From the Department of Chemistry, Philipps-Universität Marburg, D-35032 Marburg, Germany
Received for publication, October 6, 2000, and in revised form, December 6, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacillus subtilis was reported
to produce the catecholic siderophore itoic acid (2,3-dihydroxybenzoate
(DHB)-glycine) in response to iron deprivation. However, by inspecting
the DNA sequences of the genes dhbE, dhbB, and
dhbF as annotated by the B. subtilis genome
project to encode the synthetase complex for the siderophore assembly, various sequence errors within the dhbF gene were
predicted and confirmed by re-sequencing. According to the corrected
sequence, dhbF encodes a dimodular instead of a monomodular
nonribosomal peptide synthetase. We have heterologously expressed,
purified, and assayed the substrate selectivity of the recombinant
proteins DhbB, DhbE, and DhbF. DhbE, a stand-alone adenylation domain
of 59.9 kDa, activates, in an ATP-dependent reaction, DHB,
which is subsequently transferred to the free thiol group of the
cofactor phosphopantetheine of the bifunctional isochorismate
lyase/aryl carrier protein DhbB. The third synthetase, DhbF, is a
dimodular nonribosomal peptide synthetase of 264 kDa that specifically
adenylates threonine and, to a lesser extent, glycine and that
covalently loads both amino acids onto their corresponding peptidyl
carrier domains. To functionally link the dhb gene cluster
to siderophore synthesis, we have disrupted the dhbF gene.
Comparative mass spectrometric analysis of culture extracts from both
the wild type and the dhbF mutant led to the identification
of a mass peak at m/z 881 ([M-H]1 Iron is an essential trace element. In organisms, most iron is
bound intracellularly in heme, iron storage compounds such as ferritin,
or iron-sulfur compounds. It can also be complexed to extracellular
carrier glycoproteins in the form of transferrin in serum or
lactoferrin in mussel surfaces (1-3). Under certain conditions, the
level of physiologically available iron in terrestrial as well as in
aquatic environments can drop by oxidation of ferrous to ferric iron
(2, 4). This leads to concentrations far below 1 µM iron
and becomes growth-limiting for bacteria. To survive, many bacteria
evolved specialized transport systems to retract ferric iron ions by
utilizing low molecular mass iron-chelating compounds termed
siderophores, which also play a crucial role in successful infection of
pathogens in their host (5).
The most common siderophores can be classified traditionally into
two major groups, catecholic and hydroxamate siderophores, which have
recently been extended to a third main class of carboxylate siderophores. Other important subgroups are heterocyclic and mixed-type siderophores (6). Many of the siderophores are produced nonribosomally by large multidomain enzymes termed nonribosomal peptide synthetases (NRPS)1 that can assemble
peptides of wide structural diversity and broad biological activity
(7-9). NRPS have a distinct modular structure in which each module is
responsible for the recognition, activation, and, in some cases,
modification of a single amino acid residue of the final peptide
product. The modules are aligned in a sequence that is collinear with
the sequence of the product (10) and can be subdivided into domains
that catalyze specific biochemical reactions (7, 8). The adenylation
(A) domain recognizes a specific substrate (amino or hydroxy acid) and
activates it as (amino) acyl adenylate by hydrolysis of ATP. The
activated acyl moiety is then covalently thioesterified to the
enzyme-bound cofactor 4'-phosphopantetheine of a peptidyl carrier
protein domain (11, 12). This reaction is followed by the direct
transfer of the aminoacyl-S-Ppant-PCP intermediate onto the adjacent
downstream module, a peptide bond formation mediated by the
condensation (C) domain (13). A terminal thioesterase (Te) attached to
the C-terminal module releases the peptide from the enzyme by
cyclization or hydrolysis. For post-translational modification of all
PCP domains in NRPS, a dedicated 4'-phosphopantetheinyltransferase (Ppant-transferase) is needed. It converts all PCP domains from their
inactive apo form to the active holo form by covalently linking a
phosphopantetheine moiety from coenzyme A to a highly conserved serine
residue (14). The crystal structure of Sfp, a Ppant-transferase
associated with the biosynthesis of the lipopeptide surfactin, has
recently been solved (15, 16), as well as the NMR structure of a
prototype PCP domain (17).
In the case of the catechol-type siderophore enterobactin from
Escherichia coli, biochemical data proved the current model of the NRPS assembly line mechanism. The activation of the first hydroxy acid, 2,3-dihydroxybenzoate (DHB), is catalyzed by the aminoacyl-CoA ligase EntE (18) transferring the activated aryl moiety
onto the aryl carrier protein (ArCP) encoded by entB (19, 20). Amide bond formation with serine is catalyzed by EntF, a
four-domain protein exhibiting the domain organization C-A-PCP-Te (see
Fig. 1C) (21, 22). The Te domain has been shown to be responsible for trimerization and release of the trilactone
enterobactin ((DHB-Ser)3) (23).
The only catecholic siderophore that has been described so far in
Bacillus subtilis is the itoic acid (DHB-glycine) encoded by
the dhb operon (24, 25). Transcription of the dhb
operon was found to be controlled by a single
In this study, we re-sequenced the dhbF gene from several
B. subtilis strains, including B. subtilis genome
project strain 168, and have identified several sequence errors within
the dhbF gene. We expressed the three NRPS genes
dhbB, dhbE, and dhbF and purified and
characterized the synthetases DhbB, DhbE, and DhbF in vitro.
By comparing the culture broth extracts of a dhbF knockout mutant and several B. subtilis strains, the siderophore
could be identified and characterized.
PCR Amplification, Cloning, and Sequencing--
PCR
amplifications were carried out using the Expand Long Range PCR system
(Roche Molecular Biochemicals, Mannheim, Germany) in accordance with
the manufacturer's protocol. Chromosomal DNA of B. subtilis
ATCC 21332 that had been prepared using a
DNA preparation kit (QIAGEN, Hilden, Germany) was used as a template.
