Bacteria secrete siderophores to access iron, a key nutrient poorly bioavailable and the source of strong competition between microorganisms in most biotopes. Many bacteria also use siderophores produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa, an opportunistic pathogen, produces two siderophores, pyoverdine and pyochelin, and is also able to use a panel of exosiderophores. We first investigated expression of the various iron-uptake pathways of P. aeruginosa in three different growth media using proteomic and RT-qPCR approaches and observed three different phenotypic patterns, indicating complex phenotypic plasticity in the expression of the various iron-uptake pathways. We then investigated the phenotypic plasticity of iron-uptake pathway expression in the presence of various exosiderophores (present individually or as a mixture) under planktonic growth conditions, as well as in an epithelial cell infection assay. In all growth conditions tested, catechol-type exosiderophores were clearly more efficient in inducing the expression of their corresponding transporters than the others, showing that bacteria opt for the use of catechol siderophores to access iron when they are present in the environment. In parallel, expression of the proteins of the pyochelin pathway was significantly repressed under most conditions tested, as well as that of proteins of the pyoverdine pathway, but to a lesser extent. There was no effect on the expression of the heme and ferrous uptake pathways. Overall, these data provide precise insights on how P. aeruginosa adjusts the expression of its various iron-uptake pathways (phenotypic plasticity and switching) to match varying levels of iron and competition.

Iron is a key nutrient for almost all living organisms. Paradoxically, it is poorly soluble and 3 consequently poorly bioavailable. Bacteria have thus developed multiple strategies to access 4 this metal. One of the most common consists of the use of siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able to produce their own 6 siderophores or use those produced by other microorganisms (exosiderophores) in a piracy 7 strategy. Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and 8 is also able to use a large panel of exosiderophores. We investigated the ability of P. aeruginosa 9 to use nocardamine (NOCA) and ferrioxamine B (DFOB) as exosiderophores under iron-10 limited planktonic growth conditions. Proteomic and RT-qPCR approaches showed induction 11 of the transcription and expression of the outer membrane transporter FoxA in the presence of 12 NOCA or DFO in the bacterial environment. Expression of the proteins of the heme-or 13 pyoverdine-and pyochelin-dependent iron uptake pathways was not affected by the presence 14 of these two tris-hydroxamate siderophores. 55 Fe uptake assays using foxA mutants showed 15 ferri-NOCA to be exclusively transported by FoxA, whereas ferri-DFOB was transported by 16 FoxA and at least one other unidentified transporter. The crystal structure of FoxA complexed 17 with NOCA-Fe revealed very similar siderophore binding sites between NOCA-Fe and DFOB-18 Fe. We discuss iron uptake by hydroxamate exosiderophores in P. aeruginosa cells in the light 19 of these results. 20 Page 2 of 40 ACS Paragon Plus Environment ACS Chemical Biology 1 detected the presence of ferri-exosiderophores, these transcriptional regulators activate the 2 transcription of the corresponding TBDT for iron acquisition. 9,22,29-32 3 Here, we investigated the ability of P. aeruginosa to use nocardamine (NOCA, Figure 1A) as 4 an exosiderophore. NOCA, also called desferrioxamine E, is a cyclic tris-hydroxamate 5 siderophore related to DFOB, with a higher affinity for iron: 10 32 M-1 for NOCA and 10 30 M-1 6 for DFOB 33 (Figure 1). NOCA is synthesized by various actinomycetes, such as Streptomyces, Nocardia, and Micromonospora, and bacteria, such as Streptomyces griseus, 35 Pseudomonas 8 stutzeri, 36 and Enterobacter agglomerans. 37 We show that the presence of NOCA in the P. 9 aeruginosa environment strongly induces the transcription and expression of foxA with the 10 same efficiency as DFOB. We demonstrate, using 55 Fe, that NOCA-55 Fe is exclusively 11 transported by the TBDT FoxA, whereas DFOB uses, in addition to FoxA, at least one other 12 transporter. We also determined the crystal structure of FoxA complexed with ferri-NOCA, 13 revealing a siderophore-binding site very similar to that of DFOB.

In the fission yeast , acquisition of exogenous heme is largely mediated by the cell membrane-associated Shu1. Here, we report that Str3, a member of the major facilitator superfamily of transporters, promotes cellular heme import. Using a strain that cannot synthesize heme (Δ) and lacks Shu1, we found that the heme-dependent growth deficit of this strain is rescued by hemin supplementation in the presence of Str3. Microscopic analyses of a ΔΔ Δ mutant strain in the presence of the heme analog zinc mesoporphyrin IX (ZnMP) revealed that ZnMP fails to accumulate within the mutant cells. In contrast, Str3-expressingΔ Δ cells could take up ZnMP at a 10-μm concentration. The yeast cannot efficiently transport exogenously supplied hemin. However, heterologous expression of Str3 from in resulted in ZnMP accumulation within cells. Moreover, hemin-agarose pulldown assays revealed that Str3 binds hemin. In contrast, an Str3 mutant in which Tyr and Ser residues of two putative heme-binding motifs (YY and SY) had been replaced with alanines exhibited a loss of affinity for hemin. Furthermore, this Str3 mutant failed to rescue the heme-dependent growth deficit of a ΔΔ Δ strain. Further analysis by absorbance spectroscopy disclosed that a predicted extracellular loop region in Str3 containing the two putative heme-binding motifs interacts with hemin, with a of 6.6 μm Taken together, these results indicate that Str3 is a second cell-surface membrane protein for acquisition of exogenous heme in .