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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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