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Erwinia chrysanthemi chrysobactin peptidase
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Title :
Chrysobactin-dependent iron acquisition in Erwinia chrysanthemi : functional study of an
homologue of the Escherichia coli ferric enterobactin esterase
Authors :
Lise Rauscher§, Dominique Expert§*, Berthold F. Matzanke£1, and Alfred X. Trautwein£2
From the §Laboratoire de Pathologie Végétale, UMR 217 INRA/INA P-G/Université Paris 6,
16 rue Claude Bernard, 75231 Paris cedex 05, France and the £Medical University Lübeck,
£1Institute of Physics and £2Isotope Laboratory TNF, Ratzeburger Alle160, D-23538 Lübeck,
Germany
*To whom correspondence should be addressed. Tel : 33 01 44 08 17 06 ;
Fax : 33 01 44 16 31 ; E-mail : expert@inapg.inra.fr
Running Title :
Erwinia chrysanthemi chrysobactin peptidase
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on November 1, 2001 as Manuscript M107530200 by guest on A
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Summary :
Under iron limitation, the plant pathogen E. chrysanthemi produces the catechol-type
siderophore chrysobactin that acts as a virulence factor. It can also use enterobactin as a
xenosiderophore. We began this work by sequencing the 5’ upstream region of the fct cbsCEBA
operon which encodes the ferric chrysobactin receptor and proteins involved in synthesis of the
catechol moiety. We identified a new iron-regulated gene, cbsH, transcribed divergently relative
to the fct gene, the translated sequence of which is 45.6 % identical to that of the E. coli ferric
enterobactin esterase. Insertions within this gene interrupt the chrysobactin biosynthetic
pathway by exerting a polar effect on a downstream gene with some sequence identity to the E.
coli enterobactin synthase gene. These mutations had no effect on the ability of the bacterium to
obtain iron from enterobactin, showing that a functional cbsH gene is not required for iron
removal from ferric enterobactin in E. chrysanthemi. The cbsH-negative mutants were less able
to utilise ferric chrysobactin and this effect was not caused by a defect in the transport per se. In
a non-polar cbsH-negative mutant, chrysobactin accumulated intracellularly. These defects were
rescued by the cbsH gene supplied on a plasmid. The amino acid sequence of the CbsH protein
revealed characteristics of the S9 prolyl oligopeptidase family. Ferric chrysobactin hydrolysis
was detected in cell extracts from a cbsH-positive strain, that was inhibited by diisopropyl
fluorophosphate. These data are consistent with the fact that chrysobactin is a D-lysyl-L-serine
derivative. Mössbauer spectroscopy of whole cells at various states of 57Fe chrysobactin uptake
showed that this enzyme is not required for iron removal from chrysobactin in vivo. The CbsH
protein may therefore be regarded as a peptidase preventing the bacterial cells to be
intracellularly iron-depleted by chrysobactin.
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Iron is an essential but nevertheless potentially toxic element for most living organisms.
The bioavailability of the ferric ion is extremely limited because of its poor solubility (at pH7, Ksp
of Fe(OH)3 = 10-17 M). A wide variety of microorganisms accomodate this situation by
excreting siderophores. Siderophores are high-affinity Fe(III)-scavenging-solubilizing molecules
which once loaded with iron, are specifically imported into the cell. In E. coli K-12, it has been
demonstrated that delivery of the ferric-siderophore complex into the cell implicates active
transport (1, 2). The passage through the outer membrane requires a receptor which is a pore
energized by cytoplasmic membrane-generated proton-motive force transduced by the TonB
protein. Then, the ferric complex binds to the periplasmic component of a permease belonging
to the ABC transporter family, which completes the passage to the cytosol. The fate of the
siderophore ferric complex in the cytosol is not clearly understood. As the stability constants of
siderophore ferric complexes are very high and the ferrous complexes dissociate near neutral
pH, enzymatic reduction to the ferrous state has been proposed to be a plausible mechanism for
iron removal (3, 4). Ferric siderophore reductase activity has been found in cellular extracts from
several microorganisms (5, 6, 7, 8). However, the redox potentials for hexadentate catechol
siderophores are out of the range of physiological reductants and it is assumed that ligand
degradation is required for transformation of the irreducible form of the complex into a reducible
one. In E. coli, the ester bonds of the siderophore enterobactin (enterochelin), the cyclic trimer
of 2,3-dihydroxybenzoyl-L-serine (9) (Fig.1) are hydrolysed by the ferric enterobactin esterase
encoded by the fes gene, yielding 2,3-dihydroxybenzoyl-L-serine (10, 11, 12). The redox
potential of this compound is two orders of magnitude below that of ferric enterobactin. Fes-
negative mutants fail to grow if ferric enterobactin is the only iron source (13, 14). The plant-
pathogenic enterobacterium Erwinia chrysanthemi strain 3937 provides another illustration of
this question.
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Under iron limitation, E. chrysanthemi produces the catechol siderophore chrysobactin.
This siderophore is essential for this pathogen to disseminate throughout its host plant and to
cause systemic soft-rot symptoms (15). Chrysobactin is a bidentate ligand consisting of a
monomer of 2,3-dihydroxybenzoyl-D-lysyl-L-serine (16) (Fig. 1). Ferric chrysobactin is
transported back into the cell via its specific TonB-dependent outer membrane receptor Fct (17,
18, 19) and a cytoplasmic membrane permease that is missing in a class of mutants deficient in
ferric chrysobactin uptake (20). These mutants do not acquire iron from ferric enterobactin.
Enterobactin is not synthetised by E. chrysanthemi cells but promotes growth of a chrysobactin
deficient mutant if supplied exogenously. An iron-regulated outer membrane protein with an
apparent molecular weight of 88,000 Da, immunologically related to the E. coli ferric
enterobactin receptor FepA is thought to play a similar function in E. chrysanthemi (21). In
addition, E. chrysanthemi 3937 produces another high-affinity iron-uptake system mediated by a
citrate siderophore called achromobactin (22).
Most of the proteins involved in chrysobactin-mediated iron transport are encoded by a
50-kb contiguous region of the E. chrysanthemi chromosome (20). The fctcbsCEBA operon
codes for the receptor Fct and the enzymes leading to the catechol moiety in chrysobactin
biosynthesis (23) (Fig.1). Analysis of the fct gene sequence (18) revealed a strong resemblance
of the promoter region to the bidirectional promoter controlling the expression of the fepA-entD
and fes-entF operons in E. coli (24, 25) (Fig.1). The fepA gene codes for the receptor FepA
(26, 27). The entD and entF genes (Fig. 1) encode the EntD and EntF proteins which are two
components of the enterobactin synthase multi-enzyme complex (29-31). In E. chrysanthemi
like in E. coli and many other bacterial species, control by iron is achieved via a fur gene that
encodes a protein highly similar to the E. coli Fur regulator (32). In the presence of ferrous iron
as a cofactor, the E. coli Fur protein acts as a transcriptional repressor by binding to operator-
specific sequences (Fur- or iron-boxes) (33, 34).
