Characterization of the mesB Gene and Expression of Bacteriocins by Leuconostoc mesenteroides Y105

Leuconostoc mesenteroides Y105, previously described for production of mesentericin Y105, an anti-Listeria bacteriocin, was shown to secrete a second bacteriocin. The latter was purified, and its molecular mass of 3446 Da, obtained by mass spectrometric analysis, indicates that this bacteriocin should be identical to mesenterocin 52B [Revol-Junelles et al., Lett Appl Microbiol 23:120, 1996]. This second bacteriocin produced by L. mesenteroides Y105 was named mesentericin B105. Its structural gene, mesB, was then localized by a reverse genetic approach, cloned, and sequenced. MesB was found on the pHY30 plasmid, next to mesY gene clusters. Curing experiments led to isolation of two L. mesenteroides Y105 derivatives, named L. mesenteroides Y29 and Y30. The latter had lost pHY30 plasmid, encoding bacteriocin determinants, therefore explaining its phenotype (MesY-, MesB-). On the contrary, Y29 derivative still harbors the pHY30 but did not produce any bacteriocin. Thus, its phenotype could likely result from a point mutation within a gene, probably encoding a protein involved in production of both mesentericin Y105 and mesentericin B105. Received: 9 May 1999 / Accepted: 8 June 1999     

Differences in mesentericin secretion systems from two Leuconostoc strains

Leuconostoc mesenteroides Y105 and L. mesenteroides FR52 produce both mesentericin Y105 and B105, in equal amounts. The mesentericin operons of L. mesenteroides FR52 and Y105 which are involved in mesentericin Y105 and B105 production, were both sequenced and compared. Differences were limited to the two genes, mesD and mesE, which encode the dedicated transport system of mesentericin Y105. Analysis of mesentericin non-producing mutants and complementation experiments demonstrated that the major role of the membrane fusion protein, MesE, was in bacteriocin secretion for both strains. Moreover, the secretion machinery MesDE was demonstrated to be capable of transportation and maturation of the two pre-bacteriocins, mesentericin Y105 and B105. We also demonstrate that although MesDEs from strains Y105 and FR52 have significant sequence differences, both transporters were capable of assuring secretion of either bacteriocin.     

Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides.

A Leuconostoc mesenteroides ssp. mesenteroides was isolated from goat's milk on the basis of its ability to inhibit the growth of Listeria monocytogenes. The antimicrobial effect was due to the presence in the culture medium of a compound, named mesentericin Y105, excreted by the Leuconostoc mesenteroides Y105. The compound displayed known features of bacteriocins from lactic acid bacteria. It appeared as a proteinaceous molecule exhibiting a narrow inhibitory spectrum limited to genus Listeria. The apparent relative molecular mass, as indicated by activity detection after SDS-PAGE, was 2.5-3.0 kDa. The bacteriocin was purified to homogeneity by a simple three-step procedure: a crude supernatant obtained from an early-stationary-phase culture in a defined medium was subjected to affinity chromatography on a blue agarose column, followed by ultrafiltration through a 5 kDa cut-off membrane, and finally by reverse-phase HPLC on a C4 column. Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence. However, unlike leucocin A-UAL 187, mesentericin Y105 displayed a bactericidal mode of action.     

Mutational analysis of mesentericin y105, an anti-Listeria bacteriocin, for determination of impact on bactericidal activity, in vitro secondary structure, and membrane interaction

Mesentericin Y105 is a 37-residue bacteriocin produced by Leuconostoc mesenteroides Y105 that displays antagonistic activity against gram-positive bacteria such as Enterococcus faecalis and Listeria monocytogenes. It is closely related to leucocin A, an antimicrobial peptide containing beta-sheet and alpha-helical structures. To analyze structure-function relationships and the mode of action of this bacteriocin, we generated a collection of mesentericin derivatives. Mutations were obtained mostly by PCR random mutagenesis, and the peptides were produced by an original system of heterologous expression recently described. Ten derivatives were obtained displaying modifications at eight different positions in the mesentericin Y105 sequence. Purified peptides were incorporated into lysophosphatidylcholine micelles and analyzed by circular dichroism. The alpha-helical contents of these peptides were compared and related to their respective bactericidal activities. Moreover, studies of the intrinsic fluorescence of tryptophan residues naturally occurring at positions 18 and 37 revealed information about insertion of the peptides in micelles. A model for the mode of action of mesentericin Y105 and related bacteriocins is proposed.     

