The Siderophores

Soft rot spreading depends on the efficiency of the iron uptake pathway mediated by the siderophore chrysobactin. Biosynthesis of the ferrichrysobactin outer membrane receptor (Fct) and of the chrysobactin precursor, i.e. the activated form of 2,3-dihydroxybenzoic acid, are encoded by an operon,/cr ebsCEBA [3]. 

The mutant L37 cbrA21 is affected as regards to its iron uptake pathway mediated by the siderophore achromobactin. Because this mutation results in derepression of the chrysobactin mediated iron transport pathway, the mutant is probably less susceptible to iron deprivation than wild-type cells are, when entering the host. This results in a delay in Pels production thus leading to delayed symptoms, as reported by Sauvage and Expert (1994). 

The use of microbial siderophores by dicotyledonous plants appears to involve uptake of the entire metallated chelate (42-44), or an indirect process in which the siderophore undergoes degradation to release iron (45). As demonstrated in initial studies examining this question, there was concern that iron uptake from microbial siderophores may be an artifact of microbial iron uptake in which radiolabeled iron is accumulated by root-colonizing microorganisms (46). Consequently, evidence for direct uptake of iron from microbial siderophores has required the use of axenic plants. In experiments with cucumber, it was shown that the microbial siderophore ferrioxamine B could be used as an iron source at concentrations as low as 5 pM and that the siderophore itself entered the plant (42). 

The po.ssible role of a chelate reductase for iron uptake from microbial siderophores has been examined for several plant species (30,47). With certain microbial siderophores such as rhizoferrin and rhodotorulic acid, the reductase may easily cleave iron from the siderophore to allow subsequent uptake by the ferrous iron transporter. However, with the hydroxamate siderophore, ferrioxamine B, which is produced by actinomycetes and u.sed by diverse bacteria and fungi, it has been shown that the iron stress-regulated reductase is not capable... 

Iron uptake by bacteria at sites of lateral root emergence has been further confirmed using another technique employing 7-nitrobenz-2-oxa-l,3-diazole-desferrioxamine B, which is a derivitized siderophore that becomes fluorescent after it is deferrated (78). In this case, iron uptake from the siderophore ferrox-amine B was a.ssociated primarily with microbially colonized roots, but both plant and iron uptake from this chelate occurred in the regions just behind the root tips. 

Figure 3 Root fingerprints of Pseudomimets sp. associated with barley seedlings showing the production of siderophore by actively growing bacteria located in the zone of elongation behind the root tips. Root.s were pressed on to an iron-deficient minimal medium selective for Pseudomonas. After growth of the colonies, the production of siderophore was visualized by exposure of the agar plate to ultraviolet light, which causes the siderophore to Huoresce.

Rhizobium have been reported, but to date there is little evidence that siderophores produced by pseudomonads are beneficial for promoting nodulation and nitrogen fixation. In experiments examining the role of siderophore production on nodulated clover plants, siderophore-defective mutants were shown to stimulate growth of nodulated clover plants similarly to the siderophore-producing parent strain (119). 

J. S. Buyer, M. G. Kratzke, L. J. Sikora, A method for detection of pseudobactin the siderophore produced by a plant-growth-promoting Pseudomonas strain in the barley rhizosphere. Appl. Environ. Microbiol. 59 611 (1993). 

H. A. Akers, Isolation of the siderophore schizokinen from. soil of rice field. Appl. Environ. Microbiol. 45 1704 (1983). 

While much is known about siderophore-mediated ferric-iron transport, very little is known about ferrous-iron transport and iron metabolism inside the cell. It is generally assumed that Fe3+ chelated to the siderophore must be reduced to allow removal from the strong claws of the chelator. Indeed, in some cases the siderophore transported iron was found 30 minutes later in the intracellular Fe2+ pool of the cells (Matzanke et ah, 1991). 

The ent-fes-fep gene cluster is necessary for the synthesis of enterobactin and transport of the iron loaded siderophore. The fes gene product was shown to be necessary for utilization of the siderophore-bound iron inside the cell. The protein has an esterase activity which cleaves the ester bonds of the cyclic 2,3-dihydroxybenzoylserine ester in enterobactin. However, the esterase activity of Fes does not seem to be important for iron mobilization since Fes is also necessary for the utilization of iron from enterobactin analogues which do not have ester bonds (Heidinger et ah, 1983). No reductase activity has been found in Fes (Brickman and McIntosh, 1992) or in any other protein encoded in the ent-fes-fep gene cluster. 

In some cases where the siderophore architecture permits, the tetradentate siderophore will form a dimeric Fe2L2 complex with iron instead of the monomeric FeL form. These dimeric complexes are not discernible from their monomer counterparts... 

Electrochemical experiments allow the determination of complex stability constants for Fe2+ by measuring complex redox potentials over a range of pH values. The Fe34YFe2+ redox potential of the siderophore complex, as with the spectral characteristics of the complex, is dependent on the inner coordination environment of the iron. These considerations will be addressed later (Section III.D). 

One aspect of microbial iron metabolism that remains unclear in many cases is the mechanism for iron release from tight sequestration once the siderophore complex arrives at its... 

Another factor that will affect the complex redox potential is the architecture of the siderophore. A plot of ferri-siderophore redox potentials as a function of pFe for a series of hydroxamate complexes of differing denticity (shown in Fig. 19) exhibits a trend. The trend demonstrates that hydroxamate siderophores of higher denticity will form complexes with more negative E1/2 values than analogous siderophores of lower denticity. 

Another factor that can possibly affect the redox potential in biological systems is the presence of secondary chelating agents that can participate in coupled equilibria (3). When other chelators are present, coupled equilibria involving iron-siderophore redox occur and a secondary ligand will cause the siderophore complex effective redox potential to shift. The decrease in stability of the iron-siderophore complex upon reduction results in a more facile release of the iron. Upon release, the iron(II) is available for complexation by the secondary ligand, which results in a corresponding shift in the redox equilibrium toward production of iron(II). In cases where iron(II) is stabilized by the secondary chelators, there is a shift in the redox potential to more positive values, as shown in Eqs. (42)—(45). 

In Eq. (45), KFe(II)L is the stability constant for iron(II) complexation by the competing ligand, KFe(II)sid the stability constant for the complex formed between iron(II) and the siderophore, n the number of electrons transferred, Erxn the observed redox potential for the iron(III)-siderophore system coupled with iron(II) chelation, and EFJ m sld the redox potential of the iron(III)-siderophore complex. 

Another possible route for reduction of the iron center is photoreduction. This has been studied in a variety of marine siderophore systems, such as aquachelin, marinobactin, and aerobactin (2), where it was demonstrated that photolytic reduction was due to a ligand-to-metal charge transfer band of the Fe(III)-siderophore complex, eventually resulting in reduction ofiron(III) and cleavage of the siderophore (31,154,155). This suggests a possible role for iron reduction in iron release (71,155). 

While studying the formation kinetics of complexes gives useful mechanistic information about the reactivity of the iron center when bound to a particular siderophore, it is not necessarily a good model for how environmental iron will react in the siderophore system of interest. In biological systems,... 

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