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J. Biol. Chem., Vol. 276, Issue 40, 37289-37298, October 5, 2001
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From Philipps-Universität Marburg, Fachbereich
Chemie/Biochemie, Hans-Meerwein-Str., Marburg D-35032, Germany
Received for publication, April 20, 2001, and in revised form, August 3, 2001
4'-Phosphopantetheine transferases (PPTases)
transfer the 4'-phosphopantetheine moiety of coenzyme A onto a
conserved serine residue of acyl carrier proteins (ACPs) of fatty acid
and polyketide synthases as well as peptidyl carrier proteins (PCPs) of
nonribosomal peptide synthetases. This posttranslational modification
converts ACPs and PCPs from their inactive apo into the active holo
form. We have investigated the 4'-phosphopantetheinylation reaction in
Bacillus subtilis, an organism containing in total 43 ACPs and PCPs but only two PPTases, the acyl carrier protein synthase AcpS
of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin. We identified and cloned ydcB
encoding AcpS from B. subtilis, which complemented an
Escherichia coli acps disruption mutant. B. subtilis AcpS and its substrate ACP were biochemically
characterized. AcpS also modified the D-alanyl carrier protein but failed to recognize PCP and an acyl carrier protein of secondary metabolism discovered in this study, designated AcpK, that
was not identified by the Bacillus genome project. On the other hand, Sfp was able to modify in vitro all acyl
carrier proteins tested. We thereby extend the reported broad
specificity of this enzyme to the homologous ACP. This in
vitro cross-interaction between primary and secondary metabolism
was confirmed under physiological in vivo conditions by the
construction of a ydcB deletion in a B. subtilis
sfp+ strain. The genes coding for Sfp and its homolog
Gsp from Bacillus brevis could also complement the E. coli acps disruption. These results call into question the
essential role of AcpS in strains that contain a Sfp-like PPTase and
consequently the suitability of AcpS as a microbial target in such strains.
4'-Phosphopantetheine
(Ppant)1 is an essential
prosthetic group of several acyl carrier proteins involved in pathways
of primary and secondary metabolism. These include acyl carrier
proteins (ACPs) of fatty acid synthases (FASs), ACPs of polyketide
synthases (PKSs), and peptidyl carrier proteins (PCPs) and aryl carrier proteins of nonribosomal peptide synthetases (NRPSs) (1, 2). The free
thiol moiety of Ppant serves to covalently bind the acyl reaction
intermediates as thioesters during the multistep assembly of the
monomeric precursors, typically acetyl, malonyl, and aminoacyl groups.
Ppant fulfills two demands in these biosynthetic pathways. First, the
intermediates remain covalently tethered to the multifunctional enzyme
templates in an energy-rich linkage. Second, the flexibility and length
of Ppant (about 20 Å) facilitates the transport of the intermediates
to the spatially distinct reaction centers. The Ppant moiety is derived
from coenzyme A (CoA) and posttranslationally transferred onto an
invariant serine side chain. This
Mg2+-dependent conversion of the apoproteins to
the holoproteins is catalyzed by the 4'-phosphopantetheine transferases
(PPTases) (1, 2) (see Fig. 1).
Most organisms that employ more than one Ppant-dependent
pathway also contain more than one PPTase. For example,
Escherichia coli has three PPTases, namely the acyl carrier
protein synthase AcpS involved in fatty acid synthesis, EntD involved
in synthesis of the siderophore enterobactin, and the gene product of
yhhU, with as yet unknown physiological function (1, 3).
Interestingly, PPTases of different pathways can have overlapping
selectivity for their cognate acyl carrier protein partner. In E. coli, AcpS and EntD have reciprocal specificities, with AcpS only
recognizing ACP and EntD only accepting the PCPs of the enterobactin
NRPS (1). In contrast, Sfp, the PPTase of the surfactin NRPS of Bacillus subtilis, was shown to also phosphopantetheinylate
heterologous ACPs of FASs and PKSs (1, 4, 5).
PPTases have been classified in three groups according to their
sequence homologies and substrate spectrum. The first group is the AcpS
type with AcpS of Escherichia coli as the name-giving prototype. PPTases of this type are about 120 aa in length, are found
in almost all microorganisms for the modification of fatty acid ACP,
and were shown to accept as substrate also ACPs of type II PKS systems
(6). The PPTase Sfp of B. subtilis is the prototype of the
second group. Enzymes of this type are about 240 aa in length and have
mostly been found associated with the gene clusters for nonribosomal
peptide synthesis. The well characterized Sfp exhibits an
extraordinarily broad substrate specificity and could modify all acyl
carrier protein substrates tested, including PCPs of NRPS as
well as ACPs of FAS and PKS. The third group is an integrated PPTase
found as the C-terminal domain of the multifunctional FAS2, for example
in Saccharomyces cerevisiae (7). This classification has
recently been further supported by structural studies. The monomeric
Sfp was found to fold in two domains with similar topology (8). In Sfp,
CoA is bound at the interface of this pseudohomodimer. Interestingly,
both domains exhibit sequence homology to members of the AcpS group,
which are only half the size of Sfp. Two highly similar
structures of enzymes of the AcpS group have recently been reported,
namely AcpS of B. subtilis (9) and Streptococcus pneumoniae (10). In both structures, AcpS forms a trimer. As was
already suggested from the Sfp structure, each AcpS monomer has the
same folding as the two Sfp subdomains. However, since three AcpS
subunits are present in this structure, three CoA binding sites are
generated at the subunit interfaces.
In this work, we set out to characterize the
4'-phosphopantetheinylation reactions in B. subtilis. This
organism is of special interest in this respect, because it contains a
large number of Ppant-dependent pathways of primary and
secondary metabolism but only two PPTases, AcpS and Sfp (see Fig.
2). We report on the biochemical
characterization of AcpS and the identification of a second acyl
carrier protein in B. subtilis, designated AcpK, and define
the specificity of the two PPTases for the different acyl carrier
proteins. As suggested from the in vitro data, we found that
AcpS of B. subtilis is not essential in strains that are
sfp+. From these results and from genetic
complementation studies in E. coli, we present a refined
model for the substrate spectrum of PPTases of the AcpS and Sfp type
and consequences for their suitability as microbial targets.
General Techniques--
E. coli was grown on LB
medium. B. subtilis was usually grown and maintained
on Difco sporulation medium (11); however, for preparations of
chromosomal DNA, it was also grown on LB medium. Antibiotics were used
at the following concentrations for E. coli: ampicillin, 100 µg/ml; spectinomycin, 100 µg/ml; kanamycin, 60 µg/ml (25 µg/ml
for M15/pREP4 strains). For B. subtilis, the following concentrations were used: chloramphenicol, 10 µg/ml; spectinomycin, 100 µg/ml; MLS, erythromycin (1 µg/ml) plus lincomycin (25 µg/ml).
