Stereospecificity of the Siderophore Pyochelin Outer Membrane Transporters in Fluorescent Pseudomonads

Abstract

Pyochelin (Pch) and enantio-pyochelin (EPch) are enantiomer siderophores that are produced by Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, under iron limitation. Pch promotes growth of P. aeruginosa when iron is scarce, and EPch carries out the same biological function in P. fluorescens. However, the two siderophores are unable to promote growth in the heterologous species, indicating that siderophore-mediated iron uptake is highly stereospecific. In the present work, using binding and iron uptake assays, we found that FptA, the Fe-Pch outer membrane transporter of P. aeruginosa, recognized (K d ‫؍‬ 2.5 ؎ 1.1 nM) and transported Fe-Pch but did not interact with Fe-EPch. Likewise, FetA, the Fe-EPch receptor of P. fluorescens, was specific for Fe-EPch (K d ‫؍‬ 3.7 ؎ 2.1 nM) but did not bind and transport Fe-Pch. Growth promotion experiments performed under iron-limiting conditions confirmed that FptA and FetA are highly specific for Pch and EPch, respectively. When fptA and fetA along with adjacent transport genes involved in siderophore uptake were swapped between the two bacterial species, P. aeruginosa became able to utilize Fe-EPch as an iron source, and P. fluorescens was able to grow with Fe-Pch. Docking experiments using the FptA structure and binding assays showed that the stereospecificity of Pch recognition by FptA was mostly due to the configuration of the siderophore chiral centers C4؆ and C2؆ and was only weakly dependent on the configuration of the C4 carbon atom. Together, these findings increase our understanding of the stereospecific interaction between Pch and its outer membrane receptor FptA.To access iron, aerobic bacteria produce siderophores which form complexes with Fe 3ϩ in the environment and deliver it via specific membrane transporters to the bacteria (1). The energy required for this process is provided by the proton motive force of the inner membrane by means of an inner membrane complex comprising TonB, ExbB, and ExbD (2, 3). Pyochelin (Pch), 2 which is the focus of this study, is produced as a secondary siderophore under iron limitation by almost all strains of Pseudomonas aeruginosa and some closely related bacteria (4 -7). In pseudomonads, secondary siderophores (pyochelin, thioquinolobactin, etc.) are usually produced in smaller amounts, demonstrate lower iron affinity, and are rather simple molecules compared with pyoverdin (the main siderophore). It is hypothesized that under certain environmental or physiological conditions, secondary siderophores provide sufficient iron to the cell or fulfill functions other than iron sequestration (8). Pch was isolated for the first time from P. aeruginosa ATCC 15692 by Liu and Shokrani (9), and its structure was established later by Cox et al. (10) as (4ЈR,2ЉR,4ЉR)-2Ј-(2-hydroxyphenyl)-3Љ-methyl-4Ј,5Ј,2Љ,3Љ,4Љ,5Љ-hexahydro-[4Ј,2Љ]bithiazolyl-4Љ-carboxylic acid with three chiral centers located at C4Ј, C2Љ, and C4Љ (see Fig. 1). Pch is synthesized by P. aeruginosa from salicylate and two molecules of cysteine via a thiot...

Stereospecific recognition of pyochelin and enantio-pyochelin by the PchR proteins in fluorescent pseudomonads

“…Pyochelin and enantio-pyochelin were purified from culture supernatants of P. aeruginosa and P. fluorescens, respectively, as described previously (Youard et al, 2007) and added to the growth medium at a final concentration of 20 mM. This concentration, which is at least 20-fold below the physiological concentration reached under iron starvation (Reimmann et al, 1998 and our unpublished results), fully induces the expression of pyochelin and enantio-pyochelin biosynthesis genes in P. aeruginosa and P. fluorescens, respectively (Youard et al, 2007) and restores growth under iron limitation in siderophore-negative mutants (Hoegy et al, 2009). For IPTG-induced expression of proteins in GGP medium a concentration of 1 mM was used; for IPTG-induced expression of proteins on Chrome Azurol S (CAS) agar (Schwyn & Neilands, 1987), each plate was surface-spread with 100 ml of a 500 mM IPTG stock solution.…”

“…Excision of the vector via a second crossing-over was obtained by enrichment for tetracycline-sensitive cells to generate the mutants CHA1222 (DpchR, pvd : : Tn1733) and CHA1223 (DpchR, DpchDHIEFKCBA), respectively. The previously described suicide plasmids pME7531 (Hoegy et al, 2009) and pME7152 (Michel et al, 2007) were used in a similar way to delete the enantio-pyochelin receptor gene in P. fluorescens CHA0 and the pyoverdine biosynthesis gene pvdF in P. aeruginosa PAO6274, generating the corresponding mutants CHA1086 (DfetA) and PAO6402 (pchR : : v, DpvdF), respectively.…”

“…The pyochelin and enantio-pyochelin receptors FptA and FetA are specific for binding and transport of their respective ferri-siderophores (Ankenbauer & Quan, 1994;Hoegy et al, 2009), implying that for interaction with PchR ( Fig. 3) both siderophores must also be able to enter the cells via another less specific mechanism.…”

“…The pyochelin receptor FptA does not bind and transport iron-loaded enantiopyochelin in P. aeruginosa, whereas the enantio-pyochelin receptor FetA does not bind and transport ferri-pyochelin in P. fluorescens. Swapping the pyochelin and the enantiopyochelin uptake systems between the two bacterial species inverted their siderophore preference and allowed P. aeruginosa to utilize enantio-pyochelin as an iron source while P. fluorescens was enabled to grow with iron-loaded pyochelin (Hoegy et al, 2009).…”

FvbA is required for vibriobactin utilization in Pseudomonas aeruginosa

“…The opportunistic pathogen P. aeruginosa is known to produce and utilize two chemically distinct siderophores, pyochelin (Cox, 1980) through the FptA receptor (Ankenbauer & Quan, 1994;Hoegy et al, 2009;Michel et al, 2007), and pyoverdine (Cox & Adams, 1985) through two receptors, FpvA and FpvB Schalk, 2008). In addition to its endogenous siderophores, P. aeruginosa can utilize siderophores produced by other micro-organisms.…”