Sequence analysis of the 5' upstream region of the fct gene revealed the existence of a
gene (cbsH) transcribed in the opposite direction to the fct gene, that shares identity with the E.
coli fes gene. The functional analysis of the cbsH gene product is presented.
EXPERIMENTAL PROCEDURES
Bacterial strains, plasmids and microbiological techniques
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The bacterial strains used are listed in Table 1. Plasmids are described in Table 1 and Fig. 2 A.
The cbsE-1 and acs-37 mutations were transduced into strain L2 cbsH-19 with phage PhiEC2
as described previously (35). Insertional mutagenesis with the MudI1734 prophage in plasmid
pCS2 and marker exchange recombination into the chromosome were performed as described
previously (36). Expression of the cbsH17::lacZ fusion was monitored as reported previously
(37). The rich media used were L broth and L agar (38). L agar was iron-depleted by adding
EDDHA1 purchased from Sigma Chemical Co., to give a final concentration of 100 µg/ml. Tris
medium was used as the low-iron minimal medium (35). For iron-rich conditions, it was
supplemented wih 20 µM FeCl3. Glucose (2 g/l) was used as the carbon source. The
antibacterial agents and chemicals used were as reported previously (35).
General DNA methods
All DNA manipulations were carried out as described previously (35, 36). The aphA-3 cassette,
obtained by SmaI digestion of pUC18K (39) was inserted into the AfeI-digested pCS2 and then
recombined into the chromosome. The AfeI site lies within the cbsH gene at a position
corresponding to amino acid 274.
Deletion analysis, nucleotide sequence determination, primer extension
Serial truncations (200 to 250 bp) of pDE34 (Fig. 2 A) were carried out from the SalI site, using
the Pharmacia double-stranded nested deletion kit (Pharmacia LKB Biotechnology AB, Uppsala,
Sweden). The deleted subclones were PEG-purified (40) and sequenced using the Sequenase
Kit (US Biochemical Corp.) and {alpha-35S}-dATP according to the manufacturer's instructions.
The second strand sequence was determined by extension from specific oligonucleotides (17
mers). Data were analysed using the UWGCG software package provided by BISANCE (41).
The two programs, BLAST and Kanehisa were used for amino acid sequences comparaisons.
The sequence of the E. chrysanthemi cbsH (fes) gene has been submitted to GenBank under
the accession number AF 011334.
RNA templates were isolated from E. chrysanthemi 3937 and E. coli JM101 pCS1
cultures grown in Tris medium, reaching an OD at 600 nm of 0.8 and 0.6 for high- and low-iron
conditions, respectively. RNA isolation and primer extension analysis were performed as
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described previously (18). A 32P-labelled oligonucleotide complementary to nucleotides 158-142
was used for primer extension.
Identification of the CbsH product
The procedure described by Tabor and Richardson (42) was used to produce proteins encoded
by pT7-derivative plasmid. Samples were radiolabelled as described previously (37). Proteins
were separated by electrophoresis in 8 % polyacrylamide gel in the presence of SDS.
Siderophore detection
Catechol was determined using the chemical assay of Arnow (43) using DHBA as the standard.
Siderophore activity was detected as Chrome Azurol S (CAS)-reacting material in the culture
supernatant (44), using desferrioxamine B (Desferal, Norvatis Pharma SA, France) as the
standard. The biological activity of chrysobactin and enterobactin was determined in bioassays
as described previously (16), using strains RW193 and RW818-60 for enterobactin, L2 cbsE-1
and L2 fct-34 for chrysobactin as indicators.
Quantitative determination of ferric chrysobactin in cell lysates
A culture grown exponentially in L broth of the strain to be studied was diluted 1:40 in 20 ml of
Tris medium supplemented with glucose and 5 µM of FeCl3. Cultures were grown aerobically for
12-14 hours. Cells were washed, suspended in 1 ml of Tris medium and disrupted in a Vibra
Cell apparatus (Sonics and Materials Inc. USA). The lysis mixture was centrifuged for 30 mn at
7,000 rpm and 4°C in a microfuge (Medical Scientific Equipment, Leicester UK). Pelleted cell
debris was discarded and the supernatant was checked for the presence of ferric chrysobactin
in a bioassay. The concentration of ferric chysobactin in cell lysates and in culture supernatants
equivalent to 5 x 108 C.F.U. was determined spectrophotometrically. The ferric complex, at pH
7.4, had an absorption maximum at 525 nm (ε525 = 3.2 mM cm-1) (45).
Assay for enzymatic hydrolysis of ferric chrysobactin
Hydrolysis of ferric chrysobactin was assayed in cell extracts from the bacterial strains tested
prepared as following. Cells were grown aerobically in 500 ml of Tris medium supplemented
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with glucose until an OD at 600 nm of 0.6 to 0.9 was reached. Cells were washed, suspended in
2 ml of in 0.1 M Tris-HCl, pH 7.5 and disrupted as described above. Lysates were
supplemented with DTT at a final concentration of 0.005 mM and centrifuged for 20 mn at
20000 g. The enzymatic activity in supernatants was immediately tested. The reaction mixture,
incubated at 37° C for 1 h, contained 0.300 ml of lysate, 0.430 ml of 0.1 M Tris-HCl, pH 7.5 and
0.020 ml of the bis complex of ferric chrysobactin to give a final concentration of 0.028 mM.
Ferric chrysobactin was prepared by adding FeCl3 to chrysobactin in 0.1 M Tris pH 7.5 at a
ligand to iron ratio of 4 :1. Chrysobactin, a gift from Dr Buyer, was synthetised according to the
procedure described previously (46). Protein concentration in cell lysates was determined with
the Bradford reagent. Hydrolytic activity was determined spectrophotometrically as described
above. Enzymatic activity is expressed in nanomoles of ferric chysobactin hydrolysed per mg of
protein in 1 h. Diisopropyl fluorophosphate was added to the reaction mixture at a final
concentration of 0.036 mM. For each strain, three independent experiments were performed.
Assay for ferric enterobactin esterase activity
For ferric enterobactin esterase activity, cell extracts from the bacterial strains tested were
prepared as described above. Lysates were assayed as reported by Langman et al. (13).