Heterologous expression of bacteriocins using the mesentericin Y105 dedicated transport system by Leuconostoc mesenteroides

Mesentericin Y105 (MesY105) is a class IIa anti-Listeria bacteriocin, produced by Leuconostoc (Ln.) mesenteroides Y105 and with potential food grade application. This bacterium produces a second bacteriocin, mesentericin B105 (MesB105), that does not belong to the same class. To study secretion of bacteriocins by the use of the MesY105 dedicated transport system (DTS), plasmids were constructed for heterologous expression by Ln. mesenteroides. pFBYC04 (Microbiology 144 (1998) 2845) harbours two divergent operons required for MesY105 secretion, i.e. the mesYI operon, encoding pre-MesY105 and immunity, respectively, and the mesCDE operon for secretion. A pFBYC04 derivative, pDMJF01 was constructed by divergent PCR to remove the mesY gene. Ln. mesenteroides DSM20484(pDMJF01) was unable to produce MesY105. The mesYI operon and mesB, mesH and mesF genes, encoding pre-MesB105, MesB105 immunity and a putative protein with unknown function, respectively, were cloned independently into a compatible pDMJF01 plasmid to produce, respectively, pDMJF:YI and pDMJF:BHF. DSM20484 transformed independently with these plasmids was unable to secrete any bacteriocin. MesY105 and MesB105 secretion was observed for DSM20484(pDMJF01) harbouring both pDMJF:YI and pDMJF:BHF. This indicates that the MesY105 DTS permits the transport of MesB105. MesY105 secretion machinery was used to secrete pediocin PA-1 (PedPA-1) by DSM20484 by an in-frame gene fusion strategy where the gene portions corresponding to the MesY105 leader peptide and the mature PedPA-1 were ligated. Thus, MesY105 secretion machinery appears to be a useful tool for secretion of class II bacteriocins by Leuconostoc.     

Mutational Analysis of Mesentericin Y105, an Anti-Listeria Bacteriocin, for Determination of Impact on Bactericidal Activity, In Vitro Secondary Structure, and Membrane Interaction

Mesentericin Y105 is a 37-residue bacteriocin produced by Leuconostoc mesenteroides Y105 that displays antagonistic activity against gram-positive bacteria such as Enterococcus faecalis and Listeria monocytogenes. It is closely related to leucocin A, an antimicrobial peptide containing β-sheet and α-helical structures. To analyze structure-function relationships and the mode of action of this bacteriocin, we generated a collection of mesentericin derivatives. Mutations were obtained mostly by PCR random mutagenesis, and the peptides were produced by an original system of heterologous expression recently described (D. Morisset and J. Frère, Biochimie 84:569-576, 2002). Ten derivatives were obtained displaying modifications at eight different positions in the mesentericin Y105 sequence. Purified peptides were incorporated into lysophosphatidylcholine micelles and analyzed by circular dichroism. The α-helical contents of these peptides were compared and related to their respective bactericidal activities. Moreover, studies of the intrinsic fluorescence of tryptophan residues naturally occurring at positions 18 and 37 revealed information about insertion of the peptides in micelles. A model for the mode of action of mesentericin Y105 and related bacteriocins is proposed.     

Identification of a replicon from pTXL1, a small cryptic plasmid from Leuconostoc mesenteroides subsp. mesenteroides Y110, and development of a food-grade vector

A 2,665-bp cryptic plasmid, pTXL1, isolated from Leuconostoc mesenteroides subsp. mesenteroides Y110 was identified. This plasmid harbors a replicon localized on a 1,300-bp fragment. Two observations suggested that pTXL1 does not belong to rolling-circle replication (RCR)-type plasmids and most likely replicates via a theta mechanism. These hypotheses are supported by the observation that no detectable single-stranded intermediate was found for the replicon and that, unlike in RCR-type plasmids, the pTXL1 replicon sequence lacks an open reading frame encoding a replicase. The small-sized pTXL1 plasmid is stable and, according to its origin, can be considered in the "generally recognized as safe" category. Its ability to replicate in several lactic acid bacteria was exploited to develop a vector producing mesentericin Y105, a class II anti-Listeria bacteriocin. With this new vector, a recombinant industrial Leuconostoc cremoris strain able to produce mesentericin Y105 was constructed.     

