From the Department of Genetics, Groningen Biomolecular Sciences
and Biotechnology Institute, P. O. Box 14, 9750 AA Haren, The
Netherlands and the Department of Pharmaceutical Biology,
University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
Received for publication, February 4, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In mitochondria, chloroplasts, and Gram-negative
eubacteria, Oxa1p(-like) proteins are critical for the biogenesis of
membrane proteins. Here we show that the Gram-positive eubacterium
Bacillus subtilis contains two functional Oxa1p
orthologues, denoted SpoIIIJ and YqjG. The presence of either SpoIIIJ
or YqjG is required for cell viability. Whereas SpoIIIJ is required for
sporulation, YqjG is dispensable for this developmental process. The
stability of two membrane proteins was found to be mildly affected upon
SpoIIIJ limitation in the absence of YqjG. Surprisingly, the topology and stability of other membrane proteins remained unaffected under these conditions. In contrast, SpoIIIJ- and YqjG-limiting conditions resulted in a strong post-translocational defect in the stability of
secretory proteins. Together, these data indicate that SpoIIIJ and YqjG
of B. subtilis are involved in both membrane
protein biogenesis and protein secretion. However, the reduced
stability of secretory proteins seems to be the most prominent
phenotype of SpoIIIJ/YqjG-depleted B. subtilis cells.
In conclusion, our observations show that SpoIIIJ and YqjG have
different, but overlapping functions in B. subtilis. Most importantly, it seems that different members of the Oxa1p protein family have acquired at least partly distinct, species-specific, functions that are essential for life.
In recent years, not only interesting similarities, but also
striking differences between protein transport mechanisms in eubacteria, Archaea, eukaryotes, and eukaryotic organelles have been
documented (1-3). Insights in the extents of conservation and
divergence in these mechanisms were particularly increased by the
availability of many complete genome sequences. Unfortunately, the
biological significance of such insights is often difficult to test as
the majority of organisms with sequenced genomes is poorly amenable to
biochemical or genetic approaches. In this respect, the Gram-positive
eubacterium Bacillus subtilis, the complete genome sequence
of which was published by Kunst et al. (4), turned out to be
a very useful exception. First, because this organism has a natural
system for genetic transformation and second, because it has a huge
capacity for the secretion of proteins directly into the growth medium
(5).
The functional genomic approach to dissect the protein secretion
process in B. subtilis has yielded a number of remarkable surprises. These include striking differences in the composition of the
general secretion (Sec) and
twin-arginine translocation (Tat)
pathways for the transport of secretory pre-proteins across the
membranes of B. subtilis and Escherichia
coli (6-10). Specifically, translocated pre-proteins of
B. subtilis with Sec-type or twin-arginine signal peptides
were shown to be subject to processing by the largest number of type I
signal peptidases (SPases)1
known in any organism. In addition to five chromosomally encoded SPases
(SipS, -T, -U, -V, and -W; 11), some B. subtilis strains even contain plasmid-encoded SPases (12). Furthermore, SipW was the
first known eubacterial SPase of a type that is mainly encountered in
Archaea and the eukaryotic endoplasmic reticular membrane (11, 13).
Another highly remarkable finding was that the unique type II SPase
(Lsp) of B. subtilis (14), which specifically catalyzes the
maturation of lipid-modified pre-proteins, turned out to be required
for the secretion of non-lipoproteins, such as When the negative effect of an SPase II (lsp) mutation on
non-lipoprotein secretion was first observed for the Plasmids, Bacterial Strains, and Media--
Table
I lists the plasmids and bacterial
strains used. TY medium (tryptone/yeast extract) contained
Bacto-tryptone (1%), Bacto-yeast extract (0.5%), and NaCl (1%).
Minimal medium for B. subtilis was prepared as described by
Tjalsma et al. (11). Schaeffer's sporulation medium was
prepared as described by Schaeffer et al. (26). When
required, medium for E. coli was supplemented with kanamycin
(40 µg/ml), ampicillin (50 µg/ml), or erythromycin (100 µg/ml);
media for B. subtilis were supplemented with chloramphenicol (5 µg/ml), kanamycin (10 µg/ml), tetracyclin (Tc; 6 µg/ml), or erythromycin (Em; 1 µg/ml).
DNA Techniques--
Procedures for DNA purification,
restriction, ligation, agarose gel electrophoresis, and transformation
of E. coli were carried out as described by Sambrook
et al. (32). Enzymes were from Roche Molecular Biochemicals.
B. subtilis was transformed as described by Tjalsma et
al. (11). The PCR was carried out with Pwo DNA polymerase (Roche
Molecular Biochemicals) as described by van Dijl et al.
(33).
To construct B. subtilis
To construct B. subtilis
To construct B. subtilis
To construct B. subtilis
B. subtilis Growth and Maintenance of IPTG-dependent B. subtilis
Assay for Spore Development--
The efficiency of sporulation
was determined by overnight growth in Schaeffer's sporulation medium,
killing of cells with 0.1 volume chloroform, and subsequent plating.
Western Blot Analysis and Immunodetection--
Western blotting
was performed as described by Kyhse-Andersen (36). After separation by
SDS-PAGE, proteins were transferred to Immobilon polyvinylidene
difluoride membranes (Millipore Corporation). To detect LipA, PhoA,
AmyQ(-PSBT), carboxyl-terminal Myc-tagged SecDF, SipS, PrsA, FtsH,
CtaC, or QoxA, B. subtilis cells were separated from the
growth medium. Cells were resuspended in lysis buffer (20 mM potassium phosphate, pH 7.5, 15 mM
MgCl2, 20% sucrose, 0.5 mg/ml lysozyme) and incubated for
15 min at 37 °C. Next, 1 volume of SDS sample buffer (100 mM Tris-HCl, pH 6, 4% SDS, 10% 2-mercaptoethanol, 30%
glycerol, 0.005% bromophenol blue, and 1% Triton X-100) was added and
the incubation was prolonged for 15 min at 37 °C in the presence of
CompleteTM protease inhibitors (Roche Molecular
Biochemicals). In general, proteins were visualized with the ECL
detection system, using rabbit/mouse sera and horseradish peroxidase
anti-rabbit/mouse-IgG conjugates (Amersham Biosciences).
Carboxyl-terminal Myc-tagged SecDF was visualized with monoclonal c-Myc
antibodies (Clontech), and biotinylated AmyQ-PSBT
was visualized with a streptavidin-horseradish peroxidase conjugate
(Amersham Biosciences).
Pulse-Chase Protein Labeling, Immunoprecipitation, SDS-PAGE, and
Fluorography--
B. subtilis
Protease Accessibility--
Protoplasts were
prepared from late exponentially growing cells of B. subtilis. Cells were resuspended in protoplast buffer (20 mM potassium phosphate, pH 7.5, 15 mM
MgCl2, 20% sucrose) and incubated for 30 min with 1 mg/ml
lysozyme (37 °C). Protoplasts were collected by centrifugation,
resuspended in fresh protoplast buffer, and incubated at 37 °C in
the presence of 1 mg/ml trypsin (Sigma) for 30 min. The reaction was
terminated by the addition of CompleteTM protease
inhibitors (Roche Molecular Biochemicals) and protoplasts were used for
SDS-PAGE and Western blotting. In parallel, protoplasts were incubated
without trypsin, or in the presence of trypsin and 1% Triton
X-100.
