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(Received for publication, July 7, 1995) From the
The post-translationally modified, antimicrobial peptide nisin
is secreted by strains of Lactococcus lactis that contain the
chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG. When a 4-base pair deletion is introduced into
the structural nisA gene (
Nisin is an antimicrobial peptide (1, 2, 3) widely used in the food industry as
a safe and natural preservative. The ribosomally synthesized precursor
peptide undergoes extensive post-translational modification, which
includes dehydration of serine and threonine residues and the formation
of thioether bridges called (
Figure 1:
A, organization of the nisin gene
cluster. Established (nisAIPRKFEG) and putative (nisBCT) functions of the gene products have been indicated. P denotes a mapped promoter, and IR denotes an
extensive inverted repeat sequence that could act as a rho-independent
terminator(7) . B, schematic outline of the
biosynthesis of nisin A. Rings are labeled A-E. Asterisks indicate residues that will be modified. The black arrow indicates processing of the N-terminal Met
residue, while the small white arrow indicates processing of
the leader peptide by the action of NisP(8) . Dha,
dehydroalanine; Dhb,
dehydrobutyrine.
The proteins encoded by nisR(8) and nisK(10) have shown to
be involved in the regulation of nisin
biosynthesis(8, 10) . NisR is a response regulator,
and NisK is a sensor histidine kinase which belong to the class of
two-component regulatory systems (14, 15, 16) . When the genes nisABTCIR are present on a multicopy plasmid, production of fully modified
precursor nisin is observed, indicating that overexpression of nisR alone is sufficient to activate transcription of nisA and
obviously also of the biosynthetic genes downstream by partially
reading through an inverted repeat sequence (Fig. 1A)(8) . This observation is similar to
the regulation of expression of iep and degU genes in Bacillus subtilis, where overexpression of the response
regulator activates transcription of the target genes(17) , and
to the case of overexpression of epiQ, which encodes a
response regulator involved in the biosynthesis of the lantibiotic
epidermin(18) . When only the genes nisABTCI are
present on a multicopy plasmid (pNZ9000) in Lactococcus lactis MG1614, no transcription of nisA is observed(9) .
Two gene products have been identified for the regulation of the
biosynthesis of the related lantibiotic subtilin(19) , which
also belong to the class of two-component regulators, i.e. SpaR, the response regulator, and SpaK, the sensor histidine
kinase(20, 21) . Upon disruption of either of these
genes, subtilin production was abolished, indicating the involvement of
these gene products in subtilin biosynthesis(20) . The
regulation was shown to be growth-phase dependent, but an inducing
signal was not identified(20, 21) . While the
structure and function of two-component regulators have been studied in
great detail(14, 15, 16) , the nature of the
inducing signal has remained unclear in many cases. It is demonstrated
here that fully modified nisin can induce the transcription of its own
structural gene as well as of the downstream genes by limited
read-through, via signal transduction, by acting as the extracellular
signal for the sensor histidine kinase NisK.
The nisA promoter
region including part of the nisA gene was isolated as a
1442-bp BglII-Ecl136II fragment from plasmid
pNZ9000(8) . This fragment was cloned into pNZ273, containing
the promoterless gusA gene(24) , which had been
digested with BglII and ScaI, generating plasmid
pNZ8003. Part of the upstream promoter region was deleted by digesting
pNZ8003 with BglII and Tth111I. These sites were made
blunt by Klenow polymerase and ligated, generating plasmid pNZ8008,
which eventually contained a 312-bp nisA promoter fragment in
front of the gusA gene. Another part of the nisA promoter region, including the full nisA gene and the
first part of the nisB gene, was isolated as a 1904-bp BglII-MunI fragment from plasmid pNZ9000. This
fragment was cloned into pNZ273(24) , which had been digested
with BglII and EcoRI, generating plasmid pNZ8002. A
1442-bp BglII-Ecl136II promoter fragment was deleted
in pNZ8002, generating pNZ
The nisB gene was disrupted by introducing a 162-bp in-frame deletion into
the middle of the gene. This was accomplished by cloning a 4.4-kilobase
pair BglII-EcoRI fragment, containing nisB and surrounding regions from the nisin gene cluster, into a BamHI-EcoRI-digested pUC19 vector, which harbored an
additional erythromycin resistance marker, as has been described
previously(9) . The deletion was made by removing an internal HpaI fragment from the nisB gene and subsequent
ligation. The resulting plasmid was named pNZ9135 and was used for
transformation of L. lactis NZ9700. Following transformation,
erythromycin-resistant colonies were obtained that had integrated the
plasmid by recombination of the plasmid with one of the flanking
regions of the deleted fragment. After growing for 200 generations in
the absence of erythromycin and plating, a colony was obtained that was
sensitive to erythromycin. This had apparently been caused by a second
recombination event involving the flanking region on the other side of
the deletion than the side of the first recombination event, resulting
in the replacement of nisB with
Northern blotting showed that in
strain NZ9800, the transcript of
Figure 2:
Northern blot prepared using nisA as a probe of RNA from several uninduced lactococcal cultures and
cultures induced with different amounts of nisin A or unmodified
precursor nisin A or with the lantibiotic Pep5. Lane 1, NZ9700
(nisin A producer); lane 2, MG1614; lanes 3-7,
NZ9800 with nisin A (0, 1, 2.5, 10, and 50 ng/ml, respectively); lanes 8 and 9, NZ9800
Figure 3:
Dose
response of purified (mutant) nisins as inducers of gusA expression in L. lactis strain NZ9800 harboring pNZ8008.
Nisin species were as follows:
In further experiments, the nisin-producing
strain NZ9700 with either plasmid pNZ273 (containing the promoterless gusA gene) or pNZ8008 (containing the nisA promoter
fragment followed by the gusA gene) was used in an agar
diffusion assay (8) to determine the amount of nisin produced.
