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Journal of Bacteriology, January 1999, p. 685-688, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Subunit II of Bacillus subtilis Cytochrome
c Oxidase Is a Lipoprotein
Jenny
Bengtsson,1
Harold
Tjalsma,2
Carlo
Rivolta,3 and
Lars
Hederstedt1,*
Department of Microbiology, Lund University,
Lund, Sweden1;
Department of Genetics,
University of Groningen, Groningen Biomolecular Sciences and
Biotechnology Institute, Groningen, The
Netherlands2; and
Institut de
Génétique et de Biologie Microbiennes, Université de
Lausanne, Lausanne, Switzerland3
Received 10 September 1998/Accepted 30 October 1998
 |
ABSTRACT |
The sequence of the N-terminal end of the deduced ctaC
gene product of Bacillus species has the features of a
bacterial lipoprotein. CtaC is the subunit II of cytochrome
caa3, which is a cytochrome c
oxidase. Using Bacillus subtilis mutants blocked in
lipoprotein synthesis, we show that CtaC is a lipoprotein and that
synthesis of the membrane-bound protein and covalent binding of heme to the cytochrome c domain is not dependent on processing at
the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are,
however, deficient in cytochrome caa3 enzyme
activity. Removal of the signal peptide from the CtaC polypeptide, but
not lipid modification, is seemingly required for formation of
functional enzyme.
| |
TEXT |
The gram-positive bacterium
Bacillus subtilis contains at least three different
respiratory oxidases in the cytoplasmic membrane: two quinol oxidases,
cytochrome aa3 and cytochrome bd, and
one cytochrome c oxidase, cytochrome
caa3 (24). Studies of mutants have
shown that, under aerobic conditions, cytochrome
aa3 is the major oxidase, and a deficiency in
this enzyme significantly affects cell growth (16). In
contrast, lack of cytochrome caa3 has little effect on growth of B. subtilis (21). Cytochrome
caa3 oxidase activity is required for oxidation
of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), a
property which can be used to identify mutants defective in assembly or
function of the oxidase (21).
In bacteria, lipoproteins are membrane bound. The lipid is covalently
attached to a cysteine residue at the N-terminal end. The synthesis of
lipoproteins occurs in two steps (for a review, see reference
4). First, a diacylglyceryl moiety is attached to
the sulfur of the cysteine residue of the prolipoprotein (the prolipoprotein contains a signal sequence at its N-terminal end). This
step is catalyzed by prolipoprotein diacylglyceryl transferase, encoded
by the lgt gene. In the second step, signal peptidase type
II, which is specific for lipoprotein maturation and encoded by the
lsp gene, removes the signal sequence by cleaving the
peptide bond on the N-terminal side of the diacylglyceride-cysteine
residue. Generally the N terminus is finally acylated by the action of the apolipoprotein N-acyltransferase, encoded by the
lnt gene. The B. subtilis genome contains genes
for many (>100) different putative lipoproteins and one lgt
and one lsp gene, but an lnt gene has not been
found. Only a few lipoproteins have so far been experimentally
identified in B. subtilis, e.g., PrsA, OpuAC, and KapB
(3, 8, 9).
During work with B. subtilis Lgt- and Lsp-deficient mutants,
we observed that they are deficient in oxidizing TMPD, suggesting an
effect of the mutations on cytochrome caa3. The
subunit polypeptides of cytochrome caa3 are
encoded by the ctaCDEF genes (17). Subunit I
(CtaD) harbors the two heme A groups. Subunit II (CtaC) contains the
dicopper center, CuA, and has a cytochrome c
domain at the C-terminal end. The CuA and cytochrome
c domains are exposed at the outer side of the cytoplasmic
membrane. Subunit II is anchored in the membrane by two transmembrane
-helical segments constituted by the N-terminal half of the
polypeptide, as inferred from its close similarity to Paracoccus
denitrificans cytochrome aa3, whose three-dimensional structure has been determined (7). Thus, both the N- and C-terminal ends of the mature protein are located on
the outer side of the cytoplasmic membrane. The deduced B. subtilis CtaC (pro)-polypeptide consists of 356 amino acid
residues, and the first half contains three segments with hydrophobic
residues. The most N-terminal segment is absent in the assembled
oxidase of Bacillus sp. strain PS3 (6), and it
has the features of a signal sequence typical of lipoproteins (a lipo
box: -Leu-Ala/Ser-Gly/Ala-Cys-), with a cysteine
residue at the predicted cleavage site (position 21) (Fig.