YqjG Is Not Required for Sporulation--
The yidC gene
of E. coli, specifying a SpoIIIJ/YqjG orthologue
(Fig. 1), is essential for cell
viability (24). In contrast, the spoIIIJ gene of B. subtilis is not essential for growth. In fact, spoIIIJ
was originally identified as the gene containing the spo-87
mutation that blocks sporulation of B. subtilis cells at
stage III, after the completion of prespore engulfment (25). To search
for possible functions of the YqjG protein, a yqjG
disruption strain was constructed with the integration vector pMutin2
(Fig. 2). The fact that B. subtilis The Presence of Either SpoIIIJ or YqjG Is Required for Growth of B. subtilis--
To determine whether the combined activities of SpoIIIJ
and YqjG are required for the viability of B. subtilis cells, a conditional (IPTG-dependent)
yqjG-spoIIIJ double mutant strain was constructed in two
steps. First, the largest part of the yqjG gene was replaced with a tetracyclin resistance marker via double crossover
recombination. Second, the spoIIIJ gene of the latter strain
was placed under the control of the IPTG-inducible
Pspac promoter, present on pMutin2. This was achieved by
single crossover of pMutin2 into the spoIIIJ region of the
chromosome (Fig. 2). Notably, the resulting B. subtilis Maximal spoIIIJ and yqjG Transcription in the Exponential Growth
Phase--
Previous studies showed that the spoIIIJ gene is
transcribed during the exponential growth phase and that this
transcription is shut down at about the onset of sporulation (25). To
investigate whether transcription of the yqjG gene is
regulated in a similar manner, we used the transcriptional
yqjG-lacZ and spoIIIJ-lacZ gene fusions that are
present in B. subtilis SpoIIIJ and YqjG Limitation Affects a Post-translocational Stage in
Protein Secretion--
To evaluate the importance of YqjG and SpoIIIJ
function for protein secretion, B. subtilis
To investigate the nature of the secretion defect of B. subtilis SpoIIIJ and YqjG Are Required for the Stability or Cell Wall
Passage of Translocated Secretory Proteins--
To further investigate
the possibility of a post-translocational secretion defect in
SpoIIIJ-depleted cells lacking YqjG, combined pulse-chase labeling and
fractionation experiments were performed. For this purpose,
pulse-labeled cells were separated from their growth medium by
centrifugation. Next, the collected cells were treated with lysozyme,
and the resulting protoplasts were separated from the cell wall
fraction (i.e. the protoplast supernatant) by
centrifugation. Finally, radiolabeled reporter proteins were recovered
from lysed protoplasts, the cell wall fraction, and the growth medium
by immunoprecipitation. As shown in Fig.
5A, the kinetics of synthesis
and processing of pre-AmyQ (upper panel), and release of
mature AmyQ into the cell wall (middle panel) in cells of
B. subtilis
As shown by pulse-chase labeling, the synthesis of LipA in cells of
B. subtilis
Together, these data confirm the view that the secretion of proteins
into the growth medium by SpoIIIJ/YqjG-depleted cells of B. subtilis is affected at stages that follow translocation across
the cytoplasmic membrane. Within the first 10 min of the "life" of
secretory proteins investigated here, the limitation of B. subtilis Oxa1-like proteins may either result in a significantly reduced stability (AmyQ and PhoA) or cell wall retention (LipA).
Specific role of SpoIIIJ and YqjG in Membrane Protein
Stability--
As shown by Samuelson et al. (24), the YidC
protein of E. coli is very important for the correct
insertion of various proteins into the inner membrane, but not the
export and processing of Sec-dependent pre-proteins. To
investigate whether the SpoIIIJ and YqjG functions could be required
for membrane protein biogenesis in B. subtilis, the cellular
levels of the BdbB, BdbC, FtsH, SecDF-Myc, SipS, and SPase II proteins
in the
The Oxa1p protein of yeast mitochondria was previously shown to be
required for the correct assembly of cytochrome c oxidase complexes (42, 43), and the specific export of the amino and carboxyl
termini of the precursor of cytochrome c oxidase subunit II
from the mitochondrial matrix to the intermembrane space (21, 22, 44).
Therefore, a possible role of SpoIIIJ and YqjG in the biogenesis of the
transmembrane lipoproteins CtaC and QoxA, which are orthologues of
cytochrome c oxidase subunit II (45), was
investigated. Indeed, the results showed that, upon SpoIIIJ depletion
of cells lacking YqjG, the cellular levels of CtaC were slightly
reduced, whereas a specific CtaC degradation product accumulated
concomitantly (Fig. 6). However, under the same conditions, the
cellular level and stability of QoxA were not detectably affected (Fig.
6).
Previous studies by de Gier et al. (46) have shown that
impaired membrane protein insertion does not necessarily affect the
total cellular levels of membrane proteins. To study whether membrane(-associated) proteins were properly inserted in
SpoIIIJ-depleted cells lacking YqjG, the membrane topology of
SecDF-Myc, SipS, FtsH, CtaC, QoxA, and PrsA was assessed by
protoplasting and subsequent protease mapping assays. As shown in Fig.
7, the protease accessibility of none of
the tested proteins was detectably affected in
To examine the kinetics of membrane protein insertion, a
pulse-chase/protease mapping experiment was performed with cells of
the parental B. subtilis 168 strain and
Taken together, these findings show that the stability of some membrane
proteins is affected in cells with limiting amounts of SpoIIIJ and
YqjG. However, the stability, topology, and insertion kinetics of other
membrane proteins and the essential lipoprotein PrsA is not detectably
affected under these conditions. The fact that the biogenesis of none
of the tested protein secretion machinery components is
SpoIIIJ/YqjG-dependent suggests that the defect in the
secretion of AmyQ, LipA, and PhoA is not caused by impaired membrane
assembly of individual secretion machinery components in
SpoIIIJ-depleted cells lacking YqjG.
Proteins homologous to Oxa1p are conserved in eubacteria and
eukaryotic organelles, where they appear to have important functions in
protein transport and membrane protein assembly. In fact, SpoIIIJ of B. subtilis was the first protein of this family to which
a function was assigned. Errington and co-workers (25) showed in 1992 that a spoIIIJ mutation blocks sporulation of B. subtilis cells at stage III, after the completion of forespore
engulfment. It was thought that this protein was involved in a signal
transduction pathway, coupling gene expression in the forespore to
concomitant events in the mother cell. However, the exact mechanism of
SpoIIIJ function in sporulation was not unraveled.
Our present observations demonstrate that the synthesis of either
SpoIIIJ, or its paralogue YqjG, is required for growth of the
Gram-positive eubacterium B. subtilis, which is in good
agreement with recent data from Murakami and co-workers (47).
Furthermore, the accumulation of the mature AmyQ, PhoA, and LipA
proteins in the growth medium is strongly impaired under conditions of
SpoIIIJ/YqjG-limitation. Notably, the reduction in AmyQ and PhoA levels
in the medium is not paralleled by reduced rates of pre-AmyQ or
prepro-PhoA translocation and processing, or the cellular accumulation
of the respective precursors, as previously documented for
secDF, tepA, or sip mutant strains
with translocation defects (6, 11, 48, 49). Instead, pulse-chase
labeling experiments combined with cell fractionation indicate that
AmyQ and PhoA are degraded upon release of these proteins into the
growth medium. Thus, the AmyQ and PhoA secretion defects observed for
SpoIIIJ-depleted cells lacking YqjG seem to occur during
post-translocational stages that involve the folding of secretory
proteins into their active and protease-resistant conformation. The
latter is of particular importance because the extracytoplasmic side of
the membrane, the cell wall, and the growth medium of B. subtilis are highly proteolytic (5, 16).
In a wild-type cell, SpoIIIJ and YqjG might facilitate the folding of
translocated proteins in several ways. First, it is conceivable that
SpoIIIJ and YqjG have a direct role in the correct folding of mature
proteins shortly after their translocation across the membrane, similar
to the essential function proposed for the folding catalyst PrsA (15,
17). Alternatively, SpoIIIJ and YqjG might have an indirect role in
protein secretion by modulating the activity of folding catalysts, such
as PrsA. A third possibility would be that SpoIIIJ and YqjG depletion
impacts on the composition of the cell wall. For example, it has been
shown that negatively charged teichoic acids can increase the rate of
folding and stability of certain exported proteins (50). The
observation that the release of mature LipA from the cell wall into the
growth medium is affected upon SpoIIIJ/YqjG limitation could point to
the direction that the composition of the cell wall is altered under
these conditions. However, it is also conceivable that the depletion of
SpoIIIJ and YqjG affects the folding of LipA, and that malfolded LipA interacts more strongly with cell wall components than correctly folded
LipA (51).
In recent years, the Oxa1p protein of yeast mitochondria was shown to
be required for the processing of mitochondrially encoded precursors
(52), the export of amino and carboxyl termini from precursor
cytochrome c oxidase subunit II synthesized in the
mitochondrial matrix (21, 22), and the insertion of transmembrane
domains into the mitochondrial inner membrane in a pairwise fashion
(53). Considering the facts that yeast mitochondria completely lack Sec
components, and that the Oxa1p orthologue in chloroplasts (Albino III)
is required for the Sec-independent integration of the light-harvesting
chlorophyll-binding protein into the thylakoid membrane (54), members
of the Oxa1p family might represent the key components of a novel
pathway for protein export or membrane protein assembly (55).
