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INTRODUCTION |
In recent years peptide antibiotics have gained increasing
attention as therapeutics (1, 2) and food preservatives (3). Nisin
represents the most prominent member of lantibiotics, peptide antibiotics with intramolecular lanthionine bridges (4-9). The nisin
producer Lactococcus lactis 6F3 contains a gene cluster encoding proteins for the biosynthesis and transport (10-14), immunity (15), and regulation (16-18) of nisin. Subtilin (19, 20) and
ericin S (21) produced by Bacillus subtilis ATCC 6633 and A1/3, respectively, are closely related lantibiotics. Lantibiotics form
voltage-dependent pores in the bacterial cytoplasmic
membrane that are lethal for the target cells but also for the
producer. For nisin, the mode of action was investigated in several
model systems such as black lipid bilayers and membrane vesicles
(22-25). Recent findings demonstrated that specific binding of nisin
to the cell wall precursor lipid II coincides with pore formation (26,
27). Specific self-protection (immunity) mechanisms are necessary to
protect the lantibiotic-producing organisms from the action of their
own lantibiotics. For nisin and subtilin (28) immunity is based on the
expression of lanFEG encoding ABC transporter-homologous proteins (13, 15, 17; revised sequence of subtilin immunity genes, EMBL
accession number U09819), and lanI encoding non-related lipoproteins with different sizes (for review, see Ref. 29). Only
immunity transporters and no lipoproteins were found in the epidermin
(30) or mersacidin (31) gene clusters. In contrast, for Pep5 (32),
epicidin (33), and lactocin S (34) only lipoproteins and no
transporters have been found.
Although numerous genes involved in lantibiotic immunity are known, the
mechanism by which the encoded proteins confer immunity remain unclear.
For full nisin or subtilin immunity, both are required, i.e.
the lipoprotein as well as the immunity transporter. The lack of each
component diminished the tolerance to nisin (35) or subtilin (28)
significantly. Here we report for the first time on the establishment
of nisin immunity in the heterologous host B. subtilis.
Functional analyses of its different components provided evidence that
NisI acts as a nisin-sequestering protein and that NisFEG acts as a
nisin exporter that expels nisin molecules from the cytoplasmic
membrane into the environment.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains, Plasmids, and Growth Conditions--
B.
subtilis MO1099 (36) and ATCC 6633 were grown at 37 °C on Difco
sporulation medium or M9 medium (37) supplemented with 50 µg/ml
phenylalanine, 20 µg/ml tryptophan, and 0.1% casamino acids. For
subtilin production B. subtilis ATCC 6633 was grown at
37 °C in TY medium (0.8% tryptone, 0.5% yeast extract, 0.5% NaCl). Recombinant plasmids were amplified in Escherichia
coli DH5
, TP611, or M15. E. coli strains were grown
on Luria-Bertani medium (Invitrogen). Antibiotics were used in
the following concentrations: 80 µg/ml ampicillin for E. coli, and 5 µg/ml chloramphenicol, 1 µg/ml erythromycin, and
25 µg/ml lincomycin for B. subtilis. The pDR67 vector
with the IPTG1-inducible
Bacillus promoter, Pspac (38) was
used for chromosomal integration into the amyE locus of
B. subtilis MO1099. Gene expression was induced with 1-2
mM IPTG.
