From the Department of Genetics, Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands, and the
¶ Department of Pharmaceutical Biology, University of
Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands
Received for publication, March 8, 2001
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ABSTRACT |
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The processing of secretory preproteins by signal
peptidases (SPases) is essential for cell viability. As
previously shown for Bacillus subtilis, only certain SPases
of organisms containing multiple paralogous SPases are essential. This
allows a distinction between SPases that are of major and minor
importance for cell viability. Notably, the functional
difference between major and minor SPases is not reflected clearly in
sequence alignments. Here, we have successfully used molecular
phylogeny to predict major and minor SPases. The results were verified
with SPases from various bacilli. As predicted, the latter enzymes
behaved as major or minor SPases when expressed in B. subtilis. Strikingly, molecular modeling indicated that the
active site geometry is not a critical parameter for the classification
of major and minor Bacillus SPases. Even though the
substrate binding site of the minor SPase SipV is smaller than that of
other known SPases, SipV could be converted into a major SPase without
changing this site. Instead, replacement of amino-terminal residues of
SipV with corresponding residues of the major SPase SipS was sufficient
for conversion of SipV into a major SPase. This suggests that
differences between major and minor SPases are based on activities
other than substrate cleavage site selection.
Signal peptidases
(SPases)1 play a key role in
the transport of proteins across membranes in all living organisms. The
type I SPases are integral membrane proteins that remove signal
peptides from preproteins during or shortly after translocation across the cytoplasmic membrane, thereby releasing the mature proteins from
the trans side of the membrane (for reviews, see Refs. 1 and
2).
In recent years, type I SPases from many different organisms have been
identified. Comparison of these SPases showed that they can be divided
in two sub-families: P (prokaryotic)-type and ER (endoplasmic
reticulum)-type SPases (3). The P-type SPases are found in eubacteria
and organelles of eukaryotes. In contrast, the ER-type SPases are
typical for the endoplasmic reticular membrane. Strikingly, a few
ER-type SPases were shown to be present in sporulating Gram-positive
eubacteria such as Bacillus subtilis (3). In fact, B. subtilis was the first eubacterium in which the presence of both
P- and ER-type SPases was demonstrated. With respect to SPases,
B. subtilis is not only exceptional because it contains both
P- and ER-type SPases but also because it is the organism with the
largest known number of type I SPases. These include the chromosomally
encoded P-type SPases SipS, SipT, SipU, and SipV, the plasmid-encoded
P-type SPases SipP1015 and SipP1040, and the chromosomally encoded
ER-type SPase SipW (3-8). These observations suggested that at least
some of the SPases of B. subtilis have specialized
functions. Indeed, it was shown recently that SipS, SipT, and SipP1015
are of major importance for the secretion of degradative enzymes and
cell viability, whereas SipU, SipV, and SipW have only a minor role in
protein secretion and are probably involved in specific nonessential
processes (3, 8). For example, SipW is specifically required for the
processing of two precursors, pre-TasA and pre-YqxM, but not for cell
viability (9, 10). Because the presence of a single major SPase
(i.e. SipS, SipT, or SipP1015) is sufficient for the growth
and cell viability of B. subtilis, it seems the secretory
precursor processing machinery of this organism is functionally
redundant (3, 8).
In addition to an amino-terminal membrane anchor domain (A), all
P-type SPases contain four well conserved domains (B-E) (2). These
conserved domains include residues involved in substrate recognition
and catalysis. Specifically, domain B contains a strictly conserved Ser
residue, and domain D contains a strictly conserved Lys residue.
Together, these residues form a Ser-Lys catalytic dyad (11). The
domains B-E of the P-type SPases are conserved in the ER-type SPases.
Nevertheless, instead of a Lys residue, domain D of the ER-type SPases
contains a strictly conserved His residue, which is required for
catalysis. At present, it is not known whether this His residue is part
of a Ser-His catalytic dyad or a Ser-His-Asp catalytic triad as
described for the classic serine proteases (12, 13).
