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Antibacterial activity of plantaricin SIK-83, a bacteriocin produced by Lactobacillus plantarum.

Lactobacillus plantarum SIK-83 produces a bacteriocin, designated plantaricin SIK-83, which inhibits 66 of 68 lactic acid bacteria from the genera Lactobacillus, Leuconostoc, Pediococcus and Streptococcus. A 500-fold dilution of L. plantarum SIK-83 MRS culture supernatant with phosphate buffer was sufficient to kill 10(5) cells/ml of Pediococcus pentosaceus within 120 s. The killing of a sensitive population followed exponential kinetics. It was shown that the bacteriocin binds specifically to sensitive cells but not to nonsensitive lactic acid bacteria, the producer strain or Gram-negative bacteria. Sensitive cells, after exposure to the bacteriocin, could be rescued by treatment with proteolytic enzymes. In buffer, plantaricin SIK-83 was adsorbed to the cell surface almost immediately, and morphological lesions were observed within 2 h after the cells were exposed to the bacteriocin. The lethal mode of action appeared to be due to damage to the cell membrane, resulting in cell lysis, which was detected by electron microscopy and by determination of released intracellular components.

Adsorption↗

Cloning, sequencing, and characterizing the Lactobacillus leichmannii pyrC gene encoding dihydroorotase.

The gene encoding dihydroorotase (DHOase) of Lactobacillus leichmannii, the third enzyme of the pyrimidine biosynthetic pathway (Genbank (EMBL) accession no X78999), was cloned by phenotypic complementation of an E coli pyrC deficient mutant after transformation with Lactobacillus leichmannii genomic library DNA. The open reading frame of the L leichmannii pyrC gene spans 1281 bp and codes for a 427 amino cid polypeptide with a calculated M(r) of 46,316 Da. Primer extension showed that the initiation site for transcription is 37 bp upstream of the putative start codon ATG and Northern blot analysis confirmed its independent transcription from the adjacent pyrB gene. Comparison of the deduced amino acid sequence of L leichmannii DHOase with sequences established for other organisms yielded 46.6% identity with the corresponding Bacillus subtilis enzyme. Highly conserved protein domains suggest importance for the enzyme's function.

Amino Acid Sequence↗

Expression of the lipase gene from Staphylococcus hyicus in Lactobacillus curvatus Lc2-c.

An electroporation based transformation system was developed for Lactobacillus curvatus Lc2-c, a plasmid cured potential raw sausage starter. It was transformed with plasmid pLipPS1, a staphylococcal vector plasmid containing the lipase gene from Staphylococcus hyicus. Transformants exhibited high lipase activity, whereas the wild type strain was lipase negative. The production and release into the medium of a heterologous lipase by a Lactobacillus could be shown for the first time.

Gene Expression Regulation, Bacterial↗

Structural similarity and distribution of small cryptic plasmids of Lactobacillus curvatus and L. sake.

Plasmid profiles of strains of Lactobacillus curvatus and L. sake isolated from meat or sauerkraut were analysed to investigate plasmid homology and distribution in relation to the ecology of these organisms in fermenting foods. A hybridisation probe was constructed by cloning of pLc2, a cryptic, 2.6-kbp plasmid from L. curvatus LTH683, into the Escherichia coli plasmid pRV50. In Southern hybridisations with the digoxygenine labeled pLc2 probe, pLc2-related small plasmids were frequently detected in meat-borne strains of L. casei subsp. pseudoplantarum, L. curvatus, L. sake, L. alimentarius, L. farciminis and L. halotolerans and in L. curvatus and L. sake isolated from sauerkraut. Among 27 Lactobacillus type strains originally isolated from habitats other than meat this type of homology was detected only with plasmids of L. buchneri and L. mali. Restriction-enzyme mapping of six small cryptic plasmids from L. curvatus and L. sake revealed strong structural homology but no similarity to previously characterized plasmids of lactobacilli. The presence of a variable region in addition to a conserved one and the occurrence of deletions during cloning of pLc2 suggest that vectors derived from these plasmids are likely to be structurally unstable.

