Microbiology (2012), 158, 1317–1324
DOI 10.1099/mic.0.057430-0
Inner-membrane transporters for the siderophores
pyochelin in Pseudomonas aeruginosa and
enantio-pyochelin in Pseudomonas fluorescens
display different enantioselectivities
Cornelia Reimmann
Correspondence
Cornelia Reimmann
Département de Microbiologie Fondamentale, Université de Lausanne, CH-1015 Lausanne,
Switzerland
[email protected]
Received 2 January 2012
Revised
9 February 2012
Accepted 9 February 2012
Iron uptake and transcriptional regulation by the enantiomeric siderophores pyochelin (Pch) and
enantio-pyochelin (EPch) of Pseudomonas aeruginosa and Pseudomonas fluorescens,
respectively, are stereospecific processes. The iron-loaded forms of Pch (ferriPch) and of EPch
(ferriEPch) are recognized stereospecifically (i) at the outer membrane by the siderophore
receptors FptA in P. aeruginosa and FetA in P. fluorescens and (ii) in the cytoplasm by the two
AraC-type regulators PchR, which are activated by their cognate siderophore. Here,
stereospecific siderophore recognition is shown to occur at the inner membrane also. In P.
aeruginosa, translocation of ferriPch across the inner membrane is carried out by the singlesubunit siderophore transporter FptX. In contrast, the uptake of ferriEPch into the cytoplasm of
P. fluorescens was found to involve a classical periplasmic binding protein-dependent ABC
transporter (FetCDE), which is encoded by the fetABCDEF operon. Expression of a translational
fetA–gfp fusion was repressed by ferric ions, and activated by the cognate siderophore bound to
PchR, thus resembling the analogous regulation of the P. aeruginosa ferriPch transport operon
fptABCX. The inner-membrane transporters FetCDE and FptX were expressed in combination
with either of the two siderophore receptors FetA and FptA in a siderophore-negative P.
aeruginosa mutant deleted for the fptABCX operon. Growth tests conducted under iron limitation
with ferriPch or ferriEPch as the iron source revealed that FptX was able to transport ferriPch as
well as ferriEPch, whereas FetCDE specifically transported ferriEPch. Thus, stereospecific
siderophore recognition occurs at the inner membrane by the FetCDE transporter.
INTRODUCTION
Iron is essential for most bacteria but is only poorly soluble
at neutral pH in aerobic environments and is complexed by
iron storage and transport proteins in animal hosts
(Andrews et al., 2003). To acquire the necessary iron,
bacteria have evolved a number of strategies, the most
common being the synthesis, secretion and uptake of ironscavenging molecules termed siderophores. Siderophore
production and subsequent uptake of the iron–siderophore
(ferrisiderophore) complex are tightly regulated processes,
which not only ensure efficient iron acquisition but also
avoid iron accumulation to toxic levels.
Under iron-limiting conditions, the soil bacterium Pseudomonas fluorescens CHA0 secretes two siderophores,
Abbreviations: EPch, enantio-pyochelin; ferriEPch, iron-loaded form of
EPch; ferriPch, iron-loaded form of Pch; GFP, green fluorescent protein;
PBP, periplasmic binding protein; Pch, pyochelin; Pvd, pyoverdine.
Two supplementary tables are available with the online version of this
paper.
057430 G 2012 SGM
pyoverdine (Pvd; Meyer & Abdallah, 1978) and enantiopyochelin (EPch; Youard et al., 2007), the latter being an
enantiomer of the Pseudomonas aeruginosa siderophore
pyochelin (Pch; Liu & Shokrani, 1978; Rinehart et al., 1995).
Iron uptake with Pch and its regulation have been studied in
detail and reviewed by Youard et al. (2011). The genes
responsible for Pch biosynthesis are organized in two
operons, pchDCBA and pchEFGHI, which are located next
to the pchR regulatory gene and to the ferriPch (iron-loaded
form of Pch) transport operon fptABCX (Fig. 1a). All three
operons are repressed by the ferric uptake regulator Fur in
the presence of iron and are activated by the AraC-type
regulator PchR together with Pch when iron is scarce. The
genes for EPch biosynthesis in P. fluorescens are organized
in a single transcriptional unit, pchDHIEFKCBA, which
is clustered with pchR and a putative ferriEPch transport
operon, fetABCDEF (Fig. 1b; Youard et al., 2011). By
analogy with P. aeruginosa, the PchR protein of P. fluorescens, together with EPch, activates expression of the
pchDHIEFKCBA operon (Fig. 1b; Youard & Reimmann,
2010).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:38:02
Printed in Great Britain
1317
C. Reimmann
(a)
(b)
Pch-Fe3+
EPch-Fe3+
Fe3+
Fe3+
Outer membrane
FptA
FetC
FetD
FptX
FetD
Inner membrane
FetA
FetE
PchR
Fe2+
pchDCBA
pchR
pchEFGHI
Pch
Fe2+
fptABCX
fetABCDEF
Fe2+
FetE
EPch
PchR
pchR
pchDHIEFKCBA
Fe2+
Fur
Fur
Fig. 1. Schematic representation of iron uptake and its transcriptional regulation in P. aeruginosa with Pch (a) and in P.
fluorescens with EPch (b). Note that the model for EPch is based to some extent on bioinformatics analyses and on results
obtained during this study (see text). The figure is adapted from Youard et al. (2011).