The distinction between P-type and ER-type SPases can readily be made
on the basis of the conserved Lys or His residues in domain D. In
contrast, it is not presently clear which properties determine whether
a type I SPase is a major or a minor SPase of B. subtilis. A
clear definition of these properties is important to understand the
molecular basis for SPase substrate specificity. Therefore, the present
studies were aimed at the characterization of differences between major
and minor Bacillus SPases and the identification of domains
in these enzymes that are critical for their specificity. The results
show that major and minor Bacillus SPases can be
distinguished by phylogenetic analyses and that critical information
for their role in cell viability is provided by residues that are
located amino-terminally of the catalytic Ser residue. Strikingly,
molecular modeling of the active site of major and minor P-type SPases
of B. subtilis suggests that the active site cleft of the
minor SPase SipV is significantly smaller than those of the other known
Bacillus P-type SPases. Nevertheless, this difference can
not explain why SipV is a minor SPase.
Plasmids, Bacterial Strains, and Media--
Table
I lists the plasmids and bacterial
strains used. Tryptone/yeast-extract medium contained Bacto tryptone
(1%), Bacto yeast extract (0.5%), and NaCl (1%). If required, the
media for Escherichia coli were supplemented with ampicillin
(50 µg/ml) or kanamycin (20 µg/ml); the media for B. subtilis were supplemented with kanamycin (20 µg/ml) or
chloramphenicol (Cm) (5 µg/ml).
Evolutionary Tree Computations--
Amino acid sequences of
Bacillus SPases were collected from the SubtiList and
GenBank data bases. Alignments were performed with ClustalX software
(14) using the "Gonnet 250" and "Gonnet series" matrices as the
pairwise alignment parameters and multiple alignment parameters,
respectively. Default gap opening and extension parameters were
applied. When the SPase of E. coli (Lep) was included in the
alignments using the same parameters, the aligned sequences showed
highly congruent areas that correspond to DNA Techniques--
Procedures for DNA purification,
restriction, ligation, agarose gel electrophoresis, and the
transformation of E. coli were carried out as described
(16). Enzymes were from Roche Molecular Biochemicals. The polymerase
chain reaction (PCR) was carried out with Vent DNA polymerase (New
England Biolabs) as described (17). DNA and protein sequences were
analyzed with the PCGene Analysis program (version 6.7, Intelligenetics, Inc.) and ClustalW (version 1.74) (18). B. subtilis was transformed as described (3). Correct integration of
plasmids or resistance markers into the chromosome of B. subtilis was verified by Southern blotting or PCR.
The plasmid pGDL90, specifying Sip (Bli) from Bacillus
licheniformis, was constructed by ligating an EcoRI-
and SalI-cleaved PCR-amplified fragment of sip
(Bli) into the corresponding sites of pGDL48. The sip
(Bli)-specific fragment was amplified with primers Lbl1
(5'-ACGCGTCGACTATGCTGTGACAGACTG-3') and Lbl2
(5'-CGGAATTCGCAGTGCTGGCATCA-3') using B. licheniformis
chromosomal DNA as a template. Plasmid pM0V, specifying a hybrid of
SipS and SipV from B. subtilis, was constructed by ligating
an EcoRI- and SalI-cleaved PCR-amplified fragment
of sipV into the corresponding sites of pM0. The
sipV-specific fragment was amplified with primers JBV1
(5'-TGTCGTCGACGGTGACAGTATGAACCCGACCTTCC-3') and JBV2
(5'-CGGAATTCGCTAGCGACGCCTCTTCAATTAGCA-3') using B. subtilis chromosomal DNA as a template. Note that primer JBV1 is
designed such that the resulting SipSV fusion protein contains the
amino-terminal fragment of SipS (residues 1-43) that includes the
active site Ser-43 residue fused to the carboxyl-terminal fragment
starting at the corresponding position in SipV (residues 34-168).
Western Blot Analysis--
Polyclonal antibodies against SipS of
B. subtilis were prepared by the immunization of rabbits
(Eurogentec) with a purified soluble form of this
protein (sf-SipS-His K83A). This protein consists of the catalytic
domain of SipS lacking residues 2-29. Furthermore, sf-SipS-His K83A of
B. subtilis (Bsu) contains a carboxyl-terminal
hexa-histidine tag, facilitating the purification by metal affinity
chromatography (19). Western blotting was performed as described (20).
