The C-terminal domain of pediocin-like antimicrobial peptides (class IIa bacteriocins) is involved in specific recognition of the C-terminal part of cognate immunity proteins and in determining the antimicrobial spectrum

The pediocin-like bacteriocins contain two domains: a cationic N-terminal beta-sheet domain that mediates binding of the bacteriocin to the target cell surface and a more hydrophobic C-terminal hairpin-like domain that penetrates into the hydrophobic part of the target cell membrane. The two domains are joined by a hinge, which enables movement of the domains relative to each other. In this study, 12 different hybrid bacteriocins were constructed by exchanging domains between 5 different bacteriocins. The hybrid bacteriocins were by and large highly potent (i.e. similar potencies as the parental bacteriocins) when constructed such that the recombination point was in the hinge region, indicating that the two domains function independently. The use of optimal recombination points was, however, crucial. Shifting the recombination point just one residue from the hinge could reduce the activity of the hybrid by 3-4 orders of magnitude. Most interestingly, the active hybrids displayed target cell specificities similar to those of the parental bacteriocin from which their membrane-penetrating C-terminal hairpin domain was derived. The results also indicate that the negatively charged aspartate reside in the hinge of most pediocin-like bacteriocins interacts with the C-terminal hairpin domain, perhaps by interacting with the positively charged residue that is present at one of the last three positions in the C-terminal end of most pediocin-like bacteriocins. Bacteria that produce pediocin-like bacteriocins also produce a cognate immunity protein that protects the producer from being killed by its own bacteriocin. Four different active hybrid immunity proteins constructed by exchanging regions between three different immunity proteins were tested for their ability to confer immunity to the hybrid bacteriocins. The results showed that the C-terminal half of the immunity proteins contains a region that directly or indirectly specifically recognizes the membrane-penetrating C-terminal hairpin domain of pediocin-like bacteriocins. The implications these results have on how pediocin-like bacteriocins and their immunity proteins interact with cellular specificity determinants (for instance a putative bacteriocin receptor) are discussed.     

Structure-function analysis of immunity proteins of pediocin-like bacteriocins: C-terminal parts of immunity proteins are involved in specific recognition of cognate bacteriocins

The immunity proteins of pediocin-like bacteriocins show a high degree of specificity with respect to the pediocin-like bacteriocin they recognize and confer immunity to. The aim of this study was to identify regions of the immunity proteins that are involved in this specific recognition. Six different hybrid immunity proteins were constructed from three different pediocin-like bacteriocin immunity proteins that have similar sequences but confer resistance to different bacteriocins. These hybrid immunity proteins were then tested for their ability to confer immunity to various pediocin-like bacteriocins. The specificities of the hybrid immunity proteins proved to be similar to those of the immunity proteins from which the C-terminal halves were derived, thus revealing that the C-terminal half of immunity proteins for pediocin-like bacteriocins contains a domain that is involved in specific recognition of the bacteriocins they confer immunity to. Moreover, the results also revealed that the effectiveness of an immunity protein is strain dependent and that its functionality thus depends in part on interplay with strain-dependent factors. To further investigate the structure-function relationship of these immunity proteins, the enterocin A and leucocin A immunity proteins (EntA-im and LeuA-im) were purified to homogeneity and structurally analyzed under various conditions by Circular dichroism (CD) spectroscopy. The results revealed that both immunity proteins are alpha-helical and well structured in an aqueous environment, the denaturing temperature being 78.5 degrees C for EntA-im and 58.0 degrees C for LeuA-im. The CD spectra also revealed that there was no further increase in the structuring or alpha-helical content when the immunity proteins were exposed to dodecylphosphocholine micelles or dioleoyl-L-alpha-phosphatidyl-DL-glycerol (DOPG) liposomes, indicating that the immunity proteins, in contrast to the bacteriocins, do not interact extensively with membranes. They may nevertheless be loosely associated with the membrane, possibly as peripheral membrane proteins, thus enabling them to interact with their cognate bacteriocin.     

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin.     

The continuing story of class IIa bacteriocins.

Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.     

A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum

Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37 degrees C, whereas wild-type sakacin P was approximately 10 times less potent at 37 degrees C than at 20 degrees C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys-->Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.     

Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and other cationic antimicrobial peptides from complex culture medium

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.     

Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae

The production of bacteriocins can be favorable for colonization of the host by eliminating other bacterial species that share the same environment. In Streptococcus pneumoniae, the pnc (blp) locus encoding putative bacteriocins, immunity, and export proteins is controlled by a two-component system similar to the comCDE system required for the induction of genetic competence. A detailed comparison of the pnc clusters of four genetically distinct isolates confirmed the great plasticity of this locus and documented several repeat sequences. Members of the multiple-antibiotic-resistant Spain23F-1 clone, one member of the Spain9V-3 clone, sensitive 23F strain 2306, and the TIGR4 strain produced bactericidal substances active against other gram-positive bacteria and in some cases against S. pneumoniae as well. However, other strains did not show activity against the indicator strains despite the presence of a bacteriocin cluster, indicating that other factors are required for bacteriocin activity. Analysis of strain 2306 and mutant derivatives of this strain confirmed that bacteriocin production was dependent on the two-component regulatory system and genes involved in bacteriocin transport and processing. At least one other bacteriocin gene, pncE, is located elsewhere on the chromosome and might contribute to the bacteriocin activity of this strain.     

Mechanisms of resistance to bacteriocins targeting the mannose phosphotransferase system

The membrane proteins IIC and IID of the mannose phosphotransferase system (Man-PTS) together form a membrane-located complex that serves as a receptor for several different bacteriocins, including the pediocin-like class IIa bacteriocins and the class IIc bacteriocin lactococcin A. Bacterial strains sensitive to class IIa bacteriocins readily give rise to resistant mutants upon bacteriocin exposure. In the present study, we have therefore investigated lactococcin A-resistant mutants of Lactococcus lactis as well as natural food isolates of Listeria monocytogenes with different susceptibilities to class IIa bacteriocins. We found two major mechanisms of resistance. The first involves downregulation of Man-PTS gene expression, which takes place both in spontaneous resistant mutants and in natural resistant isolates. The second involves normal expression of the Man-PTS system, but the underlying mechanism of resistance for these cells is unknown. In some cases, the resistant phenotype was linked to a shift in the metabolism; i.e., reduced growth on glucose due to reduction in Man-PTS expression was accompanied by enhanced growth on another sugar, such as galactose. The implications of these findings in terms of metabolic heterogeneity are discussed.     

Production of bacteriocins and siderophore-like activity by Azospirillum brasilense

Sixty Azospirillum strains were tested for their bacteriocin production ability; twenty-seven (45%) were able to produce bacteriocins and inhibited the growth of one or more indicator strains in solid medium. Mitomycin C treatment enhanced the proportion to 80%. Sometimes large growth inhibition zones were formed, but not when FeCl3 was added in the medium. These inhibition zones probably result from the activity of siderophores. Partially purified bacteriocins produced by four strains were inactivated at pH 4, but were very stable between pH 5 to 10; bacteriocins produced by three strains lost their activity between 55 and 80 degrees C. Loss or decrease in the bacteriocin activity was observed with pronase E treatment; trypsin, lysozyme and alpha-amylase did not have an effect on bacteriocin activity. These findings show that the antagonism among azospirilla was due principally to the bacteriocins and sometimes probably due to siderophores, but not to bacteriophages or other substances.     

1.6-Angstroms crystal structure of EntA-im. A bacterial immunity protein conferring immunity to the antimicrobial activity of the pediocin-like bacteriocin enterocin A

Many Gram-positive bacteria produce ribosomally synthesized antimicrobial peptides, often termed bacteriocins. Genes encoding pediocin-like bacteriocins are generally cotranscribed with or in close vicinity to a gene encoding a cognate immunity protein that protects the bacteriocin-producer from their own bacteriocin. We present the first crystal structure of a pediocin-like immunity protein, EntA-im, conferring immunity to the bacteriocin enterocin A. Determination of the structure of this 103-amino acid protein revealed that it folds into an antiparallel four-helix bundle with a flexible C-terminal part. The fact that the immunity protein conferring immunity to carnobacteriocin B2 also consists of a four-helix bundle (Sprules, T., Kawulka, K. E., and Vederas, J. C. (2004) Biochemistry 43, 11740-11749) strongly indicates that this is a conserved structural motif in all pediocin-like immunity proteins. The C-terminal half of the immunity protein contains a region that recognizes the C-terminal half of the cognate bacteriocin, and the flexibility in the C-terminal end of the immunity protein might thus be an important characteristic that enables the immunity protein to interact with its cognate bacteriocin. By homology modeling of three other pediocin-like immunity proteins and calculation of the surface charge distribution for EntA-im and the three structure models, different charge distributions were observed. The differences in the latter part of helix 3, the beginning of helix 4, and the loop connecting these helices might also be of importance in determining the specificity.     