Molecular Biology Techniques--
Established protocols were
followed for molecular biology techniques (37). DNA was cleaved
according to the conditions recommended by the commercial supplier
(Roche Molecular Biochemicals). DNA fragments were eluted from agarose
gels by the Gene Clean Kit III (Bio 101, Vista, CA). The alkaline
extraction procedure (39) was followed to isolate plasmids of E. coli. PCR was carried out according to standard procedures (37) in
an Eppendorf Microcycler E. DNA was sequenced by Scientific Research
and Development GmbH, Oberursel/Frankfurt, Germany. B. subtilis was transformed by the competence method (40) with slight
modifications (20). Nisin immunity genes were amplified from L. lactis chromosomal DNA or the nisIFEG-containing
plasmid pSI22 (41). A copy of nisI
(EcoRV/XbaI) was cloned into pUC19 (42),
resulting in pHZ39, and fused to nisF
(XbaI/SphI of 1214-bp PCR amplified with O1/2),
resulting in pHZ40, and nisEG
(SnaBI/SphI of 908-bp PCR amplified with O3/4), resulting in pHZ41. Derivatives of pDR67 were constructed with nisI (BglII/SphI of 936-bp PCR
amplified from pHZ40 with O5/7), nisFEG
(HindIII/SphI from pHZ41), and nisIFEG
(BglII/SphI of 2070-bp PCR amplified from pHZ40
with O5/6 into pDR67 followed by integration of
SnaBI/SphI of 908-bp PCR amplified from pHZ41
with O3/4). The primers O1
(5'-GAATAGATTCTGAAACTAGTTTTATATAC-3'), O2
(5'-AACAAATCAAGGCATGCGCAGCTAAC-3'), O3,
(5'-GGAATGTGATCTGCAGAAATAATAGC-3'), O4
(5'-ATTAGGTCGAATTAGCATGCGAAAAAATAC-3'), O5
(5'-GTTACTTAGTCTTTGCTTGGAC-3'), O6 (5'-CGCCAAGCTTGCATGCGCAGC-3'), and
O7 (5'-AATTTTTGCATGCATTATATTCCAG-3') were purchased from ARK Scientific
Biosystems (Darmstadt, Germany).
Subtilin Purification--
Supernatants of stationary grown
B. subtilis ATCC 6633 (in TY medium) were separated using
semi-preparative RP-HPLC with a C-18 Lichrospher column (particle size
10 µm, 200 × 20 mm; Merck), an analytical ODS Hypersil column
(particle size 5 µm, 250 × 2 mm; Maisch, Ammerbuch, Germany),
and linear gradients of acetonitrile. Nisin was purchased from Sigma.
Nisin Sensitivity Assay--
The nisin sensitivity of B. subtilis was determined using agar diffusion tests. Various
amounts of nisin in a volume of 60 µl were poured into holes (1.6-mm
diameter) of M9 test plates (20 ml). The plates were kept for 2 h
at 4 °C for diffusion. 300 µl of stationary grown B. subtilis cultures were inoculated into 5 ml of M9 medium and grown
to an A578 of 0.8. 100 µl of a
10
2 dilution were overlaid onto the test plates in 5 ml
of molten M9 agar (50 °C) and incubated overnight at 37 °C.
Quantitative Nisin Transport Assay--
To investigate the
molecular mechanism of nisin immunity, a quantitative in
vivo peptide release assay described by Otto et al.
(43) was used with modifications. Stationary B. subtilis strains grown overnight in TY medium containing 1% (w/v) glucose were
harvested and washed with 50 mM Tris-HCl (pH 8). The cell density was adjusted to an A578 of 10 with an
incubation buffer (50 mM sodium phosphate (pH 7), 0.5 M NaCl, and 0.5% (w/v) glucose). 1-ml aliquots of the cell
suspension were incubated with nisin for 30 min at 37 °C with gentle
shaking. After centrifugation (10,000 × g, 10 min),
quantitative HPLC analyses of the supernatants were performed on a
Beckman Gold HPLC System using an analytical ODS Hypersil column
(Maisch, Ammerbuch, Germany). Nisin was eluted using a linear gradient
of 30-40% acetonitrile containing 0.1% trifluoroacetic acid (v/v/v)
over 30-column volumes. Nisin was detected measuring the absorption at
214 nm. The flow rate (0.4 ml/min) was chosen so that a Gauss
distribution of the nisin absorption peak was obtained that allowed a
quantitative determination of the nisin amount after integration. Nisin
attached to the cell sediment was extracted by gently mixing with 1 ml
of 20% acetonitrile in water (0.1% trifluoroacetic acid) at room
temperature for 5 min. After centrifugation (10,000 × g, 10 min), nisin was quantitatively determined in the supernatant.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analyses--
SDS-PAGE and Western blot analyses were performed as
described previously (14, 44); native PAGE was without SDS. Molecular weight standards for SDS-PAGE were purchased from Sigma.
