Three genes were shown to provide functions specific for ferric enterobactin transport in Escherichia coli:fepA encoded the outer membrane receptor, fepB produced a periplasmic protein, and the fepC product was presumably a component of a cytoplasmic membrane permease system for this siderophore. A 10.6-kilobasepair E. coli chromosomal EcoRI restriction fragment containing the fepB and fepC genes was isolated from a genomic library constructed in the vector pBR328. Both cistrons were localized on this clone (pITS24) by subcloning and deletion and insertion mutagenesis to positions that were separated by approximately 2.5 kilobases. Within this region, insertion mutations defining an additional ferric enterobactin transport gene (fepD) were isolated, and polarity effects from insertions intofepB suggested thatfepD is encoded downstream on the same transcript. A 31,500-dalton FepC protein and a family of FepB polypeptides ranging from 34,000 to 37,000 daltons were identified in E. coli minicells, but the product offepD was not detectable by this system. Another insertion mutation between entF and fepC was also shown to disrupt iron transport via enterobactin and thus defined thefepE locus;fepE weakly expressed a 43,000-dalton protein in minicells. It is proposed that these newly identified genes, fepD and fepE, provide functions which act in conjunction with thefepC product to form the ferric enterobactin-specffic cytoplasmic membrane permease. An additional 44,000-dalton protein was identified and shown to be expressed from a gene that is situated between fepB and entE and that is transcribed in the direction opposite that offepB. Although the function of this protein is uncharacterized, its membrane location suggests that it too may function in iron transport.

The Escherichia coli gene cluster encoding enzymatic activities responsible for the synthesis and activation of 2,3-dihydroxybenzoic acid in the formation of the catechol siderophore enterobactin was localized to a 4.

The nucleotide sequence of a 2,137-base-pair DNA fragment expressing enterobactin biosynthesis functions defined the molecular boundaries and translational products of the entB and entA genes and identified a closely linked downstream open reading frame encoding an uncharacterized protein of approximately 15,000 daltons (P15). The sequence revealed that an independent protein-coding sequence corresponding to an EntG polypeptide was not situated in the genetic region between the entB and entA cistrons, to which the EntGphenotype had been genetically localized. As a result, the biochemical nature of the EntG function in the biosynthetic pathway requires reevaluation. The EntA polypeptide displayed significant similarities at the amino acid level to the pyridine nucleotide-binding domains of several members of a family of alcoholpolyol-sugar dehydrogenase enzymes, consistent with its function as the enzyme catalyzing the final step of dihydroxybenzoate biosynthesis. An additional role for EntA in the isochorismate synthetase activity of EntC was strongly implicated by genetic evidence. Evidence from the nucleotide sequence of this region and newly constructed ent-lacZ fusion plasmids argues strongly that these genes are linked in an iron-regulated enICEBA (P15) polycistronic operon.

The vector Mu d(Apr lac) was utilized to construct operon fusions in the Escherichia coli enterobactin (ent) biosynthetic and transport genes. Enzyme assays indicated a 5to 15-fold increase in the expression of 0-galactosidase when the fusion strains were grown under iron-deficient conditions. The polarity effects seen by Mu d insertions into entA, entC, and entE were consistent with a single operon, entA(CGB)E. The direction of transcription from iron-regulated promoters was determined by directional transfer of selected genetic markers after the insertion of F'tsll4 lac+. Regulatory mutants were isolated in the fusion strains by the selection for constitutive expression of ,B-galactosidase and the ironregulated outer membrane proteins.on July 16, 2020 by guest