J. D. Marugg

The role of siderophores in potato tuber yield increase by Pseudomonas putida in a short rotation of potato

The effect of treatment of potato seed tubers withPseudomonas putida isolate WCS358 on tuber yield was studied in different crop rotations at the Experimental Farm ‘De Schreef’, near Lelystad. With untreated, tuber yield in a 1:3 (short) rotation compared to yield in a 1:6 (long) rotation of potato was decreased by 11% at 86 days (seed tuber harvest) and by 14% at 130 days (ware potato harvest) after seeding. Seed tuber treatment with the wild-type isolate WCS358 increased tuber yield with 13% in a short rotation of potato 86 days after seeding, whereas a siderophore-negative Tn5 transposon mutant of this isolate had no effect on tuber yield. Seed tuber treatment with the wild-type isolate or the siderophore-negative mutant in a long rotation of potato had no effect on tuber yield. At 130 days after seeding no effect of any of the seed tuber treatments was found in both short and long rotations of potato.Root colonization by siderophore-producing Tn5 transposon mutants of WCS358 was decreased at the end of the growing season. No difference in root colonization between siderophore-producing and siderophore-negative Tn5 transposon mutants was found at 130 days after seeding.Siderophore production seems to be a prerequisite in potato tuber yield increase by WCS358 under field conditions. This is the first time that the involvement of siderophores in growth stimulation has been demonstrated in the field.SamenvattingDe invloed van een behandeling van aardappelpootgoed metPseudomonas putida isolaat WCS358 op de knolopbrengst werd onderzocht in verschillende gewasrotaties on een proefveld van proefboerderij ‘De Schreef’, Flevopolder. In de controlebehandelingen werd in een nauwe aardappelrotatie (1:3) een reductie van 11% in opbrengst van pootaardappelen (86 dagen na het poten) geconstateerd ten opzichte van een ruime aardappelrotatie (1:6); 130 dagen na het poten werd een vermindering met 14% gevonden in de opbrengst van consumptieaardappelen.Pootgoedbehandeling met het siderofoorproducerende isolaat WCS358 verhoogde de opbrengst van pootaardappelen in de 1:3-rotatie met 13%. Een Tn5-transposonmutant van dit isolaat die het vermogen sideroforen te produceren had verloren, had geen effect op de opbrengst. In de 1:6-rotatie had behandeling van pootgoed met WCS358 geen effect op de opbrengst van pootaardappelen.Zowel in de nauwe (1:3) als in de ruimte (1:6) rotatie werd (130 dagen na het poten), geen effect van behandeling van pootgoed met WCS358 op de opbrengst van consumptieaardappelen gevonden.Wortelkolonisatie door siderofoorproducerende Tn5-transposonmutanten van WCS358 nam aan het eind van het seizoen af. Er werd, 130 dagen na het poten, geen verschil in wortelkolonisatie geconstateerd tussen siderofoorproducerende en siderofoornegatieve Tn5-transposonmutanten.Siderofoorproduktie blijkt een voorwaarde te zijn voor verhoging van de knolopbrengst door WCS358 onder veldomstandigheden. De verhoging van de knolopbrengst treedt alleen op in de nauwe aardappelrotatie. Dit is de eerste keer dat de betrokkenheid van sideroforen bij groeistimulatie onder veldomstandigheden is aangetoond.

The ferric-pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB-dependent Escherichia coli receptors and specificity of the protein

The initial step in the uptake of iron via ferric pseudobactin by the plant‐growth‐promoting Pseudomonas putida strain WCS358 is binding to a specific outer‐membrane protein. The nucleotide sequence of the pupA structural gene, which codes for a ferric pseudobactin receptor, was determined. It contains a single open reading frame which potentially encodes a polypeptide of 819 amino acids, including a putative N‐terminal signal sequence of 47 amino acids. Significant homology, concentrated in four boxes, was found with the TonB‐dependent receptor proteins of Escherichia coli. The pupA mutant MH100 showed a residual efficiency of 30% in the uptake of 55HFe3+ complexed to pseudobactin 358, whereas the iron uptake of four other pseudobactins was not reduced at all. Cells of strain WCS374 supplemented with the pupA gene of strain WCS358 could transport ferric pseudobactin 358 but showed no affinity for three other pseudobactins. It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactins.

Characterization and structural analysis of the siderophore produced by the PGPR Pseudomonas putida strain WCS358

Under iron limitation, the plant-growth-promoting Pseudomonas putida strain WCS358 produces a yellow-green fluorescent siderophore (Geels and Schippers, 1983; Geels and Schippers, 1983; Marugg et al., in press). The absorption spectra of WCS358 culture medium before and after addition of FeCl3 and at different pHs are shown in an accompanying paper by our group (weisbeek et al., in press). They strongly resemble those of the pseudobactin/pyoverdine class of siderophores (Hider, 1984).

Genetic Analysis of the Iron-Uptake System of Two Plant Groups Promoting Pseudomonas Strains

Pseudomonas species can be found in a wide variety of environments; in association with animals and plants and in water. This is a consequence of their ability to use for their metabolism a great number of different compounds and to synthesize for different purposes many secondary metabolites like complex aromatic molecules and unusual amino acids and peptides. These properties together with the great stability of the organism are the reason why Pseudomonas spp. have become one of the major groups of colonizers of the plant-root. Many of the rhizosphere-Pseudomonas species are known plant-pathogens but more recently a number of root-associated fluorescent Pseudomonas strains have received increasing attention because of their ability to benefit plant growth. (Kloepper et al., 1980: Geels and Schippers, 1983a, Geels and Schippers, 1983b).

