The copper resistance of a strain of Escherichia coli isolated from the effluent of a piggery where pigs were fed a diet supplemented with copper sulfate was controlled by a conjugative 78-megadalton plasmid designated pRJ1004. Plasmid pRJ1004 exhibited surface exclusion and incompatibility with standard plasmids belonging to incompatibility groups I1 and K. Sensitive strains of E. coli K-12 were unable to form colonies on nutrient agar containing more than 4 mM copper, whereas transconjugants which harbored pRJ1004 were able to form colonies on medium containing up to 20 mM copper.

A mutant of Escherichia coli K-12 unable to form an essential component of the enterochelin-dependent iron transport system has been isolated. This strain carries a mutation in a gene designated fep, mapping close to two genes, entA and entD, concerned with enterochelin synthesis. Strain AN102, which carries the fepallele, accumulates large quantities of enterochelin and gives a growth response to sodium citrate. The cytochrome b1 and total iron content, and the measurement of the uptake of a5FeE+, indicate an impairment of the enterochelin-dependent iron transport system. The growth response to sodium citrate is related to the presence, in strain AN102, of an inducible citrate-dependent iron transport system. MATERIALS AND METHODS Chemicals. Chemicals used were of the highest purity obtainable and, unless otherwise specified, were not further purified. "5Fe3+ was obtained as a carrierfree solution of FeCI3 in 0.1 M HCI from The Radiochemical Centre, Amersham, England. Enterochelin and 2, 3-dihydroxy-N-benzoylserine were isolated from culture supernatants as described previously (9; O'Brien and Gibson, in press). Bacterial strains. All the strains used were derived from E. coli K-12 and are listed in Table 1. Culture media. The medium used for growth of cells, except those used in iron-uptake experiments, was double-strength medium 56 described by Monod et al. (8). To the sterilized mineral salts base was 219

A new high-affinity system for iron transport, associated with the presence of ColV plasmids, has been detected in Escherichia coli and partially characterized. The presence of such "iron-transport plasmids" in E. coli cells that are defective in enterochelin-mediated transport of iron enabled them to grow in media to which 2,2'-dipyridyl had been added to reduce availability of iron. In addition, the presence of plasmid deoxyribonucleic acid in a mutant defective in enterochelin biosynthesis was associated with a marked increase in the rate of radioactive-iron uptake. Plasmid-determined uptake of iron was distinct from previously recognized systems for iron transport in E. coli K-12, and the colicin V molecule appeared not to be directly involved. Hydroxylamine-nitrogen could be detected in cell pellets of ColV+ cultures, and similar material was detected in supernatant fluids of late log- or stationary-phase cultures. The hydroxamate material was not detected in cell pellets or culture supernatants of strains from which plasmids had been eliminated, and a 95% decrease in hydroxamate synthesis was observed when cells were grown in minimal medium containing 2 microM iron.

Mutants of Escherichia coli K-12 blocked in each of the three enzymatic reactions between chorismate and 2,3-dihydroxybenzoate, in the pathway leading to the iron-sequestering compound enterochelin, have been isolated and biochemically characterized. The three genes concerned (designated entA, entB and entC ) have been shown to be clustered on the chromosome between purE and gal and to be located near minute 14 by cotransduction with the purE, lip , and fep genes. entA, entB , and entC were shown to be the structural genes for 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, 2,3-dihydro-2,3-dihydroxybenzoate synthetase, and isochorismate synthetase, respectively.

Mutants of Escherichia coli K-12 unable to synthesize the iron-sequestering compound, enterochelin, from 2,3-dihydroxybenzoate have been isolated and divided into three classes on the basis of tests for enzymatic complementation. The genes affected (designated entD, entE , and entF ) have been mapped by cotransduction and are located at about minute 14 on the E. coli genome. They were found to be closely linked to other genes ( entA, entB , and entC ) concerned with enterochelin biosynthesis and a gene ( fep ) concerned with the uptake of the iron-enterochelin complex. No detectable diffusible intermediate in the formation of enterochelin from 2,3-dihydroxybenzoate was formed by cell extracts of mutants carrying mutations in the entD, entE , or entF genes.