1). The possibility that CtaC of
Bacillus species is a lipoprotein was previously noted by
Quirk et al. (13). In this report, we show that subunit II
of B. subtilis cytochrome caa3 is a
lipoprotein and demonstrate that covalent binding of heme to the CtaC
polypeptide and assembly of cytochrome caa3
proceed in mutants blocked in lipoprotein synthesis.
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FIG. 1.
Deduced N-terminal sequence of subunit II polypeptides
of three oxidases. CtaC, QoxA, and CyoA are subunits of cytochrome
caa3, cytochrome aa3, and
cytochrome bo3, respectively, of the specified
bacteria. The signal peptidase type II signal cleavage site, indicated
with an arrow, is supported by experimental data obtained for
Bacillus sp. strain PS3 (6), Bacillus
subtilis W23 (10), and E. coli
(11).
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In vivo labeling of Lgt- and Lsp-deficient mutants with radioactive
palmitic acid.
We have made use of three mutations that affect
lipoprotein synthesis: an lgt insertion where the downstream
genes (yvoD, yvoE, and yvoF) are
transcribed from the Pspac promoter to avoid
polar effects from the insertion (strains with this mutation were grown
in the presence of 1 mM
isopropyl-
-D-thiogalactopyranoside [IPTG] except in
the experiment represented by Table 2), an lsp deletion, and
an lsp insertion where the intact lsp gene is
transcribed from the Pspac promoter (i.e.,
transcription of lsp is inducible by IPTG) (12).
The constructions of the Lgt and Lsp knockout mutants (Table
1) are described elsewhere (15,
19a).
To radiolabel lipoproteins, strains with the respective mutations
(LUH104, LUH102, and LUH103) and the parental strain (168A)
(Table
1)
were grown in nutrient sporulation medium with phosphate
(NSMP)
(
5) supplemented with 80 nM
[9,10(
n)-
3H]palmitic acid (51 Ci/mmol)
(Amersham). In contrast to
Escherichia coli,
B. subtilis can grow in the absence of functional Lgt or
Lsp.
Membranes were isolated as previously described (
19), lipids
were extracted (
4), and lipoproteins were detected by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
followed
by autoradiography (Fig.
2). Membranes of
the wild type
showed four major and many minor radioactive polypeptide
bands.
As expected, the Lgt knockout mutant LUH104, blocked in the
first
step of the lipoprotein biosynthetic pathway, lacked these bands;
the radiolabel observed as a ladder at the front of the gel corresponds
to free lipid- and low-molecular-weight lipid-containing compounds
such
as glycolipids. The band patterns of the Lsp deletion mutant
LUH102 and
mutant LUH103 with the inducible
lsp gene grown in
the
absence of IPTG differed from those of 168A and LUH103 grown
in the
presence of IPTG. For example, a major radioactive polypeptide
of about
27 kDa migrated more slowly in the Lsp-deficient mutants,
suggesting
that the signal-peptide has not been removed. A distinct
radiolabeled
polypeptide in the 38-kDa region possibly corresponding
to CtaC could
not be detected; i.e., all bands observed in the
wild type were also
found in strain LUH15 in which the
ctaCD genes
are absent
(Fig.
2). However, it should be noted that the CtaC
polypeptide
constitutes less than 0.1% of the total membrane protein
content of
wild-type cells grown in NSMP (
21), a quantity that
might be
below the detection level in this type of experiment.
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FIG. 2.
