# Structure of the Outer Membrane Transporter FemA and Its Role in the Uptake of Ferric Dihydro-Aeruginoic Acid and Ferric Aeruginoic Acid in Pseudomonas aeruginosa

Virginie Will, Lucile Moynié, Elise Si Ahmed Charrier, Audrey Le Bas, Lauriane Kuhn, Florian Volck, Johana Chicher, Hava Aksoy, Morgan Madec, Cyril Antheaume, et al.

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Virginie Will, Lucile Moynié, Elise Si Ahmed Charrier, Audrey Le Bas, Lauriane Kuhn, et al. Structure of the Outer Membrane Transporter FemA and Its Role in the Uptake of Ferric Dihydro-Aeruginoic Acid and Ferric Aeruginoic Acid in Pseudomonas aeruginosa. ACS Chemical Biology, 2025, 20 (3), pp.690-706. 10.1021/acscchembio.4c00820. hal-04987259

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Structure of the Outer Membrane Transporter FemA and its
Role in the Uptake of Ferric Dihydro-Aeruginoic Acid and
Ferric Aeruginoic Acid in Pseudomonas aeruginosa

<table><tr><td>Journal:</td><td>ACS Chemical Biology</td></tr><tr><td>Manuscript ID</td><td>cb-2024-00820u.R1</td></tr><tr><td>Manuscript Type:</td><td>Article</td></tr><tr><td>Date Submitted by the Author:</td><td>n/a</td></tr><tr><td>Complete List of Authors:</td><td>Will, Virginie; University of Strasbourg<br>Moynié, Lucile; The Rosalind Franklin Institute<br>SI AHMED CHARRIER, Elise; University of Strasbourg<br>Kuhn, Lauriane; Institut de Biologie Moléculaire et Cellulaire, Plateforme<br>Protéomique Strasbourg-Esplanade<br>Le Bas, Audrey; The Rosalind Franklin Institute<br>Volck, Florian; University of Strasbourg<br>Chicher, Johana; IBMC<br>Aksoy, Hava; University of Strasbourg<br>MADEC, Morgan; ICube,<br>Antheaume, Cyril; University of Strasbourg, CNRS, ISIS UMR 7006, ISIS<br>UMR 7006<br>Mislin, Gaëtan; CNRS, UMR7242 Biotechnologie et Signalisation Cellulaire<br>Schalk, Isabelle; University of Strasbourg, UMR7242</td></tr></table>

SCHOLARONE™
Manuscripts
1 **Structure of the Outer Membrane Transporter FemA and**

2 **its Role in the Uptake of Ferric Dihydro-Aeruginoic Acid and Ferric Aeruginoic**

3 **Acid in *Pseudomonas aeruginosa***

4

5 Virginie WILL<sup>1,2 #</sup>, Lucile MOYNIÉ<sup>*3#</sup>, Elise SI AHMED CHARRIER<sup>1,2</sup>, Audrey LE BAS<sup>3</sup>, Lauriane KUHN<sup>4</sup>,

6 Florian VOLCK<sup>1,2</sup>, Johana CHICHER<sup>4</sup>, Hava AKSOY<sup>5</sup>, Morgan MADEC<sup>6</sup>, Cyril ANTHEAUME<sup>5</sup>; Gaëtan L. A.

7 MISLIN<sup>1,2</sup>, Isabelle J. SCHALK<sup>*1,2</sup>

8

9 <sup>1</sup> CNRS, University of Strasbourg, UMR7242, UMR7242, ESBS, Bld Sébastien Brant, F-67412 Illkirch,

10 Strasbourg, France

11 <sup>2</sup> University of Strasbourg, UMR7242, ESBS, Bld Sébastien Brant, F-67412 Illkirch, Strasbourg, France

12 <sup>3</sup> The Rosalind Franklin Institute, Harwell Campus, Oxfordshire, UK OX11 0QS.

13 <sup>4</sup> Plateforme protéomique Strasbourg-Esplanade, CNRS, Université de Strasbourg, IBMC, 2 Allée

14 Konrad Roentgen, F-67084 Strasbourg, France.

15 <sup>5</sup> Université de Strasbourg, Institut des Sciences et de l'Ingénieurie Supramoleculaire, Plateforme

16 d'analyses chimiques, 8 allée Gaspard Monge, F-67000 Strasbourg, France.

17 <sup>6</sup> ICube Laboratory, UMR 7357 (CNRS / University of Strasbourg), Bld Sébastien Brant, F-67412 Illkirch,

18 Strasbourg, France

19

20 # These authors contributed equally

21

22 *Corresponding authors: isabelle.schalk@unistra.fr and Lucile.Moynie@rfi.ac.uk

23

24 **Keywords :** Aeruginoic acid, dihydroaeruginoic acid, siderophore, *Pseudomonas aeruginosa*, FemA,

25 outer membrane transporter, iron, pyochelin.
1 ABSTRACT

2 Iron is essential for bacterial growth, and *Pseudomonas aeruginosa* synthesizes the siderophores

3 pyochelin (PCH) and pyoverdine to acquire it. PCH contains a thiazolidine ring that aids in iron chelation

4 but is prone to hydrolysis, leading to the formation of 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde

5 (IQS). Using mass spectrometry, we demonstrated that PCH undergoes hydrolysis and oxidation in

6 solution, resulting in the formation of aeruginoic acid (AA). This study used proteomic analyses and

7 fluorescent reporters to show that AA, dihydroaeruginoic acid (DHA), and PCH induce the expression

8 of *femA*, a gene encoding the ferri-mycobactin outer membrane transporter in *P. aeruginosa*. Notably,

9 the induction by AA and DHA was observed only in strains unable to produce pyoverdine, suggesting

10 their weaker iron-chelating ability compared to pyoverdine. $^{55}$Fe uptake assays demonstrated that

11 both AA-Fe and DHA-Fe complexes are transported via FemA; however, no uptake was observed for

12 PCH-Fe through this transporter. Structural studies revealed that FemA is able to bind $AA_2$-Fe or $DHA_2$-

13 Fe complexes. Key interactions are conserved between FemA and these two complexes, with

14 specificity primarily driven by one of the two siderophore molecules. Interestingly, although no iron

15 uptake was noted for PCH through FemA, the transporter also binds PCH-Fe in a similar manner. These

16 findings show that under moderate iron deficiency, when only PCH is produced by *P. aeruginosa*,

17 degradation products AA and DHA enhance iron uptake by inducing *femA* expression and facilitating

18 iron transport through FemA. This provides new insights into the pathogen's strategies for iron

19 homeostasis.

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1 INTRODUCTION

2 Iron is an essential nutrient for the growth of most microorganisms. Despite its abundance in the

3 earth's crust, its availability to bacteria is limited. Indeed, under physiological pH and aerobic

4 conditions, iron predominantly exists as poorly soluble ferric hydroxides. During infections in a

5 eukaryotic host, this scarcity is further exacerbated by host proteins sequestering iron, resulting in a

6 free iron(III) concentration as low as $10^{-18}$ to $10^{-24}$ M.¹ To overcome this, bacteria have evolved efficient

7 iron uptake systems, with siderophore-mediated iron acquisition being widespread among them.²⁻⁴

8 Siderophores, secondary metabolites secreted by bacteria, are distinguished by their exceptionally

9 high affinity for iron, enabling them to effectively facilitate iron uptake.⁵,⁶ Although their chemical

10 structures vary, siderophores typically contain chelating groups like catechol, hydroxamate, hydroxy-

11 acid, 2-hydroxyphenyl-thiazoline or 2-hydroxyphenyl-oxazoline,⁵,⁶ giving them this strong binding

12 capacity for iron (III). Once iron is chelated, siderophore-iron complexes are recognized by specific

13 TonB-dependent transporters (TBDTs) on the bacterial surface of Gram-negative bacteria.⁷ These

14 transporters import ferric complexes into the cell, with energy provided by the inner membrane TonB

15 machinery—a complex of ExbB, ExbD, and TonB proteins.⁸⁻¹⁰ Powered by the proton motive force, this

16 machinery energizes TBDTs, enabling ferric-siderophore uptake. TBDTs are highly selective,

17 recognizing specific siderophores or those with closely related structures.⁷,¹¹,¹² Iron release from

18 siderophores occurs in the periplasm or cytoplasm, depending on the bacterial species, often involving

19 iron reduction and subsequent siderophore hydrolysis or enzymatic modification.¹³

20 The opportunistic pathogen *Pseudomonas aeruginosa* produces two siderophores, pyochelin (PCH)

21 and pyoverdine.¹⁴⁻¹⁶ Additionally, it efficiently utilizes a wide range of xenosiderophores—

22 siderophores produced by other bacteria or fungi—through a process known as siderophore piracy.³

23 This is enabled by 19 genes in the *P. aeruginosa* genome encoding TBDTs dedicated to iron

24 acquisition.³,¹⁷ For example, FptA mediates ferri-PCH uptake,¹⁸,¹⁹ while FpvA and FpvB handle ferri-

25 pyoverdine uptake.²⁰⁻²² For xenosiderophores, PfeA facilitates iron uptake via enterobactin,²³,²⁴ FvbA
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1 via vibriobactin,²⁵ PirA and PiuA via monocatechol-type siderophores,²⁶⁻²⁹ and FoxA and FiuA via

2 hydroxamate siderophores such as nocardamine, desferrioxamines, and ferrichrome derivatives,¹²,³⁰⁻

3 ³³ ActA handles hydroxyacid-type siderophores like rhizoferrin,¹¹ ChtA targets mixed hydroxamate and

4 hydroxy acid siderophores,¹¹,³⁴ and FemA mediates mycobactin (MYC) uptake³⁵ (Figure 1). Finally, FpvB

5 is involved in the uptake of various hydroxamate siderophores and mixed siderophores such as

6 pyoverdine, which contains both catechol and hydroxamate groups.¹²,²¹,³⁶ *P. aeruginosa*'s ability to

7 utilize diverse siderophores from different sources is crucial to its dominance in various environments.

