Ferric enterobactin transport system in Escherichia coli K-12. Extraction, assay, and specificity of the outer membrane receptor.

An outer membrane preparation from cells of Escherichia coli K-12 grown in low iron medium was found to retain ferric enterobactin binding activity following solubilization in a Tris-HCl, Na2EDTA buffer containing Triton X-100. Activity was measured by means of a DEAE-cellulose column which separated free and receptor bound ferric enterobactin. The binding activity was greatly reduced in preparations obtained from cells grown in iron rich media or from cells of a colicin B resistant mutant grown in either high or low iron media. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis enabled correlation of this lack of activity to a single band missing in the outer membrane profile of the colicin B mutant. Evidence was obtained for in vitro competition between ferric enterobactin and colicin B for the extracted receptor. The binding specificity of the extracted receptor was examined by competition between ferric enterobactin and several iron chelates including a carbocyclic analogue of enterobactin, cis-1,5,9-tris(2,3-dihydroxybenzamido)cyclododecane. The ferric form of the latter compound supported growth of siderophore auxotrophs, apparently without hydrolysis to dihydroxybenzoic acid and resynthesis into enterobactin. These data may require revision of the accepted mechanism of enterobactin mediated iron utilization.     

Iron transport of Escherichia coli K-12: involvement of the colicin B receptor and of a citrate-inducible protein.

It was shown that feuB mutants (defective in ferric enterochelin uptake) were unable to adsorb colicin B. In addition, they were missing one of the three outer-membrane proteins which are over produced in strains grown in iron-deficient, extracted medium. Thus this protein (the feuB protein) is probably the receptor for colicin B and functions in enterochelin-mediated iron transport. The feuB gene was located by P1 transduction at approximately 72.5 min on the Escherichia coli K-12 genetic map and thus maps separately from the other genes concerned with the enterochelin system. The outer membranes of various strains grown in the presence of 1 mM citrate contained a high level of a protein which was present in very small amounts when citrate was absent from the growth medium. This protein was most easily observed in feuB mutants grown in the presence of citrate, since on polyacrylamide gels it ran in a similar position to the feuB protein, which is missing in these mutants. The relationship of this citrate-inducible protein to the inducible citrate-dependent iron uptake system is discussed.     

Genetic and physiological studies on the relationship between colicin B resistance and ferrienterochelin uptake in Escherichia coli K-12.

The Escherichia coli gene for the ferrienterochelin uptake and colicins B and D receptor protein is located at approximately 13 min, adjacent to or among genes for enterochelin biosynthetic enzymes. The two receptor functions (colicin and siderophore) are separable by mutation.     

Temperature-sensitive, colicin M-tolerant mutant of Escherichia coli.

A mutant sensitive to colicin M at 30 degrees C and tolerant at 42 degrees C to high concentrations of colicin M was isolated from Escherichia coli K-12. A temperature shift from 30 to 42 degrees C rescued all cells up to the time they started to lyse at 30 degrees C (25 min after addition of colicin M). The growth rate at 42 degrees C remained unaffected by colicin M. AT 42 degrees C the cell-bound colicin M was inactivated by trypsin, sodium dodecyl sulfate, and antiserum against colicin M. Ferrichrome competed with colicin M at 42 degrees C only during the initial adsorption to the common receptor protein in the outer membrane. Since cells lysed earlier at 30 degrees C when they had been preincubated with colicin M at 42 degrees C, we conclude that the process leading finally to cell lysis is initiated at 42 degrees C and stops at a later stage of colicin M trypsin, dodecyl sulfate, and antiserum when cells were transferred from 30 to 42 degrees C, we assume that colicin M is translocated from its target site towards the cell surface. The mutation conferring tolerance was mapped close to the rpsL gene.     

Iron uptake in colicin B-resistant mutants of Escherichia coli K-12.

Four classes of colicin B-resistant mutants of Escherichia coli K-12 were examined for defects in iron uptake. All four mutant classes (cbt, exbC, exbB, and tonB) were defective in the uptake of ferri-ennterochelin. The tonB mutant was also defective in citrate-, ferrichrome-, and rhodoturulic acid-mediated iron uptake. The defects in iron transport were reflected in increased sensitivity to iron chelators and to chromium and aluminium salts, and in hypersecretion of enterochelin. One of the mutants (cbt) was apparently defective in outer membrane ferri-enterochelin receptor activity. aroE derivatives (unable to synthesize enterochelin) of the four mutant classes and the parent strain produced increased amounts of two outer membranes polypeptides when grown under iron stress. These polypeptides are implicated in ferri-enterochelin receptor activity.     

Normal iron-enterochelin uptake in mutants lacking the colicin I outer membrane receptor protein of Escherichia coli.

The outer membranes of two independent colicin Ia-resistant mutants of Escherichia coli K-12 lack the colicin Ia receptor protein. Such mutants exhibit normal capacity for enterochelin (enterobactin)-mediated iron uptake. It is concluded that the colicin Ia receptor is not involved in iron-enterochelin uptake.     

