Nucleotide sequence and transcriptional organization of the Escherichia coli enterobactin biosynthesis cistrons entB and entA.

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 EntG- phonotype 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 alcohol-polyol-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 entCEBA (P15) polycistronic operon.

Cluster of genes controlling synthesis and activation of 2,3-dihydroxybenzoic acid in production of enterobactin in Escherichia coli.

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 4.2-kilobase chromosomal DNA fragment. Analysis of various subclones and transposon insertion mutations confirmed the previously suggested gene order as entEBG(AC) and provided evidence to suggest that these genes are organized as three independent transcriptional units, composed of entE, entBG, and entAC, with the entBG mRNA transcribed in a clockwise direction. Plasmid-specific protein expression in E. coli minicells identified EntE and EntB as 58,000- and 32,500-dalton proteins, respectively, while no protein corresponding to EntG was detected. The EntA and EntC enzymatic activities could not be separated by genetic or molecular studies. A small DNA fragment encoding both activities expressed a single 26,000-dalton polypeptide, suggesting that this protein is a multifunctional enzyme catalyzing two nonsequential reactions in the biosynthetic pathway. A protein of approximately 15,000 daltons appears to be encoded by the chromosomal region adjacent to the entAC gene, but no known function in enterobactin biosynthesis or transport can yet be ascribed to this polypeptide.

Genetic organization of multiple fep genes encoding ferric enterobactin transport functions in Escherichia coli.

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-kilobase-pair 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 into fepB suggested that fepD 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 of fepD 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 the fepE 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 the fepC product to form the ferric enterobactin-specific 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 of fepB. Although the function of this protein is uncharacterized, its membrane location suggests that it too may function in iron transport.

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12.

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.

Regulation of enterobactin iron transport in Escherichia coli: characterization of ent::Mu d(Apr lac) operon fusions.

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 5- to 15-fold increase in the expression of beta-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'ts114 lac+. Regulatory mutants were isolated in the fusion strains by the selection for constitutive expression of beta-galactosidase and the iron-regulated outer membrane proteins.