8 It detects specific xenosiderophores through mechanisms involving sigma and anti-sigma factors, two-

9 component systems, or AraC-like regulators.²⁹,³⁵,³⁷⁻⁴⁰ In response, the bacterium induces the

10 expression of the appropriate uptake system, including specific TBDTs.¹¹,¹²,³²,³⁵,³⁹,⁴¹,⁴²

11 Recent studies have indicated that dihydroaeruginoic acid (DHA) and aeruginoic acid (AA), along with

12 related compounds, serve as siderophores for iron uptake into *P. aeruginosa* cells,⁴³⁻⁴⁵ but without

13 identifying the TBDTs involved and without giving any insights under which conditions these molecules

14 could be used by *P. aeruginosa* to access iron. Like PCH and yersiniabactin (YER), DHA features a 2-

15 hydroxyphenyl-thiazoline when AA is based on a 2-hydroxyphenyl-thiazole scaffold (Figure 1). Both 2-

16 hydroxyphenyl-thiazoline and 2-hydroxyphenyl-thiazole motifs possess iron chelation properties. DHA

17 has been identified as shunt product of the PCH biosynthesis.⁴⁴,⁴⁶

18
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Structures of pyochelin (PCH), yersiniabactin (YER), mycobactin (MYC), dihydroaeruginoic acid (DHA), aeruginoic acid (AA). 2-hydroxyphenyl-thiazoline and 2-hydroxyphenyl-thiazole motives are respectively colored in red and blue.</figcaption></figure>

The goal of this study was to identify the TBDT responsible for importing iron into *P. aeruginosa* cells via AA and DHA, and to determine the conditions under which these compounds behave as siderophores for the bacterium. First, mass spectrometry confirmed that AA is produced through the hydrolysis of PCH in aqueous environments. Using differential proteomics and fluorescent reporters, we found that AA and DHA induce the expression of *femA* under iron-starvation conditions—*femA* was previously associated with iron transport by MYC siderophores.³⁵ *femA* expression was also induced by PCH. Additionally, we demonstrated that both DHA and AA, but not PCH, promote iron uptake into *P. aeruginosa* via the FemA TBDT. We also solved the X-ray structure of FemA in complex with ferri-AA, ferri-DHA, and ferri-PCH, which provided critical insights into the interaction mechanism between the transporter and these compounds.
## 1 RESULTS AND DISCUSSION

2 **Synthesis of DHA and AA.** DHA was synthesized by condensation of 2-hydroxybenzonitrile 1 with (S)-

3 Cysteine hydrochloride 2 in hydro-methanolic conditions (Phosphate buffer 1M, pH 6.4/MeOH)

4 according to a previously published procedure.⁴⁷,⁴⁸ DHA was obtained in 89% yield. Synthesis of AA was

5 performed starting from DHA. The latter compound was treated with thionyl chloride and the resulting

6 acyl chloride was not isolated and directly reacted with a solution of triethylamine in methanol.

7 Intriguingly we straightforwardly isolate the oxidized derivative 3 in 43% yield. This direct conversion

8 was never described in the literature on such a compound. A further saponification of 3 with sodium

9 hydroxide in hydro-methanolic medium led to the expected AA 5 isolated in 73% yield.

<figure><img src="image_3.png" /><figcaption>**Figure 2. Synthesis of DHA and AA.**</figcaption></figure>

15 **AA is a degradation product of PCH.** PCH, one of the two siderophores produced by *P. aeruginosa*, was chemically synthesized following a protocol developed by our team.⁴⁷,⁴⁸ PCH contains three asymmetric centers: C4', C2" and C4" (Figure 1). Partial epimerization occurs at the C4' chiral center during synthesis. The C2" chiral center within the thiazolidine ring exists as a thioaminal, making it inherently epimerizable.⁴⁹,⁵⁰ Meanwhile, the configuration of the C4" chiral center remains unchanged during synthesis. Consequently, the PCH used in this work is a mixture of four stereoisomers. Analysis of PCH samples using reversed-phase ultra-high-performance liquid chromatography and high-resolution mass spectrometry confirmed the presence of the four stereoisomers and the absence of DHA and AA. However, two aldehydes, 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde also called IQS
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1 and DHAld were detected as traces contaminants (below 1%) (Figure S7 and S8, Figure 3; Table 1). IQS

2 has been identified in previous studies.<sup>51,52</sup>

3 Using the same analytical technique, chemical stability of PCH was further assessed after incubation

4 at a concentration of 10 µM in Casamino acid medium (CAA), at 30°C for 15 h (the medium and

5 temperature used to growth *P. aeruginosa* under iron starved conditions). Mass spectrometry analysis

6 of pure CAA does not highlight the presence of PCH or any of its degradation process (Figure S9 and

7 Mass spectrometry analysis revealed that after 15 h incubation of PCH in CAA, several

8 products can be detected, notably DHAld and AA. DHA was not detected under these conditions

9 (Figure S11 et S12; Table 1). As it was already demonstrated by Hertweck and coworkers, IQS results

10 from the hydrolysis of the thioaminal moiety of PCH and a subsequent oxidation (Figure 3).<sup>53</sup> On the

11 other hand, the degradation of PCH into AA has not been described. Additional experiments are

12 needed to fully understand how CAA medium promotes hydrolysis and/or oxidation steps leading to

13 compounds DHAld, DHA, IQS and AA (Figure 3).

14 To investigate whether the presence of *P. aeruginosa* can influence the degradation process, we

15 cultivated a *P. aeruginosa* strain incapable of producing its own siderophores, pyoverdine and PCH

16 ($\Delta$pvdf$\Delta$pchA, deletion on pvdf and pchA, two genes involved in the biosynthesis of pyoverdine and

17 PCH respectively, Table S1), in CAA medium in the absence or presence of 10 µM of PCH. We

18 intentionally used a strain deficient in pyoverdine and PCH production to prevent contamination from

19 PCH produced by the strain and to ensure that the strain utilized the added PCH in the growth media

20 to access iron, rather than relying on its own siderophores. After 15 hours of incubation, IQS, DHAld,

21 and AA were detected in the growth media, while DHA was not detected (Figure S15 and S16; Table

22 1). AA appears to be the ultimate degradation product of PCH (Figure 3). As a control, the same

23 experiment was repeated with *P. aeruginosa* cells grown in CAA medium without PCH. As expected,

24 neither PCH nor the degradation compounds shown in Figure 3 were observed when the bacteria were

25 cultivated alone (Figure S13 and S14; Table 1).
These data, summarized in **Table 1**, demonstrate that PCH in solution undergoes degradation with AA as ultimate oxidation product. This degradation appears to be spontaneous and independent of the presence of bacteria. The conversion of PCH to AA involves hydrolysis, producing DHAld, followed by two successive oxidation steps (the formation of a double bond and the oxidation of an aldehyde to a carboxylate). The oxidation of thiazoline to thiazole (double bond formation) by atmospheric oxygen has been reported and shown to be significantly enhanced by the presence of an aromatic ring at position 2.⁵⁴ Similarly, the aerobic oxidation of aldehydes (e.g., IQS) to the corresponding carboxylates (e.g., AA) has also been documented.⁵⁵ The mechanisms and sequence of these two oxidation reactions remain unclear and are currently under investigation. The presence of this compound in *P. aeruginosa* culture broth raised the question of its uptake by the bacteria as it was previously demonstrated for the closely related DHA.⁴³,⁴⁴

<table><caption>**Table 1.** Qualitative detection of the degradation products of PCH in CAA medium, in the absence or in the presence of a culture of *ΔpvdFΔpchA*, using mass spectrometry. Mass spectrometry was used to qualitatively identify the degradation products of PCH after 15 hours of incubation in CAA medium or in the presence of *ΔpvdFΔpchA* cells. PCH was present at a concentration of 10 µM. Gray cells labeled 'detected' indicate the presence of compounds, while uncolored cells with a '/' signify that compounds were not detected.</caption><thead><tr><th>Conditions</th><th>PCH</th><th>DHA</th><th>AA</th><th>IQS</th><th>DHAld</th></tr></thead><tbody><tr><td>CAA</td><td>/</td><td>/</td><td>/</td><td>/</td><td>/</td></tr><tr><td>CAA, PCH</td><td>Detected</td><td>/</td><td>Detected</td><td>/</td><td>Detected</td></tr><tr><td>CAA, ΔpvdFΔpchA</td><td>/</td><td>/</td><td>/</td><td>/</td><td>/</td></tr><tr><td>CAA, ΔpvdFΔpchA, PCH</td><td>Detected</td><td>/</td><td>Detected</td><td>Detected</td><td>Detected</td></tr></tbody></table>

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<figure><img src="image_4.png" /><figcaption>Figure 3. Putative degradation process of PCH into DHAId, DHA, IQS and AA in solution.</figcaption></figure>

**The presence of PCH, AA and DHA induce the transcription and expression of femA.** An effective strategy to identify the TBDT in *P. aeruginosa* used by a xenosiderophore to import iron is to use differential proteomics to determine if any TBDT genes in the *P. aeruginosa* genome show increased expression when the bacterium is grown in the presence of the tested xenosiderophore. Indeed, it is well established that the presence of certain xenosiderophores in the environment of *P. aeruginosa* triggers the expression of their corresponding TBDTs.<sup>11,12,28,32,35,41,56</sup> For instance, the presence of ferrichrome induces *fiuA* expression,<sup>41,56</sup> nocardamine triggers *foxA* expression,<sup>32</sup> and enterobactin induces *pfeA* expression.<sup>41,57,58</sup> Given that AA and DHA can be utilized as siderophores by *P. aeruginosa*,<sup>43-45</sup> we aimed to investigate whether the presence of DHA and AA induces the expression of a specific TBDT involved in the uptake of the ferric forms of DHA and AA. To achieve this, we employed a differential proteomic approach, comparing the proteomes of *P. aeruginosa* grown in the absence and presence of 10 µM DHA, AA, or PCH. The culture medium used was CAA, an iron-restricted medium (20 nM of iron).<sup>59</sup> We utilized again the *P. aeruginosa* strain lacking endogenous siderophore production (Δ*pvdFΔpchA*, **Table S1**) to control the levels of PCH, AA and DHA in the culture. TBDT proteins, even though they are membrane proteins, are easily detected by proteomic approaches, as shown previously.<sup>28,32,41,57,60,60,61</sup> Upon comparing the proteomes of the Δ*pvdFΔpchA* strain cultured
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during 8 hours in the absence and presence of DHA or AA, we observed a substantial induction of *femA*

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expression (fold change of 7,7 and 7,4 respectively, **Figure 4A**). *femA* expression has been described

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previously as being induced by the presence of ferri-MYC,³⁵ a siderophore sharing structural and

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chemical characteristics with PCH, AA and DHA (Figure 1). No other TBDT had its expression

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significantly induced or repressed in the presence of DHA or AA.

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<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /></figure>

**Figure 4. A. Modulation of protein expression in P. aeruginosa strains unable to produce pyoverdine and PCH in the presence of AA, DHA and PCH.** Proteomic analyses were conducted on the Δ*pvdFΔpchA* strain grown for 8 hours in CAA medium supplemented with 10 µM of AA, DHA or PCH. Median values measured for the cultures supplemented with either 10 µM AA, DHA or PCH were plotted against those measured without the supplementation of AA, DHA or PCH. The three volcano plots display log2-fold changes in protein abundances and corresponding -log10-transformed p-values relative to untreated control samples. Proteins associated with the PCH pathway are denoted by orange dots, those linked to the pyoverdine pathway by green dots, and the TBDTs by blue dots. **B. Transcription of the fpvA, fptA, and femA genes in the presence of 10 µM AA, DHA or PCH.** RT-qPCR was conducted on RNA isolated from P. aeruginosa PAO1 and Δ*pvdFΔpchA* cells grown in CAA medium, both in the absence and presence of 10 µM AA, DHA or PCH, for 8 hours. The results are presented as the ratio between the values obtained in the presence of 10 µM AA, DHA or PCH and those obtained in the absence of the compounds. *fpvA* encodes the TBDT of pyoverdine-Fe, *fptA* encodes the TBDT of PCH-Fe, and *femA* encodes the TBDT of ferri-mycobactin. Data from both panels are normalized relative to the reference gene *uvrD* and are representative of three independent experiments.

