RESUMEN
The csbX gene of Azotobacter vinelandii was regulated in an iron-repressible manner from a divergent promoter upstream of the catecholate siderophore biosynthesis (csb) operon and was predicted to encode an efflux pump of the major facilitator superfamily. Other proteins that were most similar to CsbX were encoded by genes found in the catecholate siderophore biosynthesis operons of Aeromonas hydrophila and Stigmatella aurantiaca. Inactivation of csbX resulted in 57-100% decrease in the amount of catecholates released when compared to the wild-type in iron-limited medium. CsbX was most important for the export of the high affinity chelator protochelin with the majority of the catecholates released by csbX mutants being the protochelin intermediates azotochelin and aminochelin.
Asunto(s)
Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Catecoles/metabolismo , Genes Bacterianos , Sideróforos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Transporte Biológico , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Orden Génico , Hierro/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Mutagénesis Insercional , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Azotobacter vinelandii strain UA22 was produced by pTn5luxAB mutagenesis, such that the promoterless luxAB genes were transcribed in an iron-repressible manner. Tn5luxAB was localized to a fragment of chromosomal DNA encoding the thrS, infC, rpmI, rplT, pheS and pheT genes, with Tn5 inserted in the 3'-end of pheS. The isolation of this mutation in an essential gene was possible because of polyploidy in Azotobacter, such that strain UA22 carried both wild-type and mutant alleles of pheS. Phenylalanyl-tRNA synthetase activity and PHES::luxAB reporter activity was partially repressed under iron-sufficient conditions and fully derepressed under iron-limited conditions. The ferric uptake regulator (Fur) bound to a DNA sequence immediately upstream of luxAB, within the pheS gene, but PHES::luxAB reporter activity was not affected by phenylalanine availability. This suggests there is novel regulation of pheST in A. vinelandii by iron availability.
Asunto(s)
Azotobacter vinelandii/enzimología , Hierro/farmacología , Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/metabolismo , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Heterocigoto , Luciferasas/genética , Datos de Secuencia Molecular , Mutación , Fenilalanina/farmacología , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Azotobacter vinelandii forms both catecholate and azotobactin siderophores during iron-limited growth. Azotobactin is repressed by about 3 microM iron, but catecholate siderophore synthesis continues up to a maximum of 10 microM iron. This suggests that catecholate siderophore synthesis is regulated by other factors in addition to the ferric uptake repressor (Fur). In this study the first gene required for catecholate siderophore biosynthesis, which encodes an isochorismate synthase (csbC), was isolated. The region upstream of csbC contained a typical sigma(70) promoter, with an iron-box overlapping the -35 sequence and a Sox-box (Box 1) overlapping the -10 sequence. Another Sox-box was found further upstream of the -35 sequence (Box 2). Also upstream, an unidentified gene (orfA) was detected which would be transcribed from a divergent promoter, also controlled by an iron-box. The activity of csbC and a csbC::luxAB fusion was negatively regulated by iron availability and upregulated by increased aeration and by superoxide stress. The iron-box in the csbC promoter was 74% identical to the Fur-binding consensus sequence and bound the Fur protein of Escherichia coli with relatively high affinity. Both Box 1 and Box 2 were in good agreement with the consensus sequence for binding the SoxS protein of E. coli and Box 1 was in very good agreement with the Sox-box found in the fpr promoter of A. vinelandii, which is also regulated by superoxide stress. Both Sox-boxes bound a protein found in A. vinelandii cell extracts, with Box 1 exhibiting the higher binding affinity. The Sox protein identified in this assay appeared to be constitutive, rather than inducible by superoxide stress. This indicates that the Sox response in A. vinelandii is different from that in E. coli. These data support the hypothesis that catecholate siderophore biosynthesis is under dual control, repressed by a Fur-iron complex and activated by another DNA-binding protein in response to superoxide stress. The interaction between these regulators is likely to account for the delay in ferric repression of catecholate siderophore production, since these siderophores have an additional role to play in the protection of iron-limited cells against oxidative damage.