From the Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
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ABSTRACT |
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Many non-lantibiotic bacteriocins of lactic acid
bacteria are produced as precursors with N-terminal leader peptides
different from those present in preproteins exported by the general
sec-dependent (type II) secretion pathway.
These bacteriocins utilize a dedicated (type I) secretion system for
externalization. The secretion apparatus for the lactococcins A, B, and
M/N (LcnA, B, and M/N) from Lactococcus lactis is composed
of the two membrane proteins LcnC and LcnD. LcnC belongs to the
ATP-binding cassette transporters, whereas LcnD is a protein with
similarities to other accessory proteins of type I secretion systems.
This paper shows that the N-terminal part of LcnC is involved in the
processing of the precursor of LcnA. By making translational fusions of
LcnC to the reporter proteins Several strains of Lactococcus lactis secrete
antibacterial peptides or proteins with antagonistic activities, the
so-called bacteriocins (1, 2). The lactococcins LcnA, B, and M/N are bacteriocins that are synthesized as precursor proteins containing N-terminal leader peptides of the double-glycine type (3). These
leaders are removed during maturation. The secretion apparatus for all
three bacteriocins consists of the two membrane proteins LcnC and LcnD
(4). Similar secretion systems have been described for bacteriocins of
other lactic acid bacteria and for colicin V from Escherichia
coli (5-10).
LcnD is an accessory protein with similarities to other proteins known
or believed to be involved in the secretion of various (poly)peptides.
They always operate in conjunction with a protein from the family of
ATP-binding cassette (ABC)1
transporters. The accessory proteins of Gram-negative bacteria are
proposed to form a family of so-called membrane fusion proteins (11).
It is hypothesized that they connect the inner and the outer membranes
to facilitate the passage of substrates. CvaA, a member of the membrane
fusion protein family, involved in the secretion of colicin V, has been
shown to interact with both a cytoplasmic membrane protein (the ABC
transporter) and a protein present in the outer membrane (12). The
function of these accessory proteins in Gram-positive bacteria is unknown.
LcnC, a protein of 715 amino acids, belongs to the family of ABC
transporter proteins present in both prokaryotic and eukaryotic organisms and involved in the import or export of a large variety of
substrates such as antibiotics, sugars, amino acids, peptides, and
proteins. A typical ABC transporter protein consists of a hydrophobic
integral membrane domain and two ATP binding domains. These domains can
be either encoded as separate polypeptides or fused into multidomain
proteins. The hydrophobic integral membrane domain of the majority of
ABC transporter proteins is predicted to consist of two times six
transmembrane segments (TMSs) (13). In a number of cases, this topology
has been experimentally confirmed (14-17). LcnC contains only one ATP
binding domain in its C terminus, whereas the integral membrane domain
is, by computer analysis, predicted to consist of four to six TMSs (see below).
LcnC contains an N-terminal domain of approximately 160 amino acids,
which shows homology to other ABC transporter proteins involved in the
secretion of bacteriocins, and is not present in most other ABC
transporter proteins. The N-terminal parts of the ABC transporter
proteins involved in the secretion of the bacteriocins pediocin PA-1
and lactococcin G have been shown to process the precursors of the
respective bacteriocins into their mature forms. For lactococcin G,
this has been shown in in vitro experiments (3), whereas for
pediocin PA-1 this has been established in vivo in the
heterologous host E. coli (18).
In this paper we show that the N-terminal part of LcnC is involved in
the processing of the precursor of LcnA. To examine at which side of
the cytoplasmic membrane this processing takes place, we determined the
membrane topology of LcnC by making translational fusions of N-terminal
parts of LcnC with a modified alkaline phosphatase (PhoA*)2 lacking its own
signal sequence and Bacterial Strains, Plasmids, and Growth Conditions--
The
bacterial strains and plasmids used in this study are listed in Table
I. L. lactis was grown at
30 °C in 2-fold diluted M17 broth (19) supplemented with 0.5%
glucose (G1/2M17). For L. lactis, chloramphenicol was used
at a final concentration of 4 µg/ml. E. coli was grown in
TY broth (see Ref. 20; or 2× TY broth when used for enzyme activity
measurements) at 37 °C with aeration. Chloramphenicol, ampicillin,
and erythromycin were used at final concentrations of 10, 100, and 100 µg/ml for E. coli, respectively. The chromogenic
substrates
5-bromo-4-chloro-3-indolyl- Molecular Cloning, Transformation, and Nucleotide
Sequencing--
DNA techniques were performed essentially as described
by Sambrook et al. (21). Enzymes were purchased from
Boehringer Mannheim GmbH (Mannheim, Germany) and were used according to
the instructions of the supplier. DNA was isolated as described before
(22) or with the plasmid miniprep isolation kit of Boehringer Mannheim.