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2 Regarding the experiment conducted in the presence of PCH, comparing the proteomes of

3 ΔpvdfΔpchA cells grown for 8 hours with and without PCH revealed an induction of femA expression

4 but no induction of fptA expression (Figure 4A), which encodes the TBDT for ferri-PCH.18,19 This lack of

5 fptA induction is consistent with our previous findings.62 We showed that, under iron-restricted

6 conditions, fptA expression reaches its maximum in a ΔpvdfΔpchA strain, which is unable to produce

7 the siderophores pyoverdine and PCH, and adding PCH does not further increase fptA expression.62

8 The induction of femA expression in the presence of PCH is higher compared to that observed in the

9 presence of either 10 µM of AA or DHA after 8 hours of incubation (fold change of 9.3 compared to 7.4

10 and 7.7 respectively, Figure 4A).

11 In a subsequent step, using RT-qPCR, we monitored the transcription levels of genes encoding the

12 TBDTs FemA, FptA, and FpvA (FpvA being the TBDT for ferri-pyoverdine) for PAO1 (wild type) and

13 ΔpvdfΔpchA cells grown under iron-restricted conditions, both in the absence and presence of 10 µM

14 AA, DHA or PCH (Figure 4B). The data revealed an induction of femA transcription in the ΔpvdfΔpchA

15 strain in the presence of AA, DHA, and PCH, with no discernible impact on fptA transcription, thereby

16 corroborating the results obtained through the proteomic approach. The induction of femA

17 transcription and expression appears to be more efficient with DHA compared to PCH or AA in

18 ΔpvdfΔpchA cells. In the case of PAO1, which synthesizes pyoverdine and PCH, femA transcription was

19 induced only in the presence of PCH, and not in the presence of DHA and AA. This suggests that DHA

20 and AA might be less effective at chelating iron in the presence of the natural siderophores pyoverdine

21 and PCH. Conversely, the induction of femA transcription in the presence of PCH in the PAO1 strain

22 may be due either to PCH itself or to higher concentrations of DHA and AA compared to the experiment

23 with ΔpvdfΔpchA cells grown in the presence of 10 µM AA or DHA. This increase in DHA and AA

24 concentrations could result from PCH degradation, leading to levels sufficient to initiate transcription

25 despite the presence of pyoverdine.
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1 Altogether, these data demonstrate that the presence of AA and DHA in the growth media of P.

2 aeruginosa induces the transcription and expression of *femA*, a gene encoding the TBDT FemA

3 previously described to be involved in the uptake of ferri-MYC.³⁵ Regarding the induction of *femA*

4 transcription and expression observed in the presence of PCH, it is difficult at this stage to conclude

5 whether it is due to PCH itself or its degradation products.

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8 FemA transports ferri-DHA and ferri-AA complexes but not PCH-Fe into P. aeruginosa cells. Given that

9 the presence of AA or DHA in the environment of *P. aeruginosa* induces the expression of *femA*, we

10 sought to confirm that FemA serves as the TBDT responsible for the import of iron via AA and DHA into

11 the bacterial cells. To investigate this, we employed a growth test under iron-deficient conditions

12 (Figure 5). We utilized strains incapable of producing their own siderophores (pyoverdine and PCH) to

13 ensure that the only source of siderophores present would be those experimentally added.

14 Δ*pvdFΔpchA* and corresponding strains deleted for the *femA* and *fptA* genes were cultured in CAA

15 medium, in the absence or presence of 10 µM AA, DHA or PCH, and bacterial growth was monitored

16 at OD<sub>600 nm</sub>. Under these culture conditions, all iron present in the growth medium is chelated by the

17 tested chelator, and for the bacteria, the only means of acquiring iron is by importing these formed

18 complexes. As growth of Δ*pvdFΔpchA* was observed in the presence of 10 µM DHA or AA in CAA

19 medium (Figure 5), it can be inferred that these two molecules can be utilized as siderophores by P.

20 aeruginosa to access iron.

21 For DHA (Figure 5B), deletion of the *fptA* gene had no effect on growth. However, complete growth

22 inhibition was observed in the mutant not expressing *femA*, indicating that DHA imports iron into P.

23 aeruginosa cells exclusively through FemA. For AA (Figure 5A), a 46% growth inhibition was observed

24 in the Δ*pvdFΔpchAΔfemA* mutant compared to the Δ*pvdFΔpchA* strain, indicating that FemA also

25 transports iron via AA. Deletion of *fptA* in the Δ*pvdFΔpchAΔfemA* strain had no additional inhibitory

26 effect on growth, suggesting that FptA is not involved in iron import by AA. These data suggest that AA
1 imports iron into *P. aeruginosa* cells via FemA and possibly through another unidentified transporter.

2 Alternatively, since AA is a siderophore of small molecular weight, ferri-AA may also diffuse through

3 the bacterial outer membrane via porins, in addition to FemA-mediated uptake.

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<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 5.** Growth of Δ*pvdFΔpchA* in the presence of AA, DHA or PCH. Δ*pvdFΔpchA* and its corresponding Δ*fptA* and Δ*femA* deletion mutants were grown in CAA medium in the absence (growth kinetics in blue) or presence (kinetics in orange) of 10 µM DHA, AA or PCH. Growth was followed by monitoring the optical density at 600 nm. Error bars were calculated from three independent biological replicates.</figcaption></figure>

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1 For PCH (Figure 5C), no growth inhibition was observed for the single TBDT mutant Δ*pvdFΔpchAΔfemA*.

2 However, in the case of Δ*pvdFΔpchAΔfptA*, total inhibition occurred during the first 16 hours of culture,

3 after which growth resumed. The lack of growth in Δ*pvdFΔpchAΔfptA* cells during this initial period,

4 combined with the absence of any effect from the *femA* mutation, strongly suggests that *FptA* is the

5 sole TBDT responsible for ferri-PCH uptake. The subsequent growth, commencing at 16 hours, may

6 result from iron uptake by DHA and AA, which form after 16 hours as PCH degrades, or from the

7 activation of an alternative iron acquisition pathway. To verify the latter hypothesis, we performed a

8 differential proteomics analysis comparing the Δ*pvdFΔpchA* strain cultured in the absence and

9 presence of 10 µM PCH for 24 hours (Figures S17). The data showed an induction of *femA* expression

10 similar to that observed at 8 hours of culture in the experiment presented in **Figure 4A**, and no

11 induction of any other TBDT involved in iron import in *P. aeruginosa*. Moreover, as total growth

12 inhibition was observed with the double TBDT mutant Δ*pvdFΔpchAΔfemAΔfptA* in the presence of PCH

13 (Figure 5C), we conclude that the TBDT implicated in the resumed growth of the Δ*pvdFΔpchA* strain at

14 16 hours in the presence of PCH is FemA. These findings substantiate the role of *FptA* as the TBDT for

15 PCH-Fe complexes, whereas FemA facilitates the import of ferric complexes of PCH degradation

16 products, namely DHA and AA.

17 This conclusion concerning PCH is further supported by ⁵⁵Fe uptake assays conducted on Δ*pvdFΔpchA*

18 and Δ*pvdFΔpchAΔfemA*, Δ*pvdFΔpchAΔfptA*, Δ*pvdFΔpchAΔfemAΔfptA* strains (Figure 6). The strains

19 were cultured in the presence of 10 µM PCH to ensure *femA* expression. PCH was loaded with ⁵⁵Fe and

20 immediately utilized in the ⁵⁵Fe uptake assay. Consequently, we presume that there is minimal

21 degradation of PCH during the 3-hour duration of the experiment, especially since the ferric form of

22 PCH is more stable than the apo form.⁴⁹,⁵⁰ A significant inhibition of ⁵⁵Fe import was observed only with

23 the two *fptA* mutant strains, Δ*pvdFΔpchAΔfptA* and Δ*pvdFΔpchAΔfemAΔfptA*, compared to the

24 reference Δ*pvdFΔpchA*. The *femA* mutation (assay with Δ*pvdFΔpchAΔfemA*) showed no detectable

25 effect on ⁵⁵Fe import, indicating that FemA does not participate in iron import via PCH. If FemA was

26 involved in iron import through PCH, residual ⁵⁵Fe import would be expected in the Δ*pvdFΔpchAΔfptA*
1 mutant cells. This would indicate FemA taking over the PCH-⁵⁵Fe uptake in the absence of FptA.

2 Therefore, the growth observed after 16 hours in the Δ*pvdFΔpchAΔfptA* strain in the presence of PCH

3 is attributable to the presence of DHA and/or AA resulting from PCH degradation (Figure 5C).

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7 grown in iron-restricted CAA medium in the presence of 10 µM PCH, to induce expression of *fptA* and *femA*. Afterwards, bacteria were incubated with 500 nM PCH-⁵⁵Fe. The amount of ⁵⁵Fe taken up by the

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8 bacteria was measured as a function of time. As a control, the experiment was repeated in the

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9 presence of the protonophore CCCP (200 µM). The experiment was also repeated with the

10 bacteria was measured as a function of time. As a control, the experiment was repeated in the

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11 biological replicates. The same experiment was also conducted with cells grown in iron-restricted CAA

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12 corresponding Δ*fptA* and Δ*femA* deletion mutants of Δ*pvdFΔpchA* (Δ*pvdFΔpchAΔfptA*, Δ*pvdFΔpchAΔfemA* and Δ*pvdFΔpchAΔfemAΔfptA*). Error bars were calculated from three independent

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15 yielding identical results (Figure S18).