After separation by SDS-polyacrylamide gel electrophoresis, proteins
were transferred to Immobilon-polyvinylidene difluoride membranes
(Millipore). To detect SPases, B. subtilis or E. coli cells were separated from the growth medium by centrifugation (5 min, 10,000 × g, room temperature), and samples for
SDS-polyacrylamide gel electrophoresis were prepared as described
previously (19, 21). SPases were visualized with specific antibodies
and horseradish peroxidase anti-rabbit-IgG conjugates (Amersham
Pharmacia Biotech).
Molecular Modeling and Molecular Dynamics
Simulations--
Three-dimensional models of Bacillus
SPases were built on the basis of homology with the E. coli
SPase (PDB Protein Data Bank no. 1b12) using the molecular modeling
program What-If (22). The molecular dynamics program GroMacs
(University of Groningen, MD group) was used to perform a standard
energy minimization in vacuo of a pentapeptide substrate in
the three-dimensional model of SipS.
Phylogenetic Clustering of Major Bacillus Signal
Peptidases--
To investigate the relationships between major and
minor SPases of the Bacillus species, phylogenetic analyses
were performed by applying the maximum likelihood and MP methods. For
this purpose, either the complete sequences, conserved Functional Identification of Major Bacillus SPases--
Because
the distinction between major and minor SPases is based on functional
differences, we tested the outcome of the phylogenetic analysis in
complementation experiments with two representative SPases: SipC of
B. caldolyticus (23), which clusters with the minor SPases
(Fig. 1), and Sip (Bli) of B. licheniformis (24), which
clusters with the major SPases. To this purpose, the sipC gene was expressed in the B. subtilis strain
Because the antibodies raised against SipS of B. subtilis
cross-reacted with Sip (Bli) (these studies) and the major SPase SipP1015 (8), we investigated whether these antibodies could be used to
discriminate between major and minor SPases of B. subtilis. To this purpose, Western blotting experiments were performed with strains containing plasmids for the overproduction of the respective SPases. Only SipT was shown to cross-react with the antibodies raised
against SipS, which implies that the major SPases SipS, SipT, and
SipP1015 share at least one antigenic determinant that is absent from
the minor SPases SipU, SipV, and SipW (Fig.
3). This idea is supported by the
observation that the major SPases SipS (Bam), SipT (Bam), and SipP1040
cross-reacted with the antibodies against SipS (Bsu) (data not shown).
It has to be noted, however, that the antibodies against SipS also
cross-reacted with the catalytic domain of SipC (Bca) upon
overproduction in E. coli (data not shown), indicating that
these antibodies do not allow the discrimination between major and
minor Bacillus SPases in general.
SPase Active Site Modeling by Homology--
To investigate whether
the active site geometries of the known major and minor P-type SPases
of B. subtilis are significantly different,
three-dimensional models of these SPases were constructed on the basis
of the crystal structure of the E. coli SPase as determined
by Paetzel et al. (11). For this purpose, the sequences of
these SPases were aligned with the ClustalW program (Fig.
4). SipS (Bsu) and the E. coli
SPase show an overall sequence identity of 26%, which is low for
modeling by homology. However, the four conserved domains B-E of these
SPases show 62% sequence identity. Notably, the active site of the
E. coli SPase is almost entirely composed of these four
conserved domains that are typical for all P-type SPases (11). We have
therefore based our conclusions exclusively on modeled active site
regions of Bacillus SPases. The homology modeling program
What-If was used to generate the three-dimensional models of various
known SPases of bacilli. As shown for SipS of B. subtilis
(Fig. 5), Met-44 and Leu-48 (marked in
blue), Val-39 and Val-82 (marked in green), and
Lys-83 form the S1 substrate binding pocket, whereas Tyr-37, Val-54,
Val-73, and His-80 (marked in yellow) together with the
residues marked in green form the S3 pocket. These findings
are in good agreement with the structure of the S1 and S3 substrate
binding pockets of the SPase of E. coli (25). Furthermore,
the idea that the latter residues make contact with the substrate
(i.e. the SPase recognition sequence in a precursor protein)
was supported by a molecular dynamics analysis in which a pentapeptide
of five Ala residues in a
The comparison of our models for the P-type SPases of B. subtilis showed that the substrate binding pockets of SipS, SipT, SipP1015, and SipP1040 were highly similar, whereas that of SipV was
significantly smaller (Fig. 5). Conversely, the substrate binding site
of SipU seemed to have a wider S1 pocket than the equivalent sites of
the other P-type SPases of B. subtilis. Upon close
examination, the volume of the substrate binding pocket of SipV is
relatively small because the side chains of Leu-73, Ile-82, and
possibly Leu-54 (SipS numbering) protrude into the S3 pocket (Fig. 5).
