Electronic supplementary material
Switching between apparently redundant iron-uptake
mechanisms benefits bacteria in changeable environments
Zoé Dumas, Adin Ross-Gillespie and Rolf Kümmerli
Summary
This file contains: (a) information on methodologies used to simultaneously quantify
investment into pyoverdine and pyochelin in batch culture; and (b) a listing of genes involved
in siderophore synthesis, secretion and uptake in order to estimate the metabolic costs
associated with the synthesis of the two siderophore systems.
(a) Measuring pyoverdine and pyochelin investment based on natural fluorescence
We established a new method based on the fluorescent properties of the pyoverdine [1] and
pyochelin [2] molecules, which allowed us to simultaneously and accurately quantify
investment into the two siderophores in batch culture. The establishment of this method
involved three steps: (a) determination of the optimal excitation (ex) and emission (em)
wavelengths for pyoverdine and pyochelin fluorescence in CAA (figure S1); (b) application
of a correction procedure to account for overlaps between the emission spectra of two
siderophores (figure S2); (c) demonstration of a linear relationship between the relative
fluorescence unit (RFU) and the actual siderophore concentrations in the media (figure S3).
For all three steps, we required purified pyoverdine and pyochelin, which we obtained by
applying standard extraction protocols for pyoverdine [3] and pyochelin [4]. Specifically, we
grew PAO1-pchEF (to obtain pyoverdine) and PAO1-pvdD (to obtain pyochelin) in 500 ml
CAA at 37°C in a shaken incubator (180 rpm) for 24 hours. Following growth, we aliquoted
the culture into 50 ml Falcon tubes, which we centrifuged for 10 minutes at 16 000 x g to
separate cells (in the pellet) from siderophores (in the supernatant). The pyoverdine-
containing supernatant from strain PAO1-pchEF was adjusted to pH 6.0 using 1 N
hydrochlorid acid (HCl), and subsequently run through an Amberlite ®-XAD-4 column
(diameter = 29mm, volume = 80cm3) at a speed of 2 drops/second. This procedure results in
the preferential binding of pyoverdine, but no other components of the supernatant, to the
amberlite resin. The column was washed with 200 ml distilled water, followed by the elution
of pyoverdine with a 1:1 methanol/H2O mix. Pyoverdine in its powder form was obtained by
evaporation of the methanol/H2O on a sterile bench, followed by lyophilisation for 24 hours.
The pyochelin-containing supernatant from strain PAO1-pvdD was adjusted to pH 1.0 using 1
N hydrochlorid acid (HCl). We then added one volume of ethyl acetate to five volumes of
supernatant. We vigorously shook the mixture, and then let rest until it separated into two
phases. We collected the pyochelin solved in the ethyl acetate phase and let the ethyl acetate
evaporate on a sterile bench. The residue was dissolved with methanol and then lyophilized
for 24 hours.
To determine the excitation (ex) and emission (em) spectra of pyoverdine [1] and pyochelin
[2], we redissolved 5mM of pyoverdine and pyochelin in 200 µl CAA medium in a 96-well
plate. We assessed spectra using a multimode microplate reader (Infinite 200 PRO, Tecan,
Switzerland), measuring emissions as RFU (relative fluorescence units) across a wide range
of excitation/emission wavelengths. These analyses revealed maximal ex/em wavelengths of
400/450 nm for pyoverdine, and 370/435 nm for pyochelin (figure S1). To keep consistency
with previous experiments, we used ex/em = 400/460 nm for pyoverdine in all experiments [1,
5]. For pyochelin, we chose wavelengths (ex/em = 350/430 nm), somewhat lower than the
maxima in order to significantly reduce overlap with the pyoverdine spectra (figure S1).
However, because pyoverdine is still excited at 350 nm (22% of maximal excitation) and has
a much stronger fluorescence intensity than pyochelin (approx. 10 times stronger), pyochelin
RFU measures were significantly influenced in the presence of pyoverdine. To take this bias
into account, we established a correction procedure. First, we grew PAO1-pchEF in ironlimited CAA for 24h at 37°C under static conditions. We obtained supernatant containing
pyoverdine as described above, and diluted this supernatant in steps of 5% in CAA. The
different dilutions were added to wells of a 96-well microtitre plate and pyoverdine
fluorescence was measured both at ex/em = 400/460 nm and 350/430 nm. Subsequently, we
measured the proportion of the pyoverdine RFU captured at 350/430 nm relative to 400/460
nm (figure S2) – a value that ranged between 3.9% and 5.1%, and significantly increased with
higher absolute RFUs (Pearson’s product moment correlation: r = 0.936, n = 27, P < 0.0001,
figure S2). Based on these findings, we used the linear (y=ax+b) function from figure S2 (x =
RFU measured at 400/460 nm; y = proportion of the pyoverdine signal captured at 350/430
nm) to obtain an unbiased estimate of pyochelin RFU at 350/430 nm (z), which is defined as
z=w-xy, where w is the total RFU measured at 350/430 nm. Since pyochelin is marginally
excited (7% of maximal excitation) at 400 nm and fluoresces only weakly, pyoverdine RFU
measures were not significantly influenced by the presence of pyochelin, and consequently no
correction procedure was necessary.