Primers (listed in Table I) were purchased from MWG
Biotech (Ebersberg, Germany) and used for the generation of terminal
restriction sites for subsequent cloning. All PCR products were
purified and cloned into the pQE vector system (QIAGEN) using standard
techniques (27). After ligation and transformation in E. coli XL1-Blue, plasmids were prepared by the method of Birnboim
and Doly (28) and checked by restriction digest. For the heterologous
expression of cloned gene fragments, plasmids were transformed in
E. coli. Strain M15(pREP4) was used to isolate apo-PCP
proteins, whereas BL21(pREP4-gsp) produces holo-PCP proteins. The
strains used in this study are listed in Table II (13,
29-36). DNA fragments and plasmid inserts were sequenced using the
chain termination method (37).
Overproduction and Purification of Recombinant
Proteins--
Expression of dhb gene fragments was carried
out under aerobic conditions in 2× yeast tryptone medium
supplemented with 10 mM MgCl2. E. coli cells were grown at 30 °C until
A600 = 0.7. Subsequently,
isopropyl- Growth Conditions for Siderophore Extraction--
To test
various B. subtilis strains under iron deprivation, cells
were grown in Spitzien's minimal medium (39) supplemented with
0.2 (w/v) casamino acids and 0.5% (w/v) glucose. Iron was added at
various concentrations (0.1-1000 µM) from a freshly
prepared solution of FeCl3, and 10 ml of cells were
incubated at 250 rpm for 48 h at 37 °C in 50-ml polyethylene
tubes. To avoid cross-contamination with iron, all glassware was rinsed
with concentrated HCl, and solutions were stored in bottles made of
polycarbonate or polyethylene.
Siderophore Extraction--
For siderophore extraction, 200 ml
of cells from B. subtilis strains ATCC 21332, 168, and JH642
were cultured for 48 h at 37 °C in Spitzien's minimal medium
supplemented with 50 µM FeCl3. After
centrifugation (10,000 rpm for 20 min), the cultured broth was
extracted three times with equal volumes of ethyl acetate. Subsequently, the volume of the pooled organic extracts was reduced to
~100 ml in a rotary evaporator at 37 °C, and the remainder was
washed two times with 0.1 M sodium citrate buffer (pH 5.5) and water, respectively. The organic layers were dried, and the pellets
were resuspended in a small volume (200 µl) of methanol. The
resulting suspensions were cleared by centrifugation (21 °C at
13,000 rpm for 5 min), and the supernatants were further analyzed.
Detection and Analysis of the Siderophore by Ferric
Hexadecyltrimethylammonium Bromide-Chrom-Azurol-S (CAS) Assay
and Electrospray Ionization Mass Spectrometry (ESI-MS)--
For
the detection of siderophore-producing B. subtilis strains,
organic extracts of their cultured broth were applied to a CAS solution
assay as described by Schwyn and Neilands (40). Additionally, the
B. subtilis strains were stroked out on CAS plates
and tested for growth and the ability to breakdown the CAS complex. The
corresponding plates were prepared as described (40) and supplemented
with 0.2% (w/v) casamino acids.
Extracts of siderophore-producing strains were further analyzed by
ESI-MS on a Hewlett-Packard 1100 series mass spectrometric detector. 5-µl samples were flow-injected through an
autosampler at 0.3 ml/min. Scans were taken in negative-ion mode
over the m/z range of 100-1200 at a capillary voltage of
5000 V, a fragmenter voltage of 240 V, drying gas (N2) at
250 °C, and a flow rate of 8 liters/min. A solution of 100%
acetonitrile and 0.04% (v/v) formic acid was used as carrier solvent
for the detection of the peak at m/z 881 ([M-H] ATP-PPi Exchange Assay for DhbE and DhbF Substrate
Selectivity--
The ATP-PPi exchange reaction was
performed as described (41), and reaction mixtures (100-µl final
volume) normally contained 2 mM amino acid, 2 mM ATP, and 500 nM enzyme in assay buffer (50 mM HEPES (pH 7.8) and 20 mM MgCl2).
Reactions were initiated by the addition of 0.15 µCi of sodium
[32P]pyrophosphate (PerkinElmer Life Sciences) and 0.1 mM PPi at pH 7.8. ATP-PPi exchange
reactions with DhbF1-A-PCP were carried out at a slightly higher pH
value of 8.8. For DhbE, the amino acid concentration had to be lowered
to 0.5 mM to avoid nonspecific reactions. For the
determination of kinetic parameters, amino acid concentrations were
varied between 1 µM and 5 mM.
Post-translational Modification Assay of the ArCP Domain of DhbB
with [3H]Coenzyme A--
The apo-to-holo form conversion
was carried out as described by Lambalot and Walsh (42). A
typical reaction mixture (100 µl) contained 25 pmol of
[3H]coenzyme A, 225 pmol of coenzyme A, 25 pmol of DhbB,
and 10 pmol of Sfp in CoA assay buffer (14). Proteins were precipitated by the addition of 800 µl of 10% (v/v) trichloroacetic acid,
pelleted and dissolved in 200 µl of formic acid, and analyzed by
liquid scintillation counting (Packard TriCarb Model 2300TR).
Analysis of Covalent 14C-Salicylation of Holo-DhbB by
DhbE--
To test for covalent aminoacylation of holo-DhbB, 50 pmol of
carrier protein were incubated with 10 pmol of DhbE, 2 nmol of [14C]salicylate, 1 mM ATP, and 10 mM MgCl2 in a total volume of 100 µl.
Reactions were incubated for 15 min at 37 °C under moderate shaking
and quenched by the addition of 800 µl of 10% trichloroacetic acid.
The trichloroacetic acid precipitate was washed once with 1 ml of 10%
trichloroacetic acid and solubilized in 200 µl of formic acid. The
acid-stable label was quantified by liquid scintillation counting.
