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Antimicrobial Agents and Chemotherapy, May 2001, p. 1317-1322, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1317-1322.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Screening System for Xenosiderophores as Potential
Drug Delivery Agents in Mycobacteria
Gisbert
Schumann and
Ute
Möllmann*
Department of Infection Biology,
Hans-Knöll-Institut für Naturstoff-Forschung e.V., D-07708
Jena, Germany
Received 28 November 2000/Returned for modification 5 January
2001/Accepted 12 February 2001
 |
ABSTRACT |
In order to establish a screening system for xenosiderophores which
can be utilized by mycobacteria, we generated a set of mutants of
Mycobacterium smegmatis that are blocked in different steps
of the well-known iron acquisition system. One mutant with a block in
mycobactin biosynthesis was generated from strain mc2155 by
chemical mutagenesis. The exochelin biosynthesis gene fxbA and the ferric exochelin uptake gene fxuA, previously
identified by Fiss et al. (E. H. Fiss, S. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557-559, 1994), were knocked out by
gene replacement. Adjacent chromosomal fragments were used for
homologous recombination in order to replace wild-type genes by the
kanamycin resistance gene from transposon Tn903. Gene
replacement was confirmed by PCR. The isolated mutants show the
expected phenotype: fxbA mutants are defective in exochelin
biosynthesis, whereas fxuA mutants excrete a significantly
larger amount of exochelin compared to the amount excreted by the
parent strain. This is due to their defectiveness in ferriexochelin
uptake, as demonstrated in growth promotion assays. This new set of
mutants allows differentiation of siderophores that supply mycobacteria
with iron by ligand exchange with exochelin or mycobactin, by the use
of separate siderophore uptake routes, or by the use of the exochelin
permease. All these types of iron uptake routes were identified with 25 exogenous siderophores as test substances. Siderophores that act
without ligand exchange are potential candidates as drug vectors that can be used to overcome permeability-mediated resistance.
| |
INTRODUCTION |
Increasing numbers of mycobacterial
infections and the spread of resistance against the frontline
antituberculosis agents urgently demand a search for novel drugs with
new mechanisms of action. The low levels of permeability of the
extraordinarily thick and hydrophobic cell envelopes of the
mycobacteria limit the activities of antimycobacterial agents. The aim
of our study was to provide a basis for overcoming the permeability
barrier of the mycobacterial envelope by conjugation of drugs to
species-specific vectors and by the use of the concomitant transport
systems essential for the pathogens. The ability to acquire iron is
essential for the survival of mycobacteria and other pathogens and is
an important virulence factor. High-affinity iron sequestration systems
have therefore evolved (3). Siderophores are part of these
systems and are the basis for new antibiotic vectors that are useful as means of circumventing membrane-associated resistance (1). Thus, a diversity of iron-siderophore uptake routes would provide a
good basis for realization of the shuttle transport of drugs through
the bacterial membrane.
Mycobacteria produce two classes of siderophores, the mycobactins and
the extracellular hydrophilic exochelins. Mycobactin was initially
isolated from Mycobacterium phlei by Snow in 1965 (19), and a large number of mycobactins which have a
common core structure have been isolated (3). The cell
wall-associated lipophilic mycobactins are synthesized by most
mycobacterial species and are thought to facilitate the transport of
iron across the cell wall (9, 14). Structurally related
water-soluble mycobactins which are excreted into the environment bind
to iron and transfer it to the cell wall-associated mycobactins
(5, 7, 8). Water-soluble mycobactins are the only
extracellular siderophores expressed by pathogenic mycobacteria, while
the saprophytic strains Mycobacterium smegmatis and
Mycobacterium neoaurum use exochelins as main siderophores
for iron acquisition (12, 16, 17). Exochelin-mediated iron
uptake into M. smegmatis is both a receptor-dependent and an
energy-dependent process similar to the intensively studied uptake of
siderophores in Escherichia coli (2). The genes
involved in the biosynthesis and export of exochelin and in the uptake of ferriexochelin have been identified and cloned (4, 21, 22).