The Pch biosynthetic pathway is well understood and has
been fully reconstituted with purified enzymes in vitro
(Patel & Walsh, 2001). Although the enzymic steps
generating the opposite stereochemical configuration in
EPch are not yet characterized, bioinformatics predict that
the overall biosynthetic pathway is similar to that of Pch
(Youard et al., 2011). In contrast, uptake of the two ironloaded siderophore enantiomers involves different transport machineries. In P. aeruginosa, the first gene of the
ferriPch transport operon, fptA, encodes the Pch receptor,
which translocates the iron-bound siderophore across the
outer membrane (Ankenbauer & Quan, 1994). Subsequent
import into the cytoplasm is carried out by the FptX
permease, which belongs to a growing family of singlesubunit siderophore transporters (Ó Cuı́v et al., 2004). The
roles of fptB and fptC, if any, in ferriPch uptake are not
clear, as deletion of these genes did not affect Pch-mediated
iron acquisition (Michel et al., 2007). Moreover, the fptBC
genes are not conserved in some Pch-producing P.
aeruginosa strains, e.g. PA7 (Roy et al., 2010). In P.
fluorescens, transport of ferriEPch across the outer
membrane requires the FetA receptor, which is structurally
related to FptA, except for its siderophore binding pocket.
However, FetA and FptA share little sequence identity with
each other (Hoegy et al., 2009; Brillet et al., 2011).
Sequence data indicate that translocation of ferriEPch
towards the cytoplasm proceeds via a classical ABC
transporter comprising a periplasmic binding protein
(PBP), FetC, an inner-membrane permease, FetD, and an
ATP binding protein, FetE (Fig. 1b; Youard et al., 2011). It
is not known how iron is released from EPch in the
1318
cytoplasm and whether the siderophore is subsequently
recycled. Based on the operon organization of fetABCDEF
it is speculated that the PepSY-associated membrane
protein FetB and the major facilitator superfamily
transporter FetF could be involved in these processes.
Interestingly, iron uptake with Pch and EPch is strictly
stereospecific, meaning that neither siderophore is functional as an iron carrier or transcriptional inducer in the
other species (Youard et al., 2011). Genetic, biochemical
and structural data have so far revealed that stereospecificity of siderophore recognition occurs (i) at the outer
membrane, by the receptors FptA and FetA (Hoegy et al.,
2009; Brillet et al., 2011), and (ii) in the cytoplasm, by the
two different versions of the regulatory proteins PchR, each
of which is activated by its cognate siderophore only
(Youard & Reimmann, 2010). The question of whether
siderophore chirality is also important during transport
across the inner membrane has not been addressed so far,
mainly because the respective genes for ferriEPch transport
in P. fluorescens have not been studied experimentally.
Here, I show that the fetCDE genes are important in this
process and that the inner-membrane transporters for
ferriPch in P. aeruginosa and for ferriEPch in P. fluorescens
have different enantioselectivities.
METHODS
Bacterial strains, plasmids and culture conditions. Bacterial
strains and plasmids are listed in Table 1. Bacteria were cultivated on
nutrient agar and in nutrient yeast broth (Stanisich & Holloway,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:38:02
Microbiology 158
Stereospecificity of siderophore uptake
Table 1. Bacterial strains and plasmids
Strain or plasmid
E. coli strains
DH5a
P. aeruginosa strains
PAO1
PAO6331
PAO6768
PAO6769
P. fluorescens strains
CHA0
CHA1238
CHA1239
CHA1363
CHA1364
CHA1365
CHA1366
CHA1367
CHA1370
Plasmids
pME497
pME3087
pME6000
pME6032
pME7034
pME7152
pME7204
pME9253
pME9605
pME9629
pME9630
pME9646
pME9647
pME9648
pME9649
pME9650
pME10061
pME10062
pME10065
pME10067
pME10068
pME10069
pME10094
pPROBE-TT9
pUCPSK
Relevant characteristics
recA1 endA1 hsdR17 deoR thi-1 supE44 gyrA96 relA1
D(lacZYA-argF) U169 (w80dlacZDM15)
Sambrook & Russell (2001)
Wild-type
DpchDCBA DpchR DpchEFGHI
DpvdF DpchDCBA DpchR DpchEFGHI
DpvdF DpchDCBA DpchR DpchEFGHI DfptABCX
ATCC 15692
Hoegy et al. (2009)
This study
This study
Wild-type
DpvdF
DpvdF DpchDHIEFKCBA
DpvdF DpchDHIEFKCBA