For E. coli techniques, such as transformation, plasmid
preparation, and P1 phage transduction, standard protocols were used (12, 13). Vent polymerase (New England Biolabs, Schwalbach, Germany) was used to amplify gene fragments for cloning and expression purposes, and the Expand Long Range PCR system (Roche Molecular Biochemicals) was used for control PCRs and for the amplification of
fragments used for transformations. Oligonucleotides were purchased from MWG Biotech. [3H]CoA was purchased from Hartmann
Analytics (Braunschweig, Germany).
Gene Knockout and Gene Introduction into E. coli and B. subtilis--
For manipulations in the chromosome of E. coli, we used the polA strain HSK42, which is unable to
replicate ColE1-based plasmids. When transformed with such a plasmid,
integration into the chromosome must occur under selective conditions.
Double crossover transformants were identified by testing for
sensitivity against ampicillin and were confirmed by PCR analysis of
chromosomal DNA (for the nrdD locus, oligonucleotides
E1 (5'-GATTATTGCGCCACTGTTGC-3') and E2 (5'-TCATTTTCCCACACGCCGAG-3'),
annealing at the nrdD gene, were used). B. subtilis strains were transformed according to the protocol of
Klein et al. (14). For PCR analysis of new genotypes,
chromosomal DNA was prepared.
Construction of Plasmids--
All plasmids used in this study
are summarized in Table I. Construction
of the integration plasmid pUC18-nrdD::acps-spc for E. coli was as follows. The vector pET22b-acps (15) was linearized with HindIII and ligated with the spectinomycin resistance
cassette (spc+) excised from pDG1726 (16) with
HindIII. The obtained plasmid pET22b-acps-spc was cut with
BglII and SphI, and the excised fragment was
cloned into BamHI- and SphI-treated pTZ18R. The
resulting plasmid pTZ18R-acps-spc served as a template in a PCR
with oligonucleotides 5'-ATAGTTAACGCGCGTTGGCCGATTC-3' and
5'- ATAGTTAACGCCTCTTCGCTATTACGC-3' to amplify a
fragment that contained acps under control of the lac promotor of pTZ18R and spc+. This
fragment was cloned blunt-ended into pUC18-nrdD, which was treated with
EcoRV, to give pUC18-nrdD::acps-spc.
Construction of the Disruption Plasmid
pTZ18R-5'-acps::kan-3' for E. coli--
Using oligonucleotides E3
(5'-ATATCTAGACCATGACGTATCGTTATC-3') and E4
(5'-ATACCATGGTTCTACTCTGGAAGTAGAG-3'), a 2080-kb
fragment was amplified from chromosomal DNA of E. coli
K-12 that comprised 990 bp upstream and 692 bp downstream of
acps. This fragment was cloned into pTrc99a using the
NcoI and XbaI sites introduced with the
oligonucleotides. The resulting plasmid pTrc99a-5'-acps-3' was then
linearized at the single SacI site, which is localized at
position 251 of the 381-bp acps gene. The kanamycin
resistance cassette (kan+) was excised from
vector pDG782 (16) with EcoRI and BglII and cloned into pTZ18R to give pTZ18R-kan. The kan+
cassette was then amplified by PCR with oligonucleotides 5'- ATAGAGCTCGACTCACTATAGGGAATTC-3' and
5-'-ATAGAGCTCTAAAACGACGGCCAGTG-3' from pTZ18R-kan,
cut with SacI and ligated with the linearized fragment of
pTZ18R-5'-acps-3' to give pTrc99a-5'-acps::kan-3'.
Construction of pTZ18R-ydcB--
ydcB was
amplified by PCR from chromosomal DNA of B. subtilis
JH642 using oligonucleotides
5'-ATATAAGCTTCATTTAAATAGTACGTACGC-3' and 5'-
TATAAGATCTCACTATCAAATATATGAGTGG-3' and cloned
blunt-ended into pTZ18R that was linearized with HincII. The
right orientation of ydcB under control of the
lac promotor of pTZ18R-ydcB was verified by restriction
analysis and sequencing.
Construction of pDR67-ydcB--
The acps gene of
B. subtilis was cut out of pTZ18R-ydcB with
HindIII and BglII and cloned between the
HindIII and BglII sites of pDR67 to give
pDR67-ydcB. pDR67 lacks an origin of replication for B. subtilis but can integrate into the amyE locus of the
genome via the amyE front and amyE back fragments
upstream and downstream of the multiple cloning site of the plasmid.
The cat+ cassette conferring chloramphenicol
resistance, which is also located between the two amyE
fragments, serves to select for the integration. Inserts cloned into
the multiple cloning site are under the control of the weak and
isopropyl- Construction of the Disruption Plasmid for ydcB of B. subtilis
pTZ18R-5'- Construction of pTZ19-gsp--
The
HindIII-PstI fragment containing the
gsp gene was excised from pGsp+ (17) and ligated into pTZ19R
to give pTZ19-gsp.
Construction of pQE60-ACP--
The acpA gene
encoding ACP was PCR-amplified with oligonucleotides
5'-AATTCCATGGCAGACACATTAGAGCGT-3' and
5'-TTTTGGATCCTTGCTGGTTTTGTATGTAGTTCAC-3' from
chromosomal DNA of B. subtilis MR168 and ligated into the NcoI and BamHI sites of pQE60 to give the
expression plasmid pQE60-acpA. The encoded recombinant protein carries
the C-terminal tag GSRSHHHHHH.
Construction of pQE60-acpK--
The acpK gene
was amplified by PCR with oligonucleotides
5'-TATCCATGGATAAACAGAGAATCTTTG-3' and
5'-TATAGATCTGGCAGATTGCACTTTGTC-3' from chromosomal DNA of
B. subtilis MR168. The amplified fragment was digested with
NcoI and BglII and ligated into the
NcoI and BamHI sites of pQE60 to create the
expression plasmid pQE60-acpK encoding the recombinant AcpK with a
C-terminal tag GSGSHHHHHH.
Construction of pQE60-dltC--
The dltC gene
encoding the D-alanyl carrier protein (DCP) was amplified
by PCR with oligonucleotides
5'-ATACCATGGATTTTAAACAAGAGG-3' and
5'-ATAAGATCTTTTCAACTCAGACAGCT-3' from chromosomal DNA
of B. subtilis MR168 and ligated into the NcoI
and BglII sites of pQE60. The resulting plasmid pQE60-dltC
encodes the recombinant DCP with a C-terminal tag RSHHHHHH.
Overproduction and Purification of Recombinant
Proteins--
E. coli M15/pREP4 was transformed with
pTZ18-ydcB to give strain RF3. 5 ml of an overnight culture of RF3 in
LB were used to inoculate 500 ml of the same medium. Cells were grown
at 37 °C and 300 rpm until an A600 of
0.7 was reached. The culture was then induced with 0.25 mM
isopropyl-
E. coli M15/pREP4 was transformed with pQE60-acpA,
pQE60-acpK, and pQE60-dltC to give strains RF1, HM403, and RF4,
respectively, for the production of the His6 fusion
proteins ACP, AcpK, and DCP. Cells were grown, induced, harvested, and
disrupted, and the crude cell extract was centrifuged as described
above for RF3. Protein purification using Ni2+ affinity
chromatography was carried out as previously described (18). Purified
proteins were dialyzed against assay buffer, brought to 10% glycerol
(v/v), and stored at Biochemical Assays with B. subtilis AcpS and Sfp--
AcpS and
Sfp activity was assayed by using a radioactive assay method
essentially as described previously (15). This method measures the
incorporation of the 3H-labeled 4'-phosphopantetheine group
from [3H]CoA into apo-ACP or other acyl carrier proteins.