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18 Under what circumstances is P. aeruginosa able to access iron via AA and DHA? To better understand

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19 the conditions under which *P. aeruginosa* can use AA and DHA to access iron, we identified the optimal

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20 conditions for *femA* expression relative to AA, DHA, and PCH concentrations in *P. aeruginosa* strains,

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21 capable or not of producing pyoverdine and PCH. This experiment employed a fluorescent reporter

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<figure><img src="image_10.png" /><figcaption>PCH - ⁵⁵Fe</figcaption></figure>

**Figure 6.** ⁵⁵Fe uptake by *P. aeruginosa* strains mediated by PCH. *P. aeruginosa* Δ*pvdFΔpchA* cells were grown in iron-restricted CAA medium in the presence of 10 µM PCH, to induce expression of *fptA* and *femA*. Afterwards, bacteria were incubated with 500 nM PCH-⁵⁵Fe. The amount of ⁵⁵Fe taken up by the bacteria was measured as a function of time. As a control, the experiment was repeated in the presence of the protonophore CCCP (200 µM). The experiment was also repeated with the corresponding Δ*fptA* and Δ*femA* deletion mutants of Δ*pvdFΔpchA* (Δ*pvdFΔpchAΔfptA*, Δ*pvdFΔpchAΔfemA* and Δ*pvdFΔpchAΔfemAΔfptA*). Error bars were calculated from three independent biological replicates. The same experiment was also conducted with cells grown in iron-restricted CAA medium supplemented with 100 µM PCH to more strongly induce the expression of *fptA* and *femA*, yielding identical results (Figure S18).
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1 system and a data analysis method similar to our previous work.⁴² This approach enables the testing

2 of various siderophore concentrations and allows monitoring of *femA* expression throughout the

3 entire culture period. In this system, the promoter sequence of the *femA* gene is fused to the coding

4 sequence of the fluorescent protein YPet. Consequently, the transcription of *ypet* is controlled by the

5 promoter sequences of *femA*. The promoter region of *femA* contains a Fur box, which is involved in

6 transcriptional regulation via Fur (a transcriptional regulator sensitive to intracellular iron

7 concentration).⁶³⁻⁶⁶ It also includes a sequence that interacts with the sigma factor FemR. Indeed, *femA*

8 transcription is positively regulated by the anti-sigma and sigma factors FemI and FemR.³⁵ This

9 promoter region and the DNA sequence of YPet were inserted into the genomes of *P. aeruginosa* PAO1

10 and the Δ*pvdF* and Δ*pvdFΔpchA* mutants between the *glmS* and PA5548 genes as previously.⁴² The

11 generated strains are referred to as PAO1-*P<sub>femA</sub>-ypet*, Δ*pvdF*-*P<sub>femA</sub>-ypet* and Δ*pvdFΔpchA*-*P<sub>femA</sub>-ypet*,

12 respectively (Table S1). We have previously constructed similar fluorescent reporters for the promoter

13 regions of the TBDT-encoding genes *pfeA* and *foxA* in *P. aeruginosa* and demonstrated via qRT-PCR

14 that (i) inserting sequences into the *P. aeruginosa* genome between the *glmS* and PA5548 genes does

15 not significantly affect the transcription of these two genes, (ii) adding a second promoter to the

16 genome does not interfere with the transcription of *pfeA* or *foxA*, and (iii) the fluorescence of these

17 reporters is proportional to the expression of the respective TBDT of interest.⁶⁷ Here also we checked

18 using qRT-PCR that the fluorescence of YPet is proportional to *femA* transcription (Figure S19).

19 We monitored *femA* expression by tracking YPet fluorescence during the growth of PAO1-*P<sub>femA</sub>-ypet*,

20 Δ*pvdF*-*P<sub>femA</sub>-ypet* and Δ*pvdFΔpchA*-*P<sub>femA</sub>-ypet* in CAA medium with increasing concentrations of AA,

21 DHA, or PCH. Bacterial growth was measured by optical density at 600 nm (OD<sub>600 nm</sub>), while *femA*

22 expression was tracked by measuring YPet fluorescence at 540 nm (excitation wavelength: 500 nm)

23 over time (Figures S20 to S22). For each condition, the fluorescence data were processed as follows:

24 first, the fluorescence of the control (without siderophores) was subtracted from the fluorescence of

25 the test condition to account for the baseline. Then, the resulting fluorescence for each condition was

26 normalized by dividing it by the corresponding OD<sub>600 nm</sub> value (Figure 7A), providing a measure of
1 fluorescence per bacterium. Under the assumption that the fluorescence signal is directly proportional

2 to the concentration of YPet ($F = k \cdot P$, where $F$ is the fluorescence signal and $P$ the concentration of

3 YPet) the transient evolution of the fluorescence signal per bacteria can be modeled as a first-order

4 system (1) involving the gene expression $\beta$ (in $\mu M/h$) and the degradation rate of YPet $d$ (in $h^{-1}$).

$$ \frac{dP}{dt} = \beta - d \cdot P \qquad (1) $$

5 The assessment of $\beta$, which is the quantity of interest when speaking about gene expression, from the

6 data is then a two-step process. First, for each AA, DHA or PCH concentration, the temporal evolution

7 of the fluorescence per bacteria is fitted by equation (1) in order to obtain the value of $\beta$ (Figure 7A).

8 Then, the curve giving $\beta$ as a function of AA, DHA or PCH concentration (Figure 7B) are fitted by a Hill

9 equation (2) in order to obtain the quantitative features of the regulation process: basal and maximal

10 expression ($y_0$ and $y_{max}$), effective affinity or the concentration at which the transition occurs ($K$),

11 steepness of the transition ($n$).

$$ \beta(X) = y_0 + (y_{max} - y_0) \cdot \frac{X^n}{X^n + K^n} \qquad (2) $$

12 An increase in fluorescence, corresponding to femA expression, was observed in the $\Delta pvdF \Delta pchA-$ strain (the fluorescent reporter producing no pyoverdine and no PCH) in response to

13 $P_{femA-ypet}$ strain (the fluorescent reporter producing no pyoverdine and no PCH) in response to

14 AA, DHA, and PCH (Figure 7A). For AA, fluorescence began to rise at 4 $\mu M$ and peaked at 80 $\mu M$. For

15 DHA, the increase started at 10 $\mu M$, also reaching a maximum at 80 $\mu M$. For PCH, fluorescence

16 increased from 30 $\mu M$ and peaked at 200 $\mu M$. The steepness of the response varied among the

17 siderophores, as reflected in the Hill coefficients ($n$ values) of 2.0, 2.2, and 3.4 for AA, DHA, and PCH,

18 respectively. Notably, a significant difference was observed in the maximum fluorescence ($ymax$):

19 $ymax$ was twice as high for AA compared to DHA and 3.5 times higher than for PCH (Figure 7B). These

20 findings indicate that in the $\Delta pvdF \Delta pchA$ strain, which lacks endogenous pyoverdine and PCH

21 production, AA is more effective at inducing femA expression than DHA or PCH. FemA expression is
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1 initiated at lower concentrations of AA compared to DHA or PCH, the K value for AA is also lower than

2 for the other siderophores, and more FemA is expressed in the presence of AA compared to DHA and

3 PCH. The regulatory mechanisms underlying *femA* expression in response to AA, DHA, and PCH appear

4 to be very similar, as suggested by the comparable *n* values and overlapping concentration ranges at

5 which the siderophores induce expression.

6 These results seem inconsistent with those obtained from proteomic analyses and qRT-PCR (Figure 4).

7 However, the latter experiments used a single siderophore concentration (10 µM), while the

8 fluorescent reporter assay evaluated a full range of concentrations. At 10 µM, *femA* expression is

9 relatively similar for AA, DHA, and PCH, but differences become more pronounced at higher

10 concentrations (Figure 7A). Moreover, fluorescent reporters offer greater sensitivity than proteomic

11 approaches for tracking gene expression, providing real-time data and detecting low expression levels.

12 In contrast, proteomic methods require higher protein abundance for detection and generally exhibit

13 lower sensitivity.

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15 For PAO1-*P*<sub>femA-ypet</sub> (the fluorescent reporter producing both pyoverdine and PCH) (Figure 7A), an

16 increase in fluorescence was observed even in the absence of AA, DHA, or PCH, indicating that *femA* is

17 already slightly expressed under iron restriction, certainly due to the PCH which is produced by the

18 strain. However, this basal expression is much lower than the expression observed when AA or DHA is

19 added to the fluorescent reporter Δ*pvdFΔpchA-P*<sub>femA-ypet</sub>. An additional increase in fluorescence

20 occurred for PAO1-*P*<sub>femA-ypet</sub> when AA or PCH was added, but not with DHA. This increase began at

21 200 µM for AA and 30 µM for PCH. The rate of YPet synthesis, β, followed a sigmoidal curve for PCH,

22 with a *n* value of 2.5 and an *y*<sub>max</sub> of 926, which is 0.6 lower than in the same experiment with the

23 Δ*pvdFΔpchA-P*<sub>femA-ypet</sub> reporter. This indicates that *femA* expression reaches a lower maximum level

24 in PAO1 in the presence of increasing PCH concentrations, likely due to competition for iron with

25 pyoverdine. Transcription of genes encoding TBDTs, mediated by anti-sigma and sigma factors, begins

26 when the siderophore-iron complex binds to the corresponding TBDT, triggering a cascade that
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1 releases the anti-sigma factor, allowing the sigma factor to engage RNA polymerase and initiate transcription. Since pyoverdine has a higher affinity for iron than PCH (K_a of 10^32 M⁻¹, vs 10^18 M⁻² for PCH),^68,69 fewer PCH-Fe complexes form in PAO1 than in Δ*pvdFΔpchA* cultures. For AA, β values remain low, indicating a very low level of *femA* expression, likely because AA is less competitive than pyoverdine and PCH produced by PAO1-P_femA-ypet for iron chelation. No increase in YPet fluorescence was detected with DHA in PAO1-P_femA-ypet cultures, likely because DHA does not compete effectively with pyoverdine for iron. The facts that (i) a greater increase in YPet fluorescence was observed with increasing concentrations of PCH compared to AA, and (ii) no variation in fluorescence was seen with DHA, confirm that PCH can induce *femA* expression.

2 transcription. Since pyoverdine has a higher affinity for iron than PCH (K_a of 10^32 M⁻¹, vs 10^18 M⁻² for PCH),^68,69 fewer PCH-Fe complexes form in PAO1 than in Δ*pvdFΔpchA* cultures. For AA, β values remain low, indicating a very low level of *femA* expression, likely because AA is less competitive than pyoverdine and PCH produced by PAO1-P_femA-ypet for iron chelation. No increase in YPet fluorescence was detected with DHA in PAO1-P_femA-ypet cultures, likely because DHA does not compete effectively with pyoverdine for iron. The facts that (i) a greater increase in YPet fluorescence was observed with increasing concentrations of PCH compared to AA, and (ii) no variation in fluorescence was seen with DHA, confirm that PCH can induce *femA* expression.

3 PCH),^68,69 fewer PCH-Fe complexes form in PAO1 than in Δ*pvdFΔpchA* cultures. For AA, β values remain low, indicating a very low level of *femA* expression, likely because AA is less competitive than pyoverdine and PCH produced by PAO1-P_femA-ypet for iron chelation. No increase in YPet fluorescence was detected with DHA in PAO1-P_femA-ypet cultures, likely because DHA does not compete effectively with pyoverdine for iron. The facts that (i) a greater increase in YPet fluorescence was observed with increasing concentrations of PCH compared to AA, and (ii) no variation in fluorescence was seen with DHA, confirm that PCH can induce *femA* expression.

4 low, indicating a very low level of *femA* expression, likely because AA is less competitive than pyoverdine and PCH produced by PAO1-P_femA-ypet for iron chelation. No increase in YPet fluorescence was detected with DHA in PAO1-P_femA-ypet cultures, likely because DHA does not compete effectively with pyoverdine for iron. The facts that (i) a greater increase in YPet fluorescence was observed with increasing concentrations of PCH compared to AA, and (ii) no variation in fluorescence was seen with DHA, confirm that PCH can induce *femA* expression.