DpvdF DpchDHIEFKCBA
DpvdF DpchDHIEFKCBA
DpvdF DpchDHIEFKCBA
DpvdF DpchDHIEFKCBA
DpvdF DpchR
Voisard et al. (1994)
This study
Brillet et al. (2011)
This study
This study
This study
This study
This study
This study
DfetB
DfetC
DfetD
DfetE
DfetF
Mobilizing plasmid, Apr
Suicide vector, ColE1 replicon, Tcr
pBBR1-based cloning vector, Tcr
lacIq-Ptac expression vector, Tcr
pUCPSK derivative carrying fptA, Apr
pME3087 derivative carrying DpvdFPAO, Tcr
pUCPSK derivative carrying fptX under Plac control, Apr
pME3087 derivative carrying DpchRCHA0, Tcr
pME3087 derivative carrying DpchFCHA0, Tcr
pME6000 with fptABCX under Plac control, Tcr
pME6000 with fetABCDEF under Plac control, Tcr
pME3087 derivative carrying DfetB, Tcr
pME3087 derivative carrying DfetC, Tcr
pME3087 derivative carrying DfetD, Tcr
pME3087 derivative carrying DfetE, Tcr
pME3087 derivative carrying DfetF, Tcr
pME6000 with fetA under Plac control, Tcr
pME6000 with fptA under Plac control, Tcr
pME3087 derivative carrying DfptABCX, Tcr
pME6032 with fetCDE under Ptac control, Tcr
pME6032 with fetB under Ptac control, Tcr
pUCPSK with fetBCDEF under Ptac control, Tcr
Translational fusion of fetA to gfp in pPROBE-TT9, Tcr
pVS1/p15A shuttle vector for construction of gfp fusions, Tcr
ColE1-pRO1600 shuttle vector, Apr
1972) at 37 uC (P. aeruginosa and Escherichia coli) or at 30 uC (P.
fluorescens). For green fluorescent protein (GFP) reporter assays, P.
fluorescens strains were grown in GGP medium (Carmi et al., 1994),
in which iron is present but not immediately accessible, thus inducing
the expression of siderophore biosynthesis and uptake genes.
Siderophore utilization assays were performed in minimal medium
M9 (Sambrook & Russell, 2001) with 0.5 % glycerol as carbon source.
Iron limitation was achieved by adding the iron chelator 2,29dipyridyl at 500 mM. Where necessary, the following antibiotics
were added to growth media: 100 mg ampicillin ml21 and 25 mg
tetracycline ml21 for E. coli, 125 mg tetracycline ml21 for P. aeruginosa
and P. fluorescens, and 250 mg carbenicillin ml21 for P. aeruginosa. To
http://mic.sgmjournals.org
Reference/source
Voisard et al. (1988)
Voisard et al. (1988)
Maurhofer et al. (1998)
Heeb et al. (2002)
Michel et al. (2007)
Michel et al. (2007)
Michel et al. (2007)
Youard & Reimmann (2010)
Brillet et al. (2011)
Hoegy et al. (2009)
Hoegy et al. (2009)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Miller et al. (2000)
Watson et al. (1996)
counterselect E. coli donor cells during mutant construction, 10 mg
chloramphenicol ml21 was used; mutant enrichment was performed
with 20 mg tetracycline ml21 and 2 mg carbenicillin ml21 (for P.
aeruginosa) or 50 mg cycloserine ml21 (for P. fluorescens). Pch and
EPch were purified from bacterial culture supernatants as described
(Youard et al., 2007) and added to the growth medium at 20 mM.
This concentration fully induces Pch and EPch biosynthesis genes in
P. aeruginosa and P. fluorescens, respectively (Youard et al., 2007), and
restores growth under iron limitation in siderophore-negative strains
(Hoegy et al., 2009). To induce the expression of siderophore uptake
genes cloned under the tac promoter, IPTG was added to the growth
medium at 1 mM.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:38:02
1319
C. Reimmann
DNA manipulations and sequencing. Oligonucleotides are listed
in Table S1 (available with the online version of this paper). Plasmid
DNA was prepared using QIAprep Spin Miniprep (Qiagen) and
Jetstar (Genomed) kits. DNA fragments were purified from agarose
gels with MinElute and QIAquick Gel Extraction kits (Qiagen). DNA
manipulations were performed according to standard procedures
(Sambrook & Russell, 2001). Bacterial transformations were carried
out by electroporation (Farinha & Kropinski, 1990). Constructs
involving PCR techniques were verified by sequence analysis using the
BigDye Terminator Cycle Sequencing kit and an ABI-PRISM 373
automatic sequencer (Applied Biosystems). The nucleotide sequence
of fetABCDEF from P. fluorescens CHA0 was determined commercially and deposited at the National Center for Biotechnology
Information (NCBI) under GenBank accession number JQ627635.
