In an iron-limited environment Pseudomonas syringae pv. syringae B301D produces a yellow-green fluorescent siderophore called pyoverdinp. which functions in high-affinity iron transport. Two-dimensional electrophoretic comparisons of the outer membrane proteins of strain B301D identified nine proteins which were expressed at low (50 nM) but not at high (10 ,uM) iron concentrations. Except for the minor protein 8e, the iron-regulated proteins exhibited high molecular weights ranging from approximately 74,000 to 80,000. A mutant of strain B301D incapable of iron uptake (lu-) from ferric pyoverdinp. lacked the 74,000-molecularweight protein 4a, which was the major iron-regulated outer membrane protein. In contrast, a nonfluorescent mutant (Flu-) unable to synthesize pyoverdinp8 showed no quantitative or qualitative difference in its outer membrane profile from that of the wild-type strain. In plant pathogenicity tests the Iu-and Flu-strains caused typical brown necrotic and sunken lesions in immature sweet cherry fruit which were indistinguishable from those of the wild-type strain. Thus, excretion of pyoverdinps and subsequent Fe(M) uptake do not have a determinative role in the pathogenicity or virulence of P. syringae pv. syringae.Iron is an abundant element, but it exists predominantly as insoluble complexes of Fe(III) in aerobic environments. Because iron is a nutrient required by all living cells, many microorganisms acquire iron by synthesizing siderophores, which are low-molecular-weight Fe(III) chelators that deliver iron to the cell via high-affinity transport systems (33-36). Virulence in several bacteria pathogenic to animals has been correlated with the production and utilization of siderophores during invasion (9, 48). The siderophores supply the iron essential for growth and pathogenesis by sequestering Fe(III) from body fluids, in which iron is mainly complexed with high-affinity proteins such as transferrin and lactoferrin (1). Infection continues only if the pathogen efficiently extracts and assimilates iron from these relatively small and guarded sources.Despite evidence that some siderophores modulate bacterial virulence in mammalian infections, the role of siderophores in plant pathogenesis is largely unexplored (36). Siderophore production nevertheless has been demonstrated for a number of plant pathogenic bacteria including Agrobacterium tumefaciens and Erwinia carotovora, which produce the catechol siderophores agrobactin and 2,3-dihydroxybenzoic acid, respectively (28, 37). Plant pathogenic and saprophytic fluorescent pseudomonads produce pyoverdin siderophores which usually contain a hydroxamate group (12,13,26,28,36,44). The pyoverdins are the yellow-green, water-soluble fluorescent pigments produced in media when iron is limited. Leong and Neilands (27) concluded that agrobactin production was not required by A. tumefaciens for infection and crown gall formation because agrobactin could not be isolated from infected tissues and agrobactin auxotrophs were not impaired in their ability t...

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Deciduous fruit tree orchards located in the Pacific Northwest were surveyed over a 3-year period for the presence of ice nucleation-active (INA) bacteria. In the Yakima Valley, only about 30% of the fruit tree orchards contained INA bacteria (median population ca. 3 x 102 CFU/g [fresh weight]) in contrast to nearly 75% of the orchards in the Hood River Valley (median population ca. 5 x 103 CFU/g [fresh weight]). These INA populations ranged from less than 10 to over 106 CFU/g (fresh weight) of blossoms and, in Hood River Valley orchards, generally comprised over 10% of the total bacterial population. Populations of INA bacteria fluctuated during the year with highest levels developing on buds and flowers during the cool, wet spring, followed by a drop in populations during the warmer, drier, summer months and finally a gradual increase in the autumn. The INA bacteria persisted on dormant buds from which they again colonized young developing vegetative tissues. All INA bacteria were identified as Pseudomonas syringae. The frequency of ice nucleation at-5°C for these strains ranged from nearly every cell being INA to less than 1 in 107 cells. The median frequency of ice nucleation at-5°C was 104 cells per ice nucleus. The INA P. syringae strains from individual orchards were diverse with respect to bacteriocin typing and in ice nucleation frequency. The consistent absence of detectable INA bacteria or presence of low populations in most of the orchards surveyed during periods when critical temperatures (i.e.,-2 to-5°C) were common indicated a limited role for INA bacteria in frost susceptibility of most Pacific Northwest orchards.

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Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdinp.s, in large quantities under iron-limited growth conditions. Maximum yields of pyoverdinp., of approximately 50 ,ug/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of .10 ,uM. Pyoverdinp. was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdinpss and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdinp6s was structurally unique since amino acid analysis of reductive hydrolysates yielded ,3-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdinp., exhibited a relatively high affinity constant for Fe(III), with values of 1025 at pH 7.0 and 1032 at pH 10.0. Iron uptake assays with [55Fe]pyoverdin. demonstrated rapid active uptake of 55Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdinpss. The chemical and biological properties of pyoverdinp6s are discussed in relation to virulence and iron uptake during plant pathogenesis.