Domenic Castignetti

Siderophores, the iron nutrition of plants, and nitrate reductase

Iron is ubiquitous in terrestrial environments [l]. It is, however, very sparingly soluble under aerobic conditions as ferric hydroxides are formed and the solubility product approaches 10T3’ [2]. Microorganisms have solved the problem of iron acquisition under aerobiosis by synthesizing siderophores, i.e. high-affinity Fe3+-chelating compounds with formation constants ranging from approx. 10” to 10” [3]. Siderophores are organic compounds comprised of either hydroxamates or catecholates (fig. 1) [1,3,4]. A new category of siderophores, termed the ‘miscellaneous’ class, has been recently recognized [5] and this group differs in chemical structure from the previous classes of siderophores, most likely containing imino or amino carboxylic acids. The reader is referred to reviews [l-7] for a more detailed discussion of siderophoie chemistry and microbial iron nutrition. The competition for Fe3+ results in the excretion of siderophores from microbes into the environment. Powell et al. [S] noted minimal siderophore concentrations of 3 nM desferrioxamine methanesulfonate equivalents (the hydroxamate siderophore used as a standard) in each of 57 soils examined. Siderophores were thus noted in grassland, coniferous and deciduous forest, and mixed herbaceous vegetation soils from across the United States. In 19 soils, concentrations of siderophores varied from 2.7 to 34 nM desferrioxamine methanesulfonate equivalents with a mean concentration of 12 nM. A 1: 1 (soil-water) extract of a sandy clay loam [9] contained 78 nM siderophores. Similar results were noted from soils in Texas [lo] and the hydroxamate siderophore schizokinen [I l] was recovered and identified from a rice field soil. Hydroxamate siderophores are not only present in but are also capable of chelating and mobilizing iron from soil as desferrioxamine B, desferrichrome [12] and (deferri-)rhodotorulic acid [ 131 removed iron from acid and alkaline soils [12] as well as silicate rocks and Mt. St. Helens’ ash [ 131. An interesting question raised by these studies is that of iron acquisition by plants; how do plants acquire the iron they require when the soils in which they are growing contain numerous microbes and microbial siderophores?

Purification of Legiobactin and Importance of This Siderophore in Lung Infection by Legionella pneumophila

ABSTRACT 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.

Degradation Pathway and Generation of Monohydroxamic Acids from the Trihydroxamate Siderophore Deferrioxamine B

ABSTRACT 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.

Probing of Pseudomonas aeruginosa, Pseudomonas aureofaciens, Burkholderia (Pseudomonas) cepacia, Pseudomonas fluorescens, and Pseudomonas putida with the Ferripyochelin Receptor A Gene and the Synthesis of Pyochelin in Pseudomonas aureofaciens, Pseudomonas fluorescens, and Pseudomonas putida

Abstract. The ferripyochelin receptor A (fptA) gene codes for the transport of the ferrisiderophore ferripyochelin in Pseudomonas aeruginosa. A P. aeruginosa fptA internal fragment was used to probe chromosomal DNA from P. aureofaciens, B. cepacia, P. fluorescens, P. putida, and five strains of P. aeruginosa. These bacteria all contained DNA that hybridized to the fptA fragment. Four of the five P. aeruginosa strains displayed marked and identical patterns, indicating a high degree of sequence similarities among these strains. DNA from the non-P. aeruginosa bacteria, in contrast, hybridized less to the fptA fragment. Pseudomonas aeruginosa and B. cepacia synthesize pyochelin. Experiments were performed to confirm P. fluorescens pyochelin synthesis and to determine if pyochelin, cepabactin or salicylic acid were made by P. aureofaciens, P. putida, and P. fluorescens. Only pyochelin was isolated and identified from P. fluorescens. P. aureofaciens and P. putida produced none of these compounds. While all of these bacteria contain chromosomal DNA that hybridized to the fptA fragment probe, pyochelin synthesis did not occur in all, indicating that fptA fragment hybridization cannot always be correlated with pyochelin biosynthesis.