The latter side chains are larger than those of the equivalent residues
in SipS of B. subtilis (Val-73, Val-82, and Val-54,
respectively) and other SPases (Fig. 4). Taken together, these
observations indicate that the active site geometries of the minor
SPases SipU and SipV of B. subtilis are different from the
active site geometries of the known major SPases.
A SipS-SipV Fusion Protein Is a Major SPase--
To investigate
whether the active site geometry is a critical determinant for major
and minor Bacillus SPases, a SipS-SipV hybrid protein
(denoted SipSV) was constructed, which is specified by plasmid pM0V.
Notably, this fusion between SipS and SipV of B. subtilis
was made at the catalytic Ser residue of these SPases. Consequently,
SipSV consists of the first 43 residues of SipS and the
carboxyl-terminal part of SipV. The major advantage of this approach is
that the active site geometry of SipSV is nearly identical to that of
SipV (data not shown). Next, we tested whether SipSV is a major or
minor SPase by introducing pM0V into B. subtilis On the basis of their importance for cell viability, we previously
have classified the type I SPases of B. subtilis as major (SipS, SipT, and SipP) and minor SPases (SipU, SipV, and SipW) (3, 8).
Thus far, it was not clear which properties of these SPases are
important for this functional distinction, particularly with respect to
the P-type SPases. Consequently, simple amino acid sequence alignments
could not be used to predict the group to which certain
Bacillus P-type SPases would belong. In the present studies,
we show for the first time that major and minor SPases can be
distinguished via phylogenetic analyses. Surprisingly, the subsequent
molecular analyses demonstrate that the distinction between major and
minor SPases does not relate specifically to the catalytic domain of a
Bacillus P-type SPase but rather to its amino-terminal
domain, which contains the membrane anchor. The latter result was
unexpected, because it was shown recently by Carlos et al.
(26) that the transmembrane domains of P-type SPases are not important
determinants for cleavage fidelity in vitro.
The most important outcome of the phylogenetic analyses of the
Bacillus type I SPases is that the major SPases form a
distinct cluster, which is well supported by the maximum parsimony and maximum likelihood methods. Moreover, these phylogenetic analyses have
predictive value, as exemplified by the complementation experiments with SipC (Bca) and Sip (Bli), showing that these behave as minor and
major SPases, respectively, when the corresponding genes are expressed
in B. subtilis. This, however, does not exclude the possibility that SipC is a major SPase in B. caldolyticus.
Furthermore, the phylogenetic analyses indicate the existence of two
clusters of minor SPases: the SipC/SipV, and SipW clusters. The latter cluster was identified previously because it contains the known ER-type
SPases of bacilli (3, 13). Only two Bacillus SPases, SipU
(Bsu) and SipX (Ban), do not belong to the three clusters of major
SPases, SipC/SipV, or SipW. This suggests that these two SPases
represent possible evolutionary intermediates between different
clusters, which is particularly interesting in the case of SipX of
B. anthracis, because this P-type SPase might represent a
link between the P- and ER-type Bacillus SPases.
The present observation that a SipSV hybrid protein containing the
largest part of the catalytic domain of the minor SPase SipV behaves as
a major SPase indicates that the catalytic domain of the P-type SPases
is not the most important determinant for the difference between major
and minor SPases. This view is supported by the fact that, according to
our models, the active site geometry of SipSV is identical to that of
the minor SPase SipV. In this respect it is important to bear in mind
that the S3 substrate binding pockets of SipV and SipSV are relatively
small compared with those of other P-type SPases of B. subtilis, particularly SipU. Nevertheless, the possibility that
subtle changes in the active site geometry of SipSV caused by the
fusion between the SipS and SipV moieties result in the conversion of a
minor SPase into a major SPase can presently not be excluded. The idea
that the catalytic domain is not important for the difference between major and minor SPases would explain why the antibodies raised against
the catalytic domain of SipS (Bsu) can not be used to distinguish
between these two functionally defined groups of SPases.