Not only those siderophores that are produced by the organism itself
can be utilized, but also siderophores that are synthesized by other
microorganisms (xenosiderophores). Mycobacteria were found to utilize
ferrirhodotorulic acid, isolated from Rhodotorula strains
(20); ferricrocin, produced by Aspergillus and
Neurospora species (10); and rhizoferrin,
produced by Rhizopus strains (11). These
results were obtained with wild-type strains and methods that use
55Fe-labeled siderophores and in situ Mössbauer
spectroscopy. Our goal is to develop a new efficient screening method
for the identification of the iron siderophores utilized by
mycobacteria. In this context it is very important to consider the
potential ligand exchange with exochelins and mycobactins in the iron
supply of wild-type mycobacteria by xenosiderophores.
In order to identify direct uptake routes for siderophores that
circumvent ligand exchange with endogenous siderophores, it is
necessary to monitor the passage of the siderophore-bound iron from the
outside to the inside of the cell by radiolabeled Fe3+
transport experiments or by time-consuming and expensive
Mössbauer spectroscopy or electron spin resonance spectroscopy.
The latter elaborate methods are useful only for single compounds and
not for the screening of numerous samples. To overcome these
limitations, it is important to generate mutant strains which lack
endogenous siderophore genes.
In order to establish an efficient screening system that can be used to
find xenosiderophores which can be used as a possible platform for
synthesis of siderophore-antibiotic conjugates, we selected M. smegmatis as a model system because it is a nonpathogenic, fast-growing mycobacterium which is genetically more tractable than
Mycobacterium tuberculosis. In this report we describe the generation of mutants blocked in the biosynthesis of exochelin and/or
mycobactin or in the transport of exochelin. These mutants provide a
basis for the screening of effective siderophores in a growth promotion
assay. This set of strains enables determination of whether there is
ligand exchange with exochelin and/or mycobactin or uptake by an
alternative transport system.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
The mycobacterial strains
and plasmids used in this study are listed in Table
1. The E. coli strain used in
this study was DH5
(Stratagene). The standard plasmid cloning vector
pUC118 was from laboratory stock.
Media and growth conditions.
E. coli cultures
were routinely grown in Luria-Bertani medium (Difco Laboratories) at
37°C. The following antibiotics, when required, were added at the
indicated concentrations: ampicillin, 100 µg/ml for E. coli; kanamycin, 30 µg/ml for E. coli and M. smegmatis. Mycobacterium cultures were grown in Middlebrook 7H9 broth (Difco Laboratories) supplemented with glycerol (0.2%), Tween 80 (0.05%), and albumin-dextrose-catalase enrichment (10%; Difco
Laboratories) at 37°C. The solid medium for plating and maintenance
of mycobacterial strains was Middlebrook 7H10 agar (Difco Laboratories)
supplemented with glycerol (0.5%) and oleic acid-albumin-dextrose-catalase enrichment (10%; Difco Laboratories) or
Oxoid medium no. 1.
Genetic methods.
Restriction enzymes (Boehringer Mannheim),
T4 DNA ligase (Gibco BRL), and Taq DNA polymerase (Qiagen)
were used in accordance with the manufacturers' recommendations. The
DNA fragments used in the cloning procedures were gel purified with
SeaPlaque GTG low-melting-temperature agarose (FMC), followed by the
recovery of DNA from agarose gel with GenElute agarose spin columns (Supelco).
Plasmids were isolated with a QIAprep Spin kit (Qiagen) and transformed
into
E. coli strains by standard techniques or
electroporated
into
M. smegmatis strains with a Gene Pulser
(Bio-Rad Laboratories)
according to the manufacture protocol, with
modifications for
electroporation into
M. smegmatis used as
described before (
18).
Electroporation conditions were
1,000
, 2,500 V, and 25 µF in
0.2-cm-electrode-gap cuvettes with
0.2 ml of mycobacteria, which
produced time constants of 16 to 21 ms.
The mycobacteria were
grown to the mid-logarithmic phase, followed by
extensive washing
with 10% glycerol at 4°C and final resuspension in
a 1/100 volume
of ice-cold 10%
glycerol.
Chromosomal DNA from mycobacterial strains was isolated by using a
DNeasy tissue kit (Qiagen). The DNA was eluted with 100
µl of buffer
AE (5 mM Tris-HCl, pH 8.5), and 2 µl of the eluant
was used in a
100-µl PCR mixture. PCR amplification for the cloning
of the products
was carried out with
Pwo DNA polymerase (Boehringer
Mannheim) according to the manufacturer's recommendations and
with the
addition of 10% dimethyl sulfoxide (DMSO) to the mixtures.