Electrotransformation of L. lactis was performed as
described earlier (23) with modifications according to Venema et
al. (24). E. coli was transformed as described (25).
Nucleotide sequences were determined using a Vistra DNA Labstation 625 in combination with a Vistra DNA sequencer 725 (Amersham International, Little Chalfont, UK).
Construction of Plasmids--
To overexpress (pre)LcnA in
L. lactis under control of the nisA
promoter, RcaI and HindIII restriction enzyme
sites were introduced upstream of lcnA and downstream of
lciA, respectively, by PCR on the template pKV4 (26)
using the primers CMF60III
(5'-AATTCGAATTCTCATGAAAAATCAATTAAATTTTAATATTG-3') and CMF61
(5'-TCTAGATCTAGAAAGCTTAGAATAGTCGTCCCCACGACATAAT-3'). The PCR
product was digested with RcaI and HindIII and
cloned in pNZ8048 restricted with NcoI and
HindIII, resulting in plasmid pNA0.
Vector pC164 was constructed to express the first 164 amino acids of
LcnC. To this end, pPC164 was digested with BglII and Asp718. After filling-in of the sticky ends, the plasmid was
self-ligated, giving rise to pIC164. Subsequently, the
AvaII/Eco47-3 fragment of the latter plasmid was
transferred to pIL253, resulting in pC164.
To express lcnD under control of the lactococcal P32
promoter, the SacI/HindIII fragment of pUClcnD
was cloned in pMG36E, resulting in pMGlcnD. The
EcoRI/HindIII fragment of pMGlcnD was transferred
to pUC19 to obtain pP32LcnD.
Generation of Directed LacZ and PhoA* Fusions--
Based on the
working model presented in Fig. 1A, primers were designed to
introduce BglII restriction enzyme sites in lcnC at various positions flanking the sequences encoding the putative transmembrane segments (TMS). PCRs were performed on pLC as a template,
using the various mutagenic primers in combination with primer MvdG1.
The sequences of the primers were (BglII sites are indicated
in bold) as follows: MvdG1, 5'-CCT CGG GAT ATG ATA AG-3'; JT164,
5'-AGA TCT AGA TCT TGA CGG GTG ATA ATT GGG-3'; JT194, 5'-AGA TCT AGA TCT CTG TCA ATC ATG CTC TGG AGG-3'; JT222, 5'-AGA TCT AGA TCT AAG ACC TGT TGG ATA-3'; JT225,
5'-AGA TCT AGA TCT GCA AAT TCT AAG ACC TGT TGG -3'; JT246,
5'-AGA TCT AGA TCT ATA TAA GAA AGA ATG ACA TC-3'; JT266,
5'-AGA TCT AGA TCT GTA ATT TCT CCT GTT CTT CGG-3'; JT277,
5'-AGA TCT AGA TCT AAA ATA GAA CTC GCA TCG G-3'; JT303,
5'-AGA TCT AGA TCT TTT TGA AGG CCT AAA ATT AG-3'; J T327,
5'-AGA TCT AGA TCT GGC GTA AAA ATA ATA ATA AC-3'; JT355,
5'-AGA TCT AGA TCT TCA ATC CCA TTG ATA TCT TC-3'; JT375,
5'-AGA TCT AGA TCT GCA AAT TCG TAG TCA ATT TTT TG-3'; JT391,
5'-AGA TCT AGA TCT TGA ATA GCT TCT GAT TTT TG-3'; JT435,
5'-AGA TCT AGA TCT GTA AAG TAA GAA AGC AGG GC-3'; JT478,
5'-AGA TCT AGA TCT TGT GAG AGG GAC AGT TC-3'; JT584,
5'-AGA TCT AGA TCT GCA TTC TCA TTA GCT CCT AG-3'. After
digestion with EcoRI and BglII, the PCR fragments
were cloned into either pFUSLC or pFUSPC and verified at the nucleotide
sequence level.
