Environmental Microbiology (2009) 11(8), 2123–2135
doi:10.1111/j.1462-2920.2009.01932.x
Distribution and evolution of ferripyoverdine receptors
in Pseudomonas aeruginosa
Josselin Bodilis,1 Bart Ghysels,2 Julie Osayande,2
Sandra Matthijs,2 Jean-Paul Pirnay,3 Sarah Denayer,2
Daniel De Vos3 and Pierre Cornelis2*
1
Université de Rouen, Laboratoire M2C, UMR CNRS
6143, groupe microbiologie, Bâtiment IRESE B, UFR
des Sciences, 76821 Mont Saint Aignan, France.
2
Flanders Institute of Biotechnology (VIB), Laboratory of
Microbial Interactions, Department of Molecular and
Cellular Interactions, Vrije Universiteit Brussel, Building
E, room 6.6, Pleinlaan 2, B-1050 Brussels, Belgium.
3
Laboratory for Molecular and Cellular Technology, Burn
Wound Center, Queen Astrid Military Hospital,
Bruynstraat 1, B-1120 Brussels, Belgium.
Summary
emi_1932
2123..2135
Pseudomonas aeruginosa is a ubiquitous Gramnegative bacterium, which is also able to cause
severe opportunistic infections in humans. The
colonization of the host is importantly affected
by the production of the high-affinity iron (III)
scavenging peptidic siderophore pyoverdine. The
species P. aeruginosa can be divided into three
subgroups (‘siderovars’), each characterized by the
production of a specific pyoverdine and receptor
(FpvA). We used a multiplex PCR to determine the
FpvA siderovar on 345 P. aeruginosa strains from
environmental or clinical origin. We found about the
same proportion of each type in clinical strains,
while FpvA type I was slightly over-represented
(49%) in environmental strains. Our multiplex PCR
also detected the presence or absence of an additional receptor for type I pyoverdine (FpvB). The
fpvB gene was in fact present in the vast majority of
P. aeruginosa strains (93%), regardless of their siderovar or their origin. Finally, molecular analyses of
fpvA and fpvB genes highlighted a complex evolutionary history, probably linked to the central role of
iron acquisition in the ecology and virulence of
P. aeruginosa.
Received 10 March, 2009; accepted 10 March, 2009. *For correspondence. E-mail pcornel@vub.ac.be; Tel. (+32) 2 6291906; Fax
(+32) 2 6291902.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
Introduction
Like other ubiquitous aerobic microorganisms, the different Pseudomonas species produce siderophores in order
to satisfy their need for iron (Braun and Killmann, 1999).
Pseudomonas aeruginosa, the type species of the genus,
is able to thrive in very diverse environments, including
water, soil, roots, plant and animal hosts where it is
known as an opportunistic pathogen able to cause lifethreatening infections (Goldberg, 2000). The common
characteristic trait of fluorescent pseudomonads is their
capacity to produce, under conditions of iron limitation,
the yellow-green fluorescent pigment and siderophore
pyoverdine (Meyer, 2000; Ravel and Cornelis, 2003; Cornelis et al., 2007; 2009; Visca et al., 2007). Pyoverdines
are composed of a conserved dihydroxyquinoline chromophore, a variable peptide chain, comprising 6–12
amino acids, specific to a producing strain, and a sidechain, generally a dicarboxylic acid or an amide (Ravel
and Cornelis, 2003; Visca et al., 2007). Both chromophore
(Mossialos et al., 2002) and peptide chain of pyoverdines
(Ravel and Cornelis, 2003) are synthesized by nonribosomal peptide synthetases (NRPSs). A specific TonBdependent outer membrane receptor recognizes and
binds the cognate pyoverdine (Smith et al., 2005). The
genes coding for the receptor and the NRPSs responsible
for the synthesis of the peptide moiety of pyoverdine are
part of the so-called ‘variable’ locus of pyoverdine genes
(Ravel and Cornelis, 2003; Smith et al., 2005; Cornelis
et al., 2007; Visca et al., 2007). Three siderovars of
P. aeruginosa can be distinguished, producing three
structurally different types of pyoverdine (type I, II, III)
(Cornelis et al., 1989; Meyer et al., 1997; De Vos et al.,
2001; Ernst et al., 2003; Spencer et al., 2003; Smith et al.,
2005), each being recognized at the level of the outer
membrane by a specific receptor (Cornelis et al., 1989;
De Chial et al., 2003; Spencer et al., 2003). It has also
been shown that the type II ferripyoverdine receptors are
more diverse and it has been suggested that the type II
receptor gene is under positive selection (Smith et al.,
2005; Tümmler and Cornelis, 2005). This selection pressure could be due to the pyocin S3 bacteriocin which uses
type II ferripyoverdine receptors in order to enter the cell
and kill it (Baysse et al., 1999; De Chial et al., 2003).
However, another pyocin, S2, was recently found to kill
strains having the type I FpvA receptor, which does not
2124 J. Bodilis et al.
show such variability, contradicting this hypothesis
(Denayer et al., 2007). A second receptor specific for type
I pyoverdine, called FpvB, the gene of which is not part of
the pyoverdine locus, has also been identified (Ghysels
et al., 2004). The fpvB gene was also detected in other
P. aeruginosa strains, including some that produce type II
and type III pyoverdines, where it was found to confer
the capacity to utilize type I pyoverdine as a source of
iron (Ghysels et al., 2004). Here, using a multiplex PCR
(MPCR) approach, we found a slightly different proportion
of each pyoverdine receptor type between clinical and
environmental strains and report that the fpvB gene is
almost ubiquitous among P. aeruginosa strains. Moreover, sequencing and molecular analyses of fpvA and
fpvB genes from each P. aeruginosa siderotype highlighted a complex evolutionary history.
Results
1
2
3
4
5
6
7
8
9
10
1000
800
600
400
Fig. 1. Multiplex PCR-amplified fragments of four reference strains
(PAO1: lanes 2 and 7, 7NSK2: lanes 3 and 8, ATCC 27853: lanes
4 and 9, and 59.20: lanes 5 and 10) with two different primer sets,
electrophoretically separated. The first and sixth lanes contain the
molecular weight markers (sizes in bp indicated on the left). The
bands corresponding to the different PVD receptors have the
following sizes in reverse order of size for set 1 (lanes 2–5): fpvAII,
for both variants (682 bp), fpvB (562 bp), fpvAIII (505 bp) and fpvAI
(324 bp), for set 2 (lanes 7–10): fpvAIIa (908 bp), fpvAIIb (863),
fpvB (562 bp), fpvAIII (505 bp) and fpvAI (324 bp).