Reaction mixtures containing 50 mM Tris/HCl, pH 8.8, 12.5 mM MgCl2, 2 mM dithiothreitol, a
6-100 µM concentration of the respective acyl carrier
protein, 2-20 µM CoA, 119-475 nM
[3H]CoA (specific activity: 40 Ci/mmol, 0.95 mCi/ml), 2.2 µM to 5.6 nM AcpS of B. subtilis,
or 0.8 µM Sfp were incubated at 37 °C for 5-30
min. Reactions were stopped by the addition of 0.8 ml of ice-cold
trichloroacetic acid (10%) and 15 µl of bovine serum albumin (25 mg/ml). Precipitated protein was collected by centrifugation at 13,000 rpm and 4 °C for 15 min. The pellet was washed twice with 0.8 ml of
ice-cold trichloroacetic acid and resuspended in 200 µl of formic
acid. The resulting suspension was mixed with 3.5 ml of Rotiszint Eco
Plus scintillation fluid (Roth) and counted using a 1900CA Tri-Carb
liquid scintillation analyzer (Packard).
For kinetic studies, reaction mixtures contained, unless otherwise
indicated, 50 mM Tris/HCl, pH 8.8 (75 mM MES,
pH 6.0, in the case of Sfp), 12.5 mM MgCl2, 2 mM dithiothreitol, 2-200 µM apo-ACP of
B. subtilis (apo/holo mixture) or 2-60 µM
AcpK, 2-1000 µM CoA, and 5.6 nM AcpS of
B. subtilis or 10 nM Sfp and were incubated at
37 °C for 10-30 min. The reaction was stopped, and the protein was
precipitated by the addition of trichloroacetic acid (final concentration 10%). The amount of holo-ACP and AcpK formed was determined by an HPLC method. This method was adapted from a method described previously (15). Reaction mixtures (800 µl each) were, after the addition of trichloroacetic acid, centrifuged for 30 min at
13,000 rpm and 4 °C. The supernatant was discarded, and the protein
pellet resuspended in 120 µl of 50 mM Tris/HCl, pH 8.8. A
100-µl sample of this solution was injected onto an analytical Nucleosil 250 C18-column (reversed phase; Macherey & Nagel)
that had been equilibrated with 0.1% trifluoroacetic acid. Absorbance at 220 nm was monitored. The column was eluted with a 1.2-ml linear gradient to 60% solvent B (methanol in 0.1% trifluoroacetic acid) followed by a 7.2-ml linear gradient to 100% solvent B at 0.3 ml/min.
Under these conditions, holo-ACP migrated faster than apo-ACP (24.3 and
25.2 min, respectively). The amount of holo-ACP formed was determined
by comparing the peak area of the holo-ACP formed with those of both
apo- and holo-ACP and subtracting the amount of holo-ACP that was
already present after the heterologous expression of the protein in
E. coli (see "Results"). Apo-AcpK and holo-AcpK were
separated in a similar manner using the same column and a 15-ml linear
gradient to 80% solvent C (isopropyl alcohol in 0.1%
trifluoroacetic acid) followed by a 0.6-ml linear gradient to 95%
solvent C at 0.3 ml/min. Holo-AcpK and apo-AcpK eluted at retention
times of 53.9 and 54.8 min.
To determine Km and kcat
values of AcpS and Sfp for apo-ACP and apo-AcpK, reaction mixtures (in
triplicate) contained 1 mM CoA, 2-200 µM
apo-ACP, or 2-60 µM apo-AcpK and either 5.6 nM AcpS or 10 nM Sfp. For the determination of
the Km value of AcpS for CoA, the reaction
conditions were the same as described above except that the
concentration of apo-ACP was kept at 200 µM, and
concentrations of CoA were 2-500 µM. The formation of
holo carrier protein was measured by the HPLC method in all cases.
Carrier proteins were dialyzed before the assay against the respective
assay buffer (50 mM Tris/HCl, pH 8.8, in the case of AcpS
and 75 mM MES, pH 6.0, in the case of Sfp).
Construction of an E. coli acps Disruption Mutant as a Genetic
Tool--
Since acps is an essential gene in E. coli (20), a disruption mutant can only be generated when a
complementing gene is present in trans. We therefore decided
to first integrate a second copy of acps in another locus of
the E. coli chromosome and then disrupt the gene at its
natural locus (at 58 min; see Fig. 2A) with a
kan+ cassette. The
acps::kan+ genotype was then
transduced using P1 phage into other E. coli strains
carrying in trans a PPTase gene to be tested for
acps complementation activity. For manipulations of the
chromosome of E. coli, we used a polA mutant
strain, HSK 42, that is unable to replicate ColE1-based plasmids and
thus allows selection of integration into the chromosome. Double
crossover integrations were identified by marker loss and PCR analysis
with flanking primers. acps is the second gene in a
bicistronic operon with pdxJ (the former name of
acps was dpj, for downstream of
pdxJ) and is
followed by a termination loop (15, 20). A disruption of
acps was therefore not expected to exert polar effects on
neighboring genes. The nrdD locus at 96 min (see Fig.
2A) was chosen for the integration of a second copy of the
acps gene into the E. coli chromosome.
nrdD encodes the anaerobic deoxyribonucleotide reductase, which is not essential under the conditions used here (21). Transformation of HSK42 with the disruption plasmid
pTrc99a-5'-acps::kan-3' and selection on LB plates with
kanamycin yielded Kmr transformants, which were exclusively
found after restreaking to be also Apr and thus had
integrated the plasmid only by a single crossover event, leaving the
original acps gene intact. This finding confirmed the
essential nature of the acps gene and the necessity to first introduce a second copy of the gene. To this end, HSK42 was first transformed with the integration plasmid
pUC18-nrdD::acps-spc, and transformants were selected on LB
plates with spectinomycin. About 10% of these were candidates for
double crossover integration, since they were Aps. This
genotype was confirmed by PCR using oligonucleotides E1 and E2. Only
the fragment nrdD::acps-spc+ and not
the wild-type fragment nrdD was obtained. The latter was
obtained in the control using chromosomal DNA of HSK42. One transformant, HM0139 (see Table II for a
list of strains), was chosen for further work and transformed with
plasmid pTrc99a-5'-acps::kan-3' to disrupt the
acps gene. Transformants selected on LB plates containing
kanamycin were subsequently tested for Aps. Now candidates
for double crossover integration were obtained and could be confirmed
by PCR using oligonucleotides E3 and E4. One of the thus identified
strains, HM0145 (relevant genotype acps::kan+,
nrdD::acps-spc+; see Table II), was
chosen for further work. A P1 phage lysate was prepared from HM0145,
which served for transduction of the acps::kan+ genotype into E. coli strains carrying PPTase genes in trans.
