Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe³⁺complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria includingPseudomonasspecies can use it. Here, we show that thePseudomonastransporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.

Iron acquisition pathways have often been considered to be gateways for the uptake of antibiotics into bacteria. Bacteria excrete chelators, called siderophores, to access iron. Antibiotic molecules can be covalently attached to siderophores for their transport into pathogens during the iron-uptake process. P. aeruginosa produces two siderophores and is also able to use many siderophores produced by other bacteria. We investigated the phenotypic plasticity of iron-uptake pathway expression in an epithelial cell infection assay in the presence of two different siderophore–antibiotic conjugates, one with a hydroxamate siderophore and the second with a tris-catechol. Proteomic and RT-qPCR approaches showed that P. aeruginosa was able to sense the presence of both compounds in its environment and adapt the expression of its iron uptake pathways to access iron via them. Moreover, the catechol-type siderophore–antibiotic was clearly more efficient in inducing the expression of its corresponding transporter than the hydroxamate compound when both were simultaneously present. In parallel, the expression of the proteins of the two iron uptake pathways using siderophores produced by P. aeruginosa was significantly repressed in the presence of both conjugates. Altogether, the data indicate that catechol-type siderophores are more promising vectors for antibiotic vectorization using a Trojan-horse strategy.

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.

Bacteria access iron, a key nutrient, by producing siderophores or using siderophores produced by other microorganisms. The pathogen Pseudomonas aeruginosa produces two siderophores but is also able to pirate enterobactin (ENT), the siderophore produced by Escherichia coli. ENT-Fe complexes are imported across the outer membrane of P. aeruginosa by the two outer membrane transporters PfeA and PirA. Iron is released from ENT in the P. aeruginosa periplasm by hydrolysis of ENT by the esterase PfeE. We show here that pfeE gene deletion renders P. aeruginosa unable to grow in the presence of ENT because it is unable to access iron via this siderophore. Two-species co-cultures under iron-restricted conditions show that P. aeruginosa strongly represses the growth of E. coli as long it is able to produce its own siderophores. Both strains are present in similar proportions in the culture as long as the siderophore-deficient P. aeruginosa strain is able to use ENT produced by E. coli to access iron. If pfeE is deleted, E. coli has the upper hand in the culture and P. aeruginosa growth is repressed. Overall, these data show that PfeE is the Achilles’ heel of P. aeruginosa in communities with bacteria producing ENT.

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.