Existence of fpvA type II variants
With the previously developed MPCR method for identification of fpvAI, II and III receptor genes in P. aeruginosa
(De Chial et al., 2003) we failed to amplify an fpvA fragment in some isolates known to produce type II PVD (as
evidenced by IEF typing of pyoverdines), including the
type II reference strain ATCC 27853. Spencer and colleagues (2003) described a new FpvA receptor sequence
(Accession No. AAO1728) and in silico analysis indicated
that this receptor is a variant of the FpvAII receptor that
we previously described (Accession No. AAN62913) (De
Chial et al., 2003). At the nucleotide level, both genes
share 89% of the residues in an overlap of more than 90%
of their sequence. We therefore designed a primer set for
the specific amplification of a fragment of this fpvAII gene
variant and detected its presence (PCR detection) in
P. aeruginosa ATCC 27853 and other type II P. aeruginosa strains that failed to give amplification with the previously designed MPCR primer set (De Chial et al., 2003).
We therefore called this second type II receptor ‘fpvAIIb’
and the original type II receptor from 7NSK2 ‘fpvAIIa’.
Multiplex PCR for the simultaneous detection of five
P. aeruginosa ferripyoverdine receptor genes
Previously, we reported the presence of a second type I
ferripyoverdine transport mediating receptor in P. aeruginosa PAO1, encoded by fpvB (PA4168) (Ghysels et al.,
2004). We also demonstrated the presence of functional
fpvB homologues in type II and type III P. aeruginosa
strains (Ghysels et al., 2004). A primer set for fpvB detection and one for detection of fpvAIIb were therefore added
to the original MPCR primer set for detection of fpvA,
fpvAIIa and fpvAIII (De Chial et al., 2003). With this five-
primer-pair MPCR set, we were able to detect simultaneously five different ferripyoverdine receptor genes in
different P. aeruginosa strains, two in the PVD type reference strain PAO1 (fpvAI and fpvB), two in the type IIa
reference strain 7NSK2 (fpvAIIa and fpvB), one in the type
IIb reference strain ATCC 27853 (fpvAIIb) and two in the
type III reference strain 59.20 (fpvAIII and fpvB) (Fig. 1).
We also used an MPCR primer set in which the primers
for detecting fpvAIIa and fpvAIIb are replaced by a single
primer pair which detects both genes without discrimination (Fig. 1). It is important to note that even with the
bacterial cells directly inoculated as template in the PCRmix (without prior boiling), clear amplifications were
obtained.
Distribution of ferripyoverdine receptor genes in a
P. aeruginosa population
The MPCR described above was applied to study the
distribution of the currently identified ferripyoverdine
receptor genes in a P. aeruginosa population comprising
345 clinical and environmental isolates from different
locations throughout the world. The results are summarized in Table 1 and the complete list of strains with their
origin is given in Table S1 in Supporting information. From
only four isolates (1.2%) no amplification signal could be
detected, while all the other strains were positive for at
least one receptor gene (Table 1).
From these 341 MPCR-positive isolates, 122 (35.8%)
had fpvAI, 48 had fpvAIIa (14.1%), 80 (23.5%) had fpvAIIb
and 83 (24.3%) had the fpvAIII gene, while in eight strains
(2.3%) only fpvB could be amplified. It is important to note
that the distribution is slightly different according to the
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
Distribution and evolution of ferripyoverdine receptors 2125
Table 1. Results of the multiplex PCR of 345 P. aeruginosa strains.
Positive strains
fpvAI
5
117
2
46
5
75
8
75
8
4
+
+
fpvAIIa
fpvAIIb
fpvAIII
fpvB
+
+
+
+
+
+
+
+
+
ence of further type II receptor variants while the others
were pyoverdine-negative (Table S1).
+
+
origin of the strains (Table S1). The clinical strains (220
strains) showed about the same proportion of each type,
while FpvA type I was over-represented (49%) in environmental strains (79 strains).
Altogether fpvB was amplified from 317 strains (93%)
either alone or together with fpvAI, fpvAIIa, fpvAIIb or
fpvAIII. The fpvB gene could not be amplified in 4.1%,
4.2%, 6.3% and 9.6% of the strains that were positive for
fpvAI, fpvAIIa, fpvAIIb or fpvAIII respectively. Figure 2
shows a similarity tree based on AFLP patterns,
sequences of oprI, oprL and oprD, and serotypes. Results
of the MPCR are also shown for each strain. All 75 strains
in the tree are mentioned in Table 1 and Table S1, except
LMG 10643, which is not a P. aeruginosa, but a
Pseudomonas oryzihabitans.
Comparison between IEF pyoverdine determination
and receptor typing
Isoelectrofocalization of pyoverdines from the spent
medium is a technique allowing fast and accurate determination of the pyoverdine type in P. aeruginosa (Meyer
et al., 1997; De Vos et al., 2001). However, some strains
had lost the ability to produce pyoverdine, as evidenced in
some cystic fibrosis isolates, but were still able to take up
ferripyoverdine (De Vos et al., 2001; Ernst et al., 2003).
For these pyoverdine-negative mutants, growth stimulation experiments with purified pyoverdines did not provide
clear-cut answers because of the ability of some strains to
utilize more than one type of ferripyoverdine as a source
of iron (De Vos et al., 2001; Ghysels et al., 2004). This is
due to the presence of FpvB, the alternative receptor for
type I ferripyoverdine and also because the type III ferripyoverdine receptor also allows some level of utilization
of the type II ferripyoverdine (Ghysels et al., 2004). All
pyoverdine-positive strains, which were tested by IEF,
showed the same fpvA receptor type as the corresponding pyoverdine, in addition to the presence or absence of
fpvB gene (results not shown). Four of the fpvA-negative
strains (So122, Lo059, Pr332 and Br700 strains) were
found to produce type II pyoverdine, suggesting the exist-
Functionality of the fpvB gene
In some pyoverdine-negative strains fpvB was amplified,
either singly or together with an fpvA gene. The results
presented in Fig. 3 for strains Mi159 and Mi162 show that
fpvB is expressed and functional as judged by the growth
stimulation assay using the three purified pyoverdines.