Fifty times lower nisin production and severely reduced immunity were
observed when plasmid pNZ8008 was present compared with the situation
where pNZ273 was present. This can be explained by titration of the
response regulator NisR by the multicopy presence of the nisA promoter region containing the putative NisR-binding site.
Figure 4:
Model for nisin biosynthesis and
regulation. In Step 1, NisK senses the presence of nisin in the medium
and autophosphorylates. In Step 2, the phosphate group is transferred
to NisR, which acts as a transcriptional activator, followed by mRNA
synthesis and ribosomal synthesis of unmodified precursor nisin and of
biosynthetic proteins. In Step 3, the precursor is modified by the
putative enzymes NisB and NisC(7, 9) . In Step 4, the
fully modified precursor peptide is translocated across the membrane by
the putative ABC transporter NisT(7, 9) . In Step 5,
fully modified precursor nisin is extracellularly processed by
NisP(8) , resulting in the release of active nisin.
NisI(9) , together with NisF, NisE, and NisG(11) ,
protects the cell from the bacteriocidal action of nisin by a thus far
unknown mechanism.
Mutants of nisin or
precursors of nisin that have the leader peptide attached to the mature
lantibiotic (second molecule shown in Fig. 1B) can also
act as inducers, whereas other antimicrobial peptides are incapable of
induction. The presence of the modified residues is of crucial
importance for induction capacity, especially those present in the
N-terminal part of nisin. To our knowledge, this is the only report of
peptides that can induce transcription of their own structural gene via
signal transduction. Interestingly, a recent report on syndecan
biosynthesis in mice, which plays a role in wound repair, describes the
role of the antimicrobial peptide PR39 in induction of syndecan gene
transcription(44) , although the amount of inducer needed (0.5
mM) is at least a factor of 10,000 higher than for nisin (30
pM). This suggests that the role of antimicrobial peptides in
nature might be broader than just the antagonistic action because in
some cases these peptides can also act as signals for transcription
activation of their own structural gene or of other genes. There may be
several evolutionary reasons for the autoregulation of nisin gene
transcription via signal transduction, e.g. (i) to save energy
by control of the integrity of the gene cluster since any dysfunctional
biosynthetic gene will abolish inducer formation and thus expression of
biosynthetic genes; (ii) to raise immunity levels in response to high
nisin production by neighboring cells, in other words, to amplify the
response to environmental signals; or (iii) to promote cell to cell
communication that allows the production of antimicrobial peptides in
high quantities in a concerted action, thereby decreasing the chance of
resistance development in target organisms.
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 27299-27304
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
nisA), transcription
of nisA is abolished. Transcription of the nisA gene is restored by adding subinhibitory amounts of nisin, nisin
mutants, or nisin analogs to the culture medium, but not by the
unmodified precursor peptide or by several other antimicrobial
peptides. Upon disruption of the nisK gene, which encodes a
putative sensor protein that belongs to the class of two-component
regulators, transcription of nisA was no longer inducible
by nisin. Fusion of a nisA promoter fragment to the
promoterless reporter gene gusA resulted in expression of gusA in L. lactis NZ9800 (nisA) only
upon induction with nisin species. The expression level of gusA was directly related to the amount of inducer that was added
extracellularly. These results provide insight into a new mechanism of
autoregulation through signal transduction in prokaryotes and
demonstrate that antimicrobial peptides can exert a second function as
signaling molecules.
-methyl)lanthionines, resulting in
five ring structures named A, B, C, D, and E (Fig. 1B).
Peptides containing these characteristic modified residues are named
lantibiotics(4) . Eleven genes organized in a cluster have been
implicated to be involved in the complex biosynthesis of nisin, i.e. nisABTCIPRKFEG (Fig. 1A)(5, 6, 7, 8, 9, 10, 11) .
Of these genes, nisA encodes the nisin A precursor peptide of
57 amino acid residues; nisB and nisC encode putative
enzymes involved in the post-translational modification reactions
(based on homology to genes found exclusively in other lantibiotic gene
clusters); nisT encodes a putative transport protein of the
ABC translocator family that is probably involved in the extrusion of
modified precursor nisin(7, 9) ; nisP encodes
an extracellular subtilisin-like protease involved in precursor
processing (8) ; nisI encodes a lipoprotein involved
in the producer self-protection against nisin(9) ; and nisFEG encodes putative transporter proteins that have also
been implied in immunity (11) . A schematic representation of
the post-translational events yielding mature nisin A is shown in Fig. 1B. Nisin Z is a natural variant of nisin A that
contains an asparagine residue at position 27 instead of the histidine
residue found in nisin A(12) . Both nisin A- and nisin
Z-producing strains are common in nature, and both structural genes (nisA and nisZ) have been
cloned(5, 6, 13) .
Strains and Plasmids
L. lactis strains
MG1614 (22) , NZ9700 (a nisin-producing transconjugant
containing Tn5276)(23) , and NZ9800 (a derivative of
NZ9700 in which the nisA gene has been exchanged by
replacement recombination with a modified nisA gene containing
a 4-bp (
)deletion in the pronisin-encoding part (nisA) and which is therefore no longer able to produce
nisin A) have been described previously(9) . L. lactis strains were cultivated without aeration at 30 °C in M17 broth
(Difco) supplemented with 0.5% (w/v) glucose (GM17) or sucrose (SM17).
For L. lactis strains harboring pNZ273-derived
plasmids(24) , media were supplemented with 10 µg/ml
chloramphenicol. Expression plasmids pNZ9010 and
pNZ9013(9, 25) , containing the nisA and nisZ genes, respectively, under control of the efficient lac promoter, were introduced into L. lactis strain
NZ9800, leading to the production of nisin A or nisin Z in similar
amounts as in L. lactis wild-type strain NZ9700(25) .