Enzymatic activity is expressed in nanomoles of enterobactin hydrolysed per mg of protein in 1
h. For each strain, three independent experiments were performed. Ferric enterobactin was
prepared according to the procedure reported by Greenwood and Luke (10), with modifications.
The supernatant of a 10-litre culture of E. coli strain BZB1013 was lyophilised and extracted with
ethyl acetate. As a final purification step, ferric enterobactin dissolved in methanol was passed
through a column of Sephadex LH-20 (30 g) with methanol as the eluent. Fractions were
collected, evaporated and dissolved in 0.1 M phosphate buffer (pH 7). Catechol-positive
fractions were bioassayed, using strains RW193 and RW818-60 as indicators and checked by
ultraviolet spectrometry. The purest fraction yielded about 50 µmoles of ferric enterobactin.
Transport experiments
An overnight culture in L broth of the strain to be studied was diluted 1:40 in Tris medium
supplemented with glucose and incubated with shaking until the required OD at 600 nm was
reached. Bacterial cells were harvested by centrifugation, washed with Tris medium with no
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phosphate, suspended in phosphate-free Tris medium supplemented with glucose and kept on
ice until use. The transport medium was Tris medium containing 40 to 50 µM DHBA equivalents
of chrysobactin, supplemented with 1 µM of 59FeCl3 (0.1 mCi ml-1 of iron [III] chloride in 0.1 M
HCl, Amersham). For transport experiments, the bacterial suspension was diluted in transport
medium to give an OD at 600 nm of 0.4 in a total volume of 5 ml, placed in a 50 ml Erlen-meyer
flask. At intervals of 5 to 30 mn, 200 µl was withdrawn and immediately filtered through a filter
with 0.45 µ pores that had been soaked for at least 12 hours in Tris medium supplemented with
20 µM unlabelled FeCl3. Filters were immmediately washed with 20 ml of Tris medium with no
phosphate. The filters were placed in scintillation vials, air dried and radioactivity was measured
by liquid scintillation counting. Two 20 µl samples of each bacterial culture were counted to
check the total amount of radioactivity. For each strain, experiments were performed in
triplicate.
Mössbauer measurements
For each Mössbauer measurement, a 2-litre bacterial culture in 5-litre Erlenmeyer flasks was
required in order to obtain approximately 1 cm3 of packed cells. Cultures of strains L2 cbsE-1
and L2 cbsH-19 were grown in Tris medium supplemented with glucose for 12 hours. The OD at
600 nm was 0.65. 57Fe labeled chrysobactin was added to the cell suspensions at a final
concentration of 1.3 µM (ligand to iron ratio of 4 :1.3). Cells were grown for additional 30, 60 and
120 min, respectively. At 0 mn and each additional time, cells were cooled down to 4°C within 2
minutes, harvested, washed in Tris medium, and transferred to Delrin Mössbauer sample
holders. All sample volumes were about 1 ml. Sample thickness did not exceed 9 mm. The
containers were quickly frozen in liquid nitrogen and kept in a liquid nitrogen storage vessel until
measurement was done. The Mössbauer spectra were recorded in the horizontal transmission
geometry using a constant acceleration spectrometer operated in conjunction with a 512-
channel analyzer in the time-scale mode. The source was at room temperature and consisted of
1.15 GBq [57Co] diffused in Rh foil (AEA Braunchweig). The spectrometer was calibrated
against a metallic α-iron foil at room temperature yielding a standard line width of 0.24 mm/s.
The Mössbauer cryostat was a helium bath cryostat (MD306, Oxford Instruments). A small field
of 20mT perpendicular to the γ-beam was applied to the tail of the bath cryostat using a
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permanent magnet. Isomer shift δ, quadrupole splitting ∆EQ, and percentage of the total
absorption area were obtained by least-squares fits of Lorentzian lines to the experimental
spectra .
RESULTS
Characterization of the cbsH gene and its translation product
The sequence of the 2.1 kb SspI-SalI fragment (Fig. 2 A) begins at nucleotide + 3 of the fct
cbsCEBA transcript and ends 100 bp upstream from the SalI site. Two contiguous ORFs (ORF1
and ORF2) and the beginning of a third (ORF3) were identified (Fig. 2 A). ORF1 has two
putative ATG initiation codons (ATG1 position 142 and ATG2 position 277, Fig. 2 B) and
terminates with a TAG stop codon (position 1444). Potential ribosome binding sites are located
6 and 8 bp upstream from the initiation codons, respectively. ORF2 starts with an ATG codon
(1557), terminates with a TGA codon (1796) and has a ribosome binding site 9 bp upstream
from the start codon. A GTG initiation codon (position 1815), preceded by a Shine-Dalgarno
sequence (5'AGGA3', position 1804) suggests the beginning of ORF3. ORF1 is separated from
ORF2 by 100 bp and ORF2 from ORF3 by 18 bp. The nucleic acid sequence of ORF1 is 64 %
identical to the E. coli fes gene for the 871 nucleotides from position 272 to 1143. The deduced
389-amino acid polypeptide sequence is 45.6 % identical to that of the E. coli Fes protein. The
beginning of ORF3 (from position 1815 to 2023) is identical to that of the E. coli entF gene
encoding enterobactin synthase. The gene corresponding to ORF1 was designated cbsH.
Sequence analysis predicted a potential promoter P, overlapping the P' promoter of the
fct cbsCEBA operon characterised previously (Fig. 2 B). To determine the transcriptional start
site of the cbsH gene, total RNA from iron-replete and -depleted cultures of E. chrysanthemi
3937 and of E. coli JM101 harboring pCS1 was used as a template in extension reactions
primed with a 32P-labelled 17-mer oligonucleotide complementary to the sequence between
positions 158 and 142. For both strains, the reactions yielded iron-regulated cDNAs that
comigrated with an A residue (Fig. 2 C). The occurrence of a transcriptional start at a T
nucleotide (position 49) is consistent with the predicted P promoter. Two putative Fur binding
sites overlapping the -10 / -35 sequences of the P promoter (Fig. 2 B) account for the observed
iron regulation. Regulation by iron was confirmed by monitoring expression of the chromosomal
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cbsH17::lacZ fusion constructed in L2 cells (Table 1 and next section) grown in Tris medium
with and without iron supplementation (Fig. 2 C).
