Transport capabilities of environmental Pseudomonads for sulfur compounds

Abstract

Sulfur is an essential element in plant rhizospheres and microbial activity plays a key role in increasing the biological availability of sulfur in soil environments. To better understand the mechanisms facilitating the exchange of sulfur-containing molecules in soil, we profiled the binding specificities of eight previously uncharacterized ABC transporter solute-binding proteins from plant-associated Pseudomonads. A high-throughput screening procedure indicated eighteen significant organosulfur binding ligands, with at least one high-quality screening hit for each protein target. Calorimetric and spectroscopic methods were used to validate the best ligand assignments and catalog the thermodynamic properties of the protein-ligand interactions. Two novel high-affinity ligand-binding activities were identified and quantified in this set of solute-binding proteins. Bacteria were cultured in minimal media with screening library components supplied as the sole sulfur sources, demonstrating that these organosulfur compounds can be metabolized and confirming the relevance of ligand assignments. These results expand the set of experimentally validated ligands amenable to transport by this ABC transporter family and demonstrate the complex range of protein-ligand interactions that can be accomplished by solute-binding proteins. Characterizing new nutrient import pathways provides insight into Pseudomonad metabolic capabilities which can be used to further interrogate bacterial survival and participation in soil and rhizosphere communities.

Keywords: ABC transporter; Pseudomonas fluorescens; Pseudomonas protegens; organosulfur compounds; rhizosphere.

Figure 1

Heat map summary of FTS assay results for Met‐family SBPs. Chart shows ΔT _m values for protein‐ligand combinations; cells are colored so shifts of 3°C to the maximum of 17°C are shaded turquoise to orange with the gradient reflecting the magnitude of the shift. Proteins are clustered by ligand‐binding profile; ligands are arranged to highlight cluster differences. All data are an average of at least three replicates. Blank cells reflect shifts less than 3°C.

Figure 2

Representative data from determination of affinity constants for PFL_0242 interaction with AKG‐MTBAc. (A) Spectrograph of PFL_0242 intrinsic Trp fluorescence in combination with AKG‐MTBAc over a range of concentrations from 0.03 to 1000 µM. The protein was excited at 290 nm and fluorescence recorded from 290 to 360 nm. Each point represents one data point and the lines show the average values of two replicates. Quenching of PFL_0242 protein fluorescence by AKG‐MTBAc increased with ligand concentration. (B) Resulting ligand‐binding plot and non‐linear curve fit to estimate interaction K _d. Reduction of tryptophan fluorescence was used to calculate the concentration of protein‐ligand complex and plotted against the concentration of AKG‐MTBAc. The line superimposed on the plot represents an optimal fit of the data as described in the Methods section. (C) ITC thermogram and ligand‐binding plot for PFL_0242 and AKG‐MTBAc. A known amount of PFL_0242 was placed in the sample cell of a calorimeter and titrated with AKG‐MTBAc. The energy required to return the sample cell to equilibrium was recorded and used to calculate the protein‐ligand plot below; data was fitted using the OneSite binding non‐linear protocol in Origin.

Figure 3

Methionine derivatives as sulfur sources in P. protogens PF‐5 and P. fluorescens SBW25. Bacteria were cultured in minimal media with listed sole sulfur sources; percent of growth relative to cultures supplied with inorganic sulfate (Sodium Sulfate) was compared between samples with methionine derivatives or without sulfur (none) to identify biologically accessible sources. Bar charts average three independent replicates, error bars represent one standard deviation above and below the mean.