Knockout Mutant--
For generation of a dhb knockout
mutant, an integration plasmid was generated by replacement of the
coding region of the second A-PCP module in dhbF by a
kanamycin (kan) resistance cassette in plasmid pJJM305
(Table II). For this purpose, the
kan gene was amplified from pDG782 (36) using primers
Kan_EagI and Kan_ApaI. The kan
gene fragment was cut with EagI and ApaI and
cloned into EagI/ApaI-digested pJJM305, resulting
in plasmid pJJM201.
Subsequently, a lincomycin/erythromycin resistance-conferring
gene (erm) (36) was cloned at the 3'-end of dhbF
in pJJM201 to facilitate a differentiation between single and double
crossover events after integration into the B. subtilis
chromosome. The erm cassette was prepared by cutting pDG780
(36) with BamHI, and it was cloned into
BamHI-digested pJJM201. The resulting plasmid (pJJM202) was
used to disrupt the dhbF gene in B. subtilis ATCC 21332 (srfA+) following the protocol of Klein
et al. (44). The integration was checked by Southern blot
analysis (45) using the PCR fragment of dhbF2-A-PCP and the
kan gene as probes (data not shown).
Sequencing of the dhb locus in Different B. subtilis Strains and
Sequence Analysis--
We first thought that the truncated sequence of
dhbF is the reason for the failure of some B. subtilis strains to produce a siderophore. Analysis of the
neighboring sequences in B. subtilis strain 168 (46)
prompted us to assure that two other reading frames, i.e.
yukL and yukM (Fig.
1A), resulted from sequencing errors and might originally be part of a larger dhbF
gene.
We therefore sequenced this region from different Bacillus
strains. Despite a minor sequence divergence, we found three major sequence errors; these critical regions of the published sequence of
B. subtilis strain 168 are aligned in Fig.
2 against our sequence data. For the
first error region, we found that one thymidine base was missing at
position 3828 (the ATG start codon of dhbF refers to
position 1), whereas two adenosine base insertions were annotated at
positions 3831 and 3834. This leads to a premature TGA stop codon of
dhbF at position 3838. The second error consists of a
missing thymidine at position 3921, which leads to a stop codon (TGA)
at position 3927. The third error is a C
The corrected sequence results in one larger gene (7146 base pairs)
(Fig. 1B) that comprises the entire former dhbF,
yukL, and yukM genes as well as the
intergenic regions and that encodes a dimodular peptide synthetase of
2378 amino acids (264 kDa). We propose to maintain the designation
dhbF for this gene. The sequence has been deposited in the
GenBankTM/EBI Data Bank under accession number AF184977. From of this
corrected sequence, we postulate a new dhb operon
organization. The genes involved in siderophore biosynthesis would now
be more similar to those of the ent cluster from E. coli, as shown in Fig. 1 (B and C). dhbE and dhbB are similar to entE and
entB, respectively; but dhbF now encodes a
dimodular peptide synthetase with two modules composed of C, A, and PCP
domains and a terminal Te domain.
A BLAST search (47) with DhbE revealed the greatest similarity to the
stand-alone DHB-AMP ligases EntE from E. coli (47% identity), VibE from Vibrio cholerae (68% identity), PchD
from Pseudomonas aeruginosa (52% identity), and YbtE from
Yersinia pestis (49% identity). DhbB shows the greatest
similarity to the bifunctional isochorismate lyase/ArCP EntB from
E. coli (48% identity). This similarity is also extended to
other ArCP domain-containing proteins such as VibB from V. cholerae (46% identity), PchE from P. aeruginosa (38%
identity), and VenB from Vibrio vulnificus (45% identity).
DhbF shares the highest similarity with the dimodular actinomycin
synthetase II from Streptomyces chrysomallus (45% identity)
and the two last modules of the tetramodular pristinamycin I
synthetase III from Streptomyces pristinae
spiralis (41% identity).
Cloning and Expression of dhbB, dhbE, and dhbF--
To analyze the
activity and substrate selectivity of the proteins encoded by
dhbB, dhbE, and dhbF, the genes were
amplified with the primers listed in Table I and cloned into C-terminal His6-tagged vectors pQE60 and pQE70 (see "Experimental
Procedures"). Expression of dhbE, dhbB, and
dhbF was carried out in E. coli, and all proteins
were purified as described under "Experimental Procedures" (Fig.
3). The overproduction of the 264-kDa
DhbF protein substantiates the newly determined sequence of
dhbF (Fig. 3D). The two A-PCP modules encoded by
dhbF were also cloned as single modules (DhbF1-A-PCP and
DhbF2-A-PCP) to unambiguously assign their substrate selectivity.
Substrate Selectivity of DhbE--
All proteogenic amino acids
as well as DHB and salicylate were tested as substrates for
DhbE in the ATP-PPi exchange reaction. The protein
was highly specific for DHB and salicylate, and no side
specificity was detected (Fig. 4). The
Km and kcat values of 7.6 µM and 167 min Post-translational Modification of the ArCP Domain of DhbB by Sfp
with [3H]Coenzyme A and Covalent Thioesterification of
Holo-DhbB by DhbE--
We assumed that DhbB contains the ArCP domain,
which covalently binds the activated DHB moiety as a thioester. We
first checked the ability of the recombinant apo-DhbB isolated from
E. coli BL21(pREP4) to be phosphopantetheinylated by the
Ppant-transferase Sfp. In fact, Sfp stoichiometrically modified
apo-DhbB to holo-DhbB (Fig.
5A). The activity was measured
by incorporation of [3H]CoASH. In the control experiment
without Sfp, no phosphopantetheine was incorporated. By this approach,
we proved that DhbB contains an ArCP domain and that it is recognized
by the Ppant-transferase Sfp. Using holo-DhbB isolated from E. coli BL21(pREP4-gsp), we assayed the transfer of
[14C]salicylate from DhbE to DhbB (radiolabeled DHB is
not commercially available). Fig. 5B shows that 98% of DhbB
could be acylated by DhbE after 10 min as judged by the thioester
binding assay and scintillation counting of the trichloroacetic
acid-precipitable material. Thus, using recombinant DhbE and holo-DhbB,
the initial reactions of the Dhb synthetases could be
reconstituted.
