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 thesynthesis 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) andPAO1-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 gto 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 wasadjusted to pH 1.0 using 1 N hydrochlorid acid (HCl). We then added one volume of ethyl acetate tofive 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 iron-limited 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 400/460nm; 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)

(b)

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.

Figure S3.Significant linear relationship between pyoverdine (squares) and pyochelin (circles) concentration in CAA medium and the measured relative fluorescence units at ex/em = 400/460 for pyoverdine and ex/em = 350/430 for pyochelin.


(b)Estimating the metabolic costs of pyoverdine and pyochelin production

To estimate the relative metabolic costs involved inpyoverdine 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 ( 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:2-ketoglutarate 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
Total / 69729 / 23217

(b)

No. / Gene / Locus tag / Gene product / No. of nucleotides / No. of amino acids
1 / fptX / PA4218 / Innermembrane pyochelin transporter / 1244 / 414
2 / fptA / PA4221 / 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
Total / 17988 / 6401

Supplementary references

1Ankenbauer 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.

2Cox CD. 1980 Iron uptake with ferripyochelin and ferric citrate by Pseudomonas aeruginosa. J. Bacteriol.142, 581-587.

3Meyer 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. BioMetals21, 259-271. (doi:10.1007/s10534-007-9115-6).

4Cox CD, Graham R. 1979 Isolation of an iron-binding compound from Pseudomonas aeruginosa. J. Bacteriol.137, 357-364.

5Kümmerli 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.1420-9101.2008.01666.x).

6Ochsner 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.

7Visca 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).

8Visca 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.

9Imperi 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).

10Youard ZA, Wenner N, Reimmann C. 2011 Iron acquisition with the natural siderophore enantiomers pyochelin and enantio-pyochelin in Pseudomonas species. Biometals24, 513-522. (doi:10.1007/s10534-010-9399-9).