Iron transport systems of Serratia marcescens.

Serratia marcescens W225 expresses an unconventional iron(III) transport system. Uptake of Fe3+ occurs in the absence of an iron(III)-solubilizing siderophore, of an outer membrane receptor protein, and of the TonB and ExbBD proteins involved in outer membrane transport. The three SfuABC proteins found to catalyze iron(III) transport exhibit the typical features of periplasmic binding-protein-dependent systems for transport across the cytoplasmic membrane. In support of these conclusions, the periplasmic SfuA protein bound iron chloride and iron citrate but not ferrichrome, as shown by protection experiments against degradation by added V8 protease. The cloned sfuABC genes conferred upon an Escherichia coli aroB mutant unable to synthesize its own enterochelin siderophore the ability to grow under iron-limiting conditions (in the presence of 0.2 mM 2.2'-dipyridyl). Under extreme iron deficiency (0.4 mM 2.2'-dipyridyl), however, the entry rate of iron across the outer membrane was no longer sufficient for growth. Citrate had to be added in order for iron(III) to be translocated as an iron citrate complex in a FecA- and TonB-dependent manner through the outer membrane and via SfuABC across the cytoplasmic membrane. FecA- and TonB-dependent iron transport across the outer membrane could be clearly correlated with a very low concentration of iron in the medium. Expression of the sfuABC genes in E. coli was controlled by the Fur iron repressor gene. S. marcescens W225 was able to synthesize enterochelin and take up iron(III) enterochelin. It contained an iron(III) aerobactin transport system but lacked aerobactin synthesis. This strain was able to utilize the hydroxamate siderophores ferrichrome, coprogen, ferrioxamine B, rhodotorulic acid, and schizokinen as sole iron sources and grew on iron citrate as well. In contrast to E. coli K-12, S. marcescens could utilize heme. DNA fragments of the E. coli fhuA, iut, exbB, and fur genes hybridized with chromosomal S. marcescens DNA fragments, whereas no hybridization was obtained between S. marcescens chromosomal DNA and E. coli fecA, fhuE, and tonB gene fragments. The presence of multiple iron transport systems was also indicated by the increased synthesis of at least five outer membrane proteins (in the molecular weight range of 72,000 to 87,000) after growth in low-iron media. Serratia liquefaciens and Serratia ficaria produced aerobactin, showing that this siderophore also occurs in the genus Serratia.     

Relationship between the transport of iron and the amount of specific colicin Ia membrane receptors in Escherichia coli.

Strains of Escherichia coli K-12 defective in their ability to utilize exogenously supplied iron due to genetic defects in the entF, tonB, fes, or fep gene exhibited elevated levels of the specific outer-membrane receptor for colicin Ia when compared with parental strains. Although entF, fes, and fep strains showed a higher degree of Ia sensitivity than did the parental strains, tonB strains were resistant to colicin action. The colicin insensitivity of tonB strains was not due to hyperproduction of enterochelin. Growth in medium containing 101.8 muM Fe2+ led to a lowering of receptor levels in all the above strains and resulted in decreased colicin Ia sensitivity in all strains except tonB, which was already at maximal resistance. Growth in citrate plus iron (1.8 muM) or in ferrichrome resulted in a substantial reduction in both receptor levels and Ia sensitivity in ent, fes, and fep strains but had no effect on receptor levels in tonB strains. Growth in citrate did not lead to an alteration in receptor levels in a mutant specifically defective in citrate-mediated iron transport. The presence of enterochelin during growth led to a reduction in the number of receptors in the parental and ent strains but not in tonB, fes, or fep strains. Thus, in all cases examined, there was an inverse relationship between the number of colicin receptors per cell and the ability of the strain to take up iron from the growth medium. This suggests that under conditions of iron limitation there is a derepression of colicin Ia receptor biosynthesis. These results may point to a role of the colicin I receptor in iron uptake.     

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.     

Characterization of group B colicin-resistant mutants of Escherichia coli K-12: colicin resistance and the role of enterochelin.

Nine classes of group B colicin-resistant mutants were examined to study the role of enterochelin in colicin resistance. Four of the mutants studied (cbt, exbC, exbB, and tonB) hypersecreted enterochelin. Enterochelin hypersecretion was apparently responsible for resistance of the exbC mutant to colicins G and H and for resistance of the exbB mutant to colicins G, H, Ia, Ib, S1, and V. All four mutants scored as colicin B tolerant, even in the absence of enterochelin synthesis. The mutants produced substantially increased amounts of two high-molecular-weight outer membrane polypeptides when grown under limiting iron conditions. The presence of these polypeptides was correlated with increased colicin B-neutralizing activity in the outer membrane preparations.     

