Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish

Enterocins NKR-5-3A, B, C, and D were purified from the culture supernatant of Enterococcus faecium NKR-5-3 and characterized. Among the four purified peptides, enterocin NKR-5-3A (5242.3 Da) was identical to brochocin A, produced by Brochothrix campestris ATCC 43754, in mature peptides, and its putative synergistic peptide, enterocin NKR-5-3Z, was found to be encoded in ent53Z downstream of ent53A, encoding enterocin NKR-5-3A. Enterocin NKR-5-3B (6316.4 Da) showed a broad antimicrobial spectrum, and enterocin NKR-5-3C (4512.8 Da) showed high activity against Listeria. Enterocin NKR-5-3D (2843.5 Da), showing high homology to an inducing peptide produced by Lactobacillus sakei 5, induced the production of the enterocins. The enterocins showed different antimicrobial spectra and intensities. E. faecium NKR-5-3 concomitantly produced enterocins NKR-5-3A, B, C, and D which probably belong to different classes of bacteriocins. Furthermore, NKR-5-3 production was induced by enterocin NKR-5-3D.     

Biochemical and Genetic Characterization of the Two-Peptide Bacteriocin Enterocin 1071 Produced by Enterococcus faecalis FAIR-E 309

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.     

Biochemical and genetic characterization of the two-peptide bacteriocin enterocin 1071 produced by Enterococcus faecalis FAIR-E 309

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.     

Molecular and Genetic Characterization of Propionicin F, a Bacteriocin from Propionibacterium freudenreichii

This work describes the purification and characterization of propionicin F, the first bacteriocin isolated from Propionibacterium freudenreichii. The bacteriocin has a bactericidal activity and is only active against strains of P. freudenreichii. Propionicin F appears to be formed through a processing pathway new to bacteriocins. The mass of the purified bacteriocin was determined by mass spectrometry, and the N-terminal amino acid sequence was determined by Edman degradation. Sequencing of pcfA, the bacteriocin structural gene, revealed that propionicin F corresponds to a 43-amino-acid peptide in the central part of a 255-amino-acid open reading frame, suggesting that mature propionicin F is excised from the probacteriocin by N- and C-terminal proteolytic modifications. DNA sequencing and Northern blot hybridizations revealed that pcfA is cotranscribed with genes encoding a putative proline peptidase and a protein from the radical S-adenosylmethionine family. A gene encoding an ABC transporter was also identified in close proximity to the bacteriocin structural gene. The potential role of these genes in propionicin F maturation and secretion is discussed.     

Identification of enterocin NKR-5-3C, a novel class IIa bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3

The structure of enterocin NKR-5-3C, an anti-listerial bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3, was determined. Enterocin NKR-5-3C is a novel class IIa bacteriocin that possesses an YGNGL motif sequence and two disulfide bridges in its structure. It is encoded on gene ent53C together with an 18-amino-acid-residue double glycine leader peptide.     

SmbFT,a Putative ABC Transporter Complex,Confers Protection against the Lantibiotic Smb in Streptococci

Streptococcus mutans, a dental pathogen, secretes different kinds of lantibiotic and nonlantibiotic bacteriocins. For self-protection, a bacteriocin producer strain must possess one or more cognate immunity mechanisms. We report here the identification of one such immunity complex in S. mutans strain GS-5 that confers protection against Smb, a two-component lantibiotic. The immunity complex that we identified is an ABC transporter composed of two proteins: SmbF (the ATPase component) and SmbT (the permease component). Both of the protein-encoding genes are located within the smb locus. We show that GS-5 becomes sensitized to Smb upon deletion of smbT, which makes the ABC transporter nonfunctional. To establish the role SmbFT in providing immunity, we heterologously expressed this ABC transporter complex in four different sensitive streptococcal species and demonstrated that it can confer resistance against Smb. To explore the specificity of SmbFT in conferring resistance, we tested mutacin IV (a nonlantibiotic), nisin (a single peptide lantibiotics), and three peptide antibiotics (bacitracin, polymyxin B, and vancomycin). We found that SmbFT does not recognize these structurally different peptides. We then tested whether SmbFT can confer protection against haloduracin, another two-component lantibiotic that is structurally similar to Smb; SmbFT indeed conferred protection against haloduracin. SmbFT can also confer protection against an uncharacterized but structurally similar lantibiotic produced by Streptococcus gallolyticus. Our data suggest that SmbFT truly displays immunity function and confer protection against Smb and structurally similar lantibiotics.     