Consistent with this view, YidC of E. coli was shown to
facilitate the Sec-independent insertion of certain membrane proteins,
such as the M13 procoat, being of minor importance for the export of
Sec-dependent preproteins (24, 56). Nevertheless, YidC, was
also shown to be associated with the Sec machinery, indicating that
this protein has a more general role in membrane protein biogenesis in
E. coli, for example, by catalyzing the exit of membrane
proteins from the Sec translocase (23, 24, 57). Indications for such a
lateral movement were obtained by Urbanus and co-workers (58), who
showed a sequential interaction of the membrane protein FtsQ with SecY
and YidC. Recent studies by Houben et al. (59) showed that
YidC can contact a transmembrane domain during biogenesis, even when it
is not yet fully exposed on the outside of the ribosome. Thus, YidC
seems to have a role in both the reception and lipid partitioning of transmembrane segments. Our present studies support the view that SpoIIIJ and YqjG of B. subtilis are also involved in
membrane protein biogenesis as the stability of the membrane proteins
FtsH and CtaC is affected under conditions of SpoIIIJ/YqjG limitation. However, pulse-chase labeling and protease mapping experiments indicate
that not all membrane proteins of B. subtilis require SpoIIIJ and YqjG for their biogenesis. For example, the insertion kinetics of SipS and QoxA were not significantly affected upon SpoIIIJ/YqjG limitation. In this respect, it is interesting to note
that the membrane topology of QoxA of B. subtilis is similar to that of Lep of E. coli for which it has been shown that
the rate of membrane insertion is significantly affected in
YidC-depleted cells (24). Even though the post-translocational protein
secretion defect is presently the most prominent phenotype of
SpoIIIJ/YqjG-depleted cells of B. subtilis, it is important
to bear in mind that this secretion defect may be indirectly caused by
the misassembly of an as yet unidentified membrane protein.
Our present results, together with the well documented requirement of
Oxa1p and YidC for membrane protein biogenesis, suggest that SpoIIIJ
and YqjG have rather specific roles in membrane protein biogenesis in
B. subtilis. It is therefore concluded that different orthologous members of the Oxa1 protein family have acquired different species-specific functions in Sec-dependent and
Sec-independent membrane protein biogenesis and protein secretion.
Interestingly, not only orthologous Oxa1-like proteins, but also
Oxa1-like paralogues within one organism have acquired (partly)
distinct functions, as evidenced by the present observation that
SpoIIIJ, but not YqjG, is required for spore development. Similarly,
only one of the two Oxa1p orthologues of Schizosaccharomyces
pombe is essential for respiration (60). As only minor differences
in the timing of transcription have been observed for the genes
specifying the paralogous Oxa1p proteins of B. subtilis
(Fig. 3C) and S. pombe (60), the differences in
function of these protein pairs are, most likely, based on differences
in their primary structures.
Finally, what is the mechanism underlying SpoIIIJ involvement in the
sporulation process? First, in view of the fact that SpoIIIJ and YqjG
are important for post-translocational protein folding steps in the
secretion process, it is conceivable that SpoIIIJ is specifically
required for the folding of certain, as yet unknown, translocated
sporulation factors. The importance of protein transport for
sporulation is underscored by the observations that SecA, and the type
I SPases SipT and SipV, are required for this process (61,
62).3 Second, SpoIIIJ might be involved in the insertion of
specific membrane proteins that are essential for the sporulation
process after stage III. Possibly, these membrane proteins are involved in the communication between the forespore and the mother cell as
originally proposed by Errington and co-workers (25). Our ongoing
research is focused on the identification of specific substrates of
SpoIIIJ, and determinants for the different specificities of SpoIIIJ
and YqjG. This will provide better insights into the molecular
mode-of-action of the members of the Oxa1 protein family.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase,
chitosanase, and lipase (15,
16).2
-amylase AmyQ, it was largely attributed to a possible malfunctioning of the lipoprotein PrsA, which is essential for the proper folding of various
translocated proteins, such as AmyQ. An unexplained observation was,
however, that B. subtilis cells lacking SPase II accumulated significantly increased levels of pre-AmyQ (15). As the latter effect
was never observed in prsA mutant strains (17, 18), it was
concluded that the increased pre-AmyQ accumulation in lsp mutant strains was because of the malfunction of one or more
lipoproteins other than PrsA. This formed the incentive to search for
lipoproteins of B. subtilis with an as yet unknown role in
protein secretion. For this purpose, the amino acid sequences of all
114 (predicted) lipoproteins of B. subtilis (15) were used
for similarity searches in public databases. Strikingly, the predicted
lipoproteins SpoIIIJ and YqjG both showed significant similarity to the
Oxa1 protein of yeast mitochondria (Fig. 1). This inner membrane
protein has been implicated in the export of the amino and carboxyl
termini of the mitochondrially encoded precursor of cytochrome
c oxidase subunit II from the mitochondrial matrix
(21, 22). Furthermore, it was shown that the E. coli
orthologue of Oxa1p, denoted YidC, is associated with the Sec
translocase (23). Thus, it was conceivable that SpoIIIJ and YqjG are
involved in protein secretion by B. subtilis. On the other
hand, Samuelson and co-workers (24) clearly demonstrated that YidC of
E. coli is of major importance for the biogenesis of several
membrane proteins, whereas the export of periplasmic and outer membrane
proteins was hardly affected in YidC-depleted cells. Therefore, the
present studies were aimed at the identification of possible roles of
SpoIIIJ and YqjG in protein secretion and/or membrane protein
biogenesis. The results show that, even though the phenotype of
SpoIIIJ- and YqjG-depleted cells is quite different from that of SPase
II mutant cells, SpoIIIJ and YqjG have an important impact on the
accumulation of secretory proteins in the growth medium. Consistent
with an important role in secretion, the presence of at
least one of these two Oxa1p orthologues is essential for cell growth.
Notably, under SpoIIIJ- and YqjG-limiting conditions that strongly
affect the stability of secretory proteins, the stability of various
membrane proteins is not or only mildly affected. Also, whereas SpoIIIJ
is essential for sporulation (25), its paralogue YqjG is not required
for this developmental process. Taken together, these data imply that SpoIIIJ and YqjG have acquired functions that are at least partly different from those of other members of the Oxa1 family.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Plasmids and bacterial strains
spoIIIJ, first an
internal fragment of the spoIIIJ gene was amplified with the
primers spoIIIJ-1 (5'-GAGAATTCGACGGGAGATAACTACGGGC-3') and
spoIIIJ-2 (5'-ATGGATCCTATGCTCTGAAATCGCCTGGG-3'). The
amplified fragment was cleaved with EcoRI and
BamHI, and ligated into the corresponding sites of pMutin2,
resulting in pMutin-
spoIIIJ. Next, B. subtilis
spoIIIJ was obtained by a single crossover (Campbell-type) integration of pMutin-
spoIIIJ into the
spoIIIJ gene of B. subtilis 168, in such a way
that the spoIIIJ gene was disrupted and the
spoVG-lacZ reporter gene of pMutin2 was placed under the transcriptional control of the spoIIIJ promoter
region (Fig. 2). Simultaneously, the jag gene located
downstream of spoIIIJ was placed under the control of the
isopropyl-
-D-thiogalacto-pyranoside (IPTG)-dependent Pspac promoter.
yqjG, first an
internal fragment of the yqjG gene was amplified with
primers yqjG-3 (5'-TGAAGCTTGCCGGGCTGTTTCACGG-3') and
yqjG-2 (5'-ATGGATCCATCGTCATCATCACAGGGAAGATG-3'). The
amplified fragment was cleaved with HindIII and
BamHI, and ligated into the corresponding sites of pMutin2,
resulting in pMutin-
yqjG. Next, B. subtilis
yqjG was obtained by a single crossover integration of
pMutin-
yqjG into the yqjG gene of B. subtilis 168, in such a way that the yqjG gene was
disrupted and the spoVG-lacZ reporter gene of
pMutin2 was placed under the transcriptional control of the
yqjG promoter region (Fig. 2).
