Previous Article | Next Article 
Appl Environ Microbiol, June 1998, p. 1997-2005, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Isolation and Characterization of Pediocin AcH
Chimeric Protein Mutants with Altered Bactericidal Activity
Kurt W.
Miller,1,*
Robin
Schamber,1
Ozlem
Osmanagaoglu,2,
and
Bibek
Ray2,*
Departments of Molecular
Biology1 and
Animal
Science2, University of Wyoming, Laramie,
Wyoming 82071
Received 9 January 1998/Accepted 24 March 1998
 |
ABSTRACT |
A collection of pediocin AcH amino acid substitution mutants was
generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an
Escherichia coli malE plasmid that directs the secretion of
maltose binding protein-pediocin AcH chimeric proteins and by screening
transformant colonies for bactericidal activity against
Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol.
64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S,
C14Y, G37E, G37R, and C44W) were completely inactive against the
pediocin AcH-sensitive strains L. plantarum NCDO955,
Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc
mesenteroides Ly. A C24S substitution mutant constructed by other
means also was inactive against these bacteria. Nine other mutants
(K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to ~60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed ~2.8-fold-higher
activity against this indicator. About one half of the mutations mapped
to amino acids that are conserved in the pediocin-like family of
bacteriocins. All four cysteines were found to be required for
activity, although only C9 and C14 are conserved among pediocin-like
bacteriocins. Several basic amino acids as well as nonpolar amino acids
located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.
| |
INTRODUCTION |
Pediocin AcH (same sequence as
pediocin PA-1) is a ribosomally synthesized bacteriocin that is
produced by certain strains of Pediococcus acidilactici
(24, 27). Synthesis is conferred by the papABCD
operon, which encodes the pediocin AcH structural gene
(papA) and ancillary genes used for production (4,
26). The bacteriocin is translated as a 62-amino-acid preprotein
and is converted by the PapD protein to a 44-amino-acid mature form by
enzymatic processing of an 18-amino-acid leader peptide (4, 33). The PapC and PapD proteins belong to the ABC export system family of proteins (11). Although an ABC export system is
used for production in Pediococcus, the mature sequence
region of pediocin AcH can be secreted via the Escherichia coli
sec machinery when its N terminus is fused to the E. coli secretory protein maltose binding protein (MBP)
(25). These results indicate that PapD is necessary for
recognition and processing of the leader peptide rather than for
accommodating the mature region as it passes through the membrane. Once
secreted, pediocin AcH becomes fully active after the formation of two
intramolecular disulfide bonds (14, 19).
Independent studies performed with pediocins PA-1 and AcH have begun to
reveal their mode of action and structural requirements for activity.
These bacteriocins kill susceptible bacteria by permeabilizing the
cytoplasmic membrane, causing leakage of ions and small molecules
(2, 5, 7, 19). The interaction of pediocin PA-1 with
membranes is promoted by acidic phospholipids (5), and
positively charged amino acids in this cationic peptide appear to be
important for membrane binding (6, 19). In fact, both
lysines and histidines may mediate membrane binding in the low-pH
environment (pH, 
5.0) in which Pediococcus strains can grow (3, 19, 35). Other pediocin-like bacteriocins, such as
sakacin P (32) (same as sakacin 674) (17),
leucocin A (13), and curvacin A (31) (same as
sakacin A) (16), also are cationic and may rely in part on
electrostatic interactions between basic amino acids and negatively
charged phospholipid head groups for membrane adsorption.
Pediocins PA-1 and AcH contain two structurally distinct sequence
regions (5, 12, 19). The N-terminal 20 amino acids are polar
and are highly conserved among pediocin-like bacteriocins. Located
within this region is a -Y3-G4-N5-G6-V7- sequence that is present in
all family members. While the function of this sequence is unknown, its
deletion from pediocin AcH completely inactivates the molecule
(25). Based on secondary structure analysis of pediocin
PA-1, it has been proposed that the first 18 amino acids fold into a
two-strand 
hairpin that is stabilized by a turn at position
-G4-N5-G6-V7- (5) and the C9---C14 disulfide bond (5,
14). Solution nuclear magnetic resonance analysis of a related
bacteriocin, leucocin A, indicated that its conformation indeed is
ordered near the C9---C14 disulfide bond, but specific H-bond
interactions expected for a -hairpin structure were not detected
(28). Perhaps due to the short length of these molecules, regions of defined secondary structure may not form until after they
have adsorbed to a membrane (20).
The C-terminal sequence region of pediocins PA-1 and AcH (residues A21
to C44) is much less polar and conserved than the N-terminal sequence
region (19). The C-terminal region is proposed to contain a
hydrophobic membrane interaction domain (12), and analysis of the properties of hybrid peptides constructed from pediocin-like bacteriocins (12) and MBP-pediocin AcH chimeric proteins
(25) supports this hypothesis. It also has been shown that
the membrane interaction domain becomes amphipathic if folded into an
helix (12). While it is well established that membrane
binding is promoted by amphipathic helices (9, 20), it
should be noted that the C24---C44 disulfide bond which is unique to
pediocins PA-1 and AcH (19) bends the C-terminal region into
a loop, and it is unknown whether an amphipathic structure can be
formed in the presence of the loop.
To facilitate analysis of the structure and mode of action of pediocin
AcH, we generated a collection of pediocin AcH substitution mutants
that display altered bactericidal activity. Mutants were produced and
detected with the E. coli MBP-pediocin AcH chimeric protein
secretion system and colony overlay screening methods (25).
Over one third of the amino acids in pediocin AcH were found to be
important for activity.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
Bacterial strains and plasmids used in this study are listed in Table
1. Plasmid pMBR1.0 carries the
papABCD operon from the P. acidilactici LB42-923
plasmid pSMB74. pMBR1.0 derivatives encoding pediocin AcH cysteine
substitution mutants were constructed as described below. E. coli JM109 served as the host for these plasmids and was grown at
37°C in Luria-Bertani (LB) broth or agar containing 30 µg of
chloramphenicol per ml. Plasmid pPR682 was used to construct mutant
malE-papA translational fusion genes. Transcription of the
chimeric genes was controlled by the
isopropyl--D-thiogalactopyranoside (IPTG)-inducible
tac promoter. E. coli E609L was used as the host for the malE-papA plasmids, and transformed E609L strains
were grown at 37°C in LB broth or agar containing 12.5 µg of
tetracycline and 100 µg of ampicillin per ml. MBP-pediocin AcH
mutants were tested for activity against the bacterial indicator
strains listed in Table 1. Lactic acid-producing bacterial strains were
grown in tryptone-glucose-yeast extract (TGE) broth without Tween 80 (3, 35). Listeria innocua Lin11 was grown in
tryptic soy broth (Difco) at 30°C.