What could be the role of the amino-terminal residues of SipS in
determining its role as a major SPase? Carlos et al. (26) have recently provided compelling evidence that the transmembrane segments of type I SPases such as SipS are not important for substrate cleavage site selection. Furthermore, we have shown recently that the
membrane anchor of SipS is not required for its activity (23). Together
with our present results, these observations imply that the major-minor
difference of SPases is not based on the recognition of residues at the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Plasmids and bacterial strains
-helices,
-strands, and
previously defined conserved domains (D) of Lep and other type I
SPases (2, 11). Therefore, the complete data set, predicted
-helices,
-strands, and conserved domains were used in the
phylogenetic analyses. Autapomorphic insertions or deletions were
removed from all data sets. Tree reconstructions were performed according to two different methods. First, the maximum likelihood method was used as implemented in the PUZZLE 4.02 software (15). The
variable time matrix was applied with four
rates. One
thousand replications were used to calculate the quartet puzzling
values. Second, the maximum parsimony (MP) method was used as
implemented in the program PAUP 4.03b. The MP tree
reconstruction was done with the
branch-swapping/tree-bisection-reconnection algorithm by applying 10 random additions of sequences. One thousand replications were used to
calculate the bootstrap values.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices,
-strands, or domains were used (Table
II). Consistent with the fact that only
few
-helices are present in type I SPases (11), the maximum likelihood analysis that was based on
-helices resulted in a tree
with a poorly resolved topology, and the equivalent MP analysis in 108 "most parsimonious trees" (112 steps long, CI excluding uninformative characters = 0.8077, RI = 0.7101, RC = 0.5833). Far better results were obtained when complete sequences,
-strands, or conserved domains were used in the maximum likelihood
and MP analyses (Table II). In fact, the MP analysis with complete
sequences resulted in one most parsimonious tree (805 steps, CI
excluding uninformative characters = 0.8188, RI = 0.7152, RC = 0.5988) (Fig. 1). One most
parsimonious tree was obtained when conserved
-strands were used
(283 steps, CI excluding uninformative characters = 0.8618, RI = 0.8046, RC = 0.7079), and three most parsimonious trees
were obtained with the conserved domains (134 steps, CI excluding
uninformative characters = 0.8319, RI = 0.7938, RC = 0.6753). Notably, all data sets are congruent with respect to the
clustering of the four best supported groups of Bacillus
SPases: (i) the SipW group, (ii) the SipV group, (iii) the SipT of Bsu + SipT of the Bacillus amyloliquefaciens (Bam) group, and
(iv) the SipS group (Table II). As shown in Fig. 1, SipT of
Bacillus anthracis (Ban) seems to be more closely related to
the SipW group (bootstrap percentages/quartet puzzling values are
100/not calculated for complete sequences and 100/70 for
-strands) than to the SipT (Bsu) + SipT (Bam) group. To prevent the
possible misinterpretation that SipT (Ban) is related to the major
SPases SipT (Bsu) and SipT (Bam), the SipT (Ban) protein was renamed
SipX (Ban). Furthermore, SipC of Bacillus caldolyticus (Bca)
seems to be most closely related to SipV (Bsu) and SipV (Bam). Most
importantly, the functionally defined major SPases SipS, SipT, and
SipP1015 of B. subtilis cluster together (Fig. 1,
circled). This clustering is supported by bootstrap percentages of 79/93 and 77/60 when complete sequences or
-strands were used for the analyses, respectively. This observation suggests that the other SPases in this cluster, SipP1040, SipS (Bam), SipT (Bam), and Sip (Bli), should also be classified as major SPases. In
contrast, all enzymes not included in this cluster would be minor
SPases.
Summary of quartet puzzling and bootstrap values
,
-helices;
,
-strands; D, conserved domains.
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Fig. 1.