An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley

AIMS: The aim of this study was to perform a detailed characterization of bacteriocins produced by lactic acid bacteria (LAB) isolated from malted barley. METHODS AND RESULTS: Bacteriocin activities produced by eight LAB, isolated from various types of malted barley, were purified to homogeneity by ammonium sulphate precipitation, cation exchange, hydrophobic interaction and reverse-phase liquid chromatography. Molecular mass analysis and N-terminal amino acid sequencing of the purified bacteriocins showed that four non-identical Lactobacillus sakei strains produced sakacin P, while four Leuconostoc mesenteroides strains were shown to produce bacteriocins highly similar or identical to leucocin A, leucocin C or mesenterocin Y105. Two of these bacteriocin-producing strains, Lb. sakei 5 and Leuc. mesenteroides 6, were shown to produce more than one bacteriocin. Lactobacillus sakei 5 produced sakacin P as well as two novel bacteriocins, which were termed sakacin 5X and sakacin 5T. The inhibitory spectrum of each purified bacteriocin was analysed and demonstrated that sakacin 5X was capable of inhibiting the widest range of beer spoilage organisms. CONCLUSION: All bacteriocins purified in this study were class II bacteriocins. Two of the bacteriocins have not been described previously in the literature while the remaining purified bacteriocins have been isolated from environments other than malted barley. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a thorough analysis of bacteriocin-producing LAB from malt and demonstrates, for the first time, the variety of previously identified and novel inhibitory peptides produced by isolates from this environment. It also highlights the potential of these LAB cultures to be used as biological controlling agents in the brewing industry.     

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.     

Purification and cloning of piscicolin 61, a bacteriocin fromCarnobacterium piscicola LV61

Piscicolin 61, a bacteriocin produced byCarnobacterium piscicola LV61, inhibits the growth of strains ofCarnobacterium, Lactobacillus, andEnterococcus. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential hydrophobic interaction and reversed-phase chromatography. The N-terminal amino acid sequence of piscicolin 61 was determined by Edman degradation. The plasmid-located structural gene encoding piscicolin (psc61) was cloned and sequenced. It encoded a primary translation product of 71 amino acid residues, which is cleaved between amino acid residues 18 and 19 to yield the active bacteriocin. The calculatedM _r from the deduced protein sequence, 5052.6, agreed with that obtained by mass spectrometry. Piscicolin 61 did not show any sequence similarities to other known bacteriocins. However, the leader sequence resembled those of the pediocin-like bacteriocins. Piscicolin 61 may be able to form amphiphilic helices and may thus act on the membrane of sensitive cells.     

Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins

Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.     

Diversity and dynamics of bacteriocins from human microbiome

Human commensal microbiota are an important determinant of health and disease of the host. Different human body sites harbour different bacterial microbiota, bacterial communities that maintain a stable balance. However, many of the factors influencing the stabilities of bacterial communities associated with humans remain unknown. In this study, we identified putative bacteriocins produced by human commensal microbiota. Bacteriocins are peptides or proteins with antimicrobial activity that contribute to the stability and dynamics of microbial communities. We employed bioinformatic analyses to identify putative bacteriocin sequences in metagenomic sequences obtained from different human body sites. Prevailing bacterial taxa of the putative bacteriocins producers matched the most abundant organisms in each human body site. Remarkably, we found that samples from different body sites contain different density of putative bacteriocin genes, with the highest in samples from the vagina, the airway, and the oral cavity and the lowest in those from gut. Inherent differences of different body sites thus influence the density and types of bacteriocins produced by commensal bacteria. Our results suggest that bacteriocins play important roles to allow different bacteria to occupy several human body sites, and to establish a long‐term commensal relationship with human hosts.     

Native and heterologous production of bacteriocins from gram-positive microorganisms

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.     

Partial characterization of bacteriocins produced by environmental strain Enterococcus faecium EK13

AIMS: The partial characterization of bacteriocins produced by an environmental strain Enterococcus faecium EK13, isolated from cattle dung water. METHODS AND RESULTS: A bacteriocin was partially purified by ammonium sulphate precipitation, followed by a SP-Sepharose column, reverse-phase chromatography and N-terminal region sequenced. The anti-microbial substance produced was found to be a heat-stable polypeptide with molecular mass 4.83 kDa, which was determined by N-terminal amino acid sequencing to be enterocin A. A second substance was specified by PCR as enterocin P. Bacteriocins were stable at 4 and -20 degrees C for long storage periods. The optimum of bacteriocin production was observed in the range of pH 5.0-6.5 at 30 and 37 degrees C. The most active substances are produced by strain EK13 in logarithmic growth phase and bacteriocins are produced after 1 h of fermentation. The highest activity detected in fermentation experiments was 51 200 AU ml(-1) and the most sensitive indicator strain was found to be Listeria innocua LMG 13568. Differences in bacteriocin activity against two indicators could be explained by more than one type of enterocin production by strain EK13, or with different mode of action or in different sensitivity of strains. CONCLUSION: Enterococcus faecium strain EK13 isolated from cattle dung water produces two bacteriocins, enterocin A and P, with an inhibitory effect against the strain of the genera Enterococcus, Leuconostoc, Lactobacillus, Streptococcus, Staphylococcus, Bacillus and Listeria (in different origin). SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecium EK13 environmental strain is a new producer of enterocin A and P. The E. faecium EK13, isolated from cattle dung water, is presented with the further aim to utilize it for waste treatment by biotechnological processes.     