5 pyoverdine and PCH produced by PAO1-P_femA-ypet for iron chelation. No increase in YPet fluorescence was detected with DHA in PAO1-P_femA-ypet cultures, likely because DHA does not compete effectively with pyoverdine for iron. The facts that (i) a greater increase in YPet fluorescence was observed with increasing concentrations of PCH compared to AA, and (ii) no variation in fluorescence was seen with DHA, confirm that PCH can induce *femA* expression.

6 was detected with DHA in PAO1-P_femA-ypet cultures, likely because DHA does not compete effectively with pyoverdine for iron. The facts that (i) a greater increase in YPet fluorescence was observed with increasing concentrations of PCH compared to AA, and (ii) no variation in fluorescence was seen with DHA, confirm that PCH can induce *femA* expression.

7 with pyoverdine for iron. The facts that (i) a greater increase in YPet fluorescence was observed with increasing concentrations of PCH compared to AA, and (ii) no variation in fluorescence was seen with DHA, confirm that PCH can induce *femA* expression.

8 increasing concentrations of PCH compared to AA, and (ii) no variation in fluorescence was seen with DHA, confirm that PCH can induce *femA* expression.

9 DHA, confirm that PCH can induce *femA* expression.

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<figure><img src="image_11.png" /><figcaption>**Figure 7.** femA expression as a function of AA, DHA and PCH concentrations in PAO1-P<sub>femaA</sub>-ypet, ΔpvdF-P<sub>femaA</sub>-ypet and ΔpvdFΔpchA-P<sub>femaA</sub>-ypet strains. A. PAO1-P<sub>femaA</sub>-ypet, ΔpvdF-P<sub>femaA</sub>-ypet and ΔpvdFΔpchA-P<sub>femaA</sub>-ypet strains were grown in CAA medium in the absence or presence of increasing concentrations of AA, DHA, or PCH. The OD<sub>600 nm</sub> and YPet fluorescence (excitation at 500 nm and emission at 540 nm) were measured (see **Figure S20 to S22**). For each concentration of siderophore, the fluorescence data of the control (without siderophore) was subtracted from the fluorescence of the condition of interest, and the resulting fluorescence was normalized by dividing by the corresponding OD<sub>600 nm</sub> value. Three independent experiments, each with three technical replicates, were performed for each concentration of AA, DHA, and PCH tested (n = 3). The mean and the standard deviation of the three biological replicates are shown. The y-axis is not the same for the 3 reporters, nor is the concentration gradient of AA, DHA, and PCH used. The arrow indicates the direction of fluorescence variation depending on the concentration of AA, DHA, or PCH tested. **B. β** values. The β values of the PAO1-P<sub>femaA</sub>-ypet, ΔpvdF-P<sub>femaA</sub>-ypet and ΔpvdFΔpchA-P<sub>femaA</sub>-ypet strains for each biological</figcaption></figure>
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1 replicate are plotted against the concentrations of AA, DHA, and PCH. The sigmoid curves were fitted

2 using the Hill equation (2), and the obtained parameters are summarized in the Table. y₀, minimum of

3 expression; yₘₐₓ, maximum of expression; n, the Hill coefficient, which describes the cooperativity in

4 the studied biological system; K, the concentration of ligand (µM) at which half of the maximum

5 expression is observed.

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7 When the Δ*pvdF*-*P*<sub>femA</sub>-ypet reporter, which lacks pyoverdine production but still produces PCH, is

8 grown in the presence of increasing concentrations of PCH (Figure 7A), the *P*<sub>femA</sub>-ypet reporter remains

9 active, yielding results closely resembling to those obtained for the Δ*pvdF*Δ*pchA*-*P*<sub>femA</sub>-ypet strain. This

10 similarity occurs because, in both cases, PCH does not compete with pyoverdine for iron, as seen in

11 the PAO1-*P*<sub>femA</sub>-ypet strain. In the experiment where Δ*pvdF*-*P*<sub>femA</sub>-ypet was cultured in the presence of

12 either AA or DHA, we observed intermediate levels of *femA* expression compared to those seen with

13 PAO1-*P*<sub>femA</sub>-ypet and Δ*pvdF*Δ*pchA*-*P*<sub>femA</sub>-ypet (Figure 7). Both AA and DHA compete for iron with

14 pyoverdine and PCH in the assay with PAO1-*P*<sub>femA</sub>-ypet, but only with PCH when Δ*pvdF*-*P*<sub>femA</sub>-ypet

15 reporter is used. In contrast, there is no siderophore competition in the experiment with the

16 Δ*pvdF*Δ*pchA*-*P*<sub>femA</sub>-ypet reporter. These findings suggest that in mutants incapable of producing

17 pyoverdine, if PCH degrades into AA and DHA, these compounds can induce *femA* expression.

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19 In conclusion, these findings demonstrate that the presence of AA and DHA in the environment of *P*.

20 *aeruginosa* induces *femA* expression only in strains that do not produce pyoverdine. This is likely

21 because AA and DHA have a lower affinity for iron, preventing them from effectively competing with

22 pyoverdine for metal chelation. The results further indicate that AA is more effective than DHA at

23 inducing *femA* expression in Δ*pvdF*Δ*pchA* or Δ*pvdF* strains. While PCH also induces *femA* expression, it

24 is less effective than both AA and DHA in these strains, where pyoverdine-mediated iron competition

25 is absent.

26 These observations suggest that *P. aeruginosa* utilizes AA and DHA to acquire iron in mutants incapable

27 of producing pyoverdine, as these degradation products facilitate *femA* expression and subsequent

28 iron uptake. It is well established that pyoverdine production enhances the virulence of *P. aeruginosa*
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1 by promoting iron uptake, given its higher affinity for iron compared to pyochelin. However,

2 pyoverdine-deficient *P. aeruginosa* mutants have been isolated from cystic fibrosis lung infections.⁷⁰⁻

3 ⁷² Our data indicate that such mutants may exploit the degradation of PCH into DHA and AA as an alternative strategy to access iron. However, a key question remains whether AA and DHA have

4 sufficient affinity for iron to effectively sequester it from the host. Similarly, in plant infection models involving wheat and *Arabidopsis*, pyoverdine-deficient *Pseudomonas fluorescens* and *Pseudomonas syringae* strains exhibit reduced fitness but can remain infectious, depending on environmental iron availability.⁷³ In such systems, if pyoverdine production is impaired by mutation, AA and DHA may

5 contribute to iron acquisition, thereby compensating for the absence of pyoverdine.

6 ⁷² Our data indicate that such mutants may exploit the degradation of PCH into DHA and AA as an alternative strategy to access iron. However, a key question remains whether AA and DHA have sufficient affinity for iron to effectively sequester it from the host. Similarly, in plant infection models involving wheat and *Arabidopsis*, pyoverdine-deficient *Pseudomonas fluorescens* and *Pseudomonas syringae* strains exhibit reduced fitness but can remain infectious, depending on environmental iron availability.⁷³ In such systems, if pyoverdine production is impaired by mutation, AA and DHA may

8 availability.⁷³ In such systems, if pyoverdine production is impaired by mutation, AA and DHA may contribute to iron acquisition, thereby compensating for the absence of pyoverdine.

9 Moreover, this mechanism of iron acquisition through DHA and AA may also operate in wild-type *PAO1* strains capable of producing both siderophores, pyoverdine and pyochelin, particularly under

10 conditions of moderate iron deficiency. Indeed, we previously demonstrated that *P. aeruginosa* *PAO1* produces significant amounts of both pyoverdine and PCH in highly iron-restricted medium, such as

11 CAA, which contains 21 nM Fe.⁵⁹ In contrast, in less iron-restricted medium, such as succinate medium containing around 320 nM Fe, *P. aeruginosa* reduces pyoverdine production while continuing to

12 produce PCH.⁵⁹ Under such less iron-restricted conditions, PCH induces *femA* expression, and its degradation into AA or DHA further enhances *femA* expression, improving iron acquisition efficiency without requiring pyoverdine production, as demonstrated using our fluorescent reporter approach.

13 The ability to utilize AA and DHA, degradation products of PCH, highlights a strategy by which *P. aeruginosa* maximizes the efficiency of PCH synthesis and utilization. When PCH itself is depleted, its breakdown products can still function as iron chelators, maintaining iron acquisition. A similar

14 phenomenon is observed in *E. coli*, where the siderophore enterobactin and its degradation product, 2,3-dihydroxybenzoylserine (DHBS), serve as iron-chelating molecules.²⁴ Notably, *P. aeruginosa* can exploit enterobactin as a xenosiderophore using PfeA as the TBDT.⁷⁴ Similarly, DHBS chelates iron and is used by *P. aeruginosa*, though the TBDT responsible for its uptake remains unidentified.

15 degradation into AA or DHA further enhances *femA* expression, improving iron acquisition efficiency without requiring pyoverdine production, as demonstrated using our fluorescent reporter approach. The ability to utilize AA and DHA, degradation products of PCH, highlights a strategy by which *P. aeruginosa* maximizes the efficiency of PCH synthesis and utilization. When PCH itself is depleted, its breakdown products can still function as iron chelators, maintaining iron acquisition. A similar

16 ²,3-dihydroxybenzoylserine (DHBS), serve as iron-chelating molecules.²⁴ Notably, *P. aeruginosa* can exploit enterobactin as a xenosiderophore using PfeA as the TBDT.⁷⁴ Similarly, DHBS chelates iron and is used by *P. aeruginosa*, though the TBDT responsible for its uptake remains unidentified.

17 degradation into AA or DHA further enhances *femA* expression, improving iron acquisition efficiency without requiring pyoverdine production, as demonstrated using our fluorescent reporter approach. The ability to utilize AA and DHA, degradation products of PCH, highlights a strategy by which *P. aeruginosa* maximizes the efficiency of PCH synthesis and utilization. When PCH itself is depleted, its breakdown products can still function as iron chelators, maintaining iron acquisition. A similar

18 without requiring pyoverdine production, as demonstrated using our fluorescent reporter approach. The ability to utilize AA and DHA, degradation products of PCH, highlights a strategy by which *P. aeruginosa* maximizes the efficiency of PCH synthesis and utilization. When PCH itself is depleted, its breakdown products can still function as iron chelators, maintaining iron acquisition. A similar

19 phenomenon is observed in *E. coli*, where the siderophore enterobactin and its degradation product, 2,3-dihydroxybenzoylserine (DHBS), serve as iron-chelating molecules.²⁴ Notably, *P. aeruginosa* can exploit enterobactin as a xenosiderophore using PfeA as the TBDT.⁷⁴ Similarly, DHBS chelates iron and is used by *P. aeruginosa*, though the TBDT responsible for its uptake remains unidentified.

