Bacteriophage Mu-mediated gene transposition and in vitro cloning of the enterochelin gene cluster of Escherichia coli

Laird, Alan; Ribbons, Douglas W.; Woodrow, Graeme; Young, Ian G.

doi:10.1016/0378-1119(80)90074-8

Cited by 45 publications

(24 citation statements)

References 30 publications

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“…The two internal HindlIl sites, 6.6 kb apart in pITS24, were used to construct the subclone pITS26. Since previous reports have shown that the genetic region between entF and entE encodes proteins required for iron transport by the enterobactin system (30,40), a number of transportdefective mutants were examined for complementation by pITS21 (which contains the outer membrane receptor gene fepA), pITS24, or pITS26. Each mutant was transformed with these plasmids and examined for the ability to grow on iron-depleted medium and to transport enterobactin ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…Luria broth (LB) was utilized as complete medium (36). Antibiotics were used at the following concentrations: ampicillin (Ap), 25 ,ug/ml; chloramphenicol (Cm), 30 ,ug/ml; tetracycline (Tc), 10 pITS24 was mutagenized with Tn1000 by the procedure of Guyer (23). Briefly, pITS24 was transformed into strain W1485.…”

Section: Methodsmentioning

confidence: 99%

“…One cytoplasmic membrane component, FepC, was recently described (40). The genes encoding FepB and FepC were mapped clockwise from fepA at the locus originally described asfep (12,30).…”

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confidence: 99%

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

Ozenberger¹,

Nahlik²,

McIntosh³

1987

J Bacteriol

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Ozenberger¹,

Nahlik²,

McIntosh³

1987

J Bacteriol

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“…Genes encoding the three enzymatic activities (EntC, EntB, and EntA) catalyzing the synthesis of 2,3-dihydroxybenzoic acid from chorismic acid have been localized to the right end of the enterobactin gene cluster at min 13 on the Escherichia coli chromosome by molecular cloning (12,16,22) and transposon mutagenesis (13,16) studies. Interspersed among these genes were two additional loci encoding activities (EntE and EntG) necessary to convert 2,3-dihydroxybenzoic acid and L-serine to the functional catechol siderophore enterobactin (also designated enterochelin), a cyclic triester of 2,3-dihydroxybenzoylserine.…”

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confidence: 99%

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

et al. 1989

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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.

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“…Although 2 different classes of phenotype have been shownforfep (A t B) mutants [51], btu (B t A) mutants [71] and fee (A + B) mutants [72] and these classes have different cotransduction frequencies with neighbouring genetic markers, complementation analysis has yielded only single classes with fep [73] and btu mutants [48]. While 2 fhu genes have been shown by merodiploid analysis, only negative complementation results have been obtained with fep and btu mutants.…”

Section: Hypothesismentioning

confidence: 99%

The tonB gene product in Escherichia coli

Wookey

1982

FEBS Letters

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Bacteriophage Mu-mediated gene transposition and in vitro cloning of the enterochelin gene cluster of Escherichia coli

Cited by 45 publications

References 30 publications

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

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

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

The tonB gene product in Escherichia coli

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