Cloning, Molecular↗

Cloning, sequencing and expression of the L-2-hydroxyisocaproate dehydrogenase-encoding gene of Lactobacillus confusus in Escherichia coli.

The gene (L-HicDH) encoding L-2-hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confusus was cloned in Escherichia coli. A 69-mer oligodeoxyribonucleotide probe, derived to be complementary to the N-terminal amino acid (aa) coding sequence, was used for screening. The complete nucleotide (nt) sequence of the L-HicDH gene was determined. The 5'-end of the mRNA was mapped by primer extension and the promoter identified. Downstream from the L-HicDH gene is a typical Rho-independent terminator. The aa sequence of L-HicDH, deduced from the nt sequence, has an overall similarity of 30% to the aa sequence of L-lactate dehydrogenase (L-LDH) from Lactobacillus casei. The aa residues involved in binding of coenzyme and substrate are highly conserved in L-HicDH with respect to prokaryotic and eukaryotic L-LDHs. The L-HicDH gene could be expressed under control of phage lambda 'Leftward' and 'rightward' promoters in E. coli up to 35% of total cell protein. The enzyme produced under these conditions exhibits full specific activity and is found exclusively in soluble form.

Alcohol Oxidoreductases↗

The molecular cloning, sequence and expression of the hdcB gene from Lactobacillus 30A.

We previously cloned the structural gene hdcA, which encodes the enzyme histidine decarboxylase (HDC; EC 4.1.1.22), from Lactobacillus 30a and found what appeared to be the start of a second gene 59 nucleotide (nt) downstream from the hdcA stop codon [Vanderslice et al., J. Biol. Chem. 32 (1986) 15186-15191]. Here we report the complete nt sequence of this second gene, which we have named hdcB, and show that it encodes a 20-kDa protein, HDCB, which was purified from Escherichia coli. The hdcA and hdcB genes together comprise an operon, the transcription from which is shown to be increased threefold by the presence of histidine in the growth medium. Western blots were used to quantitate the rise in concentrations of both gene products during histidine induction of the hdc operon. This increase was found to be proportional to the observed threefold increase in the concentration of the respective mRNAs. Transcription of the hdc operon in the mutant-3 strain of Lactobacillus 30a [Recsei and Snell, Biochemistry 12 (1973) 365-371] was shown to be constitutively 15-fold greater than in uninduced wild type cells and was unaffected by histidine. The transcription start point was defined as a guanine 73 nt 5' to the start codon of the hdcA gene. Of the transcripts initiated at this promoter, 15% include both hdcA and hdcB sequences, the remainder terminate in the intergenic region and thus encode only hdcA.

Amino Acid Sequence↗

Effects of feeding lactobacillus GG on lethal irradiation in mice.

Mice exposed to 1400 rads of total body irradiation experienced 80%-100% mortality in 2 wk. Bacteremia was demonstrated in all dead animals. Feeding Lactobacillus GG strain reduced Pseudomonas bacteremia and prolonged survival time in animals colonized with this organism. In animals not colonized with Pseudomonas, feeding Lactobacillus GG also produced some reduction in early deaths, and there was less Gram-negative bacteremia in these animals compared with controls.

Animals↗

Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22.

The sequence and genetic organization of the 1,600-bp replication region of the Lactobacillus vector pPSC22, a plasmid derived from a 7-kb cryptic plasmid of L. plantarum used for the cloning of heterologous genes in several lactobacilli, were determined. Sequence analysis revealed the presence of a plus origin of replication containing the two functional elements nic and bind, required for initiation of the leading strands typical of the rolling circle (RC)-replicating plasmids belonging to the pLS1 family. Two open reading frames (copA and repA) were located within the Lactobacillus portion of pPSC22. The repA gene product, a 234-amino acid protein, showed homologies with the Rep protein of the streptococcal plasmid pLS1 and contained the three conserved domains detected in most Rep proteins of RC-replicating plasmids and ss-coliphages. The genetic organization of the replication region of pPSC22 shared relevant homologies with the lactococcal plasmids pWVO1 and pFX2.

Base Sequence↗

Lactobacillus helveticus fermented milk lowers blood pressure in hypertensive subjects in 24-h ambulatory blood pressure measurement.

