Effects of exogenous pyoverdines on Fe availability and their impacts on Mn(II) oxidation by Pseudomonas putida GB-1

Abstract

Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium that produces pyoverdine-type siderophores (PVDs), which facilitate the uptake of Fe(III) but also influence MnO2 formation. Recently, a non-ribosomal peptide synthetase mutant that does not synthesize PVD was described. Here we identified a gene encoding the PVDGB-1 (PVD produced by strain GB-1) uptake receptor (PputGB1_4082) of strain GB-1 and confirmed its function by in-frame mutagenesis. Growth and other physiological responses of these two mutants and of wild type were compared during cultivation in the presence of three chemically distinct sets of PVDs (siderotypes n°1, n°2, and n°4) derived from various pseudomonads. Under iron-limiting conditions, Fe(III) complexes of various siderotype n°1 PVDs (including PVDGB-1) allowed growth of wild type and the synthetase mutant, but not the receptor mutant, confirming that iron uptake with any tested siderotype n°1 PVD depended on PputGB1_4082. Fe(III) complexes of a siderotype n°2 PVD were not utilized by any strain and strongly induced PVD synthesis. In contrast, Fe(III) complexes of siderotype n°4 PVDs promoted the growth of all three strains and did not induce PVD synthesis by the wild type, implying these complexes were utilized for iron uptake independent of PputGB1_4082. These differing properties of the three PVD types provided a way to differentiate between effects on MnO2 formation that resulted from iron limitation and others that required participation of the PVDGB-1 receptor. Specifically, MnO2 production was inhibited by siderotype n°1 but not n°4 PVDs indicating PVD synthesis or PputGB1_4082 involvement rather than iron-limitation caused the inhibition. In contrast, iron limitation was sufficient to explain the inhibition of Mn(II) oxidation by siderotype n°2 PVDs. Collectively, our results provide insight into how competition for iron via siderophores influences growth, iron nutrition and MnO2 formation in more complex environmental systems.

Keywords: Fe availability; MnO2; biofilm; iron limitation; iron requirement; pyoverdine; pyoverdine receptor; siderotyping.

Figure 1

MnO₂ formation by P. putida GB-1 after exposure to purified PVD_GB-1 (A,B) and to PVD released during the growth of another siderotype n°1 strain, P. putida CFML90-49 (E,F). (A,C,E) Visible light illumination to show brown MnO₂. (B,D,F) UV light illumination to detect blue-green PVD fluorescence. (A,B) Effect of 75 μL of purified PVD_GB-1 (200 mM, siderotype n°1) applied at one edge of a plate containing 20 ml of solid medium and a single streak of P. putida GB-1. The plate was incubated vertically so that the liquid remained localized, although it did spread as a crescent at the edge of the plate on the downward side, taking some inoculum with it. Note that areas of the strain GB-1 streak line near the zone of PVD_GB-1 application showed considerable blue-green PVD fluorescence, whereas growth zones further from the PVD drop contained brown MnO₂ with no visible fluorescence. (C,D) Full MnO₂ production when strain GB-1 was cultured alone. (E,F) Inhibited MnO₂ production when strain GB-1 was co-cultivated with P. putida CFML90-49, which cannot oxidize Mn(II) and produces a siderotype n°1 PVD resembling PVD_GB-1. Note the increased PVD fluorescence and decreased MnO₂ within strain GB-1 growth zones in the presence of strain CFML90-49 (E,F) as compared to the control without strain CFML90-49 (C,D).

Figure 2

The P. putida GB-1 NRPS mutant KG163, which does not produce PVD, was overlayed on CAA plates supplemented with 100 μM dipyridyl, an agent that strongly complexes Fe. 20 μl of PVD solutions from P. putida GB-1, P. putida KT2440, and Pseudomonas sp. PCP1 or MilliQ water were put on filter disks to determine the ability of exogenous PVDs to release Fe from it dipyridyl complex and to support the growth (observed as visual turbidity) of a P. putida GB-1 mutant that lacks the ability to produce PVD.

Figure 3

Effect of exogenous PVDs on the growth of the P. putida GB-1 mutant ΔPputGB1_4082. The medium was supplemented with 2 μM FeCl₃ and 10 μM of either: (i) no PVDs, (ii) PVD_GB-1, (iii) PVD_KT2440, or (iv) PVD_PCP1.

Figure 4

The effect of exogenous PVDs on biofilm formation by P. putida GB-1. Biofilm formation was estimated using crystal violet staining. [Fe(III)] = 2 μM (added as FeCl₃) and [PVD] = 10 μM (if added). Error bars indicate the standard deviation of measurements of triplicate samples.

Figure 5

Concentration of PVD observed 2 days after the addition of various exogenous PVDs to P. putida GB-1 cultures. [Fe] = 2 μM [added as FeCl₃ (dark bars) or FeSO₄•7H₂O (light bars)] and added [PVD] = 10 μM (if added). The exogenous PVDs were: siderotype n°1, PVD_GB-1 and PVD_CFML90−15; siderotype n°2, PVD_KT2440; siderotype n°4, PVD_PCP1 and PVD_ISO6. Error bars indicate the standard deviation of measurements of triplicate samples.

Figure 6

Concentration of PVD present 2 days after the addition of various exogenous PVDs to each of four different siderotype n°1-producing strains: P. putida GB-1 (open), P. putida CFML90-49 (horizontal lines), P. putida MnB1 (dots), and P. putida BC (vertical lines). [Fe(III)] = 2 μM (added as FeCl₃) and added [PVD] = 10 μM. Error bars indicate the standard deviation of triplicate samples.

Figure 7

Concentration of PVD observed 2 days after the addition of varying amounts of PVD_GB-1 to P. putida GB-1 cultures. [Fe(II)] = 2 μM (added as FeSO₄•7H₂O). The concentration of the added PVD was varied from 0 to 10 μM. Error bars indicate the standard deviation of measurement of triplicate samples. Fe(II) was added to fully repress PVD synthesis at the condition without added PVD. For all samples containing PVD, however, the initial Fe(II)-PVD complex was expected to be rapidly air oxidized to Fe(III)-PVD (Xiao and Kisaalita, 1998), consistent with data in Figure 5.

Figure 8

Concentration of PVD observed 2 days after the addition of varying concentrations of FeCl₃ to P. putida GB-1 cultures supplemented with 10 μM of each indicated PVD. No PVD (open bars), PVD_GB-1 (horizontal lines), PVD_KT2440 (diagonal lines), and PVD_PCP1 (filled). [Fe(III)] = 0, 2, 5, and 15 μM (added as FeCl₃) and added [PVD] = 10 μM. Error bars indicate the standard deviation of PVD assays of triplicate samples.