20 ²,3-dihydroxybenzoylserine (DHBS), serve as iron-chelating molecules.²⁴ Notably, *P. aeruginosa* can exploit enterobactin as a xenosiderophore using PfeA as the TBDT.⁷⁴ Similarly, DHBS chelates iron and is used by *P. aeruginosa*, though the TBDT responsible for its uptake remains unidentified.

21 ²,3-dihydroxybenzoylserine (DHBS), serve as iron-chelating molecules.²⁴ Notably, *P. aeruginosa* can exploit enterobactin as a xenosiderophore using PfeA as the TBDT.⁷⁴ Similarly, DHBS chelates iron and is used by *P. aeruginosa*, though the TBDT responsible for its uptake remains unidentified.

22 ²,3-dihydroxybenzoylserine (DHBS), serve as iron-chelating molecules.²⁴ Notably, *P. aeruginosa* can exploit enterobactin as a xenosiderophore using PfeA as the TBDT.⁷⁴ Similarly, DHBS chelates iron and is used by *P. aeruginosa*, though the TBDT responsible for its uptake remains unidentified.
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2 **X-ray structures of FemA loaded with AA-Fe, DHA-Fe or PCH-Fe.** To better understand the binding interaction of Ferri-AA, Ferri-DHA and Ferri-PCH with FemA, the structure of FemA in the absence of siderophores was solved to a resolution of 1.9 Å by molecular replacement using the structure of Acinetobacter baumanii ferri-preacinetobactin TBDT BauA (pdb code 6H7V) which has the closest sequence similarity⁷⁵ (Table S4). The asymmetric unit contains two highly similar monomers (Root-Mean-Square Deviation of 0.236 Å for 670 amino acid). FemA arbores the typical outer membrane TBDT fold with a 22 antiparallel strands β-barrel and a plug domain folded inside of the barrel (Figure 8A). The first 107 residues, including the TonB box, are flexible. Similar to BauA, the folding of the extracellular loops toward the outer edge of the β-barrel leaves the NL1-NL3 loops of the plug domain accessible, which are typically involved in siderophore binding. One molecule of Polyethylene glycol or of detergent (C8E4) is bound near these loops.

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1 (Pro335, Leu337, Phe460, Tyr 760) (Figure 9Aa). Interactions between the hydroxyl group of the

2 phenol of one molecule with the carboxylate of the other molecule may play a role in the stabilization

3 of the complex. In the FemA-DHA₂-Fe complex structure, the second molecule is flipped by 180° with

4 carboxylates occupying the hydroxyl position and vice versa, as the result this molecule makes fewer

5 interactions (one hydrogen bond with Gln417 and two hydrophobic interactions with Asn400 and

6 Phe460) (Figure 9Ab). Residual densities at the end of the refinement suggest the second molecule

7 might adopt partially an alternative conformation like AA in chain B confirming recognition by FemA is

8 mainly driven by the first siderophore molecule of the complex (Figure S26A). There are no significant

9 changes in the β-barrel or plug domain regions as a result of the ligand binding but in the FemA-AA₂-

10 Fe structure, the N-terminal residues 104-107 are well defined in the electron density map compared

11 to the apo and FemA-DHA₂-Fe structures which could be linked to a beginning of rearrangement in this

12 region involved in the TonB recognition upon binding.

13 PCH-Fe binds to the same site on FemA as AA-Fe and DHA-Fe. As previously observed with the structure

14 of the PCH outer membrane receptor FptA⁷⁶, PCH chelates iron with a 1:1 stoichiometry, despite the

15 complex being prepared with a twofold excess of PCH (Figures 9Ac, Figure S24C). The nitrogen atoms

16 of the thiazoline and thiazolidine rings, along with the oxygen atoms of the phenolate and carboxylate

17 groups, are involved in chelation. One acetylacetonate molecule, originating from the buffer,

18 completes the coordination sphere. The binding mode of PCH-Fe in FemA differs from its binding to

19 FptA in *P. aeruginosa*.⁷⁶ Unlike FptA, interactions with FemA are primarily driven by the recognition of

20 the phenol and thiazoline groups, which occupy the same position as the first molecule of AA and DHA

21 (Figure 10, 9Ac). The thiazolidine and carboxylate groups, more exposed to the solvent, are stabilized

22 by a hydrogen bond with Gln417 and a hydrophobic interaction with Phe460 (Figure 9Ac, 9Bc). On the

23 contrary, binding of PCH in FptA binding site is primarily driven by polar interactions and the

24 carboxylate is stabilized by three hydrogen bonds with the loops of the plug domain (Figure S27).

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<figure><img src="image_12.png" /><figcaption>**Figure 8.** A. Overall structure of FemA. The N-ter plug domain is colored in dark blue and the β-barrel in light blue. NL1-NL3 loops of the plug domain that are typically involved in siderophore recognition have been labelled. B. View from the extracellular side of FemA in complex with AA-Fe represented as a surface coloured by electrostatic charges calculated in CCP4MG. To localize the biding site, AA is shown as sticks with carbon atoms colored cyan, sulphur yellow, nitrogen blue and oxygen red. The Fe³⁺ is represented an orange sphere. C. FemA siderophore-Fe binding site. Secondary structure elements involved in the binding site have been labelled.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Figure 9.** Comparison of FemA structures in complex with AA₂-Fe, DHA₂ and PCH. **A.** Comparison of the binding interaction between FemA and AA₂-Fe, DHA₂-Fe and PCH/neoPCH. Residues within 4.0 Å of the siderophores are displayed in light blue for residues of the β-barrel and dark blue for the residues of the plug domain. Carbon atoms of AA, DHA, PCH and NeoPCH are colored as cyan, dark green, pink and purple, respectively. Hydrogen bonds are shown as black broken lines and Fe³⁺ is represented as an orange sphere. In AA and PCH structures, one molecule of acetylacetonate coming from the source of iron is present in the binding site (colored in sand and yellow respectively). In PCH structure, this molecule completes the coordination sphere of the Fe³⁺. **B.** View from the extracellular side of the binding pocket of FemA in complex with AA₂-Fe (a), DHA₂-Fe (b) and PCH-Fe (c) represented as a surface coloured by electrostatic charges calculated in CCP4MG. Positively charged electrostatic potential is coloured blue and negatively charged electrostatic potential is coloured red. For clarity neoPCH-Fe has been removed. Siderophore are shown as sticks with carbon atoms colored cyan for AA, in green for DHA and pink for PCH, sulphur yellow, nitrogen blue and oxygen red. The Fe³⁺ is represented an orange sphere. One molecule of acetyl acetonate (yellow) completes the coordination sphere of the Fe³⁺ in PCH structure.</figcaption></figure>

Most of the key interactions observed in AA and DHA complexes are conserved, with the phenolate and thiozoline groups buried in the binding pocket. However, the surface of interaction of the PCH
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1 molecule with the protein is smaller compared of those of the two molecules of AA or DHA (846 Å² of

2 buried surface for DHA₂, 834 Å² for AA₂, and 498 Å² for PCH) (Table 2, Figure 9Bc). Furthermore, PCH

3 is unable to interact with the key Arg334 residue, unlike the second molecule of AA and PCH, and the
4 overall charge of the ferric complex differs (1+ for Fe-PCH vs 1- for Fe-AA₂ and Fe-DHA₂). As a

5 consequence, the presence of a positively charged residue in the binding pocket of FemA could be see
6 as unfavorable for interactions with PCH and a more hydrophobic binding site, like FptA, could be more

7 optimal. We postulate that, despite being able to bind to FemA in a similar manner as AA and DHA,
8 PCH is not transported due to its reduced interaction with key residues at the binding site, which might

9 fail to initiate conformational changes needed for the transport. Additionally, the difference in the
10 overall charge of the ferric complex could also affect also transport of PCH inside de β-barrel, which

11 could be facilitated by polar residues.
12 Due to issues with the protein stability and the solubility of the siderophore molecules, we were unable

13 to determine accurate binding parameters by isothermal titration calorimetry (Figure S28). Interface
14 analysis of the complexes using QtPISA⁷⁷ (Table 2) suggests that in addition of a reduction of polar

15 interactions, the slightly more hydrophilic DHA and PCH molecules are more penalised by desolvation
16 upon binding compared to AA. This is reflected in the low enthalpy observed during the titrations.
17 During refinement, the maps revealed the presence of a PCH stereoisomer at position C4' (neoPCH)

18 (Figure S26C), which did not alter the recognition. NeoPCH is one of the minor PCH stereoisomers
19 produced during the organic synthesis of the PCH.⁴⁷,⁷⁸ NeoPCH was shown to be recognized and

20 transported by FptA,⁴⁷,⁷⁸ raising therefore the question of a potential uptake though FemA. NeoPCH
21 was tested to determine whether, like PCH, it could transport iron via FemA and induce *femA*
22 expression. The data clearly show that PCH and neoPCH behave similarly: like PCH, neoPCH was unable

23 to import ⁵⁵Fe into *P. aeruginosa* cells via FemA but did induce *femA* expression (Figure S23).
24 These data collectively demonstrate that FemA binds the ferric forms of AA, DHA, and PCH, albeit
25 through subtle different binding modes (Figure 9, 10). For PCH, ⁵⁵Fe import data indicate that although

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PCH-Fe binds to the FemA binding site, it does not proceed to transport. The unique binding mechanisms of AA-Fe and DHA-Fe with FemA enable transport, unlike PCH-Fe. However, PCH-Fe binding still activates the signaling cascade via the anti-sigma and sigma factors FemI and FemR, leading to *femA* transcription, albeit less efficiently than with AA-Fe and DHA-Fe. These findings further suggest that binding of a siderophore-Fe complex to a TBDT binding site does not necessarily result in transport. The reason why PCH-Fe is not transported through the outer membrane via FemA remains unclear, but it may be due to incomplete or partial conformational changes in the transporter required for import or to the inability of PCH to form some key polar interactions during transport.