Analysis of B. subtilis strains for
membrane-bound lipoproteins and covalently bound heme by
[3H]palmitate and [14C]ALA (heme) labeling,
respectively. Fluorographs (2) of SDS gels (16% [wt/vol]
acrylamide) (18) are shown. Growth conditions and
preparation of extracts were as described in the text. Forty micrograms
of membrane protein was loaded in each lane. The samples were as
follows: wt, strain 168A; CtaC, LUH15; Lgt, LUH104; Lsp, LUH102; ILsp,
LUH103. + and  indicate that the extract was prepared from cells
grown in the presence or absence, respectively, of 1 mM IPTG. The
numbers on the left indicate the positions of size markers. The
specific 14C label in heme in the Lgt- and Lsp-deficient
mutants was much lower than in the wild type for reasons that are
discussed in the text. The fluorographs shown are overexposed to
demonstrate that lipoproteins are absent from strain LUH104 and to more
clearly show the CtaC polypeptide(s) in mutants defective in
lipoprotein modification. The amount of CtaC polypeptide in LUH102 and
LUH104 was about 60% of that in the wild type, as calculated from the
[14C]heme in CtaC relative to that in QcrC; i.e., heme in
QcrC was used as an internal standard with the assumption that the
amount of QcrC polypeptide was the same in membranes from all
strains.
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Cytochromes c in mutants blocked in lipoprotein
modification.
Cytochromes of the c type can be
analyzed by growing cells in the presence of radioactive
5-aminolevulinic acid (ALA)
a heme biosynthetic precursorand by
SDS-PAGE of extracts followed by autoradiography (19). In
contrast to other heme proteins, heme in cytochrome c is
covalently bound to the polypeptide and therefore remains attached to
polypeptide after electrophoresis in the presence of SDS. Membranes of
B. subtilis wild-type strains contain four main proteins
with covalently bound heme: CtaC, QcrC, QcrB, and CccA. The QcrC and
QcrB polypeptides are subunits of the cytochrome bc complex,
and CccA is cytochrome c550 (21, 25).
All four cytochromes were found in membranes of Lgt- and Lsp-deficient
mutants, as determined by labeling with [
14C]ALA
(Fig.
2). However, the Lgt and Lsp knockout mutants as well
as the
mutant with the inducible
lsp gene grown without IPTG
exhibited
two differences compared to the wild type and the latter
mutant
grown in the presence of IPTG. (i) The incorporation of
radioactivity
into different cytochrome polypeptides was lower (e.g.,
compare
QcrB band intensities in samples wt and Lgt), a phenomenon
probably
due to a deficiency in uptake of [
14C]ALA. ALA
is taken up by dipeptide transport systems (
22).
Polypeptide
components of these transporters in
B. subtilis are
predicted lipoproteins (AppA, DciAE, and OppA) and might be defective
in Lgt- and Lsp-deficient mutants. Furthermore, cytochrome
c550 is sensitive to proteolysis, and the weak
CccA band of the mutants
may be a result of increased proteolytic
activity, as previously
observed with certain
B. subtilis
strains (
26). (ii) The migration
of the CtaC polypeptide in
the gel varied, indicating size differences.
CtaC in the Lgt knockout
mutant appeared as a 39- and a 38-kDa
polypeptide, the latter being the
same size as the mature wild-type
protein. The observation of the
larger polypeptide is consistent
with the presence of the 20-residue
signal peptide expected in
the Lgt-deficient mutant if CtaC is a
lipoprotein. The form of
the protein that migrated at the position of
wild-type CtaC is
most likely due to removal of the signal peptide from
a fraction
of the population of propolypeptide, possibly catalyzed by
one
or more of the five type I signal peptidases present in
B. subtilis (
20). CtaC from the Lsp knockout mutant
appeared as a single
40-kDa band, i.e., migrating more slowly than both
CtaC polypeptides
present in the Lgt-deficient mutant. This is in
agreement with
a lack of signal peptidase type II; i.e., the
polypeptide is lipid
modified but the signal peptide is not removed.
Some CtaC migrating
as mature, wild-type CtaC was observed in the
mutant with the
inducible
lsp gene grown in the absence of
added IPTG. This can
be explained by the low level of transcription of
the
lsp gene
in the absence of added inducer. From the
electrophoretic mobility
of the CtaC polypeptide synthesized in Lgt-
and Lsp-deficient
mutants, we conclude that subunit II of cytochrome
caa3 is a
lipoprotein.
Role of lipid modification of subunit II.
The analysis of
cytochromes c by radioactive labeling demonstrated that the
CtaC polypeptide in Lgt- and Lsp-deficient mutants contains covalently
bound heme, i.e., that the cytochrome c domain is assembled
and located on the outer side of the membrane. However, the relative
amount of CtaC compared to QcrC and QcrB, as estimated from the heme
content (radioactivity), was significantly lower in the mutants.