Biochemical Analysis of DhbF--
The entire dimodular NRPS DhbF
was produced as a 264-kDa protein, supporting the revised
dhbF sequence (Fig. 3D). The separate DhbF
modules DhbF1-A-PCP and DhbF2-A-PCP were also produced as individual
proteins and were assayed for substrate selectivity using the
ATP-PPi exchange reaction. For the DhbF1-A-PCP construct (Fig. 6), a low but specific
glycine-dependent ATP-PPi exchange was
observed. This reaction was dependent on a slightly higher pH value (pH
8.8) than the standard assay condition of pH 7.8. In contrast, for the
second module, DhbF2-A-PCP, a highly specific ATP-PPi
exchange dependent on L-threonine was observed (Fig.
7). D-Threonine was not
recognized as a substrate, whereas the stereoisomeric L-allo-Thr covered 47% activity. Upon testing
all other proteogenic amino acids, only side specificity for
phenylalanine (9%) could be observed. The activation pattern of
full-length DhbF in the ATP-PPi exchange assay was, as
expected, a sum of the activities from the single domains (data not
shown).
dhbF Disruption Mutant--
To identify the siderophore and to
correlate the dhb gene cluster with its synthesis, we
generated a dhbF disruption mutant from B. subtilis ATCC 21332 (see "Experimental Procedures"). The integration and the double crossover event in the resulting B. subtilis strain, JJM405, were proved by Southern blot analysis (data not shown). JJM405 was subsequently investigated for siderophore production.
Cross-reaction of Surfactin in the CAS Assay--
As described
above, most of the known siderophores are able to retract ferric ions
from the CAS complex, a process that is monitored by the change in
color of agar or solution from blue to orange (see "Experimental
Procedures"). First, we tested the growth and color change of
different B. subtilis strains on CAS plates. The B. subtilis strains 168 and JH642 showed no halo. The failure of
these strains to produce a siderophore can be attributed to a defective
sfp gene. Surprisingly, not only the surfactin producer
B. subtilis ATCC 21332, but also the
In a second attempt, we searched for an efficient culture broth
siderophore extraction method to analyze putative siderophores by
ESI-MS. To avoid any surfactin cross-reaction with our CAS assay,
we used the surfactin non-producer B. subtilis strain
6051 and found ethyl acetate suitable for extraction of the putative siderophores.
Mass Spectrometric Analysis of the Siderophore--
As shown in
Fig. 8 (50), ESI-MS in negative-ion mode allowed us to
identify the mass of the siderophore by
comparing the culture broth extracts from the
Variations of the fragmenter voltage and carrier solvent
induced the following fragmentation patterns (data not shown). (i) m/z 745.2 can be attributed to a single loss of DHB at the
amide bond ([M-DHB]1-), i.e.
[C32H37N6O15]1
Summarizing these mass spectrometric data and the biochemistry of the
DhbEBF synthetases, we deduce the siderophore to be a trilactone
((DHB-Gly-Thr)3). In analogy with the enterobactin biosynthesis, we suggest that the function of the terminal thioesterase domain (DhbF-Te) is to catalyze the intermolecular condensation of
three DHB-Gly-Thr units and to release bacillibactin by intramolecular condensation (shown in Fig. 9).
In this report, we show that the Gram-positive bacterium B. subtilis produces the catecholic siderophore bacillibactin, which is structurally related to enterobactin isolated from the Gram-negative bacterium E. coli. Both siderophores contain three
2,3-dihydroxybenzoate moieties for octahedral iron complexation that
are coupled to a cyclic amino acid core synthesized by multimodular
nonribosomal peptide synthetases.
The three genes entE, entB, and entF
encoding the EntEBF synthetase complex for enterobactin assembly in
E. coli were initially used to identify putative homologs
named dhbE, dhbB, and dhbF in B. subtilis. We revised the erroneous dhbF sequence and
characterized DhbF as dimodular NRPS in contrast to the monomodular
EntF synthetase.
Although the three dhb genes encoding the synthetases for
the assembly of bacillibactin reside in one operon, this is not the
case for the enterobactin synthetase genes (Fig. 1), where entF is located ~7 kilobases upstream of the
entCEBA operon. The homologous vibriobactin synthetase genes
are furthermore divided into two gene clusters (51, 52) separated by 1 megabase on the larger of the two chromosomes of V. cholerae
(53). Nevertheless, the dhb genes and homologs support par
excellence the idea of horizontal gene transfer between Gram-negative
and Gram-positive bacteria and genetic rearrangement of NRPS, leading
to a variety of structurally related siderophores in different
organisms (5). Although the sequence of the DhbEBF modules is collinear
to the sequence of the product, some domains need further attention.
The first step in nonribosomal peptide synthesis is the adenylation of
the cognate substrate (amino) acid. Determination of the substrate
selectivity of the catalyzing A domains can be accomplished either by
the ATP-PPi exchange assay or by analysis of the
selectivity-conferring residues as guided by the nonribosomal code of A
domains from NRPS (41). In the case of DhbEF, both studies
independently led to the determination of DHB, Gly, and Thr selectivity
for DhbE, DhbF1, and DhbF2, respectively. Intriguingly, regarding activation of DHB and salicylate by DhbE, the selectivity code cannot
distinguish between both aryl substrates (Table
IV) (41), although the various
adenylating enzymes (EntE, VibE, and YbtE) (see Table III) reveal a
clear preference for one or the other substrate. Likewise, we were able
to show that DhbE is selective for DHB, activating salicylate with a
6-fold lower catalytic efficiency. The same kind of analysis revealed
Gly and Thr selectivity for the first and second modules of DhbF,
respectively. The latter studies were performed with the constructs
DhbF1-A-PCP and DhbF2-A-PCP, although we could produce the dimodular
full-length protein DhbF. Unfortunately, none of the recombinant
constructs allowed a precise determination of kinetic constants because
each of them was unstable, losing activity within <12 h after
purification. Nonetheless, we were able to biochemically prove (to our
knowledge, for the first time) a low but significant activation of the
predicted glycine substrate by DhbF1-A-PCP. However, probably due to
the intrinsic betaine character, this activation could be observed only
at slightly higher pH values. In contrast, the ATP-PPi
exchange assay with DhbF2-A-PCP revealed a distinct selectivity for the predicted substrate, L-Thr, and some nonspecific activation
of the miscognate substrates L-allo-Thr and
L-Phe (47 and 9%, respectively). These moderately differ
from the activation pattern as determined for the homologous VibF (48),
and both enzymes show a clear stereospecificity for the activation of
an L-configured amino acid (D-Thr,
2%).