Isolation of Membrane Vesicles--
Membrane vesicles from
B. subtilis were prepared as described previously (45).
Construction and Isolation of Hexahistidine-tagged NisI--
A
nisI copy was PCR amplified from pHZ39 with primers O8
(5'-GTTTATCAGGATCCTATCAAACAAGTC-3') and O9
(5'-GAATTTTCTGCAGTCTAGTTTCCTAC-3') (ARK) and inserted into the pQE9
vector (Qiagen, Hilden, Germany). The E. coli strain M15
(pREP4) was transformed with the resulting plasmid and grown in LB to
an A600 of 0.5. After IPTG induction, the
cells were incubated for additional 4 h. The cells were harvested, suspended in lysis buffer (50 mM
NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and disrupted by sonication. After
removal of cell debris by centrifugation at 17,000 × g
(30 min, 4 °C) the supernatant was incubated with
nickel-nitrilotriacetic acid-agarose (Qiagen) with gentle shaking (1 h,
4 °C). The protein was eluted using the same buffer containing 100 mM imidazole and dialyzed against the storage buffer (20 mM Tris-HCl, 10% glycerol and 5 mM
dithiothreitol, pH 6.5).
Interaction between NisI and Nisin--
NisI membrane vesicles
were incubated under gently shaking with 0.17 volumes of 3% (w/v)
laurylmaltoside (dodecyl-
-D-maltoside, Roche Molecular
Biochemicals) and 0.17 volumes of 2 M 6-aminocaproic acid
(Fluka) at room temperature for 2 h. After centrifugation (30 min,
18,000 × g, 4 °C), 15% (w/v) glycerol was added to
the supernatant, which contained the dissolved membrane proteins. From
this fraction, 30 µl were incubated with different amounts of nisin
or subtilin (10 min, 37 °C). The samples were divided 70:30, and the
larger sample was loaded onto 9% polyacrylamide gels without SDS,
separated at 20 mA for 6 h, and electroblotted to nitrocellulose
by using standard buffers with 0.1% SDS. The smaller sample was loaded
onto a denaturing 14% SDS-PAGE, followed by electroblotting. Finally,
NisI was visualized with NisI-antisera (41).
Different amounts of purified His6-NisI (0-160 µg) were
incubated with 20 µg of nisin in a final volume of 20 µl in 20 mM Tris-HCl, pH 6.5, and 1 mM dithiothreitol
(37 °C, 30 min). After centrifugation (10 min, 13,000 rpm), the
pellets were dissolved in 20 µl of 0.02 N HCl. 5-µl
aliquots of the supernatants and solubilized pellets were analyzed in a
agar diffusion tests using Micrococcus luteus ATCC 9341 as
the test organism.
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RESULTS |
Fusion of nisI to nisFEG and Transfer to the Chromosome of B. subtilis--
In contrast to subtilin immunity (Fig.
1A) (46), the L. lactis nisin immunity genes nisI and nisFEG
reside on different transcriptional units (Fig. 1B) (18,
47). To study nisin immunity without the influence of nisin production,
we coexpressed different combinations of nisin immunity genes in the
nisin-sensitive B. subtilis hosts MO1099 and ATCC 6633, as
exemplified for the nisIFEG construct in Fig.
1C.

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Fig. 1.
Lantibiotic gene clusters and transfer of
nisin immunity genes. A and B, genetic
organization of the closely related subtilin (46) and nisin (47) gene
clusters. Whereas nisI was found on a transcriptional unit
nisABTCIP together with the structural gene
(nisA) and genes involved in posttranslational modifications
(nisBC), the processing (nisP) and transport
(nisT) of nisin expression of nisFEG is
controlled by its own promoter (47). C, nisI and
nisFEG were fused into a single transcriptional unit under
the control of the Pspac promoter (pHZ51) and
integrated into the chromosome of B. subtilis MO1099. Double
recombination is accompanied by resistance marker exchange,
i.e. macrolide-lincomycin-streptogramin (MLS)
versus chloramphenicol (cat). The incomplete
amyE genes were represented as amyE' (5'-end) and
'amyE (3'-end).
