and the protein concentration. At pH below 5, AS-48 is in the monomeric state at protein concentrations below 0.55.mg ml⁻¹, but it also forms dimers above this protein concentration. This bacteriocin forms oligomers at pH values above 5, in agreement with the observation that it is also more hydrophobic at neutral pH. AS-48 is stable to mild heat treatments irrespectively of pH. At 120°C it is more heat resistant under acidic conditions, but it inactivates at neutral pH. Activity of AS-48 against E. faecalis is highest at neutral pH, but it is highest at pH 4 for E. coli. The influence of pH on bacteriocin activity could be owing to changes in the conformation/oligomerization of the bacteriocin peptide as well as to changes in the surface charge of the target bacteria. Received: 3 July 2000\t/\tAccepted: 11 August 2000     

Bacteriocin AS-48 binding to model membranes and pore formation as revealed by coarse-grained simulations

Bacteriocin AS-48 is a membrane-interacting peptide that acts as a broad-spectrum antimicrobial against Gram-positive and Gram-negative bacteria. Prior Nuclear Magnetic Resonance experiments and the high resolution crystal structure of AS-48 have suggested a mechanism for the molecular activity of AS-48 whereby the peptide undergoes transition from a water-soluble to a membrane-bound state upon membrane binding. To help interpret experimental results, we here simulate the molecular dynamics of this binding mechanism at the coarse-grained level. By simulating the self-assembly of the peptide, we predict induction by the bacteriocin of different pore types consistent with a “leaky slit” model.     

Characterization of linear forms of the circular enterocin AS-48 obtained by limited proteolysis

AS-48 is a 70-residue circular peptide from Enterococcus faecalis with a broad antibacterial activity. Here, we produced by limited proteolysis a protein species carrying a single nicking and fragments of 55 and 38 residues. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that circularization is not required for the bactericidal activity, but it is important to stabilize the native structure. Moreover, it is possible to reduce the sequence to a minimal AS-48 domain without causing inactivation of this bacteriocin.     

Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4.

A simple two-step procedure was developed to obtain pure enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4. Chemical and genetic characterization revealed that the primary structure of enterocin 4 is identical to that of peptide antibiotic AS-48 from Enterococcus faecalis S-48. In contrast to the reported inhibitory spectrum of AS-48, enterocin 4 displayed no activity against gram-negative bacteria.     

Characterization of a new operon,as-48EFGH,from the as-48 gene cluster involved in immunity to enterocin AS-48

Enterocin AS-48 is a cyclic peptide produced by Enterococcus faecalis S-48 whose genetic determinants have been identified in the conjugative plasmid pMB2. A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52). In this region, the as-48A structural gene and as-48B, as-48C, as-48C(1), as-48D, and as-48D(1) genes and open reading frame 6 (ORF6) and ORF7 had been identified. The sequence analysis carried out in this work in the BglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs, as-48G and as-48H, necessary for full AS-48 expression. Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level. Tn5 disruption experiments in the last genes, as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid. The as-48EFGH operon encodes a new ABC transporter that could be involved in producer self-protection. On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed. This cluster of genes is expressed by two polycistronic mRNAs, T(2) and T(3), in JH2-2(pAM401-81) in coordinate expression. Our results also suggest that expression of T(3) could be regulated, because in JH2-2(pAM401(EH)) transformants, T(3) was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for the as-48D(1) gene for immunity against AS-48.     

Sequence-specific 1H assignment and secondary structure of the bacteriocin AS-48 cyclic peptide

The bacteriocin AS-48 is a cationic peptide (7149 Da) having a broad antimicrobial spectrum, encoded by the 68 kb conjugative plasmid pMB2 from Enterococcus faecalis S-48. It is a unique peptide since it has a cyclic structure, which is achieved by the formation of a tail–head peptide bond after ribosomal synthesis (Gálvez et al., 1989; Martínez-Bueno et al., 1994; Samyn et al., 1994). Preliminary CD and calorimetric studies (data not shown) pointed towards a highly helical and very stable three dimensional structure.All the information gathered until now indicates that the target of AS-48 is the cytoplasmic membrane in which it opens channels or pores, leading to dissipation of the proton motive force and cell death, which in some cases is also followed by bacterial lysis (Gálvez et al., 1991). This peptide is a suitable tool for studying protein–membrane interactions, and it also offers promising perspectives for biotechnological applications.Knowledge of the 3D structure of AS-48 is a first step in the conduct of further structure–function studies. Here we report the complete¹ H NMR assignment of its proton resonances together with the resulting secondary structure pattern as prerequisites for the determination of a high-resolution 3D solution structure.