Genetic features of circular bacteriocins produced by Gram-positive bacteria

This review highlights the main genetic features of circular bacteriocins, which require the co-ordinated expression of several genetic determinants. In general terms, it has been demonstrated that the expression of such structural genes must be combined with the activity of proteins involved in maturation (cleavage/circularization) and secretion outside the cell via different transporter systems, as well as multifaceted immunity mechanisms essential to ensuring the bacteria's self-protection against such strong inhibitors. Several circular antibacterial peptides produced by Gram-positive bacteria have been described to date, including enterocin AS-48, from Enterococcus faecalis S-48 (the first one characterized), gassericin A, from Lactobacillus gasseri LA39, and a similar one, reutericin 6, from Lactobacillus reuteri LA6, butyrivibriocin AR10, from the ruminal anaerobe Butyrivibrio fibrisolvens AR10, uberolysin, from Streptococcus uberis, circularin A, from Clostridium beijerinckii ATCC 25752, and subtilosin A, from Bacillus subtilis. We summarize here the progress made in the understanding of their principal genetic features over the last few years, during which the functional roles of circular proteins with wide biological activity have become clearer.     

HPLC purification and re‐evaluation of chemical identity of two circular bacteriocins,gassericin A and reutericin 6

Aim: The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Methods and Results: Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse‐phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2‐propanol. Mass analysis, N‐terminal sequencing and bacteriocin assay of the HPLC‐purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H]⁺ and both had the same specific activity. d/l‐ amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)‐1‐(9‐fluorenyl)ethyl chloroformate and o‐phthalaldehyde/N‐acetyl‐l ‐cystein revealed neither gassericin A nor reutericin 6 contained d ‐alanine residues contrary to our previous results. Conclusion: Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no d ‐alanine residues. Significance and Impact of the Study: The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.     

Inhibition of food-borne pathogenic bacteria by bacteriocins from Lactobacillus gasseri

Seventy-three strains of the Lactobacillus acidophilus group and a Lact. reuteri isolated from human faeces were examined for production of antimicrobial agents against 16 strains of six species of food-borne enteric pathogenic bacteria. Several strains of Lact. gasseri showed wide inhibitory activity against the tested bacteria. Gassericin A produced by Lact. gasseri LA39 was one of the most widely active bacteriocins. It was bactericidal without causing cell lysis.     

Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055

A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.     

Conjugative Plasmid from Lactobacillus gasseri LA39 That Carries Genes for Production of and Immunity to the Circular Bacteriocin Gassericin A