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Effect of Nisin Immunity Genes in B. subtilis--
The nisin
sensitivity levels of the recipient B. subtilis strains were
analyzed (Fig. 2). The highest level of
tolerance was obtained for B. subtilis MO1099
containing all four immunity genes, nisI, and
nisFEG (Fig. 2, A, plate 4,
and B, semi-quantitative analyses). This strain tolerates 8 µg of nisin, a quantity that already induced large growth inhibition
zones for the control strain without any immunity genes (Fig.
2A, plate 1). Remarkably, after
expression of the lipoprotein NisI alone, a significant nisin tolerance
level was obtained (Fig. 2A, plate 2)
that was comparable with B. subtilis cells expressing
nisFEG (Fig. 2A, plate 3).
These data clearly showed that B. subtilis MO1099 represents a surrogate host for the functional expression of nisin immunity genes
and that the action of two immunity systems, the lipoprotein NisI and
the ABC transporter-homologous protein NisFEG, is needed to obtain full
nisin tolerance.

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Fig. 2.
Functional analysis of nisin
immunity in B. subtilis MO1099. Lantibiotic
sensitivity of B. subtilis strains expressing different
combinations of nisin immunity genes. Strains were investigated in agar
diffusion tests as indicated under "Experimental Procedures."
A, nisin sensitivity of B. subtilis MO1099
transformed with the vector plasmid pDR67 without immunity genes
(plate 1) and expressing nisI (plate
2), nisFEG (plate 3), and nisIFEG
(plate 4). Starting from the arrow and moving
clockwise, the applied amounts of nisin are 2, 4, 8, 15, 25, and 35 µg. According to the Second Law of Diffusion (also referred to as
Fick's Law), the square of the diffusion distance of a given solute
into a liquid is directly proportional to the natural logarithm of its
initial concentration. Thus, using standard volumes (60 µl) and
sufficient diffusion times, linear dependences between the square of
the halos and the natural logarithm of the applied nisin amounts were
obtained. B, B. subtilis MO1099
(filled circles) expressing nisI
(open triangles), nisFEG (filled
triangles), and nisIFEG (open circles).
C, nisin sensitivity of B. subtilis ATCC 6633 (filled circles) expressing NisIFEG
(open circles). D, subtilin sensitivity of
B. subtilis MO1099 (filled circles)
expressing nisIFEG (open circles) and B. subtilis ATCC 6633 (filled triangles) expressing
nisIFEG (open triangles). S.E. was <15% for
each given value in panels B and C
(means of three independent assays).
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Additionally, the nisin immunity components were transferred to the
subtilin-producing B. subtilis strain ATCC 6633. After coordinated expression of nisIFEG, the recipient cells
exhibited significant tolerance to nisin (Fig. 2C). This
showed that the immunity system from L. lactis 6F3 is also
effective in B. subtilis cells, which express two
lantibiotic immunity systems, nisIFEG and
spaIFEG. The different tolerance levels obtained for the
nisIFEG-expressing B. subtilis strains MO1099 and
the subtilin producer ATCC 6633 were based on the faster growth rate of
the ATCC 6633 strain. Remarkably, the expression of nisin immunity
genes nisIFEG (Fig. 2D) as well as other
combinations of nisin immunity genes (not shown) in B. subtilis strains MO1099 and ATCC 6633 had no effect on subtilin tolerance.
Cellular Localization of NisI--
NisI (25.8 kDa, calculated
without signal sequence) expressed in B. subtilis was
detected in both the membrane fraction (Fig. 3A, lane 3) and the
soluble cell extract (lane 4). Two bands were obtained for
NisI (shown in Fig. 3A, and more pronounced in Fig. 4A, see below), suggesting
that the upper band (I) corresponds to membrane-associated
NisI and the lower band (II) to NisI without the membrane
anchor. Thus, at least approximately half of NisI seemed to be
correctly anchored in the cytoplasmic membrane of B. subtilis. Probably, degradation and/or incomplete modification with the lipid moiety led to a protein with faster migration after SDS-PAGE. In accordance with this probability, prolonged incubation times of nisI-expressing cells led to the observation of
NisI within the culture supernatant (not shown).