) that
corresponds to a cyclic trimeric ester of
DHB-glycine-threonine.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A-dependent promoter that comprises a
fur box-binding site, an iron regulatory element (26).
According to the published data derived from B. subtilis
genome strain 168, DhbF would end within a C domain. Consequently, no
terminal Te domain that is normally fused to the C-terminal NRPS module
had been annotated, indicating a nonfunctional enzyme. This unusual
truncation of dhbF led us to take a closer look at the
neighboring sequences and the entire biosynthetic operon for this siderophore.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used in this study
-D-thiogalactopyranoside was added to a final
concentration of 0.5 mM, and the cells were cultured for an
additional 2-3 h. For E. coli strain JJM304, the protocol
was slightly modified. Cells were grown at 25 °C, and 0.2 mM isopropyl-
-D-thiogalactopyranoside was
added at A600 = 0.4. Following expression, cells
were harvested and resuspended in buffer A (50 mM HEPES and
100 mM NaCl (pH 7.8)). Cells were lysed by three passages
through a French pressure cell (1100 p.s.i.), and cell debris was
removed by centrifugation for 30 min at 17,500 rpm. Supernatants were
applied to a Ni2+-nitrilotriacetic acid column (QIAGEN) and
purified by immobilized metal ion affinity chromatography on an
Amersham Pharmacia Biotech FPLC system. Purity was analyzed by
SDS-polyacrylamide gel electrophoresis, and fractions containing the
recombinant proteins were pooled and dialyzed against buffer A. Concentration of proteins was determined by the method of Bradford
(38).
1). For the detection of fragment
ions, a carrier solvent of 50% methanol, water, and 0.04% (v/v)
formic acid; a capillary voltage of 4000 V, and varying fragmenter
voltages (up to 350 V) were used.
Bacterial strains and plasmids
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 1.
Siderophore biosynthesis gene clusters from
B. subtilis (dhb) and E. coli (ent) and their corresponding domain
organization of synthetase modules. A, the
dhb gene cluster of B. subtilis strain 168 as
annotated by the genome sequencing project (46). Arrows
indicate the positions of sequencing primers. B, the
corrected dhb gene cluster of B. subtilis strain
168. C, the ent gene cluster involved in
enterobactin biosynthesis. ICL, isochorismate lyase;
kb, kilobases.
T transition at position
4406, resulting in the end of yukM.
View larger version (49K):
[in a new window]
Fig. 2.
Sequence alignment of the three critical
dhbF regions derived from different B. subtilis strains. Sequences were obtained by direct
sequencing of PCR-amplified genes from chromosomal DNA.
Mismatches are shaded, and premature stop codons resulting
from sequence errors are boxed. Base numbering refers to the
ATG start codon of DhbF.
View larger version (47K):
[in a new window]
Fig. 3.
SDS-polyacrylamide gel electrophoresis of
recombinant apoproteins DhbB, DhbE, DhbF, DhbF1-A-PCP, and DhbF2-A-PCP
overproduced in E. coli M15(pREP4) and purified using
immobilized metal ion affinity chromatography. A,
DhbF1-A-PCP and DhbF2-A-PCP; B, DhbE; C, DhbB;
D, DhbF.
1 (Table III)
(18, 49),2
respectively, clearly indicate that DHB
is the preferred substrate. These data
are in accordance with those reported for
EntE and VibE for enterobactin and vibriobactin, respectively
(18).2
View larger version (10K):
[in a new window]
Fig. 4.
Acid-dependent
ATP-PPi exchange for DhbE. To determine the substrate
selectivity of DhbE, assays were performed with all 20 proteinogenic
amino acids plus DHB and salicylate. Only four representative amino
acids and the substrate acids DHB and salicylate (Sal) are shown. The
highest activity was set at 100%; in this case, it corresponds to 36 µM label exchanged by 500 nM DhbE with 0.5 mM DHB in 5 min.
Substrate-dependent ATP-PPi exchange catalyzed by
DhbE and homologs
View larger version (16K):
[in a new window]
Fig. 5.
Characterization of the ArCP domain of
DhbB. A, post-translational modification of 25 pmol of
apo-DhbB using 250 pmol of [3H]coenzyme A and 10 pmol of
Ppant-transferase Sfp. Maximum modification was achieved after 15 min
at 37 °C. B, aminoacylation of 50 pmol of DhbB with 10 mol of [14C]salicylate using 25 pmol of DhbE and 200 mmol
of ATP.
View larger version (11K):
[in a new window]
Fig. 6.
Amino acid-dependent
ATP-PPi exchange for DhbF1-A-PCP. DhbF1 substrate
selectivity was determined at pH 8.8. The highest activity was set at
100%.
View larger version (11K):
[in a new window]
Fig. 7.
Amino acid-dependent
ATP-PPi exchange for DhbF2-A-PCP. The activation
pattern indicates a stereospecific selectivity for L-Thr as
proposed before. L-allo-Thr showed an unexpected
high specificity, and Phe only a minor side specificity. All cognate
amino acids were tested, but only representative amino acids above
background activation are shown. The highest activity was set at
100%.
dhbF mutant JJM405 produced significant orange
halos. We expected that strain JJM405 would produce no halo and
therefore deduced from this experiment that surfactin interferes with
the CAS assay. To prove this idea, surfactin was spotted onto CAS
plates, resulting in significant orange halos. From these data, we
deduce the CAS assay to be an inadequate method for siderophore
detection when surfactin is present.
dhbF mutant JJM405 with those made from the
parental strain ATTC 21332 (see "Experimental Procedures" for
details). The mass peak at m/z 881.2 ([M-H]1
) found in the wild-type extract matches three
DHB-Gly-Thr units minus 3H2O
([C39H41N6O18]1-),
which corresponds to a cyclic trimeric ester,
(DHB-Gly-Thr)3. The calculated exact mass of
[C39H41N6O18]1
is 881.25.