Both isolates are pyoverdine-negative, but in Mi159 both
fpvAIII and fpvB were amplified by PCR and only fpvB in
Mi162. The growth of Mi159 was stimulated by the three
pyoverdines, showing a good correlation with the presence of FpvAIII and of FpvB. As already mentioned,
FpvAIII allows the uptake not only of type III, but also, to
some extent, of type II ferripyoverdine, and FpvB is
responsible for the uptake of type I ferripyoverdine
(Ghysels et al., 2004). In Mi162 only FpvB seems to be
functional.
In strains SG17M, C2 and C19, which can be typed by
IEF as type II pyoverdine producers (although their
pyoverdine production is low), both fpvAIIb and fpvB can
be amplified, although only in the case of SG17M could
the growth be stimulated by type I pyoverdine, indicating
either that in C2 and C19 the fpvB gene is not expressed
or that its product is not functional (results not shown).
Nature of a supplementary 450 bp PCR fragment
detected in some P. aeruginosa strains
In a minority of the strains (8.9%) we obtained, in addition
to the expected fragment associated with the different
fpvA receptors, an additional amplicon of around 450 bp
(Fig. 4). Closer analysis revealed that this fragment was
the PCR product of the primer pair fpvAIf and fpvBf. A
BLASTX search of Pseudomonas genomes revealed that
the translated product had 94% identity with the products
of two genes from PA7, PSPA7_0713 and PSPA7_5043
which are annotated as coding putative phage proteins.
The fragment also appeared to be more frequently amplified in type III strains (18%) than in type II (9%) and type
I strains (2.5%).
Phylogeny of PVD receptors
In order to investigate the evolutionary history of the ferripyoverdine receptor genes in P. aeruginosa, we carried
out a phylogenetic analysis with 8 fpvAI, 10 fpvAII (4 IIa
and 6 IIb), 8 fpvAIII and 15 fpvB genes from 22 strains
(Fig. 5A). While the dendrogram shows a great variability
between fpvA and fpvB clusters, the variability within each
fpvA and fpvB cluster is much lower, as highlighted by the
scales on the dendrograms and the overall mean variabil-
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
2126 J. Bodilis et al.
fpvAI
fpvAIIa
fpvAIIb
fpvAIII
fpvB
100
96
98
94
92
90
88
86
84
82
80
78
74
76
Pyoverdine receptors
72
70
68
66
64
62
fAFLP+oprD+oprI+oprL
.
Us448
.
Lw1047
.
.
Is580
.
.
Br670
.
.
Br257
.
.
Aa249
.
.
ATCC
27853
.
.
Mi159
.
.
Is579
.
.
Br776
.
.
Lw1048
.
.
.
PAO1
.
.
LMG
14083
.
.
Br908
.
Lo053
.
.
Mi162
.
.
Bu004
.
.
C17
(clone C)
.
.
SG17M
(clone C) .
.
C
.
. (clone C)
C1
.
. (clone C)
C18
(clone C)
.
.
PT31M
(clone C) .
.
C2
.
. (clone C)
SG50M
(clone C) .
.
C19
(clone C)
.
.
C13
(clone C)
.
.
Pr335
.
.
Li012
.
.
.
Li010
.
LMG
14085
.
.
Br667
.
.
Lo049
.
.
Br229
.
.
Pa6
.
.
Tu863
.
.
Us451
.
.
Us449
.
.
Us450
.
.
Li009
.
.
Bo548
.
.
So095
.
.
Us447
.
.
So099
.
.
Br692
.
.
Is573
.
.
Bu007
.
.
Aa245
.
.
.
So092
.
.
Be128
.
Pr317
.
.
.
Br735
.
Br642
.
.
Ro124
.
.
.
LMG
2107
.
Mi151
.
.
TuD199
.
.
.
.
Br906
.
.
Be136
.
TuD47
.
.
.
Lo050
.
.
Li004
.
.
Bo546
.
PhDW6
.
.
PAO23
.
.
.
PAO29
.
.
Be133
.
.
Bo559
.
.
LMG
14084
.
.
Aa246
.
.
LMG
5031
.
.
CPHL
11451
.
.
Br680
.
.
PA7
.
.
LMG
10643 (P. oryzihabitans)
Fig. 2. Dendrogram (UPGMA, BioNumerics v5.2) based on the comparison of the composite data set consisting of the AFLP pattern, the oprI,
oprL and oprD nucleotide sequences and the serotype of 75 diverse P. aeruginosa strains isolated from different clinical and environmental
sites across the world. Black squares represent the type of receptor identified by MPCR. Strain name, geographical origin, isolation site and
year and pyoverdine receptor profiles are shown in Table S1. The PA7 clade is highlighted in grey.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
Distribution and evolution of ferripyoverdine receptors 2127
Mi159
III
Mi162
II
III
I
I
fpvAIII
PCR fpvB
II
PCR
fpvB
Fig. 3. Result of growth stimulation assays using disks
impregnated with 2 mM purified pyoverdines (types I–III as
indicated on the picture) for strains Mi159 and Mi162. The insert
below shows the result of the multiplex PCR amplification.
ity (Pi) in Table 2. Among these clusters, the fpvAII cluster
is the most discriminatory (Pi is about twofold higher), with
two robust subclusters (Bayesian posterior probabilities ⱖ 98%). The other fpv clusters contain more similar
sequences revealing ambiguous topologies with statistical supports frequently lower than 50%.
To explain the presence of these three very different
types at the fpvA locus, it seems likely that some lateral
transfers have occurred during the evolutionary history
of the ferripyoverdine receptor genes of P. aeruginosa.
Accordingly, several articles already suggested that
several lateral transfers occurred in P. aeruginosa, especially at pyoverdine locus (Pirnay et al., 2005; Smith et al.,
2005; Wiehlmann et al., 2007).
In order to get insight into the evolutionary history of
pyoverdine genes, we used several approaches. First, we
carried out a comparative analysis between the ferripyoverdine receptor gene and organism phylogenies.
Second, we looked at the synonymous codon usage
[codon adaptation index (CAI index)] and the GC
content.