Enzyme Activity Assays--
Alkaline phosphatase and
Protein Electrophoresis and Western Hybridization--
After
harvesting and washing, the cells were resuspended in the appropriate
buffer for either alkaline phosphatase or Overexpression of (Pre)LcnA/LciA in L. lactis--
Induction of
genes under control of the nisA promoter were carried out in
L. lactis NZ9000 containing either pIL253 or pC164. Overnight cultures were diluted 100-fold into fresh medium and grown
until an A600 between 0.4 and 0.5. Subsequently,
the cultures were split in half, and one of these was induced with
nisin at a final concentration of 0.5 ng/ml. Both cultures were grown
for another 2 h. Purified nisin A was kindly provided by Dr. Oscar Kuipers (NIZO, Ede, The Netherlands).
Overlay Test L--
L. lactis cells were disrupted by
sonication using the Soniprep 150 (MSE, Crawley, UK) at 8 µm (eight
cycles of 15 s with 45-s intervals, all on ice), boiled in 2×
sample buffer, and run on Tricine/SDS-16% PAA gels. Gels were
overlayered with the indicator strain L. lactis IL1403
(18).
Computer Predictions of LcnC Membrane Topology--
To obtain a
working model for the membrane topology of LcnC, four different
computer programs were used that predict the topology of membrane
proteins. Due to the various algorithms used by these programs (SOAP
(32, 33), Rao and Argos (34), Helixmem (35), and TopPred (36, 37)) all
four programs arrived at a different topological model for LcnC. The
model obtained with SOAP, contains six transmembrane segments (TMSs)
and is shown in Fig. 1A.
TopPred predicts five of the six TMSs indicated by SOAP (namely I, II, IV, V, and VI), whereas both Rao and Argos (I, II, IV, and V) and
Helixmem (II, IV, V, and VI) only predict four.
LcnC Membrane Topology by Protein Fusion Studies--
The
contradictory results obtained with the various computer programs
warranted the analysis of the membrane topology of LcnC by protein
fusion studies. N-terminal fusions of LcnC were made with the reporter
proteins alkaline phosphatase (PhoA*) and Translational Fusions of N-terminal Parts of LcnC with
PhoA*--
From the data obtained in E. coli (Table II), it
is clear that at least the N-terminal 164 amino acids of LcnC reside in
the cytoplasm; the alkaline phosphatase activity of the fusion protein in which these amino acids are fused to PhoA* (PC164) is low, indicating an intracellular location of the PhoA* moiety. The alkaline
phosphatase activity of PC194 is high, indicating that the PhoA* moiety
in this chimera is in the periplasm. This strongly suggests the
presence of a TMS (TMS I) between the amino acids at positions 164 and
194. The chimeras PC222, PC225, PC246, PC266, and PC277 all have low
alkaline phosphatase activities in E. coli, indicating that
in all these chimeras the PhoA* moiety is intracellular. In accordance
with all computer programs tested, these results suggest that TMS II is
present and traverses the cytoplasmic membrane, whereas TMS III, only
predicted by SOAP, does not exist. The alkaline phosphatase activity of
PC303 is high compared with PC277, indicating that TMS IV is present.
The same applies to the fusions PC327, PC355, PC375, and PC390, which
argues against the existence of TMS V. As the alkaline phosphatase
activities of PC478 and PC584 are low, the C-terminal part of LcnC is
located intracellularly. This must be facilitated by the presence of
TMS VI. However, the high alkaline phosphatase activity of PC435 would
argue against the existence of TMS VI.
To examine the expression of the LcnC-PhoA* chimeras, all samples used
for the activity assays were subjected to SDS-PAGE and Western
hybridizations (Fig. 2, A and
B). As for all chimeras, bands of the expected sizes were
detected, lack of activity of certain chimeras cannot be explained by
lack of expression of that fusion protein (e.g. compare the
alkaline phosphatase activities and expression levels of PC194 and
PC222 in Table II and Fig. 2A, respectively). However, the
amount of full-sized fusion protein generally decreased as the size of
the chimera increased.
