When cultured in a low-iron medium, Legionella pneumophila secretes a siderophore (legiobactin) that is both reactive in the chrome azurol S (CAS) assay and capable of stimulating the growth of iron-starved legionellae. Using anion-exchange high-pressure liquid chromatography (HPLC), we purified legiobactin from culture supernatants of a virulent strain of L. pneumophila. In the process, we detected the ferrated form of legiobactin as well as other CAS-reactive substances. Purified legiobactin had a yellow-gold color and absorbed primarily from 220 nm and below. In accordance, nuclear magnetic resonance spectroscopy revealed that legiobactin lacks aromatic carbons, and among the 13 aliphatics present, there were 3 carbonyls. When examined by HPLC, supernatants from L. pneumophila mutants inactivated for lbtA and lbtB completely lacked legiobactin, indicating that the LbtA and LbtB proteins are absolutely required for siderophore activity. Independently derived lbtA mutants, but not a complemented derivative, displayed a reduced ability to infect the lungs of A/J mice after intratracheal inoculation, indicating that legiobactin is required for optimal intrapulmonary survival by L. pneumophila. This defect, however, was not evident when the lbtA mutant and its parental strain were coinoculated into the lung, indicating that legiobactin secreted by the wild type can promote growth of the mutant in trans. Legiobactin mutants grew normally in murine lung macrophages and alveolar epithelial cells, suggesting that legiobactin promotes something other than intracellular infection of resident lung cells. Overall, these data represent the first documentation of a role for siderophore expression in the virulence of L. pneumophila.

Twelve denitrifying bacteria representing six genera were tested for an ability to nitrify pyruvic oxime heterotrophically. Six of these bacteria exhibited appreciable nitrification activity, yielding as much as 5.8 mM nitrite and little or no nitrate when grown in a mineral salts medium containing 7 mM pyruvic oxime and 0.05% yeast extract. Of the six active bacteria, four (Pseudomonas denitrificans, Pseudomonas aeruginosa, and two strains of Pseudomonas fluorescens) could grow on yeast extract but not pyruvic oxime, one (Pseudomonas aureofaciens) could grow slowly on pyruvic oxime, and one (Alcaligenes faecalis) could apparently grow on pyruvic oxime in the presence of yeast extract but not in its absence. Eight of the twelve bacteria in the resting state could oxidize hydroxylamine to nitrite, and P. aiureofaciens was remarkably active in this regard. In general, those denitrifiers active in the nitrification of pyruvic oxime or hydroxylamine or both are abundant in soils. A possible advantage of having nitrification and denitrification capabilities in the same organism is discussed.

Sputum samples from the lungs of cystic fibrosis patients harboring Pseudomonas aeruginosa infections were coUlected and examined for the presence of the siderophore pyoverdine. Fluorescence quenching, due to the addition of ferric ion, as wel as column and thin-layer chromatography results indicated that all samples contained the siderophore. Six samples furnished sufficient material after purification to allow us to obtain visible absorbance spectra. These spectra were characteristic of the ferrated analog of the P. aeruginosa pyoverdine, that is, ferripyoverdine, and in all cases they indicated a degree of ferration in excess of 50%. P. aeruginosa in the cystic fibrosis lung is thus iron stressed and responds by synthesizing pyoverdine, which subsequently binds ferric ion.

Three bacteria, two of which were previously noted as active heterotrophic nitrifiers, were examined for their ability to grow and nitrify with the siderophore deferrioxamine B as the carbon source. Pseudomonas aureofaciens displayed limited growth and nitrification while a heterotrophic nitrifying Alcaligenes sp. was without action concerning its metabolism of deferrioxamine B. The third bacterium, a unique Gram-negative soil isolate, was unable to nitrify deferrioxamine B but grew well when the siderophore was employed as the sole C source. The Gram-negative bacterium removed deferrioxamine B from the medium and left only residual amounts of the compound in solution at the termination of its growth. The organism was without action when the ferrated analogue of deferrioxamine B, ferrioxamine B, served as either the C source for growth, for metabolism by resting cells, or as the substrate for cell-free extracts. Deferrioxamine B, by contrast, was rapidly metabolized by resting cells. Cell-free extracts of the bacterium synthesized a monohydroxamate(s) when deferrioxamine B was the substrate. The catabolism of deferrioxamine B, which is synthesized by soil microbes, suggests that soil microflora have the ability to return deferrioxamine B, and perhaps other, siderophores to the C cycle.