Construction of mutagenized malE-papA plasmids.
Base substitutions were introduced into the papA gene by PCR
random mutagenesis (22) (Fig.
1). Mutagenesis reaction mixtures contained 25 U of Taq DNA polymerase (GIBCO-BRL) per ml, 0.1 µg of pPR6821 template per ml, 5 mM MgCl2, 0.5 mM
MnCl2, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide, 10 mM each nucleoside triphosphate, and 0.07 µg of each primer per ml. A
30-cycle repeated protocol consisting of 90 s of strand
denaturation at 94°C, 60 s of primer annealing at 55°C, and
60 s of primer extension at 72°C was used to amplify papA DNA. The 5' PCR primer
(5'-CGGGGATCCATCGAGGGTAGG-3') binds immediately upstream of
the papA gene residing in pPR6821 and primes DNA synthesis
beginning with the K1 codon of the mature sequence region. A
BamHI restriction endonuclease digestion site is contained
in the primer and was used for cloning purposes. The 3' PCR primer
(5'-CAAGCTTGCCTGCAGGTCGACCTA-3') binds immediately downstream of codon C44 of the papA gene and contains a
SalI restriction endonuclease digestion site. Amplified
papA DNA fragments were treated with the Klenow fragment of
E. coli DNA polymerase I to repair ragged ends, digested
with BamHI and SalI restriction endonucleases, gel purified, and ligated into
BamHI-SalI-digested pPR682 (29). Fusion genes in which the malE and mutagenized
papA coding sequences were joined in frame were created by
ligation.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 1.
Construction of mutagenized malE-papA
plasmids. papA DNA fragments were synthesized by PCR
amplification under random mutagenesis conditions with plasmid pPR6821
as a template. Subsequently, papA DNA fragments were
digested with BamHI and SalI restriction
endonucleases and ligated into plasmid pPR682 to construct
malE-papA translational fusion genes. Plasmids containing
mutant MBP-pediocin AcH chimeric proteins were identified by colony
overlay screening, as explained in the text. Plasmids were named based
on the location of the mutation, e.g., plasmid pKN1 contains the K1N
mutation (indicated by an asterisk). Abbreviations:
ampr, gene encoding -lactamase;
lacIq, gene encoding the lac
repressor; lacZ', gene encoding truncated -galactosidase;
malE, gene encoding MBP; mcs, multiple cloning site;
ori, origin of replication; Ptac,
tac promoter.
|
|
The mutagenized pool of
malE-papA plasmids was transformed
into strain E609L, and transformants were selected by plating on
LB
agar containing 12.5 µg of tetracycline and 100 µg of ampicillin
per ml. Because the indicator strain
Lactobacillus plantarum
NCDO955
is sensitive to these antibiotics, the activity of chimeric
proteins
synthesized by the clones could not be screened directly on
the
primary transformant plates. Instead, E609L colonies were collected
by scraping into LB medium containing antibiotics and were grown
overnight at 37°C. On the following day, cells were pelleted by
centrifugation and resuspended in LB broth without antibiotics.
Aliquots of the suspension containing ~100 cells were plated on
5 ml
of TGE soft agar as described previously (
25). The plates
were incubated for 24 h at 37°C until colonies had formed and
then were overlaid with 5 ml of TGE soft agar containing
~10
6 cells of
L. plantarum NCDO955 and 1 mM
IPTG. Overlaid plates
were incubated for an additional 24 h until
zones of growth inhibition
had formed around E609L producer colonies.
Nonproducer mutant
colonies were picked from the plates and grown in LB
medium containing
antibiotics, and plasmid DNA was isolated for
transformation into
E. coli XL1-Blue to obtain
sequencing-quality DNA. Dideoxy terminator
double-stranded DNA
sequencing was performed with Sequenase T7
DNA polymerase (Amersham).
Mutagenized plasmids were designated
based on the location of the
substitution site, e.g., pKN1, pNK5,
and so forth.
Construction of pediocin AcH cysteine substitution mutant
plasmids.
Cysteine substitution mutations were introduced into the
papA gene of plasmid pMBR1.0 by three different methods. The
C9S and C24S codon changes were created by use of a phosphorothioate oligonucleotide-directed in vitro mutagenesis procedure
(30). An M13mp18 bacteriophage vector (36)
containing the subcloned papA sequence served as the
mutagenesis template. The sequences of oligonucleotides used to create
cysteine codons were 5'-GGGGTTACTAGTGGCAA-3' (oligo C9S) and 5'-CTACCACTAGCATAATC-3'
(oligo C24S); bases changed from the wild type are underlined.
After in vitro mutagenesis procedures, modified double-stranded
bacteriophage DNAs were transformed into E. coli TG1, and
cysteine substitution mutants were identified by plaque hybridization
with the 32P-labeled mutagenic oligonucleotides that were
used to screen nitrocellulose filters (29). The sequences of
mutagenized papA inserts were confirmed by single-stranded
DNA sequencing with Sequenase T7 DNA polymerase. The C9S and C24S
papA genes were excised from the replicative forms of the
bacteriophage vectors by digestion with MscI and
Bpu1102I restriction endonucleases and were cloned into
pMBR1.0, replacing the wild-type papA coding region. The
papA sequences of the resulting pMBR1.0 cysteine
substitution mutant plasmids (named pMCS9 and pMCS24; Table 1) were
subjected to double-stranded DNA sequencing to confirm the swap of
mutant for wild-type DNA.
The C44S substitution mutation was created in the
papA gene
by PCR amplification with pMBR1.0 as a template. The C44S codon
change
was incorporated at the underlined base into the 3' PCR
primer
(5'-CAGCTCAGCATAATGC
TAGCTTTTATG-3') used in the
amplification
reaction. The primer also contains a
Bpu1102I
restriction endonuclease
digestion site for cloning into pMBR1.0. The
5' PCR primer (5'-AGAAATGGCCAATATCATTGGTGGTAAA-3')
used in
the amplification reaction contains an
MscI restriction
endonuclease digestion site. The reaction conditions used to synthesize
the C44S
papA DNA fragment have been described before
(
25).
The C44S DNA product was treated with the Klenow
fragment of DNA
polymerase I, digested with
MscI and
Bpu1102I restriction endonucleases,
gel purified, and
ligated into pMBR1.0. The substitution of the
mutant for the wild-type
sequence in the resulting plasmid, pMCS44,
was confirmed by
double-stranded DNA sequencing.