As a host strain for cloning experiments, Escherichia coli strain MC1061 (26) was used.8002, by making the BglII site
blunt with Klenow polymerase and subsequent ligation to the Ecl136II site. All constructs were initially made in E.
coli MC1061(26) . Plasmids pNZ8008, pNZ8002, and
pNZ8002 were used to transform L. lactis NZ9700 and L. lactis NZ9800(9) , and transformants were obtained
by selecting for resistance to chloramphenicol.DNA Techniques and DNA Sequence
Analysis
Restriction enzymes and other DNA-modifying enzymes
were purchased from Life Technologies, Inc. or U. S. Biochemical Corp.
and used as recommended by the manufacturers. The DNA sequence of the nisZ region was determined on the double-stranded plasmid DNA
with the HindIII primer by the chain termination
method(27) . Transcription analyses of the nisA and nisA genes were performed by isolation of RNA from L.
lactis strains NZ9700 and NZ9800, Northern blotting, and
subsequent hybridization with a radiolabeled nisA probe as
described previously(9) . RNA isolation was performed 2 h after
induction of a culture with an A of 0.5. RNA
(20 µg) was loaded in each lane, and the amounts were estimated by
comparing the intensity of the 16 S and 23 S RNA bands.
Inactivation of Chromosomal nisK and nisB by Gene
Replacement
The chromosomal copy of the nisK gene was
inactivated by introduction of an erythromycin resistance gene (28) into the open reading frame of nisK. For this
purpose, a 2.8-kilobase pair HindIII-EcoRI
chromosomal DNA fragment from strain NZ9700 containing the 3`-part of
the nisP gene and the intact nisR and nisK genes was cloned into pUC19. The erythromycin resistance gene was
introduced into a unique SalI site, resulting in the
interruption of the nisK open reading frame between the
encoded amino acids 9 and 10 of NisK and leaving 1.1 and 1.7 kilobase
pairs of flanking regions at the 5`- and 3`-ends, respectively. This
construct was designated pNZ9150. Strain NZ9800 was transformed with
the nonreplicating plasmid pNZ9150, and integrants were selected on
plates containing erythromycin (2.5 µg/ml). The selected integrants
were analyzed by polymerase chain reaction using primers
5`-CGGTCAATCTCGGAG-3` and 5`-CGCTTTGTAATCATTTTCATC-3` and by Southern
hybridization using pUC19 DNA (29) as a probe. In one of the
strains (NZ9850), the erythromycin resistance gene had been integrated
via gene replacement at the 3`- and 5`-flanking regions, introducing
this gene into the open reading frame of the chromosomal copy of the nisK gene, in the absence of any pUC19 sequences. The
resulting construct was further analyzed by polymerase chain reaction
of the nisK region and by Southern blotting using nisK as a probe to confirm the desired configuration.nisB on the
chromosome. The configuration of the desired construct was confirmed by
polymerase chain reaction analysis of the nisB region with the
deletion and by Southern analysis of BglII-digested
chromosomal DNA. The desired strain was called NZ9700 nisB.Production, Purification, and Characterization of Nisin
Mutants
Mutants of nisin Z were produced as described previously (25) . The primers used for site-directed mutagenesis of the nisZ gene were as follows:
5`-CAGGTGCATCACCACGCTGGACAAGTATTTCGCTATGTAC-3` (I1W),
5`-CACCACGCATTTCAAGTATTTCGCTATG-3` (T2S),
5`-CACCACGCATTACAACAATTTCGCTATGTACACCC-3` (S3T),and
5`-AACAGGAGCTCTGTGGGGTTGTAACATG-3` (M17W) (mutated nucleotides
are indicated in boldface). All mutants were purified to homogeneity,
and the structures of the modified residues were confirmed by one- and
two-dimensional H NMR(25) . It was established that
T2S nisin Z contains a dehydroalanine residue at position 2, S3T nisin
Z has a -methyllanthionine ring between residues 3 and 7 instead
of a lanthionine, and I1W nisin Z and M17W nisin Z contain a Trp
residue at positions 1 and 17, respectively. Precursor nisin Z,
containing the subtilin leader peptide (sl-nisin Z), was obtained as
described before(30) . Purified lacticin 481 was isolated as
described previously(31, 32) . Purified Pep5 (33) was obtained from Dr. H.-G. Sahl (Bonn, Germany).
Unmodified precursor nisin A was obtained from the laboratory of Dr. G.
Jung (Tübingen, Germany). Preparations of subtilin (19) and lactococcin A (34) consisted of culture
supernatants of producing strains, which were confirmed to possess
substantial antimicrobial activity in agar diffusion assays.
Antimicrobial activities against L. lactis MG1614 were
determined essentially as described before for Micrococcus
flavus(25) . L. lactis was cultured in GM17 broth
at 30 °C with an initial A of 0.025, and
outgrowth was measured when the culture without nisin had reached an A
of 0.8.
Lactococcal cells (1
ml) were harvested at 1.5 h after induction with nisin (or mutants or
fragments or other antimicrobial species) and adjusted in NaP-Glucuronidase Assays
buffer (50 mM NaHPO
, pH 7.0) to a final A of 2.0. The cells were permeabilized by
adding 50 µl of acetone/toluene (9:1) per ml of cells followed by
10 min of incubation at 37 °C. The extracts were used immediately
in the assay. For the determination of
-glucuronidase activity, 40
µl of extract was added to 950 µl of buffer (50 mM NaHPO, pH 7.0, 10 mM -mercaptoethanol, 1
mM EDTA, 0.1% Triton X-100) and 10 µl of 100 mMp-nitro--D-glucuronic acid (CLONTECH, Palo Alto,
CA). The mixture was incubated, and the increase in A was measured at 37 °C. Histochemical screening for gusA positive colonies was performed by including
5-bromo-4-chloro-3-indolyl glucuronide (Research Organics Inc.,
Cleveland, OH) at a final concentration of 0.5 mM in GM17
plates(24) . Purified nisin fragments (35, 36, 37, 38, 39) were
obtained from Prof. T. Shiba (Protein Research Foundation, Osaka,
Japan).
Transcription Analyses of
The promoter sequence and the
transcription start site of nisA have been identified, and a
transcript of nisA in the Presence and
Absence of Nisin or Nisin Mutants260 nucleotides has been demonstrated in L.
lactis strain NZ9700, which contains Tn5276(9) .