The two potential translation start codons ATG1 and ATG2 (Fig. 2 B) have good
matches to the Shine-Dalgarno sequence (5'GGAGG' and 5'GGACG3', respectively). To
determine which of these codons is functional, we analysed the translation products of two
constructs, pCS3 and pLR1, placed under the control of the T7 phi10 promoter in SDS-PAGE.
pCS3, containing the 2.1 kb DraI-SalI fragment (Fig. 2 A), includes both ATG codons and the 5'
untranslated region. pLR1, in which the 158 bp Ssp1-EcoNI fragment present in pCS3 has been
deleted (Fig. 2 A B), lacks the ATG1 codon. For both constructs, a same polypeptide migrating
in the 43,000 Da range, induced at 42°C only was identified (data not shown). Thus, in the
conditions described, ATG2 (position 277, Fig. 2 B) is the functional translation start codon for
the cbsH gene.
A cbsH mutation has no effect on iron acquisition from enterobactin in E. chrysanthemi
We investigated the protein encoded by the cbsH gene by isolating mutants (L2 cbsH-17 and L2
cbsH-19, Table 1), using insertional mutagenesis. Insertion cbsH-19 was mapped to position
943 by sequencing and was analysed further. As prophage MudI1734 generates polar
mutations, we first investigated whether the mutant was able to produce chrysobactin. It did not
grow on EDDHA-L agar medium. It produced catechol compounds but did not release a
functional siderophore, as shown by the CAS assay and growth stimulation experiments (data
not shown). The introduction of pCS2 which carries the cbsH gene and ORF2, did not enable
the mutant cells to grow on EDDHA-L agar medium. We therefore concluded that the mutant did
not synthetise chrysobactin because of the polar effect on the downstream gene that shares
identity with the E. coli entF gene.
As ferric enterobactin esterase is an essential component of the enterobactin-mediated
iron-transport pathway in E. coli, we investigated whether the L2 cbsH-19 mutant could use
ferric enterobactin as an iron source (Table 2). Ferric enterobactin promoted the growth of this
mutant as efficiently as for a cbsH-positive strain. This mutant may have been able to utilise
ferric enterobactin because it produced achromobactin or catechol. We therefore transduced
the mutant with mutations acs-37 and cbsE-1. The transductants L2 cbsH-19 acs-37 cbsE-1
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utilised ferric enterobactin as efficiently as did the simple mutant (Table 2). This shows that a
functional cbsH gene is not required in E. chrysanthemi for iron acquisition from enterobactin.
Lack of functional complementation of the E. coli fes mutation by the cbsH gene
These data led us to verify whether the cbsH gene could functionally complement an E. coli fes
mutant. MM272-60 cells were transformed with plasmid pCS2 or pLR2. The transformants did
not grow on EDDHA-L agar medium and their growth was not stimulated by ferric enterobactin
(Table 2). A similar result was obtained with MM272-60 cells harboring pTF12, which contains
the cbsH gene on a low-copy-number vector (Table 2). We checked that the lack of
complementation did not result from the production of non-physiological levels of the CbsH
protein, by testing low-iron cultures of cells harboring pLR2 for the presence of enterobactin. All
culture supernatants tested were strongly positive in the bioassay (data not shown).
A cbsH mutation affects iron acquisition from chrysobactin in E. chysanthemi
To know whether the CbsH protein was a component of the chrysobactin-dependent iron
transport pathway, we assessed the stimulation of growth of the L2 cbsH-19 acs-37 mutant by
chrysobactin. After 24 hours of incubation, the mutant had not grown, but after 72 hours, a halo
of growth became visible (Table 2). The introduction of pCS2 into the mutant restored its growth
in 24 hours. Rescue was also observed after the introduction of pLR2, which carries the cbsH
gene only (Table 2). Thus, the mutant phenotype did not result from a polar effect of the
mutation on downstream genes of the same operon. In contrast, the E. coli fes mutant (MM272-
60) in which the ferric chrysobactin receptor fct gene is present on plasmid pLR3 (unlike pLR2)
grew normally if supplied with ferric chrysobactin as an iron source (Table 2). The halo of growth
was similar with strain MM272-60 carrying pTF12 which contains the cbsH gene (Table 2).
Thus, the protein encoded by the cbsH gene is not required in E. coli cells, if ferric chrysobactin
is the iron source. One possible interpretation of these data is that a cbsH-negative mutant of E.
chrysanthemi was affected in the transport of ferric chrysobactin.
We therefore determined the ability of the mutant to transport 59Fe-chrysobactin, and
compared it with that of the parental strain (Fig. 3). Strains were grown in Tris medium and
uptake experiments were conducted with cells harvested at an optical density at 600 nm of 0.6.
After 5 hours, the growth of mutant L2 cbsH-19 acs-37 had slowered considerably, indicating
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that iron was poorly assimilated (Fig. 3 A). The ferric chrysobactin transport rate was higher in
the mutant than in the parental strain (Fig. 3 B). Thus, the mutation has no effect on ferric
chrysobactin transport per se. The transport rate seems to depend on intracellular metabolic
state and presumably reflects the level of derepression by iron of the entire protein machinery
involved in transport.
Accumulation of ferric chrysobactin in a non polar cbsH-negative mutant
We investigated the protein encoded by the cbsH gene, by constructing a non-polar mutant with
the aphA-3 cassette (39). Mutant L2 cbsH::aphA-3 acs-37 gave rise to colonies with a red color
that was not observed in polar mutants. The E. coli fes mutant is also red on L agar medium.
The mutant did not grow on EDDHA-L agar medium. If supplied with ferric chrysobactin, there
was a time lag before it started growing as observed with polar mutants (Table 2). This mutant
grew very slowly in Tris medium, but transported 59Fe-chrysobactin very quickly, indicating that
the bacterial cells were severely iron-depleted (Fig. 3 B). These observations suggest that an
iron binding compound accumulated inside the cells. To know whether this compound was the
ferric chysobactin complex, cellular extracts of L2 cbsH-apha3 acs37 were compared with those
of a fur mutant (L37 acs1 fur) that also overexpresses chrysobactin biosynthesis and transport
proteins in Tris medium supplemented with FeCl3. A bioassay shows that extracts from cbsH-
negative cells promoted the growth of a chrysobactin deficient strain very efficiently (Fig. 4 A).
This effect was not observed with extracts from fur deficient cells. The ferric chrysobactin
complex was quantitatively determined in culture supernatants and cell lysates (Fig. 4 B). The
cbsH mutant accumulated ferric chrysobactin intracellularly, unlike the fur strain for which most
of the ferric complex was present in the culture supernatant.