The C14S codon change was introduced into the
papA gene by
extension overlap PCR mutagenesis (
15). The
papA
coding sequence
first was amplified as two DNA fragments in which the
3' end of
the upstream fragment overlapped the 5' end of the downstream
fragment. The targeted C14 codon resided in the overlapping region.
Plasmid pMBR1.0 served as a template, and PCR conditions were
the same
as those used for synthesis of the C44S DNA fragment.
The primers used
to synthesize the upstream DNA fragment were
5'-AGAAATGGCCAATATCATTGGTGGTAAA-3' (5' PCR primer) and
5'-CCAGTCAACAGAGC
TGGAATGTTTG-3'
(3' PCR primer),
and the primers used to synthesize the downstream
DNA fragment were
5'-CAAACATTCC
AGCTCTGTTGACTGG-3' (5' PCR primer)
and 5'-ATTGATGCCAGCTCAGCATAATGCTA-3' (3' PCR primer); the
bases
changed to create the C14S substitution in the region of overlap
are underlined. After the two primary PCR products were synthesized,
they were gel purified and combined at a concentration of 0.1
µg/ml
each in the extension overlap PCR mixture. Conditions used
to
synthesize the extension overlap secondary PCR product were
identical
to those used to synthesize the primary products, except
that only the
two outermost primers were used. The extension overlap
papA
DNA fragment was treated with the Klenow fragment of DNA
polymerase I,
digested with
MscI and
Bpu1102I restriction
enzymes,
gel purified, and ligated into pMBR1.0. Again, transfer of the
mutant sequence to the resulting plasmid, pMCS14, was confirmed
by
double-stranded DNA sequencing.
Comparison of MBP-pediocin AcH chimeric protein synthesis levels.
E. coli E609L cells transformed with the mutant
malE-papA plasmids were grown at 37°C in LB medium
containing tetracycline and ampicillin. At the mid-log phase, IPTG (1 mM final concentration) was added to induce the synthesis of chimeric
proteins. After 3 h of continuous growth, culture broths were
subjected to trichloroacetic acid precipitation, and precipitated
proteins were solubilized in sample loading buffer containing 3%
sodium dodecyl sulfate (SDS), boiled, and analyzed on 10%
acrylamide-bisacrylamide-SDS gels (21). Proteins were
visualized by Coomassie brilliant blue dye staining, and relative
levels of synthesis of wild-type and mutant chimeric proteins were
compared by scanning densitometry with a Bio-Rad Gel Dock laser
densitometer (1, 25).
Measurement of the bactericidal activities of MBP-pediocin AcH
chimeric proteins.
Chimeric protein-expressing strains were grown
at 37°C in LB medium without antibiotics and induced at the mid-log
phase for 3 h by the addition of 1 mM IPTG. Culture broths were
boiled, and aliquots were tested against the indicator strains listed in Table 1. Two types of activity tests were performed. The first assay
(the well assay) was used to establish if the mutants exhibited any
activity. In this assay, 100-µl aliquots of the culture broths were
placed into wells cut in TGE agar plates on which soft-agar lawns of
the indicator bacteria had been spread. Plates were incubated overnight
and examined for zones of growth inhibition around the wells. The
second assay (the titration assay) was used to determine the activity
levels of mutants that were shown by the well assay to be partially
active. In this assay, aliquots of boiled, 3-h IPTG-induced culture
broths were applied to plates containing an overlay of L. innocua Lin11, and the minimum volume necessary to produce a zone
of growth inhibition in the lawn was determined (3, 35). It
should be noted that 1 µl of a boiled, 3-h IPTG-induced culture broth
from the wild-type MBP-pediocin AcH-producing strain (E609L/pPR6821)
was sufficient to form a small zone of growth inhibition against
L. innocua Lin11. After titration of the activities of the
broths, the activities of mutant chimeric proteins relative to that of
the wild-type MBP-pediocin AcH chimeric protein were calculated.
Activities were corrected for differences in culture optical density at
600 nm (OD600) and the relative levels of proteins in the
culture broths.
Analysis of pediocin AcH sequence hydrophobicity, amphipathicity,
and -turn potentials.
The average sequence hydrophobicities and
average sequence hydrophobic moments of wild-type and mutant pediocin
AcH sequence regions were calculated by use of the normalized amino
acid hydrophobicity scale and hydrophobic moment calculation method of
Eisenberg et al. (9). -Turn potentials of selected
pediocin AcH sequence regions also were calculated by use of published
methods (18).
| |
RESULTS |
Isolation of MBP-pediocin AcH chimeric protein random mutants.
An MBP-pediocin AcH chimeric protein secretion system (25)
was used for the generation and detection of pediocin AcH mutants. Mutations were introduced into DNA encoding the mature sequence region
of the papA gene (codons K1 to C44) by PCR random
mutagenesis (22). The conditions used typically create about
1 base substitution per 100 bp of DNA. Subsequently, mutagenized
papA DNA fragments were cloned into the pPR682
malE expression plasmid (Fig. 1), and the pool of plasmid
DNAs was transformed into the periplasmic leaky E. coli
host, E609L. About one third of the MBP-pediocin AcH chimeric proteins
synthesized by this strain are secreted into the periplasm and released
into the culture medium (25). All chimeric protein
substitution mutants described in this study were isolated by colony
overlay screening against the pediocin AcH-sensitive indicator strain
L. plantarum NCDO955 (3, 35).
Approximately 2,500 colonies were obtained from transformation of E609L
with the pool of mutagenized plasmid DNAs. Of these
transformants,
about one half displayed reduced or no activity.
Plasmid DNAs from 53 mutant colonies were sequenced, and 17 unique
substitution mutations
affecting 14 codons in the
papA gene were
identified (Table
2). Two substitution mutations (G37R and
C44W)
occurred frequently (>10 times each), perhaps due to a tendency
of
Taq DNA polymerase to misincorporate bases at these
codons
under the mutagenesis conditions used. Only a few
multiple-substitution
mutants were obtained (data not shown),
confirming the bias of
the technique for generating single base
substitutions in short
DNA sequences (
22). Other mutants
might have been recovered
if more of the inactive clones in the pool
had been sequenced.
About one half of the mutations (K1N, N5K, C9R, K11E, C14S/Y, W18R, and
A34D) mapped to amino acids that are conserved in
the pediocin-like
family of bacteriocins (
19). Other mutations
(I26T, M31T,
G37E/R, N41K, H42L, K43N/E, and C44W) affected amino
acids that either
are not conserved or are minimally conserved.
As expected, the side
chains of substituted amino acids typically
differed substantially from
those of wild-type residues. Because
mutations often mapped to
conserved residues and residues such
as cysteines that are known by
experimental means to be important
for activity (
7,
19), we
concluded that the mutagenesis and
screening procedures were highly
effective in identifying important
amino acids in the pediocin AcH
sequence.