Gassericin A is a circular bacteriocin produced by Lactobacillus gasseri strain LA39. We found a 33,333-bp plasmid, designated pLgLA39, in this strain. pLgLA39 contained 44 open reading frames, including seven genes related to gassericin A production/immunity (gaa), as well as genes for replication, plasmid maintenance, and conjugative transfer. pLgLA39 was transferred from LA39 to the type strain of L. gasseri (JCM 1131) by filter mating. The transconjugant exhibited >30-fold-higher more resistance to gassericin A and produced antibacterial activity. Lactobacillus reuteri LA6, the producer of reutericin 6, was proved to harbor a plasmid indistinguishable from pLgLA39 and carrying seven genes 100% identical to gaa. This suggests that pLgLA39 might have been transferred naturally between L. gasseri LA39 and L. reuteri LA6. The seven gaa genes of pLgLA39 were cloned into a plasmid vector to construct pGAA. JCM 1131^T transformed with pGAA expressed antibacterial activity and resistance to gassericin A. pGAA was segregationally more stable than a pGAA derivative plasmid from which gaaA was deleted and even was more stable than the vector. This suggests the occurrence of postsegregational host killing by the gaa genes. pLgLA39 carried a pemIK homolog, and segregational stabilization of a plasmid by the pLgLA39-type pemIK genes was also confirmed. Thus, pLgLA39 was proved to carry the genes for at least two plasmid maintenance mechanisms, i.e., gaa and pemIK. Plasmids containing a repA gene similar to pLgLA39 repA were distributed in several L. gasseri strains.Lactobacillus species are normal inhabitants of the human gastrointestinal tract, and Lactobacillus gasseri is one of the most commonly detected of these species (37, 47). Health-promoting effects of this species, such as immunomodulation (35), suppression of Helicobacter pylori-induced interleukin-8 production (44), and improvement of intestinal conditions (34), have been reported, and some L. gasseri strains are used in commercial probiotic products.Bacteriocins are antimicrobial peptides, proteins, or protein complexes produced by bacteria and active mainly against related bacterial species (38). Several bacteriocins also inhibit the growth of food-borne pathogens, such as Listeria, Bacillus cereus, and Clostridium perfringens. Production of bacteriocin is thought to be a desired feature for probiotic strains, since bacteriocin is believed to provide an advantage for survival in the ecological niche and to prevent the growth of pathogens. Several L. gasseri strains are known to produce bacteriocins (18). The classification of bacteriocins remains controversial. We use the definition proposed by Maqueda et al. (30), where bacteriocins are classified into class I (lantibiotics), class II (nonlantibiotics), class III (large heat-labile bacteriocins), and class IV (circular bacteriocins linked at the N- and C-terminal ends). Among these, the class IV circular bacteriocins have attracted increasing attention, since they are the simplest prokaryotic representatives of the ubiquitous circular peptides with various physiological activities (6). Enterocin AS-48 from Enterococcus faecalis strain S-48 is the first and most vigorously characterized member of the class IV bacteriocins (30). L. gasseri strain LA39 (JCM 11657) produces a 58-amino-acid (aa) circular bacteriocin, gassericin A (18). Gassericin A is a representative of the non-AS-48-like circular bacteriocin group including butyrivibriocin AR10 from Butyrivibrio fibrisolvens AR10 (15) and carnocyclin A from Carnobacterium maltaromaticum UAL307 (32), as well as reutericin 6 from Lactobacillus reuteri LA6 (17) and acidocin B from Lactobacillus acidophilus M46 (26). The last two bacteriocins have nearly identical amino acid sequences to that of gassericin A. Though the number of reported circular bacteriocins has been increasing, their primary sequences and the genes responsible for production of and immunity to them are diversified (for a review, see reference 31). Recently, we isolated and sequenced seven genes (gaaBCADITE) from LA39 deduced to be responsible for production of and immunity to gassericin A (20). The gaa genes add new information to the complex world of the class IV bacteriocin genes.The structural gene of gassericin A, gaaA, was reported to be located on the chromosome of LA39 (19). However, the high amino acid sequence identity of gassericin A to reutericin 6 (100%) and to acidocin B (98%) suggests recent horizontal gene transfers of the relevant bacteriocin genes, possibly via mobile elements. In fact, the acidocin B genes were reported to be located on a plasmid, namely, pCV461 (26). Many Lactobacillus strains are known to harbor one or more plasmids of various sizes, and several Lactobacillus plasmids have been reported to contain genes for production of bacteriocins (48). To our knowledge, however, only three have been sequenced entirely: these are pLA103 from Lactobacillus acidophilus TK8912 (16), pRC18 from Lactobacillus curvatus (previously known as Lactobacillus casei) CRL705 (7), and pMP118 from Lactobacillus salivarius subsp. salivarius UCC118 (5). Thus, genetic information about bacteriocin-producing Lactobacillus plasmids is still limited. Furthermore, little has been known about plasmids of L. gasseri, even though the existence of plasmids in a few strains has been reported, including a 26.5-kb anonymous plasmid in strain ADH (27) and pK7 in strain K7 (28).Here we describe a 33.3-kb plasmid, designated pLgLA39, from L. gasseri LA39. The gaa genes are located on this plasmid. pLgLA39 carries a set of genes for conjugative transfer and was shown to be transmitted to another L. gasseri strain. L. reuteri LA6 also harbors a plasmid almost identical to pLgLA39. We demonstrated that production of gassericin A increased the apparent segregational stability of a plasmid carrying the gaa genes. A pemIK homolog in pLgLA39 was also functional as a plasmid-stabilizing mechanism. This is the first report describing the entire nucleotide sequence and detailed genetic analysis of an L. gasseri plasmid, which contains functional genes for circular bacteriocin production, conjugation, and plasmid maintenance.     