<figure><img src="image_14.png" /><figcaption>**Figure 10.** Comparison of the binding mode of AA₂-Fe, DHA₂-Fe and PCH-Fe. Structures have been superposed using coordinates of the protein residues. Only atoms of the siderophore have been displayed for more clarity. Carbon atoms of DHA, AA, PCH are colored as green, cyan and pink. Fe³⁺ are represented as an orange sphere. Portion of the siderophores that superpose well (phenolate and thiazole or thiazoline rings) has been highlighted by a black circle. The two molecules of AA and DHA have been labelled I and II.</figcaption></figure>
<table><caption>**Table 2:** PISA analysis of interfaces.77 Value reported are an average from chain A and B. Interactions between the two molecules of DHA or AA are not detailed in the table but included in the assembly analysis.</caption><thead><tr><th rowspan="2"></th><th colspan="3">AA</th><th colspan="2">DHA</th><th colspan="2">PCH</th></tr><tr><th colspan="2">Interfaces</th><th>Assembly</th><th colspan="2">Interfaces</th><th>Interface</th><th>Assembly</th></tr><tr><th></th><th>AA (I) /FemA</th><th>AA (II) /FemA</th><th>2AA- FemA</th><th>DHA (I) /Fema</th><th>DHA (II) /Fema</th><th>2DHA- FemA</th><th>PCH/FemA</th><th>PCH-FemA</th></tr></thead><tbody><tr><td>Interface area Å<sup>2</sup></td><td>184</td><td>161</td><td>417</td><td>195</td><td>153</td><td>423</td><td>249</td><td>249</td></tr><tr><td>Ligand buried surface area Å<sup>2</sup></td><td>250 (69%)</td><td>201 (55%)</td><td>-</td><td>260 (69%)</td><td>177 (48%)</td><td>-</td><td>304 (66%)</td><td>-</td></tr><tr><td>Protein buried surface Å<sup>2</sup></td><td>119 (0.5%)</td><td>122 (0.5%)</td><td>-</td><td>131 (0.5%)</td><td>129 (0.5%)</td><td>-</td><td>194 (0.7%)</td><td>-</td></tr><tr><td>Assembly buried surface Å<sup>2</sup></td><td>-</td><td>-</td><td>834</td><td>-</td><td>-</td><td>846</td><td>-</td><td>498</td></tr><tr><td>Δ<sup>i</sup>G<sup>a</sup>(kcal/mol)</td><td>0.1</td><td>-0.7</td><td></td><td>6.1</td><td>2.2</td><td></td><td>1.0</td><td></td></tr><tr><td>P-value<sup>b</sup></td><td>-1.0</td><td>-1</td><td></td><td>0.138</td><td>0.0023</td><td></td><td>0.80595</td><td></td></tr><tr><td>Polar interactions</td><td>1</td><td>2</td><td>3</td><td>1</td><td>1</td><td>2</td><td>2</td><td>2</td></tr><tr><td>No. of residues involved</td><td>13</td><td>9</td><td>18</td><td>13</td><td>8</td><td>17</td><td>15</td><td>15</td></tr></tbody></table>

<sup>a</sup>:Δ<sup>i</sup>G, solvation energy gain upon formation of the interface.

<sup>b</sup>:P-value : probability to get a Δ<sup>i</sup>G lower.

## CONCLUSION

Our findings provide valuable new insights into the intricate iron acquisition strategies employed by *Pseudomonas aeruginosa*, particularly under conditions of moderate iron deficiency. We demonstrate that PCH degrades in solution to form AA as the final oxidation product, and that the presence of AA and DHA in bacterial growth media induces the transcription and expression of *femA*. This gene encodes the transporter FemA, previously characterized as the TBDT for ferri-MYC. While FptA
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1 selectively imports ferri-PCH, FemA is responsible for importing ferri-AA and ferri-DHA, showcasing

2 distinct siderophore-transporter pairings.

3 Structural analysis of FemA bound to ferri-AA, ferri-DHA, and ferri-PCH reveals similar binding

4 interactions, yet only ferri-AA and ferri-DHA are transported. This indicates that the binding mode

5 involves highly precise interactions to trigger the uptake process. Structural studies we reported show

6 that PCH binding is similar to AA and DHA to some extent, with the phenolate and thiazoline groups

7 forming the same interactions as the first molecule of AA and DHA. However, we postulate that slight

8 differences in PCH binding interactions (surface of interaction with the protein, lack of interaction with

9 the key residue Arg334) and the difference in the overall charge of the ferric complex (PCH-Fe vs AA₂-Fe and DHA₂-Fe) might affect the transport. This could occur either by failing to initiate the

10 conformational changes necessary for transport or by being unable to interact with polar residues

11 inside the β-barrel during the transport process.

12 Nonetheless, all three complexes induce *femA* expression. Importantly, AA and DHA can only induce

13 *femA* expression in strains lacking pyoverdine production. This is due to their lower affinity for iron,

14 which prevents them from effectively competing with pyoverdine for metal chelation. Under moderate

15 iron limitation—when PCH is produced but pyoverdine is absent⁵⁹—*femA* expression is upregulated by

16 PCH and its degradation products, AA or DHA. This enables iron uptake through these compounds,

17 enhancing overall iron acquisition efficiency.

18 This adaptive mechanism parallels strategies observed in other bacteria, such as *Escherichia coli* with

19 enterobactin, where siderophore degradation products are repurposed to optimize iron acquisition.

20 Furthermore, our results suggest that binding of a siderophore-iron complex to a TBDT does not always

21 result in transport. For instance, while FemA binds the ferri-PCH complex, transport does not occur,

22 possibly due to incomplete conformational changes in the transporter.

23 Overall, these findings deepen our understanding of siderophore transport specificity and regulation,

24 shedding light on how *P. aeruginosa* tailors its iron uptake systems to adapt to varying environmental

25 iron conditions.

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## MATERIALS AND METHODS

4 **Chemicals.** The protonophore carbonyl cyanide *m*-chlorophenylhydrazone (CCCP) was obtained from Sigma-Aldrich, the $FeCl_3$ was purchased from VWR and $^{55}$FeCl$_3$ was sourced from Perkin Elmer.

6 Synthesis of AA and DHA are described in Supplemental informations. PCH and neoPCH were synthesized using previously published protocols.⁴⁷

9 **Bacterial strains, plasmids, and growth conditions.** The *P. aeruginosa* strains utilized in this investigation are delineated in Table S1 in the Supplementary Materials. The iron-deficient CAA medium employed comprises 5g L⁻¹ low-iron CAA (Difco), 1.46 g L⁻¹ $K_2HPO_4$ 3H₂O, and 0.25 g L⁻¹ $MgSO_4$ 7H₂O.

14 **Plasmid and strain construction.** Enzymes were procured from ThermoFisher Scientific. Plasmid construction and mutagenesis in the chromosomal genome of *P. aeruginosa* were executed as previously outlined⁴¹ and the nucleotides used are described in Table S2. In essence, the blunt-end ligation strategy was employed to integrate 1400 bp of flanking sequences of the genes *femA* and *fptA* for the deletion mutants, into the suicide vector pEXG2. For the fluorescent reporters *PAO1-PfemA-ypet*, $\Delta pvdF-PfemA-ypet$ and $\Delta pvdF\Delta pchA-PfemA-ypet$, the plasmid pEXG2 $PfemA-ypet$ was constructed as previously described.⁴² The resultant plasmids, following sequencing, were utilized to introduce chromosomal mutations into the *P. aeruginosa* PAO1, $\Delta pvdF$ and $\Delta pvdF\Delta pchA$ strains. The insertion of the $PfemA-ypet$ sequence was executed in the intergenic region between *glms* and *PA5548* genes as described previously.⁴² All generated mutants were corroborated via PCR and sequencing.

24
1 **Mass spectrometry metabolic profiling.** Mass spectrometry profiling was performed on UHPLC

2 ThermoFisher Ultimate3000 with Scientific Vanquish Flex. Detector: Vanquish PDA (VF-XX).

3 ThermoFisher Orbitrap EMR series: Exactive Plus with Extend Mass Range: Source HESI II. Column: Thermo Fisher RP-MS:150x2.1 mm, 1.9 mm. Mobile phase are A(H₂O) and B(acetonitrile) both with

5 0,1% Formic acid. 2.5 µL was injected by sample followed with a control (CAA) and a blank of MeOH (same solvent used for sample preparation) with the following chromatographic conditions: 1) 0 to 0.5

7 min starting conditions: 2% B. 2) 0.5 to 9 min: 2% to 100%. 3) 9 to 11 min: 100% B. 4) 11-11.5 min back to starting conditions. Processing: specific targeted compound was followed by extracted-ion

9 chromatogram using Xcalibure Qual browser software (ThermoFisher Scientific).

10 P. aeruginosa Δ*pvdFΔpchA* cells were initially grown in 5 mL LB followed by an overnight culture in CAA medium at 30°C. Bacteria were pelleted, diluted in CAA medium to an OD₆₀₀ nm of 0.1, and grown for 15 hours in the absence or presence of 10 µM PCH at 30°C. Subsequently, bacteria were pelleted to collect the supernatant. The supernatant was filtered using 0.22 µm membrane, acidified and extracted using dichloromethane. The organic layer was dried over Na₂SO₄, filtered and evaporated under vacuum. The resulting samples were dissolved with 1mL MeOH LC/MS grade, and a volume of 2.5mL was injected for metabolic profiling. The qualitative analysis of a group of related compounds was performed by monitoring positive and negative extracted-ion chromatogram, see **Figure S7 to S11**. Each targeted ion was extracted with a maximum variation of 0.04 atomic mass unit (amu). Extracted-ion chromatogram was therefore enough to determine the presence or absence of a specific compound, specifically for PCH, DHA, AA 5, AAld and DHAld, see **Table 1**. To assume the presence or absence of targeted ion a theoretical fitting was achieved with the experimental mono-isotopic pattern at the resolution given by the mass spectrometer.

23 **Proteomic analysis.** P. aeruginosa Δ*pvdFΔpchA* cells were initially cultured overnight in LB followed by a culture in CAA medium. Subsequently, bacteria were pelleted, diluted in CAA medium to an OD₆₀₀ nm of 0.1, and grown for 8 hours with or without 10 µM PCH, DHA or AA at 30°C. Cell harvest involved 5 x
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1 10⁸ cells, and a biological triplicate was prepared for each sample under each cell-culture condition.

2 Cell pellets were resuspended in 200 µL lysis buffer (UTCT buffer containing 7 M urea, 2 M thiourea,

3 4% CHAPS, and 20 mM Tris-HCl pH 7.6) supplemented with nuclease and DNase. Protein

4 concentrations were determined using the Bradford assay with bovine serum albumin as the standard.

5 Proteins were subsequently precipitated overnight with glacial 0.1 M ammonium acetate in 100%

6 methanol (5 volumes, -20°C). After centrifugation at 12,000 x g and 4°C for 15 minutes, resulting pellets

7 were washed twice with 0.1 M ammonium acetate in 80% methanol and further dried under vacuum

8 (Speed-Vac concentrator). Pellets were resuspended in 100 µL 50 mM ammonium bicarbonate and

9 subjected to reduction (5 mM dithiothreitol, 95°C, 10 minutes) and alkylation (10 mM iodoacetamide,

10 room temperature, 20 minutes). Proteins were finally digested overnight with 150 ng sequencing-

11 grade trypsin (Promega).

