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Journal of Bacteriology, June 1999, p. 3610-3612, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Salmonella typhimurium IroN and FepA
Proteins Mediate Uptake of Enterobactin but Differ in Their
Specificity for Other Siderophores
Wolfgang
Rabsch,1
Wolfgang
Voigt,1
Rolf
Reissbrodt,1
Renée M.
Tsolis,2 and
Andreas
J.
Bäumler3,*
National Reference Center for Salmonellae and
Other Enterics, Robert Koch-Institute, D-38855 Wernigerode,
Germany1; Department of Veterinary
Pathobiology, College of Veterinary Medicine, Texas A&M University,
College Station, Texas 77843-44672; and
Department of Medical Microbiology and Immunology, College
of Medicine, Texas A&M University Health Science Center, College
Station, Texas 77843-11143
Received 19 January 1999/Accepted 25 March 1999
 |
ABSTRACT |
Salmonella typhimurium possesses two outer membrane
receptor proteins, IroN and FepA, which have been implicated in the
uptake of enterobactin. To determine whether both receptors have
identical substrate specificities, fepA and
iroN mutants and a double mutant were characterized. While
both receptors transported enterobactin, the uptake of
corynebactin and myxochelin C was selectively mediated by IroN and
FepA, respectively.
| |
TEXT |
Iron is essential for the
multiplication of enterobacteria, since it is a component of enzymes
(e.g., ribonucleotide reductase) which are required for the
biosynthesis of macromolecules (e.g., DNA) and energy-generating
electron transport processes. In order to obtain iron from insoluble
Fe(III) complexes present under aerobic growth conditions,
enterobacteria release low-molecular-weight compounds, designated
siderophores, which bind this metal ion with high affinity
(4). The Fe(III)-siderophore complexes are then internalized
by iron-regulated outer membrane receptor proteins which display
substrate specificity. The primary siderophore produced by
enterobacteria is enterobactin, a cyclic trimer of
N-(2,3-dihydroxybenzoyl)-L-serine (DBS). In
Escherichia coli, transport of enterobactin across the outer
membrane is mediated by the FepA outer membrane receptor protein
(13).
An orthologue of fepA is present in Salmonella
enterica, but the phenotype of a strain carrying a mutation in
this gene has not been described to date (7, 19, 21).
Recently, iroN, a gene absent from the E. coli
K-12 genome, has been shown to encode an outer membrane siderophore
receptor of S. enterica serotype Typhimurium (S. typhimurium) (3). When introduced into an E. coli
fepA aroB mutant, the cloned iroN gene mediates the
utilization of enterobactin as the sole source of iron
(20a). However, an S. typhimurium iroN aroA
mutant is not deficient in enterobactin uptake, presumably because it
is still able to utilize this siderophore via FepA. These data suggest
that S. typhimurium possesses two enterobactin receptor
proteins, IroN and FepA.
The ability to produce siderophores is one of the main strategies by
which microbes acquire Fe(III) in the environment. In addition to
receptors for the utilization of enterobactin, S. typhimurium possesses several outer membrane receptors, including FhuA, FhuE, and FoxA, which are involved in the utilization of siderophores that are not produced by this organism (12,
21). Thus, a second strategy apparently used by S. typhimurium to acquire Fe(III) is to steal siderophores produced
by other microbes. This siderophore piracy (6) may explain
why S. typhimurium possesses two receptors for the
utilization of enterobactin. The presence of these two outer membrane
enterobactin receptors may be advantageous under conditions when the
available siderophores can be utilized only through either FepA or
IroN. An important assumption in this line of reasoning is that
although FepA and IroN both serve as enterobactin receptors, each
receptor may in addition mediate the uptake of substrates that are not
recognized and transported by any other siderophore receptor in
S. typhimurium. To test this hypothesis, we constructed
S. typhimurium strains lacking FepA or IroN or both
receptors. Analysis of the siderophore profile utilized by these
mutants enabled us to determine that the enterobactin receptors IroN
and FepA differ in their substrate specificities.
Isolation of an S. typhimurium fepA mutant.