The rpoN Gene of Enterococcus faecalis Directs Sensitivity to Subclass IIa Bacteriocins

The sigma54 factor has been previously described to be involved in Listeria monocytogenes sensitivity to mesentericin Y105, a subclass IIa bacteriocin. Here, we identified the rpoN gene, encoding sigma54, of Enterococcus faecalis JH2-2 and showed that its interruption leads to E. faecalis resistance to different subclass IIa bacteriocins. Moreover, this rpoN mutant remained sensitive to nisin, a class I bacteriocin, suggesting that sigma54 is especially involved in sensitivity to subclass IIa bacteriocins. Received: 5 May 2000 / Accepted 28 June 2000     

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0–12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain’s genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.

     

The putative immunity protein of the Gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment

Abstract Immunity proteins are thought to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin. The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105³⁷ bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein. The antibodies recognized the immunity proteins of various strains of Leuconostoc , including Leuconostoc mesenteroides and Leuconostoc gelidum . This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria. This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.     

Properties and Characteristics of a Bacteriocin from Serratia marcescens

A strain of Serratia marcescens was found to produce a bacteriocin that inhibits the growth of certain Escherichia coli strains. This inhibition was bacteriocidal rather than bacteriostatic and was not caused by a bacteriophage. Whereas the bacteriocin was inactive on the 7 Serratia strains tested, it killed 11 of the 20 E. coli strains tested for sensitivity. A relationship of the bacteriocin to a possible colicin cannot as yet be excluded, although E. coli mutants resistant to 1 or 2 of 15 different colicins remained sensitive to the bacteriocin. The bacteriocidal effect by the bacteriocin could be interrupted in a substantial fraction of the treated cell population by the addition of trypsin. The synthesis of the bacteriocin was inducible by ultraviolet light or by starvation for thymidine. Both procedures led to a similar increase in maximum bacteriocin titer relative to noninduced cultures.     

Study of structure and orientation of mesentericin Y105, a bacteriocin from Gram-positive Leuconostoc mesenteroides, and its Trp-substituted analogues in phospholipid environments

Mesentericin Y105 (Mes-Y105) is a bacteriocin secreted by Leuconostoc mesenteroides which is particularly active on Listeria. It is constituted by 37 residues and reticulated by one disulfide bridge. It has two W residues, W18 and W37, which can be studied by fluorescence. Two single substituted W/F analogues were synthesized (Mes-Y105/W18 and Mes-Y105/W37) to differentiate the local environment around each W and to study their changes in the presence of lipid vesicles. Fluorescence experiments show that, for the pure Trp-analogues, W18 and W37 are fully exposed to solvent whatever pH and buffer conditions. In the presence of lipid vesicles, both became buried. Lipid affinities were estimated: they are weak for zwitterionic phospholipids but an order of magnitude higher for negatively charged phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids. On negatively charged PG lipids, Mes-Y105 and Mes-Y105/W37 display comparable lipid affinities. A decrease in lipid affinity is observed for Mes-Y105/W18 compared to Mes-Y105, which means that W37 would seem to be required for increased lipid selectivity. In the lipid-bound state W18 is strongly dehydrated, probably embedded into the acyl chains, while W37 stands more at the interface. Mes-Y105 was also studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS), alone and in various phospholipid environments, to obtain structural information and to assess lipid perturbations. At nanomolar concentrations close to those required for anti-Listeria activity, Mes-Y105 forms films at the air/water interface and inserts into negatively charged lipid monolayers. In situ infrared data show that Mes-Y105 binding only affects the polar head group vibrations while the lipid order of the acyl chains remains unaffected. The PMIRRAS show that Mes-Y105 folds into an N-terminal antiparallel beta-sheet followed by an alpha-helix, both structures being tilted (40 degrees) compared to the normal at the interface, which is in agreement with the thickness estimated by Brewster angle microscopy (BAM). All these data support the proposal of a new model for Mes-Y105 at the membrane interface.     

The biological activity and physicochemical properties of a new bacteriocin from a strain of Pseudomonas cepacia 5779

Pseudomonas cepacia 5779 bacteriocin (cepaciacin) whose producer was revealed due to application of the special screening system has been studied for its certain biological and physicochemical properties. Possessing a narrow range of action, it inhibits only the P. cepacia strains. Its biosynthesis occurs more intensely on the rich nutrient media, the highest quantities of cepaciacine being revealed at the terminal stage of the produced log growth. UV irradiation or mitomycin C introduction into the medium stimulated biosynthesis of this bacteriocin. Cepaciacin P. cepacia 5779 is a complex consisting of several protein subunits and carbon part. The protein-carbohydrate ratio is 3:1. The molecular weight of the complex is 1.8 x 10(6) Da. Lipopolysaccharides isolated from the indicator strain being added, cepaciacine loses its activity. This bacteriocin is stable in the narrow range of pH, thermolabile, decomposes under the effect of proteases and is, evidently, a representative of a new type of the bacteriocin-like substances.     