It has also been found that transcription of nisA is dependent
on the integrity of nisA itself since a 4-bp deletion in the
middle of the nisA gene (
nisA) on the chromosome
of L. lactis strain NZ9800 completely abolishes transcription
of this gene(9) . For further transcription analyses of the
structural and biosynthetic genes, a series of isogenic lactococcal
strains was used, including the nisin-producing NZ9700 and
non-nisin-producing NZ9800 strains.nisA was absent, but
after adding small amounts of nisin A to the culture medium at an A of 0.5, nisA transcripts appeared
again (Fig. 2). Interestingly, the amount of these transcripts
was dependent on the amount of nisin A added (Fig. 2, lanes
3-7). Several other related peptides were able to induce
transcription, such as nisin Z and various nisin Z mutants, i.e. T2S nisin Z, S5T nisin Z(25) , M17W nisin Z, S3T nisin Z,
and sl-nisin Z, a fully modified nisin Z species that has the subtilin
leader peptide still attached(30) . However, the last two
species were >100-fold less effective inducers compared with nisin Z
(data not shown). In contrast, the T2S and M17W nisin Z mutants were
more potent inducers than nisin Z. These findings demonstrate that the
modified lantibiotic part plays an important role in the induction
process. Interestingly, several less related peptides evoked no
restoration of transcription, i.e. the unmodified synthetic
nisin A precursor of 57 amino acid residues (Fig. 1B and Fig. 2, lane 12), the 56% homologous
lantibiotic subtilin (19) , the lantibiotic lacticin
481(31, 32) , the lantibiotic Pep5 (Fig. 2, lane 13)(33) , and the antimicrobial peptide
lactococcin A (34) (data not shown for subtilin, lacticin 481,
and lactococcin A).
nisK with nisin A
(0 and 2.5 ng/ml, respectively); lanes 10 and 11,
NZ9700 nisB with nisin A (0 and 2.5 ng/ml, respectively); lane 12, NZ9800 with unmodified precursor nisin A (1000
ng/ml); lane 13, NZ9800 with Pep5 (1000
ng/ml).
Determination of the Induction Capacity of Nisin
(Mutants) by Use of the Nisin Promoter Fragment Fused to the Reporter
Gene gusA
To obtain a more quantitative assay of induction
capacity and to investigate whether the nisA promoter could be
used to regulate expression of heterologous genes in L.
lactis, a nisin promoter fragment of 312 bp containing part of the nisA structural gene was fused to the promoterless reporter
gene gusA of E. coli on plasmid pNZ273(24) .
This construct, named pNZ8008, was used to transform strain NZ9800. The
resulting strain was assayed for -glucuronidase activity with and
without induction by (mutant) nisins or other antimicrobial peptides.
Without induction, -glucuronidase activity could not be
demonstrated, whereas wild-type nisin A and nisin Z effectively induced
-glucuronidase activity (Fig. 3). Moreover, the T2S and
M17W nisin Z mutants were found to induce higher expression of gusA compared with wild-type nisin A and nisin Z, whereas the S3T and
I1W nisin Z mutants were found to have lower induction capacity (Fig. 3). It was calculated that <5 molecules of the best
inducer (T2S nisin Z) per cell are sufficient to activate
transcription, which illustrates the high efficiency characteristic of
signal transduction processes. Concordant with the transcription
analyses, some of the antimicrobial peptides tested did not elicit
induction of gusA expression (i.e. the unmodified
synthetic nisin A precursor peptide, subtilin, lacticin 481, and
lactococcin A). There is no direct relationship between antimicrobial
activity of the nisin mutants against L. lactis strain MG1614
and their induction capacity (Table 1). The difference in potency
can be attributed to the observation that antimicrobial activity is
dependent on pore-forming activity in
membranes(40, 41, 42) , while induction
capacity is likely to be dependent on interaction (directly or
indirectly) with NisK.
, T2S nisin Z; 
, M17W
nisin Z; 
, wild-type nisin Z; , S3T nisin Z;
, I1W
nisin Z. Standard errors were <20% for each given value. A.U., arbitrary units.
Structural Requirements of the Inducer Molecule Tested by
Use of Synthetic Nisin Fragments
More detailed insight into the
minimal structural requirements of the inducer molecule was obtained by
using synthetic nisin A fragments (35, 36, 37, 38, 39) in the gusA reporter assay (Table 1). The minimal requirement
for retaining induction capacity (2% induction of that of nisin A) was
the presence of residues 1-11 of nisin A, comprising the first
two rings. Addition of the third ring enhanced induction (8-30%
induction), whereas a severe decrease in induction was caused by
deleting the N-terminal residues Ile-1 and dehydrobutyrine 2
(0-1% induction) (Table 1). Fragments that contained rings
B and C or rings D and E (for nomenclature of rings, see Fig. 1B) were not capable of acting as a signal
effector. Thus, the most probable site of molecular interaction with
the sensor protein NisK will be residues 1-11 of the nisin
molecule.Requirement of nisK Expression for Signal
Transduction
The sequence of the nisK gene located on
Tn5276 has been reported (43) and was found to be
identical to that of nisK from L. lactis 6F3(10) . The chromosomal nisK gene was
insertionally inactivated by introduction of an erythromycin resistance
gene (28) into strain NZ9800, yielding strain NZ9850. As
expected, transcription of nisA was no longer inducible
by any of the nisin species (Fig. 2, lanes 8 and 9). Nisin production in strain NZ9850 could not be restored by
introduction of plasmid pNZ9010 (nisA) or pNZ9013 (nisZ), whereas it could be restored in strain NZ9800. Since
the immunity level of strain NZ9850 is similar to that of strain MG1614
(0.01 µg of nisin A/ml), induction experiments were performed with
amounts of nisin well below this level. Under these conditions, normal
growth of the cells was observed. Strain NZ9850 was also transformed
with pNZ8008, but after induction with 0.0005-0.0025 µg of
nisin A/ml, no -glucuronidase activity could be measured (<0.3
arbitrary unit), indicating at least 200 times lower expression than in
strain NZ9800 containing pNZ8008, with the same inducer concentrations (Fig. 3). No polar effects of the nisK disruption on
expression of the nisFEG genes downstream of nisK are
expected since a promoter has been indicated in front of nisFEG(11) . (
)Moreover, the nisR and nisK genes have been integrated on the chromosome of
strain MG1614 by replacement recombination, and the resulting strain
was transformed by pNZ8008. In this strain, gusA expression
was inducible by nisin species (data not shown), proving that only nisR and nisK are required for signal transduction.