Analysis of the bactericidal activities of MBP-pediocin AcH
chimeric protein mutants.
The mutants listed in Table 2 were
tested for activity against the gram-positive pediocin AcH-sensitive
strains L. plantarum NCDO955, P. acidilactici
LB42, Leuconostoc mesenteroides Ly, Enterococcus faecalis M1, and L. innocua Lin11. Five strains were
used to determine how broadly the mutations inactivated pediocin AcH.
In this regard, a mutation that interferes with a fundamental property
such as membrane binding could inactivate pediocin AcH for a variety of bacteria.
The mutants first were classified as inactive or active by well assay
testing against the five indicator strains. Examples
of plates on which
mutants were tested against
L. innocua Lin11
and
E. faecalis M1 are presented in Fig.
2.
Based on testing against
the five indicator strains, 7 mutants (N5K,
C9R, C14S, C14Y, G37E,
G37R, and C44W) were classified as inactive and
10 mutants (K1N,
K11E, W18R, I26T, M31T, A34D, N41K, H42L, K43E, and
K43N) were
classified as partially to fully active compared to the wild
type
(Table
2). The test strains were uniformly sensitive or
insensitive
to all mutants except A34D. The A34D mutant retained some
activity
against
L. plantarum NCDO955,
E. faecalis M1, and
L. innocua Lin11
but was inactive
against
P. acidilactici LB42 and
L. mesenteroides Ly with the method used. Note that a 100-µl sample of the wild-type
E609L/pPR6821 culture broth contained ~100-fold more chimeric
protein
than was necessary to inhibit the growth of these bacteria.
View larger version (124K):
[in this window]
[in a new window]
|
FIG. 2.
Well assay analysis of MBP-pediocin AcH substitution
mutant and pediocin AcH cysteine substitution mutant antibacterial
activities. Screening was performed against L. innocua Lin11
(A) and E. faecalis M1 (B) with 100 µl of boiled,
IPTG-induced (3 h) culture broths (chimeric protein strains) or 100 µl of boiled, overnight-grown culture broths (pediocin AcH cysteine
substitution mutant strains) per well. The analysis of only culture
samples 1 to 12 is shown for E. faecalis M1. Samples: 1, E609L; 2, E609L/pPR6821; 3, E609L/pKN1; 4, E609L/pNK5; 5, E609L/pCR9;
6, E609L/pKE11; 7, E609L/pCS14; 8, E609L/pCY14; 9, E609L/pWR18; 10, E609L/pIT26; 11, E609L/pMT31; 12, E609L/pAD34; 13, E609L/pGE37; 14, E609L/pGR37; 15, E609L/pNK41; 16, E609L/pHL42; 17, E609L/pKE43; 18, E609L/pKN43; 19, E609L/pCW44; 20, JM109/pMBR1.0; 21, JM109/pMCS9; 22, JM109/pMCS14; 23, JM109/pMCS24; 24, JM109/pMCS44; 25, 1 µl of boiled
P. acidilactici LB42-923 culture broth containing wild-type
pediocin AcH. E609L/pKN1 and E609L/pAD34 had very slight activities
against both indicator strains.
|
|
The active mutants subsequently were subjected to titration assays to
measure levels of activity relative to that of the wild-type
chimeric
protein. As shown in Table
3, the active
mutants retained
from <1% (K1N, W18R, M31T, and A34D) to ~60%
(K43N) wild-type
activity against
L. innocua Lin11. The K11E
mutant actually exhibited
2.75-fold-greater activity than the wild-type
protein against
this strain.
L. innocua Lin11 was used in
titration assays because
it is relatively more sensitive to pediocin
AcH than the other
strains. The level of activity of the wild-type
culture broth
was calculated to be 1,000 activity units/ml because 1 µl was
sufficient to produce a small zone of growth inhibition
against
this indicator strain (Table
3). The activities of the mutants
were corrected for differences in the optical densities of cultures
and
levels of chimeric proteins in culture broths. Relative protein
synthesis levels varied by about twofold (Table
3), based on
an
analysis of SDS-polyacrylamide gels like the one shown in Fig.
3. Protein synthesis levels were lowest
for the K11E and H42L
mutants and highest for the I26T, M31T, and A34D
mutants.
View larger version (111K):
[in this window]
[in a new window]
|
FIG. 3.
SDS-polyacrylamide gel analysis of mutant MBP-pediocin
AcH chimeric protein synthesis levels. Proteins were visualized by
staining with Coomassie brilliant blue dye. The arrow marks the
position of migration of the MBP-pediocin AcH chimeric proteins.
Equivalent masses of total culture proteins were analyzed in each lane.
Lanes: 1, prestained standards; 2, E609L; 3, E609L/pPR6821; 4, E609L/pKN1; 5, E609L/pNK5; 6, E609L/pCR9; 7, E609L/pKE11; 8, E609L/pCS14; 9, E609L/pCY14; 10, E609L/pWR18; 11, E609L/pIT26; 12, E609L/pMT31; 13, E609L/pAD34; 14, E609L/pGE37; 15, E609L/pGR37; 16, E609L/pNK41; 17, E609L/pHL42; 18, E609L/pKE43; 19, E609L/pKN43; 20, E609L/pCW44. Molecular weights (in thousands) of prestained standards
are shown at the left. The levels of synthesis of chimeric proteins
varied by about twofold among the strains.
|
|
Experiments were performed to determine why the highly active K11E
mutant originally was picked up by colony overlay screening
as a null
mutant. As shown in Fig.
4, zones of
growth inhibition
typically do not form around E609L/pKE11 colonies
when IPTG is
added to the primary plating agar instead of to the agar
overlay.
This result indicates that zones of growth inhibition
typically
do not form when these colonies are growing at the time at
which
they are exposed to IPTG. In contrast, colonies of strains that
synthesize less potent chimeric proteins, such as the wild-type
or K43N
mutant proteins, actually show larger zones of growth
inhibition when
IPTG is added to the primary plating agar (Fig.
4). Taken together, the
data indicate that high-level synthesis
of the K11E mutant during the
growth of colonies causes a null
activity phenotype. It seems likely
that under these conditions,
cells within the colony lose the plasmid
and stop synthesizing
the K11E protein, which is toxic for the host
(data not shown).
Possibly, the original K11E mutant colony was
recovered from a
plate on which it was growing well at the time at
which it was
overlaid with top agar containing IPTG.
View larger version (101K):
[in this window]
[in a new window]
|
FIG. 4.