The
fepA gene is located at 14 min on the E. coli
genetic map in a DNA region containing enterobactin biosynthesis
(ent) genes (11, 13-15). According to a recent
analysis of the 5' end of the fepA gene and the adjacent DNA
region, this genetic organization is conserved in S. typhimurium with regard to gene order and map location
(21). Since it is transcribed as a monocistronic messenger, mutational inactivation of fepA has no effect on the
biosynthesis of enterobactin in E. coli. Based on these
data, we reasoned that a selectable marker in fepA could be
used to move enterobactin biosynthesis genes by cotransduction
(16). Our strategy to identify a fepA mutant was
therefore to characterize transposon insertions, which are cotransduced
with enterobactin biosynthesis genes. A phage lysate of a random bank
of S. typhimurium Tn10dTc mutants was transduced
into an S. typhimurium mutant (TA2700) which is defective
for enterobactin production (17). Transductions were performed by using the high-frequency generalized transducing phage
mutant P22HT 105/1 int 201. Transductants were selected on chrome
azurol S (CAS) agar plates, on which colonies producing enterobactin
can be readily detected by their ability to form a halo
(20). A total of 130 colonies producing a halo on CAS agar
plates were isolated, and enterobactin production was confirmed in a
cross-feeding test with the S. typhimurium
ent mutant (TA2700) (18). Outer membranes were
isolated from these mutants as described previously by Hantke
(8), and proteins were separated by using sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Protein bands were
visualized by Coomassie blue staining. One mutant, WR1316, lacked an
83-kDa outer membrane protein, the size reported for FepA (data not
shown). The Tn10dTc insertion in WR1315 was mapped to the
enterobactin biosynthesis region by cotransduction with
entB::MudJ (cotransduction frequency,
2%) and ent::MudJ from strain AR8439
(cotransduction frequency, 5%) (21). To confirm transposon
insertion in fepA, a Southern blot of
EcoRI-restricted genomic DNA of strains TA2700 and WR1315
was probed with the labeled (nonradioactive labeling kit from NEN)
insert of pFT17, a plasmid carrying the 5' ends of the S. typhimurium fepA gene (21). The probe hybridized with
restriction fragments of 10 and 3 kb in TA2700 and WR1315,
respectively. Thus, inactivation of fepA in strain WR1315
was suggested by Southern hybridization and the lack of an 83-kDa outer
membrane protein.
Utilization of siderophores by FepA and IroN.
In order to
study siderophore utilization of the fepA mutant, the
Tn10dTc insertion in WR1315 was transduced into TA2700
(ent), and a transductant (WR1316) which was tetracycline
resistant and unable to produce enterobactin was selected on CAS agar
plates. The ability of strain WR1316 to utilize enterobactin and
structurally related siderophores was
tested. For this purpose, enterobactin was prepared and purified from E. coli AN311 according to a
protocol reported previously (22). Myxochelin
derivatives were synthesized and characterized by W. Trowitzsch-Kienast and H. D. Ambrosi, Technische
Fachhochschule Berlin, Berlin, Germany (1, 10). The
siderophore corynebactin was isolated from Corynebacterium glutamicum (5) and kindly provided by H. Budzikiewicz,
Institute of Organic Chemistry, University of Cologne, Cologne,
Germany. Utilization of siderophores was detected by an agar diffusion assay (18). To create iron-limiting growth conditions, 0.15 mM 2,2'-dipyridyl was added to Vogel Bonner medium (VBD)
(18). The strain to be tested was poured into 3 ml of VBD
top agar (VBD with 2% agar)-Noble agar onto a VBD agar plate. Filter
paper disks impregnated with 5 µl of a 1-mg/ml solution of the
respective siderophores were laid onto the top agar and, after
incubation overnight at 37°C, growth stimulation around the filter
disk was recorded. Comparison of the profiles of the siderophores
utilized by WR1316 (ent fepA) and its parent, TA2700
(ent), revealed that myxochelin C utilization was abolished
in the fepA mutant (Table 1).
To quantify growth stimulation by siderophores, strains TA2700 (ent) and WR1316 (ent fepA) were cultured in VBD
broth, and bacterial growth was recorded by measuring the absorbance at
620 nm. Myxochelin C stimulated the growth of strain TA2700, but not
that of strain WR1316, in VBD broth.
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FIG. 1.