Siderophore reduction catalyzed by higher plant NADH: Nitrate reductase

Squash cotyledon NADH:nitrate reductase catalyzes the reduction of the siderophore ferrioxamine B. The enzyme also reduced ferric ion in a buffer system containing the chelators oxalate and maleate. Ferrioxamine B reduction was maximal at pH 4; ferric ion reduction was maximal at pH 8. The present study indicates that iron assimilation by higher plants may occur with microbial siderophores serving as ferric ion sources and nitrate reductase functioning as the siderophore reductase.

The catabolism and heterotrophic nitrification of the siderophore deferrioxamine B

SummaryThree 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 nitrifyingAlcaligenes 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, sereved 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.

Effect of Eastern Oysters (Crassostrea virginica) and Seasonality on Nitrite Reductase Gene Abundance (nirS, nirK, nrfA) in an Urban Estuary

The influence of oysters on nitrogen (N) cycling has received increased research attention. Previous work focused on fluxes of N solutes and gases, but the effects on microbes responsible for N transformations are unknown. In May 2010, we deployed eastern oysters (Crassostrea virginica) in mesh cages above sand-filled boxes at four sites across a nutrient gradient in Jamaica Bay, New York City. In fall and winter, we used quantitative PCR to measure abundance of 16S rRNA and nitrite reductase genes for denitrification (nirS and nirK) and dissimilatory nitrate reduction to ammonium (nrfA) in sediment. We measured water column nutrients and chlorophyll a, sediment C:N and organic matter (OM), exchangeable ammonium (NH4+), ammonification, nitrification, and denitrification potential (DNP). Oysters did not affect gene abundance in fall, when we predicted that their influence would be strongest, or in winter. However, gene abundance was significantly different among sites and seasons. Factors which explained 16S rRNA, nirS, and nirK gene abundance included sediment OM, water column N, and chlorophyll a, similar to previous research. Abundance of nrfA was lower than that of nir genes and positively related to sediment C:N, suggesting OM lability may drive the balance between nir and nrfA. Finally, nirS and nirK abundance was unrelated to DNP, which is consistent with variable results from the literature. More studies that combine molecular techniques with N transformation rates in the context of oyster reefs are needed. Results will advance models which predict the ecosystem effects of reef conservation and restoration under variable environmental conditions.

Pathway of oxidation of pyruvic oxime by a heterotrophic nitrifier of the genus Alcaligenes: evidence against hydrolysis to pyruvate and hydroxylamine

A heterotrophic nitrifier of the genus Alcaligenes, which grows vigorously on pyruvic oxime, was tested by several methods for possible differences or similarities in metabolic performance between pyruvic oxime and its hydrolysis products, pyruvate and hydroxylamine. Major differences were observed between pyruvic oxime and one or both of the other reductants with regard to growth yield, rates of reductant uptake, rates of oxygen uptake, sensitivity of their oxidations to inhibition by thiocyanate, and performance in reductant pulse experiments. Other oximes, some of which are structural analogs of pyruvic oxime and all of which are potential sources of hydroxylamine, were not metabolized by cells or cell-free extract. Collectively the results indicate a pathway of oxidation of pyruvic oxime to nitrite and CO2 that does not involve its initial hydrolysis, but probably involves the oxidation of N and/or C before C-N bond breakage.

Iron acquisition by plants: the reduction of ferrisiderophores by higher plant NADH: Nitrate reductase

Abstract Siderophores are avid Fe 3+ -chelators of microbial origin. Plant roots are colonized by fungi and bacteria which synthesize siderophores, and plants have been shown to metabolize these substances to obtain iron. We have previously shown that nitrate reductase from squash catalyzed the reduction of the ferrisiderophore ferrioxamine B with the subsequent loss of Fe 2+ . Using a spectrophotometric assay which traps Fe 2+ in a ferrozine complex, we have noted that the substrate diversity of nitrate reductase as a ferrisiderophore reductase includes ferrichrome A, ferrichrome, ferrirhodotorulic acid, ferrischizokinen, and the novel siderophore ferri-‘AAHS’. These reductions were inhibited by polyclonal antibodies against nitrate reductase, but ferrisiderophore reductase activity, as evidenced with ferrirhodotorulic acid, was unaffected by low concentrations of azide. In addition, maximal activity occurred between pH 4 and 5, and appaarent K m values were approx. 100 μmolar. Thus, we suggest that plant nitrate reductases might be involved in iron assimilation as well as nitrate reduction.

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N2Hinf5sup+) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.