Effects of timing of IPTG induction on sizes of zones of
growth inhibition formed by MBP-pediocin AcH expression strains against
L. innocua Lin11. IPTG (1 mM) was added either in the
primary agar containing E. coli colonies or ~24 h later
(indicated by prime symbol) with the agar overlay containing the
indicator strain. Strains tested were E609L/pPR6821 (wild type; WT),
E609L/pKE11 (K11E mutant), E609L/pGE37 (G37E mutant), and E609L/pKN43
(K43N mutant). The diameters of zones of growth inhibition formed by
wild-type and mutant colonies differed depending on the time of
induction (see the text).
|
|
Analysis of the bactericidal activities of pediocin AcH cysteine
substitution mutants.
A serine was substituted for each of the
four cysteines in the native pediocin AcH molecule to determine if C24
is important for activity and to verify that C9, C14, and C44 are
required in both native and chimeric proteins. Substitution mutations
were introduced into the papA gene of plasmid pMBR1.0 by PCR
or oligonucleotide-directed site-specific mutagenesis. Mutant plasmids
were named pMCS9, pMCS14, pMCS24, and pMCS44 based on the location of
the substitution site. Pediocin AcH mutants were expressed in E. coli JM109, in which secretion is directed via the PapC-PapD
export machinery carried by pMBR1.0 (4).
All four substitution mutant strains grew well and grew at rates
comparable to that of the wild-type JM109/pMBR1.0 strain.
However, only
the wild-type strain showed zones of growth inhibition
in colony
overlay screening (data not shown) and in well assay
testing of culture
broths (Fig.
2). These results showed that
C24 is required for
activity. They also indicated that substitution
of cysteines causes the
same inhibitory effects in native and
chimeric forms of the
bacteriocin. Thus, it appears that the effects
of the mutations in
chimeric proteins can be extrapolated to the
native molecule. It is
possible, however, that the extent of inactivation
caused by some
mutations may vary between native and chimeric
molecules due to the
presence of the MBP domain.
| |
DISCUSSION |
Fourteen amino acids that are important for the activity of
pediocin AcH have been identified by random mutagenesis. The amino acids are distributed across the entire peptide chain, indicating that
little of its sequence may be dispensable for function. Mutations were
obtained at residues anticipated to be required based on sequence
conservation as well as at residues that could not be predicted to be
necessary. Some mutations within the N-terminal region (N5K, C9R, and
C14S/Y) mapped to amino acids that are proposed to stabilize the
structure of the molecule (5, 19). Several mutations were
obtained at positively charged amino acids (K1N, H42L, and K43N/E) and
at amino acids contained within a hydrophobic sequence in the
C-terminal region (I26T, M31T, A34D, and G37E/R) that may mediate
membrane binding (5, 12, 19). In about one half of the
cases, the mutants were completely inactive against several bacterial
strains, suggesting that the affected residues play central roles in
the mode of action of the bacteriocin. Only one mutation (A34D) may
have a species-specific effect on activity.
The experiments showed that all four cysteines in pediocin AcH are
necessary for activity. It seems likely that cysteines are required for
the formation of disulfide bonds, which stabilize the structure of this
short peptide. For example, the C9---C14 disulfide bond may be required
to establish the conformation of the intervening -G10-K11-H12-S13-
sequence that forms the apex of the putative hairpin within the
N-terminal region (5). As indicated in Table
4, this sequence exhibits weak homology
to six known types of consensus turns (18) and therefore
may not be able to maintain an active conformation without the
assistance of the C9---C14 disulfide bond. It may be possible to
construct an active pediocin AcH molecule lacking the C9---C14
disulfide bond if a strong consensus -turn sequence is substituted
for the G10-to-S13 region. There are consensus-type turns in which
lysine frequently occurs (18). It should be noted that the
substitution of glutamate for K11 neither creates nor destroys a strong
consensus turn in this sequence region (Table 4). The possible role
of the C24---C44 disulfide bond is discussed below.
The N5K mutation maps to a putative -turn sequence (-G4-N5-G6-V7-)
(5) within the hairpin that is 79% identical to a type
I' turn (Table 4). We have determined that two other sequences in
this region (-Y2-Y3-G4-N5- and -Y3-G4-N5-G6-) also have a high potential to form turns. Of the three sequences, -Y3-G4-N5-G6- exhibits the highest identity (97%) to a known type of turn, in
this case, a type II' turn (Table 4). While the calculations do not
definitively identify the type of turn present, they do support the
hypothesis that the N-terminal region is folded into a hairpin,
since both type I' and II' turns occur frequently in these
structures (18). In addition, the calculations show that the
substitution of lysine for asparagine moderately changes the identity
scores of the three turns (Table 4) and perhaps does not prohibit
their formation per se. However, the N5K mutation may perturb the
structure of this region due to charge repulsion between lysine and
other positively charged residues that may be located nearby in the
three-dimensional structure.
Experiments performed with magainin (34) and model peptides
(23) have indicated that positively charged amino acids are particularly important for the binding of moderately hydrophobic peptides, such as pediocin AcH (see below), to phospholipid bilayers. Of the four lysines and three histidines in the molecule, three residues (K1, H42, and K43) were found to be important for activity. As
shown in Table 3, the K1N substitution nearly fully inactivated the
molecule, whereas the H42L and K43N/E substitutions decreased activity
by ~40 to 80%. The results indicate that if these residues help
mediate membrane binding, then K1 plays an essential role whereas H42
and K43 do not. Instead, H42 and K43 may only augment the binding of
pediocin AcH to membranes. It should be possible to define the
functions of these positively charged residues by performing membrane
binding assays with the mutants. Binding experiments have been used to
show that K11 modulates the binding of pediocin PA-1 fragments to
membranes (6). While the experiments suggested that K11
participates in membrane binding, the finding that high activity was
conserved when glutamate was substituted for K11 indicated that K11
plays another, more important role in the function of the bacteriocin.
The C-terminal sequence region of pediocin AcH was subjected to
hydrophobicity analysis to locate nonpolar sequences that could mediate
membrane binding. The most hydrophobic stretch of amino acids in this
region is the 13-residue I25-to-G37 sequence, for which the average
sequence hydrophobicity is 0.45 (Table 5) (9). A 17-amino-acid sequence (A21 to G37) is only slightly less hydrophobic (hydrophobicity, 0.39). The hydrophobicities for both
sequences are slightly higher than those exhibited by a number of
surface-active peptides and are quite low compared to those exhibited
by sequences that are inserted individually into membranes with a
transmembrane orientation (9). However, either sequence
should be sufficiently hydrophobic to localize to the interior of a
phospholipid bilayer if two or more molecules are inserted into the
membrane together (9, 10), as may occur during the assembly
of bacteriocin pore complexes (19). While many questions
remain about the structure and function of the C-terminal region, the
mutagenesis results strongly suggest that the hydrophobicity of this
region is important for activity. In this regard, all mutations within
the I25-to-G37 region increased polarity (Table 5) and resulted in a
complete loss of activity (Table 3).