A Siderophore Peptide Synthetase Gene from Plant-growth-promoting Pseudomonas putida WCS358

Under iron limiting conditions, Pseudomonas putida WCS358 produces and secretes a fluorescent siderophore called pseudobactin 358 which consists of a nonapeptide linked to a fluorescent dihydroxy quinoline moiety. Previous studies have identified a major gene cluster involved in pseudobactin 358 biosynthesis and several regulators responsible for the activation of biosynthetic genes under iron starving conditions. In this study, we identified the promoter transcribing the pseudobactin 358 synthetase gene. Promoter deletion experiments have demonstrated that the DNA region downstream of the initiation of transcription site is necessary for proper promoter functioning. This promoter controls the expression of a gene designated ppsD which encodes a 2,247-residue protein, PpsD, which has a predicted molecular weight of 247,610 Da and contains two highly homologous domains of approximately 1000 amino acids each. ppsD::Tn5 mutants of strain WCS358 are unable to synthesise pseudobactin 358 and can be complemented when ppsD is provided in trans. It is concluded that ppsD is a peptide synthetase involved in the biosynthesis of the peptide moiety of pseudobactin 358. PpsD displays a very high degree of similarity (52% aa identity) with PvdD from P. aeruginosa, a non-ribosomal peptide synthetase involved in the biosynthesis of pyoverdine, the fluorescent siderophore produced by P. aeruginosa. It also displayed homology with other peptide synthetases from other micro-organisms involved in the biosynthesis of siderophores and peptide antibiotics.

Genetics of iron transport in plant growth-promoting Pseudomonas putida WCS358

Root-colonizing Pseudomonas putida WCS358 enhances growth of potato in part by producing under iron-limiting conditions a yellow-green, fluorescent siderophore designated pseudobactin 358. This siderophore efficiently complexes iron(III) in the rhizosphere, making it less available to certain endemic microorganisms, including phytopathogens, thus inhibiting their growth. At least 15 genes distributed over five gene clusters are required for the biosynthesis of pseudobactin 358. The transcriptional organization and the iron-regulated expression of a major gene cluster (A) involved in the biosynthesis of pseudobactin 358 and transport of iron(III) via pseudobactin 358 were analyzed. The DNA sequence of the gene encoding the outer membrane receptor protein for ferric pseudobactin 358 has been determined; the mature protein consists of 772 amino acids (86.01 kilodaltons) with a signal sequence of 47 amino acids, which is extremely long for prokaryotes. The receptor protein, which appears to be specific for ferric pseudobactin 358, shares strong homology with four regions of TonB-dependent receptor proteins of Escherichia coli, which suggests the presence of a TonB-like protein in strain WCS358 required for iron(III) transport. Strain WCS358 also possesses a low-affinity iron uptake system for ferric pseudobactin 358 and ferric pseudobactins from many other fluorescent pseudomonads. Genes coding for the biosynthesis of pseudobactin 358 and the high-affinity receptor protein for ferric pseudobactin 358 are regulated transcriptionally by iron(III).

Siderophore Biosynthesis, Uptake and Effect on Potato Growth of Rhizosphere Strains

Treatment of seed potatoes with certain root-colonizing Pseudomonas putida and fluorescens strains has resulted in protection of the potato tuber yield against the effects of narrow rotation cropping (1:3) and against the effects of certain microbial pathogens. This protective activity of the bacteria is thought to be caused by the production and excretion of large quantities of siderophores with high affinity for binding of iron(III) and uptake of the siderophore-iron(III) complex. The subsequent decrease in iron(III) around the root-surface prevents or delays the growth of other (pathogenic) micro-organisms.

The Iron-Uptake System of the Plant-Growth-Stimulating Pseudomonas Putida WCS358: Genetic Analysis and Properties and Structure Analysis of Its Siderophore

Treatment of potato seed tubers with certain root-colonizing Pseudomonos putida and Pseudomonas fluorescens strains protects the plants from deleterious microorganisms which are present in the root environment during narrow rotation cropping, resulting in an increase of tuber yield in these soils (Schippers et al 1985). The plant-growth-stimulating activity is thought to be the consequence of their very efficient iron (III)-binding capacity. Under iron-limiting conditions which exist in most soils, the pseudomonads produce and excrete large quantities of yellow-green, fluorescent siderophores which chelate iron (III) with high affinity. The pseudomonads take up the iron(III)-siderophore complex specifically and with high efficiency. The deleterious microorganisms, however, are not able to do so and thus are prevented to grow efficiently due to iron (III)-depletion.

Molecular Aspects of Plant Growth Affecting Pseudomonas Species

Organic compounds released by roots stimulate the activity of a variety of micro-organisms in the root environment, the rhizosphere (Rovira and Davey 1974). Of these micro-organisms, soil-borne plant pathogens and nitrogen fixing micro-organisms have obtained most attention. Relatively little is known about the majority of the rhizosphere micro-organisms, the saprophytes and their influence on root functioning, plant development and crop yield.