Immunoblot analysis with antibodies raised against the C-terminal
peptide of CtaC (Fig. 3) showed that this
difference was due to decreased amounts of the CtaC polypeptide in
membranes of the mutants rather than to a deficiency in the synthesis
of cytochrome c. An autoradiogram to detect 14C
on the blotted membrane (not shown) showed a pattern with relative intensities of bands identical to that of the immunoblot. The 40-kDa
form of Cta in the Lsp-deficient mutant membranes was not transferred
from the acrylamide gel to the immunoblot membrane as efficiently as
the 38-kDa form, accounting for the difference in relative band
intensities in Fig. 2 and 3.
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FIG. 3.
Immunoblot analysis for CtaC polypeptide in membranes.
The samples are the same as those analyzed for covalently bound heme in
Fig. 2. SDS-PAGE was carried out as described for Fig. 2 except that
about 35 µg of protein was loaded in each lane. This gel did not
resolve the two forms of CtaC in the Lgt-deficient mutant. Proteins
were electroblotted onto a polyvinylidene difluoride membrane under
semidry conditions with Tris-glycine buffer with 20% methanol. The
anti-CtaC serum, used at a 500-fold dilution, had been obtained by
immunizing a rabbit with the peptide MLNALTEKRTRGC, corresponding to
the C-terminal end of B. subtilis CtaC, conjugated to bovine
serum albumin. The ECL Western blot system (Amersham) was used to
detect antigens. The position of a 28-kDa size marker is indicated on
the left.
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The immunoblot analysis also showed that a heme-containing polypeptide
of about 28 kDa (migrating just in front of QcrC) present
in the Lgt-
and Lsp-deficient mutants is a fragment of CtaC. The
apparent size of
this fragment, its localization in the membrane,
and its reactivity
with the peptide antiserum suggest that it
corresponds to CtaC
polypeptide truncated at the N-terminal end
but containing at least one
transmembrane
segment.
As mentioned, colonies of Lgt- and Lsp-deficient mutants have reduced
TMPD oxidation activity (Table
2). This
phenotype was
more pronounced in colonies grown on Tryptose blood agar
base
(TBAB) (Difco) plates than in those grown on NSMP plates.
Probably,
the steady-state amount of cytochrome
caa3 is higher in cells
grown on NSMP than on
TBAB. To determine the activity of cytochrome
caa3 in Lgt- and Lsp-deficient mutants,
cytochrome
c oxidation
activity of isolated membranes (from
cells grown in NSMP in the
presence of IPTG) was measured
spectrophotometrically as described
before (
21) except that
we used 20 µM reduced
Saccharomyces cerevisiae cytochrome
c as a substrate. Membranes of the Lgt (LUH104)-
and Lsp
(LUH102)-deficient mutants contained 26% ± 8% and 5% ±
2% of the
cytochrome
c oxidase activity, respectively, of the
parental
strain 168A (0.20 µmol of cytochrome
c oxidized per mg
of
membrane protein per min). Membranes of strain LUH15, devoid
of
cytochrome
caa3, showed no cytochrome
c oxidase activity. The
results demonstrate that a
deficiency in Lsp has a much more drastic
effect on cytochrome
caa3 activity than a deficiency in Lgt.
To analyze the amount of assembled cytochrome
caa3 in mutants defective in lipoprotein
synthesis, we constructed Lgt- and
Lsp-deficient strains also lacking
the major cytochrome
a, i.e.,
cytochrome
aa3, encoded by the
qoxABCD operon.
The different strains
(LUH108, LUH109, and LUH110) as well as the
control, strain LUH17
lacking both cytochrome
aa3 and cytochrome
caa3,
were grown in
NSMP medium to the end of the exponential growth phase.
In these
strains, cytochrome
bd functions as a terminal
oxidase to support
growth. Isolated membranes (
5) were
analyzed for cytochrome
a by light absorption spectroscopy
(
19). Only an assembled cytochrome
caa3 containing both subunits I and II shows
absorption at 605
nm in ascorbate- or dithionite-reduced minus
ferricyanide-oxidized
difference spectra. The mutants lacking Lgt or
Lsp contained approximately
50% of the wild-type level of cytochrome
a. This amount correlated
with the relative amounts of CtaC
polypeptide, determined by immunoblot
analysis of membrane preparations
(spectra and blot not shown)
and calculated from
[
14C]heme (Fig.