A comparative study of three bacteriocins of Bacteroides fragilis

Three different bacteriocins produced by strains of Bacteroides fragilis were compared in terms of their production kinetics, physico-chemical nature, and action on macromolecular synthesis in a common indicator strain. Bacteriocin 78/438 was produced during the logarithmic growth phase, was thermolabile and stable between pH 5 and 9. It was susceptible to trypsin and pepsin, and affected DNA, RNA and protein syntheses in susceptible cells. Bacteriocin A49 was produced during the stationary growth phase, was thermolabile and stable between pH 7 and 9. This bacteriocin was also susceptible to trypsin and pepsin, but only RNA synthesis was affected in the indicator strain. Bacteriocin A55 differed markedly from both 78/438 and A49, and was found to be predominantly cell-bound, resistant to inactivation by high temperatures and stable over a wide pH range of 2 to 12. It was susceptible to trypsin but resistant to pepsin. A55 had a delayed effect on macromolecular synthesis with DNA synthesis being inhibited after 60 min. With all three bacteriocins, killing of the indicator strain followed single hit kinetics with the interaction of bacteriocin and target cell occurring in two stages. Killing by bacteriocin A55 was much slower than the other two and this may be related to its effect on macromolecular synthesis. The killing action of all three bacteriocins was dependent on the growth phase of the susceptible cells.     

Mutational analysis of residues in the helical region of the class IIa bacteriocin pediocin PA-1

A 15-mer fragment that is derived from the helical region in the C-terminal half of pediocin PA-1 inhibited the activity of pediocin PA-1. Of 13 other pediocin-like (hybrid) bacteriocins, only the hybrid bacteriocin Sak/Ped was markedly inhibited by the 15-mer fragment. Sak/Ped was the only one of these bacteriocins that had a sequence (in the C-terminal helix-containing half) identical to that of the 15-mer fragment, indicating that the fragment inhibits pediocin-like bacteriocins in a sequence-dependent manner. By replacing (one at a time) all 15 residues in the fragment with Ala or Leu, five residues (K1, A2, T4, N8, and A15) were identified as being especially important for the inhibitory action of the fragment. The results suggest that the corresponding residues (K20, A21, T23, N27, and A34, respectively) in pediocin PA-1 might be involved in interactions between pediocin PA-1 and its receptor. To characterize the environment surrounding these five residues when pediocin PA-1 interacts with target cells, these residues were replaced (one at a time) with a hydrophobic large (Leu) residue, a hydrophilic charged (Asp or Arg) residue, and a small (Ala or Gly) residue. The results revealed that residues A21 and A34 are in a spatially constrained environment, since the replacement with a small (Gly) residue was the only substitution that did not markedly reduce the bacteriocin activity. The positive charge in K20 and the polar amide group in N27 appeared to interact with electronegative groups, since the replacement of these two residues with a positive (Arg) residue was well tolerated, while replacement with a negative (Asp) residue was detrimental to the bacteriocin activity. K20 was in a less constrained environment than N27, since the replacement of K20 with a large hydrophobic (Leu) residue was tolerated fairly well and to a greater extent than N27. T23 seemed to be in an environment that was not restricted with respect to size, polarity, and charge, since replacements with large (Leu) and small (Ala) hydrophobic residues and a hydrophilic negative (Asp) residue were tolerated fairly well (2- to 6-fold reduction in activity). Moreover, the replacement of T23 with a large positive (Arg) residue resulted in wild-type or better-than-wild-type activity.     

Purification and partial amino acid sequence of curvaticin FS47, a heat-stable bacteriocin produced by Lactobacillus curvatus FS47.

Curvaticin FS47, a bacteriocin produced by Lactobacillus curvatus FS47, is inhibitory to Listeria monocytogenes, as well as Lactobacillus, Pediococcus, Enterococcus, and Bacillus spp. The bacteriocin was purified by 40% ammonium sulfate precipitation, solid-phase extraction, and reversed-phase high-pressure liquid chromatography. Purified curvaticin FS47 was determined to be 4.07 kDa by mass spectrometry and was partially sequenced. Thirty-one N-terminal amino acids were identified; the curvaticin FS47 protein sequence did not show homology to the pediocin-like group of bacteriocins.