Comparison between PVD receptor and
organism phylogenies
We investigated the evolutionary history of organisms in a
fine resolution by using a dendrogram (UPGMA, BioNumerics v5.2) based on the comparison of the composite data
set consisting of AFLP patterns, oprI, oprL and oprD
nucleotide sequences and serotypes (Fig. 2). In general,
we can see that the closely related strains (i.e. with a
similarity superior to 85%) presented the same fpvA/fpvB
distribution, especially when the strains were epidemiologically related (e.g. clone C) but also when they were
not (e.g. Br692 versus Is573 strains). In contrast, some
closely related strains (e.g. PAO1 versus LMG14083
strains or Lo053 versus Mi162 strains) showed different
fpvA/fpvB distribution, corresponding likely to some lateral
transfers. Because the profiles become probably too different when the strains are not closely related, almost all
the dendrograms constructed from molecular fingerprints
lose resolution in the deeper nodes.
In order to compensate for this putative limitation, we
estimated an organism phylogeny at a larger resolution
by using a set of 34 ribosomal concatenated genes from
seven sequenced P. aeruginosa genomes (Fig. 5B). Interestingly, while the topology between six closely related
strains (less than 0.3% of difference) has not been fully
resolved (some weak statistical supports), the PA7 strain
is well separated from the other strains in the organism
phylogeny (maximum Bayesian posterior probabilities).
We cannot formally exclude a faster evolution of the PA7
strain, as happens with a mutator strain. However, the
position of the root as highlighted by out-grouping with
Pseudomonas mendocina and Azotobacter vinelandii
shows clearly an early divergence of this strain in the
P. aeruginosa species (Fig. 5B). Moreover, from the last
P. aeruginosa common ancestor, about the same evolutionary distance is observed to each strain.
Because both phylogenies (of ferripyoverdine receptor
genes and concatenated ribosomal genes) have not been
fully resolved, it is difficult to compare them. However, two
observations can be made. (i) The presence of the same
1
2
3
4
5 6
7
8
9 10 11 12 13 14
1000
800
600
400
200
Fig. 4. Multiplex PCR-amplified fragments of six strains with two
different primer sets, and separated by electrophoresis on 1%
agarose. The first and 14th lanes contain the molecular weight
markers as in Fig. 1. The bands corresponding to the different PVD
receptors have the following sizes in reverse order of molecular
weight for set 1 (lanes 2–7): fpvAIIa (908 bp), fpvAIIb (863 bp),
fpvB (562 bp), fpvAIII (505 bp) and fpvAI (324 bp), for set 2 (lanes
8–13): fpvAII, for both variants (682 bp), fpvB (562 bp), fpvAIII
(505 bp) and fpvAI (324 bp). The four pyoverdine type reference
strains are: PAO1 (lanes 2 and 8), 7NSK2 (lanes 3 and 9), ATCC
27853 (lanes 4 and 10) and 59.20 (lanes 5 and 11). In strains
Br667 (lanes 6 and 12) and Is573 (lanes 7 and 13) we amplified, in
addition to the bands corresponding to their receptor types (which
is fpvAIII and fpvB for Br667 and fpvAIIb and fpvB for Is573),
another band of around 450 bp (indicated by arrows) which
appeared to be the result of amplification of a genomic fragment,
probably of phage origin that is present in a small fraction of the
P. aeruginosa population.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
2128 J. Bodilis et al.
I PAO1 fpvB
I W15Aug21 fpvB (EU348658)
III 59.20 fpvB (EU348655)
IIa W15Aug15 fpvB (EU348656)
58
IIb W15Dec11 fpvB (EU348663)
A
III LES fpvB
99
IIa PACS2 fpvB
100
IIb C3719 fpvB
71
fpvB
III W15Dec9 fpvB (EU348661)
87
III W15Dec10 fpvB (EU348662)
I PA14 fpvB
75
I 2192 fpvB
94
100
54
I W15Dec6 fpvB (EU348660)
I W15Dec1 fpvB (EU348659)
99
0.0005
III W15Aug16 fpvB (EU348657)
I PA14 fpvA
79
100
I W15Aug21 fpvA (EU348649)
fpvAI
I W15Dec6 fpvA (EU348651)
I 10-15 fpvA (AY765259)
57
I PAO1 fpvA
91
I PAO29p fpvA (EU348646)
0.0005
I 2192 fpvA, I W15Dec1 fpvA (EU348650)
III 206-12 fpvA (AY765261)
100
III W15Dec9 fpvA (EU348652)
fpvAIII
III W15Dec10 fpvA (EU348653)
89
III LES fpvA, III W15Aug16 fpvA (EU348648)
III 59.20 fpvA (AF537094)
III ATCCO13 fpvA (AY765262)
88
IV Rprime fpvA (AY765260)
0.0005
IIa PACS2 fpvA
IIa W15Aug15 fpvA (EU348647)
100
IIa 7NSK2 fpvA (AF537095)
91
IIa MSH fpvA (AY765263)
IIb PA7 fpvA
100
IIb C3719 fpvA
IIb W15Dec11 fpvA (EU348654)
IIb 1-60 fpvA (AF540992),
IIb 2-164 fpvA (AF540993),
IIb ATCC27853 fpvA (EU348645)
100
100
fpvAII
100
98
0.1
0.005
B
fpvAIIa
subcluster
fpvAIIb
subcluster
PA7
PAO1
100
LES
78
100
C3719
79
2192
0.001
99
PACS2
PA14
Fig. 5. Phylogenetic relationships among ferripyoverdine receptor genes (A) and 34 concatenated ribosomal genes (B) from P. aeruginosa
isolates. Strains for which the genome is sequenced are in bold. For the phylogenetic tree of ferripyoverdine receptor genes (A), each cluster
(fpvAI, fpvAII, fpvAIII, fpvB) is highlighted separately, because of a great difference between intra- and inter-cluster variabilities. The names of
the sequences on the subtrees correspond, respectively, to the fpvA type (I, IIa, IIb or III) of the corresponding strain, the strain name, the
gene name (fpvA or fpvB), and the GenBank accession number (expect for genes from sequenced genome). For the phylogenetic tree of 34
concatenated ribosomal genes (B), the position of the root was determined by using P. mendocina and Azotobacter vinelandii (ymp and AvOP
strains respectively) as out-group. All the dendrograms were generated using Bayesian inference under a GTR +g model of evolution.
Numbers on tree branches report Bayesian posterior probabilities (expressed in percentage). Only statistical support ⱖ 50% are reported
(majority rule consensus tree). The horizontal length of branches is proportional to the estimated number of substitutions. For each tree, the
scale corresponds to the number of substitutions per site.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
Distribution and evolution of ferripyoverdine receptors 2129
Table 2. Properties of the genes analysed in this study.