Mutant constructions. All gene replacement mutants of P.
aeruginosa and P. fluorescens listed in Table 1 were generated with
suicide plasmids as described previously (Schnider et al., 1995; Ye
et al., 1995; Laville et al., 1998). Apart from pME7152 (Michel et al.,
2007), pME9253 (Youard & Reimmann, 2010) and pME9605 (Brillet
et al., 2011), all new suicide plasmids were constructed in a similar
way. First, two PCR fragments were generated from chromosomal
DNA using primer pairs 1/2 and 3/4 (see Table S1 for nucleotide
sequences). The two fragments were then combined by overlap
extension PCR using an overhang on the second fragment. The
combined fragment, cleaved with BamHI and HindIII, was cloned
into the suicide vector pME3087 between corresponding sites.
Suicide plasmids were then mobilized from E. coli DH5a into P.
fluorescens and P. aeruginosa recipients using the helper plasmid
pME497 and chromosomally integrated with selection for tetracycline resistance. Excision of the vector via a second crossing-over was
obtained by enrichment for tetracycline-sensitive cells and gene
replacement mutants were subsequently identified by PCR. Table S2
lists the primer pairs used for generating the different suicide plasmids,
the P. aeruginosa and P. fluorescens recipient strains used for mutant
construction, and the names of the corresponding mutants.
Construction of plasmids for complementation and transporter
swapping experiments. The P. fluorescens and P. aeruginosa
receptor genes fetA and fptA, respectively, were placed under the
control of the constitutive lac promoter on pME6000. To generate
pME10061, fetA was excised on a 2.4 kb XhoI–EcoRV fragment from
the fetABCDEF plasmid pME9630 and cloned into pME6000 between
the XhoI and BamHI sites; the latter was blunted by T4 DNA
polymerase treatment. To obtain pME10062, a 2.8 kb HindIII–
BamHI fragment carrying fptA was excised from pME7034 and cloned
into pME6000 between the corresponding sites.
Plasmids pME10067 and pME10068 expressing fetCDE and fetB,
respectively, under the control of the IPTG-inducible tac promoter
(Ptac) were constructed as follows. For the fetCDE construct, a 3.1 kb
PCR fragment was generated from chromosomal DNA of strain
CHA0 with primers fetCDE-1 and fetCDE-2, trimmed with SacI and
XhoI, and inserted between the corresponding sites on pME6032. For
fetB, a 0.95 kb fragment was amplified by PCR from DNA of strain
CHA0 with primers fetB-7 and fetB-6, trimmed with EcoRI and BglII,
and cloned into pME6032 between the corresponding sites.
A 1.9 kb BamHI–ClaI fragment of pME10068 (carrying the lac
repressor and the 59 part of fetB under Ptac control) was ligated to a
4.8 kb ClaI–HindIII fragment from pME9630 (carrying the 39 part of
fetB and fetCDEF) and inserted into pUCPSK between BamHI and
HindIII. This generated plasmid pME10069, which expresses
fetBCDEF under Ptac control.
Construction of a translational fetA–gfp reporter. A translational
fusion of fetA with gfp was constructed at the first codon of both
1320
genes by overlap extension as follows. A 0.6 kb PCR fragment 1 was
amplified from chromosomal DNA of strain CHA0 using primers
fetAgfp-1 and fetAgfp-2 and a PCR fragment 2 was amplified from
pPROBE-TT9 using primers fetAgfp-3 and PB4. Fragments 1 and 2
were mixed in equimolar amounts, elongated by 10 PCR cycles with
Taq polymerase and dNTPs and subsequently amplified by 20 PCR
cycles with primers fetAgfp-1 and PB4. The resulting PCR fragment
was cleaved with EcoRI and NdeI and cloned into the corresponding
sites of pPROBE-TT9 to generate pME10094.
RESULTS
Utilization of ferriEPch as an iron source requires
the P. fluorescens genes fetCDE
To evaluate whether the genes downstream of fetA were
required for ferriEPch utilization, individual in-frame
deletion mutants were constructed in a Pvd- and EPchnegative background and tested for their ability to grow
with ferriEPch as the sole iron source. As shown in Fig.
2(a), the Pvd- and EPch-negative parent strain (CHA1239)
was not able to grow in M9-glycerol medium containing
the iron chelator 2,29-dipyridyl. When EPch was added,
growth was restored (Fig. 2b), confirming that EPch is a
stronger iron chelator than 2,29-dipyridyl and is able to
promote growth of P. fluorescens when iron is scarce. In
contrast, the Pvd-negative but EPch-producing precursor
strain (CHA1238) grew well regardless of whether the
medium contained EPch.
Similar growth promotion experiments were then performed with CHA1239-derived fetB (CHA1363), fetC
(CHA1364), fetD (CHA1365), fetE (CHA1366) and fetF
(CHA1367) mutants, respectively. As expected, none of
these siderophore-negative strains grew in the presence of
2,29-dipyridyl (Fig. 2a). When EPch was added to the
medium, growth of the fetB and fetF mutants was restored
(Fig. 2b), indicating either that these genes are not essential for ferriEPch utilization or that their function is
redundant in P. fluorescens. In contrast, no growth promotion by ferriEPch occurred in the fetC (CHA1364), fetD
(CHA1365) or fetE (CHA1366) mutants, suggesting that the
putative ABC transporter encoded by fetCDE is responsible
for ferrisiderophore translocation across the inner membrane.