DNA Sequencing and Homologous Expression of a Small Peptide Conferring Immunity to Gassericin A,a Circular Bacteriocin Produced by Lactobacillus gasseri LA39

Gassericin A, produced by Lactobacillus gasseri LA39, is a hydrophobic circular bacteriocin. The DNA region surrounding the gassericin A structural gene, gaaA, was sequenced, and seven open reading frames (ORFs) of 3.5 kbp (gaaBCADITE) were found with possible functions in gassericin A production, secretion, and immunity. The deduced products of the five consecutive ORFs gaaADITE have homology to those of genes involved in butyrivibriocin AR10 production, although the genetic arrangements are different in the two circular bacteriocin genes. GaaI is a small, positively charged hydrophobic peptide of 53 amino acids containing a putative transmembrane segment. Heterologous expression and homologous expression of GaaI in Lactococcus lactis subsp. cremoris MG1363 and L. gasseri JCM1131^T, respectively, were studied. GaaI-expressing strains exhibited at least sevenfold-higher resistance to gassericin A than corresponding control strains, indicating that gaaI encodes an immunity peptide for gassericin A. Comparison of GaaI to peptides with similar characteristics found in the circular bacteriocin gene loci is discussed.Bacteriocins are antimicrobial peptides that act primarily against related bacterial species. The classification of bacteriocins remains controversial. Here, we use the classification of Maqueda et al. (30): class I (lantibiotics); class II (nonlantibiotics) with subclasses IIa (antilisteral pediocin-like bacteriocins), IIb (two-peptide bacteriocins), and IIc (leaderless bacteriocins); class III (large heat-labile bacteriocins); and class IV (circular bacteriocins linked at the N- and C-terminal amino acids).Nine class IV circular bacteriocins have been reported to date. They can be further divided into two major groups by using their primary structures, biochemical characteristics, and genetic arrangements. One group is the family of enterocin AS-48 (32), the first circular bacteriocin described (in 1994), which includes circularin A (25) and uberolysin (40). The other group is the family of gassericin A (19, 21), the second bacteriocin found (in 1998), which includes acidocin B (28), reutericin 6 (with a primary structure 100% identical to that of gassericin A) (22, 23), butyrivibriocin AR10 (17), and carnocyclin A, from Carnobacterium maltaromaticum UAL307 (33). The lantibiotic-like subtilosin A produced by Bacillus subtilis subsp. subtilis strain 168 (24) is an orphan member of the class IV bacteriocins. The gassericin A family of bacteriocins have been isolated from various bacterial species in several countries, suggesting the bacteriocin genes may be associated with transferable genetic elements.The bacteriocins of lactic acid bacteria (LAB) and bacteriocin-producing LAB strains isolated from foods are promising food preservative candidates, and strains of human origin are expected to be probiotics that could help to prevent the growth of harmful bacteria in food and the human intestine. Lactobacillus gasseri belongs to the Lactobacillus acidophilus group of LAB, which are natural inhabitants of the human intestinal tract (35), and many L. gasseri strains have been shown to produce bacteriocins (16, 20). Gassericin A was produced by L. gasseri LA39 isolated from the feces of a human infant; it has bactericidal activity against the food-borne pathogens Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus (16). Recently, using proteose peptone, some strains of L. gasseri containing LA39 were successfully cultured in reconstituted skim milk and cheese whey, where L. gasseri LA39 produced gassericin A; these low-cost, safe media could be used to improve the safety of biopreservation (1). Gassericin A has been purified and characterized, and its structural gene (gaaA) has been cloned and sequenced (21, 22). Determination of the complete chemical structure of gassericin A showed that the bacteriocin belongs to class IV and consists of 58 amino acid residues linked at the N and C termini (19). Little is known about the mechanisms of secretion and circularization of gassericin A and immunity to the circular bacteriocin.Here, we sequenced six genes surrounding gaaA thought to be related to production of and immunity to gassericin A and examined the homologous and heterologous expression of a small hydrophobic peptide, GaaI; we found that gaaI is an immunity gene providing protection against gassericin A.     