Substrate specificity of the S. typhimurium
IroN and FepA outer membrane (OM) receptor proteins. PP, periplasmic
space. The structures of enterobactin, corynebactin, and myxochelin C
are shown above.
|
|
We next tested whether the defect in myxochelin C utilization in WR1316
could be restored by introducing the cloned fepA gene from
E. coli. A plasmid (pITS449) (2) carrying the
E. coli fepA gene was kindly provided by Phil E. Klebba, University of Oklahoma. Introduction of plasmid pITS449
(E. coli fepA) restored the ability of strain WR1316 to
utilize myxochelin C as an iron source (Table 1). These data showed
that FepA can mediate the uptake of myxochelin C in S. typhimurium. Furthermore, the inability of WR1316 (ent
fepA) to utilize myxochelin C suggested that FepA is the only
outer membrane receptor involved in the transport of this substrate in
S. typhimurium.
Unlike an E. coli fepA mutant, the S. typhimurium
fepA mutant (WR1316) was able to utilize enterobactin, presumably
because of the presence of a second enterobactin receptor (Table 1). To
test the hypothesis that S. typhimurium possesses two outer membrane enterobactin receptors, strain TA2700 (ent) was
used to generate isogenic ent iroN (WR1223) and ent
iroN fepA (WR1332) mutants. A iroN allele which carries
an insertion of suicide vector pGP704 (9) was moved by P22
transduction from S. typhimurium AJB64 (3). The
parent (TA2700, ent), the ent fepA mutant
(WR1316), and the ent iroN mutant (WR1223) were able to
utilize enterobactin. However, utilization of enterobactin and DBS were
abolished in the ent iroN fepA mutant (WR1332), suggesting
that both IroN and FepA can mediate uptake of these siderophores in
S. typhimurium (Table 1).
In contrast to the ent mutant (TA2700) and the ent
fepA mutant (WR1316), strains WR1223 (ent iroN) and
WR1332 (ent iroN fepA) were unable to utilize corynebactin,
suggesting that this siderophore is transported via IroN in S. typhimurium (Table 1). To quantify growth stimulation by
siderophores, bacterial strains were cultured in VBD broth supplemented
with corynebactin, myxochelin C, or enterobactin. As a control, VBD
broth was supplemented with myxochelin B, a siderophore, which is not
transported by FepA or IroN. VBD broth supplemented with corynebactin
did not support the growth of strain WR1223 (ent iroN) or
WR1332 (ent iroN fepA). Furthermore, strain WR1332 was
unable to grow in VBD broth supplemented with myxochelin C. While
strain TA2700 and its derivatives (WR1332 and WR1223) were unable to
grow in VBD broth lacking siderophore supplements, all strains were
able to grow in VBD broth supplemented with myxochelin B. Enterobactin
promoted the growth of strains TA2700 (ent), WR1223
(ent iroN), and WR1316 (ent fepA) but not that of
strain WR1332 (ent iroN fepA). While cultures of strains TA2700 (ent) and WR1223 (ent iroN) reached the
same density (optical density at 620 nm [OD620] = 0.5),
the growth of strain WR1316 (ent fepA) was reduced
(OD620 = 0.4), suggesting that enterobactin is utilized
more effectively by FepA than by IroN.
Our data show that S. typhimurium possesses two outer
membrane enterobactin receptor proteins, IroN and FepA. These receptor proteins differ in their specificities for other substrates, such as
myxochelin C and corynebactin (Fig. 1). Corynebactin is excreted by
Corynebacterium glutamicum, an organism found in soil, an
environment frequently encountered during the fecal-oral transmission
of S. typhimurium. Growth in this environment may require
the ability to utilize a wide spectrum of siderophores produced by the
bacteria in the soil. It could therefore be speculated that IroN and
FepA facilitate growth in soil, since the presence of these receptor proteins may increase the capability of S. typhimurium to
obtain iron by siderophore piracy.
| |
ACKNOWLEDGMENTS |
We thank K. E. Sanderson for providing S. typhimurium strains and K. Hantke and J. R. Roth for helpful
discussions and support of the genetic work. We thank P. E. Klebba
for kindly providing us with the fepA plasmid. Ute Strutz is
gratefully acknowledged for her excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology and Immunology, College of Medicine, Texas A&M
Univ. Hlth. Sci. Ctr., 407 Reynolds Medical Building, College Station, TX 77843-1114. Phone: (409) 862-7756. Fax: (409) 845-3479. E-mail: abaumler{at}tamu.edu.
| |
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Journal of Bacteriology, June 1999, p. 3610-3612, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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