yqjG-Tc, first a
fragment comprising the yqjG gene and upstream and
downstream sequences was amplified with the primers yqjG-1
(5'-GCTTTGGATTTCTTTTGCCGTCTC-3') and yqjG-4 (5'-GGTTCGTGAGCATAAAGGGAAGC-3'). The amplified fragment was cleaved with XbaI and KpnI, and ligated into the
corresponding sites of pUK21. Next, the 714-bp EcoRI and
PstI fragment, containing the 5' sequences of the
yqjG gene, was replaced with a tetracyclin resistance marker
resulting in plasmid pUKyqjG-Tc. Finally, the chromosomal
yqjG gene of B. subtilis 168 was largely deleted
by a double crossover recombination event with linearized
pUKyqjG-Tc, resulting in B. subtilis
yqjG-Tc (see Fig. 2).
yqjG-IspoIIIJ, first a fragment comprising the
ribosome binding site, start codon, and the 5' region of the
spoIIIJ gene, but not the spoIIIJ promoter(s),
was amplified with primers spoIIIJ-3
(5'-GGAATTCTAGAGTGTAAAGATTAATTATAGGAGGAAATGTTG-3') and
spoIIIJ-2. The amplified fragment was cleaved with
EcoRI and BamHI, and ligated into the
corresponding sites of pMutin2, resulting in
pMutin-IspoIIIJ. Finally, B. subtilis
yqjG-IspoIIIJ was obtained by Campbell-type
integration of pMutin-IspoIIIJ into the spoIIIJ gene of B. subtilis
yqjG-Tc, in such a way
that the spoIIIJ gene and the downstream jag gene
were placed under the control of the IPTG-dependent
Pspac promoter, whereas the
spoVG-lacZ reporter gene of pMutin2 was
placed under the transcriptional control of the spoIIIJ
promoter region (Fig. 2).
yqjG-IspoIIIJ
containing a xylose-inducible secDF-Myc gene was obtained by
transformation of B. subtilis
yqjG-IspoIIIJ with chromosomal DNA of the
xSecDF-Myc strain (6). All constructed strains were selected on plates
with the proper antibiotics, and checked by PCR analyses for correct
integration of plasmids into the chromosome.
yqjG-IspoIIIJ Strains--
The IPTG-dependent strain
B. subtilis
yqjG-IspoIIIJ, and
derivatives thereof, were grown in media containing 500 nM
IPTG, Em (1 µg/ml), and Tc (6 µg/ml). It should be noted that in
the absence of IPTG, such strains sometimes start to grow again after a
lag period. This is probably because of the occurrence of a point
mutation in the Pspac promoter, causing constitutive
expression of the downstream genes (34). To avoid this potential
problem, prior to each experiment, individual colonies were
replica-plated on plates without IPTG and colonies displaying no growth
on the latter plates were used for SpoIIIJ/YqjG depletion experiments. To this purpose, B. subtilis
yqjG-IspoIIIJ was grown overnight in TY medium
supplemented with 500 nM IPTG, Em (1 µg/ml), and Tc (6 µg/ml). Cells were washed in fresh TY medium without IPTG and diluted
1:20 in fresh TY medium without, or with limiting concentrations (50 nM) of IPTG. After 3 h of growth, cells were harvested.
yqjG-IspoIIIJ strains stop growing
after about 2-3 h in the absence of IPTG (Fig. 3).
-Galactosidase Activity Assay--
Overnight cultures were
diluted 100-fold in fresh medium and samples were taken at hourly
intervals for optical density (OD) readings and
-galactosidase
activity determinations. The assays and the calculations of
-galactosidase units (expressed as units per
A600) were carried out as described by Miller
(35).
yqjG-IspoIIIJ was grown overnight in S7 medium
supplemented with 500 nM IPTG, Em, and Tc. Cells were
washed with fresh S7 medium without IPTG and diluted 1:10 in fresh S7
medium containing Em, with or without IPTG. After 2 h of growth,
cells were resuspended in methionine and cysteine-free S7 (S7
starvation) medium with or without 500 nM IPTG and grown
for another hour prior to pulse-chase labeling with
[35S]methionine/cysteine ([35S]Pro-Mix;
Amersham Biosciences). Immunoprecipitation, SDS-PAGE, and fluorography
were performed as described previously (37, 38).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
yqjG could be obtained showed that YqjG, like SpoIIIJ, is not essential for growth (Fig.
3A). Notably, disruption of
the yqjG gene did not detectably affect sporulation, whereas
a
spoIIIJ control strain constructed with pMutin2 was unable to develop viable spores (data not shown). Thus, YqjG has no
essential function in the sporulation process, in contrast to
SpoIIIJ.
View larger version (39K):
[in a new window]
Fig. 1.
Conservation of Oxa1p(-like) proteins in
B. subtilis, E. coli, and
mitochondria of the yeast S. cerevisiae.
A, the amino acid sequences of SpoIIIJ and YqjG, and the
partial sequences of YidC and Oxa1p, comprising the conserved part as
depicted in part B of this figure. Identical residues are
indicated in bold. The conserved transmembrane segments I-V
(marked in gray shading) were predicted as described by
Sipos and von Heijne (19). The putative SPase II cleavage sites
(lipoboxes) in SpoIIIJ and YqjG are underlined.
Notably, SpoIIIJ/YqjG orthologues with putative lipoprotein signal
peptides have been found in several Gram-positive eubacteria, such as
Bacillus anthracis, Bacillus halodurans,
Bacillus stearothermophilus, Lactococcus
lactis, and Staphylococcus aureus (H. Tjalsma, S. Bron, and J. M. van Dijl, unpublished observations).
Numbers refer to the position of amino acids in the
corresponding protein sequence. B, predicted membrane
topologies of SpoIIIJ and YqjG of B. subtilis, YidC of
E. coli, and Oxa1p of S. cerevisiae. Only the
membrane topology of YidC has been verified experimentally (20). Note
that the YidC protein has a large amino-terminal loop located in the
periplasm that is absent from SpoIIIJ, YqjG, and Oxa1p. C,
carboxyl terminus; the cytoplasmic, cell wall, periplasmic, matrix, or
inter membrane space (IMS) sides of the membranes, and the
"conserved" parts of the Oxa1p(-like) proteins are indicated. Note
that the amino termini of SpoIIIJ and YqjG are most likely
lipid-modified.
View larger version (23K):
[in a new window]
Fig. 2.
Construction of spoIIIJ
and/or yqjG mutant strains of B. subtilis. B. subtilis spoIIIJ and
B. subtilis
yqjG were, respectively,
constructed by the single crossover integration of
pMutin-
spoIIIJ and pMutin-
yqjG into the
chromosome B. subtilis 168. In these strains, the respective
spoIIIJ or yqjG genes are disrupted while the
lacZ gene of pMutin2 is placed under the transcriptional
control of the promoter regions of these genes. B. subtilis
yqjG-Tc was constructed by replacement of the 5' part of
the yqjG gene with a tetracyclin resistance marker by double
crossover recombination. B. subtilis
yqjG-IspoIIIJ was constructed by integration
of pMutin-IspoIIIJ into the spoIIIJ region of
B. subtilis
yqjG-Tc. By this approach, the
spoIIIJ gene was placed under the control of the
IPTG-dependent Pspac promoter. Notably, growth
and viability of the latter strain is dependent on the presence of
IPTG. The relative positions of open reading frames in the
spoIIIJ and yqjG regions are shown. PCR-amplified
DNA fragments that were used to direct the integration of pMutin2 into
the B. subtilis chromosome are indicated with black
bars. Restriction sites relevant for the construction are
indicated: B, BamHI; dco, double
crossover; E, EcoRI; H,
HindIII; jag, gene of unknown function specifying
a predicted cytoplasmic protein; P, PstI.
Ori pBR322, replication functions of pBR322;
Emr, erythromycin resistance marker;
sco, single crossover; T1T2,
transcriptional terminators on pMutin2; spoIIIJ', 3'
truncated spoIIIJ gene; 'spoIIIJ, 5' truncated
spoIIIJ gene; yqjG', 3' truncated yqjG
gene; 'yqjG, 5' truncated yqjG gene.
View larger version (19K):
[in a new window]
Fig. 3.
Properties of spoIIIJ and/or
yqjG mutant strains of B. subtilis.