Set of genes
Number of
strainsa
Length of
sequences (bp)
GC content
(mol%)
CAI
Overall mean
variability (Pi)
Mean Ks (Ka/Ks)
Concatenated ribosomal genesb
fpvAI
fpvAII
fpvAIII
fpvB
7
7
8
7
15
12 735
2 367
2 382
2 265
2 343
59.7–59.9
60.8–61.2
62.2–64.0
59.4–59.6
66.1–66.7
0.77
0.81–0.82
0.83–0.87
0.76
0.86–0.87
0.006
0.003
0.073
0.003
0.005
0.017 (0.060)
0.009 (0.034)
0.161 (0.253)
0.007 (0.159)
0.011 (0.229)
a. See the name of strains in Fig. 5A. Identical sequences were removed for this analysis.
b. Corresponding to 34 concatenated ribosomal genes (see Experimental procedures).
hypothesis would be a lateral transfer of fpvB gene before
the divergence of the PA7 clade, followed by a deletion
after this divergence. In both scenarios, the fpvB gene
could have been introduced just before, during or just
after the speciation event. It is worth to mention again
that all strains in PA7 clade had the fpvAIIb gene. By
studying the genomic context of the fpvB gene (http://
v2.pseudomonas.com), it can be observed that the
regions upstream and downstream of fpvB are conserved
in PAO1, LES and PA14. These genomic regions are also
conserved in the genome of PA7 strain (Fig. 6). Interestingly, in PA7, the two genes flanking fpvB are conserved,
and in place of fpvB a fragment of about 100 nucleotides
showing 93% of identity with the end of the gene can be
detected, highlighting an ancient deletion of the fpvB gene
in the PA7 clade.
Study of the synonymous codon usage and
the GC content
PA14_10050
PA14_10040
PA14_10020
PA14_09990
PA14_10010
PA7_0929
PA7_0928
PA7_0926
PA7_0925
‘fpvB
PA7_0927
PA14_09980
Finally, we studied the synonymous codon usage (CAI
index) and the GC content to investigate the occurrence
of lateral transfers during the evolutionary history of
the ferripyoverdine receptor genes in P. aeruginosa. As
expected, for each gene or set of genes, the CAI index
fpvB
PA7_0924
PA14_09950
PA7_0922
PA7_0923
PA14_09960
PA14_09940
xth2
PA14_09920
PA14_09930
PA7_0920
PA7_0919
prpL
PA14_09910
fpvA type (IIb) in two evolutionary-distant strains (PA7 and
C3719 strains) added to the presence of all the possible
fpvA types in closely related strains (PAO1, C3719, LES,
2192 and PACS2) confirmed that some lateral transfers
have likely occurred at the fpvA locus. (ii) The second
observation concerns the presence or absence of the
fpvB gene in the seven sequenced genomes. Since the
fpvB gene was detected in about 93% of our set of 345
P. aeruginosa strains and not in other Pseudomonas
species (even in the close species P. mendocina), it might
be useful to know whether the insertion of the fpvB gene
was correlated with the P. aeruginosa speciation event,
followed by some deletion events, or whether the insertion
of the fpvB gene occurred after the speciation event,
highlighted by an ancestral state of some P. aeruginosa
strains without the fpvB gene. Interestingly, the peculiar
PA7 strain is the only strain with a sequenced genome
without the fpvB gene. Moreover, fpvB was not detected in
three other strains forming a cluster with the PA7 strain
(denominated ‘PA7 clade’ as highlighted in Fig. 2) in the
composite dendrogram analysis. Since these three
strains were not temporally and spatially related, we wondered whether the fpvB insertion event occurred after the
divergence of the PA7 clade, which could have inherited
the ancestral state without the fpvB gene. An alternative
Fig. 6. Schematic representation of the genomic region around the (complete or partial) fpvB gene in the PA14 (above) and PA7 (bellow)
strains. ORFs oriented in the right are in the leading strand. Orthologous genes (according to the Pseudomonas Genome Project) are in grey
and linked by double arrows.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
2130 J. Bodilis et al.
and GC content were conserved between the sequenced
P. aeruginosa strains for a given gene, while these
features varied between the genes for a given strain
(Table 2). However, it should be noted that, again, the PA7
strain had the highest value of GC content and CAI index
for ribosomal genes and the fpvAIIb gene. When comparing the GC content of fpvA genes we can see a decrease
in % GC from fpvAII to fpvAIII (Table 2). Since many
driving forces are responsible for variations in GC content
(e.g. position in the genome) or in synonymous codon
usage (e.g. level of gene expression), it is usually difficult
to compare these features between different genes.
However, as expected, the concatenated ribosomal
genes showed a GC content of about 60%, a classically
lower value than the GC content calculated from the core
genome of PAO1 strain (67.1%) (Wolfgang et al., 2003;
Bodilis and Barray, 2006). Interestingly, the fpvB gene
showed the same GC content as for the core genome and
high CAI values, typical for a gene present in the lineage
for a long time. These features are in agreement with both
the evolutionary scenario described above, suggesting
that the fpvB gene was introduced early in the P. aeruginosa lineage and subsequently lost in some strains such
as those representing the PA7 clade. Concerning the fpvA
genes, since the different alleles are roughly at the same
locus, code a similar function and present a priori the
same level of expression, we could expect the same CAI
index and GC content values between the three fpvA
types. Because this was not the case, we deduced that
some inter-species lateral transfers occurred at different
times and/or from different organisms, lateral transfer of
the fpvAII gene being the more ancient event and/or from
a closely related organism, followed by the fpvAI gene
and finally the fpvAIII gene.
The fpvAIII gene of LES strain is triplicated
Analysis of the recently annotated genome of the
Liverpool epidemic strain (LES) revealed that three
identical copies of fpvAIII are present (http://www.
pseudomonas.com). Also the pvdE gene is triplicated and
there are two incomplete pvdF genes before each fpvAIII.
The sequences of pvdE and fpvAIII are all identical to
each other.
Discussion
Because most of the studies about the population structure of P. aeruginosa had an epidemiological goal and
focused on recent clonal expansions, geographic localizations and links with virulence factors and pathogenicity,
little was known about the early evolutionary history of the
P. aeruginosa species. In this article, we have approached
this aspect in order to study the distribution of ferripyover-
dine receptor genes from an evolutionary point of view.