Enterocin 96, a Novel Class II Bacteriocin Produced by Enterococcus faecalis WHE 96, Isolated from Munster Cheese

Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.Bacteriocins are a heterogeneous group of ribosomally synthesized antibacterial peptides that inhibit strains and species that are usually, but not always, closely related to the producing bacteria (16). Enterococcal bacteriocins, often termed enterocins, have been widely investigated, mainly because they are active against gram-positive food-borne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. The vast majority of enterocins are active only against gram-positive bacteria (10, 17); however, some exceptions with broad activity spectra described in recent years showed the ability to inhibit the growth of gram-negative microorganisms (5, 11, 13).The increasing number of enterocins reported in the literature and the emergence of novel structures that could not be included in classical bacteriocin classifications (12, 14, 17) prompted the grouping of enterocins into a new four-class scheme by Franz et al. (8). Most enterocins known so far were included in class II (small, nonlantibiotic peptides), which was divided into three subgroups: class II.1, enterocins of the pediocin family; class II.2, enterocins synthesized without a leader peptide; and class II.3, other linear, non-pediocin-like enterocins.The fact that numerous Enterococcus strains found in a variety of fermented and nonfermented foods produce bacteriocins, often more than one per strain, has sparked interest in their use in food preservation (4). Despite the concerns over enterococci as opportunistic pathogens and indicators of fecal contamination, they are indigenous species in the gastrointestinal tract and have long been used as human and/or animal probiotics (1, 2, 7).In this work, we describe and characterize a new class II enterocin produced by Enterococcus faecalis WHE 96, previously isolated from Munster cheese, for its anti-Listeria properties. The amino acid sequence, the structural gene, and the spectrum of activity of this bacteriocin are reported.     

Garvicin A,a Novel Class IId Bacteriocin from Lactococcus garvieae That Inhibits Septum Formation in L. garvieae Strains

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.     

The Expression of Superoxide Dismutase (SOD) and a Putative ABC Transporter Permease Is Inversely Correlated during Biofilm Formation in Listeria monocytogenes 4b G

Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant Δsod, and double deletion mutant of the sod and lm. G_1771 genes Δ1771Δsod were used to investigate the role of SOD and its relationship to the expression of the putative ABC transporter permease in biofilm formation. Our results showed that the ability to form a biofilm was significantly reduced in the Δsod mutant and the Δ1771Δsod double mutant. Both Δsod and Δ1771Δsod mutants exhibited slow growth phenotypes and produced more reactive oxygen species (ROS). The growth was inhibited in the mutants by methyl viologen (MV, internal oxygen radical generator) treatment. In addition, the expression of one oxidation resistance gene (kat), two stress regulators encoding genes (perR and sigB), and one DNA repair gene (recA) were analyzed in both the wild-type L. monocytogenes 4b G and the deletion mutants by RT-qPCR. The expression levels of the four genes were increased in the deletion mutants when biofilms were formed. Taken together, our data indicated that SOD played an important role in biofilm formation through coping with the oxidant burden in deficient antioxidant defenses.     

Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal

The main objective of this study was to detect the antimicrobial activity and the presence of bacteriocin structural genes in 224 enterococcal isolates from fecal origin obtained from humans, pets, wild animals and birds. Direct antimicrobial activity against Listeria monocytogenes CECT4032 was detected in 102 (45.6%) of the tested isolates. From these, only 22 displayed bacteriocin activity against this indicator. The bacteriocinogenic strains contained one or more of the bacteriocin structural genes tested in this study, with those of enterocins P, A and L50 (L50A and L50B) being the most abundant. Our results show a high occurrence of the combination of different bacteriocin structural genes in the enterococcal isolates analyzed, indicating an elevated genetic potential of these strains to produce various bacteriocins.     