Reactions
took place as follows: denaturation in a 50-µl mixture
containing
DNA,
Pwo buffer, primer, and DMSO at 98°C for 10 min
and
addition of 50 µl of a second mixture containing
Pwo
buffer,
deoxynucleoside triphosphates, and
Pwo DNA
polymerase, followed
by 30 cycles of denaturation at 96°C for 1 min,
primer annealing
at 45 to 56°C for 1 min, and primer extension at
72°C for 1 min.
PCR amplification for the analysis of transformants
was carried
out with
Taq DNA polymerase (Qiagen) and
Q-solution (Qiagen) instead
of DMSO. Oligionucleotide primers were
obtained from MWG-Biotech
AG, Ebersberg, Germany, and are listed in
Table
2.
The fidelities of the PCR-amplified DNA fragments were established by
nucleotide sequencing after subcloning. Automated sequencing
was
performed with a Licor DNA sequencer with dye-primer cycle
sequencing
chemistry.
Chemical mutagenesis of
M. smegmatis mc
2155 was
carried out with a culture grown for 2 days in medium TL, which
contained (per
liter) glycerol, 10 g; meat extract, 5 g;
peptone, 5 g; and NaCl,
3 g (pH 7.0). Cells that had been
harvested by centrifugation
were washed in sterile water and
resuspended in medium TL to the
density of a no. 3 McFarland standard
(9 × 10
8 CFU per ml; BioMerieux).
N-Methyl-
N'-nitro-
N-nitrosoguanidine
was added at 1.5 mg per ml, and the suspension was incubated for
1 h at 37°C. After appropriate dilution the cells were plated
onto
Oxoid no. 1 agar
plates.
Assays.
The production of mycobactin was determined by
measurement of the UV fluorescence at 264 nm of patched colonies grown
for 3 days at 37°C on the iron-limiting medium GHE, which contained (per liter) yeast extract (Difco), 10 g; glucose, 10 g;
ethylendiamin-di-(o-hydroxyphenylacetic acid) (EDDHA), 3.6 mg; and agar (Difco), 15 g.
For the detection of exochelin production, chrome azurol S (CAS) medium
(
15) was used with the modifications described previously
(
4).
The utilization of exochelin and heterologous siderophores was
determined by growth promotion assays.
M. smegmatis mutants
were grown for 2 days on Oxoid no. 1 agar, suspended in medium
VA
(glycerol, 50 ml; NaCl, 8.5 g per liter [pH 7.2]), and diluted
to the density of a no. 1 McFarland standard (3 × 10
8
CFU per ml). A total of 1.5 ml of this suspension was mixed with
99 ml
of test medium prepared as described by Hall and Ratledge
(
6) (glycerol, 20 ml;
L-asparagine, 5 g;
KH
2PO
4, 5 g per liter
of distilled water
[pH 7.5]). After the addition of 20 g of
Al
2O
3 and sterilization at 121°C for 15 min,
the suspension was filtered
and the pH was adjusted to 6.8. A total of
12 g agar (Oxoid no.
1) was added, and the medium was sterilized
again at 121°C for
15 min. Prior to the inoculation,
ZnSO
4 · 7H
2O, 2.03 mg;
MnSO
4 · 4H
2O, 0.405 mg;
MgSO
4, 0.2 mg; CaCl
2 · 2H
2O,
1 mg; Na
2MoO
4 ·
2H
2O, 0.2 mg; CuSO
4 · 5H
2O, 0.2 mg;
CoCl
2 · 6H
2O, 0.4 mg; and
EDDHA,
3.6 g, were added to the medium as a filter-sterilized
solution.
Siderophore solutions were applied to assay disks (diameter,
6 mm) the
disks were placed onto the surfaces of the inoculated
test agar plates,
and the zones of growth surrounding the disks
were estimated after
incubation for 2 days at 37°C.
Siderophores.
The siderophores were kindly supplied as
indicated in Table 4.
| |
RESULTS |
Construction of mutants.