View larger version (13K):
[in a new window]
Fig. 8.
Comparative mass spectrometric analysis of
culture broth extracts from B. subtilis ATCC 21332 and
dhbF (JJM405). ESI-MS in
negative-ion mode allowed the siderophore identification at
m/z 881.2 ([M-H]1
), corresponding to a
trimer of DHB-Gly-Thr units minus 3H2O. Formation of a
trilactone was further substantiated by analysis of daughter ions (data
not shown; see "Results"). The m/z series 992-1062
correspond to surfactin ([M-H]1
) differing in fatty
acid chain length (50).
,
with a calculated mass of 745.23. (ii) m/z 587.3 corresponds to (DHB-Gly-Thr)2 minus 2H2O
([M-(DHB-Gly-Thr)-H]1-), i.e.
[C26H27N4O12]1
,
with a calculated mass of 587.16. Note that no m/z 605, i.e. (DHB-Gly-Thr)2 minus 1H2O, was
found. (iii) m/z 293.1 corresponds to
(DHB-Gly-Thr)1 minus 2H2O
([M-(DHB-Gly-Thr)2-H]1
), i.e.
[C13H13N2O6]1
,
with a calculated mass of 293.08. No m/z 311, i.e. (DHB-Gly-Thr)1 minus 1H2O, was
detected. (iv) m/z 249.1 can be attributed to the
decarboxylation of (DHB-Gly-Thr)3 minus 3CO2.
The resulting three fragments have the same mass
([C12H13N2O4]1
)
with a calculated mass of 249.09.
View larger version (22K):
[in a new window]
Fig. 9.
Model for the assembly of bacillibactin.
In analogy with the enterobactin synthetase holo-EntEBF, (amino) acids
are activated and bound as acyl-S-Ppant intermediates on the
corresponding carrier proteins. Condensation results in formation of
the DHB-Gly-Thr unit on the last PCP domain. The peptide chain is then
transferred to a conserved serine residue of the terminal Te domain. Te
catalyzes the condensation of three DHB-Gly-Thr units and finally
releases bacillibactin by intramolecular cyclization (see
"Discussion" for details). ICL, isochorismate
lyase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Selectivity-conferring amino acids of adenylation domains
The second step in nonribosomal peptide biosynthesis is the transfer of the activated aminoacyl moiety from the adenylate to the thiol group of the adjacent carrier protein domain. In this study, this so-called thiolation reaction could be reconstituted in vitro for the formation of DHB-S-Ppant-DhbB, which represents the initial step in bacillibactin biosynthesis (Fig. 9) (1). Here, DhbE activates the substrate carboxyl acid DHB as DHB-O-AMP; and subsequently, the aryl moiety is transferred to holo-DhbB, yielding the DHB-S-Ppant thioester. The reaction necessarily relies on the presence of a functional HS-Ppant holo-carrier protein, which is usually generated by the action of a dedicated Ppant-transferase. We demonstrated that Sfp, the Ppant-transferase known to be associated with lipopeptide antibiotic surfactin biosynthesis (54), is capable of converting the ArCP domain of DhbB in vitro from the inactive apo form to the HS-Ppant holo form. The obvious presumption that Sfp may also represent the dedicated Ppant-transferase of the bacillibactin system is corroborated by the fact that its in vivo biosynthesis in B. subtilis inevitably depends on the presence of a functional Sfp protein. Notably, the genome project B. subtilis strain 168 carries the complete NRPS gene clusters for the biosynthesis of surfactin and bacillibactin, but has a defect in the sfp gene. Consequently, all carrier protein domains remain in their inactive apo form, and the strain produces neither of the two NRPS products.
Although DhbB could be characterized biochemically (apo-to-holo conversion and thiolation), the two carrier protein domains of DhbF were determined by sequence homology. In this context, it is worthwhile to notice that carrier proteins can be distinguished not only by the moiety they have to carry (acyl, acyl carrier protein; aryl, ArCP; and peptidyl, PCP), but also by the signature of their highly conserved Ppant-binding site. Up to now, the following signature sequences could be observed: acyl carrier proteins, (ED)LGXDSL(DAT); ArCPs, (DN)LXXXGLDSXR (43); PCPs preceding a C or Te domain, DXFFXXLGGHSLK; PCPs preceding an epimerization domain, DXFFXXLGGDSIK; and C-terminal PCPs, FF(ED)XGGNSLK. According to this classification, the carrier protein of DhbB is clearly an ArCP, whereas the two carrier proteins of DhbF belong to the first class of PCP domains. The significance of having different types of carrier protein domains has yet to be established, and they might have emerged to mediate specific protein-protein interaction with a certain Ppant-transferase or to facilitate the correct interplay with various upstream and downstream partner domains. In our opinion, the latter represents the more likely variant since, in the first scenario, one had to suppose the existence of type-specific Ppant-transferases. However, it has been shown that EntD, the Ppant-transferase associated with enterobactin biosynthesis, modifies both the ArCP domain of EntB (20) and the PCP domain of EntF (21). Likewise, Sfp has been noticed to be rather nonselective, modifying all kinds of carrier protein domains with high catalytic efficiency (54).