Complementation of CHA1364, CHA1365 and CHA1366
with the fetCDE plasmid pME10067 fully restored ferriEPchmediated growth promotion (data not shown).
Expression of the EPch transport operon is
repressed by iron and activated by EPch via PchR
Inspection of the fetABCDEF promoter region revealed
potential binding sites for the Fur repressor [consensus
sequence GATAATGATAATCATTATC (de Lorenzo et al.,
1987)] and for the transcriptional activator PchR [consensus sequence TGCATCGAAAGAAAAAGCCCSGCAATCGAAA (Michel et al., 2005)] (Fig. 3), suggesting that
expression of the ferriEPch transport operon is repressed
by iron with Fur and is induced by EPch via PchR. This
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:38:02
Microbiology 158
Stereospecificity of siderophore uptake
0.5
(a)
(b)
_ EPch
+ EPch
Cell density (OD600)
0.4
0.3
0.2
Pvd-, EPch-
prediction was tested using the translational fetA–gfp
reporter plasmid pME10094. To avoid signal interference
of GFP with Pvd, experiments were carried out in a Pvdnegative non-fluorescent background. As shown in Fig. 4,
expression of fetA–gfp was strongly repressed in the Pvdnegative strain CHA1238 when GGP medium was
supplemented with 100 mM FeCl3. Full expression under
iron-limiting conditions required PchR and EPch, as
only low activities were measured in the Pvd-negative
strains CHA1370 and CHA1239, which lacked pchR and
pchDHIEFKCBA, respectively. I conclude from this experiment that the fetABCDEF operon is negatively regulated by
iron and positively regulated by EPch via PchR. Regulation
of the ferriEPch transport operon thus resembles the
regulation of the ferriPch transport operon fptABCX of P.
aeruginosa, which is repressed by iron via Fur and induced
by Pch via PchR (Michel et al., 2007).
FetCDE is specific for ferriEPch whereas FptX
translocates both enantiomers to the cytoplasm
The enantioselectivity of the inner-membrane transporters
FetCDE and FptX was evaluated (Table 2). Experiments
tF
tE
fe
Pvd-, EPch-
fe
tD
fe
tC
fe
tB
_
h
Pc
fe
_
Pv
d
Pv
d
_
,E
tF
fe
tE
tD
fe
fe
tC
fe
tB
h
_
fe
_
Pc
Pv
d
_
,E
0
Pv
d
0.1
Fig. 2. Role of fetB, fetC, fetD, fetE and
fetF in ferriEPch-mediated growth promotion.
The P. fluorescens strains CHA1238 (Pvd”),
CHA1239 (Pvd”, EPch”), and derivatives
of CHA1239 deleted for fetB (CHA1263),
fetC (CHA1364), fetD (CHA1365), fetE
(CHA1366) and fetF (CHA1367), respectively, were grown in 200 ml M9-glycerol
medium containing the iron chelator 2,29dipyridyl at 500 mM in the absence (a) or
presence (b) of 20 mM HPLC-purified EPch.
Growth in 96-well microtitre plates was
assessed after 100 h. OD600 values represent
the means±SD from three parallel cultures.
The experiment was repeated twice with very
similar results.
were performed with the P. aeruginosa mutant PAO6769,
which lacks pvdF, pchDCBA, pchR, pchEFGHI and
fptABCX. This mutant is unable to grow in iron-limited
M9-glycerol medium due to its inability to produce Pvd
and Pch. When the growth medium was supplemented
with 20 mM Pch or EPch as an iron source, no growth
occurred because the mutant lacks the ferriPch transport
operon fptABCX and because P. aeruginosa is not able to
grow with ferriEPch, as shown previously (Hoegy et al.,
2009). When the Pch receptor gene fptA and the innermembrane permease gene fptX were introduced jointly on
plasmid pME9629 (fptABCX) or separately on plasmids
pME10062 (fptA) and pME7204 (fptX), growth was
restored with Pch but not with EPch. As expected, the
fptA plasmid alone did not allow siderophore-dependent
growth promotion. When the fptA plasmid was combined
with the fetBCDEF plasmid pME10069, growth was not
restored by either Pch or EPch. I conclude from this that
(i) in agreement with previous findings (Hoegy et al.,
2009), the P. aeruginosa receptor FptA cannot transport
iron-loaded EPch across the outer membrane and (ii)
the P. fluorescens ABC transporter FetCDE is unable to
translocate FptA-delivered ferriPch from the periplasm
StopPchR
PchR box
Fur box
S.D.
StartFetA
Fig. 3. Location of potential binding sites for the regulatory proteins Fur and PchR in the fetABCDEF promoter region.