Most parsimonious tree of known
Bacillus SPases. A most parsimonious tree of
different Bacillus SPases is shown. The calculated maximum
parsimony (top) and maximum likelihood (bottom)
values are shown at the junctions. The length of the branches reflects
the number of amino acid changes between different SPases, as indicated
by the bar. See "Experimental Procedures" for details
concerning the calculations. The cluster of major SPases is
circled. The following SPases are shown: SipS, SipT, SipU,
SipV, SipW, SipP1015, and SipP1040 from B. subtilis; SipS,
SipT, SipV, and SipW from B. amyloliquefaciens; SipC from
B. caldolyticus; Sip from B. licheniformis; and
SipX and SipW from B. anthracis. Note that SipX was
originally annotated as SipT (NCBI accession no. AAF13664), but to
avoid possible misinterpretations this SPase was renamed.
nc, not calculated.
S, which
lacks the sipS gene, by transformation with the
pGDL48-derived plasmid pGDL46.36. Subsequently, we tried to disrupt the
sipT gene of the resulting strain with a Cm resistance
marker by transformation with chromosomal DNA of B. subtilis
T-Cm. Even though this experiment was repeated several times, no
Cm-resistant transformants were obtained, indicating that SipC of
B. caldolyticus behaves as a minor SPase in B. subtilis that cannot replace SipS and SipT. A completely different
result was obtained in parallel experiments with Sip (Bli). To test
whether this SPase could replace SipS and SipT of B. subtilis, the sip (Bli) gene was amplified by PCR and
cloned. Next, B. subtilis
S was transformed with the
pGDL48-derived plasmid pGDL90, which carries the sip (Bli)
gene, and the sipT gene of the resulting strain was
disrupted with a Cm resistance marker by transformation with the
chromosomal DNA of B. subtilis
T-Cm. Viable Cm-resistant transformants were obtained and shown to have disrupted the
sipS and sipT genes (data not shown). As shown by
Western blotting, these transformants produce Sip (Bli), which
cross-reacts with the antibodies raised against the catalytic domain of
SipS of B. subtilis (Fig. 2).
Taken together, these observations strongly suggest that Sip (Bli)
behaves as a major SPase and SipC (Bca) as a minor SPase in B. subtilis. The view that all SPases clustering with SipS (Bsu),
SipT (Bsu), SipP1015, and Sip (Bli) behave as major SPases was finally
confirmed by similar complementation experiments, demonstrating that
SipS (Bam), SipT (Bam), and SipP1040 can replace SipS and SipT of
B. subtilis.2
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Fig. 2.
Functional identification of major
SPases. Western blot analysis of B. subtilis 8G5 (BS), B. subtilis S (BS
S), B. subtilis
ST (BS
ST), and E. coli MC1061
(EC) producing Sip (Bli) as specified by plasmid pGDL90 or
the hybrid SPase SipSV as specified by plasmid pM0V. As a control,
strains containing the empty vector pGDL48 (ev) were used.
The presence of SipS (Bsu), SipT (Bsu), and Sip (Bli) was visualized
with polyclonal antibodies against the catalytic domain of SipS. Note
that the hybrid SPase SipSV does not cross-react with these
antibodies.
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Fig. 3.
Limited cross-reactivity of SipS-specific
antibodies. Western blot analysis of B. subtilis S
strains containing plasmids for the overproduction of type I SPases of
B. subtilis. Plasmid pGDL41 was used for the overproduction
of SipS, pGDL100 for SipT, pGDL121 for SipU, pGDL131 for SipV, and
pGDL140 for SipW.
-strand conformation was modeled into the
substrate binding pockets of SipS (Fig. 5). This pentapeptide was
placed at the position that corresponds to that of the penem
inhibitor in the crystal structure of the E. coli SPase I
(11).
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Fig. 4.
Alignment of the substrate binding regions of
known Bacillus SPases. The conserved domains B,
C, and D from P-type SPases of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. caldolyticus, B. anthracis, and E. coli,
which contain residues that form the S1 and S3 substrate binding
regions, were aligned. Residues predicted to belong to the S1 or S3
pockets are labeled with 1 or 3, respectively.
Residue numbers below the alignment are derived from SipS
(Bsu).
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Fig. 5.
Active site models for SipS and SipV.
