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Cloning and expression of a plasmid-linked pediocin determinant trait of Pediococcus acidilactici F.

Plasmid DNA from Pediococcus acidilactici F was prepared by lysozyme-mutanolysin method and purified by cesium chloride-ethidium bromide (CsCl-EtBr) density gradient ultracentrifugation. Agarose gel electrophoresis of plasmid DNA and plasmid-curing experiments suggested that bacteriocin activity was harboured on a small plasmid of approximately 9.1 kb (kilobasepair) in Pediococcus acidilactici F. Plasmid encoding bacteriocin production in P. acidilactici F was examined for restriction enzyme cleavage patterns and its map has been constructed. An Escherichia coli strain transformed with the recombinant plasmid, pQE322, produced and, most probably, secreted pediocin F.

Bacteriocins↗

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria.

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin (dso) and a region with palindromic sequences that could function as single strand origin (sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55-58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.

Base Sequence↗

Influence of Pediococcus-based probiotic on coccidiosis in broiler chickens.

Coccidiosis is the major parasitic disease of poultry and is caused by the apicomplexan parasites Eimeria. Drugs and live vaccines are the 2 main control measures of the disease; however, due to increasing concerns with prophylactic drug use and the high cost of vaccines, alternative control methods are needed. Recent evidence that various dietary and live microbial supplements can influence host immunity against enteric diseases prompted us to investigate the role of a Pediococcus-based probiotic on coccidiosis in broiler chickens. In the present study, we examined BW gains, oocyst shedding, and antibody responses of broilers fed the commercial probiotic MitoGrow. Day-old chicks were fed either a regular broiler diet or 1 of 2 probiotic diets supplemented with 0.1% (MG 0.1) or 0.2% MitoGrow. Chicks were orally challenged with 5,000 or 10,000 sporulated oocysts of Eimeria acervulina or with 5,000 Eimeria tenella oocysts on d 10 or 12 of age, respectively. In E. acervulina-infected birds, the MG 0.1 group improved (P < 0.05) weight gain as compared with the other 2 groups and reduced (P < 0.05) oocyst shedding in birds infected with 5,000 E. acervulina oocysts. In E. tenella-infected birds, Eimeria-specific antibody levels were higher (P < 0.05) in the Mito-Grow-fed groups, especially in the MG 0.1 birds, compared with the regular diet group, although their oocyst shedding and weight gains were not clearly improved. These results demonstrate that this Pediococcus acidilactici-based probiotic effectively enhances the resistance of birds and partially protects against the negative growth effects associated with coccidiosis, particularly when supplemented at 0.1% MitoGrow of the diet.

Animal Feed↗

Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61.

Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61, was purified and partially characterized. The purification was achieved by dialysis against PEG 20,000, butanol extraction and electroendosmotic preparative electrophoresis. The protocol led to a 7843-fold increase in the specific activity, with 3.9% activity recovered. SDS-PAGE of pediocin A resulted in a single 80 kDa protein band. The antimicrobial compound was sensitive to proteolytic enzymes and heat (10 min at 100 degrees C). It exhibited inhibition against species of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Staphylococcus, Enterococcus, Listeria and Clostridium.

Bacteriocins↗

Detection and quantification of Brettanomyces bruxellensis and 'ropy' Pediococcus damnosus strains in wine by real-time polymerase chain reaction.

AIMS: Brettanomyces bruxellensis is a well-known wine spoilage yeast that causes undesirable off-flavours. Likewise, glucan-producing strains of ropy Pediococcus damnosus are considered as spoilage micro-organisms because the synthesis of glucan leads to an unacceptable viscosity of wine. METHODS AND RESULTS: We developed a real-time PCR method to detect and quantify these two spoilage micro-organisms in wine. It is based on specific primer pairs for amplification of target DNA, and includes a melting-curve analysis of PCR products as a confirmatory test. CONCLUSIONS: The detection limit in wine was 10(4) CFU ml(-1) for B. bruxellensis and 40 CFU ml(-1) for ropy Pediococcus damnosus. The real-time PCR proved to be reliable for the early, sensitive detection and quantification of B. bruxellensis and ropy P. damnosus in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR-based method described in this study provides a new tool for monitoring spoilage micro-organisms in wine. Time-consuming culture and colony isolation steps are no longer needed, so winemakers can intervene before spoilage occurs.

Ascomycota↗

Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme.

The bacteriocin pediocin PA-1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA-1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N-terminal domains of PedD and a subgroup of bacteriocin ABC-transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.

