Essential to the biological activity of many proteins mediating electron transfer and redox reactions, iron is absolutely necessary for most forms of life. However, in aerobic conditions, its bioavailability is severely compromised by its poor solubility. At pH 7, ferric ion is available at 10−17 M, while microorganisms require micromolar concentrations of this metal to multiply (Braun and Killmann, 1999). Iron catalyses the formation of reactive free radicals in the presence of oxygen and can trigger chain reactions that are harmful to macromolecules (Luo et al., 1994). This high reactivity means that iron is generally not present in biological tissues as a free element and not readily accessible to invading microorganisms. A wide variety of microorganisms solubilize iron and control their intracellular iron levels by excreting siderophores. Siderophores are high-affinity Fe(III)-scavenging/solubilizing molecules that, once loaded with iron, are specifically imported into the cell ( Neilands, 1995). In Gram-negative bacteria, production of a siderophore and proteins involved in uptake of its ferric complex is accurately controlled by the sensory and regulatory protein Fur. Fur protein acts as a dimer, each monomer containing a non-haem ferrous iron site (Hantke, 2001). If the cellular iron level becomes too low, the active Fur repressor losses Fe2+, its co-repressor, and is no longer able to bind to its operator sites (Escolar et al., 1999; Gonzalez de Peredo et al., 2001). The production of siderophores by pathogenic bacteria can greatly contribute to their virulence, because these molecules can remove iron from a wide variety of organic substrates (Ratledge and Dover, 2000). The role of siderophores in microbial pathogenesis has been extensively studied in infectious diseases of mammals, which express an array of defences aimed at withholding nutritional iron from the pathogen (Weinberg, 1993; 2000).
Interestingly, the virulence of the plant pathogenic enterobacterium Erwinia chrysanthemi strain 3937 on African violets (Saintpaulia ionantha) depends on the production of the siderophore chrysobactin (Enard et al., 1988; Neema et al., 1993; Masclaux and Expert, 1995). Chrysobactin is a bidentate ligand consisting of a monomer of 2,3-dihydroxybenzoyle-d-lysyl-l-serine (Persmark et al., 1989) and thus is a less powerful ferric ion ligand than hexadentate siderophores like the tris-catecholate enterobactin. Siderophores are compared by calculating their pFe values at a concentration of ligand of 10−5 M and pH of 7.4 (pFe is - log[Fe3+]): a higher pFe value corresponds to lower free Fe3+ concentration and stronger binding of the iron by the siderophore. The pFe values of chysobactin and enterobactin are 17.1 and 35.5 respectively (A.-M. Albrecht-Gary, pers. comm.). Chrysobactin is essential for E. chrysanthemi cells to cause systemic infection (Expert, 1999). The soft rot symptom produced by E. chrysanthemi consists of a progressive disorganization of parenchymatous tissues called maceration and results from several bacterial enzymatic activities, including pectinases and endoglucanases which work in concert to degrade plant cell walls (Pérombelon, 2002). E. chrysanthemi cells colonize intercellular spaces of leaf tissues and progress intercellularly. Deconstruction of the cell walls causes cell lysis and allows bacteria free access to cellular nutrients (Murdoch et al., 1999).
Most of the proteins involved in chrysobactin-mediated iron transport are encoded by a 25 kb contiguous region of the E. chrysanthemi chromosome. The presence of four operons divergently transcribed from two bidirectional promoters is reminiscent of the genetic organization of the enterobactin region in Escherichia coli K12 (Expert et al., 2004). In particular, the fct-cbsCEBA operon encodes the receptor Fct and the enzymes leading to the catechol moiety in chysobactin biosynthesis (Franza and Expert, 1991). The promoter region for this operon is controlled by iron via a direct interaction with the ferric uptake regulatory protein Fur, which is highly similar to the E. coli Fur repressor (Sauvage et al., 1996; Franza et al., 1999; 2002). In addition, the E. chrysanthemi Fur protein negatively controls the transcription of several pectinase encoding genes, according to an anti-activation mechanism. Indeed, the Fur binding sites are located in the close vicinity of the sequences recognized by the cAMP receptor protein CRP that plays a crucial role in expression of the pectinolysis genes (Nasser et al., 1997). Fur could thus inhibit the binding of CRP and the subsequent gene transcription activation (Franza et al., 2002). Fur behaves as a global regulator coupling two functions important for pathogenicity.
However, in response to iron limitation, E. chrysanthemi synthesizes a second siderophore, achomobactin. Achromobactin belongs to a new class of siderophores derived from citrate (Münzinger et al., 2000), based on carboxylate and hydroxy donor groups rather than the commonly encountered hydroxamates and catecholates (Fig. 1). Achromobactin was uncovered on the basis of the phenotype of chrysobactin biosynthetic mutants, which are still able to form a halo of discoloration on Chrome Azurol S (CAS) agar medium, used to detect siderophore production. In a previous work, we isolated mutants accumulating achromobactin in the medium that are derepressed for the production of chrysobactin. The mutations were designated as cbr (for chrysobactin regulation), and were shown unexpectedly to map in an operon, cbrABCD, encoding the ABC permease for ferric achromobactin (Mahéet al., 1995).
In this study, we sequenced a region of 15 kb extending 5′ upstream to the cbrA gene. We uncovered a cluster of eight genes involved in the biosynthesis and transport functions of achromobactin. We reported a functional analysis of this second iron transport pathway and investigated its role in pathogenicity of E. chrysanthemi.