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The two peptide lantibiotic lacticin 3147 is effective in bacteriocin induced lysis

The two peptide lantibiotic lacticin 3147 is effective in bacteriocin induced lysis. Girbe Buist. Department of Dairy Science and Technology, Instituto del Frío (CSIC), Ciudad Universitaria, Madrid, Spain Carmen Martínez-Cuesta Teresa Requena Elisabet Herranz Carmen Peláez

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The two peptide lantibiotic lacticin 3147 is effective in bacteriocin induced lysis

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  1. The two peptide lantibiotic lacticin 3147 is effective in bacteriocin induced lysis Girbe Buist Department of Dairy Science and Technology, Instituto del Frío (CSIC), Ciudad Universitaria, Madrid, Spain Carmen Martínez-Cuesta Teresa Requena Elisabet Herranz Carmen Peláez Department of Biochemistry, University of Oslo, NorwayHavard Hauge Jon Nissen-Meyer Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen Jan Kok

  2. Bacteriocincharacteristics • produced by L. lactis IFPL105, isolated from Spanish raw goats’ milk • heat stable • broad spectrum of inhibitory activity • hydrolyzed by chymotrypsin, proteinase K and pancreatin • resistant to trypsin and pepsin • genes for immunity and production located on 46 kb plasmid pBAC105

  3. Purification

  4. 0.3 14 0.3 14 12 12 10 10 0.2 0.2 60 8 8 10 3 x Activity (BU ml-1) 10 3 x Activity (BU ml-1) A A 280 280 6 6 40 2-Propanol (%) 0.1 0.1 4 4 20 2 2 0 0 0 0 0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 Elution Volume (ml) Elution Volume (ml) a b Reverse-phasechromatography •  peptide is 3322 Da •  peptide is 2848 Da • complementary action needed for activity

  5. N-termini blocked •  peptide cleft by chymotrypsin • Edman: Ile-(Ser)-Thr-Asn-Thr-(Glu)-Pro • homologous to a peptide of lacticin 3147 Amino acid and nucleotide sequence • ltnA1 encodes a peptide of 59 aa • ltnA2 encodes a peptide of 62 aa • ‘double-Gly’ type leader peptides

  6. Amino acidcomposition • LtnA1 and LtnA2 are post-translational modified • Ser > D-Ala conversion shown and lanthionine present

  7. ~ 13 Kb ltn operon • gene organization in pBAC105 and pMRC01 are identical

  8. Lacticin 3147 • produced by L. lactis DPC3147 isolated from Irish kefir grain • PMF promotes the interaction with membrane • pores are selective for K+ ions and inorganic phosphate • dissipation of the membrane potential > hydrolysis of ATP > collapse of the pH gradient > cell death • induces cell lysis • lytic response is strain dependent

  9. 10 Growth (OD600) 1 0.1 0 2 4 6 Time (h) 0 hours 3 hours % lysis 1.0  109 1.0  108 14.9 (1.5) 6.0  105 8.7  103 25.7 (0.8) 0 hours 3 hours % lysis 3.3  109 1.0  108 32.5 (2.3) 5.3  107 4.8  103 51.3 (1.2) 0 hours 3 hours % lysis 6.8  108 1.0  108 15.6 (1.3) 5.4  108 3.0  105 74.5 (3.7) 10 10 Growth (OD600) 1 Growth (OD600) 1 0.1 0.1 0 2 4 6 0 2 4 6 Time (h) Time (h) Lb. rhamnosus JCL1211 L. lactis IFPL359 Lb. casei IFPL731 Lacticin 3147induced lysis • loss of viability not concurrent with cell lysis • addition of Co2+ or SDS reduces lysis

  10. 5 4 MG1363acmA 3 OD[600nm] 2 MG1363 1 0 0 30 60 90 120 150 180 Time in hours AcmA: responsible for autolysis

  11. 10 0.035 0.030 0.025 0.020 600 1 OD MG1363 Units of PepX / ml 0.015 0.010 0.005 0.1 0 0 2 4 6 8 10 Time (h) Involvement of AcmA in lacticin induced lysis

  12. 10 0.035 0.030 0.025 Bac 0.020 600 1 OD MG1363 Units of PepX / ml 0.015 MG1363 (+Bac) 0.010 0.005 0.1 0 0 2 4 6 8 10 Time (h) Involvement of AcmA in lacticin induced lysis

  13. 10 0.035 0.030 0.025 Bac 0.020 600 1 OD MG1363 Units of PepX / ml 0.015 MG1363 (+Bac) 0.010 MG1363acmA (+Bac) 0.005 0.1 0 0 2 4 6 8 10 Time (h) Involvement of AcmA in lacticin induced lysis • reduction of viability identical • AcmA responsible for lacticin 3147 induced lysis

  14. Lacticin 3147 does not activate AcmA • similar results obtained when using Nisin or a mixture of lactococcins A, B, and M Cell wall degradation

  15. Depletion of cellular energy causes an imbalance in the control of the action of the autolysin, resulting in cell wall degradation and, thus, cell lysis. Conclusion

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