Pseudomonas aeruginosa zinc uptake in chelating environment is primarily mediated by the metallophore pseudopaline

Abstract

Metal uptake is vital for all living organisms. In metal scarce conditions a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here we characterize a four-gene operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated with an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The pseudopaline system is involved in nickel uptake in poor media, and, most importantly, in zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as the main zinc importer under these metal scarce conditions.

Figure 1

Determination of the cnt operon of Pseudomonas aeruginosa. Schematic representation of the genetic environment of the four cnt genes recovered on the P. aeruginosa PA14 genome. The upstream DNA sequence carrying the −35 and −10 boxes of the σ 70 promoter are highlighted in pink and the predicted palindromic Zur box is underlined. The first initiating ATG codon of cntO gene is indicated in italic. RT-PCR were performed on total RNA isolated from P. aeruginosa PA14 strain grown in MS medium. PCR were performed without template (A) or with RNA (B), genomic DNA (C) or cDNA (D) as template. PCR corresponding to the four intragenic regions (1 to 4) and the five intergenic regions (5 to 9) tested are indicated in gray.

Figure 2

PaCntL production under various growth conditions. (a) Immunoblotting using antibody directed against the V5 epitope for revealing PaCntL_V5 production under poor (MS) and rich (LB) media. (b) Dot-blot revealing the PaCntL_V5 production in MS medium supplemented by divalent metals. (c) Immunoblot detection of PaCntL_V5 production in PA14 WT and Zur deficient strains (zur ⁻) in various growth conditions.

Figure 3

In vivo PaCntL-dependent detection of a nickel or zinc-bound metallophore in the extracellular fraction of P. aeuginosa. (a) HILIC/ICP-MS chromatogram of metal-bound metabolites found in the extracellular fraction of P. aeruginosa grown in MS medium. (b) HILIC-ESI/MS mass spectrum of a Ni-metallophore complex in the extracellular fraction of the WT strain but absent in the ΔcntL mutant (both grown in MS medium). (c) HILIC-ESI/MS mass spectrum of a Zn-metallophore complex in the extracellular fraction of the WT strain but absent in the ΔcntL mutant. The empirical molecular formula of the CntL-dependent Ni- or Zn-metallophore complexes were deduced from the exact mass.

Figure 4

In vitro reconstitution of the pseudopaline biosynthesis pathway. (a) TLC experiment using PaCntL and [¹⁴C]-SAM showing that PaCntL discriminates between D- and L-histidine substrate with the production of the reaction intermediate (noted yNA) only visible when using L-histidine. (b) Titration of NADPH (blue) and NADH (red) binding to PaCntM (5 µM) followed by fluorescence resonance energy transfer. Fitting of the data obtained for NADH led to a K_d of 30 µM. (c) TLC separation of reaction products after incubation of [¹⁴C]-SAM with purified enzymes (PaCntL and PaCntM), different source of α-ketoacid (pyruvate or α-KG), cofactor (NADH or NADPH) and histidine (L-His or D-His). (d) HILIC/ESI-MS chromatograms of putative reaction products using PaCntL incubated with L-histidine, revealing the production of the yNA intermediate (top), and a mix of PaCntL and PaCntM incubated with all their putative substrate (SAM, L-histidine, NADH and α-Ketaoglutarate), revealing the specific detection of pseudopaline in this case (red trace, bottom). (e) Summary of the PaCntL/M-dependent biosynthesis pathway for the assembly of pseudopaline from L-his, SAM, NADH and α-KG.

Figure 5

Pseudopaline is involved in nickel uptake in minimal media and in zinc uptake in chelating media. Intracellular nickel (a) or zinc (b) levels measured by ICP-MS in WT, ΔcntL and ΔcntL::cntL strains grown in MS medium supplemented or not with 10 or 100 µM EDTA. Error bars, mean ± s.d. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to the WT.

Figure 6

Model of pseudopaline synthesis, secretion and metal uptake in P. aeruginosa. (a) Detection of pseudopaline in the extracellular fraction of WT and mutant strains grown in MS medium. Error bars, mean ± s.d. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to the WT. (b) Detection of pseudopaline in the intracellular fraction of WT and mutant strains. ND: Not Detectable. (c) Model of pseudopaline production, secretion and recovery of nickel or zinc. Outer membrane (OM), inner membrane (IM), periplasm (P).