Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12. Four polypeptides involved in the conversion of 2,3-dihydroxybenzoate to enterochelin.

Four gene products involved in the enzymatic synthesis of enterochelin from 2,3-dihydroxybenzoate, L-serine and ATP (Luke, R.K.L. and Gibson, F. (1971) J. Bacteriol. 107,557-562; Woodrow, G.C., Young, I.G. and Gibson, F. (1975) J. Bacteriol. 124, 1-6) have been partially purified using a previously reported fractionation procedure (Bryce, G.F. and Brot, N. (1972) Biochemistry 11, 1708-1715). The products of genes E, F and G have been separated from each other and correspond to the E1, E2 and E3 activities described by Bryce and Brot. These three gene products were not completely separated from the product of gene D. We refer to these gene products as components E, F, G and D of the enzymic apparatus for biosynthesis of enterochelin. Certain properties and functions of the four semi-purified components have been investigated. The E component is involved in the activation of 2,3-dihydroxybenzoate and the F component in the activation of L-serine. The D component physically associates with the F and G components during gel filtration and chromatography on DEAE Sephadex. It is proposed that the synthesis of enterochelin from L-serine and 2,3-dihydroxybenzoic acid is catalysed in vivo by a multienzyme complex, enterochelin synthetase.

Enzymatic hydrolysis of enterochelin and its iron complex in Escherichia Coli K-12. Properties of enterochelin esterase.

Properties of the enzyme which hydrolyses enterochelin (a cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine) to 2,3-dihydroxybenzoylserine have been investigated with a view to resolving discrepancies between earlier reports. Enterochelin esterase, previously reported to consists of two components (O'Brien, I.G., Cox, G.B. and Gibson, F. (1971) Biochim. Biophys. Acta 237, 537-549), has been shown to be fully active in the absence of the so-called A component. The hydrolase described previously (Bryce, G.F. and Brot, N. (1972) Biochemistry 11, 1708-1715) as being able to break down enterochelin but not its iron complex, ferric-enterochelin, appears to be identical with the B component of enterochelin esterase. The single component enterochelin esterase corresponding to what was previously described as component B, hydrolyses both enterochelin and ferric-enterochelin. Under the assay conditions used, enterochelin is hydrolysed 2.5 times faster than the complex. Enzymatic activity is inhibited by N-ethylmaleimide and is lost rapidly at 37 degrees C. Activity is stabilized in the presence of ferric-enterochelin, enterochelin, dithiothreitol or certain protein fractions.

Regulation of enterochelin synthetase in Escherichia coli K-12.

Enterochelin synthetase activity is controlled by both repression and feed-back inhibition mechanisms. Inclusion of iron in growth media results in synthesis of all four (D, E, F and G) components of enterochelin synthetase being repressed. The specific inhibition of L-serine activation (partial reaction catalyzed by the F component) by the end products, ferric-enterochelin and 2,3-dihydroxybenzoylserine, is shown to inhibit overall enterochelin synthetase activity.

Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12, Salmonella typhimurium and Klebsiella pneumoniae. Physical association of enterochelin synthetase components in vitro.

Further evidence is presented in support of the proposal made previously (Greenwood, K.T. and Luke, R.K.J. (1976) Biochim. Biophys. Acta 454, 285-297) that components of the Escherichia coli enterochelin synthetase system physicaloly associate to form enzyme complexes. Evidence for the existence of three enzyme complexes, designated in order of increasing stability G-D < F-D < F-D-G, has been obtained following gel filtration and chromatography on DEAE-Sephadex. Persistence of the F-D and G-D complexes during chromatography appears to depend on the flow rate of the column. On the basis of complementation with appropriate ent mutants of E. coli, activities corresponding to those of the D, E, F and G components of enterochelin synthetase in E. coli have been detected in cell-extracts of both Salmonella typhimurium and Klebsiella pneumoniae (formerly Aerobacter aerogenes) strains. These are designated D', E', F' and G' activities. Components E' and G' are eluted from Sephadex G-100 in similar fashion to their E. coli counterparts. Peaks of F' and D' activities however, are eluted together at a position corresponding to that of the E. coli F component. We suggest that in S. typhimurium and K. pneumoniae, either a single polypeptide combines the functions of the E. coli F and D components, or that separate F' and D' components form a stable complex and that activity of uncomplexed D' and component was not detected under the conditions used during chromatography and assay.

Hydroxamate-mediated transport of iron controlled by ColV plasmids.

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.

Iron-suppressible production of hydroxamate by Escherichia coli isolates.

A total of 476 strains of Escherichia coli isolated from humans, pigs, cattle, poultry, potable water, or effluent were examined for iron-suppressible ability to produce hydroxamate. Isolates able to produce such material (Hyd+ isolates) are presumed to be able to carry out hydroxamate-dependent transport of iron. The percentages of Hyd+ isolates found among E. coli isolated from the feces of breast-fed babies (71%), adults (46%), milk-fed calves (32%), or poultry (28%) were significantly greater (P less than 0.01) than the percentages isolated from potable water and effluent (6%) or from the feces of suckling piglets (6%), weaned pigs (6%), or weaned cattle (4%). The percentages of Hyd+ isolates found among E. coli associated with diarrhea in humans (51%), weaned pigs (7%) or calves (25%) were not significantly different (P greater than 0.1) from those found among strains isolated from corresponding nondiarrheic hosts. Many of the E. coli isolated from cases of E. coli bacteremia in humans and poultry were Hyd+ (64% and 83%, respectively). We conclude that ability to carry out hydroxamate-mediated transport of iron is widely distributed among natural isolates of E. coli but that the distribution of Hyd+ E. coli is not random. E. coli isolated from sources where levels of available iron might be expected to be low tend to be Hyd+. It seems that a link may exist between prevalence of Hyd+ E. coli and active host-defense based on restricted availability of iron.