In the first step, a
mycobactin-deficient mutant was isolated from strain mc2155
after chemical mutagenesis with nitrosoguanidine. Mutagenized cells
were screened for clones that lacked UV fluorescence on iron-depleted
GHE medium. Of 72 colonies tested, one mutant (mutant M24) that lacked
UV fluorescence was identified. The loss of mycobactin biosynthesis was
confirmed by high-pressure liquid chromatography and mass spectroscopy
of ethanolic biomass extracts (data not shown). Because
nitrosoguanidine is known to produce multiple mutations, we cannot
exclude the possibility that M24 has additional defects other than that
which precludes mycobactin synthesis; but our tests have shown that the
strain is not affected in relevant characteristics such as growth,
production, and excretion of exochelin or utilization of mycobactin-
and exochelin-bound iron.
In the second step, mutant strain M24 and wild-type strain
mc
2155 were used to generate defined mutants with a block
in exochelin
biosynthesis or ferric exochelin uptake by gene
replacement. The
exochelin biosynthesis gene
fxbA and the
ferric exochelin uptake
gene
fxuA, previously identified by
Fiss et al. (
4), were replaced
by the aminoglycoside
phosphotransferase (
aph) gene of Tn
903,
which
confers resistance to kanamycin in
M. smegmatis. The gene
was obtained as a 1,264-bp
BamHI fragment from pUC4K.
Chromosomal
fragments adjacent to the target genes (Fig.
1) were amplified
by PCR and cloned into
the
E. coli vector pUC118 on flanking sites
of the
aph gene. Restriction sites in the primers used to amplify
these fragments (Table
2) allowed targeted insertion in the orientation
of the chromosomal context.
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FIG. 1.
Restriction map and genetic organization of the part of
exochelin gene cluster of M. smegmatis mc2155
relevant to the present study. The arrows indicate the direction of
gene transcription. Boxes represent chromosomal fragments cloned in
pGS22 (fragments B and C) and pGS31 (fragments A and B). Fragment A is
the 507-bp part upstream of fxbA gene, fragment B (758 bp)
contains the entire fxuB gene, and fragment C (768 bp)
contains a part of the fxuC gene.
|
|
The resulting constructs contain an origin of replication that permits
autonomous propagation in
E. coli but not in
M. smegmatis.
The cloned chromosomal fragments permit integration
into
M. smegmatis genomic DNA by homologous recombination,
and a double crossover
in both fragments results in the replacement of
the target gene
by the
aph gene (Fig.
2A and
3A).
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FIG. 2.
Replacement of the fxbA gene in M. smegmatis mc2155. (A) Strategy used to replace the
fxbA gene by double crossover. The horizontal arrows
indicate annealing sites of primers used to confirm the replacement
(B1-K1, B9-K2) by using the chromosomal DNA of mutant strain B1 as the
template and to generate control fragments by using plamid pGS31 as the
template. The expected sizes of the fragments are indicated between the
arrows. (B) Confirmation of replacement of the fxbA gene by
PCR analysis of DNA from the B3  fxbA mutant.
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FIG. 3.
Replacement of the fxuA gene in M. smegmatis mc2155 mutant strain M24. (A) Strategy used
to replace the fxuA gene by double crossover. The horizontal
arrows indicate annealing sites of primers used to confirm the
replacement (U3-K1, U9-K2) by using the chromosomal DNA of mutant
strain U3 as the template and to generate control fragments by using
plasmid pGS22 as the template. The expected sizes of the fragments are
indicated between the arrows. (B) Confirmation of replacement of the
fxuA gene by PCR analysis of DNA from the U3
fxuA mutant.
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|
M. smegmatis mc
2155 and M24 were transformed
with these plasmids by electroporation, and kanamycin-resistant clones
were selected.
For the identification of mutants blocked in exochelin
biosynthesis
by replacement of the
fxbA gene,
kanamycin-resistant clones were
selected after transformation with
pGS31 (Table
1) and were screened
on CAS medium (
5). A
total of 145 transformants of both strains
were analyzed, and 4 mutants
that lacked an orange halo on CAS
medium were identified (Fig.
4). Replacement of
fxbA by
homologous
recombination by double crossover was confirmed by PCR (Fig.
2B).