Several LcnC-PhoA* chimeras were expressed in L. lactis, and
their alkaline phosphatase activities were determined. The results, presented in Table II, are in accordance with those obtained in E. coli taking into consideration that, as we have shown
previously, alkaline phosphatase activity is high when the PhoA* moiety
is in the cytoplasm of L. lactis, whereas it is low when
PhoA* is extracellularly located.2 Western analysis showed
that the amount of fusion proteins in L. lactis with low
alkaline phosphatase activity, indicative of an extracellular location
of the PhoA* moiety, was much lower than the amount of a chimera with
high enzymatic activity (data not shown). This is in accordance with
similar topology studies of LcnD (38).
In conclusion, the results obtained with the LcnC-PhoA* fusions
indicate that both the N- and C-terminal parts of LcnC are located in
the cytoplasm and that four TMSs (I, II, IV, and VI) span the
cytoplasmic membrane (Fig. 1B).
Translational Fusions of N-terminal Parts of LcnC to
LacZ--
Genetic instability was observed during construction and
maintenance of several of the lcnC::lacZ plasmids.
Although all cultures were started from a single blue
(LacZ+) colony, several of these cultures appeared to
consist of mixtures of LacZ+ and LacZ
The differences in the
The expression levels of all LcnC-LacZ chimeras were examined by
Western hybridizations (data not shown). Bands of the expected sizes
were detected for all chimeras, whereas the amounts of full-sized fusion proteins varied between the LcnC-LacZ chimeras, and proteolytic breakdown was more severe than for the full-sized LcnC-PhoA* chimeras.
In conclusion, the results of the LcnC-LacZ fusions indicate that both
the N- and C-terminal part of LcnC are located in the cytoplasm and
that four TMSs span the cytoplasmic membrane. Although the data of the
LcnC-PhoA* fusions suggest the presence of TMSs I, II, IV, and VI (Fig.
1B), the results obtained with the LcnC-LacZ chimeras
favor the presence in the cytoplasmic membrane of the TMSs I,
II, IV, and V (Fig. 1C).
Influence of LcnD on the Membrane Topology of LcnC--
To examine
whether the membrane topology of LcnC was influenced by the presence of
its accessory protein LcnD, pUC19 or pP32LcnD was introduced into the
various E. coli strains expressing LcnC-LacZ or LcnC-PhoA*
fusions. No significant differences were observed in the
PreLcnA Is Processed by the N-terminal Domain of LcnC--
To
examine whether the N-terminal domain of LcnC, as postulated earlier
(3), is involved in the maturation of preLcnA into its mature form,
plasmids pC164 and pNA0 were jointly introduced into L. lactis NZ9000. A control strain carried pIL253 and pNA0. Production of preLcnA was induced from pNA0, either in the presence (pC164) or absence (pIL253) of the N-terminal 164 amino acids of LcnC.
Overexpression of preLcnA was clearly observed in both cases (Fig.
3). No effect of the presence of the N
terminus of LcnC on preLcnA overexpression was observed in Coomassie
Brilliant Blue-stained SDS-15% PAA gels.
Samples of sonicated cells were subjected to Tricine/SDS-PAGE and were
analyzed by overlayer assays (Fig. 4).
PreLcnA activity was only observed upon nisin induction of the
lcnA gene present on pNA0. In the presence of the N-terminal
164 amino acids of LcnC, activity of the mature form of LcnA was
clearly present, indicating that the processing domain of LcnC resides
in the N-terminal part.
To elucidate the membrane topology of LcnC, PhoA* and LacZ protein
fusions to various N-terminal parts of LcnC were studied. Both the
N-terminal domain of approximately 160 amino acid residues and the
C-terminal domain of LcnC, containing the ATP binding domain, were
shown to be located in the cytoplasm. Therefore, an even number of TMSs
should span the cytoplasmic membrane. Indeed, both the PhoA* and LacZ
studies indicate that LcnC contains four TMSs in LcnC, but they differ
in the exact positioning of these domains. Both reporter enzymes show
that TMSs I, II, and IV exist, whereas TMS III, predicted only by the
program SOAP, is not present. Ambiguity arises as to whether TMS V or
TMS VI is present; although the PhoA* fusions point to the existence of
TMS VI, the results obtained with the LacZ chimeras indicate that TMS V
traverses the cytoplasmic membrane. These conflicting results may be
explained in several ways. First, they may be ascribed to differences
in enzymatic activities caused by proteolytic breakdown of the various chimeras. As judged from Western hybridizations (Fig. 2 and data not
shown), high levels of protein of the expected size were observed for
those chimeras in which either of the reporter proteins had been fused
to amino acids within loops 1, 2, and 3 of LcnC (Fig. 1). The levels
were significantly lower for those chimeras with fusion points in loops
4 and 5 and for chimeras in which the reporter protein had been fused
to LcnC downstream of the predicted TMS VI. This applied in particular
to the chimeras in which the reporter protein was fused with amino acid
residues 435, 478, or 584 of LcnC, respectively (see Fig.