A, IPTG-dependent growth of B. subtilis yqjG-IspoIIIJ on plates at 15 and 37 °C. Individual colonies of B. subtilis
yqjG-IspoIIIJ were transferred to fresh
TY-agar plates containing 1000, 500, 100, 50, or 0 nM IPTG.
Next, the plates were incubated overnight at 37 °C (upper
panel), or for 5 days at 15 °C (lower panel).
B, IPTG-dependent growth of B. subtilis
yqjG-IspoIIIJ. Overnight
cultures of B. subtilis 168 (parental;
), B. subtilis
spoIIIJ (
), B. subtilis
yqjG (
), and B. subtilis
yqjG-IspoIIIJ (
), grown in TY medium at
37 °C, were washed and diluted 20-fold in fresh TY medium without
IPTG and incubated at 37 °C. In addition, B. subtilis
yqjG-IspoIIIJ was diluted in fresh TY medium
containing 50 (
) or 500 (
) nM IPTG. Zero time
(t = 0) indicates the transition point between the
exponential and post-exponential growth phases. C,
transcription profiles of the spoIIIJ and yqjG
genes. Time courses of the transcription of the
spoIIIJ-lacZ and yqjG-lacZ gene fusions
in B. subtilis
spoIIIJ (
) and B. subtilis
yqjG (
) were determined in cells growing
at 37 °C in TY medium.
-Galactosidase activities were determined
in units per A600. Zero time (t = 0) indicates the transition point between the exponential and
post-exponential growth phases.
yqjG-IspoIIIJ strain could be obtained only in
the presence of IPTG, indicating that at least the spoIIIJ
gene had to be transcribed for the growth of this strain. In fact,
about 500 nM IPTG was required for this strain to display
unimpaired growth on TY agar plates at 37 °C (Fig. 3A).
Upon dilution in fresh TY medium without IPTG, B. subtilis
yqjG-IspoIIIJ stopped growing after 2-3 h of incubation (Fig. 3B). Similar to the
IPTG-dependent growth of this strain on TY plates, the
presence of 500 nM IPTG was required to support wild-type
growth of B. subtilis
yqjG-IspoIIIJ
in liquid TY medium at 37 °C. In contrast, growth of B. subtilis
yqjG-IspoIIIJ in the presence of
50 nM IPTG was significantly reduced as compared with that
of the parental strain 168, indicating that SpoIIIJ was synthesized at
limiting levels. Importantly, growth of B. subtilis
yqjG-IspoIIIJ in the absence of IPTG could be
restored by the ectopic expression of the spoIIIJ
gene,3 showing that the
growth inhibition of this strain is because of SpoIIIJ limitation, and
not polar effects on the expression of the downstream located
jag gene. Together with the observation that
spoIIIJ and yqjG single mutants are viable, these
findings show that the presence of either SpoIIIJ or YqjG is required
for growth of B. subtilis. Notably, the IPTG dependence of
B. subtilis
yqjG-IspoIIIJ was
strongly increased at 15 °C (Fig. 3A). As protein transport via the Sec pathways of B. subtilis and E. coli is intrinsically cold-sensitive (6, 39, 40), the observed
temperature effect suggests that SpoIIIJ and YqjG have a role in
Sec-dependent protein transport.
yqjG and
spoIIIJ, respectively (Fig. 2). Both strains were grown
in TY medium, minimal medium, or sporulation medium, and samples
withdrawn at hourly intervals were assayed for
-galactosidase
activity. The results showed that, irrespective of the growth medium,
the
-galactosidase levels in both strains reached a maximum in the
exponential phase (Fig. 3C, only the results obtained with
cells grown in TY medium are documented). The
-galactosidase levels
were strongly decreased upon the transition (t = 0)
into the post-exponential growth phase. As the transcription profiles
of the yqjG-lacZ gene fusion were very similar to those of
the spoIIIJ-lacZ fusion under all conditions tested (Fig.
3C; data not shown), it seems highly unlikely that the lack
of effect of a yqjG mutation on sporulation is because of
differences in the transcription of these genes.
yqjG,
spoIIIJ, and
yqjG-IspoIIIJ, as well as parental strain 168, were transformed with plasmid pLip2031 for the secretion of the
B. subtilis esterase LipA (29), pPSPhoA5 for the secretion
of the alkaline phosphatase PhoA of E. coli fused to the
prepro-region of the lipase gene from Staphylococcus hyicus
(7), or pKTH10 for the secretion of the
-amylase AmyQ (30). As no
secretion defects were detectable in the single mutant strains (data
not shown), we focused our attention on the
yqjG-IspoIIIJ double mutant. To deplete
B. subtilis
yqjG-IspoIIIJ of
SpoIIIJ, this strain was grown for 3 h in TY medium without IPTG,
as described under "Experimental Procedures." As a control, TY
medium with 50 nM IPTG (limiting amounts of SpoIIIJ) or 500 nM IPTG (full induction of SpoIIIJ) was used (see Fig. 3,
A and B). Next, the secretion of LipA, PhoA, and
AmyQ was analyzed by Western blotting. As shown in Fig.
4A (lower panels),
the levels of LipA, PhoA, and AmyQ in the medium of SpoIIIJ-depleted
cells of B. subtilis
yqjG-IspoIIIJ
(no IPTG) were significantly reduced compared with those in the media
of the fully induced double mutant (500 nM IPTG), or the
parental strain 168. Moreover, SpoIIIJ-depleted double mutant cells
containing pPSPhoA5 or pKTH10 also contained significantly decreased
levels of mature PhoA or AmyQ, respectively (Fig. 4A,
upper panels). In contrast, the cellular levels of mature LipA, and the precursor forms of LipA and AmyQ were not affected by
SpoIIIJ depletion in the absence of YqjG (Fig. 4A).
Interestingly, the levels of LipA and PhoA in the media of
yqjG-IspoIIIJ strains that were fully induced
with IPTG (500 nM) were higher than those in the media of
the parental control strains. The latter finding suggests that
overexpression of the spoIIIJ gene can result in improved protein secretion in B. subtilis.
View larger version (47K):
[in a new window]
Fig. 4.
SpoIIIJ and YqjG are required for
extracellular accumulation of secretory proteins. A,
precultures of B. subtilis
yqjG-IspoIIIJ and the parental
strain B. subtilis 168, both transformed with plasmid
pLip2031 (specifying LipA), pPSPhoA5 (specifying PhoA), or pKTH10
(specifying AmyQ), were prepared by overnight growth at 37 °C in TY
medium containing 500 nM IPTG. Next, cells were washed with
fresh TY medium without IPTG, diluted 20-fold in fresh TY medium
containing 500, 50 nM, or no (0) IPTG, and incubated for
3 h at 37 °C before sampling for SDS-PAGE and Western blotting.
Specific antibodies were used to detect the cellular (pre-)LipA, PhoA,
or (pre-)AmyQ levels (upper panels), and the levels of
secreted LipA, PhoA, or AmyQ in the growth medium (lower
panels). The positions of pre-LipA and pre-AmyQ (
), mature
LipA, PhoA, and AmyQ (
), or degradation products of PhoA (
) are
indicated. B, processing of pre-AmyQ in B. subtilis
yqjG-IspoIIIJ was analyzed by
pulse-chase labeling at 37 °C in S7 medium with (500 nM;
upper panel) or without (0) IPTG (lower panel),
subsequent immunoprecipitation, SDS-PAGE, and fluorography. Cells were
labeled with [35S]methionine/cysteine for 1 min prior to
chase with excess non-radioactive methionine/cysteine. Samples were
withdrawn after the chase at the times indicated. Because the
incorporation of label into (pre-)AmyQ cannot be stopped
instantaneously by the addition of non-radioactive methionine, samples
withdrawn at t = 0, and t = 1 contain
lower amounts of labeled AmyQ than the sample withdrawn at
t = 5. The positions of pre-AmyQ (
) and mature AmyQ
(
) are indicated. C, translocation of pre-AmyQ-PSBT in
SpoIIIJ-depleted cells lacking YqjG. To analyze pre-AmyQ translocation,
cells of B. subtilis
yqjG-IspoIIIJ,
B. subtilis
secDF (positive control), and the
parental strain B. subtilis 168 (negative control) were
transformed with plasmid pKTH10-BT (specifying AmyQ-PSBT), and grown as
described for panel A of this figure. Cellular
(biotinylated) AmyQ-PSBT was visualized by SDS-PAGE and Western
blotting using AmyQ-specific antibodies (upper panel), or a
streptavidin-horseradish peroxidase conjugate (lower panel).