The CbsH protein is a peptidase hydrolysing chrysobactin
The accumulation of the ferric chrysobactin complex in the cytosol of cbsH-negative cells
indicates that this molecule was not degraded following its transport. As chrysobactin possesses
a peptide bond, one possiblility was that the cbsH gene encodes a peptidase. Indeed, the
catalytic mechanism of certain esterases involving the formation of an acetyl-enzyme
intermediate during the reaction is analogous to that of serine proteases (47). The alignement of
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ferric enterobactin esterase-like protein sequences from various bacterial genera present in data
banks (Fig. 5) reveals the presence of a common signature, GXSXGG-D-H found in the family
of prolyl oligopeptidases (48). These residues are within about 130 residues of the C-terminus
and the N-terminal parts of the molecule are more or less variable. Therefore, we investigated
whether cbsH-positive cells had an enzyme enabling them to catalyse the hydrolysis of ferric
chrysobactin, that was lacking in mutant cells. Enzymatic activity was determined in cell extracts
from low-iron cultures of the parental strain L2 cbsE-1 and the mutant L2 cbsH-19 (Table 3). We
observed the disappearance of the ferric chysobactin complex only in cbsH-positive cells. This
enzymatic activity was thiol dependent like a number of other cytosolic peptidases. The addition
of diisopropyl fluorophosphate, an inhibitor of serine proteases totally blocked the reaction.
These results show that the CbsH protein has a ferric chrysobactin peptidase activity. To also
determine whether this enzyme has a ferric enterobactin esterase activity, we used ferric
enterobactin as a substrate. Although cbsH-positive cells had a significant level of ferric
enterobactin esterase activity as compared to mutant cells, the specific enzyme activity was 10
times lower than that found for the hydrolysis of ferric chrysobactin, under the same conditions.
Iron removal from chrysobactin in vivo does not require a functional cbsH gene
To get basic information on the metabolic utilization of chrysobactin bound iron in situ
Mössbauer spectroscopy of whole cells was performed at various states of chrysobactin uptake.
In situ Mössbauer spectroscopy enables in principle simultaneous identification of all main iron
metabolites on a qualitative as well as on a quantitative level without destruction of the cellular
assembly (49, 50). Moreover, time dependent changes can be followed the resolution of which
is merely limited by the time required for sample preparation (50).
As expected, samples of either strain (L2 cbsE-1 and L2 cbsH-19) taken directly after
addition of 57Fe chrysobactin yielded Mössbauer spectra with very poor resolution. The cbsH-
positive sample exhibits a single doublet of high-spin ferrous iron in a octahedral oxygen or
nitrogen environment: δ = 1.26 (6) mm s-1, ∆EQ = 3.19 (11) mm s-1, Γ = 0.512 mm s-1. This
component accounts for most of the Mössbauer absorption (84%). Based on the evolution of a
second component visible after growth with 57Fe-chrysobactin, this second component was fitted
to the experimental data δ = 0.38 (6) mm s-1, ∆EQ = 0,65 (5) mm s-1, Γ = 0.27 mm s-1, (16%). For
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the cbsH-negative strain a featureless absorption is found. Nevertheless, we tried a fit δ1 = 0.48
(8) mm s-1, ∆EQ = 0,65 (11) mm s-1, Γ = 0.7 mm s-1 (75%), δ2 = 0.97 (6) mm s-1 , ∆EQ = 1.54 (11)
mm s-1, Γ = 0.27 mm s-1. After 30, 60, and 120 min, the Mössbauer spectra are well resolved
and allowed unambiguous analysis (Fig. 6, Table 4). The Mössbauer parameters of the cbsH-
positive strain and its mutant after 57Fe-chrysobactin uptake are summarized in Table 4. Like
other catechol-type siderophores, chrysobactin exhibits a typical magnetically split S=5 /2
pattern (49, 51, Fig. 7A). No ferric chrysobactin is detectable in Mössbauer spectra of whole
cells. In contrast, there is almost exclusively ferrous iron found at t=0 (Fig. 6A, Table 4) and
even after 30 mn of uptake (Fig. 6B, Table 4), the majority of the transported iron is present in
its ferrous form. (Fig. 6B, Table 4).
The second component observed spectroscopically corresponds to a ferric high spin
species. The 57Fe-content of the cells is growing with increasing incubation time (increasing total
absorption area), although slightly slower for the cbsH-positive strain. Whereas after 30 min of
incubation the ferric iron species contributes only little to the Mössbauer absorption, it
represents the major component after 2 hours. This species exhibits Mössbauer parameters
very similar to bacterioferritin found in E.coli (54-55). Comparison of the Mössbauer parameters
obtained from a spectrum measured at 86 K (data not shown) with those derived from a
spectrum at 4.3 K (Fig. 6) reveals a significant increase of Γ (from 0.505 mms-1 to 0.814 mms-1)
and a concomittant decrease of relative transmission. This considerable line broadening is
typically found at temperatures close to superparamagnetic transitions (53, 54). E.coli-type
bacterioferritins (Bfr) display magnetic broadening below 4.3 K and show eventually
magnetically split spectra at temperatures below 1 K (56). Fig. 7B displays the Mössbauer
spectrum of cbsH-negative mutant cells measured at 1.8 K. Indeed, the ferric iron species is
missing in this spectrum, instead, a magentically broadened absorption is visible as expected for
a Bfr-type protein. Therefore, we attribute the ferric iron species to a Bfr-like compound.
DISCUSSION
In this study, we report the functional analysis of a new gene, cbsH, that belongs to the
chrysobactin-dependent iron transport gene cluster of E. chrysanthemi 3937. The cbsH gene is
the first gene of an operon involved in chrysobactin biosynthesis, transcribed from the iron-
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regulated divergent promoter, fct-cbsH, which controls in opposite orientation the transcription of
the fct cbsCEBA operon, identified ealier (23). The cbsH gene is 64 % identical to the E. coli fes
gene for the 871 nucleotides from position 272 to 1143. It encodes a polypeptide with an
apparent molecular weight of 43,000 daltons, a size similar to that reported for the E. coli Fes
protein (25). The CbsH protein is 45.6 % identical to the Fes protein of E. coli, 46 % identical to
the Fes protein of Yersinia enterocolitica (57), and 42 % identical to S. enterocolitica IroD (iroD
gene: GenBank accession number U 97227), with the level of identity uniform over the entire
amino acid sequence (according to the program BLAST)
The presence in E. chrysanthemi of an homologue of the ferric enterobactin esterase of
E. coli was expected. E. chrysanthemi has a ferric enterobactin transport system that supplies
the cell with iron. As the hydrolysis of ferric enterobactin is essential in E. coli cells, we thought it
likely that this molecule would have the same fate in E. chrysanthemi. We show that the CbsH
protein is not required for the removal of iron from ferric enterobactin in E. chrysanthemi. These
data indicate that cleavage of the ester bonds of ferric enterobactin is not required in E.
chrysanthemi for iron reduction and release. This was not due to the presence of an additional
fes-like gene on the E. chrysanthemi chromosome, as shown by DNA/DNA hybridization
analysis (data not shown). In contrast, the Fes homologue from Y. enterocolitica appears to be
absolutely required for ferric enterobactin utilisation in this bacterium (57). In addition, the viuB
gene from Vibrio cholerae, which is involved in vibriobactin processing, can complement the E.
coli fes mutation (58). No functional complementation of the E. coli fes mutation was observed
with the E. chrysanthemi cbsH gene. These results show that iron release from enterobactin is
not CbsH-dependent. Instead, a Fes/CbsH-independent mechanism has to be considered.