These results clearly demonstrate that NisK is essential in the signal
transduction pathway and probably interacts directly with nisin itself. Effects of Disruption of nisB on Transcription of nisA
and Downstream Genes
An in-frame deletion in the nisB gene of L. lactis strain NZ9700, made by replacement
recombination, abolished nisin production as well as transcription of nisA, demonstrating that a hampered biosynthesis of nisin
abolishes transcription of nisA. In this case, transcription
of nisA could be restored by addition of nisin to the cells (Fig. 2, lane 11), probably because of the presence of
intact nisR and nisK genes, which have their own
promoter. The transcription start site of nisRK was mapped by
primer extension and shown to be an A nucleotide 26 nucleotides
upstream of the start codon of nisR (position 2117 in the
nucleotide sequence published in (8) ). To probe the influence
of a large inverted repeat sequence located in the intergenic region
between nisA and nisB on expression of genes
downstream of nisA (Fig. 1A), another
plasmid was constructed (pNZ8002) in which the nisin promoter fragment
including nisA as well as the intergenic region and the
first part of nisB was fused to the gusA gene. This
plasmid was able to direct expression of gusA in strain NZ9800
only after induction with nisin species, albeit to an 50-fold
reduced level relative to gusA expression in pNZ8008 in strain
NZ9800. When the nisin promoter fragment was removed from pNZ8002,
yielding pNZ
8002, -glucuronidase activity was completely
abolished, even in the presence of an inducer. These results show that
expression of at least one downstream gene, i.e. nisB, is
coregulated and is dependent on the presence of the nisA promoter. Most likely, expression of the other downstream genes nisTCIP limited read-through is also dependent on the nisA promoter since a significant increase in immunity levels, for
which NisI is partially responsible(9) , was found in the
induced state relative to the uninduced state of strain NZ9800.
Moreover, no apparent promoter sequences were found in front of any of
the genes nisBTCIP.Conclusion
We have demonstrated that transcription
of nisA is autoregulated, not intracellularly by its direct
translation product, but extracellularly by the secreted and fully
modified peptide via signal transduction by a two-component regulatory
system. A model based on previous work(5, 6, 7, 8, 9, 10, 11) and on
this study shows the possible sequence of events with regard to nisin
biosynthesis and regulation (Fig. 4).
))
We thank Dr. T. Shiba, Dr. K. Fukase, and Dr. T.
Wakamiya for the generous gift of synthetic nisin fragments; Dr. G.
Jung and Dr. A. Beck-Sickinger for the generous gift of unmodified
synthetic precursor nisin A; and Dr. H.-G. Sahl for the generous gift
of Pep5. We thank Roger Bongers and Patrick van den Bogaard for
construction of several mutant nisin genes, Hans Kosters for purifying
and characterizing some of the mutants, and Lilian Vreede for
determination of antimicrobial activities of mutants against L.
lactis MG1614. We are grateful to Bruce Chassy, Roland Siezen,
Joey Marugg, and Elaine Vaughan for critically reading the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Y. Liu, L. Zeng, and R. A. Burne AguR Is Required for Induction of the Streptococcus mutans Agmatine Deiminase System by Low pH and Agmatine Appl. Envir. Microbiol., May 1, 2009; 75(9): 2629 - 2637. [Abstract] [Full Text] [PDF]
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 G. Liu, J. Zhong, J. Ni, M. Chen, H. Xiao, and L. Huan Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50 Microbiology, February 1, 2009; 155(2): 584 - 593. [Abstract] [Full Text] [PDF]
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A. Fernandez, N. Horn, U. Wegmann, C. Nicoletti, M. J. Gasson, and A. Narbad Enhanced Secretion of Biologically Active Murine Interleukin-12 by Lactococcus lactis Appl. Envir. Microbiol., February 1, 2009; 75(3): 869 - 871. [Abstract] [Full Text] [PDF]
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H. B. van den Berg van Saparoea, P. J. Bakkes, G. N. Moll, and A. J. M. Driessen Distinct Contributions of the Nisin Biosynthesis Enzymes NisB and NisC and Transporter NisT to Prenisin Production by Lactococcus lactis Appl. Envir. Microbiol., September 1, 2008; 74(17): 5541 - 5548. [Abstract] [Full Text] [PDF]
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R. Rink, J. Wierenga, A. Kuipers, L. D. Kluskens, A. J. M. Driessen, O. P. Kuipers, and G. N. Moll Dissection and Modulation of the Four Distinct Activities of Nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation Appl. Envir. Microbiol., September 15, 2007; 73(18): 5809 - 5816. [Abstract] [Full Text] [PDF]
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 P. Williams, K. Winzer, W. C Chan, and M. Camara Look who's talking: communication and quorum sensing in the bacterial world Phil Trans R Soc B, July 29, 2007; 362(1483): 1119 - 1134. [Abstract] [Full Text] [PDF]
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 B. Li and W. A. van der Donk Identification of Essential Catalytic Residues of the Cyclase NisC Involved in the Biosynthesis of Nisin J. Biol. Chem., July 20, 2007; 282(29): 21169 - 21175. [Abstract] [Full Text] [PDF]
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I. Hernandez, D. Molenaar, J. Beekwilder, H. Bouwmeester, and J. E. T. van Hylckama Vlieg Expression of Plant Flavor Genes in Lactococcus lactis Appl. Envir. Microbiol., March 1, 2007; 73(5): 1544 - 1552. [Abstract] [Full Text] [PDF]