DNA isolated from the mutants was subjected to PCR amplification
with primers that anneal at both ends of the
aph cassette
and
at chromosomal sites that flank the cloned fragments. Control
fragments were generated with pGS31 as a template and primers
which
bound at the ends of the cloned fragments.
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FIG. 4.
CAS plate assay of isolated mutants. The presence of an
orange halo indicates the exochelin production. 1, mc2155
wild type; 2, mutant strain M24 (mycobactin negative); 3, mutant strain
U3 fxuA; 4, mutant strain B3 fxbA.
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|
To screen mutants blocked in ferric exochelin uptake,
kanamycin-resistant clones selected after transformation with pGS22
(Table
1) were tested in a growth promotion assay (Fig.
5). Among
the 75 transformants tested
from strain M24 only, 1 mutant which
did not grow in the presence of
ferric exochelin could be identified.
The replacement of
fxuA gene, as confirmed by PCR (Fig.
3B), resulted
in
exochelin hyperproduction, as monitored on CAS agar (Fig.
4).
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FIG. 5.
Growth promotion assay with an fxuA
mutant. (A) Control plate inoculated with wild-type strain
mc2155. (B) Test plate inoculated with mutant strain U3
fxuA. Zones of growth surrounding the disks containing
exochelin or mycobactin indicate utilization of siderophores by the
tested strains. 1, mycobactin; 2, exochelin.
|
|
The various mutants generated during our study showed different growth
characteristics. Compared to parent strain mc
2155, mutants
M24 and B1 are able to grow under conditions of iron
depletion with a
slightly increased lag phase because M24 can
take up exochelin directly
via the permease and B1 can use low-molecular-weight
iron chelators
like citrate via mycobactin (
10). B3 and U3 are
nearly
completely unable to grow in the absence of an alternative
iron supply
because of their lack of mycobactin and exochelin
or exochelin
permease. Both strains, however, can grow under conditions
of iron
depletion when xenosiderophores are added. This observation
indicates
the existence of additional mechanisms of direct uptake
of siderophores
besides the exochelin-mycobactin
route.
These new mutants and wild-type strains differ in the presence or
absence of the endogenous siderophores exochelin and mycobactin
as well
as in the presence or absence of the exochelin permease
(Table
3). By use of these strains in growth
promotion experiments,
it is possible to differentiate between
siderophore iron supply
processes that include ligand exchange with
exochelin and/or mycobactin
and those that exclude ligand exchange. In
addition, it is now
possible to differentiate between uptake via the
exochelin permease
and uptake via an alternative transport system.
Consequently,
the mutants can be used to generate an ideal screening
system
for the selection of siderophores that are able to enter the
mycobacterial
cell directly on the basis of simple and efficient growth
promotion
assays.
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TABLE 3.
Phenotypic characteristics of the various strains of
M. smegmatis generated and used in the present
studya
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|
Several exogenous siderophores of different structural classes were
tested with this new screening model. Exochelin and mycobactin
were
used as controls. The results are shown in Table
4. Six
xenosiderophores showed the same
effectiveness in growth promotion
in all strains tested, indicating the
utilization of the siderophore-bound
iron which is independent of
ligand exchange or exochelin permease.
With the other 12 xenosiderophores tested, the growth responses
of the wild-type strain
and the mutants used in the test were
different. These findings imply
that ligand exchange with exochelin
and/or mycobactin accounts for the
process of iron uptake from
these substrates or that the exochelin
permease is involved in
the uptake of the xenosiderophore.
| |
DISCUSSION |
Previously, Matzanke et al. (10) have shown that
mycobacteria utilize a variety of xenosiderophores. Utilization of
siderophore-bound iron occurs by diverse mechanisms which include
ligand exchange between citrate and mycobactin or rapid reductive
removal from ferricrocin. Whether ferricrocin is transported into the
cytoplasm before reduction or the reduction occurs at the membrane
remained unclear. The results presented by Matzanke et al.
(11) imply a third uptake mechanism for rhizoferrin which
excludes mycobactin as a temporary iron carrier. Moreover, the
intracellular presence of small amounts of ferric rhizoferrin strongly
suggests that the complex was transported into the cytoplasm as a
whole. The complete permeation of the cell envelope by the siderophore
is a prerequisite for the possible use of siderophore-antibiotic conjugates to circumvent the barrier problem of the relatively impermeable, hydrophobic cell envelopes of mycobacteria. These results
were obtained by Mössbauer or electron spin resonance spectroscopy, which is usable only for single compounds.