BACKGROUND: The present study was carried out to evaluate the blood pressure (BP)-lowering effect and the safety aspects of Lactobacillus helveticus LBK-16H fermented milk with high tripeptide doses on hypertensive subjects using 24-h ambulatory measurements (ABPM). METHODS: In a randomized, double blinded placebo-controlled parallel group study, 94 hypertensive patients not receiving any drug treatment were given 150 mL twice daily of either L. helveticus LBK-16H fermented milk with a high concentration of tripeptides (Ile-Pro-Pro 7.5 mg/100 g and Val-Pro-Pro 10 mg/100 g) or a control product, for 10 weeks after a 4-week run-in period. Twenty-four-hour ABPM were taken at the beginning and at the end of the intervention period. The average baseline systolic and diastolic BP values were 132.6 +/- 9.9/83.0 +/- 8.0 mm Hg in the L. helveticus group and 130.3 +/- 9.6 /80.2 +/- 7.0 mm Hg in the control group. RESULTS: There was a mean difference of -4.1 +/- 0.9 mm Hg in systolic (P = .001) and a -1.8 +/- 0.7 mm Hg in diastolic BP (P = .048) between the L. helveticus group and the control group. There was no difference in the sum of the adverse events (P = .820). CONCLUSIONS: Lactobacillus helveticus LBK-16H fermented milk containing bioactive peptides, in daily use, does have a BP-lowering effect in hypertensive subjects and is thus a potential for the dietary treatment of hypertension.

Analysis of Variance↗

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.

Animals↗

Kinetic growth parameters of different amylolytic and non-amylolytic Lactobacillus strains under various salt and pH conditions.

Four Lactobacillus species were studied for their ability to grow at high NaCl concentrations and different initial pH values. Lactobacillus plantarum 541 and A6 could ferment glucose and produce lactic acid in the presence of 8% salt in the medium. For strain 541, the specific rate of lactate production (q(lac)) and the yield of lactic acid relative to substrate (Y(p/s)) remained constant, whereas the yield of biomass relative to substrate (Y(x/s)) decreased up to 6% salt. In contrast, for strain A6, Y(p/s) decreased up to 6% salt whereas Y(x/s) did not vary markedly. Combined effects of salt and pH studied through a factorial design did not show significant interaction between salt and pH. The pH was the dominant factor in glucose fermentation for both the strains. Considering overall performance, 4% salt and pH between 6.0 and 6.6 can be taken as appropriate conditions, for the use of both strains as starters in processes where higher salt concentrations are required.

Bioreactors↗

Induction of local protective immunity to Eimeria acervulina by a Lactobacillus-based probiotic.

Previously we have shown that resistance to Eimeria acervulina (EA) infection in broiler chickens was enhanced by a probiotic treatment. In the present studies, we examined cytokine and oocyst production under similar conditions using a commercial Lactobacillus-based probiotic. Day-old male broiler chicks were fed control or probiotic diets and were orally challenged with either 2x10(4) (Experiment 1) or 1x10(4) (Experiment 2) oocysts of EA at 3 weeks of age. For the first experiment, fecal oocyst shedding and IFN-gamma levels in the culture supernatants of ConA-stimulated spleen lymphocytes were evaluated. Humoral and local cell-mediated immunity in the second experiment were assessed by evaluating antibody and cytokine (IFN-gamma and IL-2) levels in sera and intestinal secretions on a 3-day interval post inoculation. Results showed small but significant (P<0.05) differences in cytokine levels and oocyst production but not antibody levels between the probiotic-treated and control groups. Collectively, these data suggest a positive impact of the probiotic on cellular immune responses of infected broilers as compared to control chickens resulting in enhanced resistance to EA as shown in reduced fecal oocyst shedding. The results showed an immunoregulatory effect of probiotic diets on the local cell-mediated immunity in poultry and provide a rationale for further study to investigate the beneficial effects of Lactobacillus-based probiotics in food animals.

Animals↗

A pilot study evaluating the safety and effectiveness of Lactobacillus vaginal suppositories in patients with recurrent urinary tract infection.

