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.

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The development of new antibiotics against Gram-negative bacteria has to deal with the low permeability of the outer membrane. This obstacle can be overcome by utilizing siderophore-dependent iron uptake pathways as entrance routes for antibiotic uptake. Iron-chelating siderophores are actively imported by bacteria, and their conjugation to antibiotics allows smuggling the latter into bacterial cells. Synthetic siderophore mimetics based on MECAM (1,3,5-N,N′,N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) and DOTAM (1,4,7,10-tetrakis­(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) cores, both chelating iron via catechol groups, have been recently applied as versatile carriers of functional cargo. In the present study, we show that MECAM and the MECAM-ampicillin conjugate 3 transport iron into Pseudomonas aeruginosa cells via the catechol-type outer membrane transporters PfeA and PirA and DOTAM solely via PirA. Differential proteomics and quantitative real-time polymerase chain reaction (qRT-PCR) showed that MECAM import induced the expression of pfeA, whereas 3 led to an increase in the expression of pfeA and ampc, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of pirA but upregulated the expression of two zinc transporters (cntO and PA0781), pointing out that bacteria become zinc starved in the presence of this compound. Iron uptake experiments with radioactive 55Fe demonstrated that import of this nutrient by MECAM and DOTAM was as efficient as with the natural siderophore enterobactin. The study provides a functional validation for DOTAM- and MECAM-based artificial siderophore mimetics as vehicles for the delivery of cargo into Gram-negative bacteria.