Siderophore-mediated iron uptake is essential for the survival of many bacteria in soil and water and during infection. Pyoverdine (PVD) is a siderophore produced by fluorescent pseudomonads [1]. Using the soil bacterium Pseudomonas putida KT2440 as a model system, we previously identified the tripartite efflux pumps PvdRT-OpmQ (ABC type) and MdtABC-OpmB (RND type) as being involved in PVD secretion [2]. Here, we aim to (1) identify additional efflux systems contributing to PVD secretion and (2) provide biochemical evidence for the interaction of PVD with PvdRT-OpmQ.

In a screen, genes encoding components of other tripartite efflux systems were individually deleted in a P. putida KT2440 strain lacking functional PvdRT-OpmQ and MdtABC-OpmB systems. The consequences of the deletions on PVD secretion were analyzed. Furthermore, the inner membrane component PvdT and the periplasmic adaptor protein PvdR were purified and characterized with respect to ATPase activity and interactions using DRaCALA and surface plasmon resonance (SPR).

The screen revealed that the RND efflux system ParXY is critical for growth under iron limitation when PvdRT-OpmQ and MdtABC-OpmB are inactivated. Additional inactivation of ParXY increases the amount of PVD accumulated in the periplasm and further inhibits PVD secretion. In contrast, individual inactivation of ParXY has no significant effect [3]. DRaCALA and SPR revealed that PVD directly interacts with PvdRT. Furthermore, the ATPase activity of solubilized PvdT was stimulated by interaction with PvdR but not by addition of PVD [4].

We conclude that PvdRT-OpmQ is the major system for PVD secretion in P. putida KT2440, whereas MdtABC-OpmB and ParXY contribute only conditionally to this process. Given the importance of iron uptake for cellular metabolism, these overlapping activities ensure the survival and growth of bacteria under the common conditions of iron deficiency.

[1] Ringel, MT & Brüser, T (2018) Microb Cell 5, 424-437.

[2] Henriquez, T et al. (2019) Environ Microbiol Rep 11, 98-106.

[3] Stein, NV et al. (2023) Microbiol. Spectr. 11, e02300-02323

[4] Stein, NV et al. (2023) FEBS Lett. 597, 1403-1414.