Transcript The two peptide lantibiotic LtnA is effective in bacteriocin

The two peptide lantibiotic lacticin 3147
is effective in bacteriocin induced lysis
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
Girbe Buist
Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen
Jan Kok
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Bacteriocin
characteristics

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
Fraction
Volume
Total
Total activity
Specific
Purification
Yield
(ml)
A280a
(BU)b
activityc
(fold)
(%)
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)
BU; bacteriocin units
c
Specific activity is total activity divided by total A280
b
Purification


6
0.1
4
2
0
0
2
4
6
8
10
12
Elution Volume (ml)
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
0
14
4
0
2
0
8
6
0.1
4
10 3 x Activity (BU ml-1)
A280
8
10
0.2
6
0
A280
10
0.2
14
12
2-Propanol (%)
12
Reverse-phase
chromatography
0.3
14
10 3 x Activity (BU ml-1)
0.3
2
0
0
0
2
4
6
8
10
12
0
14
Elution Volume (ml)
 peptide is 3322 Da
 peptide is 2848 Da
complementary action needed for activity
N-termini blocked
 peptide cleft by chymotrypsin
Edman: Ile-(Ser)-Thr-Asn-Thr-(Glu)-Pro
homologous to a peptide of lacticin 3147
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Amino acid and
nucleotide
sequence
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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
Amino acid
composition
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LtnA1
-peptide
LtnA2
Ala
Arg
Asx
Cys
Glx
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
Total
3-4
0
4
3
1-2
2-3
1
0
2
1
1
1
0
0
0
ND
1
0
20-23
2
0
4
4
1
2
1
0
2
1
1
1
0
3
4
3
1
0
30
7
1
1
3
0
0
0
3
1-2
1
0
0
3-4
0
3
ND
1
0
24-26
4
1
1
3
0
0
0
3
1
1
0
0
3
3
8
0
1
0
29
MM
3322
3428.4
2848
2985.5
LtnA1 and LtnA2 are post-translational
modified
Ser > D-Ala conversion shown and
lanthionine present
~ 13 Kb
Protein
LtnE
LtnF
LtnI
LtnR
LtnA1
LtnA2
LtnM1
LtnT
LtnM2
LtnD
ltn operon
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Function
ABC-transporter for immunity
ABC-transporter for immunity
Immunity protein
Transcription repressor
-peptide
-peptide
Dehydration/thioether-forming reactions
ABC-transporter
Dehydration/thioether-forming reactions
dehydrogenase
gene organization in pBAC105 and
pMRC01 are identical
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Lacticin 3147
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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
Lb. casei IFPL731
1
10
L. lactis IFPL359
Growth (OD600)
10
Growth (OD600)
Growth (OD600)
10
1
0.1
0
Lb. rhamnosus JCL1211
1
0.1
0.1
0
2
0 hours
3 hours
% lysis
0 hours
3 hours
% lysis
0 hours
3 hours
% lysis
1.0  109
6.0  105
1.0  108
8.7  103
14.9 (1.5)
25.7 (0.8)
3.3  109
5.3  107
1.0  108
4.8  103
32.5 (2.3)
51.3 (1.2)
6.8  108
5.4  108
1.0  108
3.0  105
15.6 (1.3)
74.5 (3.7)
4
Time (h)
Lacticin 3147
induced lysis
6
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
2
4
Time (h)
6
0
2
4
Time (h)
loss of viability not concurrent with cell
lysis
addition of Co2+ or SDS reduces lysis
6
5
4
MG1363acmA
OD[600nm]
3
2
MG1363
1
AcmA:
responsible
for autolysis
0
0
30
60
90
Time in hours
120
150
180
10
0.035
0.030
0.020
1
0.015
0.010
0.005
Involvement of 0.1
0
AcmA in lacticin
induced lysis
0
2
4
6
Time (h)
8
10
Units of PepX / ml
OD 600
0.025
MG1363
10
0.035
0.030
Bac
0.020
1
0.015
0.010
0.005
Involvement of 0.1
0
AcmA in lacticin
induced lysis
0
2
4
6
Time (h)
8
10
Units of PepX / ml
OD 600
0.025
MG1363
MG1363 (+Bac)
10
0.035
0.030
Bac
0.020
1
0.015
0.010
0.005
Involvement of 0.1
0
AcmA in lacticin
induced lysis
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0
2
4
6
8
Units of PepX / ml
OD 600
0.025
MG1363
MG1363 (+Bac)
MG1363acmA
(+Bac)
10
Time (h)
reduction of viability identical
AcmA responsible for lacticin 3147
induced lysis
Cell wall degradation (%)
Cell walls
AcmA
Control
Bacteriocin
(300 AU/ml)
L. lactis MG1363
-
21.9 (0.2)
23.1 (1.5)
L. lactis MG1363acmA1
+
22.8 (3.5)
20.9 (0.9)
L. lactis MG1363acmA1
-
3.8 (1.4)
3.8 (1.8)
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Cell wall
degradation
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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