Finally, we assessed whether measures of RFU increase linearly with pyoverdine and
pyochelin concentrations in CAA. Specifically, we redissolved 2, 5, 10, 20, 40, 60, 80, 100,
200 µM of pyoverdine and pyochelin in 200 µl CAA and measured the RFU as described
above. The range of concentrations chosen resulted in a range of RFUs that were also
observed in our experiments. Within this range, we found that there were nearly perfect linear
relationships between RFU measures and the concentrations of pyoverdine (R2 = 0.991, F1,8 =
991, P < 0.0001) and pyochelin (R2 = 0.989, F1,8 = 812, P < 0.0001) (figure S3).
Figure S1. Excitation (black lines) and emission (grey lines) spectra of (a) pyoverdine and (b)
pyochelin. Spectra of purified pyoverdine and pyochelin (both at a concentration of 5mM)
were measured in CAA. Dots represent the excitation (black dots) and emission (grey dots)
wavelengths chosen for all experiments. Fluorescence intensities are scaled relative to the
maximum fluorescence intensity.
(a)
Fluorescence intensity
1.0
0.8
0.6
0.4
0.2
0.0
300 320 340 360 380 400 420 440 460 480 500 520
Wavelength (nm)
1.0
Fluorescence intensity
(b)
0.8
0.6
0.4
0.2
0.0
300 320 340 360 380 400 420 440 460 480 500 520
Wavelength (nm)
Figure S2. Proportion of the pyoverdine RFU (relative fluorescence unit) captured at ex/em =
350/430 nm relative to 400/460 nm. The function of the linear regression (solid line) was used
to obtain an unbiased estimate of pyochelin RFU measured at ex/em = 350/430 nm.
0.08
y = 3E-07x + 0.0409
Proportion of pyoverdine RFU
captured at ex/em = 350/430 nm
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0
10000
20000
30000
Pyoverdine RFU measured with ex/em = 400/460 nm
Figure S3. Significant linear relationship between pyoverdine (squares) and pyochelin
(circles) concentration in CAA medium and the measured relative fluorescence units at ex/em
Scaled siderophore fluorescense (RFU)
= 400/460 for pyoverdine and ex/em = 350/430 for pyochelin.
1.0
0.8
0.6
0.4
0.2
0.0
0
50
100
150
200
Siderophore concentration (! M) in CAA media
(b) Estimating the metabolic costs of pyoverdine and pyochelin production
To estimate the relative metabolic costs involved in pyoverdine and pyochelin production, we
counted the number of genes involved in pyoverdine and pyochelin synthesis as well as the
number of nucleotides and amino acids needed to transcribe and translate these genes (table
S1). Data were obtained from the Pseudomonas Genome Database (http://pseudomonas.com/)
and from the following publications [6-10]. Note that this analysis does not take into account
the variation in expression levels among genes [6]. Moreover, because not all genes involved
in pyoverdine/pyochelin synthesis have been characterized so far, our analysis must be
understood as a rough proxy of the actual metabolic costs involved in the synthesis of the two
siderophores.
Table S1. Genes involved in pyoverdine (a) and pyochelin (b) synthesis and secretion.
(a)
No.