Changes in the indigenous vaginal microflora with uropathogenic bacteria can predispose women to frequently recurring bacterial cystitis. Lactobacilli used as probiotics have played an important role in preventing the colonization of pathogenic bacteria in the vagina. A prospective clinical pilot study was performed to confirm the safety and effectiveness of Lactobacillus vaginal suppositories against the recurrence of bacterial urinary tract infection (UTI). The patients enrolled in the study were instructed to administer vaginal suppositories containing the strain Lactobacillus crispatus GAI 98322. A significant reduction in the number of recurrences was noted, without any adverse complication (P=0.0007). The administration of vaginal suppositories containing L. crispatus GAI 98332 seemed to be a safe and promising treatment for the prevention of recurrent UTI.

Adult↗

Expression of the catalase gene katA in starter culture Lactobacillus plantarum TISTR850 tolerates oxidative stress and reduces lipid oxidation in fermented meat product.

The catalase gene katA of Lactobacillus sakei SR911 was cloned and expressed in Escherichia coli UM2 and Lactobacillus plantarum TISTR850 under strong lactococcal promoter P59 in E. coli-lactococcus expression vector pIL1020. The L. plantarum TISTR850 is a catalase-deficient strain isolated from local fermented meat product. The recombinant L. plantarum TISTR850 was shown to decompose hydrogen peroxide, and catalase activity approximately three times higher that of natural catalase-producing strain L. sakei SR911. The recombinant protein was also detected by in situ activity staining of the catalase enzyme. The recombinant L. plantarum TISTR850 did not accumulate hydrogen peroxide under glucose-limited aerobic conditions and remained viable after 60 h of incubation. The recombinant and host strain L. plantarum TISTR850 were used as starter cultures in the fermented meat product, and lipid oxidation was monitored over a 7-day storage at 20 degrees C determined as thiobarbituric acid-reactive substances (TBARS) value. The lipid oxidation level in the fermented meat product seeded with the catalase genetically modified starter culture L. plantarum TISTR850 was significantly lower than that of the natural catalase-deficient strain.

Catalase↗

Molecular properties of the two-component cell lysis system encoded by prophage phigaY of Lactobacillus gasseri JCM 1131T: cloning, sequencing, and expression in Escherichia coli.

Shotgun cloning of the Lactobacillus gasseri JCM 1131T whole DNA yielded two recombinant plasmids, p118gaY1 and p118gaY2, which directed cell lysis activity. Sequencing analysis revealed that the two plasmids carried almost identical inserted genes in following orders (truncated genes, in parentheses): in p118gaY1, (orf149)-orf92-holgaY-lysgaY-orf35-attL-(mnaAgaY1); in p118gaY2, (orfXgaY1)-orf169-orf149-orf92-holgaY-lysgaY-orf35-attP-(intgaY). The lysgaY-encoded protein (designated as LysgaY, 33.7 kDa) showed significant homology with putative muramidases (peptidoglycan-degrading enzyme) of the Lactobacillus phage phiadh, Lj965, Lj928, LL-H, mv4, and mv1. By zymogram analysis, LysgaY overproduced in Escherichia coli exhibited lytic activity towards 17 Gram-positive bacterial strains, including lactobacilli, lactococci, and staphylococci. The holgaY-encoded protein (15.7 kDa) contained three potential transmembrane helices, resembling putative holins (cytoplasmic membrane-disrupting protein) of Lj928 and Lj965. On the other hand, another clone p118gaYR obtained by EcoRI-shotgun cloning carried the (ptsCgaY1)-attR-(intgaY) genes. Three sequences, attL, attP, and attR, had a 47-bp common (core) sequence, and the core of attR was located in 3' region of a potential tRNA(Arg) gene. These results suggested that (i) attL and attR are phage-host junctions, left- and right-arms, respectively, (ii) attP is a phage attachment site, and (iii) intgaY is an integrase gene for phage integration and/or excision. After mitomycin C-induction, phage particles were demonstrated by electron microscopy. The prophage (phigaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY-LysgaY), closely resembling that of Lj928 as well as Lj965.