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Fig. 3.
Expression of NisI in
heterologous hosts. A, NisI immunoblot of
SDS-PAGE-separated B. subtilis MO1099 extracts. B. subtilis cells expressing NisI (lane 2) were disrupted
by sonication. After centrifugation, the supernatant (lane
4) and the membrane pellet suspended into a comparable volume of
lysis buffer (lane 3) were analyzed. Lane 1,
B. subtilis MO1099 without NisI genes. I and
II, NisI with and without lipo-modification, respectively.
Approximately 40 µg of total protein was loaded onto each lane.
B, bromphenol blue stain of SDS-PAGE-separated extracts of
E. coli M15 (pREP4) cells overexpressing
His6-NisI without membrane anchor (lane 1) and
after IPTG induction (lane 2). After lysis and
centrifugation (17,000 × g, 30 min),
His6-NisI was found in the supernatant (lane 3)
and was adsorbed to nickel-agarose. Purified His6-NisI
after elution from the nickel-agarose beads with 100 mM
imidazole is shown in lane 4). M, molecular mass
marker. The position of NisI is indicated by an
arrow.
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Fig. 4.
Functional analysis of NisI: Interaction with
nisin. A, protein fractions solubilized from membrane
vesicles (60 µg) of NisI-expressing B. subtilis MO1099
were incubated with different amounts of nisin or subtilin. After
splitting into two parts (70:30), the larger part was separated by
native PAGE, and the minor part was separated by SDS-PAGE. NisI
immunoblots of both gels are shown. The lanes identical for both gels.
Solubilized membrane proteins were incubated with a culture supernatant
of a subtilin-negative mutant (lane 1), 2, 3, or 4 µg of
nisin (lanes 2-4), and 4 or 6 µg of subtilin (lanes
5-6). The position of NisI is indicated by arrows.
B, NisI immunoblots of His6-NisI (5 µg) after
incubation with increasing amounts of nisin and subsequent native PAGE.
Lanes 1-6, 0.05, 5, 10, 20, and 30 µg of nisin.
C, after incubation of His6-NisI with nisin, a
pellet was formed. The antimicrobial activities within the supernatant
and the solubilized pellet were analyzed using M. luteus as
the test organism. The complete assays are described under
"Experimental Procedures." Assays 1-5 contained 20 µg of nisin
and 0, 20, 40, 80, and 160 µg of His6-NisI, respectively;
assay 6 contained 160 µg of His6-NisI without
nisin.
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Construction and Isolation of Hexahistidine-tagged NisI--
NisI
is a typical lipoprotein, possessing both a N-terminal signal sequence
and a membrane-anchoring Cys residue immediately proceeding the
cleavage site. To avoid possible anchoring and export of NisI in
E. coli, the NisI lipoprotein signal sequence and the
anchoring Cys residue (amino acids 1-20) were substituted by the
sequence MRSGSHHHHHH, resulting in the protein His6-NisI. Recombinant His6-NisI from E. coli (Fig.
3B, lane 2) was found in the soluble protein
fraction (lane 3) after cell lysis. His6-NisI was purified by nickel-agarose affinity chromatography (lane
4) and used for interaction studies with nisin (see below) and
immunoaffinity purification of the polyclonal NisI antibody (48). The
purified antibody showed no cross-reactivity with components of an
SDS-PAGE-separated B. subtilis total cell extract (Fig.
2A, lane 1), demonstrating its high selectivity.
Specific Interaction between NisI and Nisin--
To unravel the
function of NisI in nisin immunity, we investigated possible
interactions between NisI and nisin. NisI was solubilized from
nisI expressing B. subtilis MO1099 membrane
vesicles and incubated with different amounts of nisin and subtilin.
Subsequently, the samples were analyzed by parallel native and SDS-PAGE
(Fig. 4A). Incubation of NisI with nisin (lanes
1-4) led to a depletion of the NisI signal, which was not
observed if subtilin was used (lanes 5-6). Regardless of a
former incubation with nisin (lanes 1-4) or subtilin
(lanes 5-6), all samples revealed the same NisI signal
intensity after separation under denaturing conditions (SDS-PAGE in
Fig. 4A). A similar signal depletion was observed for
His6-NisI isolated from E. coli (Fig.