We should point out that the role of the E. coli ferric enterobactin esterase has been
much debated. In particular, several experimental aspects have remained unexplained (10, 11,
59). For instance, this enzyme is required for the removal of iron from enterobactin analogs
devoid of ester bonds (60, 61). The redox potential of a ferric siderophore depends on the
binding constant for iron and thus on the capacity of the molecule to be protonated at neutral pH
(62). If the internal pH of E. chrysanthemi were slightly lower than that of E. coli, then iron would
be easier to extract in E. chrysanthemi than in E. coli. Cohen et al. (59) have reported that
enterobactin, like synthetic analogues such as TRENSAM, may adopt a tris-salycilate mode of
binding if sequencially protonated, with iron release facilitated by a biological reductant.
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On the basis of amino acid sequences comparisons, we found that the CbsH protein
displays characteristics of the S9 prolyl oligopeptidase family (48), namely the conservation of
amino acids around the catalytic triad Ser, Asp and His (Fig.5). Family S9 contains serine
peptidases with a varied range of restricted specificities including oligopeptidase B from
eubacteria, which cleaves arginyl and lysyl bonds. In agreement with sequence predictions, we
showed that the CbsH protein is an enzyme able to degrade ferric chrysobactin in the cytosol.
This hydrolytic activity is thiol dependent and inhibited by fluorophosphates such as DFP. Given
the chrysobactin structure (Fig. 1), it is very likely that this enzyme cleaves the lysyl bond thus
forming DHB-lysine and serine.
To further understand the role of this enzyme, we analysed the cellular distribution and
redox state of iron following the transport of ferric chrysobactin in the cytosol, using Mössbauer
spectroscopy. After 30 min of incubation with 57Fe chrysobactin, the Mössbauer spectra of the
cbsH-positive strain and of a cbsH-negative mutant show mainly ferrous iron. The total lack of
ferric chrysobactin and the initially observed high ferrous iron contribution in the cell spectra of
Erwinia clearly demonstrate that ferric chrysobactin transport is followed by a very rapid
intracellular enzymatic iron reduction. Because ferric chrysobactin is transported across the cell
membranes via a highly specific receptor-mediated pathway (18), the reduction obviously occurs
at the level of the cytoplasmic membrane or in the cytosol. The affinity of catecholate
siderophores for ferrous iron is very low. Even water is a better chelator of ferrous high-spin iron
than these siderophores. Therefore, the presence of ferrous iron provides evidence for a rapid
reductive release of the metal from its carrier preventing an observable intracellular
concentration of 57Fe-chrysobactin. Although a ferrous hexaquo complex is stable in a strict
reductive (and anaerobic) environment it is very likely that the reduced metal is complexed by a
specific intracellular chelator in order to prevent Haber-Weiss-Fenton chemistry (50). Previously,
we found that ferrous iron constitutes one of the major cellular iron species in many
microorganisms under conditions of siderophore controlled growth (52, 53). The corresponding
compound has been isolated from E.coli and from Pantoea agglomerans2 and partially
characterized as an oligomeric sugar phosphate (52). It was termed ferrochelatin (53). Based on
the previous studies we attribute the detected ferrous iron to ferrochelatin. Whereas
ferrochelatin bound iron keeps its intracellular concentration at a certain level (approximately
0.3% effect) the second component of the Mössbauer spectra increases its contribution by time
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and represents the major component after 2 hours of incubation. Based on the temperature
dependent Mössbauer spectra and their parameters the conclusion must be drawn that this
component represents a bacterioferritin-like iron storage compound. Thus, ferrous iron released
from chrysobactin is immediately transferred into the iron storage form where it is oxidized again
at the ferroxidase site (71, 72). In summary, the Mössbauer spectroscopic analysis does neither
show significant differences of the chrysobactin mediated iron uptake between the parental
strain and its mutant nor of the metabolic distribution pattern. Based on the results of this
investigation it is safe to state that metabolic utilization of both enterobactin and chrysobactin
bound iron is not cbsH-dependent (see scheme shown in Fig. 8).
As described above there is good evidence for hydrolytic cleavage of chrysobactin by
CbsH. In addition, longterm growth inhibition is observed in cbsH-negative mutants. This ligand
hydrolysis occurs obviously after iron removal and lack of hydrolysis in the mutants results in
growth inhibition. This unexpected finding might be linked either to a utilization of the aromatic
systems of chrysobactin for anabolic reactions or to a role of cbsH in intracellular iron
homeostasis. Within the rationale of bacterial iron metabolism we favor the latter line of thought.
The free chrysobactin ligand is thermodynamically capable of extracting ferric iron from all
intracellular ferric iron sources exhibiting a lower complex formation constant than ferric
chrysobactin. In addition, recent studies on the uptake of iron(III) by chrysobactin have shown
that the carboxyl group of the serine residue in chrysobactin strongly influences the kinetics of
formation of the ferric complex3. In order to prevent iron removal from metabolically active
enzymes or from any accessible intracellular iron pool, the ligand has either to be reexcreted -
which is known for some bacterial siderophore uptake systems - or it must be degraded or
modified. At this point it is important to note that the non polar cbsH-negative mutant behaves as
if it was severely iron-depleted although it contains high levels of ferric chrysobactin. This finding
fits well into our hypothesis because there seems to occur an intracellular post-transport
recomplexation of iron by the non-degraded ligand. In summary, taking all pieces of
circumstantial evidence together, we suggest that hydrolytic degradation of chrysobactin by
cbsH is aimed on keeping the intracellular iron distribution on a well regulated level (iron
homeostasis) in E. chrysanthemi 3937.
