Generation of Azotobacter vinelandii strains defective in siderophore production and characterization of a strain unable to produce known siderophores

Summary: Siderophore-negative mutants of Azotobacter vinelandii were generated by insertional mutagenesis with a Tn5 construct containing a promoterless luxAB fusion. The use of this construct, delivered on a suicide plasmid by conjugation, allowed the selection of mutations in iron-repressible genes by virtue of the expression of iron-regulated bioluminescence. Although many iron-regulated mutants were selected, only a few could be easily identified as defective in siderophore production. These included a non-fluorescent azotobactin-negative phenotype (strain D27), and strain F196, which had lost the ability to produce the catechol siderophores azotochelin and aminochelin as well as the lower-affinity chelator 2,3-dihydroxybenzoic acid. Strain D27 had normal production of catechol siderophores, while strain F196 produced 2·5 times as much azotobactin as the wild-type. Two other mutants demonstrated normal catechol levels and either low or relatively unrepressed azotobactin levels. Transformation of the DNA from strain F196 into another spontaneously obtained azotobactin-negative strain (UA1) resulted in strain P100, which was unable to produce the known siderophores. Unlike the wild-type and other siderophore-deficient mutants, this strain was unable to grow in the presence of the iron chelator ethylenediamine di-(o-hydroxyphenylacetic acid) (EDDHA; 50 mUg ml-1) unless stored iron was carried over in the inoculum. Strain P100 did grow on iron-limited medium containing EDDHA when the catechol or azotobactin siderophores were provided exogenously. However, strain P100 gave a positive result in the chrome azurol-S assay (CAS), a non-specific assay for siderophores. The CAS activity was iron-repressible and strain P100 was able to grow and accumulate more iron from the insoluble iron minerals FeS, vivianite and Fe3O4 than was available by simple diffusion or exchange. Therefore, it appears that iron-limited A. vinelandii produces an as yet unidentified low-affinity non-conventional (non-catechol, non-hydroxamate) siderophore.