2), indicating that most of the CtaC
polypeptides
in the membrane were present in assembled
enzyme.
Conclusion.
Cytochrome caa3 belongs to
a family of heme-copper containing oxidases that includes cytochrome
c oxidases and quinol oxidases (1). Based on the
content of metal centers and the sequence in the C-terminal half of the
polypeptide, subunit II proteins fall into three classes: those that
contain the CuA center (e.g., cytochrome
aa3 of mammalian mitochondria and P. denitrificans); those that contain both CuA and a
cytochrome c domain (e.g., cytochrome caa3 of Bacillus species); and those
that lack metal centers (e.g. E. coli cytochrome
bo3). Subunit II of various oxidases in the family can now be further classified according to the chemical composition of its N-terminal end. It was recently demonstrated that
subunit II (CyoA) of E. coli cytochrome
bo3 is a lipoprotein (11), and in
this work we show that subunit II (CtaC) of B. subtilis
cytochrome caa3 is a lipoprotein.
The N-terminal sequence of CyoA, like those of CtaC from several
Bacillus species, has a motif characteristic of
lipoproteins.
This motif is also present in the N-terminal sequence of
B. subtilis QoxA, subunit II of cytochrome
aa3 (Fig.
1). Functional cytochrome
aa3 is assembled in
B. subtilis Lgt-
and Lsp-deficient mutants,
as judged from growth properties and
cytochrome absorption spectra
(data not shown). The presequence of
subunit II of
P. denitrificans cytochrome
c
oxidase (cytochrome
aa3), which clearly is not a
lipoprotein, does not contain a lipobox. Intriguingly, the N-terminal
sequence of subunit II of
P. denitrificans quinol oxidase
(cytochrome
ba3) is processed and the mature
protein contains a cysteine residue
at its N-terminus, but it is not
modified by lipid (
14,
27),
showing that lipoproteins cannot
be predicted with certainty from
sequence
data.
Lipid modification of subunit II of
E. coli cytochrome
bo3 is not required for assembly or function of
the oxidase, since
replacement of the relevant cysteine residue by
serine has no
drastic effect (
11). In the case of
B. subtilis CtaC, posttranslational
modification at the N-terminal
end does not seem to be required
for assembly but may be important for
oxidase activity. The Lgt
and Lsp knockout mutants both contained about
50% cytochrome
caa3 chromophore in membranes
compared to the wild type but showed
ca. 26% and only ca. 5%
cytochrome
c oxidase activity, respectively.
The activity in
the Lgt mutant may be explained by the removal
of the signal peptide
from a fraction (about 50%) of the CtaC
polypeptides. Thus, it seems
as though removal of the signal peptide
from, but not the lipid
modification of, subunit II is required
for the formation of active
cytochrome
caa3 in
B. subtilis.
Membranes
of strain LUH103 with the inducible
lsp gene grown
in liquid medium
without IPTG contained some 38-kDa CtaC (without
signal peptide)
(Fig.
2) but were TMPD oxidation negative on plates.
This difference
can be explained by the different growth conditions,
i.e., that
little of the 38-kDa form is formed in
colonies.
| |
ACKNOWLEDGMENTS |
We are grateful to Matti Saraste for providing antiserum against CtaC.
This work was supported by grants from the Swedish Natural Science
Research Council to L.H.; by grants from Genencor International (Rijswijk, The Netherlands) and Gist-brocades BV (Delft, The
Netherlands) to H.T., Sierd Bron, and Jan Maarten van Dijl; and by
grant 96.0245 from the Office Fédéral de l'Education et de
la Science (Switzerland) to Dimitri Karamata.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Lund University, Sölvegatan 12, S-223 62 Lund, Sweden. Phone: 46 (46) 2228622. Fax: 46 (46) 157839. E-mail: Lars.Hederstedt{at}mikrbiol.lu.se.
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Journal of Bacteriology, January 1999, p. 685-688, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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