Identification of the ydcB Gene Encoding AcpS of B. subtilis and
Its Genetic Characterization--
At the beginning of this work, the
gene encoding the ACP synthase (AcpS) of B. subtilis was not
identified or characterized. By homology searches, we identified
ydcB, which was revealed by the B. subtilis
genome sequencing project (22) as the putative gene coding for AcpS
(33% identity to E. coli AcpS). ydcB is located at 44° of the Bacillus genome (see Fig. 2B),
366 bp in length, and encodes a protein of 121 aa (13.7 kDa).
We assumed that AcpS of B. subtilis should complement the
corresponding activity in E. coli and therefore attempted to
disrupt acps of E. coli expressing
ydcB in trans. For this purpose, E. coli K-12 strain MG1655 was transformed with pTZ18R-ydcB to give strain HM0172. To test for complementation activity, the
acps::kan+ genotype was then
transduced with the P1 lysate of HM0145, and transductants were
selected on LB plates containing ampicillin and kanamycin. Indeed,
Kmr colonies could be obtained overnight using HM0172 as
recipient strain, whereas no Kmr-colonies appeared in the
control (strain HM0169, MG1655 with plasmid pTZ18R) under these
conditions. The Kmr colonies were Sps,
indicating that the
nrdD::acps-spc+
genotype was not co-transduced. Both genotypes were confirmed by PCR
(not shown).
Sfp and Other Members of the Sfp Family Can Also Complement AcpS of
E. coli in Vivo--
We also tested the ability of PPTases of the Sfp
type to modify noncognate ACP substrates under heterologous in
vivo conditions. To this end, plasmids pUC8-sfp and pTZ19-gsp were
transformed into E. coli K-12 to give the strains HM0170 and
HM0171, respectively. As described above, the P1 lysate of HM0145 was
then used to transduce the acps::kan+
genotype. In both cases, using HM0170 and HM0171, Kmr
transductants (which were SpS) were obtained overnight,
whereas no Kmr colonies could be observed with the control
strain HM0169. Further analysis of the transductants by PCR confirmed
their acps::kan+ genotype. Thus, the
PPTases Sfp and Gsp of secondary metabolism from B. subtilis
and Bacillus brevis can also complement AcpS of E. coli's primary metabolism.
Interestingly, Kmr and Sps colonies also
appeared in the experiments using control strain HM0169 after a
prolonged time period (after 2-3 days at 37 °C). These colonies
were confirmed by PCR to be true transductants, since they were
acps::kan+. Obviously, the same
suppressor mutations that complement the lethal acps
disruption were selected as previously described by Lam et
al. for a conditional mutant of the acps gene (formerly dpj) under nonpermissive conditions (20, 23). In agreement with the results by these authors, most of the transductants displayed a very mucous morphology on LB plates. Suppressor mutations can occur
in the lon gene and at another location in the chromosome (20, 23), presumably in the gene yhhU encoding a PPTase of unknown function (3).
Overproduction and Purification of B. subtilis AcpS, ACP, AcpK, and
DCP--
Pure B. subtilis AcpS was obtained by heterologous
overexpression of the ydcB gene in E. coli and
subsequent four-column purification as described under "Experimental
Procedures." ACP, DCP, and AcpK (see below) of B. subtilis
were produced as C-terminal His6 tag fusion proteins and
purified by affinity chromatography. SDS-polyacrylamide gel
electrophoresis analysis (not shown) showed that two bands were
obtained in the case of ACP and DCP, pointing to partial apo to holo
conversion by E. coli AcpS during heterologous expression. In contrast to a report on the isolation and cloning of the
acpA gene in E. coli (24), neither the production
of ACP nor of the other proteins seemed to significantly inhibit growth
of E. coli cells.
Biochemical Characterization of B. subtilis AcpS--
To determine
the catalytic activity of B. subtilis AcpS, an HPLC assay
was applied. The recombinant substrate ACP of B. subtilis was determined by the HPLC method to be present in a ratio of 84% apo
to 16% holo form after heterologous production in E. coli. Kinetic constants were determined through a Michaelis-Menten fit of the
data sets (see Fig. 3 and Table
III). Interestingly, first saturation
occurred between 2 and 8 µM apo-ACP (Fig. 3A),
but velocity values began to increase when the apo-ACP concentration was raised to 20 µM apo-ACP. Therefore, two different
Km and kcat values could be
determined. The first Km for apo-ACP concentrations
between 2 and 8 µM was 0.2 ± 0.3 µM,
with the kcat being 22 ± 2 min Protein Partners of AcpS and Sfp: ACP, PCP, and DCP--
Since Sfp
was reported to be of broad specificity, we assumed that it would also
modify the ACP of B. subtilis. However, this experiment was
intriguing, because a higher degree of specialization would be
conceivable for the homologous substrates of the same organism. All
cases of broad specificity of Sfp were demonstrated for heterologous
acyl carrier proteins. Nevertheless, as shown in Fig.
4, Sfp also efficiently recognizes and
modifies ACP of B. subtilis. The dispensable PPTase Sfp of
the secondary metabolism can thus convert the ACP of primary metabolism
into its active holo form in vitro. Determination of kinetic
constants revealed saturation at low and high apo-ACP concentration as
observed for AcpS. In the ACP range of 2-8 µM, a
Km of 1.4 ± 0.3 µM and a
kcat of 1.7 ± 0.1 min
We next tested the D-alanyl carrier protein DCP and a PCP
for their ability to serve as substrates for AcpS and/or Sfp.
Therefore, the dltC gene encoding DCP was cloned and
expressed as a His6 tag fusion. As a representative of
PCPs, we chose the excised TycC3-PCP domain of the multimodular
tyrocidine NRPS (19). As shown in Fig. 4, Sfp recognized both acyl
carrier proteins as substrate, whereas AcpS could only modify DCP.
Identification of a Second ACP in B. subtilis, AcpK, Which Is Only
Modified by Sfp--
The B. subtilis genome project has
confirmed the presence of the previously described gene acpA
coding for ACP of fatty acid synthesis (22). We have reexamined the
regions of the genome that harbor the clusters for secondary metabolite
production in a search for possible misannotations of the genome
project data. The pksX cluster contains several genes that
encode enzymes homologous to fatty acid or polyketide synthases of type
II (pksB-I), to polyketide synthases of type I
(pksKLMPR), and to nonribosomal peptide synthetases
(pksJKNR) (25) (and GenBankTM accession number
Z99113). A relatively large gap between pksE and
pksF led us to reexamine this region in detail (see Fig.