Diversity of bacteriocinogenic lactic acid bacteria isolated from Mediterranean fish viscera

Nine lactic acid bacteria strains showing bacteriocin-like activity were isolated from various fresh fish viscera. The following species were identified based on 16S rDNA sequences: Enterococcus durans (7 isolates), Lactococcus lactis (1) and Enterococcus faecium (1). These strains were active against Listeria innocua and other LAB. Random amplified polymorphic DNA analyses showed four major patterns for the E. durans species. PCR analyses revealed a nisin gene in the genome of the Lc. lactis strain. Genes coding enterocins A, B and P were found in the genome of the E. faecium isolate. Enterocins A and B genes were also present in the genome of E. durans GM19. Hence, this is the first report describing E. durans strains producing enterocins A and B. Electrospray ionization mass spectrometry revealed that the purified bacteriocin produced by the E. durans GMT18 strain had an exact molecular mass of 6,316.89 Da. This bacteriocin was designated as durancin GMT18. Edman sequencing failed to proceed; suggesting that durancin GTM18 may contain terminal lanthionine residues. Overall, the results obtained revealed the presence of a variety of enterococci in Mediterranean fish viscera, as evidenced by their genetic profiles and abilities to produce different bacteriocins. These strains could be useful for food biopreservation or as probiotics.     

Isolation and characterization of bacteriocin‐producing bacteria from the gastrointestinal tract of broiler chickens for probiotic use

Aims: To isolate and characterize the bacteriocin‐producing bacteria (BPB) from the gastrointestinal tract of broiler chickens for probiotic use. Methods and Results: In total, 291 bacterial strains were isolated from broilers and screened for bacteriocin‐producing ability. The bacteriocins produced by Enterococcus faecium SH 528, Ent. faecium SH 632 and Pediococcus pentosaceus SH 740 displayed inhibitory activity against pathogens including Clostridium perfringens and Listeria monocytogenes. Activity of the bacteriocins remained unchanged after 30 min of heat treatment at 60°C or exposure to organic solvents, but diminished after treatment with proteolytic enzymes. PCR was used to detect the structural genes enterocin A and B in SH 528, enterocin L50 and P in SH 632, and pediocin PA‐1 in SH 740. Most of them were resistant to 0·5% bile salts and remained viable after 2 h at pH 3·0. Ent. faecium SH 528 exhibited the highest amylase activity among the strains tested. Conclusions: We selected Ent. faecium SH 528 and SH 632 and Ped. pentosaceus SH 740 by probiotic selection criteria including inhibition activity against pathogens. Significance and Impact of the Study: The isolated BPB could potentially be used in the poultry industry as probiotics to control pathogens.     

Characterization of Mundticin L,a Class IIa Anti-Listeria Bacteriocin from Enterococcus mundtii CUGF08