Stereo pictures of residues in the modeled active site regions of SipS
(top) and SipV (bottom). Residues rendered
in spheres are likely to be involved in the formation of the
S1 pocket (Met-44 and Leu-48 of SipS, blue), S1 and S3
pockets (Val-39 and Val-82 of SipS, green), or S3 pocket
(Tyr-37, Val-54, Val-73, and His-80 of SipS, yellow) for
substrate binding. The SipS model contains a pentapeptide of Ala
residues, which are docked in the substrate binding pocket. The P1 and
P3 residues of this model substrate are shown in orange. The
active site Ser-43 and Lys-83 residues as well as Tyr-81 of SipS and
the corresponding Leu residue of SipV are shown as "ball and stick"
models. The latter residues are probably involved in substrate
stabilization by interaction with P2 residues of the substrate.
ST as
described above for the sipC (Bca) and sip (Bli)
genes. Strikingly, viable
ST transformants containing pM0V were
obtained, showing that SipSV can replace SipS and SipT. As shown in
Fig. 2, SipSV is not recognized by the antibodies raised against SipS. In conclusion, these observations show that SipSV is a major SPase and
that SipV is not a minor SPase because of the geometry of its catalytic
site but rather that some residues of its amino-terminal stretch
determine SipV to belong to the class of minor SPases. Furthermore, the
antibodies against SipS do not distinguish between major and minor SPases.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
3 positions, relative to the scissile peptide bond per
se. This leaves at least three alternative possibilities open.
First, the amino-terminal residues might position the catalytic site of
a major SPase in such a way in the membrane that it can interact with
the cleavage site of one or more as yet unidentified preproteins that
have to be processed for cell viability. Second, the amino-terminal
residues might be required for an as yet unidentified essential
interaction of a major SPase with preprotein translocases. Third, the
amino-terminal residues might target the respective SPases to
topologically distinct regions of the membrane such as the septa of
dividing cells. Notably, the regions preceding the active site Ser
residues of Bacillus SPases, which include their membrane
anchor, show a relatively high degree of sequence variation (23). To
elucidate the role of the amino-terminal region in SPase function, we
are presently investigating the role of the first 42 residues of SipS
by site-directed mutagenesis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gert Vriend and Dr. Rob Veltman for critical discussions on the modeling of Bacillus SPases, Drs. Danilo Roccatano and Giorgio Colombo for the molecular dynamics simulations, and Dr. Harold Tjalsma, Dr. O. Kuipers, and other members of the European Bacillus Secretion Group for stimulating discussions.
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FOOTNOTES |
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* This work was supported by the Dutch Ministry of Economic Affairs through Associatie Biologische Onderzoeksscholen Nederland (to M. L. v.R.), Stichting Levenswetenschappen Grant 805-33.605 (to J. D. H. J), and European Union Grants QLK3-CT-1999-00413 and QLK3-CT-1999-00917 (to S. B, J. D. H. J., J. Y. D., and J. M. v.D.).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.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
31-50-363-3079; Fax: 31-50-363-2348; E-mail:
J.M.VAN.DIJL@FARM.RUG.NL.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M102099200
2 J. D. H. Jongbloed and H. Tjalsma, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SPase, signal peptidase; P, prokaryotic; ER, endoplasmic reticulum; Cm, chloramphenicol; D, conserved domains; MP, maximum parsimony; PCR, polymerase chain reaction; Bli, B. licheniformis; Bsu, B. subtilis; CI, consistency index; RI, retention index; RC, rescaled consistency index; Bam, B. amyloliquefaciens; Ban, B. anthracis; Bca, B. caldolyticus.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Pugsley, A. P. (1993) Microbiol. Rev. 57, 50-108[Abstract] |
2. |
Dalbey, R. E.,
Lively, M. O.,
Bron, S.,
and van Dijl, J. M.
(1997)
Protein Sci.
6,
1129-1138 |
3. |
Tjalsma, H.,
Bolhuis, A.,
van Roosmalen, M. L.,
Wiegert, T.,
Schumann, W.,
Broekhuizen, C. P.,
Quax, W. J.,
Venema, G.,
Bron, S.,
and van Dijl, J. M.
(1998)
Genes Dev.