5A). Surprisingly, we detected
an open reading frame, thereafter designated acpK, that has
a 20-bp overlap with the 5'-end of pksF. The putative gene
product AcpK (82 aa, 9.251 kDa, pI 4.2) showed significant similarities to ACPs, in particular around the conserved serine residue, which serves as the Ppant attachment site (see Fig.
5B). The highest similarity was found to two ACPs, TaB and
TaE, that are obviously involved in synthesis of the antibiotic TA in
Myxococcus xanthus (53 and 33% identity) (26). The
similarity to B. subtilis and E. coli ACPs was
only 18 and 22%, respectively. Fig. 5B shows an alignment
of AcpK with these acyl carrier proteins. We were interested to know
whether AcpK is a substrate for AcpS or Sfp or even for both. As shown
in Fig. 4, only Sfp, but not AcpS, was capable of converting AcpK into
its holo form. Determination of kinetic constants revealed a normal
Michaelis-Menten behavior with a Km of 7.9 ± 2.1 µM and a kcat of 3.2 ± 0.2 min Deletion Mutant of B. subtilis acps--
The gene ydcB
(363 bp) is predicted to be organized in an operon with the downstream
gene ydcC (1,119 bp) encoding a putative protein (42.2 kDa)
of unknown function. It is not known whether ydcC is
essential or not. We decided to attempt deletion of ydcB regardless of possible polar effects on ydcC. For the
deletion, we followed a similar strategy as for E. coli
acps. First, a second copy of ydcB was introduced into
the amyE locus of the B. subtilis chromosome (see
Fig. 2B) by transforming B. subtilis Mo1099 with pDR67-ydcB and selecting for Cmr transformants. These were
subsequently checked for MLSs by restreaking. One of the
Cmr and MLSs colonies, HM0451, was chosen for
further work. HM0451 was then transformed with a PCR product containing
5' and 3' regions of the original ydcB gene flanking a
spc+ cassette that substituted the deleted
ydcB (obtained by PCR amplification using
pTZ18-5'-
Since Sfp was shown in this study to in vitro modify ACP of
B. subtilis, we speculated that ydcB might not be
essential in sfp+ strains. B. subtilis OKB105 (11) was chosen as a test strain expressing the
intact sfp gene at physiological levels (another prominent
sfp+ strain, the surfactin producer ATCC21332,
was found to be already Spr). OKB105 was transformed with
the PCR product as described above. Indeed, Spr
transformants could be obtained. Several of these strains were chosen
for PCR analysis, which confirmed their
ydcB::spc+ genotype (see Fig. 6). One
of these strains was designated HM0489. HM0489 still produced surfactin
in amounts similar to the parent strain OKB105, as judged by analysis
of hemolytic activity on blood agar plates (data not shown).
Furthermore, the growth curve patterns of OKB105 and HM0489 were
indistinguishable in both rich and minimal (glucose) media (data not
shown). These results prove that ydcB is dispensable in
sfp+ strains. The PPTase of secondary metabolism
in B. subtilis can substitute for AcpS of primary metabolism
under physiological conditions.
We have investigated the phosphopantetheinylation reaction in
Ppant-dependent pathways of primary and secondary
metabolism of B. subtilis. This organism employs many acyl
carrier proteins that need to be converted from the inactive apo
into the active holo form by the action of a PPTase. Central to
primary metabolism and thus essential for survival of the cell is
holo-ACP, which acts as the carrier for intermediates of fatty acid
synthesis. Another acyl carrier protein-dependent route
involved in cell wall synthesis in B. subtilis is the
attachment of D-alanyl to free hydroxyl groups of teichoic
acid. The D-alanyl moiety is provided by
D-alanyl-Ppant-DCP, which is generated from holo-DCP and
the D-alanyl carrier protein ligase DclA. This process,
which modulates the overall charge of the cell wall, is not essential for cell survival (27); however, as a component of the cell wall
synthesis, it should rather be assigned to primary than to secondary
metabolism. In addition to ACP and DCP, the B. subtilis genome harbors three large clusters, srfA, pps,
and pksX, for the production of secondary metabolites as
well as the dhb cluster for the synthesis of the siderophore
bacillibactin (28) that is expressed under iron-limiting conditions.
These genes make up about 4% of the entire chromosome. The
multifunctional NRPSs and PKSs encoded comprise a total of 40 acyl
carrier proteins of type I that are embedded as domains within the
multidomain enzymes. Upstream of the pksX cluster, we have
identified a second type II ACP, designated AcpK, which raises the
number of Ppant-dependent acyl carrier proteins to 43 in
B. subtilis (see Fig. 2B).
In contrast to the large number of acyl carrier protein, only two
PPTases are present in B. subtilis for their conversion into
the holo forms. Sfp of the surfactin NRPS was previously biochemically
characterized as an enzyme with broad specificity for its protein
partner and therefore found manifold applications for the in
vitro or in vivo modification of recombinant NRPSs or
PKS. Sfp was able to modify every acyl carrier protein tested so far
in vitro including ACP of E. coli FAS. It has
been shown to modify the surfactin NRPS (1, 5), the bacillibactin NRPS (28), and the fengycin or plipastatin NRPS of B. subtilis.
Sfp is thus responsible for the 4'-phosphopantetheinylation of all 40 integrated acyl carrier protein of B. subtilis.
At the outset of this work, the second PPTase, encoded by the gene
ydcB, was not yet described or characterized. We have
identified the gene by sequence homology of the gene product to
E. coli AcpS; genetically verified its function as the ACP
synthase of B. subtilis by complementation of E. coli
acps; and finally overproduced, purified, and biochemically
characterized the recombinant protein with its substrates ACP and CoA
(see Figs. 3 and 4). In agreement with the studies on AcpS of the
Gram-positive S. pneumoniae (29) we found two
Km values for the substrate apo-ACP, due to a first
substrate saturation at low (2-8 µM) ACP concentrations. The first Km of 0.2 ± 0.3 µM
(kcat = 22 ± 2 min The homologous E. coli AcpS (33% identity) was previously
demonstrated to modify not only its natural substrate ACP but also various heterologous ACPs of type II PKSs (6) and DCP of
Lactobacillus casei (31). These results contributed to the
idea that PPTases of the AcpS type can modify type II ACPs of either
fatty acid or polyketide synthesis, and E. coli AcpS was
discussed as a means to modify and misprime type II PKSs (6). We found
that B. subtilis AcpS can also modify the DCP substrate of
this organism but not the tested PCP substrate, as expected.
Surprisingly, however, we found that B. subtilis AcpS was
also unable to modify the second acyl carrier protein of B. subtilis, AcpK (see Fig. 4). The ability of Sfp to modify AcpK
in vitro further corroborates the promiscuity of this enzyme
in choosing its protein substrate. Thus, the simple interpretation of
this data would suggest that in B. subtilis AcpS is
dedicated to the two acyl carrier protein substrate of primary
metabolism, ACP and DCP, whereas Sfp provides the catalytic Ppant for
all acyl carrier proteins of the secondary metabolism, whether they are
integrated domains or distinct enzymes such as AcpK. This model would
correspond to the situation in E. coli, where AcpS and EntD
selectively recognize their substrates ACP of primary and EntB/EntF of
secondary metabolism, respectively, but do not cross-interact (the
third PPTase encoded by yhhU can replace acps
only when expressed under nonphysiological conditions from a high copy
plasmid (3) or, possibly, when suppressor mutations occur (23)).