In the present study, we have generated a set of mycobacterial mutant
strains blocked in the biosynthesis of exochelin and mycobactin or in
the transport of exochelin in order to establish a screening system.
This set of strains allows testing of siderophores in a growth
promotion assay for ligand exchange with exochelin and/or mycobactin or
uptake by an alternative transport system.
Previously, Fiss et al. (4) identified four contiguous
genes that may be involved in exochelin biosynthesis and uptake by
M. smegmatis. One of these genes (fxbA) was shown
to be involved in exochelin biosynthesis by complementation of a
blocked mutant. On the basis of the similarities of the amino acid
sequences of known cytoplasm membrane permeases to the deduced amino
acid sequences of the three other genes (fxuA, fxuB, fxuC),
the investigators suggested that these genes code for the subunits of
the ferric exochelin permease.
The published sequences were used to replace fxbA and
fxuA by the aminoglycoside phosphotransferase
(aph) gene of Tn903. Functional studies confirmed
that the isolated mutants represent the expected phenotypes.
fxbA mutants are defective in exochelin biosynthesis, whereas fxuA mutants excrete significantly larger amounts of
exochelin than the parent strain. This effect is due to defective
ferriexochelin uptake, as demonstrated in growth promotion tests.
This new set of mutants were used in combination with their parent
strain as well as a mutant blocked in biosynthesis of mycobactin in a
growth promotion assay to 25 natural xenosiderophores which exhibit a
wide range of different structural building blocks.
On the basis of the results obtained with this assay system, the tested
siderophores can be divided into six different groups. (i) Coprogen,
ferrichrysin, ferrirubin, and myxochelin E are not utilized by M. smegmatis mc2155. (ii) Iron utilization mediated by
corynebactin, ferricrocin, ferrioxamine B, and ferrioxamine D1 depends
on ligand exchange with exochelin or mycobactin. Strain B3, which lacks
the ability to synthesize exochelin and mycobactin, and strain U3,
which lacks the ability to synthesize mycobactin and exochelin
permease, are unable to use these substrates. (iii) Iron bound to
ferrioxamine D2 and ferrioxamine E could not be used by the strains
that lack exochelin biosynthesis or exochelin permease. This indicates
ligand exchange with exochelin but not with mycobactin. (iv) The
results obtained with rhodotorulic acid and ferrioxamin G also suggest ligand exchange with exochelin but, additionally, suggest
mycobactin-dependent utilization. (v) The growth of all strains except
strain U3 showed excellent responses to amycolachrom and ferrithiocin.
This finding implies that iron utilization via amycolachrom and
ferrithiocin depends on the action of exochelin permease. (vi) Iron
bound to aerobactin, arthrobactin, enterobactin, ferrichrome,
ferrichrome A, myxochelin C, omibactin, rhizoferrin, and
triacetylfusarinine was utilized by all strains, although with
different levels of effectiveness. These results indicate that no
ligand exchange with exochelin or mycobactin takes place and that the
exochelin permease is not involved in uptake of these siderophores. In
conclusion, this suggests the existence of specific uptake systems for
these xenosiderophores, as already indicated for rhizoferrin
(11). All these siderophores represent potential
candidates for synthetic derivatization toward new antibiotic shuttle vectors.
| |
ACKNOWLEDGMENTS |
This work was supported by the German Ministry of Education and
Research (BMBF grant 0311232) and the Grünenthal GmbH, Aachen, Germany
We thank G. Mrotzek and K. Hartung from the HKI Department of Cell and
Molecular Biology for help with sequencing. We also thank B. F. Matzanke and P. F. Zipfel for helpful discussions and critical
reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Hans-Knöll-Institut für Naturstoff-Forschung,
Beutenbergstr. 11, D-07745 Jena, Germany. Phone: 49 3641 656656. Fax:
49 3641 656652. E-mail: moellman{at}pmail.hki-jena.de.
| |
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Antimicrobial Agents and Chemotherapy, May 2001, p. 1317-1322, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1317-1322.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
