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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
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