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O. Hyink, P. A. Wescombe, M. Upton, N. Ragland, J. P. Burton, and J. R. Tagg Salivaricin A2 and the Novel Lantibiotic Salivaricin B Are Encoded at Adjacent Loci on a 190-Kilobase Transmissible Megaplasmid in the Oral Probiotic Strain Streptococcus salivarius K12 Appl. Envir. Microbiol., February 15, 2007; 73(4): 1107 - 1113. [Abstract] [Full Text] [PDF]
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 H. Li and D. J. O'Sullivan Identification of a nisI Promoter within the nisABCTIP Operon That May Enable Establishment of Nisin Immunity Prior to Induction of the Operon via Signal Transduction J. Bacteriol., December 15, 2006; 188(24): 8496 - 8503. [Abstract] [Full Text] [PDF]
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A. Kuipers, J. Wierenga, R. Rink, L. D. Kluskens, A. J. M. Driessen, O. P. Kuipers, and G. N. Moll Sec-Mediated Transport of Posttranslationally Dehydrated Peptides in Lactococcus lactis Appl. Envir. Microbiol., December 1, 2006; 72(12): 7626 - 7633. [Abstract] [Full Text] [PDF]
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S. Schmitz, A. Hoffmann, C. Szekat, B. Rudd, and G. Bierbaum The Lantibiotic Mersacidin Is an Autoinducing Peptide Appl. Envir. Microbiol., November 1, 2006; 72(11): 7270 - 7277. [Abstract] [Full Text] [PDF]
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S. Leimanis, N. Hoyez, S. Hubert, M. Laschet, E. Sauvage, R. Brasseur, and J. Coyette PBP5 Complementation of a PBP3 Deficiency in Enterococcus hirae. J. Bacteriol., September 1, 2006; 188(17): 6298 - 6307. [Abstract] [Full Text] [PDF]
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S. C. J. De Keersmaecker, K. Braeken, T. L. A. Verhoeven, M. Perea Velez, S. Lebeer, J. Vanderleyden, and P. Hols Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins. Appl. Envir. Microbiol., July 1, 2006; 72(7): 4923 - 4930. [Abstract] [Full Text] [PDF]
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H. T. A. Hilmi, K. Kyla-Nikkila, R. Ra, and P. E. J. Saris Nisin induction without nisin secretion. Microbiology, May 1, 2006; 152(Pt 5): 1489 - 1496. [Abstract] [Full Text] [PDF]
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R. E. Wirawan, N. A. Klesse, R. W. Jack, and J. R. Tagg Molecular and Genetic Characterization of a Novel Nisin Variant Produced by Streptococcus uberis Appl. Envir. Microbiol., February 1, 2006; 72(2): 1148 - 1156. [Abstract] [Full Text] [PDF]
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T. G. Kloosterman, J. J. E. Bijlsma, J. Kok, and O. P. Kuipers To have neighbour's fare: extending the molecular toolbox for Streptococcus pneumoniae Microbiology, February 1, 2006; 152(2): 351 - 359. [Abstract] [Full Text] [PDF]
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K. Fiola, J.-P. Perreault, and B. Cousineau Gene Targeting in the Gram-Positive Bacterium Lactococcus lactis, Using Various Delta Ribozymes Appl. Envir. Microbiol., January 1, 2006; 72(1): 869 - 879. [Abstract] [Full Text] [PDF]
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R. S. Bongers, J.-W. Veening, M. Van Wieringen, O. P. Kuipers, and M. Kleerebezem Development and Characterization of a Subtilin-Regulated Expression System in Bacillus subtilis: Strict Control of Gene Expression by Addition of Subtilin Appl. Envir. Microbiol., December 1, 2005; 71(12): 8818 - 8824. [Abstract] [Full Text] [PDF]
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E. Sorvig, G. Mathiesen, K. Naterstad, V. G. H. Eijsink, and L. Axelsson High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors Microbiology, July 1, 2005; 151(7): 2439 - 2449. [Abstract] [Full Text] [PDF]
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 B. Wang and H. K. Kuramitsu Inducible Antisense RNA Expression in the Characterization of Gene Functions in Streptococcus mutans Infect. Immun., June 1, 2005; 73(6): 3568 - 3576. [Abstract] [Full Text] [PDF]
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T. Stein, S. Heinzmann, S. Dusterhus, S. Borchert, and K.-D. Entian Expression and Functional Analysis of the Subtilin Immunity Genes spaIFEG in the Subtilin-Sensitive Host Bacillus subtilis MO1099 J. Bacteriol., February 1, 2005; 187(3): 822 - 828. [Abstract] [Full Text] [PDF]
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N. Golic, M. Schliekelmann, M. Fernandez, M. Kleerebezem, and R. van Kranenburg Molecular characterization of the CmbR activator-binding site in the metC-cysK promoter region in Lactococcus lactis Microbiology, February 1, 2005; 151(2): 439 - 446. [Abstract] [Full Text] [PDF]
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D. A. Brede, T. Faye, O. Johnsborg, I. Odegard, I. F. Nes, and H. Holo Molecular and Genetic Characterization of Propionicin F, a Bacteriocin from Propionibacterium freudenreichii Appl. Envir. Microbiol., December 1, 2004; 70(12): 7303 - 7310. [Abstract] [Full Text] [PDF]
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D. Llull and I. Poquet New Expression System Tightly Controlled by Zinc Availability in Lactococcus lactis Appl. Envir. Microbiol., September 1, 2004; 70(9): 5398 - 5406. [Abstract] [Full Text] [PDF]
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C. Richard, D. Drider, K. Elmorjani, D. Marion, and H. Prevost Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli J. Bacteriol., July 1, 2004; 186(13): 4276 - 4284. [Abstract] [Full Text] [PDF]
