Department of Dairy Science and Technology, Instituto del Frío (CSIC), Ciudad Universitaria, Madrid, Spain

Carmen Martínez-CuestaTeresa RequenaElisabet HerranzCarmen Peláez

Department of Biochemistry, University of Oslo, NorwayHavard HaugeJon Nissen-Meyer

Groningen Biomolecular Sciences and Biotechnology Institute, University of GroningenJan Kok

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

Girbe Buist

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

Purification

Fraction Volume

(ml)

Total

A280a

Total activity

(BU)b

Specific

activityc

Purification

(fold)

Yield

(%)

Culture supernatant 2 000 71 800 4.44 105 6.2 1 100

I (Ammonium sulphate precipitation) 250 2 597 2.77 105 106.6 17 63

II (Anion exchange) 260 4 316 3.33 105 77.2 12 75

III (cation exchange) 270 3 375 3.30 105 97.7 16 74

IV (Phenyl sepharose) 20 57.6 1.50 105 2 604 421 34

V (C2/C18 column) 2 1.2 0.57 105 21 250 3 438 11

a Total A280 is A280 multiplied by the volume (ml)b BU; bacteriocin unitsc Specific activity is total activity divided by total A280

Reverse-phasechromatography

peptide is 3322 Da peptide is 2848 Da complementary action needed for activity

0 2 4 6 8 10 12 140

2

4

6

8

10

12

140.3

0

0.1

0.2

10

3 x

Act

ivity

(B

U m

l-1)

0.1

0.2

0.3

00 2 4 6 8 10 12 14

0

2

4

6

8

10

12

14

0

20

40

60

2-P

ropa

nol (

%)

A2

80

A2

80

10

3 x

Act

ivity

(B

U m

l-1)

Elution Volume (ml) Elution Volume (ml)

Amino acid and nucleotide sequence

N-termini blocked peptide cleft by chymotrypsin Edman: Ile-(Ser)-Thr-Asn-Thr-(Glu)-Pro homologous to a peptide of lacticin 3147

ltnA1 encodes a peptide of 59 aa ltnA2 encodes a peptide of 62 aa ‘double-Gly’ type leader peptides

Number of residues per molecule

Amino acid -peptide LtnA1 -peptide LtnA2

Ala 3-4 2 7 4Arg 0 0 1 1Asx 4 4 1 1Cys 3 4 3 3Glx 1-2 1 0 0Gly 2-3 2 0 0His 1 1 0 0Ile 0 0 3 3

Leu 2 2 1-2 1Lys 1 1 1 1Met 1 1 0 0Phe 1 1 0 0Pro 0 0 3-4 3Ser 0 3 0 3Thr 0 4 3 8Trp ND 3 ND 0Tyr 1 1 1 1Val 0 0 0 0

Total 20-23 30 24-26 29

MM 3322 3428.4 2848 2985.5Amino acidcomposition LtnA1 and LtnA2 are post-translational

modified Ser > D-Ala conversion shown and

lanthionine present

ltn operon

Protein FunctionLtnE ABC-transporter for immunityLtnF ABC-transporter for immunityLtnI Immunity proteinLtnR Transcription repressorLtnA1 -peptideLtnA2 -peptideLtnM1 Dehydration/thioether-forming reactionsLtnT ABC-transporterLtnM2 Dehydration/thioether-forming reactionsLtnD dehydrogenase

~ 13 Kb

gene organization in pBAC105 and pMRC01 are identical

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

Gro

wth

(O

D60

0)

Time (h)

0.1

1

10

0 2 4 6

Lb. casei IFPL731 L. lactis IFPL359

0.1

1

10

0 2 4 6

Gro

wth

(O

D60

0)Time (h)

0.1

1

10

0 2 4 6

Gro

wth

(O

D60

0)

Time (h)

Lb. rhamnosus JCL1211

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)

loss of viability not concurrent with cell lysis

addition of Co2+ or SDS reduces lysis

Lacticin 3147induced lysis

AcmA: responsible for autolysis

180

0

1

2

3

4

5

0 30 60 90 120 150

Time in hours

OD

[600

nm

]

MG1363

MG1363acmA

0.1

1

10

0 2 4 6 8 10

Time (h)

OD

600

0

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Uni

ts o

f P

epX

/ m

l

MG1363

Involvement of AcmA in lacticin induced lysis

0.1

1

10

0 2 4 6 8 10

Time (h)

OD

600

0

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Uni

ts o

f P

epX

/ m

l

Bac

MG1363 (+Bac)

MG1363

Involvement of AcmA in lacticin induced lysis

0.1

1

10

0 2 4 6 8 10

Time (h)

OD

600

0

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Uni

ts o

f P

epX

/ m

l

Bac

MG1363acmA(+Bac)

MG1363 (+Bac)

MG1363

reduction of viability identical AcmA responsible for lacticin 3147

induced lysis

Involvement of AcmA in lacticin induced lysis

Cell wall degradation

Cell wall degradation (%)

Cell walls AcmA Control Bacteriocin

(300 AU/ml)

L. lactis MG1363 - 21.9 (0.2) 23.1 (1.5)

L. lactis MG1363acmA1 + 22.8 (3.5) 20.9 (0.9)

L. lactis MG1363acmA1 - 3.8 (1.4) 3.8 (1.8)

Lacticin 3147 does not activate AcmA

similar results obtained when using Nisin or a mixture of lactococcins A, B, and M

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