Precursor (
) and mature (
) forms of AmyQ-PSBT are
indicated.
yqjG-IspoIIIJ, further
experiments were performed with AmyQ and an AmyQ variant (AmyQ-PSBT) as
described previously by Bolhuis et al. (6). As shown by
pulse-chase labeling experiments, within the period of labeling (zero
time), the pre-AmyQ synthesis and processing in cells of B. subtilis
yqjG-IspoIIIJ depleted of SpoIIIJ (no IPTG) was not significantly different from that observed in
cells of this strain in which SpoIIIJ was fully induced (500 nM IPTG; Fig. 4B), or the parental strain 168 (data not shown). However, in particular after 5 min of chase,
significantly reduced amounts of mature AmyQ were detectable in the
yqjG-IspoIIIJ cells depleted of SpoIIIJ. This
suggests that these cells have a defect in post-translocational protein
folding into a stable conformation rather than a defect in protein
translocation. To verify that SpoIIIJ-depleted cells lacking YqjG have
no translocation defect, experiments were performed with AmyQ-PSBT,
which contains a carboxyl-terminal biotin-accepting domain (PSBT) of a
transcarboxylase from Propionibacterium shermannii (11). The
rationale of this experiment is that pre-AmyQ-PSBT can only be
biotinylated by the cytoplasmic biotin ligase when the PSBT domain
folds into its native three-dimensional structure prior to
translocation. Consequently, biotinylation of pre-AmyQ-PSBT occurs at
significantly increased levels when pre-AmyQ-PSBT translocation is
slowed-down, for example, by the disruption of the secDF
gene (6). As shown in Fig. 4C, SpoIIIJ-depleted cells
lacking YqjG did not accumulate increased amounts of biotinylated
pre-AmyQ-PSBT as compared with cells in which SpoIIIJ synthesis was
induced with IPTG, or the parental strain 168. In contrast, B. subtilis cells with a disrupted secDF gene (positive
control) accumulated strongly increased amounts of biotinylated
AmyQ-PSBT (Fig. 4C). Notably, the biotinylated AmyQ-PSBT in
the
secDF cells was present both in the precursor and
mature forms. Taken together, these observations support the view that
the translocation and processing of pre-AmyQ is not affected in
SpoIIIJ-depleted cells lacking YqjG. Instead, SpoIIIJ and YqjG appear
to be important for the post-translocational folding stages in protein secretion.
yqjG-IspoIIIJ depleted
of SpoIIIJ (no IPTG), or the parental strain, were not significantly
different. However, the amount of mature AmyQ released into the growth
medium after 10 min of chase (lower panel) was about 3-fold
reduced in the case of
yqjG-IspoIIIJ cells
depleted of SpoIIIJ. Moreover, a specific AmyQ degradation product was
detectable in the medium fractions of pulse-labeled
yqjG-IspoIIIJ cells depleted of SpoIIIJ, but
not in the corresponding fractions of the parental strain. Similarly,
the prepro-PhoA synthesis and processing (Fig. 5B, upper panel), as well as the release of pro-PhoA into the
cell wall (not shown) was not significantly different in cells of
B. subtilis
yqjG-IspoIIIJ depleted
of SpoIIIJ, or the parental strain. Importantly, significantly reduced
amounts of (pro-)PhoA were detected in all medium fractions of
pulse-labeled B. subtilis
yqjG-IspoIIIJ depleted of SpoIIIJ (Fig.
5B, lower panel). Also, a specific PhoA
degradation product was present in the medium of
yqjG-IspoIIIJ cells depleted of SpoIIIJ, but
not in the medium of the parental strain (lower panel).
These observations imply that mature AmyQ and (pro-)PhoA are subject to
proteolysis upon their release from SpoIIIJ/YqjG-depleted cells into
the growth medium. This suggests that SpoIIIJ and YqjG are (directly or
indirectly) required for the folding of AmyQ and PhoA into a stable,
protease-resistant conformation that allows the accumulation of these
proteins in the growth medium.
View larger version (45K):
[in a new window]
Fig. 5.
SpoIIIJ and YqjG are required for stability
or release of secretory proteins. The secretion of AmyQ
(A), PhoA (B), and LipA (C) was
analyzed in B. subtilis
yqjG-IspoIIIJ and the parental strain 168 by
pulse-chase labeling in S7 medium at 37 °C, fractionation
(protoplasts, cell wall and medium), immunoprecipitation, SDS-PAGE, and
fluorography. Cells were labeled with
[35S]methionine/cysteine for 1 min prior to chase with
excess non-radioactive methionine/cysteine. Samples were withdrawn at
1, 2, and 10 min after the chase. Cells and medium were separated by
centrifugation, and protoplast were subsequently prepared as described
under "Experimental Procedures." Finally, the cell wall and
protoplast fractions were separated by centrifugation. The positions of
pre-AmyQ and prepro-PhoA (
), degradation products of (pro-)PhoA and
AmyQ (*), and mature AmyQ, PhoA, and LipA (
) are indicated.
yqjG-IspoIIIJ depleted
of SpoIIIJ (no IPTG) was not affected (Fig. 5C, upper
panel; note that processing of pre-LipA is complete after 1 min of
chase). However, significant amounts of mature LipA were retained in
the cell wall of
yqjG-IspoIIIJ cells depleted
of SpoIIIJ, whereas this protein was rapidly chased from the cell wall
of the parental strain (Fig. 5C, middle panel). These findings indicate that the post-translocational release of mature
LipA from the cell wall into the growth medium is significantly affected in SpoIIIJ/YqjG-depleted cells. Compared with AmyQ and (pro-)PhoA, the effect of SpoIIIJ depletion on the kinetics of LipA
secretion into the growth medium by cells of B. subtilis
yqjG-IspoIIIJ was relatively mild (Fig. 5,
compare A, B, and C).
yqjG-IspoIIIJ strain were monitored by
Western blotting. In addition, the lipoprotein PrsA was included in the
analyses. These seven membrane(-associated) proteins were primarily
selected because they have different membrane topologies and different
numbers of transmembrane segments (Fig. 6; not shown for BdbB/C and SPase II,
which have four transmembrane segments and an
Nin-Cin topology). Furthermore, these proteins, which are known to be involved in protein secretion by B. subtilis (5), were selected to investigate whether the secretion
defects of SpoIIIJ-depleted cells lacking YqjG might be indirectly
caused by the impaired membrane biogenesis of secretion machinery
components. Interestingly, the levels of SecDF-Myc, SipS, and PrsA were
not detectably affected in SpoIIIJ-depleted cells lacking YqjG that were grown at 37 °C (Fig. 6), or 15 °C (data not shown).
Similarly, the cellular amounts of BdbB, BdbC, and SPase II remained
unchanged (data not shown). In fact, FtsH was the only protein involved in protein secretion (41) that was (mildly) affected upon SpoIIIJ depletion in the absence of YqjG. As shown in Fig. 6, low amounts of an
FtsH degradation product (lower panel) accumulated in
yqjG-IspoIIIJ cells depleted of SpoIIIJ.
View larger version (32K):
[in a new window]
Fig. 6.
Specific role of SpoIIIJ and YqjG in membrane
protein biogenesis. Cells of B. subtilis
yqjG-IspoIIIJ xSecDF-Myc
(
yqjG-IspoIIIJ) and the control strain
B. subtilis xSecDF-Myc (parental) were grown overnight at
37 °C in TY medium containing 500 nM IPTG. Next, cells
were washed with fresh TY medium without IPTG, diluted 20-fold in fresh
TY medium containing 1% xylose (production SecDF-Myc) and 500, 50 nM, or no (0) IPTG, and incubated at 37 °C for 3 h.
Samples for SDS-PAGE and Western blotting were prepared from cells and
specific antibodies were used to detect the cellular levels of
SecDF-Myc, SipS, FtsH, CtaC, QoxA, and PrsA. The positions of the
native proteins (
) and their degradation products (*) are
indicated. It should be noted that FtsH-derived degradation products
(FtsH, lower panel) were only visible after prolonged
fluorography. Putative membrane topologies of these proteins are
depicted (N, amino terminus; C, carboxyl
terminus; in, cytoplasmic side of the membrane;
out, cell wall-exposed side of the membrane). Note that the
amino termini of CtaC, QoxA, and PrsA are lipid-modified.
yqjG-IspoIIIJ cells upon SpoIIIJ depletion.