Amino Acid Sequence↗

Modelling Yersinia enterocolitica inactivation in coculture experiments with Lactobacillus sakei as based on pH and lactic acid profiles.

In food processing and preservation technology, models describing microbial proliferation in food products are a helpful tool to predict the microbial food safety and shelf life. In general, the available models consider microorganisms in pure culture. Thus, microbial interactions are ignored, which may lead to a discrepancy between model predictions and the actual microbial evolution, particularly for fermented and minimally processed food products in which a background flora is often present. In this study, the lactic acid mediated negative microbial interaction between the lactic acid bacterium Lactobacillus sakei and the psychrotrophic food pathogen Yersinia enterocolitica was examined. A model describing the lactic acid induced inhibition (i.e., early induction of the stationary phase) of the pathogen [Vereecken, K.M., Devlieghere, F., Bockstaele, A., Debevere, J., Van Impe, J.F., 2003. A model for lactic acid induced inhibition of Yersinia enterocolitica in mono- and coculture with Lactobacillus sakei. Food Microbiology 20, 701-713.] was extended to describe the subsequent inactivation (i.e., decrease of the cell concentration to values below the detection limit). In the development of a suitable model structure to describe the inactivation process, critical points in the variation of the specific evolution rate mu [1/h] with the dynamic (time-varying) pH and undissociated lactic acid profiles were taken into account. Thus, biological knowledge, namely, both pH and undissociated lactic acid have an influence on the microbial evolution, was incorporated. The extended model was carefully validated on new data. As a result, the newly developed model is able to accurately predict the growth, inhibition and subsequent inactivation of Y. enterocolitica in coculture as based on the dynamic pH and lactic acid profiles of the medium.

Antibiosis↗

Characterization of new bacteriocinogenic lactic acid bacteria isolated using a medium designed to simulate inhibition of Listeria by Lactobacillus sakei 2512 on meat.

Bacteriocinogenic bacteria have been proposed to protect food products from Listeria contamination as bioprotective cultures. Lactobacillus sakei 2512 was demonstrated to inhibit the growth of Listeria on sliced cooked ham by challenge test. A liquid medium simulating ham, BHI5L200, was designed in order to select bioprotective strains for meat protection. Two strains were selected, from the 201 lactic acid bacteria screened, that produced bacteriocins at pH 5.8 in BHI5L200. The first one, Leuconostoc pseudomesenteroides 2733, produced a new bacteriocin which was purified and partially characterized. The second, Lactobacillus curvatus 2711, produced sakacin X and was shown to contain sakacin T and sakacin P structural genes. Co-culture experiments in BHI5L200 demonstrated that growth of Listeria was inhibited by L. sakei 2512 as well as by L. curvatus 2711.

Animals↗

Purification and characterisation of acidocin D20079, a bacteriocin produced by Lactobacillus acidophilus DSM 20079.

Bacteriocins are natural antimicrobial agents produced by food fermentative bacteria. Lactobacillus acidophilus DSM 20079 produces a small bacteriocin, with a molecular mass of 6.6 kDa, designated acidocin D20079. This antimicrobial peptide was extremely heat-stable (30 min at 121 degrees C) and was active over a wide pH range. It was found to be sensitive to proteolytic enzymes (trypsin, ficin, pepsin, papain, and proteinase K). Acidocin D20079 has a narrow inhibitory spectrum restricted to the genus Lactobacillus which includes L. sakei NCDO 2714, an organism known to cause anaerobic spoilage of vacuum-packaged meat. Maximum production of acidocin D20079 in MRS broth was detected at pH 6.0, and the peptide was purified by ammonium sulphate precipitation followed by sequential cation exchange and hydrophobic interaction chromatography. Purified acidocin D20079 spontaneously formed spherulite crystals during dialysis. As the N-terminus was found to be blocked for sequencing, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used to determine a partial sequence, and the molecular mass of the bacteriocin in the formed crystals (6.6 kDa). Estimates of the molecular weight of the partially purified peptide, using tricine-SDS-PAGE, in which bacteriocin activity was confirmed by overlayer techniques were in accordance with this value.

Amino Acid Sequence↗