12 Proteomic data sets were obtained by injecting 1000 ng of each peptide mixture into a TripleTOF 5600

13 mass spectrometer (Sciex) coupled to an U3000-RSLC liquid chromatography (Thermo-Fisher

14 Scientific), as described previously. For each differential proteomic analyses, data were searched

15 against the *P. aeruginosa* UniprotKB database (UniprotKB release 2020_05, taxon 208964, *P. aeruginosa* strain PAO1, 5564 forward protein sequences). Peptides and proteins were identified using

16 the Mascot algorithm (version 2.8, Matrix Science, London, UK). The following parameters were used:

17 (i) Trypsin/P was selected as the enzyme, (ii) two missed cleavages were allowed, (iii) methionine

18 oxidation and acetylation of the protein N-terminus were set as variable modifications and

19 carbamidomethylation of cysteine as a fixed modification, (iv) mass tolerance for precursor ions was

20 set to 20 ppm and 0.5 Da for fragment ions. Mascot data were then imported into Proline software

21 v2.0.⁷⁹ Proteins were validated against a Mascot pretty rank equal to 1 and a 1% FDR for both peptide

22 spectrum matches (PSM score) and protein sets (Protein Set score).

23

24 All MS/MS fragmentation spectra were used to quantify each protein from at least three independent

25 biological replicates. After a total number of spectra normalization of the data matrix, the "BasicSC"

26 spectral counts were subjected to a negative-binomial test using edgeR GLM regression of R (R v3.2.5)
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1 through the IPinquiry package.⁸⁰ The statistical test was based on the published msmsTests R package

2 available in Bioconductor to process label-free LC-MS/MS data by spectral counts.⁸¹ For each identified

3 protein, an adjusted P-value (adjp), corrected by the Benjamini–Hochberg procedure, was calculated,

4 as well as the protein fold-change (FC).

5 The MS data were deposited in the ProteomeXchange Consortium database via the PRIDE⁸² partner

6 repository with the dataset identifier PXD058246 (Reviewer account details: Username:

7 reviewer_pxd058246@ebi.ac.uk, Password: j58fqZfdC273).

8

9 **Quantitative real-time PCR.** The transcription of specific genes of interest was assessed using

10 quantitative reverse transcription PCR (qRT-PCR), following previously described methods.⁴¹,⁵⁷ Cultures

11 were conducted identically to those used for the proteomic analyses. After 8 hours of culture, 2.5 X

12 10⁸ cells were harvested, and two volumes of RNAprotect Bacteria Reagent (Qiagen) were added. Total

13 RNA was isolated from the pellet using the RNeasy Plus Mini Kit (Qiagen) according to the

14 manufacturer's instructions. Subsequently, 1 µg of total RNA was reverse transcribed using the RNA-

15 to-cDNA Kit (Applied Biosystems). Specific cDNA in our samples was quantified using a StrepOne Plus

16 instrument (Applied Biosystems), along with the appropriate primers (Table S3), and the Power Sybr

17 Green PCR Master Mix (Applied Biosystems). The transcript level for each gene was normalized against

18 those of *uvrD* and expressed as a log2 ratio (fold-change) relative to the reference condition.

19

20 **Growth assays under iron-restricted conditions.** For *P. aeruginosa* growth assays in microplates under

21 iron-restricted conditions, bacteria (*ΔpvdFΔpchA* and it corresponding *fptA* and *femA* mutants as well

22 as the fluorescent reporters *PAO1-P<sub>femA</sub>-ypet*, *ΔpvdF-P<sub>femA</sub>-ypet* and *ΔpvdFΔpchA-P<sub>femA</sub>-ypet*) were

23 cultured an initial overnight at 30°C in 5 mL LB broth, followed by bacterial washing and a second

24 overnight culture in 10 mL CAA medium at 30°C. Subsequently, bacteria were washed, resuspended in

25 CAA medium to an optical density of 0.01 at 600 nm, and dispensed into a 96-well plate (Greiner, U-
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1 bottom microplate) in the presence or absence of 10 µM PCH, DHA, AA, or of increasing concentrations

2 of PCH (0-800 µM), DHA (0-1000 µM), AA (0-2000 µM) or neoPCH (0-800 µM). Plates were then

3 incubated at 30°C with shaking in a TECAN microplate reader (Infinite M200, Tecan) and the bacterial

4 growth (OD at 600 nm) were monitored every 30 minutes, for 24h. For the experiments with the

5 fluorescent reporters, YPet fluorescence (excitation at 500 nm and emission at 540 nm) and the

6 bacterial growth (OD at 600 nm) were monitored every 30 minutes, for 24h.

7

8 Iron uptake. The PCH<sup>-55</sup>Fe and Neo-PCH<sup>-55</sup>Fe complexes were prepared at <sup>55</sup>Fe concentrations of 50

9 µM, with a chelator:iron (mol:mol) ratio of 20:1, following established protocols.<sup>83,84</sup> *P. aeruginosa*

10 strains were sequentially cultured overnight in LB broth, followed by overnight growth in CAA medium,

11 and finally, overnight growth in CAA medium supplemented with either 10 µM PCH or 100 µM neoPCH

12 at 30°C. The bacteria were subsequently utilized to investigate <sup>55</sup>Fe uptake kinetics, as previously

13 described, in the presence or absence of 200 µM CCCP (a proton motive force inhibitor) at a

14 concentration of chelator<sup>-55</sup>Fe complexes of 500 nM.<sup>83,84</sup>

15

**Cloning, and over-expression of femA in E. coli and purification for the structural biology.** The coding

17 sequence of the mature protein FemA (uniprot Q9I2J4) was amplified using the primers 5'-

18 GTCATGACCCCCGCCCTGCTTGCC and 5'- GGATCCTCAGAAGTCCGCCGTGAGGGAAA and Q5 DNA

19 polymerase. PCR product has been digested by BspHI and BamHI restriction enzymes and inserted in

20 pTAMAHISTEV vector using Ncol and BamHI restriction sites generating a construct with an N-terminal

21 tobacco etch virus (TEV) protease cleavable His7-tag.

22 The protein was over-expressed in *E. coli* C43 (DE3) cells. Cells were grown at 37 °C in LB medium

23 containing 100 µg.mL<sup>-1</sup> ampicillin until an OD<sub>600 nm</sub> of 0.7 and then induced with 0.4 mM IPTG at 25°C

24 overnight. The proteins were purified following the protocol used for PfeA described in Moynié *et al.*

25 <sup>24</sup>. Isolated outer-membrane pellets were solubilized with 7% octylpolyoxyethylene (octylPOE) and the
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1 proteins were purified with a Ni²⁺-NTA affinity chromatography followed by the His-tag cleavage by
2 TEV protease and a second IMAC column. Finally, the protein was loaded on a Superdex S200 gel
3 filtration chromatography (10 mM Tris pH 7.5, 150 mM NaCl, 0.45% C8E4) and concentrated to 16
4 mg.mL⁻¹.

5

6 **Crystallization and structure determination.** Apo crystals of FemA appeared at 20°C after a few days by mixing 200 nL of protein solution (16 mg.mL⁻¹) with 100 nL of reservoir solution containing 29% PEG 400, 0.05 M calcium acetate pH 5, 0.1M sodium Chloride. FemA-AA-Fe and FemA-DHA-Fe complex structures were obtained by adding 1 mM siderophore-Fe with a stoichiometry of 1:2 (siderophore:Fe³⁺) to the protein solution (16 mg.mL⁻¹) prior setting up the plates. Crystals appeared with a reservoir solution containing 33% PEG 400, 0.1 M Hepes pH 7.5 and 22% PEG 4000, 0.1 MES pH 6, 0.1 M magnesium acetate respectively. Complex structure with PCH has been obtained by soaking crystals with a solution freshly prepared of 2 mM Fe³⁺-acetylacetonate PCH in a 1:2 ratio (Fe³⁺: siderophore) for few minutes with the mother liquor containing 26% PEG 400 before freezing. Data were collected at the beamline I24, I04 at Diamond Light Source. Data were processed with xia2.<sup>85-87</sup> The apo structure have been solved by molecular replacement using coordinate of the *Acinetobacter baumanii* preacinetobactin TBDT (6H7V). Structures of the complexes have been solved using the apo structure, models were adjusted with COOT<sup>88</sup> and refinement was carried out using Phenix<sup>89</sup> or REFMAC5<sup>90</sup> and topologies of ligands were generated by AceDRG.<sup>91</sup> Final refinement statistics are given in Table S4. Atomic coordinates and structure factors have been deposited in the Protein Data Bank (8S34, 9EX3, 9F2T, 9FVQ). Figures were drawn using PyMOL Molecular Graphics System, Version 2.3.5 Schrödinger, LLC and CCP4MG version 2.11.0.

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25 FUNDING SOURCES
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1 IJS and GLAM warmly thank *Vaincre la Mucoviscidose* and the *Association Grégory Lemarchal* (French

2 associations against cystic fibrosis) for repeated financial support. IJS and GLAM also acknowledge the

3 Interdisciplinary Thematic Institute (ITI) InnoVec (Innovative Vectorization of Biomolecules, IdEx, ANR-

4 10-IDEX-0002), the SFRI (ANR-20-SFRI-0012) and the *Centre National de la Recherche Scientifique*

5 (CNRS) for financial support and for the Master fellowship of EC. The presented work was also

6 supported by a grant from the CNRS (80/Prime 2020), as well as by two grants from the Agence

7 Nationale de la Recherche (IRUPP, ANR-22-CE44-0024-01, and VECTRIUM project, ANR 19-CE18-0011-

8 02) for financial support. VW acknowledges the *Ministère de la Recherche et des Nouvelles Technologies* for providing a PhD fellowship. CA acknowledges CNRS and the *Institut de Science et d'Ingénierie Supramoléculaire* (ISIS) for providing a Master Degree fellowship for HA. The Rosalind

9 Franklin Institute is funded by UK Research and Innovation through the Engineering and Physical

10 Sciences Research Council. We would like to thank the Membrane Protein Laboratory at Diamond Light

11 Source (funded by Welcome Trust grant 223727/Z/21/Z) for help and support.

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## AUTHORS CONTRIBUTIONS

16 IJS, GLAM and LM wrote the manuscript with input from the others authors. IJS supervised biology experiments, GLAM supervised chemistry experiments, CA supervised the mass spectrometry studies, MM supervised modelisation of the fluorescent data and LM supervised structural biology. GLAM synthesized PCH and neoPCH, EC synthesized AA and DHA. HA performed the mass spectrometry analysis. VW did microbiology experiments and radioactive iron uptake assays, LK and JC performed and analyzed the proteomic experiments, FV performed with VW the modelisation of the fluorescent data. ALB and LM performed all the structural biology. IJS and GLAM found the grants.

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## COMPETING INTEREST

25
1 Authors declare no competing interests

2

3

## SUPPORTING INFORMATION

5 Synthesis protocols and spectral characterization (including ¹H, ¹³C NMR spectra and HRMS) of

6 compounds **3** and **AA**. UV total scans, total and extracted -ion chromatogram (positive and negative

7 mode) used for mass spectrometry metabolic profiling. Additional figures in microbiology and

8 structural biology. Lists of *P. aeruginosa* strains and of primers used for this study. Crystallographic

9 data and refinement statistics concerning FemA structures.

10

11

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<figure><img src="image_15.png" /></figure>

Fe uptake in *P. aeruginosa* by pyochelin, DHA and AA