2B). Moreover, for those chimeras with a fusion to amino
acid residues 327, 355, 375, or 390, a strong additional protein band
was observed that decreased in mobility concomitantly with the
corresponding band of the full-length chimeras (Fig. 2B).
This may be the result of the presence of one or more specific
proteinase cleavage sites in loop 5. If certain of these breakdown
products are enzymatically active, the resulting background activities
might lead to a false topological assignment. If correct, this
explanation would favor the model based on PhoA* fusions, as PhoA*
first needs to be translocated to the periplasm of E. coli
to be active. PhoA* retained in the cytoplasm of E. coli
after proteolytic degradation of an intracellularly located chimera
would be inactive due to the absence of disulfide bridges, which are
normally formed in the periplasm by DsbA (39) and, therefore, would not
lead to a false background activity. LacZ is less reliable in this
respect since breakdown products that stay in the cytoplasm could give
rise to improper (background) activities.
Alternatively, it is conceivable that LcnC alternates between the two
different topological states and that both are, in fact, intermediate
stages of LcnC in the bacteriocin secretion process. Our results
suggest that loop 5 (situated between the putative TMSs V and VI) of
LcnC can either be found at the intracellular or extracellular side of
the cytoplasmic membrane. Interestingly, it has been described that the
multidrug transporter P-glycoprotein can exist in (at least) two
conformations, either with "two times four" or with "two times
six" TMSs spanning the cytoplasmic membrane (40, 41). P-glycoprotein
is a member of the ABC transporter family, in which two
nucleotide-binding sites and 12 putative TMSs are fused together in one
polypeptide. Striking similarity is observed between the topological
model of the N-terminal part of P-glycoprotein in its two times four
conformation and our model of LcnC based on the LcnC-PhoA* fusions.
The fact that the chimeras LC327, LC435, and PC435 were enzymatically
active may be due to the circumstance that the reporter protein had
been fused immediately downstream of a TMS. It is known that LacZ and
PhoA fusions can introduce biases into membrane protein topology
analysis (42, 43), especially when positively charged residues in the
amino acid sequence downstream of the TMS are absent in the fusion
protein, as is the case in these chimeras (44-48).
Apart from these possibilities, the results presented here do not
completely exclude the possibility that six membrane helices are
present in wild-type LcnC as all constructs lack the ATP binding domain, which may be involved in topological changes concurrent with
bacteriocin secretion.
To investigate whether loop 5 is able to alternate between a
cytoplasmic and extramembranous location, we attempted to raise antibodies against loop five. However, we were not able to overexpress this loop fused to an N-terminal His tag using various systems. Moreover, we were not able to generate reporter protein fusions at the
extreme C terminus of LcnC, as lcnC appeared to be
unclonable in E. coli and L. lactis under control
of P32.
The presence of LcnD in cells producing the various LcnC chimeras had
no effect on their enzymatic activities. Apparently, although LcnC and
LcnD form a complex in the
membrane,3 LcnD does not
influence the membrane topology of these LcnC chimeras.
We have shown here that the N terminus of LcnC is located in the
cytoplasm of L. lactis. The N-terminal part of LcnC was
expected to cleave prelactococcin to the mature active bacteriocin. As prebacteriocin maturation has only been shown in vitro or in
a heterologous host, we examined whether this could occur in
vivo in the natural producer organism, in this case L. lactis. In the presence of the N-terminal 164 amino acids of LcnC
(C164), processing of nisin-induced preLcnA into its mature form was
observed in the cytoplasm of L. lactis. The tiny amount of
mature LcnA present in the absence of C164 may be ascribed to general
proteolytic breakdown of the high amounts of prebacteriocin formed
inside the cell.