View this table:
[in this window]
[in a new window]
|
TABLE 5.
Effects of substitution mutations on average
hydrophobicities and average hydrophobic moments of selected
pediocin AcH sequence regionsa
|
|
As mentioned above, the nonpolar C-terminal region, which includes the
I25-to-G37 sequence, would be amphipathic if it were folded into an helix (12). Although the C24---C44 disulfide bond may
interfere with the formation of an helix, we nonetheless thought it
might be informative to calculate hydrophobic moments (9)
for putative -helical sequences within the C-terminal region (Table
5). The calculations indicated that the wild-type I25-to-G37 sequence
and the much less hydrophobic I25-to-H42 sequence are moderately
amphipathic if they are folded into helices. Although the
hydrophobic moments of these sequences are fairly low compared to those
of many surface-active peptides (18), this fact would not
necessarily preclude adsorption of these helices to a membrane,
since the hydrophobicities (0.75 to 0.78) of their nonpolar faces are
quite high. Interestingly, all mutations fell on one half of a helical
wheel diagram representing this sequence region and would overlap both
the nonpolar and the polar faces of the helices (Fig.
5). In all but one case (N41K),
substitutions changed the hydrophobic moments of the sequences (Table
5) and therefore could change the depth or angle of contact at which pediocin AcH adsorbs to the membrane interface. While the calculations and data make it tempting to conclude that the amphipathicity of this
region is important for activity, the effect of the C24---C44 disulfide
bond on the structure of this region must be determined before
conclusions about the role of sequence amphipathicity can be drawn.
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 5.
Schiffer-Edmundsen helical wheel analysis of the
I25-to-H42 sequence region of pediocin AcH. Amino acids in the
wild-type sequence are shown next to the wheel, and substitutions are
indicated in parentheses. Note that all substitution sites are located
on the bottom half of the helix and that the sites fall on both the
polar (white) and nonpolar (shaded) faces.
|
|
The final mutation to be discussed, W18R, occurred at a site located
between the N- and C-terminal regions of the molecule. The tryptophan
residue at this position is conserved in several pediocin-like
bacteriocins. W18 may penetrate into the acyl-chain region of membrane
phospholipids when the bacteriocin adsorbs to the interface
(7). We speculate that the W18R mutation could alter the
depth to which pediocin AcH adsorbs to the membrane interface. In this
regard, tryptophans located near the ends of integral membrane protein
transmembrane segments are thought to help establish the interfacial
boundaries of these segments (8).
In the future, models for the structure of pediocin AcH will be tested
further with these and related mutants. Biochemical analysis of the
mutants also should help clarify the mode of action of pediocin AcH and
may explain why it has greater potency and spectrum of activity than
other pediocin-like bacteriocins (19). In this regard, the
structure of pediocin AcH may differ substantially from those of other
pediocin-like bacteriocins due to its unique C24---C44 disulfide bond.
Finally, the finding that the K11E mutation increased potency
demonstrates that it is possible to improve the properties of pediocin
AcH and perhaps other bacteriocins by mutagenesis.
| |
ACKNOWLEDGMENTS |
We thank investigators for providing bacterial strains.
Financial support was provided by the National Science Foundation, the
state of Wyoming, and The Michigan Biotechnology Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address for Kurt W. Miller: Department of Molecular Biology, P.O. Box 3944, University of
Wyoming, Laramie, WY 82071-3944. Phone: (307) 766-2037. Fax: (307)
766-5098. E-mail: kwmiller{at}uwyo.edu. Mailing address for Bibek
Ray: Department of Animal Science, P.O. Box 3684, University of
Wyoming, Laramie, WY 82071-3684. Phone: (307) 766-3140. Fax: (307)
766-2350. E-mail: labcin{at}uwyo.edu.
Present address: Middle East Technical University, Ankara,
Turkey.
| |
REFERENCES |
| 1.
|
Amann, E.,
J. Brosius, and M. Ptashne.
1983.
Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli.
Gene
25:167-178[Medline].
|
| 2.
|
Bhunia, A. K.,
M. C. Johnson,
B. Ray, and N. Kalchayanand.
1991.
Mode of action of pediocin AcH from Pediococcus acidilactici H on sensitive bacterial strains.
J. Appl. Bacteriol.
70:25-30.
|
| 3.
|
Biswas, S. R.,
P. Ray,
M. C. Johnson, and B. Ray.
1991.
Influence of growth conditions on the production of bacteriocin, pediocin AcH, by Pediococcus acidilactici H.
Appl. Environ. Microbiol.
57:1265-1267[Abstract/Free Full Text].
|
| 4.
|
Bukhtiyarova, M.,
R. Yang, and B. Ray.
1994.
Analysis of the pediocin AcH gene cluster from plasmid pSMB74 and its expression in a pediocin-negative Pediococcus acidilactici strain.
Appl. Environ. Microbiol.
60:3405-3408[Abstract/Free Full Text].
|
| 5.
|
Chen, Y.,
R. Shapira,
M. Eisenstein, and T. J. Montville.
1997.
Functional characterization of pediocin PA-1 binding to liposomes in the absence of a protein receptor and its relationship to a predicted tertiary structure.
Appl. Environ. Microbiol.
63:524-531[Abstract].
|
| 6.
|
Chen, Y.,
R. D. Ludescher, and T. J. Montville.
1997.
Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles.
Appl. Environ. Microbiol.
63:4770-4777[Abstract].
|
| 7.
|
Chikindas, M. L.,
M. J. Garcia-Garcera,
A. J. M. Driessen,
A. M. Ledeboer,
J. Nissen-Meyer,
I. F. Nes,
T. Abee,
W. N. Konings, and G. Venema.
1993.
Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC 1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells.
Appl. Environ. Microbiol.
59:3577-3584[Abstract/Free Full Text].
|
| 8.
|
Cowan, S. W., and J. P. Rosenbusch.
1994.
Folding pattern diversity of integral membrane proteins.
Science
264:914-916[Free Full Text].
|
| 9.
|
Eisenberg, D.,
E. Schwarz,
M. Komaromy, and R. Wall.
1984.
Analysis of membrane and surface protein sequences with the hydrophobic moment plot.
J. Mol. Biol.
179:125-142[Medline].
|
| 10.
|
Engelman, D. M., and T. A. Steitz.
1981.
The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis.