The ecological importance of heterotrophic nitrification has been difficult to assess because of low levels of nitrification associated with this phenomenon. Nitrification by a soil isolate, an Alcaligenes sp., which oxidizes pyruvic oxime to produce up to 1867 mg nitrite-nitrogen/L, is described. Sequential nitrification with the chemoautotroph Nitrobacter agilis, ATCC 14123, resulted in nitrate accumulation and a concomitant decrease of the nitrite produced by the Alcaligenes sp. when the bacteria were jointly cultured. The ecological significance of such a sequential system is discussed.

Siderophores are avid ferric ion-chelating molecules that sequester the metal for microbes. Microbes elicit siderophores in numerous and different environments, but the means by which these molecules reenter the carbon and nitrogen cycles is poorly understood. The metabolism of the trihydroxamic acid siderophore deferrioxamine B by a Mesorhizobium loti isolated from soil was investigated. Specifically, the pathway by which the compound is cleaved into its constituent monohydroxamates was examined. High-performance liquid chromatography and mass-spectroscopy analyses demonstrated that M. loti enzyme preparations degraded deferrioxamine B, yielding a mass-to-charge (m/z) 361 dihydroxamic acid intermediate and an m/z 219 monohydroxamate. The dihydroxamic acid was further degraded to yield a second molecule of the m/z 219 monohydroxamate as well as an m/z 161 monohydroxamate. These studies indicate that the dissimilation of deferrioxamine B by M. loti proceeds by a specific, achiral degradation and likely represents the reversal by which hydroxamate siderophores are thought to be synthesized.Elemental iron (Fe) is required to synthesize a number of key enzymes and biomolecules (1,8,14,(24)(25)(26)). Iron's biological availability, however, is restricted in those environments where oxygen gas is present and the prevailing pH is near neutrality or is alkaline. As the solution to the problem of acquiring sufficient amounts of Fe, numerous microbes employ siderophores, that is, avid, organic, microbial ferric ion chelators which sequester iron from environments where it is in short supply. Siderophores are thus essential to the nutrition of microbes existing in environments that would otherwise limit their growth (1,8,14,24,25). Such environments include fresh and marine waters, divergent soils, and living organisms (8,10,14,16,(27)(28)(29). In these ecosystems, micromolar concentrations of siderophores have been noted (16,27,28).Siderophores are virulence factors for both animal and plant pathogens (2, 3, 9-11, 18, 23). Indeed, the sequestration of iron by the host is an innate immune mechanism that may limit the course of pathogenic infections (8,14). Much research has been conducted to investigate the biosynthesis, iron chelation, iron assimilation, and genetics which allow microbes to acquire iron via siderophores. Far less attention, however, has been given to the study of how siderophores are mineralized and returned to the carbon and nitrogen cycles.Three siderophore-degrading microbes, a pseudomonad (33-35), Azospirillum irakense (36, 37), and Mesorhizobium loti (4,7,17,39), catabolize siderophores concomitant with their growth. Neilands and colleagues (33-35) observed that their microbe, named Pseudomonas FC1, degraded the hydroxamate siderophores ferrichrome, ferrichrome A, and coprogen, with ferrichrome A being the most facile to degrade. Pseudomonas FC1 siderophore degradation was due to inducible enzymes and could occur with either the deferrisiderophore or the ferrisiderophore. The enzymes required to degr...

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