We therefore estimated an organism phylogeny in the
scale of the P. aeruginosa species from parts of the core
genome of the seven P. aeruginosa sequenced genomes.
The phylogenetic tree obtained from ribosomal genes
showed an early divergence of PA7 strain that was
strongly distant from the six other closely related
P. aeruginosa strains (Figs 2 and 5B). These six strains
were not clearly evolutionarily distinct from each other,
with a not fully supported topology, probably because of a
very limited variability. In contrast, a composite dendrogram (including AFLP pattern, oprI, oprL and oprD gene
sequences, and serotype) was useful to discriminate
between those more closely related strains but may have
some limitations on a larger scale. Altogether, the use of
these two phylogenic approaches permitted us to study
the evolutionary history in the whole P. aeruginosa
species.
The ribosomal genes have already been shown to be
useful for constructing a robust phylogeny among
Pseudomonas (Bodilis and Barray, 2006). It is important
to note that there are some discussions about methods for
estimating phylogeny from a set of genes (Gadagkar
et al., 2005). Phylogeny could be estimated either from
concatenated genes (as we did), or by carrying out a
consensus from individual trees. The principal argument
against phylogeny from concatenated genes is the variation of the evolutionary rate between functionally distinct
genes. However, because the ribosomal genes code for
functionally linked proteins and have likely evolved slowly
at the same evolutionary rate (independent of environmental changes), we argue that this argument against
phylogeny from concatenated genes is not valuable here.
Second, from the 34 (generally not well supported) trees
constructed from individual ribosomal genes, we arrived
to the same conclusions, i.e. a strong separation of the
PA7 strain and variable topologies for the six other closely
related strains (data not shown).
Pseudomonas aeruginosa is a ubiquitous microorganism, which is endowed with a high capacity for adaptation to different niches (Goldberg, 2000). This is
reflected in its capacity to take up different siderophores
next to the uptake of its own siderophores, pyoverdine
and pyochelin (Cornelis and Matthijs, 2002; Cornelis
et al., 2007; 2009). Pseudomonas aeruginosa strains
can be subdivided into three groups based on the type
of pyoverdine they produce (Cornelis et al., 1989; Meyer
et al., 1997; De Vos et al., 2001). The receptors corresponding to these three ferripyoverdines have now been
identified by different teams (Poole et al., 1993; De Chial
et al., 2003; Spencer et al., 2003; Smith et al., 2005).
Here, by using an MPCR, we typed 345 clinical and
environmental isolates from different locations throughout the world and found a similar distribution of each
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
Distribution and evolution of ferripyoverdine receptors 2131
receptor type, type I being slightly over-represented in
environmental strains. Interestingly, in a recent work on
240 P. aeruginosa strains (only a few strains were
common with our study), Wiehlmann and colleagues
(2007) found about the same proportion for each ferripyoverdine receptor type. Since competition for iron plays
an important role for the fitness of Pseudomonas (Griffin
et al., 2004) a link between the distribution of the FpvA
types and the ecological niches could be expected. The
slightly different proportion of each type between environmental and clinical strains would be interesting to
investigate further by studying the coexistence of strains
with different FpvA types, in terms of cooperation (which
would tend to limit the number of different PVD type)
and competition (which would tend to increase the
number of different PVD type).
Another important observation concerns the conservation of the fpvB gene among P. aeruginosa strains, suggesting that the ability to utilize type I ferripyoverdine as a
source of iron is a common trait of the vast majority of
P. aeruginosa strains (Ghysels et al., 2004). Although we
did not investigate the functionality of FpvB in a large
number of strains, it is evident that there are some
instances where the gene is present (or at least the part
we amplified with the primers used in this study), but the
ability to utilize the heterologous type I pyoverdine could
not be observed, perhaps because fpvB is not expressed
in these strains. In the study of Wiehlmann and colleagues (2007), the authors found that 10% of the
P. aeruginosa tested do not have the fpvB gene, which is
close to the 7% we found. Moreover, it could be deduced
from both the study of Wiehlmann and colleagues (2007)
and ours (Fig. 2) that at least a few deletions of fpvB
genes have occurred as evidenced in PA7 (Fig. 6).
Because fpvB was only found in P. aeruginosa and was
absent in other Pseudomonas spp., we formulate the
hypothesis of an ancestral state of some P. aeruginosa
strains before the insertion of the fpvB gene. So, the fpvB
gene was likely introduced early in the P. aeruginosa
species (or just before the speciation event), and lost in
the PA7 clade. The deletion of fpvB would therefore have
occurred in the PA7 clade soon after its insertion. This
observation refutes thus the most parsimonious hypothesis of an ancestral state without fpvB inherited by the
PA7 clade.
Finally, the fact that the great majority (more than 90%)
of P. aeruginosa have fpvB could highlight a fundamental
role of this gene in the ecology of this species. Nevertheless, it cannot be excluded that introduction of fpvB in the
P. aeruginosa species would be concomitant with a transfer of a more important gene and so, would result from a
genetic hitchhiking.
In their interesting study on the evolution of pyoverdine biosynthesis and uptake genes, Smith and col-
leagues (2005) propose that the pyoverdine region has
been acquired by horizontal transfer, since the codon
usage of the corresponding genes is unusual. Within the
P. aeruginosa pyoverdine region, some genes show high
divergence between types. These genes include the
NRPS genes involved in the biosynthesis of the pyoverdine peptide chain, the pvdE gene coding for an ABC
transporter, and the fpvA gene encoding the receptor
(Ravel and Cornelis, 2003; Smith et al., 2005; Visca
et al., 2007). Based on large strain collections, this study
and two previous studies (Pirnay et al., 2005; Wiehlmann et al., 2007) have arrived at the same conclusion
of frequent intra-species lateral transfers of fpvA genes,
correlated with the important role of the FpvA type in the
fitness of P. aeruginosa. It is interesting to mention that
in other fluorescent pseudomonads the genes involved
in the biosynthesis and uptake of pyoverdine are also
clustered, suggesting that horizontal gene transfers have
also occurred in these species (Ravel and Cornelis,
2003). According to the study of Smith and colleagues
(2005), from GC content and synonymous codon usage
it seems that the type III ferripyoverdine receptor gene
was transferred more recently or from a more distant
organism than the other two types, in agreement with
the low GC content of this gene (59%), the lowest of all
other TonB-dependent receptor genes, which have an
average value of 67% (P. Cornelis and J. Bodilis, in
preparation). In contrast, the type IIb ferripyoverdine
receptor gene was probably transferred before the other
two types or from a more closely related organism. Interestingly, since FpvAIIb is the receptor of the peculiar
PA7 clade, it may be the first fpvA type of the P. aeruginosa species.