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Acknowledgments
We thank Céline Masclaux and Chrystèle Sauvage for the construction of recombinant plasmids
and their interest in this work, Pr. Kenneth Raymond and Thierry Franza for helpful discussions,
Dr. Anne-Marie Albrecht-Gary for communicating data prior to publication, and Alex Edelman for
reading of the English of the manuscript. This work was supported by grants of the Institut
National de la Recherche Agronomique (INRA). D.E. is a researcher from the Centre national de
la Recherche Scientifique (CNRS).
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Footnotes :
1The abbreviations used are : EDDHA, ethylenediamine-N,N'-bis(2-hydroxy-phenylacetic acid) ;
C.F.U., colony forming unit ; DHBA, dihydroxybenzoic acid ; DTT, dithiothreitol ; 2 unpublished
observations ; 3 personal communication
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Figure legends :
Fig. 1: Enterobactin and chrysobactin mediated iron transport in E. coli K-12 (70) and E.
chrysanthemi 3937, respectively.
In A, gene clusters specifically involved in transport and biosynthetic pathways are shown
Details are in the text. Arrows indicate the direction of transcription. Filled arrows correspond the
genes referred in the text. In B, structures of enterobactin and chrsobactin are shown.
Enzymatic cleavage sites are indicated by arrows.
Fig. 2 : The chrysobactin fct-cbsH region and the regulatory role of iron.
A shows the physical map of the fct-cbsH region (bold line) carried by the various plasmids.
Restriction sites: E, EcoRI; K, KpnI; S, SalI; P, PstI; H, HindIII; B, BamHI; D, DraI; Ss, SspI; Hp,
HpaI; En, EcoNI; Sa, SacII. Plasmid pDE34 includes a BamHI-HindIII fragment from the Tn5-
B20 transposon (Simon et al., 1989) containing the fct34::lacZ fusion. BamHI and EcoRI sites as
the right-hand side in pDE34 are those of the pUC18 polylinker. Arrows indicate the direction of
transcription of the various genes. In B, -10 / -35 determinants of promoters directing the
transcription of fct (P') ( ref) and cbsH (P) are indicated. Potential Shine-Dalgarno sequences
and start codons are boxed. Relevant restriction sites shown on plasmid pDE34 are underlined.
In C, the autoradiograph shows the results of primer extension reactions in iron-replete (+) and
iron-depleted (-) conditions, showing that P is the functional promoter of cbsH. Lanes A, C, G, T
are sequencing ladders. To the right of the autoradiograph is the DNA sequence of the region
with position of the migrating mRNA indicated in bold. The graph shows the ß-galactosidase
activity of the cbsH17::lacZ fusion assayed during the growth of bacterial cells in Tris medium
supplemented (filled circles) or not (open circles) with FeCl3.
Fig. 3: Ferric chrysobactin transport in cbsH-negative mutants.
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A shows growth curves in Tris medium for the various strains tested. Transport assays were
performed with bacterial cells collected at an OD (600 nm) of 0.6, as indicated by the arrow. In
B, 59Fe-chrysobactin transport was analysed as described in Experimental Procedures.
Symbols in A and B indicate the cbsH genotype of the L2 acs-37 cells analysed; circles: wild-
type, squares: cbsH-19, triangles: cbsH::aphA-3.. Transport data are the means of
measurements obtained in three separate experiments with error bars representing the
standard deviation.
Fig. 4: Accumulation of ferric chrysobactin in a cbsH mutant
In A, ferric chysobactin was determined in a bioassay. Genotypes of indicator strains are
reported at the top of photographs; at the bottom, the mutant strains from which the lysates
were prepared are indicated. In B, the ferric chrysobactin complex in culture supernatants and
cell lysates was determined spectrophotometrically. Details are in Experimental procedures.
Data are the means of measurements obtained in three separate experiments with error bars
representing the standard deviation.
Fig. 5: Multiple alignment of Fes related proteins from various bacterial species.
Stars indicate identical amino acids and dots indicate residues with similar chemical properties.
Bold characters with stars correspond to amino acids conserved in the S9 prolyl oligopeptidase
family (48). His and Asp residues in bold face can potentially belong to the catalytic site.
Fig. 6: Mössbauer spectra of frozen E.chrysanthemii cells measured at 4.3 K in a perpendicular
field of 20 mT.
Cells were grown in iron depleted medium as described in Experimental Procedures and
harvested : directly after addition of 5 µM 57Fe-chrysobactin (A) and after 30 min (B), 60 min,
(C), 120 min (D) of additional growth. Solid and broken lines were obtained by least-squares fits
of Lorentzian lines to the experimental spectra yielding the Mössbauer paramaters listed in
Table 4 A and B.
Fig. 7: Mössbauer spectra of a frozen aqueous solution (Tris buffer pH 7) of 57Fe chrysobactin
(1:4) measured at 4.3 K (A) and of frozen E.chrysanthemi cbsH–negative mutant cells
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Erwinia chrysanthemi chrysobactin peptidase
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measured at 1.8 K (B) in a field of 20mT perpendicular to the the γ−rays. The cell suspension
was supplied with 5 µM of 57Fe-chrysobactin at an OD600 of 0.65. Cells were harvested after
120 min of additional growth. The Mössbauer parameters and the corresponding percentage of
the absorption areas are listed in Table 4 A and B.
Fig. 8: Schematic drawing of ferric chrysobactin (Fe [Cb]3) uptake and its metabolic utilization in
