Abstract
BackgroundPseudomonas syringae pv. phaseolicola 1448a (P. syringae 1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches. Under conditions of iron starvation P. syringae 1448a secretes siderophores for active uptake of iron. The primary siderophore of P. syringae 1448a is pyoverdine, a fluorescent molecule that is assembled from amino acid precursors by non-ribosomal peptide synthetase (NRPS) enzymes. Whereas other species of Pseudomonas often exhibit structural variations in the pyoverdine produced by different strains, all P. syringae pathovars previously tested have been found to make an identical pyoverdine molecule. P. syringae 1448a also appears to have the genetic potential to make two secondary siderophores, achromobactin and yersiniabactin, each of which has previously been detected in different P. syringae pathovars.ResultsFive putative pyoverdine NRPS genes in P. syringae 1448a were characterized in-silico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Pyoverdine was purified from P. syringae 1448a and analyzed by MALDI-TOF and MS/MS spectroscopy. Peaks were detected corresponding to the expected sizes for the pyoverdine structure previously found in other P. syringae pathovars, but surprisingly P. syringae 1448a appears to also produce a variant pyoverdine species that has an additional 71 Da monomer incorporated into the peptide side chain. Creation of pyoverdine null mutants of P. syringae 1448a revealed that this strain also produces achromobactin as a temperature-regulated secondary siderophore, but does not appear to make yersiniabactin. Pyoverdine and achromobactin null mutants were characterized in regard to siderophore production, iron uptake, virulence and growth in iron limited conditions.ConclusionsThis study provides the first evidence of a P. syringae pathovar producing a side chain variant form of pyoverdine. We also describe novel IC50 and liquid CAS assays to quantify the contribution of different siderophores across a range of iron starvation conditions, and show that although achromobactin has potential to contribute to fitness its contribution is masked by the presence of pyoverdine, which is a significantly more effective siderophore. Neither pyoverdine nor achromobactin appear to be required for P. syringae 1448a to cause bean halo blight, indicating that these siderophores are not promising targets for crop protection strategies.
Highlights
Pseudomonas syringae pv. phaseolicola 1448a (P. syringae 1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches
Identification and in silico characterization of the P. syringae 1448a pyoverdine locus The biosynthesis of pyoverdine has been most extensively studied in P. aeruginosa PAO1 and most, if not all, of the genes required for pyoverdine synthesis in this strain have been identified [6,10,26]
The first concerns the only PAO1 gene that clearly lacks an ortholog in P. syringae, pvdF, which encodes an enzyme required for generating the N5-formyl-N5-hydroxyornithine residues that are present in the PAO1 pyoverdine side chain
Summary
Pseudomonas syringae pv. phaseolicola 1448a (P. syringae 1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches. Due to insolubility at neutral pH the bioavailability of iron is extremely low in most natural environments. To circumvent this problem many bacteria respond to iron starvation by synthesizing high affinity iron-chelating molecules known as siderophores. These siderophores are secreted into the extra-cellular environment where they bind ferric iron and are actively transported back into the cell via specific ferricsiderophore receptors [1]. Fluorescent pseudomonads synthesize the peptide-derived molecule pyoverdine as their primary siderophore, together with secondary siderophores that have lower affinity for iron [2]. As iron restriction is a key host defense mechanism, pyoverdine is frequently implicated as an important virulence factor [5,6]
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