The cytoplasmic protein GroEL was not degraded by extracellular
trypsin, indicating that lysis of protoplasts did not occur during the
assay. It should be noted that the degradation product of FtsH, which
accumulates in
yqjG-IspoIIIJ cells depleted of
SpoIIIJ (Fig. 6), was also detectable in the parental strain upon
protoplasting. The degradation product of CtaC, which accumulates in
yqjG-IspoIIIJ cells depleted of SpoIIIJ (Fig.
6), was not detectable upon protoplasting of these cells.
View larger version (44K):
[in a new window]
Fig. 7.
Protease mapping of membrane proteins in
SpoIIIJ-depleted cells lacking YqjG. To analyze the insertion of
the membrane proteins SecDF-Myc, SipS, FtsH, CtaC, QoxA, and PrsA,
cells of B. subtilis yqjG-IspoIIIJ
xSecDF-Myc (
yqjG-IspoIIIJ) and the control
strain B. subtilis xSecDF-Myc (parental) were grown
overnight at 37 °C in TY medium containing 500 nM IPTG.
Cells were washed with fresh TY medium without IPTG, diluted 20-fold in
fresh TY medium without IPTG, and incubated at 37 °C for 3 h.
Next, the production of SecDF-Myc was induced by the addition of 1%
xylose 15 min prior to protoplasting. Protoplasts were incubated for 30 min without further additions, in the presence of trypsin (1 mg/ml), or
trypsin and Triton X-100 (1%). This procedure was performed in
parallel with cells of B. subtilis
yqjG-IspoIIIJ and B. subtilis 168 (parental), both containing pKTH10 for production of AmyQ. Samples were
used for SDS-PAGE, Western blotting, and specific antibodies were used
to detect SecDF-Myc, SipS, FtsH, CtaC, QoxA, PrsA, GroEL, or AmyQ. The
positions of intact proteins and pre-AmyQ (
), degradation products
because of the incubation with trypsin (*), mature AmyQ (
), and
trypsin-resistant fragments (
) are indicated.
yqjG-IspoIIIJ cells upon SpoIIIJ depletion. To
this purpose, pulse-labeled cells were subject to protoplasting and,
subsequently, protoplasts were incubated in the presence of trypsin,
plus or minus Triton X-100. As shown in Fig.
8, the protease accessibility of SipS,
QoxA, and PrsA at the surface of intact protoplasts was not detectably
affected in SpoIIIJ-depleted
yqjG-IspoIIIJ
cells after a chase of 1 min. GroEL was not degraded by extracellular
trypsin, indicating that no lysis of protoplasts occurred during this
assay. Consistent with the lack of effect on the insertion of PrsA in
the membrane, the processing of pre-PrsA by SPase II, as verified by
pulse-chase labeling, was not affected in
yqjG-IspoIIIJ cells upon SpoIIIJ depletion
(data not shown). Unfortunately, we could not use SecDF-Myc, CtaC, and
FtsH for pulse-chase labeling experiments as we were unable to
immunoprecipitate these proteins from labeled cell extracts.
View larger version (46K):
[in a new window]
Fig. 8.
Protease mapping of pulse-labeled membrane
proteins in SpoIIIJ-depleted cells lacking YqjG. To analyze the
kinetics of SipS, QoxA, and PrsA biogenesis, cells of B. subtilis yqjG-IspoIIIJ and the parental
strain 168 were grown in S7 medium at 37 °C, and labeled with
[35S]methionine/cysteine for 1 min followed by a chase of
1 min with excess non-radioactive methionine/cysteine. Cells were
collected by centrifugation, and protoplasts were subsequently prepared
as described under "Experimental Procedures." Protoplasts were
incubated for 30 min without further additions, in the presence of
trypsin (1 mg/ml), or trypsin and Triton X-100 (1%) prior to
immunoprecipitation, SDS-PAGE, and fluorography. Immunoprecipitation
with anti-GroEL antibodies was used to check protoplast integrity. The
positions of intact proteins (
) and trypsin-resistant fragments
(
) are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. H. Antelmann, R. Dorenbos, A. J. M. Driessen, J. D. H. Jongbloed, J. Swaving, and other members of the Groningen and European Bacillus Secretion Groups (see www.ncl.ac.uk/ebsg) for stimulating discussions, and Drs. M. Sarvas, T. Wiegert, V. P. Kontinen, J. Bengtsson, and A. Kröger for providing specific antibodies against AmyQ, FtsH, PrsA, CtaC, and QoxA, respectively.
![]() |
FOOTNOTES |
---|
* 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.
Supported by Genencor International (Leiden, The Netherlands).
§ Supported in part by European Union Grants QLK3-CT-1999-00413 and QLK3-CT-1999-00917.
¶ To whom correspondence should be addressed. Tel.: 31503632105; Fax: 31503632348; E-mail: S.Bron@biol.rug.nl.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M301205200
2 H. Antelmann, H. Tjalsma, J. M. van Dijl, and M. Hecker, unpublished observations.
3 H. Tjalsma, S. Bron, and J. M. van Dijl, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SPase, signal
peptidase;
IPTG, isopropyl--D-thiogalactopyranoside;
Em, erythromycin;
Tc, tetracyclin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Pohlschröder, M., Prinz, W. A., Hartmann, E., and Beckwith, J. (1997) Cell 91, 563-566[Medline] [Order article via Infotrieve] |
2. | Dalbey, R. E., and Robinson, C. (1999) Trends Biochem. Sci. 24, 17-22[CrossRef][Medline] [Order article via Infotrieve] |
3. | Dalbey, R. E., and Kuhn, A. (2000) Annu. Rev. Cell Dev. Biol. 16, 51-87[CrossRef][Medline] [Order article via Infotrieve] |
4. | Kunst, F., et al.. (1997) Nature 390, 249-256[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Tjalsma, H.,
Bolhuis, A.,
Jongbloed, J. D. H.,
Bron, S.,
and van Dijl, J. M.
(2000)
Microbiol. Mol. Biol. Rev.
64,
515-547 |
6. |
Bolhuis, A.,
Broekhuizen, C. P.,
Sorokin, A.,
van Roosmalen, M. L.,
Venema, G.,
Bron, S.,
Quax, W. J.,
and van Dijl, J. M.
(1998)
J. Biol. Chem.
273,
21217-21224 |
7. |
Bolhuis, A.,
Venema, G.,
Quax, W. J.,
Bron, S.,
and van Dijl, J. M.
(1999)
J. Biol. Chem.
274,
24531-24538 |
8. |
Jongbloed, J. D. H.,
Martin, U.,
Antelmann, H.,
Hecker, M.,
Tjalsma, H.,
Venema, G.,
Bron, S.,
van Dijl, J. M.,
and Müller, J.
(2000)
J. Biol. Chem.
275,
41350-41357 |
9. | Robinson, C., and Bolhuis, A. (2001) Nat. Rev. Mol. Cell. Biol. 2, 350-356[CrossRef][Medline] [Order article via Infotrieve] |
10. |
van Wely, K. H. M.,
Swaving, J.,
Klein, M.,
Freudl, R.,
and Driessen, A. J. M.
(2000)
Microbiology
146,
2573-2581 |
11. |
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 |
12. | 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] |
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. | Prágai, Z., Tjalsma, H., Bolhuis, A., van Dijl, J. M., Venema, G., and Bron, S. (1997) Microbiology 143, 1327-1333[Abstract] |
15. |
Tjalsma, H.,
Kontinen, V. P.,
Prágai, Z.,
Wu, H.,
Meima, R.,
Venema, G.,
Bron, S.,
Sarvas, M.,
and van Dijl, J. M.
(1999)
J. Biol. Chem.
274,
1698-1707 |
16. |
Antelmann, H.,
Tjalsma, H.,
Voigt, B.,
Ohlmeier, S.,
Bron, S.,
van Dijl, J. M.,
and Hecker, M.
(2001)
Genome Res.