4B), suggesting similar properties for NisI without a lipid anchor.
Remarkably, after incubation of nisin with His6-NisI, a
small pellet was observed. The pellet contained a portion of the nisin molecules that are easily solubilized under aqueous conditions as
demonstrated by M. luteus agar diffusion tests (Fig.
4C, lanes 2-5, pellet). In
accordance, after incubation of nisin with increasing amounts of
His6-NisI, a slight decrease of the activity in the supernatant was observed (Fig. 4C, lanes 2-5,
supernatant). With equal amounts of subtilin, no pellet was
formed (not shown). This argues for a specific complex formation
between NisI and nisin that reduces the quantity of free nisin molecules.
The Function of NisFEG--
The activity of NisFEG was
investigated with a series of quantitative in vivo peptide
release assays. After incubation of B. subtilis cells with
nisin, the quantities of the nisin in the culture supernatant and the
nisin associated with the cell-pellet were determined by quantitative
RP-HPLC (Fig. 5A). For
B. subtilis MO1099 and MO1099 expressing NisI, ~7.5 µg
of nisin were found attached to the cells independently of the quantity
of applied nisin (9 or 12 µg, Fig. 5, B and C). After
expression of nisFEG or nisIFEG, the quantity of
cell-associated nisin was significantly reduced to about 5 µg. In
accordance, the nisin quantity in the supernatant increased about
4-fold if 9 µg of nisin was applied (Fig. 5B) and about
2.5-fold if 12 µg of nisin was applied (Fig. 5C),
suggesting an export function of the transporter-homologous system
NisFEG. Remarkably, in all experiments >90% of the applied nisin
could be recovered. The structural identities of the nisin in the
supernatant and the nisin attached to cells were verified after RP-HPLC
and MALDI-TOF mass spectrometry analyses (m/z
3354, not shown). Thus, no nisin modification or degradation systems were present for lowering its toxicity. After application of subtilin (12 µg) in the peptide release assay, about two-thirds (7.5-8 µg)
were found cell-associated and about one-third (3-4 µg) in the
supernatant, regardless of whether B. subtilis
MO1099 wild-type or nisFEG-expressing cells were
analyzed.

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Fig. 5.
Functional analysis of NisFEG; quantitative
nisin transport assay. A, stationary grown B. subtilis cells were incubated with different amounts of nisin.
After centrifugation, the quantity of nisin in the supernatant and the
quantity of the cell-associated nisin were determined by quantitative
RP-HPLC. B and C, the quantity of nisin
determined by the nisin transport assay with 9 µg (B) and
12 µg (C) of applied nisin; white bars,
supernatant; black bars, extracted from cells. The presented
values represent the means of three independent assays for which all
determinations were performed twice, respectively. S.E. of <20% was
obtained for each given value.
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DISCUSSION |
To study the molecular mechanism of nisin self-protection provided
by NisIFEG, different combinations of the immunity genes were
integrated into the genome of nisin-sensitive B. subtilis strains. After expression of either the lipoprotein NisI alone or the
ABC transporter-homologous system NisFEG, the recipient B. subtilis cells acquired a significant level of nisin tolerance. The strongest tolerance was obtained after coexpression of
nisI and nisFEG, as previously suggested from
deletion analyses in the nisin producer L. lactis 6F3 (15,
35). Although B. subtilis expressing the nisin immunity gene
nisIFEG acquired a >3-fold nisin tolerance level as
compared with wild-type B. subtilis (Fig. 2), only ~30%
of the nisin immunity level of L. lactis 6F3 was achieved
(15). However, in the nisin-producing L. lactis strain, the
establishment of nisin immunity is based on two operons,
nisABTCIP and nisFEG. The coordinated and
IPTG-induced expression of nisIFEG in the B. subtilis host cells is quite different from the autoregulatory control of nisin immunity in L. lactis (18). Remarkably, the acquired nisin immunity levels of the heterologous host B. subtilis were comparable with the immunity level of nisin
non-producing L. lactis strains expressing
plasmid-encoded nisI and nisFEG. Also for these
cells, an immunity level of only 20% compared with the nisin producer
was obtained (49). The B. subtilis nisin tolerance level
provided by the nisin immunity genes suggests the additive action of
two independent systems, the lipoprotein NisI and the transporter
NisFEG. Nevertheless, we cannot exclude the effect of additional
factors that contribute to nisin immunity in the nisin producer
L. lactis, for which a rather cooperative effect of NisI and
NisFEG was discussed (35, 50). Remarkably, the transfer of all
components of the nisin immunity system to a subtilin-producing
B. subtilis host was successful. The functional expression
of two closely related self-protection systems, nisIFEG and
spaIFEG (Fig. 2, C and D),
demonstrated that cross-immunity between both systems is less likely.