12,
2318-2331 |
4. | van Dijl, J. M., de Jong, A., Vehmaanperä, J., Venema, G., and Bron, S. (1992) EMBO J. 11, 2819-2828[Abstract] |
5. | Bolhuis, A., Sorokin, A., Azevedo, V., Ehrlich, S. D., Braun, P. G., de Jong, A., Venema, G., Bron, S., and van Dijl, J. M. (1996) Mol. Microbiol. 22, 605-618[Medline] [Order article via Infotrieve] |
6. |
Tjalsma, H.,
Noback, M. A.,
Bron, S.,
Venema, G.,
Yamane, K.,
and van Dijl, J. M.
(1997)
J. Biol. Chem.
272,
25983-25992 |
7. | Meijer, W. J. J., de Jong, A., Wisman, G. B. A., Tjalsma, H., Venema, G., Bron, S., and van Dijl, J. M. (1995) Mol. Microbiol. 17, 621-631[Medline] [Order article via Infotrieve] |
8. |
Tjalsma, H.,
van den Dolder, J.,
Meijer, W. J. J.,
Venema, G.,
Bron, S.,
and van Dijl, J. M.
(1999)
J. Bacteriol.
181,
2448-2454 |
9. |
Stöver, A. G.,
and Driks, A.
(1999)
J. Bacteriol.
181,
7065-7069 |
10. |
Stöver, A. G.,
and Driks, A.
(1999)
J. Bacteriol.
181,
1664-1672 |
11. | Paetzel, M., Dalbey, R. E., and Strynadka, N.-C. J. (1998) Nature 396, 186-190[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Van Valkenburgh, C.,
Chen, X. M.,
Mullins, C.,
Fang, H.,
and Green, N.
(1999)
J. Biol. Chem.
274,
11519-11525 |
13. |
Tjalsma, H.,
Stöver, A. G.,
Driks, A.,
Venema, G.,
Bron, S.,
and van Dijl, J. M.
(2000)
J. Biol. Chem.
275,
25102-25108 |
14. |
Thompson, J. D.,
Gibson, T. J.,
Plewniak, F.,
Jeanmougin, F.,
and Higgins, D. G.
(1997)
Nucleic Acids Res.
25,
4876-4882 |
15. |
Strimmer, K.,
and Von Haeseler, A.
(1996)
Mol. Biol. Evol.
13,
964-969 |
16. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
17. |
van Dijl, J. M.,
de Jong, A.,
Venema, G.,
and Bron, S.
(1995)
J. Biol. Chem.
270,
3611-3618 |
18. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |
19. |
van Roosmalen, M. L.,
Jongbloed, J. D. H.,
Kuipers, A.,
Venema, G.,
Bron, S.,
and van Dijl, J. M.
(2000)
J. Bacteriol.
182,
5765-5770 |
20. | Kyhse-Andersen, J. (1984) J. Biochem. Biophys. Methods 10, 203-209[CrossRef][Medline] [Order article via Infotrieve] |
21. | van Dijl, J. M., de Jong, A., Smith, H., Bron, S., and Venema, G. (1991) Mol. Gen. Genet. 227, 40-48[Medline] [Order article via Infotrieve] |
22. | Vriend, G. (1990) J. Mol. Graph. 8, 52-56[CrossRef][Medline] [Order article via Infotrieve] |
23. |
van Roosmalen, M. L.,
Jongbloed, J. D. H.,
de Jong, A.,
van Eerden, J.,
Venema, G.,
Bron, S.,
and van Dijl, J. M.
(2001)
Microbiology
147,
909-917 |
24. | Hoang, V., and Hofemeister, J. (1995) Biochim. Biophys. Acta 1269, 64-68[Medline] [Order article via Infotrieve] |
25. | Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (2000) Pharmacol. Ther. 87, 27-49[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Carlos, J. L.,
Paetzel, M.,
Brubaker, G.,
Karla, A.,
Ashwell, C. M.,
Lively, M. O.,
Cao, G. Q.,
Bullinger, P.,
and Dalbey, R. E.
(2000)
J. Biol. Chem.
275,
38813-38822 |
27. | Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 6, 60-89[CrossRef] |
28. | Bron, S., and Venema, G. (1972) Mutat. Res. 15, 395-409[Medline] [Order article via Infotrieve] |