Enterococcus mundtii CUGF08, a lactic acid bacterium isolated from alfalfa sprouts, was found to produce mundticin L, a new class IIa bacteriocin that has a high level of inhibitory activity against the genus Listeria. The plasmid-associated operons containing genes for the mundticin L precursor, the ATP binding cassette (ABC) transporter, and immunity were cloned and sequenced. The fifth residue of the conservative consensus sequence YGNGX in the mature bacteriocin is leucine instead of valine in the sequences of the homologous molecules mundticin KS (ATO6) and enterocin CRL35. The primary structures of the ABC transporter and the immunity protein are homologous but unique.Bacteriocins are ribosomally synthesized proteinaceous compounds that inhibit closely related bacteria (19). Due to consumer concerns with chemical and irradiation preservation methods and due to the rising demand for minimally processed food products, alternative methods for shelf life extension and enhanced safety are needed. Bacteriocins are considered “natural” antimicrobials since many bacteriocins are produced by food grade lactic acid bacteria, which are generally recognized as safe. Bacteriocins can be divided into three main classes: the class I lanthionine-containing lantibiotics, exemplified by nisin; the class II non-lanthionine-containing bacteriocins; and the class III heat-labile, large proteins (6). Class III bacteriocins have limited application due to their thermal instability and cytolytic activity against eukaryotic cells. Class II can be further divided into class IIa containing pediocin-like bacteriocins, class IIb containing two-peptide bacteriocins, and class IIc containing other bacteriocins (8). Class IIa bacteriocins have been extensively studied since pediocin PA-1 was first discovered (12) and characterized (20). Currently, only nisin in class I has been approved by the FDA as a natural food additive. Bacteriocins belonging to class IIa are promising alternative antimicrobials since they are more stable over a broader range of heating regimens and pH conditions. In addition, these bacteriocins exhibit stronger antimicrobial activity against the genus Listeria than nisin (17) but have a narrower antimicrobial spectrum.The potential applications of class IIa bacteriocins in both meat and plant-based foods as a means to provide protection against potential food-borne pathogens and extend shelf life continue to expand. In an attempt to use biological methods for controlling food-borne pathogens on fresh sprouts, a number of food grade lactic acid bacteria were isolated from the indigenous microbiota on alfalfa sprouts. Some of these isolates were found to be bacteriocinogenic. This study describes a new class IIa bacteriocin, mundticin L produced by Enterococcus mundtii CUGF08 isolated from alfalfa sprouts.     

Properties of the strains<Emphasis Type="Italic">Enterococcus haemoperoxidus</Emphasis> and<Emphasis Type="Italic">E. moraviensis</Emphasis>, new species among enterococci

Antibiotic susceptibility or resistance, urease activity, detection of the structural genes for bacteriocin production, bacteriocin activity as well as sensitivity of the isolates to enterocins (Ent) A and M were determined in 23 isolates of new species Enterococcus haemoperoxidus and E. moraviensis. The majority of the strains were antibiotic sensitive and exhibited low urease activity (< 10 nkat/mL). The most frequently detected genes for Ent were entA and entP. However, only the strain 466 of E. haemoperoxidus produced an antibacterial substance with inhibitory activity against 21 G+ indicators. It was partially purified reaching an activity of up to 12 800 AU/mL. This bacteriocin active strain also possessed the genes for EntA and EntP. The other strains did not inhibit the indicator strains. The substance produced by the 466 strain was active even after a 5-months storage at +4 and -20 degrees C. This substance has proteolytic and hydrophilic character, pH optimum of bacteriocin production by this strain being between 4 and 7. While E. moraviensis strains showed sensitivity to EntA (produced by E. faecium EK13) and to EntM (produced by E. faecium AL41), E. haemoperoxidus strains were sensitive to EntA (except strain 382) but less sensitive to the treatment by EntM.     

Enterocin X,a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.Bacteriocins are gene-encoded antibacterial peptides and proteins. Because of their natural ability to preserve food, they are of particular interest to researchers in the food industry. Bacteriocins are grouped into three main classes according to their physical properties and compositions (11, 12). Of these, class IIb bacteriocins are thermostable non-lanthionine-containing two-peptide bacteriocins whose full antibacterial activity requires the interaction of two complementary peptides (8, 19). Therefore, two-peptide bacteriocins are considered to function together as one antibacterial entity (14).Enterocins A and B, first discovered and identified about 12 years ago (2, 3), are frequently present in Enterococcus faecium strains from various sources (3, 5, 6, 9, 13, 16). So far, no other bacteriocins have been identified in these strains, except the enterocin P-like bacteriocin from E. faecium JCM 5804^T (18). Here, we describe the characterization and genetic identification of enterocin X in E. faecium KU-B5. Enterocin X (identified after the enterocin P-like bacteriocin was discovered) is a newly found class IIb bacteriocin in E. faecium strains that produce enterocins A and B.     