ATP-Binding Cassette Transporters↗

pUCL287 plasmid from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 represents a new theta-type replicon family of lactic acid bacteria.

A cryptic plasmid, pUCL287, was isolated from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315. It had a theta-type mechanism of replication in its natural host. Its minimal replicon, Rep287, was isolated on a 1.6-kb EcoRI fragment. The Rep287 host range included the genera Pediococcus, Enterococcus, Lactobacillus and Leuconostoc but not genus Lactococcus. Plasmids hybridizing to pUCL287 are rare among lactic acid bacteria. As assessed by hybridization, Rep287 is dissimilar to pAM beta 1, pIP501 and pUCL22, representatives of the most common theta-type replicon groups in Gram-positive bacteria. Therefore, pUCL287 appears to represent a new theta-type replicon family from lactic acid bacteria.

Base Sequence↗

Isolation and characterization of Pediococcus halophilus from salted anchovies (Engraulis anchoita).

The presence of bacteria in salted anchovies during and at the end of the curing process was investigated. Attempts to isolate bacteria under aerobic or anaerobic conditions led to the isolation of only bacteria of the genus Pediococcus which were identified as Pediococcus halophilus. The isolates correspond to a rather heterogeneous group in which some of the members differ in some biochemical tests from the types described in the literature.

Animals↗

Detection of Pediococcus spp. in brewing yeast by a rapid immunoassay.

A membrane immunofluorescent-antibody test was developed to detect diacetyl-producing Pediococcus contaminants in brewery pitching yeast (yeast [Saccharomyces cerevisiae] slurry collected for reinoculation). Centrifugations at 11 and 5,100 x g separate yeast cells from bacteria and concentrate the bacteria, respectively. Pelleted bacteria resuspended and trapped on a black membrane filter are reacted with monoclonal antibodies specific for cell surface antigens and then with fluorescein-conjugated indicator antibodies. Whether pitching yeast is contaminated with pediococci at 0.001% is determined in less than 4 h. The sensitivity of the assay is 2 orders of magnitude below the Pediococcus detection limit of direct microscopy.

Fluorescent Antibody Technique↗

Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells.

Pediocin PA-1 is a bacteriocin which is produced by Pediococcus acidilactici PAC1.0. We demonstrate that pediocin PA-1 kills sensitive Pediococcus cells and acts on the cytoplasmic membrane. In contrast to its lack of impact on immune cells, pediocin PA-1 dissipates the transmembrane electrical potential and inhibits amino acid transport in sensitive cells. Pediocin interferes with the uptake of amino acids by cytoplasmic membrane vesicles derived from sensitive cells, while it is less effective with membranes derived from immune cells. In liposomes fused with membrane vesicles derived from both sensitive and immune cells, pediocin PA-1 elicits an efflux of small ions and, at higher concentrations, an efflux of molecules having molecular weights of up to 9,400. Our data suggest that pediocin PA-1 functions in a voltage-independent manner but requires a specific protein in the target membrane.

Amino Acids↗

Monoclonal antibody-based enzyme immunoassay for pediocins of Pediococcus acidilactici.

Monoclonal antibody (MAb) R2-AR against pediocin RS2 was developed. Mice were immunized for 12 weeks with pediocin RS2 conjugated to a polyacrylamide gel. Two hybridoma fusions yielded an MAb that in Western blots (immunoblots) reacted only with pediocins RS2 and AcH (3 kDa) from Pediococcus acidilactici RS2 and H, respectively, and did not react with any other bacteriocin, including sakacin A from Lactobacillus sake Lb 706, leuconocin LCM1 from Leuconostoc carnosum LM1, nisin from Lactococcus lactis ATCC 11454, and pediocin A from Pediococcus pentosaceus FBB61. Each of the bacteriocin bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was confirmed to be biologically active by a gel overlay test performed with sensitive indicator organisms. In dot immunoblot assays, the MAb could detect a minimum of 32,000 arbitrary units of pediocin RS2 or AcH per ml. In colony immunoblot assays, the MAb was used successfully to differentiate bac+ and bac- variants of P. acidilactici RS2 strains.

Acrylic Resins↗

Production of active chimeric pediocin AcH in Escherichia coli in the absence of processing and secretion genes from the Pediococcus pap operon.