In conclusion, the N-terminal domain of LcnC is involved in the
processing of LcnA, a process that takes place at the cytosolic side of
the membrane. Based on the results presented in this article on LcnC
and those obtained by others with the analogous proteins LagD and PedD
(3, 18), we propose to classify a new subfamily of ABC transporters
called the ABC-containing Maturation and
Secretion Proteins (AMS proteins). Members of this ABC
transporter protein subfamily are involved in the secretion of
proteinaceous compounds and contain an additional N-terminal domain,
involved in the processing of their substrates.
-galactosidase (LacZ) and alkaline
phosphatase (PhoA*), it was shown that both the N- and C-terminal parts
of LcnC are located in the cytoplasm. As the N terminus of LcnC is
required for LcnA maturation and is localized in the cytoplasm, we
conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase (LacZ). The results indicate that
four membrane helices of LcnC span the cytoplasmic membrane and that
both the N- and C termini are cytoplasmic.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside and 5-bromo-4-chloro-3-indolylphosphate were used at final
concentrations of 5 µg/ml.
Bacterial strains and plasmids used in this study
-galactosidase assays were performed as described earlier (see Refs.
27 and 28, respectively). Independent cultures were started from three
single colonies of each construct. Plasmid stability was checked by
plating dilutions of the cultures on plates containing the proper
chromogenic substrate prior to the activity measurements. Enzyme assays
were performed in triplicate on 2 ml of exponential cultures of
E. coli and L. lactis. All samples used for the
enzymatic assays were also subjected to analysis by Western hybridizations.
-galactosidase activity
assays. Parts of the same samples were prepared for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by diluting aliquots of the suspensions 1:1 in 2× sample buffer (29) containing 2.5% SDS and subsequent boiling for 10 min. SDS-PAGE and Tricine/SDS-PAGE were carried out as
described earlier (29, 30). Fusion protein samples were run on SDS-5 or
10% PAA gels, whereas samples containing (pre)LcnA were run on either
SDS-15% PAA gels or Tricine/SDS-16% PAA gels. Proteins were blotted
onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany)
according to standard protocols (31). LacZ and PhoA* fusion proteins
were detected using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate (Promega, Madison, WI). Polyclonal antibodies against LacZ
and PhoA were obtained from 5 Prime
3 Prime, Inc. (Boulder, CO). Alkaline phosphatase-conjugated mouse anti-rabbit immunoglobulins (Promega) were used as secondary antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Topology models of the L. lactis
membrane protein LcnC, as predicted by the computer program SOAP
(A), alkaline phosphatase (B),
or -galactosidase (C)
activities of LcnC-PhoA* and LcnC-LacZ chimeras, respectively.
Positively and negatively charged amino acids are indicated by plus and
minus, respectively. Putative transmembrane segments are presented as
open boxes with roman numerals. The
numbered open circles identify the position of the amino
acid residues in LcnC to which the reporter proteins LacZ and PhoA*
have been fused. N, N-terminal domain (164 amino acids);
C, C-terminal domain (of 256 amino acids) containing the ATP
binding motifs; L, loop; CM, cytoplasmic
membrane.
-galactosidase (LacZ)
using the pFUSP/LC plasmid system that we have previously described.2 The alkaline phosphatase and
-galactosidase
activities of exponentially growing cultures of E. coli and
L. lactis carrying the constructed plasmids are presented in
Table II. Fig. 1A gives an
overview of the positions in LcnC to which PhoA* or LacZ were
fused.
Alkaline phosphatase (PhoA*) and -galactosidase (LacZ) activities in
exponentially growing E. coli and L. lactis cells expressing protein
chimeras specified by the indicated plasmids
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Fig. 2.
Western immunoblots of cell extracts of
exponentially growing E. coli cultures expressing
various LcnC-PhoA* fusion proteins. The plasmids contained in
cells of the individual cultures are indicated above each
lane. The sizes (in kDa) of the proteins in the protein molecular
weight marker in lane M are shown in the left
margins. The LcnC-PhoA* chimeras were detected using
anti-PhoA antibodies. The most pronounced slowest migrating band in
each lane represents the chimera of the expected size. The band
indicated by an arrow in the right margins
corresponds to a protein from the E. coli host. Blot
B has been developed twice as long as blot A in order
to visualize all the full-sized chimeric proteins.
cells.