Surprisingly, however, our finding that Sfp could also modify in
vitro the acyl carrier proteins of primary metabolism, ACP and
DCP, and had not evolved a discrimination against these homologous
substrates suggested 4'-Ppant transfer to be different in the
Gram-positive B. subtilis. Sfp exhibited Km values for ACP comparable with AcpS (for low
apo-ACP concentrations 8-fold higher, and for higher apo-ACP
concentrations about 2-fold lower) and only about 10-fold reduced
kcat values. This corresponds to a 110-fold drop
in kcat over Km for low
apo-ACP concentrations, but only in an about 6-fold drop for higher
apo-ACP concentrations, suggesting that Sfp should be catalytically competent enough to serve as a functionally redundant ACP synthase activity in B. subtilis (see Table III). Furthermore, it was
reported for E. coli strain MP4, which is conditionally
defective in acps, that apo-ACP could comprise 70% of the
total ACP pool under growth-permitting conditions (32), indicating a
tolerance toward significant amounts of the inactive form. We therefore
attempted disruption of the ydcB gene encoding AcpS in
B. subtilis to answer the question of whether Sfp is also
capable of carrying out the same reaction under more stringent,
physiological in vivo conditions. The sfp gene is
under a very weak constitutive promotor (33). In perfect agreement with
these considerations and the in vitro results, we could
delete ydcB in the sfp+ strain OKB105
(see Fig. 6) but failed to do so in sfp We have collected further evidence for the ability of Sfp-like PPTases
from the secondary metabolism to complement in vivo the
essential function of AcpS. Both Gsp from B. brevis and Sfp could complement E. coli AcpS in vivo. This also
indicates that Sfp is probably not the only promiscuous PPTase but
rather that this broad specificity is a general feature of enzymes of
the Sfp family or at least often encountered here. In fact, our
laboratory routinely uses Gsp and Sfp in co-expression experiments in
E. coli for modification of various recombinant NRPSs (18,
34).
The ability of PPTases of the Sfp type, and especially of Sfp itself,
to complement the activity of AcpS raises questions both about the
evolution of PPTase types and about the need for the presence of an
AcpS type enzyme in strains that also contain an enzyme of the Sfp
type. From sequence and structural considerations, it seems plausible
to hypothesize that the Sfp family evolved from the AcpS family
by a gene fusion event with subsequent diversification of the two
halves. This evolution probably took place when the acyl carrier
proteins were fused with the other enzymatic units to become integrated
domains of FASs, PKSs, or NRPSs. The new architecture of the resulting
large protein templates presumably was inaccessible to AcpS for steric
or electrostatic reasons, as was suggested from structural data (9).
Another explanation could be the need for a well defined regulation of
the CoA pool (35), possibly to selectively switch on and off primary
and secondary metabolism. This argument could still be valid for
E. coli, where Ppant transfer of primary and secondary
metabolism are separated, but seems irrelevant for B. subtilis in the light of our results. If regulatory aspects are
not crucial, then one could expect that PPTases of the AcpS type should
gradually get lost from strains also harboring an Sfp type enzyme, as
we have experimentally simulated with the acps deletion
mutant of B. subtilis. In fact, such strains can be
encountered in the pool of microorganisms whose complete genome
sequence has been determined. The entire genome of the Gram-negative
Pseudomonas aeruginosa, for example, obviously contains only
one PPTase encoding gene, whose gene product belongs to the Sfp type
(242 aa, 12% identical to Sfp, accession number AAG04554). The
Pseudomonas genome is rich in ACPs and NRPSs. The
cyanobacterium Synochocystis PCC6803 also seems to lack an
enzyme of the AcpS type but contains one of the Sfp type (246 aa, 21%
identical to Sfp, accession number 1001183). Strikingly, however, this
organism is obviously devoid of any secondary metabolism genes encoding
PKSs or NRPSs, since we could only detect the homologue to ACP. The
genome of Bacillus halodurans reveals, similar to B. subtilis, one PPTase gene of each group, encoding an AcpS and a
Sfp homologue (119 and 214 aa, accession numbers BAB04327 and BAB05571,
48 and 21% identical to AcpS and Sfp, respectively), but again, the
fatty acid ACP seems to be the only acyl carrier protein present.
We note that the wide distribution and the ability of PPTases of the
Sfp type to complement the essential function of AcpS in
vivo may pose significant problems in approaches to direct new
inhibitors against AcpS, which was proposed as an attractive antimicrobial target (9, 10). Therefore, a potential inhibitor would
need to be of broad enough specificity toward both types of PPTases.
The role of AcpK in B. subtilis is unknown, but a function
in polyketide assembly can be deduced from its location within the
large pksX cluster (see Fig. 5). There are striking
similarities between the pksX cluster, which is completely
uncharacterized except for its sequence, and the reported parts of the
cluster responsible for the biosynthesis of the antibiotic TA in
M. xanthus (26, 36). Not only does AcpK display the highest
similarities to the two ACPs, TaB and TaE, but also PksG is highly
similar to TaC and TaF, all of which are predicted from sequence
analysis to encode 3-hydroxy-3-methyl-glutaryl-CoA synthases.
Furthermore, the enzyme PksK, a mixed NRPS/PKS, has an identical domain
organization (T-C-A-T-PKS) as the fragment that is known from the
enzyme TA1 (-C-A-T-PKS; the N terminus has not yet been determined)
(26). We found that the signature sequences of the A-domains of both NRPS enzymes are almost identical and are predicted to confer glycine
specificity (37), which would fit well with the glycine residue found
in the antibiotic TA. If this analogy extends further, one could
speculate that the starter in the biosynthesis of the pksX
product is the same as in antibiotic TA (26). At present, however, it
cannot be ruled out that AcpK is the donor of the fatty acid for the
biosynthesis of surfactin and fengycin. Another candidate for this role
would be the fatty acid ACP itself, since the fatty acid moiety of
these latter antibiotics is an intermediate in fatty acid biosynthesis.
In conclusion, the many acyl carrier proteins in B. subtilis
are converted into their active holo form by only two PPTases, AcpS and Sfp. Obviously, in this Gram-positive bacterium, a strict separation of the different biosynthetic pathways on the level of Ppant
transfer, as reported for E. coli, is not necessary. AcpS is
not essential for cell survival, because Sfp of secondary metabolism
can complement its function in primary metabolism.
We thank Torsten Stachelhaus for
critical comments on the manuscript and Mohammad R. Mofid for
discussions. We thank Mary Berlyn from the E. coli Genetic
Stock Center for providing strains, Wolfgang Klein for providing strain
HSK42, Donald L. Court for providing strain HT253, Christopher T. Walsh
for providing plasmid pET22b-acps, and Philippe Marlière for
providing plasmid pUC18-nrdD.