Minimum requirements have been determined for synthesis and secretion of the Pediococcus antimicrobial peptide, pediocin AcH, in Escherichia coli. The functional mature domain of pediocin AcH (Lys+1 to Cys+44) is targeted into the E. coli sec machinery and secreted to the periplasm in active form when fused in frame to the COOH terminus of the secretory protein maltose-binding protein (MBP). The PapC-PapD specialized secretion machinery is not required for secretion of the MBP-pediocin AcH chimeric protein, indicating that in Pediococcus, PapC and PapD probably are required for recognition and processing of the leader peptide rather than for translocation of the mature pediocin AcH domain across the cytoplasmic membrane. The chimeric protein displays bactericidal activity, suggesting that the NH2 terminus of pediocin AcH does not span the phospholipid bilayer in the membrane-interactive form of the molecule. However, the conserved Lys(+1)-Tyr-Tyr-Gly-Asn-Gly-Val(+7)-sequence at the NH2 terminus is important because deletion of this sequence abolishes activity. The secreted chimeric protein is released into the culture medium when expressed in a periplasmic leaky E. coli host. The MBP fusion-periplasmic leaky expression system should be generally advantageous for production and screening of the activity of bioactive peptides.

ATP-Binding Cassette Transporters↗

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Adult↗

Genomic diversity within the genus Pediococcus as revealed by randomly amplified polymorphic DNA PCR and pulsed-field gel electrophoresis.

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.

Animals↗

Phenotypic and genotypic characterization of Pediococcus strains isolated from human clinical sources.

Seventy-two strains of pediococci isolated from human clinical sources were characterized by conventional physiological tests, chromogenic enzymatic tests, analysis of whole-cell protein profiles (WCPP) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analysis of chromosomal DNA restriction profiles by pulsed-field gel electrophoresis (PFGE). Conventional tests allowed identification of 67 isolates: 52 strains were identified as Pediococcus acidilactici, 15 strains were identified as Pediococcus pentosaceus, and 5 strains were not identified because of atypical reactions. Analysis of WCPP identified all isolates since each species had a unique WCPP. By the WCPP method, the atypical strains were identified as P. acidilactici (two strains) and P. pentosaceus (three strains). The chromogenic substrate test with o-nitrophenyl-beta-D-glucopyranoside differentiated all 54 strains of P. acidilactici (negative reactions) and 13 (72%) of 18 strains of P. pentosaceus (positive reactions). Isolates of both species were shown to be nonclonal as revealed by the genetic diversity when chromosomal DNA was analyzed by PFGE. Using WCPP as the definitive identification procedure, P. acidilactici (28 of 54 strains; 51.8%) was more likely than P. pentosaceus (4 of 18 strains; 22.3%) to be isolated from blood cultures.

Bacterial Typing Techniques↗

Bacteremic infection with Pediococcus: vancomycin-resistant opportunist.

Pediococci are recently recognized Gram-positive human pathogens, resistant to vancomycin and generally susceptible to penicillin. Infection in adults has been seen in patients with chronic underlying conditions as well as those with previous abdominal surgery. Two previous infants with congenital gastrointestinal malformations requiring surgical correction have been reported with sepsis attributable to Pediococcus sp. We report a third infant born with gastroschisis who developed Pediococcus bacteremia and meningitis 3 months after surgery, and speculate regarding the role of probiotics in the pathogenesis of this infection.

Bacteremia↗

Pediococcus parvulus gtf gene encoding the GTF glycosyltransferase and its application for specific PCR detection of beta-D-glucan-producing bacteria in foods and beverages.

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, beta-D-glucan-producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize beta-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the beta-D-glucan producers Lactobacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of beta-D-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 X 10(2) CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

Agglutination Tests↗

Purification and characterization of the HPR protein of Pediococcus halophilus.

HPr protein, the ptsH gene product, of the phosphoenolpyruvate:sugar phosphotransferase system of Pediococcus halophilus was purified to homogeneity using heat and acid treatments, DEAE-Sepharose CL-6B and hydroxyapatite column chromatographies. The purified protein complemented the HPr activity of the phosphotransferase system in Staphylococcus aureus ptsH mutant cell lysate. The molecular weight was estimated as 6,500 by SDS polyacrylamide gel electrophoresis. The amino acid sequence of the amino-terminal part (1-44) of the native purified protein was highly homologous to those of HPr proteins from gram-positive bacteria. Antiserum raised against the purified HPr protein specifically reacted with the Pediococcus halophilus HPr protein and did not cross-react with Staphylococcus aureus or Escherichia coli HPr proteins.

Amino Acid Sequence↗