E. chrysanthemi.
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Table 1 : Bacterial strains, bacteriophages, plasmids used
________________________________________________________________________________
Strain / Plasmid Relevant characteristics Source / Reference
________________________________________________________________________________
Strain
Erwinia chrysanthemi
3937 Wild type isolated from African violet Our collection
L2, L37 Lac- derivatives of 3937 (63)
L37 acsA1 fur acsA1::MudI1734, fur::�� .PR, SpecR (32)
achromobactin synthesis deficient, Acs-, Fur-
3937 cbsE-1 cbsE::�� 6SHFR, chrysobactin synthesis deficient, Cbs- (37)
L2 fct-34 fct34::lacZ, fct::Tn5-B20, KmR (64)
ferric chrysobactin transport deficient, Fct-
L2 cbsH-17 cbsH17::lacZ , cbsH::MudI1734, KmR,CbsH-, Cbs- This work
L2 cbsH-19 cbsH19::MudI1734, KmR,CbsH-, Cbs- This work
L2 acs-37 acs::MudIIpR13, Acs-, CmR (36)
L2 cbsE-1 Cbs-, SpecR This work
L2 acs-37 cbsE-1 Acs-, Cbs-, CmR, SpecR This work
L2 cbsH-19 Acs-, Cbs-, KmR, CmR, SpecR This work
acs-37 cbsE-1
L2 cbsH::apha-3 CbsH-, Acs-, KmR, CmR This work
acs-37
Escherichia coli K-12
TG1 supE hsd¨� thi ¨�lac-proAB) F' (42)
[traD36 proAB+ lacIq lacZ¨0��@
JM101 supE thi ¨�lac-proAB) F' (65)
[traD36 proAB+ lacIq lacZ¨0��@
JM109 recA1 supE44 endA1 hsdR17 gyrA 96 relA1 (65)
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thi ¨�lac-proAB) F' [traD36 proAB+lacIq lacZ¨0��@
M8820 F- Lac- araD 139 ¨�DUD-leu)7697 (66)
¨�proAB-argFlacIPOZYA)XIII rpsL, SmR
POI1734 F- MudI1734 (lac, KmR) ara::(mucts)3 (66)
D(proAB-argFlacIPOZYA)XIII rpsL, SmR
RW193 F- entA thi trpE proC leuB lacY mtl xyl T. Pugsley
galK ara rpsL azi tsx supE
RW818-60 F- entA fepA thi trpE proC leuB lacY mtl M. McIntosh
xyl galK ara rpsL azi tsx supE
MM272-60 F- fes thi trpE proC leuB lacY mtl xyl (25)
galK ara rpsL azi tsx supE recA
BZB1013 F- fepA thyA-36 deoC2 IN1 T. Pugsley
Phage
øEC2 Generalised transducing phage from (67)
E. chrysanthemi strain 3690
Plasmid
pUC19 2.7 kb vector, AmpR (68)
pT7.6 pT7.1 derivative, ApR (42)
pWSK29 pSC101 derivative, AmpR (69)
pUC18K 850-bp aphA-3 cassette in the SmaI (39)
site of pUC18, KmR, AmpR
pTF12 8.1 kb PstI-EcoRI fragment (23)
in pRK767, TcR
pTF6.34 pTF6 derivative with (64)
fct-34::Tn5-B20, TcR, KmR
pDE34 6.1 kb HindIII-SalI fragment from (18)
pTF6.34 in pUC18, ApR
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pCS1 Truncated derivative from the SalI This work
site of pDE34 (6 kb)
pCS2 1.9 kb HpaI-BamHI fragment from This work
pDE34 cloned into BamHI HincII sites of pUC19
pCS3 2.1 kb DraI-EcoRI fragment from This work
pDE34 cloned into pT7.6
pLR1 Derivative of pCS3 with a 160 bp This work
SspI-EcoNI deletion
pLR2 Derivative of pCS2 with a 545 bp This work
SacII-BamHI deletion
pLR3 3.9 kb EcoRI-EcoNI fragment from This work
pTF12 cloned into pWSK29
pLR4 850-bp aphA-3 cassette from pUC18K This work
cloned into pCS2
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Table 2 : Stimulation of the growth of E. chrysanthemi and E. coli mutants by enterobactin and
chrysobactin
____________________________________________________________________________
Strain and genotype Iron source (µM DHB)
Enterobactin (50 µM) Chrysobactin (50 µM)
24 H 24 H > 72 H
____________________________________________________________________________
E. chrysanthemi L2
fct-34 ++ - -
acs-37 cbsE-1 ++ ++ +++
cbsH-19 ++ - ++
cbsH-19 acs-37 cbsE-1 ++ - ++
cbsH-19 acs-37 cbsE-1 pLR2 ++ +++ +++
cbsH::aphA-3 acs-37 ++ - ++
cbsH::aphA-3 acs-37 pLR2 ++ +++ +++
E. coli MM272-60
fes - -
fes pCS2 - -
fes pLR2 - -
fes pLR3 - ++
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fes pTF12 - ++
______________________________________________________________________________
The growth of the mutants on EDDHA-L agar medium was scored in the presence of 10 µl of
enterobactin or chrysobactin corresponding to 50 µM DHB equivalents, as described in
Experimental Procedures. Diameters of growth zones (in mm) are indicated by signs: + (13 ± 2),
++ (18 ± 2), +++ (24 ± 2), - no growth. Experiments were carried out at least in duplicate.
Table 3 : Hydrolytic activities determined in cell lysates from cbsH positive and negative strains
____________________________________________________________________________
Strain FeCb2 hydrolysed FeEnt hydrolysed
____________________________________________________________________________
L2 cbsE-1 29.4 (3.64) 2.30 (0.75)
L2 cbsH-19 1.03 (0.6) 0.64 (0.33)
____________________________________________________________________________
Enzymatic activities were assayed in whole cell extracts from cultures grown in conditions as
described in Experimental Procedures. Specific enzyme activity is expressed in nanomoles of
ferric chrysobactin (FeCb2) or ferric enterobactin (FeEnt) degraded per mg of protein per hour.
For each strain, three batches of cells were prepared and numbers in brackets represent the
standard deviation.
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Table 4 : Mössbauer parameters of chsH+ (Fig. 6) cells and cbsH cells at various incubation
times determined by least-squares-fits of Lorentzians. (A) represent values of the ferric iron
component and (B) of the ferrous iron component.
(A)
strain
genotype
δ (mm.s-1) ∆(mm.s-1) Γ(mm.s-1) relative
area/%
time
(min)
cbsH+
0.48
0.65
0.58
13
0
cbsH 0.42 0.72 0.75 70 0
cbsH+ 0.48 0.65 0.59 22 30
cbsH 0,50 0.71 0.57 24 30
cbsH+ 0.47 0.65 0.46 61 60
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cbsH 0.49 0.69 0.78 53 60
cbsH+ 0.49 0.708 0.66 64 120
cbsH 0.50 0.68 0.5 78 120
(B) strain
genotype
δ (mm.s-1) ∆ (mm.s-1) Γ(mm.s-1) relative
area/%
time
(min)
cbsH+ 1.28 3.14 0.58 87 0
cbsH 0.96 ( ?) 1.54 ( ?) 0.39 ( ?) 30 0
cbsH+ 1.25 3.01 0.64 78 30
cbsH 1.26 3.02 0.71 76 30
cbsH+ 1.20 2.86 0.55 39 60
cbsH 1.27 3.03 0.72 47 60
cbsH+ 1.22 3.04 0.76 35 120
cbsH 1.25 2.95 0.52 22 120
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Lise Rauscher, Dominique Expert, Berthold F. Matzanke and Alfred X. Trautweinof an homologue of the Escherichia coli ferric enterobactin esterase
Chrysobactin-dependent iron acquisition in Erwinia chrysanthemi: Functional study
published online November 1, 2001J. Biol. Chem.
10.1074/jbc.M107530200Access the most updated version of this article at doi:
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