Salmochelin, a new siderophore of E. coli: biosynthesis, transport and utilization [Taschenbuch]

The siderophore salmochelin is produced under iron-poor conditions by Salmonella enterica and many uropathogenic Escherichia coli strains. The production of salmochelin, a C-glucosylated enterobactin, is dependent on the iroBCDEN gene cluster. IroB transfers 2 glucose residues to enterobactin to form cyclic salmochelin S4. These two glucose residues make the molecule more hydrophilic and may be important in the pathogenicity of S. enterica. IroC showed similarity to eukaryotic multidrug resistance proteins (MDRs). IroD was predicted to be a Fes-like esterase located in the cytoplasm; and IroE was predicted to be a hydrolase located in the periplasm. IroN was shown to be a catecholate siderophore receptor in the outer membrane. In this work, the cloned IroD protein was purified from inclusion bodies with an N-terminal His-tag. NHis-IroD cleaved salmochelin S4 into the linear trimer salmochelin S2, the dimer salmochelin S1, and the monomer dihydroxybenzoylserine and C-glucosylated dihydroxybenzoylserine (salmochelin SX; pacifarinic acid). IroD did not split ferric-salmochelin S4 unless Fe2+-complexing agents such as ferrozine were provided. The iroD gene complemented a fes mutation in E. coli since IroD could also split enterobactin into (DHBS)3 trimer, (DHBS)2 dimer, and DHBS monomer.

The hydrolase IroE was cloned and purified as MalE-IroE fusion protein. In vitro, MalE-IroE degraded salmochelin S4 and ferric salmochelin S4 to the linear trimer salmochelin S2 and ferric-salmochelin S2, respectively. No further degradation to the dimeric salmochelin S1 or the monomeric salmochelin SX was observd. MalE-IroE did not split gallium-salmochelin S4. This indicated that beside the ester bond, other factors are recognized by the enzyme. MalE-IroE cleaved enterobactin to the linear (DHBS)3 trimer.

Uptake of ferric-salmochelin S4 was dependent on the cleavage by IroE and independent of the FepBCDG transport system in the cytoplasmic membrane. IroE did not function as a binding protein since salmochelin S2 was taken up in the absence of a functional IroE protein. IroC was necessary for the uptake of salmochelin S2 from the periplasm into the cytoplasm. IroC mediated uptake of iron-enterobactin in a fepB mutant. The spectrum of salmochelins produced by the fepB mutant, complemented with different mutated iro plasmids confirmed that IroC transports linear salmochelins.

The iroE homolog pfeE of Pseudomonas aeruginosa was cloned. The MalE-PfeE fusion protein showed an enzymatic activity very similar to that of IroE. It is suggested that homologs in other bacteria are also periplasmic IroE-type siderophore esterases.

In a transport assay, it was confirmed that IroN is the major receptor of salmochelin S4 since its deletion reduced the uptake of S4.

To study the regulation of the iro gene cluster the iroB and iroN promoter regions were fused with the lac gene. Both promoters were derepressed by low iron, the iroN promoter more strongly than the iroB promoter. However, only a twofold derepression was observed, possibly because the promoter regions were cloned in a high copy number vector. Keywords: salmochelin, enterobactin, siderophore, esterase, iro gene cluster