11,
1484-1502 |
17. | Kontinen, V. P., and Sarvas, M. (1993) Mol. Microbiol. 8, 727-737[Medline] [Order article via Infotrieve] |
18. | Jacobs, M., Andersen, J. B., Kontinen, V. P., and Sarvas, M. (1993) Mol. Microbiol. 8, 957-966[Medline] [Order article via Infotrieve] |
19. | Sipos, L., and von Heijne, G. (1993) Eur. J. Biochem. 213, 1333-1340[Abstract] |
20. |
Saaf, A.,
Monne, M.,
de Gier, J. W.,
and von Heijne, G.
(1998)
J. Biol. Chem.
273,
30415-30418 |
21. | Hell, K., Herrmann, J. M., Pratje, E., Neupert, W., and Stuart, R. A. (1997) FEBS Lett. 418, 367-370[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Hell, K.,
Herrmann, J. M.,
Pratje, E.,
Neupert, W.,
and Stuart, R. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2250-2255 |
23. |
Scotti, P. A.,
Urbanus, M. L.,
Brunner, J.,
de Gier, J. W.,
von Heijne, G.,
van der Does, C.,
Driessen, A. J. M.,
Oudega, B.,
and Luirink, J.
(2000)
EMBO J.
19,
542-549 |
24. | Samuelson, J. C., Chen, M., Jiang, F., Moller, I., Wiedmann, M., Kuhn, A., Phillips, G. J., and Dalbey, R. E. (2000) Nature 406, 637-641[CrossRef][Medline] [Order article via Infotrieve] |
25. | Errington, J., Appleby, L., Danie, R. A., Goodfellow, H., Partridge, S. R., and Yudkin, M. D. (1992) J. Gen. Microbiol. 138, 2609-2618[Medline] [Order article via Infotrieve] |
26. | Schaeffer, P., Millet, J., and Aubert, P. J. (1965) Proc. Natl. Acad. Sci. U. S. A. 54, 704-711[Medline] [Order article via Infotrieve] |
27. | Vagner, V., Dervyn, E., and Ehrlich, S. D. (1998) Microbiology 144, 3097-3104[Abstract] |
28. | Vieria, J., and Messing, J. (1990) Gene (Amst.) 100, 189-194[CrossRef] |
29. | Dartois, V., Copée, J. Y., Colson, C., and Baulard, A. (1994) Appl. Environ. Microbiol. 60, 1670-1673[Abstract] |
30. | Palva, I. (1982) Gene (Amst.) 19, 81-87[CrossRef][Medline] [Order article via Infotrieve] |
31. | Wertman, K. F., Wyman, A. R., and Botstein, D. (1986) Gene (Amst.) 49, 253-262[CrossRef][Medline] [Order article via Infotrieve] |
32. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
33. | van Dijl, J. M., de Jong, A., Venema, G., and Bron, S. (1995) J. Biol. Chem. 270, 3611-3618[CrossRef] |
34. |
Pragai, Z.,
and Harwood, C. R.
(2000)
J. Bacteriol.
182,
6819-6823 |
35. | Miller, J. H. (1982) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
36. | Kyhse-Andersen, J. (1984) J. Biochem. Biophys. Methods 10, 203-209[CrossRef][Medline] [Order article via Infotrieve] |
37. | van Dijl, J. M., de Jong, A., Smith, H., Bron, S., and Venema, G. (1991) J. Gen. Microbiol. 137, 2073-2083[Medline] [Order article via Infotrieve] |
38. | 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] |
39. |
Pogliano, J. A.,
and Beckwith, J.
(1993)
Genetics
133,
763-773 |
40. |
van Wely, K. H. M.,
Swaving, J.,
Broekhuizen, C. P.,
Rose, M.,
Quax, W. J.,
and Driessen, A. J. M.
(1999)
J. Bacteriol.
181,
1786-1792 |
41. | Deuerling, E., Mogk, A., Richter, C., Purucker, M., and Schumann, W. (1997) Mol. Microbiol. 23, 921-933[Medline] [Order article via Infotrieve] |
42. |
Bonnefoy, N.,
Kermorgan, M.,
Groudinsky, O.,
Minet, M.,
Slonimski, P. P.,
and Dujardin, G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11978-11982 |
43. | Altemura, N., Capitanio, N., Bonnefoy, N., Papa, S., and Dujardin, G. (1996) FEBS Lett. 382, 111-115[CrossRef][Medline] [Order article via Infotrieve] |
44. | He, S., and Fox, T. D. (1997) Mol. Biol. Cell 8, 1449-1460[Abstract] |
45. |
Bengtsson, J.,
Tjalsma, H.,
Rivolta, C.,
and Hederstedt, L.
(1999)
J. Bacteriol.
181,
685-688 |
46. | de Gier, J. W., Mansournia, P., Valent, Q. A., Phillips, G. J., Luirink, J., and von Heijne, G. (1996) FEBS Lett. 399, 307-309[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Murakami, T.,
Haga, K.,
Takeuchi, M.,
and Sato, T.
(2002)
J. Bacteriol.
184,
1998-2004 |
48. |
Bolhuis, A.,
Matzen, A.,
Hyyrylaïnen, H. L.,
Kontinen, V. P.,
Meima, R.,
Chapuis, J.,
Venema, G.,
Bron, S.,
Freudl, R.,
and van Dijl, J. M.
(1999)
J. Biol. Chem.
274,
24585-24592 |
49. |
Tjalsma, H.,
Noback, M. A.,
Bron, S.,
Venema, G.,
Yamane, K.,
and van Dijl, J. M.
(1997)
J. Biol. Chem.
272,
25983-25992 |
50. |
Hyyrylainen, H. L.,
Vitikainen, M.,
Thwaite, J.,
Wu, H.,
Sarvas, M.,
Harwood, C. R.,
Kontinen, V. P.,
and Stephenson, K.
(2000)
J. Biol. Chem.
275,
26696-266703 |
51. | van Dijl, J. M., Bolhuis, A., Tjalsma, H., Jongbloed, J. D. H., de Jong, A., and Bron, S. (2001) in Bacillus subtilis and Its Closest Relatives (Sonenshein, A. L. , Hoch, J. A. , and Losick, R., eds) , pp. 337-355, ASM Press, Washington, D. C. |
52. | Bauer, M., Behrens, M., Esser, K., Michaelis, G., and Pratje, E. (1994) Mol. Gen. Genet. 245, 272-278[Medline] [Order article via Infotrieve] |
53. |
Herrmann, J. M.,
Neupert, W.,
and Stuart, R. A.
(1997)
EMBO J.
16,
2217-2226 |
54. |
Moore, M.,
Harrison, M. S.,
Peterson, E. C.,
and Henry, R.
(2000)
J. Biol. Chem.
275,
1529-1532 |
55. | Stuart, R. A., and Neupert, W. (2000) Nature 406, 575-577[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Samuelson, J. C.,
Jiang, F.,
Yi, L.,
Chen, M.,
de Gier, J. W.,
Kuhn, A.,
and Dalbey, R. E.
(2001)
J. Biol. Chem.
276,
34847-34852 |
57. | Houben, E. N., Scotti, P. A., Valent, Q. A., Brunner, J., de Gier, J. L., Oudega, B., and Luirink, J. (2000) FEBS Lett. 476, 229-233[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Urbanus, M. L.,
Scotti, P. A.,
Froderberg, L.,
Saaf, A.,
de Gier, J. W.,
Brunner, J.,
Samuelson, J. C.,
Dalbey, R. E.,
Oudega, B.,
and Luirink, J.
(2001)
EMBO Rep.
2,
524-529 |
59. |
Houben, E. N.,
Urbanus, M. L.,
Van Der Laan, M.,
Ten Hagen-Jongman, C. M.,
Driessen, A. J. M.,
Brunner, J.,
Oudega, B.,
and Luirink, J.
(2002)
J. Biol. Chem.
277,
35880-35886 |
60. | Bonnefoy, N., Kermorgant, M., Grodinsky, O., and Dujardin, G. (2000) Mol. Microbiol. 35, 1135-1145[CrossRef][Medline] [Order article via Infotrieve] |
61. | Asai, K., Kawamura, F., Sadaie, Y., and Takahashi, H. J. (1997) J. Bacteriol. 179, 544-547[Abstract] |
62. |
Jiang, M.,
Grau, R.,
and Perego, M.
(2000)
J. Bacteriol.
182,
303-310 |