*
Work in the laboratory of M. A. M. was supported by
Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF260727.
§
These authors contributed equally to this work.
¶
To whom correspondence should be addressed:
Philipps-Universität Marburg, Fachbereich Chemie/Biochemie,
Hans-Meerwein-Str., D-35032 Marburg, Germany. Tel.:
49-6421-2825722; Fax: 49-6421-2822191; E-mail:
Marahiel@chemie.uni-marburg.de.
Published, JBC Papers in Press, August 6, 2001, DOI 10.1074/jbc.M103556200
The abbreviations used are:
Ppant, 4'-phosphopantetheine;
ACP, acyl carrier protein;
AcpK, putative acyl
carrier protein localized in the pksX cluster;
AcpS, acyl
carrier protein synthase;
CoA, coenzyme A;
DCP, D-alanyl
carrier protein;
FAS, fatty acid synthase;
NRPS, nonribosomal peptide
synthetase;
PCP, peptidyl carrier protein;
PCR, polymerase chain
reaction;
PKS, polyketide synthase;
PPTase, 4'-phosphopantetheine
transferase;
Sfp, PPTase involved in surfactin production;
aa, amino acid(s);
bp, base pair(s);
kb, kilobase pair(s);
HPLC, high pressure
liquid chromatography;
MES, 4-morpholineethanesulfonic acid.
4'-Phosphopantetheine Transfer in Primary and Secondary
Metabolism of Bacillus subtilis*
§,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activity of PPTases. PPTases catalyze
the posttranslational transfer of the 4'-phosphopantetheine moiety of
CoA onto a conserved serine residue within ACPs or PCPs. Thereby, the
acyl carrier protein is converted from its inactive apo form into the
active holo form. The reaction is dependent on Mg2+ and
yields 3',5'-ADP as a second product.
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Fig. 2.
Genetic maps of the E. coli
and B. subtilis genomes. The genetic loci
relevant for this study are shown on the genetic maps of E. coli (A) and B. subtilis (B).
Genes encoding PPTases as well as the nrdD and
amyE loci, that were used to introduce second copies of
acps and ydcB, respectively, are marked
outside the circle. The insertion and deletion
strategy for the disruption of E. coli acps and B. subtilis ydcB is indicated above these gene loci. Genes
and gene cluster-encoding acyl carrier proteins, their numbers given in
parentheses, are shown inside the
circle.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Plasmids used in this study
-D-thiogalactopyranoside-inducible spac promotor, which is leaky in rich media according to our experience.
ydcB::spc-3'--
To clone flanking regions of
ydcB, a fragment from 992 bp upstream to 994 bp downstream
of ydcB was amplified from chromosomal DNA of B. subtilis MR168 by PCR with oligonucleotides
5'-ATAGGATCCAGCCTTCATTTTAAAGTGG-3' (primer 1 in Fig. 6) and
5'-AATTCTGCAGCAATCTGGGCTTTTTCCTG-3' (primer 2 in Fig. 6)
and cloned into the BamHI and PstI sites of
pTZ18R. The resulting plasmid pTZ18-5'-ydcB-3' then served as a
template for an inverse PCR with oligonucleotides
5'-ATAGATATCATGTATGATAACCTCC-3' and
5'-ATAGATATCCTAGTCTGCATATTAGGG-3' (introducing
EcoRV restriction sites) to replace the entire
ydcB with the spc+ cassette of
pDG1726 (16), which was excised with EcoRV and HincII, to give pTZ18R-5'-ydcB::spc-3'.
-D-thiogalactopyranoside and grown at 37 °C
and 300 rpm for 3 h. Cells were harvested by centrifugation at
4,500 × g at 4 °C, resuspended in 50 mM
Tris/HCl (pH 7.8), and disrupted by three passages through a cooled
French pressure cell. The resulting cell extract was centrifuged at
36,000 × g at 4 °C for 30 min. Ammonium sulfate was
added to the supernatant to a final concentration of 25% saturation.
The resulting protein suspension was stirred at 4 °C for 1 h
and centrifuged at 36,000 × g and 4 °C for 45 min.
Subsequently, the pellet was discarded, and ammonium sulfate was added
to the supernatant to a final concentration of 40% saturation. The
suspension was stirred again at 4 °C for 1 h and centrifuged as
described above. The resulting pellet was resuspended in 50 mM Tris/HCl, 1 M
(NH4)2SO4 (HIC buffer A, pH 7.8)
and centrifuged as described above, and the supernatant was applied to
a High-LoadTM 26/10 phenyl-Sepharose column (Amersham
Pharmacia Biotech) that had been equilibrated with HIC buffer A. The
column was washed with buffer A at a flow rate of 1 ml/min, and the
protein was eluted with a linear gradient of 1.0 to 0 M
(NH4)2SO4 in HIC buffer A; 4-ml
fractions were collected. The presence of AcpS in the fractions was
detected by SDS-polyacrylamide gel electrophoresis analysis (15%
Laemmli gels). Fractions containing AcpS were pooled and concentrated
by 40% (NH4)2SO4 precipitation as
described above. The resulting pellet was resuspended in 50 mM NaH2PO4/NaHPO4 (pH 7.0), centrifuged as described above, and applied to a
SuperdexTM G75 26/60 gel filtration column (Amersham
Pharmacia Biotech) that had been equilibrated with 50 mM
NaH2PO4/NaHPO4 (pH 7.0); 4-ml
fractions were collected. Fractions containing AcpS were collected,
pooled, and applied to a 6-ml ResourceTM 15 S column
(Amersham Pharmacia Biotech) that had been equilibrated with 50 mM NaH2PO4/NaHPO4 (CAT
buffer A, pH 7.0). The column was washed with CAT buffer A, and the
protein was eluted with a linear gradient of 0-0.5 M NaCl
in CAT buffer A while collecting 2-ml fractions. The AcpS-containing
fractions were collected and applied to a SuperdexTM G75
26/60 gel filtration column that had been equilibrated with 50 mM Tris/HCl, 2 mM dithiothreitol (assay buffer,
pH 8.8). Fractions containing AcpS were collected, analyzed by
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry, adjusted with glycerol to a final concentration of 10%
(v/v), and stored in small aliquots at 
80 °C.
80 °C. TycC3-PCP, hereafter referred to as
PCP, and Sfp-His6 were produced and purified as previously
described (8, 19). Protein concentrations were determined based on the
calculated extinction coefficient at 280 nm: AcpS, 6,520 M
1 cm1; ACP-His6,
1,280 M1 cm1;
DCP-His6, 5,810 M1
cm1; AcpK-His6, 1,400 M1 cm1.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strains used in this study
1 (Fig. 3A), and the second
Km was determined as 68 ± 11 µM
with a kcat of 125 ± 9 min1
for apo-ACP concentrations between 20 and 200 µM (Fig.