Characterization of the amylovorin locus of Lactobacillus amylovorus DCE 471, producer of a bacteriocin active against Pseudomonas aeruginosa, in combination with colistin and pyocins

Lactobacillus amylovorus DCE 471 produces amylovorin L, a bacteriocin with an antibacterial activity against some strains of the Lactobacillus lineage. Based on the sequence of one active peptide, a gene encoding active amylovorin L was cloned and sequenced. Genome walking allowed us to sequence a larger fragment of 7577 bp of genomic DNA, with 12 predicted ORFs. The previously characterized amylovorin L peptide-encoding gene is preceded by another gene encoding a small polypeptide with a typical bacteriocin-processing double-glycine site, suggesting that amylovorin L is a two-component class IIb bacteriocin (amylovorin Lalpha/beta). Lalpha and Lbeta show the highest similarity to gassericin T from Lactobacillus gasseri SBT2055 and BlpN from Streptococcus pneumoniae R6, respectively, and to LafA and LafX, which form the lactacin F bacteriocin of Lactobacillus johnsonii NCC 533. As for other lactic acid bacteria bacteriocins, amylovorin L showed no activity against the Gram-negative opportunistic pathogen Pseudomonas aeruginosa on its own, but showed synergistic inhibitory activity when used in combination with the peptide antibiotic colistin, and, remarkably, with the P. aeruginosa soluble bacteriocins, pyocins S1 and S2.     

Wide-Inhibitory Spectra Bacteriocins Produced by Lactobacillus gasseri K7

The aim of our study was to determine the genetic characterization and classification of Lb. gasseri K7 bacteriocins, comparison with bacteriocins of the Lb. gasseri LF221 strain and other related strains. Bacteriocin-encoding genes were amplified by PCR, subjected to DNA sequencing, and BLAST sequence analysis was performed to search the database for homologous peptides. Lb. gasseri K7 produces two two-peptide bacteriocins, named gassericin K7 A and gassericin K7 B. Their nucleotide sequences were deposited at GenBank, under accession numbers EF392861 for the gassericin K7 A and AY307382 for the gassericin K7 B. Analysis of gene clusters of bacteriocins in Lb. gasseri K7 strain revealed a 100 percent sequence identity with bacteriocins in LF221 strain. An active peptide of gassericin K7 B is homologous to the complementary peptide of gassericin T, and a complementary peptide of gassericin K7 B is homologous to the active peptide of gassericin T. Another surprising finding was that the sakacin T-beta peptide is partly homologous to the active peptide of gassericin K7 A, while the other sakacin T peptide (alfa) is partly homologous to the complementary peptide of gassericin K7 B. Gassericins of Lb. gasseri K7 strain were both classified as two-peptide bacteriocins. Human probiotic strains Lb. gasseri K7 and LF221 are different isolates but with identical bacteriocin genes. They produce wide-inhibitory spectra bacteriocins that are new members of two-peptide bacteriocins with some homologies to other bacteriocins in this group. Described bacteriocins offer a great potential in applications in food industry, pharmacy and biomedicine.     

Bacteriocins of Lactobacillus gasseri K7 – Monitoring of gassericin K7 A and B genes’ expression and isolation of an active component

The genome of Lactobacillus gasseri K7, isolated from baby's faeces, contains gene regions encoding two-component bacteriocins named gassericin K7 A (GenBank EF392861) and gassericin K7 B (GenBank AY307382). The strain has been known to exhibit bacteriocin activity in vitro, however, no data exist on the expression of particular genes of bacteriocins’ operons or on the activity of individual components of this bacteriocin complex, which has not been isolated so far. The objectives of this study were to examine bacteriocin genes’ expression during the growth of L. gasseri K7 and to isolate individual components in order to reveal the contribution of individual peptides to the overall bacteriocin activity. All eight target genes were expressed during exponential phase of growth in MRS broth. Mass spectrometry analysis revealed that the amino acid sequence of isolated peptide matched the deduced amino acid sequence of putative active peptide of gassericin K7 B (Gas K7 B_AcP) and GatX, a complementary peptide of gassericin T, previously supposed to have no antimicrobial activity. The isolated peptide showed a broad spectrum of antimicrobial activity. Furthermore, the isolation protocol developed in this study will enable to obtain a considerable amount of purified bacteriocins needed for further investigation of their functionality.     