Gene
Locus tag
Gene product
No. of
nucleotides
No. of
amino acids
1
pvcA
PA2254
Paerucumarin biosynthesis
protein for chromophore synthesis
987
328
2
pvcB
PA2255
Paerucumarin biosynthesis
protein for chromophore synthesis
876
291
3
pvcC
PA2256
Paerucumarin biosynthesis
protein for chromophore synthesis
1503
500
4
pvcD
PA2257
Paerucumarin biosynthesis
protein for chromophore synthesis
648
215
5
ptxR
PA2258
Transcriptional regulator PtxR for
chromophore synthesis genes
939
312
6
pvdQ
PA2385
3-oxo-C12-homoserine lactone
acylase
2289
762
7
pvdA
PA2386
L-ornithine N5-oxygenase
1332
443
8
fpvI
PA2387
ECF sigma factor required for
expression of fpvA
480
159
9
fpvR
PA2388
Antisigma factor for PvdS and
FpvI
996
331
10
pvdR
PA2389
Element of tripartite efflux system
involved in pyoverdine recycling
1176
391
11
pvdT
PA2390
Element of tripartite efflux system
involved in pyoverdine recycling
1992
663
12
opmQ
PA2391
Element of tripartite efflux system
involved in pyoverdine recycling
1425
474
13
pvdP
PA2392
PvdP
1635
544
14
pvdM
PA2393
Probable dipeptidase precursor
1347
448
15
pvdN
PA2394
PvdN
1284
427
16
pvdO
PA2395
PvdO
855
284
17
pvdF
PA2396
N5-hydroxyornithine
transformylase
828
275
18
pvdE
PA2397
ABC transporter
1650
549
19
fpvA
PA2398
Ferripyoverdine receptor
2448
815
20
pvdD
PA2399
Non-ribosomal peptide synthetase
7347
2448
21
pvdJ
PA2400
Non-ribosomal peptide synthetase
6474
2157
22
pvdI
PA2402
Non-ribosomal peptide synthetase
15450
5149
23
pvdH
PA2413
L-2,4-diaminobutyrate:2ketoglutarate 4-aminotransferase
1410
469
24
pvdL
PA2424
PvdL
13029
4342
25
pvdG
PA2425
PvdG
765
254
26
pvdS
PA2426
ESC sigma factor required for
expression of pvd genes
564
187
69729
23217
Total
(b)
No.
Gene
Locus tag
Gene product
No. of
nucleotides
No. of
amino acids
1
fptX
PA4218
1244
414
2
fptA
PA4221
Innermembrane pyochelin
transporter
Ferripyochelin receptor
2163
720
3
pchG
PA4224
Pyochelin biosynthetic protein
1050
349
4
pchF
PA4225
Pyochelin synthetase
5430
1809
5
pchE
PA4226
Dihydroaeruginoic acid
synthetase
4317
1438
6
pchR
PA4227
Transcriptional regulator for
pyochelin synthesis genes
891
296
7
pchD
PA4228
Salicylate biosynthesis protein
1644
547
8
pchC
PA4229
Salicylate biosynthesis protein
756
251
9
pchB
PA4230
Salicylate biosynthesis protein
306
101
10
pchA
PA4231
Salicylate biosynthesis
isochorismate synthase
1431
476
17988
6401
Total
Supplementary references
1
2
3
4
5
6
7
8
9
10
Ankenbauer R, Sriyosachati S, Cox CD. 1985 Effects of siderophores on the growth of
Pseudomonas aeruginosa in human serum and transferrin. Infect. Immun. 49, 132-140.
Cox CD. 1980 Iron uptake with ferripyochelin and ferric citrate by Pseudomonas
aeruginosa. J. Bacteriol. 142, 581-587.
Meyer JM, Gruffaz C, Raharinosy V, Bezverbnaya I, Schäfer M, Budzikiewicz H. 2008
Siderotyping of fluorescent Pseudomonas: molecular mass determination by mass
spectrometry as a powerful pyoverdine siderotyping method. BioMetals 21, 259-271.
(doi:10.1007/s10534-007-9115-6).
Cox CD, Graham R. 1979 Isolation of an iron-binding compound from Pseudomonas
aeruginosa. J. Bacteriol. 137, 357-364.
Kümmerli R, Jiricny N, Clarke LS, West SA, Griffin AS. 2009 Phenotypic plasticity of
a cooperative behaviour in bacteria. J. Evol. Biol. 22, 589-598. (doi:10.1111/j.14209101.2008.01666.x).
Ochsner UA, Wilderman PJ, Vasil AI, Vasil ML. 2002 GeneChip® expression analysis
of the iron starvation response in Pseudomonas aeruginosa: identification of novel
pyoverdine biosynthesis genes. Mol. Microbiol. 45, 1277-1287.
Visca P, Imperi F, Lamont IL. 2007 Pyoverdine siderophores: from biogenesis to
biosignificance. Trends Microbiol. 15, 22-30. (doi:10.1016/j.tim.2006.11.004).
Visca P, Imperi F, Lamont IL. 2007 Pyoverdine synthesis and its regulation in
fluorescent Pseudomonads. In Microbial Siderophores (eds Varma A, Chincholkar S),
pp. 135-163. Berlin: Springer-Verlag.
Imperi F, Tiburzi F, Visca P. 2009 Molecular basis of pyoverdine siderophore recycling
in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. 106, 20440-20445.
(doi:10.1073/pnas.0908760106).
Youard ZA, Wenner N, Reimmann C. 2011 Iron acquisition with the natural
siderophore enantiomers pyochelin and enantio-pyochelin in Pseudomonas species.
Biometals 24, 513-522. (doi:10.1007/s10534-010-9399-9).