Intra-type variability and tests for positive selection have
highlighted a diversifying selection of the fpvAII gene
(Smith et al., 2005; Tümmler and Cornelis, 2005). Smith
and colleagues (2005) made the suggestion that the more
rapid evolution of this gene might be driven by the need to
resist killing by pyocin S3, for which FpvAII is the receptor
(Baysse et al., 1999; De Chial et al., 2003). Although we
also think that a Darwinian selection most likely occurred
for the fpvA gene, we do not totally agree with this hypothesis of driving force proposed by Smith and colleagues
(2005). First, we have recently shown that another soluble
pyocin, S2, kills strains having the type I ferripyoverdine
receptor, but sequences of different fpvAI alleles from
S2-sensitive and S2-resistant strains did not reveal such a
diversifying selection (Denayer et al., 2007). The second
argument is the sensitivity to pyocin S3 of strains with both
FpvAII receptor subtypes (IIa and IIb), highlighting that this
positive selection gives no particular advantage for resistance to pyocin S3 (data not shown). So, the driving force
may be unknown yet, e.g. the use of FpvAII as a phage
receptor or the need to escape to the immune system. To
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
2132 J. Bodilis et al.
explain this observed positive selection and more generally to explain the great diversity of the PVD/FpvA pairs, we
suggest an alternative scenario where the evolution of the
receptor is driven essentially by changes in pyoverdine
structure. In the competition for iron, new pyoverdine structures could offer a selective advantage. In this context, we
hypothesize that the changes occur first in just one or only
a few modules of the NRPS for the biosynthesis of a given
pyoverdine. Since a receptor can sometimes recognize
heterologous pyoverdines (Ghysels et al., 2004), a new
pyoverdine variant could still be recognized by the receptor, although with lower efficiency. This could now drive the
evolution of the receptor towards a finer specificity, by a
positive selection. In this scenario, the type II pyoverdine
would result from relatively recent modifications in its structure (in fact, perhaps concomitant with the speciation
event) and the recognition of the pyoverdine by the receptor would not yet be optimized. In this regard, it is important
to mention that type II FpvA is the receptor showing the
highest specificity, since it does not allow the transport of
the other two P. aeruginosa pyoverdines (Ghysels et al.,
2004). In order to check this hypothesis, it would be interesting to study the competition between bacteria with type
IIa and those with type IIb FpvA in conditions of iron
limitation, with or without pyocin S3. Since evolution of
receptors could also be facilitated by gene duplications, it is
of interest to notice that three copies of pvdE and fpvAIII
exist in the LES strain. However, the three copies are
identical, suggesting that this is a recent event. In
Pseudomonas syringae genomes there are two copies of
fpvA in tandem, but the two proteins are only 73% identical
(P. Cornelis and J. Bodilis, in preparation).
Finally, in addition to changing or diversifying their
pyoverdine and their associated FpvA receptor, acquisition of alternative receptors (without the PVD genes), like
FpvB but also like the 35 other putative TonB-dependent
receptors identified in the PAO1 genome (Cornelis et al.,
2007), can be considered as a cheap (and cheat) strategy
to increase the fitness.
The MPCR described in this study allows a more rapid
and accurate identification of the pyoverdine type com-
pared with the IEF-based method for siderotyping (Meyer
et al., 1997) and should also be useful for the typing of
pyoverdine-negative strains that are often isolated from
Cystic Fibrosis (CF) lungs (De Vos et al., 2001). Since
nine patterns are possible (fpvAI, fpvAIIa, fpvAIIb, fpvAIII,
fpvAI-fpvB, fpvAIIa-fpvB, fpvAIIb-fpvB, fpvAIII-fpvB, fpvB),
this MPCR could be useful as a complementary technique
for typing P. aeruginosa isolates. Since it appears that
several typing methods, with different degrees of resolution, are necessary for the study of P. aeruginosa, similar
MPCR assays could be designed by including other
receptor genes, such as fptA for pyochelin (Ankenbauer
and Quan, 1994) or pfeA and pirA for ferrienterobactin
(Ghysels et al., 2005).
Experimental procedures
Bacterial strains used in this study
The P. aeruginosa strains used for reference in this MPCR
are PAO1, a type I pyoverdine producer (Stover et al., 2000),
7NSK2 and ATCC27853, both type II pyoverdine producers
(De Chial et al., 2003), and 59.20 as an example of a type III
pyoverdine producer (De Chial et al., 2003). Some (75
strains) of the 345 strains used in this study are reported in
Fig. 2. A list of all the strains used for this study as well as
their origin is available in Table S1.
Primers and PCR conditions
The primers used for this MPCR are listed in Table 3. The
PCR was performed using ™Ex-Taq polymerase (Takara),
supplied with buffer and dNTPs, according to the following
cycling parameters: 94°C (5 min) followed by 30 cycles [94°C
(30 s)-52°C (30 s)-72°C (2 min)] and a final extension [72°C
(10 min step)]. All the primers were manufactured by Eurogentec (Seraing, Belgium). The template for the PCR-mix
was either a pipette tip of bacterial cells (without prior boiling),
or 2 ml of a chromosomal DNA preparation. Double-stranded
DNA sequencing of some fpvA and fpvB genes was carried
out by the VIB sequence facility. The nucleotide sequences
determined in this study have been deposited in the GenBank
database.
Table 3. Primers used in this study.