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A. Kuipers, E. de Boef, R. Rink, S. Fekken, L. D. Kluskens, A. J. M. Driessen, K. Leenhouts, O. P. Kuipers, and G. N. Moll NisT, the Transporter of the Lantibiotic Nisin, Can Transport Fully Modified, Dehydrated, and Unmodified Prenisin and Fusions of the Leader Peptide with Non-lantibiotic Peptides J. Biol. Chem., May 21, 2004; 279(21): 22176 - 22182. [Abstract] [Full Text] [PDF]
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 L. Xie, L. M. Miller, C. Chatterjee, O. Averin, N. L. Kelleher, and W. A. van der Donk Lacticin 481: In Vitro Reconstitution of Lantibiotic Synthetase Activity Science, January 30, 2004; 303(5658): 679 - 681. [Abstract] [Full Text] [PDF]
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T. Inaoka, K. Takahashi, H. Yada, M. Yoshida, and K. Ochi RNA Polymerase Mutation Activates the Production of a Dormant Antibiotic 3,3'-Neotrehalosadiamine via an Autoinduction Mechanism in Bacillus subtilis J. Biol. Chem., January 30, 2004; 279(5): 3885 - 3892. [Abstract] [Full Text] [PDF]
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I. Pastar, I. Tonic, N. Golic, M. Kojic, R. van Kranenburg, M. Kleerebezem, L. Topisirovic, and G. Jovanovic Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate Lactobacillus rhamnosus BGT10 Appl. Envir. Microbiol., October 1, 2003; 69(10): 5802 - 5811. [Abstract] [Full Text] [PDF]
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J. Reunanen and P. E. J. Saris Microplate Bioassay for Nisin in Foods, Based on Nisin-Induced Green Fluorescent Protein Fluorescence Appl. Envir. Microbiol., July 1, 2003; 69(7): 4214 - 4218. [Abstract] [Full Text] [PDF]
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P. A. Wescombe and J. R. Tagg Purification and Characterization of Streptin, a Type A1 Lantibiotic Produced by Streptococcus pyogenes Appl. Envir. Microbiol., May 1, 2003; 69(5): 2737 - 2747. [Abstract] [Full Text] [PDF]
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T. Stein, S. Heinzmann, I. Solovieva, and K.-D. Entian Function of Lactococcus lactis Nisin Immunity Genes nisI and nisFEG after Coordinated Expression in the Surrogate Host Bacillus subtilis J. Biol. Chem., January 3, 2003; 278(1): 89 - 94. [Abstract] [Full Text] [PDF]
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O. Koponen, M. Tolonen, M. Qiao, G. Wahlstrom, J. Helin, and P. E. J. Saris NisB is required for the dehydration and NisC for the lanthionine formation in the post-translational modification of nisin Microbiology, November 1, 2002; 148(11): 3561 - 3568. [Abstract] [Full Text] [PDF]
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H. Li and D. J. O'Sullivan Heterologous Expression of the Lactococcus lactis Bacteriocin, Nisin, in a Dairy Enterococcus Strain Appl. Envir. Microbiol., July 1, 2002; 68(7): 3392 - 3400. [Abstract] [Full Text] [PDF]
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L. Nilsson, M. K. Nielsen, Y. Ng, and L. Gram Role of Acetate in Production of an Autoinducible Class IIa Bacteriocin in Carnobacterium piscicola A9b Appl. Envir. Microbiol., May 1, 2002; 68(5): 2251 - 2260. [Abstract] [Full Text] [PDF]
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M. H. N. Hoefnagel, M. J. C. Starrenburg, D. E. Martens, J. Hugenholtz, M. Kleerebezem, I. I. Van Swam, R. Bongers, H. V. Westerhoff, and J. L. Snoep Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis Microbiology, April 1, 2002; 148(4): 1003 - 1013. [Abstract] [Full Text] [PDF]
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T. Stein, S. Borchert, B. Conrad, J. Feesche, B. Hofemeister, J. Hofemeister, and K.-D. Entian Two Different Lantibiotic-Like Peptides Originate from the Ericin Gene Cluster of Bacillus subtilis A1/3 J. Bacteriol., March 15, 2002; 184(6): 1703 - 1711. [Abstract] [Full Text] [PDF]
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A. Gomez, M. Ladire, F. Marcille, and M. Fons Trypsin Mediates Growth Phase-Dependent Transcriptional Regulation of Genes Involved in Biosynthesis of Ruminococcin A, a Lantibiotic Produced by a Ruminococcus gnavus Strain from a Human Intestinal Microbiota J. Bacteriol., January 1, 2002; 184(1): 18 - 28. [Abstract] [Full Text] [PDF]
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A. Guder, T. Schmitter, I. Wiedemann, H.-G. Sahl, and G. Bierbaum Role of the Single Regulator MrsR1 and the Two-Component System MrsR2/K2 in the Regulation of Mersacidin Production and Immunity Appl. Envir. Microbiol., January 1, 2002; 68(1): 106 - 113. [Abstract] [Full Text] [PDF]
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 L. M. Cintas, M. P. Casaus, C. Herranz, I. F. Nes, and P. E. Hernandez Review: Bacteriocins of Lactic Acid Bacteria Food Science and Technology International, August 1, 2001; 7(4): 281 - 305. [Abstract] [PDF]
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M. Upton, J. R. Tagg, P. Wescombe, and H. F. Jenkinson Intra- and Interspecies Signaling between Streptococcus salivarius and Streptococcus pyogenes Mediated by SalA and SalA1 Lantibiotic Peptides J. Bacteriol., July 1, 2001; 183(13): 3931 - 3938. [Abstract] [Full Text] [PDF]
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L. M. Cintas, P. Casaus, C. Herranz, L. S. Håvarstein, H. Holo, P. E. Hernández, and I. F. Nes Biochemical and Genetic Evidence that Enterococcus faecium L50 Produces Enterocins L50A and L50B, the sec-Dependent Enterocin P, and a Novel Bacteriocin Secreted without an N-Terminal Extension Termed Enterocin Q J. Bacteriol., December 1, 2000; 182(23): 6806 - 6814. [Abstract] [Full Text]