Reutericin 6, a new bacteriocin produced by Lactobacillus reuteri LA 6

Lactobacillus reuteri LA 6, isolated from infant faeces, produced an antimicrobial agent active against Lactobacillus acidophilus JCM 2125, Lactobacillus delbrueckii subsp. bulgaricus JCM 1002 and Lactobacillus delbrueckii subsp. lactis JCM 1148 and JCM 1248. The agent was sensitive to proteolytic enzymes and retained activity after heating at 100°C for 20 min. This agent was a bacteriocin and has been designated as reutericin 6. Reutericin 6 exceeds 200 kDa as determined by ultrafiltration studies. Activity against sensitive cells was both bacteriocidal and bacteriolytic.     

Nutritional Significance of Dietary Cystine for Maintaining the Hepatic Albumin mRNA Level in Rats Fed on a Soybean Diet

A new bacteriocin, gassericin A, was purified from the culture fluid of Lactobacillus gasseri LA39 mainly by reverse-phase (RP) chromatography. The purification of gassericin A from a modified MRS broth, in which Tween 80 had been replaced by oleic acid, resulted in a 4500-fold increase in specific activity with a 6% recovery. Gassericin A was eluted as a single peak on the chromatogram from RP-HPLC and migrated by SDS-PAGE as a single band with a molecular weight of ca. 3.8 kDa. Gassericin A, a highly hydrophobic bacteriocin, was slightly soluble in water, but its solubility was increased by adding alcohol and acetonitrile. An amino acid analysis revealed that it was composed of 45.7% hydrophobic amino acids in the total residues of 35 amino acids. Gassericin A produced in the MRS broth associated strongly with Tween 80, although several further trials of dissociation were unsuccessful.     

Inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus gasseri isolated from the vaginal tract of cattle

Infectious diseases are the major reproductive complication during postpartum. The bacteria that colonize the postpartum uterus of cattle are specific as well as opportunistic pathogens that include Staphylococcus aureus. The antibiotics and antiseptic agents used in the treatment of postpartum acute infections induce residues in foods, spread of bacterial resistance, increase in financial costs and failures in defense mechanisms of the host. Preventive treatment with probiotic products could decrease the use of antibiotics in dairy farming systems. Lactobacilli are present in the vaginal microflora of healthy cows. They can prevent pathogen colonization by mechanisms such as the production of antagonistic substances as lactic acid, H2O2, or bacteriocins. The aim of the present study was the selection of H2O2 generating lactobacilli from a group of 72 strains isolated from the vagina of cattle. Both Lactobacillus gasseri CRL1421 and Lactobacillus gasseri CRL1412, which share some probiotic properties, produce H2O2, detected by the plate colorimetric method. They were chosen to study the kinetics of H2O2 production under different culture conditions. Both microorganisms produced greater amounts of H2O2 in aerated than in static cultures. As L. gasseri CRL1421 had a greater capacity to generate H2O2, associative cultures with this strain and S. aureus were conducted. A significant decrease in the growth of the pathogen was detected after culture for 6h, this effect being greater under aerated conditions. The addition of catalase to mixed cultures partially abolished the inhibition, an effect that could be attributed to the combined action of H2O2 and other antagonistic metabolites. The simultaneous addition of catalase and NaOH to these cultures restored S. aureus growth. This observation suggests that the inhibition was produced by the combination of H2O2 and lactic acid, both released by the lactobacilli. Electron microphotographs showed the damage caused by the lactobacilli supernatant on the pathogenic cells. The treatment of S. aureus with lactic acid and hydrogen peroxide evidenced that each metabolite produced a different type of morphological damage. The number of viable cells obtained agrees with the electron microscopy observations. The results support the idea that L. gasseri CRL1421 could be successfully included in a probiotic product to prevent S. aureus infection in cows.     