Conserved nucleotides are highlighted in bold. S.D., Shine–Dalgarno sequence.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:38:02
1321
C. Reimmann
70 000
alone. However, when the fetA plasmid pME10061 was
combined with the fptX plasmid pME7204, PAO6769 was
able to grow with ferriEPch (but not with ferriPch) as
an iron source. This experiment confirms that the FetA
receptor is specific for ferriEPch as reported previously
(Hoegy et al., 2009) and shows that FptX is not only a
transporter for ferriPch, but is also perfectly capable of
transporting ferriEPch across the inner membrane.
FetA expression
(RFU)
60 000
50 000
40 000
30 000
20 000
10 000
0
0
10
20
30
40
50
Time (h)
Fig. 4. Regulation of the ferriEPch transport operon. The P.
fluorescens strains CHA1238 (DpvdF), CHA1370 (DpvdF,
DpchR) and CHA1239 (DpvdF, DpchDHIEFKCBA) containing
the reporter plasmid pME10094 with a fetA–gfp translational
fusion were grown in microtitre wells at 30 6C and 500 r.p.m., and
green fluorescence was measured over 48 h from three parallel
cultures. Strains were grown in GGP medium (e, CHA1238; #,
CHA1370; h, CHA1239) or GGP medium with 100 mM FeCl3
(X, CHA1238; $, CHA1370; &, CHA1239). Fluorescence is
expressed as relative fluorescence units (RFU; mean±SD).
to the cytoplasm, indicating that this inner-membrane
transporter is highly stereospecific.
In contrast with FetCDE, stereospecificity of inner-membrane transport was not observed with FptX (Table 2). As
expected, growth promotion of PAO6769 was restored with
EPch (but not with Pch), when the mutant was complemented with either the fetABCDEF plasmid pME9630
or a combination of pME10061 (fetA) and pME10069
(fetBCDEF). Again, no complementation occurred with fetA
DISCUSSION
Siderophore-mediated iron uptake in Gram-negative
bacteria can be viewed as a two-step process in which the
iron–siderophore complex is first recognized at the cell
surface by a TonB-dependent receptor and transported
into the periplasm. There, the complex is usually bound by
a PBP which delivers its cargo to the cytoplasm via a
cognate ABC transporter. The genes for the siderophore
receptor and the corresponding PBP-dependent ABC
transporter are often clustered in bacterial chromosomes,
allowing their functional connection to be predicted. In P.
fluorescens CHA0, genes for a putative PBP-dependent ABC
transporter are present downstream of the EPch receptor
gene fetA and I have shown here that these genes, which we
termed fetCDE (Youard et al., 2011), are indeed required
for utilization of ferriEPch as an iron source. In contrast,
fetB and fetF, two genes flanking fetCDE, were not required
for EPch-dependent growth promotion under the experimental conditions used. Their potential importance in
reductive iron release from the siderophore and for
subsequent siderophore recycling was not investigated
during this study.
The genetic organization of fetA, fetB, fetC, fetD, fetE and
fetF suggests that the six genes are cotranscribed, forming a
Table 2. Stereospecificity of FetCDE and FptX
P. aeruginosa PAO6769 carrying the plasmids indicated was grown at 37 uC and 500 r.p.m. in microtitre wells containing 200 ml iron-limited M9glycerol medium. Where necessary, carbenicillin (but not tetracycline) was added for plasmid maintenance and IPTG was used to induce expression
from Ptac. Iron limitation was achieved with 2,29-dipyridyl at 500 mM. HPLC-purified siderophores were added at a final concentration of 20 mM.
Growth was measured after 4 days. Mean OD600 values±SD are from one typical experiment performed with three parallel cultures. The experiment
was repeated twice with similar results. ND, No growth detected (OD600 ¡0.03).
Plasmid 1
No plasmid
pME9629
pME10062
pME10062
pME10062
pME9630
pME10061
pME10061
pME10061
1322
Genes of plasmid 1
fptABCX
fptA
fptA
fptA
fetABCDEF
fetA
fetA
fetA
Plasmid 2
pME7204
pME10069
Genes of plasmid 2
Growth (OD600) in iron-limited medium
with:
fptX
fetBCDEF
pME10069
fetBCDEF
pME7204
fptX
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:38:02
Pch
EPch
ND
ND
0.38±0.03
0.53±0.03
ND
ND
ND
ND
ND
ND
ND
0.42±0.03
0.38±0.02
ND
ND
ND
ND
0.37±0.01
Microbiology 158
Stereospecificity of siderophore uptake
ferriEPch transport operon (Youard et al., 2011). The fetA
promoter region contains potential Fur and PchR binding
sites, suggesting that the ferriEPch transport operon could
be regulated like the ferriPch transport operon fptABCX in
P. aeruginosa (Michel et al., 2007). Expression studies
confirmed this hypothesis and showed that fetABCDEF was
repressed by iron and activated by EPch via PchR. I
conclude that the regulatory circuits governing iron uptake
with Pch and EPch are conserved in the two Pseudomonas
species.
outer-membrane siderophore transporters in fluorescent pseudomonads: structural bases for unique enantiospecific recognition. J Am
Chem Soc 133, 16503–16509.