Cell
23:79-88[Medline].
|
| 11.
|
Fath, M. J., and R. Kolter.
1993.
ABC-transporters: bacterial exporters.
Microbiol. Rev.
57:995-1017[Abstract/Free Full Text].
|
| 12.
|
Fimland, G.,
O. R. Blingsmo,
K. Sletten,
G. Jung,
I. F. Nes, and J. Nissen-Meyer.
1996.
New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity.
Appl. Environ. Microbiol.
62:3313-3318[Abstract].
|
| 13.
|
Hastings, J. W.,
M. Sailer,
K. Johnson,
K. L. Roy,
J. C. Vederas, and M. E. Stiles.
1991.
Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum.
J. Bacteriol.
173:7491-7500[Abstract/Free Full Text].
|
| 14.
|
Henderson, J. T.,
A. L. Chopko, and P. D. van Wassenaar.
1992.
Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0.
Arch. Biochem. Biophys.
295:5-12[Medline].
|
| 15.
|
Higuchi, R.
1990.
Recombinant PCR, p. 177-183.
In
M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. A guide to methods and applications. Academic Press, Inc., San Diego, Calif.
|
| 16.
|
Holck, A.,
L. Axelsson,
S.-E. Birkeland,
T. Aukrust, and H. Bloom.
1992.
Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake LB706.
J. Gen. Microbiol.
138:2715-2720[Abstract/Free Full Text].
|
| 17.
|
Holck, A.,
L. Axelsson,
K. Huhne, and L. Krockel.
1994.
Purification and cloning of sakacin 674, a bacteriocin from Lactobacillus sake LB674.
FEMS Microbiol. Lett.
115:143-150[Medline].
|
| 18.
|
Hutchinson, E. G., and J. M. Thornton.
1994.
A revised set of potentials for -turn formation in proteins.
Protein Sci.
3:2207-2216[Medline].
|
| 19.
|
Jack, R. W.,
J. R. Tagg, and B. Ray.
1995.
Bacteriocins of gram-positive bacteria.
Microbiol. Rev.
59:171-200[Abstract/Free Full Text].
|
| 20.
|
Kaiser, E. T., and F. J. Kezdy.
1984.
Amphiphilic secondary structure: design of peptide hormones.
Science
223:249-255[Abstract/Free Full Text].
|
| 21.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
|
| 22.
|
Leung, D. W.,
E. Chen, and D. V. Goeddel.
1989.
A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction.
Technique
1:11-15.
|
| 23.
|
Liu, L.-P., and C. M. Deber.
1997.
Anionic phospholipids modulate peptide insertion into membranes.
Biochemistry
36:5476-5482[Medline].
|
| 24.
|
Marugg, J. D.,
C. F. Gonzalez,
B. S. Kunka,
A. M. Ledeboer,
M. J. Pucci,
M. Y. Toonen,
S. A. Walker,
L. C. M. Zoetmulder, and P. A. Vandenbergh.
1992.
Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0.
Appl. Environ. Microbiol.
58:2360-2367[Abstract/Free Full Text].
|
| 25.
|
Miller, K. W.,
R. Schamber,
Y. Chen, and B. Ray.
1998.
Production of active chimeric pediocin AcH in Escherichia coli in the absence of processing and secretion genes from the Pediococcus pap operon.
Appl. Environ. Microbiol.
64:14-20[Abstract/Free Full Text].
|
| 26.
|
Motlagh, A. M.,
M. Bukhtiyarova, and B. Ray.
1994.
Complete nucleotide sequence of pSMB74, a plasmid encoding the production of pediocin AcH in Pediococcus acidilactici.
Lett. Appl. Microbiol.
18:305-312[Medline].
|
| 27.
|
Ray, B.
1996.
Characteristics and application of pediocin(s) of Pediococcus acidilactici: pediocin PA-1/AcH, p. 155-203.
In
T. F. Bozoglu, and B. Ray (ed.), Lactic acid bacteria: current advances in metabolism, genetics, and applications. Springer, New York, N.Y.
|
| 28.
|
Sailer, M.,
G. L. Helms,
T. Henkel,
W. P. Niemczura,
M. E. Stiles, and J. C. Vederas.
1993.
15N- and 13C-labeled media from Anabaena sp. for universal isotopic labeling of bacteriocins: NMR resonance assignments of leucocin A from Leuconostoc gelidum and nisin A from Lactococcus lactis.
Biochemistry
32:310-318[Medline].
|
| 29.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
In
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 30.
|
Taylor, J. W.,
J. Ott, and F. Eckstein.
1985.
The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.
Nucleic Acids Res.
13:8765-8785[Abstract/Free Full Text].
|
| 31.
|
Tichaczek, P. S.,
R. F. Vogel, and W. P. Hammes.
1993.
Cloning and sequencing of curA encoding curvacin A, the bacteriocin produced by Lactobacillus curvatus LTH1174.
Arch. Microbiol.
160:279-283[Medline].
|
| 32.
|
Tichaczek, P. S.,
R. F. Vogel, and W. Hammes.
1994.
Cloning and sequencing sakP encoding sakacin P, the bacteriocin produced by Lactobacillus sake LTH673.
Microbiology
140:361-367[Abstract/Free Full Text].
|
| 33.
|
Venema, K.,
J. Kok,
J. D. Marugg,
M. Y. Toonen,
A. M. Ledeboer,
G. Venema, and M. L. Chikindas.
1995.
Functional analysis of the pediocin operon of Pediococcus acidilactici PAC 1.0: PedB is the immunity protein and PedD is the precursor processing enzyme.
Mol. Microbiol.
17:515-522[Medline].
|
| 34.
|
Wieprecht, T.,
M. Dathe,
M. Beyermann,
E. Krause,
W. L. Maloy,
D. L. MacDonald, and M. Bienert.
1997.
Peptide hydrophobicity controls the activity and selectivity of magainin 2 amide in interaction with membranes.
Biochemistry
36:6124-6132[Medline].
|
| 35.
|
Yang, R.,
M. C. Johnson, and B. Ray.
1992.
Novel method to extract large amounts of bacteriocins from lactic acid bacteria.
Appl. Environ. Microbiol.
58:3355-3359[Abstract/Free Full Text].
|
| 36.
|
Yanisch-Perron, C.,
J. Vieira, and J. Messing.
1985.
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
Gene
33:103-119[Medline].
|
Appl Environ Microbiol, June 1998, p. 1997-2005, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Rihakova, J., Petit, V. W., Demnerova, K., Prevost, H., Rebuffat, S., Drider, D.