3B). The Km of AcpS for CoA was
determined in essentially the same fashion, except that the apo-ACP
concentration was kept constant at 200 µM, while the CoA
concentration was varied between 5 and 500 µM. The
Michaelis-Menten fit of the experimental data set yielded a
Km of 5.4 ± 1.5 µM and a
kcat of 109 ± 5 min1 (Fig.
3C).
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Fig. 3.
Determination of kinetic constants of
B. subtilis Acps for its substrates apo-ACP and CoA
and of Sfp for apo-ACP and apo-AcpK. Reaction mixtures were
incubated for 10 min in the case of AcpS (5.6 nM) and 30 min in the case of Sfp (10 nM). For the fit of the kinetic
data, a hyperbolic Michaelis-Menten function was used. The kinetic
constants toward the carrier proteins are summarized in Table III.
A, plot of velocity of AcpS against apo-ACP concentration
between 2 and 8 µM; the CoA concentration was held
constant at 1 mM. B, when the apo-ACP
concentration was further increased beyond 20 µM, a
second Km value of AcpS for apo-ACP could be
determined. C, the Km value of AcpS for
CoA was determined by varying the CoA concentration between 5 and 500 µM with the apo-ACP concentration being constant at 200 µM. Shown are kinetic data for Sfp with apo-ACP
concentrations between 2 and 15 µM (D) and
between 20 and 150 µM (E) apo-ACP.
F, Km values for Sfp with apo-AcpK were
measured between 2 and 60 µM apo-AcpK.
Kinetic constants of B. subtilis AcpS and Sfp toward ACP and AcpK
1 were
determined. For ACP concentrations from 20 to 150 µM, the Km was found to be 38 ± 8 µM
with a kcat of 12.5 ± 1.0 min1 (see Fig. 3, D and E, and
Table III). The values for low ACP concentrations are in good agreement
with those determined for the interaction of Sfp with E. coli ACP (Km of 6 µM and
kcat of 5.8 min1) (5).
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Fig. 4.
Protein partners of AcpS and Sfp. The
different acyl carrier proteins (6 µM) of primary and
secondary metabolism of B. subtilis were incubated with
[3H]CoA and AcpS (0.22 µM) or Sfp (0.8 µM). Shown is the modification in percent of the
respective acyl carrier protein by AcpS (white
column) and Sfp (black column) in a
qualitative analysis after 30 min of reaction time. Sfp recognizes all
acyl carrier proteins tested, whereas AcpS only modifies ACP and DCP of
primary metabolism and not AcpK and PCP of secondary metabolism.
1 (see Fig. 3F and Table III).
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Fig. 5.
Identification of a second acyl carrier
protein in B. subtilis, designated AcpK.
A, localization of the acpK gene in the
pksX cluster of B. subtilis. Putative promotors
and termination loops are indicated as suggested by Ref. 22.
B, alignment of AcpK with the acyl carrier proteins TaB and
TaE involved in synthesis of the antibiotic TA and with the acyl
carrier proteins involved in fatty acid synthesis of E. coli
and B. subtilis. The asterisks mark the residues
that provide contacts between AcpS and ACP of B. subtilis
(9).
ydcB::spc-3' as template; see Fig.
6B). By using the PCR product,
only a double crossover recombination, and thus a deletion of
ydcB, can lead to Spr transformants. B. subtilis MR168, JH642, and Mo1099, which are all
sfp0, were transformed with this PCR product for
comparison. As expected, Spr transformants were only
obtained using HM0451, and one of these was named HM0492 (see Fig. 6
for confirmation of genotype by PCR), but not for MR168, JH642, and
Mo1099. In control experiments using the circular plasmid
pTZ18-5'-ydcB::spc-3' for the transformation, high
numbers of Spr transformants were observed for all strains.
However, PCR analysis of several of these transformants confirmed that
they all resulted only from a single crossover event, leaving the
ydcB gene intact (see Fig. 6). Importantly, the successful
deletion of ydcB in strain HM0492 showed that an eventual
polar effect on the downstream gene ydcC is not lethal.
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Fig. 6.
Deletion of the ydcB gene
encoding AcpS in B. subtilis. A,
chromosomal organization around the ydcB gene in wild-type
B. subtilis OKB105. A spectinomycin resistance cassette
(spc+) was cloned between the 5'- and
3'-flanking regions of the ydcB gene (plasmid
pTZ18-5'- ydcB::spc-3'). This plasmid served as a template
to amplify the PCR fragment shown using oligonucleotides 3 and 4, which
was then used for transformation. Double crossover recombination
resulted in deletion of the ydcB gene from the chromosome
(B). Selection of SpR transformants yielded
strain HM0489 (ydcB::spc+)
(C). D and E show the PCR analysis
from chromosomal DNA of HM0489 and various controls: HM0489
(lane 1); HM0490 (a second ydcB
deletion strain isolated) (lane 2); HM0491,
resulting from transformation of OKB105 with circular plasmid
pTZ18-5'-ydcB::spc-3', which integrated with a single
crossover recombination (lane 3); OKB105
(lane 4); HM0492, carrying a second copy of
ydcB in the amyE site (compare Fig. 2)
(lane 5); and plasmid
pTZ18-5'-ydcB::spc-3' (lane 6).
D shows the PCR analysis using oligonucleotides 3 and 4. The
ydcB wild-type locus yields a 2.1-kb PCR amplificate, and
the ydcB::spc+ locus gives a 2.9-kb
fragment. E shows the PCR analysis using oligonucleotides 1 and 2. The intact ydcB gene yields a 0.4-kb PCR fragment,
and the ydcB::spc+ locus results in
amplification of the 1.2-kb spectinomycin cassette.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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1) is
comparable with the Km of E. coli AcpS
for its natural substrate (Km = 0.5 µM; kcat = 68 min1)
(30). At higher ACP concentration, the first saturation was overcome,
and the second Km of 68 ± 11 µM
(kcat = 125 ± 9 min1) was
determined. This behavior may also point to a positive cooperativity in
binding apo-ACP. The co-crystal structures of AcpS with apo-ACP and CoA
suggested that AcpS binds first its co-substrate CoA and then forms the
catalytic complex with apo-ACP (9). It seems reasonable in this light
that we find a Km for CoA of 5.4 ± 1.5 µM that is lower than the (second) Km
for apo-ACP. However, further work will be necessary to understand the
mechanistic details and the reported differences between the two AcpS
enzymes from Gram-positive organisms and that of the Gram-negative
E. coli. Interestingly, also with the PPTase Sfp, two
similar saturation concentrations of apo-ACP were found. Apo-AcpK
displayed normal Michaelis-Menten kinetics as reported for Sfp with
other (heterologous) ACPs and PCPs (5).
strains MR168 and Mo1099. Thus, Sfp can function as a redundant ACP
synthase in B. subtilis.
ACKNOWLEDGEMENTS
FOOTNOTES
A Ph.D. fellow of Stiftung Stipendien-Fonds des Verbandes der
Chemischen Industrie.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
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