Functional Genetic Analysis of the GarML Gene Cluster in Lactococcus garvieae DCC43 Gives New Insights into Circular Bacteriocin Biosynthesis

Garvicin ML (GarML) is a circular bacteriocin produced by Lactococcus garvieae DCC43. The recently published draft genome of this strain allowed determination of the genetic background for bacteriocin production. Bioinformatic analysis identified a gene cluster consisting of nine open reading frames likely involved in the production of and immunity to GarML. The garA gene encodes the bacteriocin precursor, garX a large transmembrane protein, garBCDE a putative immunity protein (garB) followed by an ATPase and two transmembrane proteins, and garFGH a putative ABC transporter complex. Functional genetic analysis revealed that deletion of garFGH had no effect on sensitivity to or production of GarML. In contrast, deletion of garBCDE or inactivation of garX resulted in high-level sensitivity to GarML and completely abolished production of active bacteriocin. Mass spectrometry of culture supernatants revealed that wild-type cultures contained the mature circular form as well as the linear forms of the bacteriocin, both with and without the three-amino-acid leader sequence, while bacteriocin-negative mutants contained only the linear forms. These results indicate that cleavage of the leader peptide precedes circularization and is likely performed by a functional entity separate from the GarML gene cluster. To our knowledge, this is the first conclusive evidence for these processes being separated in time. Loss of immunity and antimicrobial activity in addition to our inability to detect the circular bacteriocin in the ΔgarBCDE and garX::pCG47 mutants demonstrate that both these units are indispensable for GarML biosynthesis as well as immunity. Furthermore, the results indicate that these genes are implicated in the circularization of the bacteriocin and that their functions are probably interlinked.     

Two Subpopulations of Listeria monocytogenes Occur at Subinhibitory Concentrations of Leucocin 4010 and Nisin

In situ analyses of single Listeria monocytogenes cells at subinhibitory concentrations of leucocin 4010 and nisin revealed two subpopulations when measured by fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. One subpopulation consisted of cells with a dissipated pH gradient (ΔpH), and the other consisted of cells that maintained ΔpH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate ΔpH for 90% of the cell population (ED₉₀) was predicted. ED₉₀ increased after the addition of sodium chloride (1 to 3% [wt/vol]) to the bacteriocin solutions, while ED₉₀ decreased by the addition of sodium nitrite (60 and 100 ppm). Other meat additives, including sodium phosphate, sodium lactate, sodium citrate, and sodium acetate slightly increased ED₉₀. The inhibitory effect of sodium chloride on the antilisterial activity of leucocin 4010 and nisin was confirmed on the surfaces of meat sausages. This study highlights the important practical implications of applying subinhibitory concentrations of bacteriocins, which results in unaffected target cells. In situ analyses by FRIM in combination with modeling of single-cell data can be applied to ensure that sufficient concentrations of bacteriocins are used in food preservation.     

Bacteriocinogenic Potential of <Emphasis Type="Italic">Enterococcus faecium</Emphasis> Isolated from Wine

A total of 145 lactic acid bacteria isolated from a variety of Turkish red wines during malolactic fermentation were screened to find bacteriocin-producing strains. Among them, 14 isolates of Enterococcus faecium were identified to produce bacteriocins. PCR screening revealed that some isolates harbored entA and entB genes while some harbored entA, entB and entP genes. An isolate designated as Ent. faecium H46 was selected to characterize its bacteriocins. The bacteriocins were purified to homogeneity from culture supernatant by Amberlite XAD-16, cation-exchange and reverse-phase chromatography. MALDI-TOF mass spectrometry analysis identified the bacteriocins as enterocin A and enterocin B. The presence of Ent. faecium is noteworthy since it is not associated with wine fermentation. However, it has been reported as an important wine spoilage organism due to its potential to produce tyramine. Although species of Enterococcus is not known as wine bacteria, contamination by Ent. faecium may arise from grapes or wineries equipments used for wine production.