(2009). Insights into Structure-Activity Relationships in the C-Terminal Region of Divercin V41, a Class IIa Bacteriocin with High-Level Antilisterial Activity. Appl. Environ. Microbiol.
75: 1811-1819
[Abstract]
[Full Text]
-
Tominaga, T., Hatakeyama, Y.
(2007). Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins. Appl. Environ. Microbiol.
73: 5292-5299
[Abstract]
[Full Text]
-
Drider, D., Fimland, G., Hechard, Y., McMullen, L. M., Prevost, H.
(2006). The Continuing Story of Class IIa Bacteriocins. Microbiol. Mol. Biol. Rev.
70: 564-582
[Abstract]
[Full Text]
-
Tominaga, T., Hatakeyama, Y.
(2006). Determination of Essential and Variable Residues in Pediocin PA-1 by NNK Scanning. Appl. Environ. Microbiol.
72: 1141-1147
[Abstract]
[Full Text]
-
Brede, D. A., Faye, T., Stierli, M. P., Dasen, G., Theiler, A., Nes, I. F., Meile, L., Holo, H.
(2005). Heterologous Production of Antimicrobial Peptides in Propionibacterium freudenreichii. Appl. Environ. Microbiol.
71: 8077-8084
[Abstract]
[Full Text]
-
Johnsen, L., Dalhus, B., Leiros, I., Nissen-Meyer, J.
(2005). 1.6-A Crystal Structure of EntA-im: A BACTERIAL IMMUNITY PROTEIN CONFERRING IMMUNITY TO THE ANTIMICROBIAL ACTIVITY OF THE PEDIOCIN-LIKE BACTERIOCIN ENTEROCIN A. J. Biol. Chem.
280: 19045-19050
[Abstract]
[Full Text]
-
Johnsen, L., Fimland, G., Nissen-Meyer, J.
(2005). The C-terminal Domain of Pediocin-like Antimicrobial Peptides (Class IIa Bacteriocins) Is Involved in Specific Recognition of the C-terminal Part of Cognate Immunity Proteins and in Determining the Antimicrobial Spectrum. J. Biol. Chem.
280: 9243-9250
[Abstract]
[Full Text]
-
Xue, J., Hunter, I., Steinmetz, T., Peters, A., Ray, B., Miller, K. W.
(2005). Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in Listeria innocua, Identified by Screening for Pediocin AcH Resistance. Appl. Environ. Microbiol.
71: 1283-1290
[Abstract]
[Full Text]
-
Yamazaki, K., Suzuki, M., Kawai, Y., Inoue, N., Montville, T. J.
(2005). Purification and Characterization of a Novel Class IIa Bacteriocin, Piscicocin CS526, from Surimi-Associated Carnobacterium piscicola CS526. Appl. Environ. Microbiol.
71: 554-557
[Abstract]
[Full Text]
-
Brede, D. A., Faye, T., Johnsborg, O., Odegard, I., Nes, I. F., Holo, H.
(2004). Molecular and Genetic Characterization of Propionicin F, a Bacteriocin from Propionibacterium freudenreichii. Appl. Environ. Microbiol.
70: 7303-7310
[Abstract]
[Full Text]
-
Morisset, D., Berjeaud, J.-M., Marion, D., Lacombe, C., Frere, J.
(2004). Mutational Analysis of Mesentericin Y105, an Anti-Listeria Bacteriocin, for Determination of Impact on Bactericidal Activity, In Vitro Secondary Structure, and Membrane Interaction. Appl. Environ. Microbiol.
70: 4672-4680
[Abstract]
[Full Text]
-
Gibbs, G. M., Davidson, B. E., Hillier, A. J.
(2004). Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126. Appl. Environ. Microbiol.
70: 3292-3297
[Abstract]
[Full Text]
-
Johnsen, L., Fimland, G., Mantzilas, D., Nissen-Meyer, J.
(2004). Structure-Function Analysis of Immunity Proteins of Pediocin-Like Bacteriocins: C-Terminal Parts of Immunity Proteins Are Involved in Specific Recognition of Cognate Bacteriocins. Appl. Environ. Microbiol.
70: 2647-2652
[Abstract]
[Full Text]
-
Simon, L., Fremaux, C., Cenatiempo, Y., Berjeaud, J. M.
(2002). Sakacin G, a New Type of Antilisterial Bacteriocin. Appl. Environ. Microbiol.
68: 6416-6420
[Abstract]
[Full Text]
-
Fimland, G., Eijsink, V. G. H., Nissen-Meyer, J.
(2002). Comparative studies of immunity proteins of pediocin-like bacteriocins. Microbiology
148: 3661-3670
[Abstract]
[Full Text]
-
Kazazic, M., Nissen-Meyer, J., Fimland, G.
(2002). Mutational analysis of the role of charged residues in target-cell binding, potency and specificity of the pediocin-like bacteriocin sakacin P. Microbiology
148: 2019-2027
[Abstract]
[Full Text]
-
Uteng, M., Hauge, H. H., Brondz, I., Nissen-Meyer, J., Fimland, G.
(2002). Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium. Appl. Environ. Microbiol.
68: 952-956
[Abstract]
[Full Text]
-
Le Marrec, C., Hyronimus, B., Bressollier, P., Verneuil, B., Urdaci, M. C.
(2000). Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by Bacillus coagulans I4. Appl. Environ. Microbiol.
66: 5213-5220
[Abstract]
[Full Text]
-
Johnsen, L., Fimland, G., Eijsink, V., Nissen-Meyer, J.
(2000). Engineering Increased Stability in the Antimicrobial Peptide Pediocin PA-1. Appl. Environ. Microbiol.
66: 4798-4802
[Abstract]
[Full Text]
-
Mora, D., Fortina, M. G., Parini, C., Daffonchio, D., Manachini, P. L.
(2000). Genomic subpopulations within the species Pediococcus acidilactici detected by multilocus typing analysis: relationships between pediocin AcH/PA-1 producing and non-producing strains. Microbiology
146: 2027-2038
[Abstract]
[Full Text]
-
Fimland, G., Johnsen, L., Axelsson, L., Brurberg, M. B., Nes, I. F., Eijsink, V. G. H., Nissen-Meyer, J.
(2000). A C-Terminal Disulfide Bridge in Pediocin-Like Bacteriocins Renders Bacteriocin Activity Less Temperature Dependent and Is a Major Determinant of the Antimicrobial Spectrum. J. Bacteriol.
182: 2643-2648
[Abstract]
[Full Text]
-
Ray, B., Schamber, R., Miller, K. W.
(1999). The Pediocin AcH Precursor Is Biologically Active. Appl. Environ. Microbiol.
65: 2281-2286
[Abstract]
[Full Text]
Top
Abstract
Introduction