PBP-dependent ABC transporters are not the only
transporters able to translocate iron–siderophore complexes across the inner membrane of Gram-negative
bacteria. In Yersinia pestis, an ABC transporter for
yersiniabactin-mediated iron uptake has been described
which appears to be independent of a PBP (Fetherston
et al., 1999). Moreover, single-subunit transporters such as
RhtX of Sinorhizobium meliloti and FptX of P. aeruginosa
were found to transport iron-loaded rhizobactin 1021 and
Pch, respectively, across the inner membrane (Ó Cuı́v
et al., 2004). The NCBI database reveals potential homologues of RhtX and FptX in many Gram-negative bacterial
species, suggesting that this transporter family may be
more widespread than currently appreciated.
Farinha, M. A. & Kropinski, A. M. (1990). High efficiency
As the inner-membrane transporters for ferriPch in P.
aeruginosa and for ferriEPch in P. fluorescens belong to
different protein families, I investigated whether they
would display different enantioselectivities. FptX of P.
aeruginosa was found to translocate not only ferriPch but
also ferriEPch, indicating that this single-subunit siderophore transporter was unable to distinguish between the
two enantiomers. In contrast, the PBP-dependent ABC
transporter FetCDE of P. fluorescens was found to display
stereospecificity as no growth promotion was observed
with ferriPch as the iron source. Thus, in this instance, the
PBP-dependent ABC transporter was intolerant towards
stereochemical variation of its cargo, although related
transporters are often able to transport several structurally
related siderophores (Koster, 2005).
Carmi, R., Carmeli, S., Levy, E. & Gough, F. J. (1994). (+)-(S)-
dihydroaeruginoic acid, an inhibitor of Septoria tritici and other
phytopathogenic fungi and bacteria, produced by Pseudomonas
fluorescens. J Nat Prod 57, 1200–1205.
de Lorenzo, V., Wee, S., Herrero, M. & Neilands, J. B. (1987).
Operator sequences of the aerobactin operon of plasmid ColV-K30
binding the ferric uptake regulation (fur) repressor. J Bacteriol 169,
2624–2630.
electroporation of Pseudomonas aeruginosa using frozen cell suspensions. FEMS Microbiol Lett 58, 221–225.
Fetherston, J. D., Bertolino, V. J. & Perry, R. D. (1999). YbtP and
YbtQ: two ABC transporters required for iron uptake in Yersinia
pestis. Mol Microbiol 32, 289–299.
Heeb, S., Blumer, C. & Haas, D. (2002). Regulatory RNA as mediator
in GacA/RsmA-dependent global control of exoproduct formation in
Pseudomonas fluorescens CHA0. J Bacteriol 184, 1046–1056.
Hoegy, F., Lee, X., Noel, S., Rognan, D., Mislin, G. L., Reimmann, C. &
Schalk, I. J. (2009). Stereospecificity of the siderophore pyochelin
outer membrane transporters in fluorescent pseudomonads. J Biol
Chem 284, 14949–14957.
Koster, W. (2005). Cytoplasmic membrane iron permease systems in
the bacterial cell envelope. Front Biosci 10, 462–477.
Laville, J., Blumer, C., Von Schroetter, C., Gaia, V., Défago, G., Keel, C.
& Haas, D. (1998). Characterization of the hcnABC gene cluster
encoding hydrogen cyanide synthase and anaerobic regulation by ANR
in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.
J Bacteriol 180, 3187–3196.
Liu, P. V. & Shokrani, F. (1978). Biological activities of pyochelins:
iron-chelating agents of Pseudomonas aeruginosa. Infect Immun 22,
878–890.
Maurhofer, M., Reimmann, C., Schmidli-Sacherer, P., Heeb, S.,
Haas, D. & Défago, G. (1998). Salicylic acid biosynthetic genes
expressed in Pseudomonas fluorescens strain P3 improve the induction
of systemic resistance in tobacco against tobacco necrosis virus.
Phytopathology 88, 678–684.
Meyer, J.-M. & Abdallah, M. A. (1978). The fluorescent pigment of
Pseudomonas fluorescens: biosynthesis, purification and physicochemical properties. J Gen Microbiol 107, 319–328.
Michel, L., González, N., Jagdeep, S., Nguyen-Ngoc, T. & Reimmann, C.
(2005). PchR-box recognition by the AraC-type regulator PchR of
ACKNOWLEDGEMENTS
I thank Angelo Espinha-Henriques for technical assistance, and
Xiaoyun Lee, Po-Chi Lin and Dieter Haas for careful reading of the
manuscript. This work was supported by the Swiss National Science
Foundation for Scientific Research (project 31003A-132998).