Primer
Position
fpvAIf
fpvAIr
fpvAIIaf
fpvAIIar
fpvAIIbf
fpvAIIbr
fpvAIIIf
fpvAIIIr
fpvBf
fpvBr
1833
2157
658
1566
865
1728
1276
1781
1561
2123
Expected size (bp)
324
908
863
505
562
Sequence
5′-CGAACCCGACGAAGGCCAGA-3′
5′-GTAGCTGGTGTAGAGGCTCAA-3′
5′-TACCTCGACGGCCTGCACAT-3′
5′-GAAGGTGAATGGCTTGCCGT-3′
5′-GAACAGGGCACCTACCTGTA-3′
5′-GATGCCGTTGCTGAACTCGTA-3′
5′-ACTGGGACAAGATCCAAGAGA-3′
5′-CTGGTAGGACGAAATGCGA-3′
5′-GCATGAAGCTCGACCAGGA-3′
5′-TTGCCCTCGTTGGCCTTG-3′
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
Distribution and evolution of ferripyoverdine receptors 2133
Phylogenetic analyses
Sequence analyses
From 22 strains (including the seven strains for which the
genomes were sequenced), nearly complete FpvA and/or
FpvB sequences (41 sequences in total) were aligned using
CLUSTALX version 1.81, with default parameters (Thompson
et al., 1997), and optimized visually. The nucleic acid alignment was deduced from the corrected protein alignment,
leading to about 2300 aligned nucleotide positions.
A set of 34 ubiquitous ribosomal genes were retrieved from
the seven (fully or partially) sequenced Pseudomonas aeruginosa genomes (PAO1, LES, 2192, PACS2, C3719, PA7 and
PA14 strains). All the genes were aligned individually and
concatenated, leading to 12 735 unambiguously aligned
nucleotide positions.
From nucleic alignments, Bayesian analysis was performed using MrBayes 3.1 (Ronquist and Huelsenbeck,
2003). The Modeltest software (Posada and Crandall, 1998)
was used to choose the evolutionary model. For both phylogenies (PVD receptor and ribosomal genes), the model
used is the complex GTR with an among-site rate heterogeneity (GTR + g). In addition, we also used a model that
takes into account rate heterogeneity among positions in
codon. Since the resulting topologies were identical for the
two models, except for two weak-supported nodes in the
fpvB cluster, only the phylogenetic analyses from the first
model were presented in Fig. 5. All analyses were carried
out with random starting trees. Four Metropolis coupled
Markov chain Monte Carlo (MCMC) chains were run, stopping after 1 or 2 million generations (for ribosomal and PVD
receptor genes respectively), when the standard deviation
of split frequencies was less than 0.01. Trees were sampled
every 100 generations and the first 25% burn-in cycles (i.e.
2500 or 5000 trees) were discarded prior to consensus
trees construction. Analyses were repeated twice to ensure
the correct topology. Consensus trees were visualized with
TreeView 1.6.6 (Page, 1996) and posterior probabilities
were employed to test the statistical support of clades. Additionally, a data set consisting of the AFLP pattern, oprI, oprL
and oprD gene sequences, and serotype of 75 P. aeruginosa isolates was analysed using biological data analysis
software. AFLP band patterns were imported into BioNumerics v5.2 software (Applied Maths, Belgium) for further
normalization (background subtraction, filtering: arithmetic
average, and band search: minimum profiling 0.5% relative
to maximum value) and cluster analysis (similarity coefficient: Pearson correlation, dendrogram type: UPGMA, optimization: 0%, position tolerance: 1%, uncertain bands were
ignored). Sequences were clustered (Pairwise alignment,
open gap penalty: 100%, unit gap penalty 0%, minimum
match sequence: 2, maximum number of gaps: 9, fast algorithm), aligned (multiple alignment, open gap penalty: 100%,
unit gap penalty: 0%, minimum match sequence: 2,
maximum number of gaps: 98) and clustered a second time
(using the same parameters) using BioNumerics v5.2 software. The serotypes were compared using the Pearson correlation. These individual comparisons resulted in individual
similarity matrices. These similarity matrices were averaged
into the similarity matrix of the composite data set. No correction for internal weights was applied. A dendrogram
(UPGMA, BioNumerics v5.2) based on the comparison of the
composite data set was built.
The synonymous and non-synonymous rates were determined using the modified Nei-Gojobori method implemented
in the MEGA v2.0 software (Kumar et al., 2001). The
transition to transversion ratio was fixed at 2 and the
Jukes–Cantor correction was used to account for multiple
substitutions at the same site. Codon adaptation index
(CAI) was calculated with the new method implemented in
DAMBE software which deals with several computational
problems (Xia and Xie, 2001; Xia, 2007). All the measurements were also carried out with the classical method as
implemented in EMBOSS.cai program (Rice et al., 2000)
and, although the values were always lower than the
ones presented here, the trends were the same (data not
shown). As CAI is a measure of the relative codon usage
bias of a gene towards the average codon usage of an
organism, a reference codon usage table of the given
organism is required. Because only the reference codon
usage table of the PAO1 strain is available in EMBOSS and
DAMBE data (Epae), we wondered whether differences in
codon usage between P. aeruginosa strains would prevent
us using the same reference table for all P. aeruginosa
strains. To deal with this problem, we estimated seven reference codon usage tables from concatenated ribosomal
genes of the seven P. aeruginosa sequenced genomes, by
using the cusp program of EMBOSS (Rice et al., 2000). Next,
we used these reference codon usage tables to calculate
CAI (with classical and new methods) for several genes and
found almost identical results, whatever the strains used to
construct the reference codon usage tables (data not
shown), highlighting almost identical optimal codon
usage between the different P. aeruginosa strains tested.
Therefore only the results obtained with the reference
codon usage table of the PAO1 strain (Epae) are presented
here.
Pyoverdine typing by IEF
For IEF typing, pyoverdines were partially purified by chromabond C18-affinity chromatography from 10 ml supernatant
of cell culture in casamino acid medium (CAA). Pyoverdine
was eluted from this matrix with a 1:1 water/methanol
mixture. Pyoverdine-IEF was carried out on Ampholine PAG
plates (pH 3.5–9.5; Pharmacia) as described previously
(Meyer et al., 1997). For growth stimulation assays, pyoverdines from the different reference strains (PAO1, 7NSK2 or
59.20) were semi-purified on a preparative scale on an
XAD-4 amberlite column as described earlier (Budzikiewicz,
1993; Ghysels et al., 2004).
Acknowledgements
This work received the support of the OZR fund from the
VUB, of the Belgian Federal Research Policy (contract No.
C3/00/13), and of the Association Française de Lutte contre
la Mucoviscidose. We thank Dr Paul De Vos (University of
Gent) for his interesting comments.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135
2134 J. Bodilis et al.
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Supporting information
Additional Supporting Information may be found in the online
version of this article:
Table S1. Strains used in this study, indicating their MPCR
results, origin, year, and source (environmental strains in
yellow).
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© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 2123–2135