A typical lipoprotein signal sequence including the lipobox sequence
LSGC (51) was found for NisI, suggesting that NisI becomes a peripheral
membrane protein after lipid modification of the lipobox Cys residue,
processing, and transport over the cytoplasmic membrane. Although it
provides significant nisin tolerance, only 50% of NisI expressed in
B. subtilis was localized in the membrane. The other portion
of NisI was found in the soluble protein extract and showed a slightly
faster migration after SDS-PAGE, suggesting that it represents an
incompletely lipid-modified or degraded NisI species (Fig.
3A). Obviously, the lipid modification is not
sufficient for complete attachment of the respective proteins to the
cytoplasmic membrane. However, for non-lipo-modified NisI protein
species significant activity is also conceivable. The essential major
B. subtilis lipoprotein PrsA in B. subtilis showed at least partial activity without lipid
modification (52).
To provide evidence for physical interaction between NisI and nisin, we
used two different approaches. NisI could be efficiently extracted from
B. subtilis membranes after treatment with laurylmaltoside and 6-aminocaproic acid. An interaction of NisI with nisin and not with
the structurally closely related subtilin could be clearly monitored by
the depletion of the NisI signal after native PAGE and immunoblotting
(Fig. 4A). The detection of NisI signals not entering the
native PAGE implies a low solubility of the NisI-nisin complex. A
similar behavior was obtained for recombinant His6-NisI from E. coli (Fig. 4B), suggesting that the
lipoprotein signal sequence and/or the lipid modification is not
necessary for NisI activity.
Further evidence for specific interaction of NisI with nisin was
provided after incubation of His6-NisI with nisin (Fig.
4C), which resulted in the formation of an insoluble
complex. This argues for an interception of a portion of soluble
nisin molecules. Obviously, the complex is easily dissociated in
aqueous conditions, thus fully recovering the activity of precipitated nisin.
The ABC transporter-homologous system NisFEG seems to work by expelling
cell-attached nisin molecules into the environment, a mechanism that is
similar to the one described for the epidermin transporter homologue
EpiFEG (43). Nevertheless, the epidermin self-protection system lacks a
lipoprotein LanI, implying a greater necessity for the epidermin
immunity transporter in S. epidermidis. Our results suggest
that the export capacity of NisFEG is independent of the lipoprotein
NisI. After extraction, we could quantitatively recover cell-associated
nisin. Even after prolonged incubation times (up to 60 min) of
nisIFEG-expressing cells with nisin, approximately all
applied nisin molecules could be recovered quantitatively. This argues
against degradation or modification of nisin either by NisIFEG or other systems.
The lethal activity of nisin can be described with a four-step
mechanism that includes membrane adhesion, membrane integration, pore
formation, and pore dissociation (25). Specific binding of nisin to the
cell wall precursor lipid II coincides with pore formation (26, 27).
Our results provided experimental evidence that nisin immunity is based
on two independently acting systems. The lipoprotein NisI is orientated
to the outside of the cytoplasmic membrane. NisI would intercept nisin
at the surface of the cytoplasmic membrane and, by sequestering nisin,
prevent it from inserting into the membrane and/or prevent high local
density of nisin molecules necessary for pore formation. The
nisin-exporting function of NisFEG would diminish the quantity of nisin
molecules that have already entered the cytoplasmic membrane
before/during pore formation.