1. 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, Norway Havard Hauge Jon Nissen-Meyer Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen Jan Kok The two peptide lantibiotic lacticin 3147 is effective in bacteriocin induced lysis Girbe Buist

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

3. Purification

4. Reverse-phase chromatography n  peptide is 3322 Da n  peptide is 2848 Da n complementary action needed for activity 

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

6. Amino acid composition n LtnA1 and LtnA2 are post-translational modified n Ser > D-Ala conversion shown and lanthionine present

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

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

9. Growth (OD 600 ) Time (h) 0.1 1 10 0246 Lb. casei IFPL731 L. lactis IFPL359 0.1 1 10 0246 Growth (OD 600 ) Time (h) 0.1 1 10 0246 Growth (OD 600 ) Time (h) Lb. rhamnosus JCL1211 0 hours3 hours% lysis 1.0  10 9 1.0  10 8 14.9 (1.5) 6.0  10 5 8.7  10 3 25.7 (0.8) 0 hours3 hours% lysis 3.3  10 9 1.0  10 8 32.5 (2.3) 5.3  10 7 4.8  10 3 51.3 (1.2) 0 hours3 hours% lysis 6.8  10 8 1.0  10 8 15.6 (1.3) 5.4  10 8 3.0  10 5 74.5 (3.7) n loss of viability not concurrent with cell lysis n addition of Co 2+ or SDS reduces lysis Lacticin 3147 induced lysis

10. AcmA: responsible for autolysis 180 0 1 2 3 4 5 0306090120150 Time in hours OD[600nm] MG1363 MG1363acmA

11. 0.1 1 10 02468 Time (h) OD 600 0 0.005 0.010 0.015 0.020 0.025 0.030 0.035 Units of PepX / ml MG1363 Involvement of AcmA in lacticin induced lysis

13. n reduction of viability identical n AcmA responsible for lacticin 3147 induced lysis Involvement of AcmA in lacticin induced lysis

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

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