Pseudomonas aeruginosa requires the siderophore pyochelin as an
effector. Mol Microbiol 58, 495–509.
Michel, L., Bachelard, A. & Reimmann, C. (2007). Ferripyochelin
uptake genes are involved in pyochelin-mediated signalling in
Pseudomonas aeruginosa. Microbiology 153, 1508–1518.
Miller, W. G., Leveau, J. H. & Lindow, S. E. (2000). Improved gfp and
inaZ broad-host-range promoter-probe vectors. Mol Plant Microbe
Interact 13, 1243–1250.
REFERENCES
Ó Cuı́v, P. O., Clarke, P., Lynch, D. & O’Connell, M. (2004).
Andrews, S. C., Robinson, A. K. & Rodrı́guez-Quiñones, F. (2003).
Bacterial iron homeostasis. FEMS Microbiol Rev 27, 215–237.
Ankenbauer, R. G. & Quan, H. N. (1994). FptA, the FeIII-pyochelin
receptor of Pseudomonas aeruginosa: a phenolate siderophore receptor
homologous to hydroxamate siderophore receptors. J Bacteriol 176,
307–319.
Brillet, K., Reimmann, C., Mislin, G. L. A., Noël, S., Rognan, D.,
Schalk, I. J. & Cobessi, D. (2011). Pyochelin enantiomers and their
http://mic.sgmjournals.org
Identification of rhtX and fptX, novel genes encoding proteins that
show homology and function in the utilization of the siderophores
rhizobactin 1021 by Sinorhizobium meliloti and pyochelin by
Pseudomonas aeruginosa, respectively. J Bacteriol 186, 2996–3005.
Patel, H. M. & Walsh, C. T. (2001). In vitro reconstitution of the
Pseudomonas aeruginosa nonribosomal peptide synthesis of pyochelin:
characterization of backbone tailoring thiazoline reductase and Nmethyltransferase activities. Biochemistry 40, 9023–9031.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:38:02
1323
C. Reimmann
Rinehart, K. L., Staley, A. L., Wilson, S. R., Ankenbauer, R. G. & Cox,
C. D. (1995). Stereochemical assignment of the pyochelins. J Org
Chem 60, 2786–2791.
Roy, P. H., Tetu, S. G., Larouche, A., Elbourne, L., Tremblay, S., Ren, Q.,
Dodson, R., Harkins, D., Shay, R. & other authors (2010). Complete
genome sequence of the multiresistant taxonomic outlier Pseudomonas
aeruginosa PA7. PLoS ONE 5, e8842.
approaches. In Molecular Ecology of Rhizosphere Microorganisms, pp.
67–89. Edited by F. O’Gara, D. N. Dowling & B. Boesten. Weinheim:
VCH Publishers.
Watson, A. A., Alm, R. A. & Mattick, J. S. (1996). Construction of
improved vectors for protein production in Pseudomonas aeruginosa.
Gene 172, 163–164.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a
Ye, R. W., Haas, D., Ka, J. O., Krishnapillai, V., Zimmermann, A.,
Baird, C. & Tiedje, J. M. (1995). Anaerobic activation of the entire
Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory.
denitrification pathway in Pseudomonas aeruginosa requires Anr, an
analog of Fnr. J Bacteriol 177, 3606–3609.
Schnider, U., Keel, C., Blumer, C., Troxler, J., Défago, G. & Haas, D.
(1995). Amplification of the housekeeping sigma factor in
Youard, Z. A. & Reimmann, C. (2010). Stereospecific recognition of
Pseudomonas fluorescens CHA0 enhances antibiotic production and
improves biocontrol abilities. J Bacteriol 177, 5387–5392.
Stanisich, V. A. & Holloway, B. W. (1972). A mutant sex factor of
Pseudomonas aeruginosa. Genet Res 19, 91–108.
Voisard, C., Rella, M. & Haas, D. (1988). Conjugative transfer of
plasmid RP1 to soil isolates of Pseudomonas fluorescens is facilitated
by certain large RP1 deletions. FEMS Microbiol Lett 55, 9–13.
Voisard, C., Bull, C. T., Keel, C., Laville, J., Maurhofer, M., Schnider, U.,
Défago, G. & Haas, D. (1994). Biocontrol of root diseases by
Pseudomonas fluorescens CHA0: current concepts and experimental
1324
pyochelin and enantio-pyochelin by the PchR proteins in fluorescent
pseudomonads. Microbiology 156, 1772–1782.
Youard, Z. A., Mislin, G. L., Majcherczyk, P. A., Schalk, I. J. &
Reimmann, C. (2007). Pseudomonas fluorescens CHA0 produces
enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin. J Biol Chem 282, 35546–35553.
Youard, Z. A., Wenner, N. & Reimmann, C. (2011). Iron acquisition
with the natural siderophore enantiomers pyochelin and enantiopyochelin in Pseudomonas species. Biometals 24, 513–522.
Edited by: H.-M. Fischer
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:38:02
Microbiology 158