Solution Structure of Acidocin B,a Circular Bacteriocin Produced by Lactobacillus acidophilus M46

Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ∼7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.     

Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7

A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.     

Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.     

The Three-dimensional Structure of Carnocyclin A Reveals That Many Circular Bacteriocins Share a Common Structural Motif

Carnocyclin A (CclA) is a potent antimicrobial peptide from Carnobacterium maltaromaticum UAL307 that displays a broad spectrum of activity against numerous Gram-positive organisms. An amide bond links the N and C termini of this bacteriocin, imparting stability and structural integrity to this 60-amino acid peptide. CclA interacts with lipid bilayers in a voltage-dependent manner and forms anion selective pores. Several other circular bacteriocins have been reported, yet only one (enterocin AS-48) has been structurally characterized. We have now determined the solution structure of CclA by NMR and further examined its anion binding and membrane channel properties. The results reveal that CclA preferentially binds halide anions and has a structure that is surprisingly similar to that of AS-48 despite low sequence identity, different oligomeric state, and disparate function. CclA folds into a compact globular bundle, comprised of four helices surrounding a hydrophobic core. NMR studies show two fluoride ion binding modes for CclA. Our findings suggest that although other circular bacteriocins are likely to have diverse mechanisms of action, many may have a common structural motif. This shared three-dimensional arrangement resembles the fold of mammalian saposins, peptides that either directly lyse membranes or serve as activators of lipid-degrading enzymes.Bacteriocins are a diverse group of ribosomally synthesized, antimicrobial peptides produced by bacteria. Those made by Gram-positive bacteria are usually cationic and typically have 30–70 residues (1–3). These peptides are substantially more active than conventional antibiotics against numerous pathogenic and drug resistant bacteria, including virulent strains of Staphylococci, Enterococci, Listeria, and Clostridia, but they display virtually no toxicity toward eukaryotic cells. The circular bacteriocins are a unique group, characterized by an amide bond linking the N and C termini of the peptide. They exhibit enhanced stability to pH and temperature variation and are resistant to numerous proteases, in contrast to many linear bacteriocins. This stability derives, in part, from the cyclic structure of the peptide (4). Interestingly, circular peptides are not unique to bacteria; they have also been discovered in plants and animals and exhibit a diverse range of bioactivities. Typically, the circular peptides from these higher organisms are shorter in length and contain at least one disulfide bond, further bracing the structure and enhancing stability (5, 6).We recently isolated carnocyclin A (CclA)² from Carnobacterium maltaromaticum UAL307 and employed tandem mass spectrometry amino acid sequencing and genetic analysis to confirm that it is a circular bacteriocin (7). This 60-residue peptide displays a broad antimicrobial spectrum and is particularly potent against the serious food pathogen Listeria monocytogenes. The producer strain has recently been approved in the United States as an additive for preservation of processed meat products.Eight other circular bacteriocins have been reported. These include enterocin AS-48 (8), butyrivbriocin AR10 (9), circularin A (10), gassericin A and reutericin 6 (11, 12), subtilosin A (13, 14), uberloysin (15), and most recently, lactocyclicin Q (16). Gassericin A and reutericin 6 have identical primary sequences but differ by the presence of one d-alanine, resulting in different spectra of activity and secondary structure profiles (11). Acidocin B is considered a putative circular protein, as it shows 98% sequence identity to gassericin A and reutericin 6, but its circular nature has not been confirmed (17). Of these bacteriocins, subtilosin A is atypical; it is significantly shorter (35 amino acids), anionic, and contains unique thioether bridges linking cysteine sulfurs to the α-carbon of other residues (13, 18, 19). As such, subtilosin A represents a unique class of bacteriocins (13) and will not be included in the present discussion of the other circular bacteriocins. These range from 58–70 amino acids in length, are cationic, and contain a large number of hydrophobic residues (4).To date, the structure of only one of these circular bacteriocins has been in reported. In 2000, González et al. (20) described the NMR solution structure of AS-48, revealing that it consists of five helices encompassing a compact hydrophobic core. The covalent bond linking the N and C termini of the peptide was found to reside within the fifth helix. In 2003, crystallographic studies supported the proposal that at physiological pH, AS-48 exists as a water soluble dimer, in which the hydrophobic faces of the individual monomers are in contact and polar interactions with the aqueous solvent are maximized (21). However, upon interaction with a membrane, this dimer undergoes a conformational change, exposing its hydrophobic faces and facilitating insertion into the membrane. The three-dimensional structure of AS-48 shows a significant charge separation across the molecule, as a cluster of lysines at one end of the molecule imparts a high degree of positive charge on the surface of the peptide. This charge localization is believed to be crucial for insertion of the peptide into the membrane through a mechanism known as molecular electroporation (20, 22, 23). Functional studies of AS-48 have shown that this peptide causes nonselective pore formation in lipid bilayers, thereby allowing for the free diffusion of ions and low molecular weight solutes across the membrane (24). A similar mode of action has been reported for gassericin A and reutericin 6 (11).We have now determined the three-dimensional solution structure of CclA by NMR. Our results reveal that CclA assumes a globular structure, consisting of four helices surrounding a compact, hydrophobic core. The global architecture of CclA and its surface features are remarkably similar to those of AS-48, suggesting a common structural motif for circular bacteriocins that closely resembles the saposin fold, a conserved structural moiety found within the saposin and saposin-like polypeptide families (25, 26). Functional studies show that CclA forms anion selective channels in lipid bilayers (27). We have further characterized these ion channels by examining the anion selectivity of the pores and the effect of pH on channel conductance. NMR studies demonstrate that CclA binds fluoride in two different ways. The results show that despite the shared structural motif of a sapsosin fold, circular bacteriocins can have unique functional properties.     

Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124

Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L. gasseri JCM 2124. The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced. Gassericin B2 did not show any sequence similarities to other known bacteriocins. The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus. However, synergistic activity upon complementation of B1 and B3 was not observed. Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3).     

Characterization of reutericyclin produced by Lactobacillus reuteri LTH2584

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C(8) chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. H?ltzel, M. G. G?nzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766-2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.     

Gassericin A: a circular bacteriocin produced by Lactic acid bacteria Lactobacillus gasseri

During the recent years extensive efforts have been made to find out bacteriocins from lactic acid bacteria (LAB) active against various food spoilage and pathogenic bacteria, and superior stabilities against heat treatments and pH variations. Bacteriocins isolated from LAB have been grouped into four classes. Circular bacteriocins which were earlier grouped among the four groups of bacteriocins, have recently been proposed to be classified into a different class, making it class V bacteriocins. Circular bacteriocins are special molecules, whose precursors must be post translationally modified to join the N to C termini with a head-to-tail peptide bond. Cyclization appears to make them less susceptible to proteolytic cleavage, high temperature and pH, and, therefore, provides enhanced stability as compared to linear bacteriocins. The advantages of circularization are also reflected by the fact that a significant number of macrocyclic natural products have found pharmaceutical applications. Circular bacteriocins were unknown two decades ago, and even to date, only a few circular bacteriocins from a diverse group of Gram positive organisms have been reported. The first example of a circular bacteriocin was enterocin AS-48, produced by Enterococcus faecalis AS-48. Gassereccin A, produced by Lactobacillus gasseri LA39, Reutericin 6 produced by Lactobacillus reuteri LA6 and Circularin A, produced by Clostridium beijerinickii ATCC 25,752, are further examples of this group of antimicrobial peptides. In the present scenario, Gassericin A can be an important tool in the food preservation owing to its properties of high pH and temperature tolerance and the fact that it is produced by LAB L. gasseri, whose many strains are proven probiotic.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.