1 JBC Papers in Press. Published on November 1, 2001 as ...

Erwinia chrysanthemi chrysobactin peptidase

1

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 : [email protected]

Running Title :


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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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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Edward Marklin

Fig. 6 Fig. 6

Edward Marklin

Edward Marklin

http://www.jbc.org/


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Edward Marklin

Fig. 7

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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.

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