Cultures in which this mixed phenotype was observed were discarded. The
enzymatic activities in cultures of cells expressing the various
LcnC-LacZ fusions are shown in Table II. The location of both the N-
and C-terminal domains of LcnC in the cytoplasm of E. coli
was confirmed by the observation that
-galactosidase activities of
the chimeras LC164, LC478, and LC584 were high. The
-galactosidase
activity of LC194 was much less than that of LC164, which is in
accordance with the presence of TMS I. However, the
-galactosidase
activities of those chimeras located in loops 2 and 3 were rather low,
whereas on the basis of the PhoA* fusions, the LacZ moiety was expected
to reside in the cytoplasm. The
-galactosidase activities of LC222
and LC225 were only slightly higher than that of LC194. The activities
of the chimeras in which LacZ had been fused to amino acids of loop 3 in LcnC showed considerable differences; fusions LC246 and LC277
suggest an extracellular location of loop 3, whereas LC266 would rather
favor a cytosolic location of loop 3. The low
-galactosidase
activity of LC303 indicated that loop 4 is exposed to the periplasm,
whereas the high activities of LC355, LC375, and LC390 are indicative
for a cytosolic location of loop 5. The low activity of LC327 may be
due to the absence of the positive charge of amino acid 331 in the
chimera, which may be important for a proper membrane location of loop
5. A similar explanation may apply to the low
-galactosidase
activity of LC435, as six positive charges immediately downstream of
the fusion point are missing in this chimera.
-galactosidase activities of various
LcnC-LacZ chimeras were more pronounced in L. lactis (Table
II). The activities of LC164, LC435, LC478, and LC584 clearly indicate that the N- and C-terminal domains of LcnC are located intracellularly. Loops 1 and 4 must be exposed at the extracellular side of the cytoplasmic membrane as the
-galactosidase activities of LC194 and
LC303 were very low. In contrast, loops 2, 3 and 5 seem to be
cytoplasmic, as judged from the high
-galactosidase activities of
LC225, LC277, and LC355.
-galactosidase or alkaline phosphatase activities, whether LcnD was
present or not (data not shown). The enzymatic activities of
representative LacZ and PhoA* chimeras in each of the loops were also
tested in L. lactis IL1403, a strain carrying the genes of
LcnC and LcnD homologues on its chromosome (26). The presence of the
lactococcin secretion apparatus did not influence the relative enzymatic activities of the various chimeras (data not shown).
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Fig. 3.
Coomassie Brilliant Blue-stained SDS-15% PAA
gel used to monitor the overexpression of preLcnA (8 kDa) and LciA (11 kDa) in L. lactis NZ9000. The plasmids present in
the cells are indicated above the lanes. The sizes (in kDa)
of the proteins in the protein molecular mass marker in lane
M are shown in the right margin. The arrow
indicates the position of the overexpressed proteins in lanes
2 and 4. Lanes 1 and 3, protein
samples of uninduced cultures ( ); lanes 2 and
4, samples of cultures induced with 0.5 ng/ml nisin A
(+).
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Fig. 4.
Tricine/SDS-16% PAA gel analysis of preLcnA
processing. After electrophoresis, the gel was washed to remove
SDS, placed on top of a G1/2M17 agar plate, and overlayered with 10 ml
of G1/2M17 top agar seeded with 10 µl of an overnight culture of
L. lactis IL1403. Lane 1, 5 µl of the
supernatant of an overnight culture of L. lactis IL1403
(pMB553) (49). Lane 2, protein molecular weight marker, the
sizes of which are shown in the right margin. Lanes
3 and 4, L. lactis NZ9000 (pNA0,pIL253)
uninduced and induced with 0.5 ng/ml nisin A, respectively. Lanes
5 and 6, L. lactis NZ9000 (pNA0,pC164)
induced and uninduced, with 0.5 ng/ml nisin A, respectively.
P, preLcnA; M, mature LcnA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Henk Mulder for preparing the photographs.
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FOOTNOTES |
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* This work was supported in part by a fellowship of the Royal Netherlands Academy of Arts and Sciences (to J. Kok).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 31 50 3632111;
Fax: 31 50 3632348; E-mail: J.Kok{at}biol.rug.nl.
2 C. M. Franke, J. Tiemersma, G. Venema, and J. Kok, submitted for publication.
3 M. Varcamonti, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: ABC, ATP-binding cassette; TMS, transmembrane segment; PAGE, polyacrylamide gel electrophoresis; PAA, polyacrylamide; PCR, polymerase chain reaction; Tricine, N-[2- hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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