The last steps in nonribosomal peptide biosynthesis consist of substrate condensation, followed by product release catalyzed by the Te domain. The peptide chain is then transferred from the last PCP domain onto a Ser residue within the highly conserved core motif TE (G(HY)SXG) (10). Two ways of Te-mediated product release have been described so far: ester hydrolysis by water or nucleophilic attack by amino or hydroxyl groups of the peptide itself, leading to a cyclic lactame or lactone. In the case of enterobactin biosynthesis, mutational analysis of EntF-Te led to reduced product release and enabled Shaw-Reid et al. (23) to identify intermediate reaction products such as linear (DHB-Ser)2 and enzyme-bound species such as (DHB-Ser)-S-PCP, (DHB-Ser)1-O-Te, and (DHB-Ser)2-O-Te by ESI-MS. These data provide evidence that EntF-Te additionally catalyzes the trimerization of the DHB-Ser units prior to cyclization.
In our case, due to rapid loss of DhbF activity, we were unable to
prove these last steps for bacillibactin biosynthesis in vitro. A clue in the determination of the catalytic role of
DhbF-Te was the product analysis by ESI-MS (Fig. 8). The ion at
m/z 881.2 ([M-H]1) and daughter ions clearly
define bacillibactin as (DHB-Gly-Thr)3 trilactone. Taking
our mass spectrometric data together with the assembly line enzymology
of EntEBF, we propose the following model for bacillibactin
biosynthesis as depicted in Fig. 9. The synthetase holo-DhbEBF
activates the substrates DHB (1), Gly, and Thr, which are
bound as acyl-S-Ppant intermediates. Subsequent condensation leads to
the products DHB-Gly (2) and DHB-Gly-Thr (3) on
the corresponding PCP domains. The nucleophilic attack of the Ser
hydroxyl group of the Te domain (G(HY)SXG) leads to an
(DHB-Gly-Thr)-O-Te ester (4). The DhbF2-PCP, set free
for a second round of DHB-Gly-Thr unit synthesis. Subsequently, the Thr
hydroxyl group of 4 attacks a second (DHB-Gly-Thr)-S-PCP (3), leading to a (DHB-Gly-Thr)2-O-Te
intermediate (5). Iteration of this reaction produces the
linear (DHB-Gly-Thr)3-O-Te (6), which is
released by cyclization as trilactone (DHB-Gly-Thr)3 (7) with a corresponding mass of 882. An identical
siderophore structure was reported for corynebactin isolated from
Corynebacterium glutamicum (48), but neither the genes nor
proteins involved in the biosynthesis of this product have been reported.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to N. Kessler for excellent practical help and to T. Stachelhaus and H. Mootz for critical reading of the manuscript. Additionally, we thank Veit Bergendahl for the initial steps in solving some analytical problems.
![]() |
FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the Graduiertenkolleg "Proteinfunktion auf atomarer Ebene."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) AF184977.
To whom correspondence should be addressed. Tel.:
49-6421-282-5722; Fax: 49-6421-282-2191; E-mail:
marahiel@chemie.uni-marburg.de.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M009140200
2 Keating, T. A., Marshall, C. G., and Walsh, C. T. (2000) Biochemistry 39, 15522-15530.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NRPS, nonribosomal peptide synthetase(s); A domain, adenylation domain; C domain, condensation domain; Te domain, thioesterase domain; Ppant, 4'-phosphopantetheinyl; PCP, peptidyl carrier protein; ArCP, aryl carrier protein domain; DHB, 2,3-dihydroxybenzoate; PCR, polymerase chain reaction; CAS, ferric hexadecyltrimethylammonium bromide-Chrom-Azurol-S; ESI-MS, electrospray ionization mass spectrometry.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Griffiths, E. (1991) Biol. Met. 4, 7-13[Medline] [Order article via Infotrieve] |
2. | Guerinot, M. L. (1994) Annu. Rev. Microbiol. 48, 743-772[CrossRef][Medline] [Order article via Infotrieve] |
3. | Payne, S. M. (1993) Trends Microbiol. 1, 66-69[CrossRef][Medline] [Order article via Infotrieve] |
4. | Neilands, J. B. (1983) Adv. Inorg. Biochem. 5, 137-166[Medline] [Order article via Infotrieve] |
5. | Quadri, L. E. N. (2000) Mol. Microbiol. 37, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
6. | Winkelmann, G, C. C. J. (1998) Transition Metals in Microbial Metabolism , pp. 1-49, Harwood Academic Publishers GmbH, Amsterdam BV, The Netherlands |
7. | Marahiel, M. A., Stachelhaus, T., and Mootz, H. D. (1997) Chem. Rev. 97, 2651-2673[CrossRef][Medline] [Order article via Infotrieve] |
8. | Konz, D., and Marahiel, M. A. (1999) Chem. Biol. 6, R39-R48[CrossRef][Medline] [Order article via Infotrieve] |
9. | von Döhren, H., Keller, U., Vater, J., and Zocher, R. (1997) Chem. Rev. 97, 2675-2705[CrossRef][Medline] [Order article via Infotrieve] |
10. | Marahiel, M. A. (1997) Chem. Biol. 4, 561-567[CrossRef][Medline] [Order article via Infotrieve] |
11. | Laland, S. G., and Zimmer, T. L. (1973) Essays Biochem. 9, 31-57[Medline] [Order article via Infotrieve] |
12. |
Stein, T.,
Vater, J.,
Kruft, V.,
Otto, A.,
Wittmann-Liebold, B.,
Franke, P.,
Panico, M.,
McDowell, R.,
and Morris, H. R.
(1996)
J. Biol. Chem.
271,
15428-15435 |
13. |
Stachelhaus, T.,
Mootz, H. D.,
Bergendahl, V.,
and Marahiel, M. A.
(1998)
J. Biol. Chem.
273,
22773-22781 |
14. | Lambalot, R. H., Gehring, A. M., Flugel, R. S., Zuber, P., LaCelle, M., Marahiel, M. A., Reid, R., Khosla, C., and Walsh, C. T. (1996) Chem. Biol. 3, 923-936[Medline] [Order article via Infotrieve] |
15. | Mofid, M. R., Marahiel, M. A., Ficner, R., and Reuter, K. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 1098-1100[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Reuter, K.,
Mofid, M. R.,
Marahiel, M. A.,
and Ficner, R.