Requirement of autolytic activity for bacteriocin-induced lysis

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co(2+) and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmADelta1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmADelta1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.     

On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.     

Polyglutamine expansion inhibits respiration by increasing reactive oxygen species in isolated mitochondria

Huntington’s disease results from expansion of the polyglutamine (PolyQ) domain in the huntingtin protein. Although the cellular mechanism by which pathologic-length PolyQ protein causes neurodegeneration is unclear, mitochondria appear central in pathogenesis. We demonstrate in isolated mitochondria that pathologic-length PolyQ protein directly inhibits ADP-dependent (state 3) mitochondrial respiration. Inhibition of mitochondrial respiration by PolyQ protein is not due to reduction in the activities of electron transport chain complexes, mitochondrial ATP synthase, or the adenine nucleotide translocase. We show that pathologic-length PolyQ protein increases the production of reactive oxygen species in isolated mitochondria. Impairment of state 3 mitochondrial respiration by PolyQ protein is reversed by addition of the antioxidants N-acetyl-l-cysteine or cytochrome c. We propose a model in which pathologic-length PolyQ protein directly inhibits mitochondrial function by inducing oxidative stress.     

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.     

Stress-related Pseudomonas genes involved in production of bacteriocin LlpA

Pseudomonas sp. BW11M1 produces a novel type of bacteriocin that inhibits the growth of Pseudomonas putida GR12-2R3 and some phytopathogenic fluorescent Pseudomonas. A collection of mutants was screened for altered bacteriocin production phenotypes. Strongly reduced bacteriocin production was found to be caused by inactivation of the recA gene or the spoT gene. Conversely, in a recJ mutant, the bacteriocin was constitutively overproduced. The same phenotype was observed for a mutant hit in a gene of unknown function. The predicted gene product belongs to a distinct subgroup of prokaryotic helicase-like proteins within the SWI/SNF family of regulatory proteins. One mutant that also exhibited a bacteriocin overproducer phenotype was deficient in the production of the peptidoglycan-associated lipoprotein OprL. This study shows that various environmental stress response pathways are involved in controlling expression of the Pseudomonas sp. BW11M1 bacteriocin.     

Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesizes two distinct bacteriocins

A.M. REVOL-JUNELLES, R. MATHIS, F. KRIER, Y. FLEURY, A. DELFOUR AND G. LEFEBVRE. 1996. Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, was purified from producing cells by the adsorption-desorption method, combined with reverse-phase high-performance liquid chromatography. The elution profile revealed the presence o two inhibitory peaks of activity, each displaying different inhibitory spectra. Mesenterocin 52A possessed a broad inhibitory spectrum, including anti- Listeria activity, while Mesenterocin 52B was only active against Leuconostoc spp. The amino acid sequence and M_r of Mesenterocin 52A appeared identical to the previously described Mesentericing Y105. In contrast, Mesenerocin 52B possessed a M_r of 3446 Da, corresponding to 32 amino acids and a sequence that shared no homology with known bacteriocins:     

Inhibition of succinate-linked respiration and complex II activity by hydrogen peroxide

Hydrogen peroxide produced from electron transport chain derived superoxide is a relatively mild oxidant, and as such, the majority of mitochondrial enzyme activities are impervious to physiological concentrations. Previous studies, however, have suggested that complex II (succinate dehydrogenase) is sensitive to H₂O₂-mediated inhibition. Nevertheless, the effects of H₂O₂ on succinate-linked respiration and complex II activity have not been examined in intact mitochondria. Results presented indicate that H₂O₂ inhibits succinate-linked state 3 mitochondrial respiration in a concentration dependent manner. H₂O₂ has no effect on complex II activity during state 2 respiration, but inhibits activity during state 3. It was found that conditions which prevent oxaloacetate accumulation during state 3 respiration, such as inclusion of rotenone, glutamate, or ATP, blunted the effect of H₂O₂ on succinate-linked respiration and complex II activity. It is concluded that H₂O₂ inhibits succinate-linked respiration indirectly by sustaining and enhancing oxaloacetate-mediated inactivation of complex II.