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S. Pavan, P. Hols, J. Delcour, M.-C. Geoffroy, C. Grangette, M. Kleerebezem, and A. Mercenier Adaptation of the Nisin-Controlled Expression System in Lactobacillus plantarum: a Tool To Study In Vivo Biological Effects Appl. Envir. Microbiol., October 1, 2000; 66(10): 4427 - 4432. [Abstract] [Full Text]
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J. A. Wouters, M. Mailhes, F. M. Rombouts, W. M. de Vos, O. P. Kuipers, and T. Abee Physiological and Regulatory Effects of Controlled Overproduction of Five Cold Shock Proteins of Lactococcus lactis MG1363 Appl. Envir. Microbiol., September 1, 2000; 66(9): 3756 - 3763. [Abstract] [Full Text]
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D. B. Diep, L. Axelsson, C. Grefsli, and I. F. Nes The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system Microbiology, September 1, 2000; 146(9): 2155 - 2160. [Abstract] [Full Text] [PDF]
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K. Altena, A. Guder, C. Cramer, and G. Bierbaum Biosynthesis of the Lantibiotic Mersacidin: Organization of a Type B Lantibiotic Gene Cluster Appl. Envir. Microbiol., June 1, 2000; 66(6): 2565 - 2571. [Abstract] [Full Text]
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H. Hirt, S. L. Erlandsen, and G. M. Dunny Heterologous Inducible Expression of Enterococcus faecalis pCF10 Aggregation Substance Asc10 in Lactococcus lactis and Streptococcus gordonii Contributes to Cell Hydrophobicity and Adhesion to Fibrin J. Bacteriol., April 15, 2000; 182(8): 2299 - 2306. [Abstract] [Full Text]
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M. O’Connell-Motherway, D. van Sinderen, F. Morel-Deville, G. F. Fitzgerald, S. D. Ehrlich, and P. Morel Six putative two-component regulatory systems isolated from Lactococcus lactis subsp. cremoris MG1363 Microbiology, April 1, 2000; 146(4): 935 - 947. [Abstract] [Full Text]
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M.-C. Geoffroy, C. Guyard, B. Quatannens, S. Pavan, M. Lange, and A. Mercenier Use of Green Fluorescent Protein To Tag Lactic Acid Bacterium Strains under Development as Live Vaccine Vectors Appl. Envir. Microbiol., January 1, 2000; 66(1): 383 - 391. [Abstract] [Full Text]
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O. McAuliffe, C. Hill, and R. P. Ross Identification and overexpression of ltnI, a novel gene which confers immunity to the two-component lantibiotic lacticin 3147 Microbiology, January 1, 2000; 146(1): 129 - 138. [Abstract] [Full Text]
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G. Zheng, L. Z. Yan, J. C. Vederas, and P. Zuber Genes of the sbo-alb Locus of Bacillus subtilis Are Required for Production of the Antilisterial Bacteriocin Subtilosin J. Bacteriol., December 1, 1999; 181(23): 7346 - 7355. [Abstract] [Full Text]
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J. A. Wouters, B. Jeynov, F. M. Rombouts, W. M. de Vos, O. P. Kuipers, and T. Abee Analysis of the role of 7 kDa cold-shock proteins of Lactococcus lactis MG1363 in cryoprotection Microbiology, November 1, 1999; 145(11): 3185 - 3194. [Abstract] [Full Text] [PDF]
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G. Wahlström and P. E. J. Saris A Nisin Bioassay Based on Bioluminescence Appl. Envir. Microbiol., August 1, 1999; 65(8): 3742 - 3745. [Abstract] [Full Text]
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T. O'Keeffe, C. Hill, and R. P. Ross Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146 Appl. Envir. Microbiol., April 1, 1999; 65(4): 1506 - 1515. [Abstract] [Full Text]
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U. Pag, C. Heidrich, G. Bierbaum, and H.-G. Sahl Molecular Analysis of Expression of the Lantibiotic Pep5 Immunity Phenotype Appl. Envir. Microbiol., February 1, 1999; 65(2): 591 - 598. [Abstract] [Full Text]
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Z. Eichenbaum, M. J. Federle, D. Marra, W. M. de Vos, O. P. Kuipers, M. Kleerebezem, and J. R. Scott Use of the Lactococcal nisA Promoter To Regulate Gene Expression in Gram-Positive Bacteria: Comparison of Induction Level and Promoter Strength Appl. Envir. Microbiol., August 1, 1998; 64(8): 2763 - 2769. [Abstract] [Full Text]
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F. Lopez de Felipe, M. Kleerebezem, W. M. de Vos, and J. Hugenholtz Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase J. Bacteriol., August 1, 1998; 180(15): 3804 - 3808. [Abstract] [Full Text]
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E. L. Anderssen, D. B. Diep, I. F. Nes, V. G. H. Eijsink, and J. Nissen-Meyer Antagonistic Activity of Lactobacillus plantarum C11: Two New Two-Peptide Bacteriocins, Plantaricins EF and JK, and the Induction Factor Plantaricin A Appl. Envir. Microbiol., June 1, 1998; 64(6): 2269 - 2272. [Abstract] [Full Text]
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S. A. Walker and T. R. Klaenhammer Molecular Characterization of a Phage-Inducible Middle Promoter and Its Transcriptional Activator from the Lactococcal Bacteriophage phi 31 J. Bacteriol., February 15, 1998; 180(4): 921 - 931. [Abstract] [Full Text]
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E. H. M. L. Heuberger, E. Smits, and B. Poolman Xyloside Transport by XylP, a Member of the Galactoside-Pentoside-Hexuronide Family J. Biol. Chem., September 7, 2001; 276(37): 34465 - 34472. [Abstract] [Full Text] [PDF]
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