(1999)
EMBO J.
18,
6823-6831 |
17. | Weber, T., Baumgartner, R., Renner, C., Marahiel, M. A., and Holak, T. A. (2000) Struct. Fold. Des. 8, 407-418[Medline] [Order article via Infotrieve] |
18. | Rusnak, F., Faraci, W. S., and Walsh, C. T. (1989) Biochemistry 28, 6827-6835[Medline] [Order article via Infotrieve] |
19. | Rusnak, F., Liu, J., Quinn, N., Berchtold, G. A., and Walsh, C. T. (1990) Biochemistry 29, 1425-1435[Medline] [Order article via Infotrieve] |
20. | Gehring, A. M., Bradley, K. A., and Walsh, C. T. (1997) Biochemistry 36, 8495-8503[CrossRef][Medline] [Order article via Infotrieve] |
21. | Gehring, A. M., Mori, I., and Walsh, C. T. (1998) Biochemistry 37, 2648-2659[CrossRef][Medline] [Order article via Infotrieve] |
22. | Rusnak, F., Sakaitani, M., Drueckhammer, D., Reichert, J., and Walsh, C. T. (1991) Biochemistry 30, 2916-2927[Medline] [Order article via Infotrieve] |
23. | Shaw-Reid, C. A., Kelleher, N. L., Losey, H. C., Gehring, A. M., Berg, C., and Walsh, C. T. (1999) Chem. Biol. 6, 385-400[CrossRef][Medline] [Order article via Infotrieve] |
24. | Ito, T., and Neilands, J. B. (1958) J. Am. Chem. Soc. 80, 4645-4647 |
25. | Ito, T. (1993) Appl. Environ. Microbiol. 59, 2343-2345[Abstract] |
26. | Rowland, B. M., and Taber, H. W. (1996) J. Bacteriol. 178, 854-861[Abstract] |
27. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
28. | Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523[Abstract] |
29. | Burkholder, P. R. (1952) Sci. Am. 40, 601-631 |
30. | Cooper, D. G., Macdonald, C. R., Duff, S. J. B., and Kosaric, N. (1981) Appl. Environ. Microbiol. 42, 408-412 |
31. | Smith, N. R., Gibson, T., Gordon, R. E., and Sneath, P. H. A. (1964) J. Gen. Microbiol. 34, 269-272[Medline] [Order article via Infotrieve] |
32. |
Hoch, J. A.,
and Mathews, J.
(1973)
Genetics
73,
215-228 |
33. | Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89[Medline] [Order article via Infotrieve] |
34. | Zamenhof, P. J., and Villarejo, M. (1972) J. Bacteriol. 110, 171-178[Medline] [Order article via Infotrieve] |
35. | Bullock, W. O., Fernandez, J. M., and Short, J. M. (1987) BioTechniques 5, 376-379 |
36. | Guerout-Fleury, A. M., Shazand, K., Frandsen, N., and Stragier, P. (1995) Gene (Amst.) 167, 335-336[CrossRef][Medline] [Order article via Infotrieve] |
37. | Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467[Abstract] |
38. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
39. | Harwood, C. R., and Cutting, S. M. (1990) Molecular Biological Methods in Bacillus subtilis , pp. 548-549, John Wiley & Sons Ltd., Chichester, United Kingdom |
40. | Schwyn, B., and Neilands, J. B. (1987) Anal. Biochem. 160, 47-56[Medline] [Order article via Infotrieve] |
41. | Stachelhaus, T., Mootz, H. D., and Marahiel, M. A. (1999) Chem. Biol. 6, 493-505[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Lambalot, R. H.,
and Walsh, C. T.
(1995)
J. Biol. Chem.
270,
24658-24661 |
43. | Gehring, A. M. (1998) Phosphopantetheinyl Transferase Catalyzed Activation of Polyketide and Nonribosomal Peptide Synthases and Deconvolution of Entrobactin and Yersiniabactin Siderophore Biosynthesis.Ph.D. Thesis , Harvard University |
44. | Klein, C., Kaletta, C., Schnell, N., and Entian, K.-D. (1992) Appl. Environ. Microbiol. 58, 132-142[Abstract] |
45. | Southern, E. M. (1975) J. Mol. Biol. 98, 503-517[Medline] [Order article via Infotrieve] |
46. | Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., Bertero, M. G., Bessieres, P., Bolotin, A., Borchert, S., Borriss, R., Boursier, L., Brans, A., Braun, M., Brignell, S. C., Bron, S., Brouillet, S., Bruschi, C. V., Caldwell, B., Capuano, V., Carter, N. M., Choi, S. K., Codani, J. J., Connerton, I. F., et al.. (1997) Nature 390, 249-256[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
48. | Budzikiewicz, H., Bössenkamp, A., Taraz, K., Pandey, A., and Meyer, J.-M. (1997) Z. Naturforsch. Sect. C Biosci. 52, 551-554 |
49. | Gehring, A. M., Mori, I., Perry, R. D., and Walsh, C. T. (1998) Biochemistry 37, 11637-11650[CrossRef][Medline] [Order article via Infotrieve] |
50. | Stachelhaus, T., Schneider, A., and Marahiel, M. A. (1995) Science 269, 69-72[Medline] [Order article via Infotrieve] |
51. | Wyckoff, E. E., Stoebner, J. A., Reed, K. E., and Payne, S. M. (1997) J. Bacteriol. 179, 7055-7062[Abstract] |
52. |
Butterton, J. R.,
Choi, M. H.,
Watnick, P. I.,
Carroll, P. A.,
and Calderwood, S. B.
(2000)
J. Bacteriol.
182,
1731-1738 |
53. |
Trucksis, M.,
Michalski, J.,
Deng, Y. K.,
and Kaper, J. B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14464-14469 |
54. | Quadri, L. E., Weinreb, P. H., Lei, M., Nakano, M. M., Zuber, P., and Walsh, C. T. (1998) Biochemistry 37, 1585-1595[CrossRef][Medline] [Order article via Infotrieve] |