Effect of osmotic shock and shearing forces on ferric enterochelin transport in Escherichia coli K12

The fes mutation in Escherichia coli K12, which inactivates enterochelin esterase, allows the cell to accumulate ferric enterochelin. The ferric complex of enterochelin was released in significant quantities from a fes mutant after osmotic shock. Analysis of the effects of the individual stages of the shock procedure in wild-type cells showed that prior exposure of cells to sucrose and EDTA was not required, careful dilution of cells into a hypo-osmolar medium being sufficient to induce efflux of Fe3+. Prior treatment with EDTA or exposure to shearing forces served either to enhance efflux or to induce efflux in isotonic media. Neither vitamin B12 nor 5'-nucleotidase was released from the periplasm by these procedures. The release observed under mild conditions was stimulated specifically by Co2+, did not occur at 0 degree C, and was inhibited by 2,4-dinitrophenol at 37 degrees C. From these observations, it was concluded that the efflux of Fe3+ represents a physiological response of the cell to exposure to a hypo-osmolar medium. Such changes may enhance survival following physicochemical stressing of the bacterial outer membrane.     

Siderophore protection against colicins M, B, V, and Ia in Escherichia coli.

A variety of natural and synthetic siderophores capable of supporting the growth of Escherichia coli K-12 on iron-limited media also protect strain RW193+ (tonA+ ent-) from the killing action of colicins B, V, and Ia. Protective activity falls into two categories. The first, characteristic of enterobactin protection against colicin B and ferrichrome protection against colicin M, has properties of a specific receptor competition between the siderophore and the colicin. Thus, enterobactin specifically protects against colicin B in fes- mutants (able to accumulate but unable to utilize enterobactin) as predicted by our proposal that the colicin B receptor functions in the specific binding for uptake of enterobactin (Wayne and Neilands, 1975). Similarly ferrichrome specifically protects against colicin M in SidA mutants (defective in hydroxamate siderophore utilization). The second category of protective response, characteristic of the more general siderophore inhibition of colicins B, V, and Ia, requires the availability or metabolism of siderophore iron. Thus, enterobactin protects against colicins V and Ia, but only when the colicin indicator strain is fes+, and hydroxamate siderophores inhibit colicins B, V, and Ia, but only when the colicin indicator strain is SidA+. Moreover, ferrichrome inhibits colicins B, V, and Ia, yet chromium (III) deferriferrichrome is inactive, and ferrichrome itself does not prevent adsorption of colicin Ia receptor material in vitro. Although the nonspecific protection against colicins B, V, and Ia requires iron, the availability of siderophore iron for cell growth is not sufficient to bring about protection. None of the siderophores tested protect cells against the killing action of colicin E1 or K, or against the energy poisons azide, 2, 4-dinitrophenol, and carbonylcyanide m-chlorophenylhydrazone. We suggest that nonspecific siderophore protection against colicins B, V, and Ia may be due either to an induction of membrane alterations in response to siderophore iron metabolism or to a direct interference by siderophore iron with some unknown step in colicin action subsequent to adsorption.     

Fuctional organization of the outer membrane of escherichia coli: Phage and colicin receptors as components of iron uptake systems

The functional interaction of outer memberane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 anf ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all colicins. The interaction of the ton A, Ton B, and feu functions apparently permits quite different “substrates” to overcome the permeablility barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of ³H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source waw used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: (1) ton A; (2) ton B mutants; (3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; (4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; (5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000–83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene. The nature of these proteins and their possible role in iron transport is disussed.     

Dihydroxybenzolyserine—a siderophore for E. coli

Dihydroxybenzoylserine, the breakdown product of enterochelin, was able to stimulate growth of Escherichia coli under iron limiting conditions by acting as a siderophore. The dihydroxybenzoylserine-iron complex was taken up via the outer membrane receptor proteins Fiu, FepA and to a minor extent via Cir. Transport of Fe3(+)-dihydroxybenzoylserine across the cytoplasmic membrane was only dependent on genes from the fep region. In addition, it was shown that dihydroxybenzoate was taken up via Fiu and Cir and less efficiently by FepA.     

Outer Membrane-Dependent Transport Systems in Escherichia coli: Effect of Repression or Cessation of Colicin Receptor Synthesis on Colicin Receptor Activities

Proteins in the outer membrane of gram-negative bacteria serve as general porins or as receptors for specific nutrient transport systems. Many of these proteins are also used as receptors initiating the processes of colicin or phage binding and uptake. The functional activities of several outer membrane proteins in Escherichia coli K-12 were followed after cessation or repression of their synthesis. Cessation of receptor synthesis was accomplished with a thermolabile suppressor activity acting on amber mutations in btuB (encoding the receptor for vitamin B(12), the E colicins, and phage BF23) and in fepA (encoding the receptor for ferric enterochelin and colicins B and D). After cessation of receptor synthesis, cells rapidly became insensitive to the colicins using that receptor. Treatment with spectinomycin or rifampin blocked appearance of insensitive cells and even increased susceptibility to colicin E1. Insensitivity to phage BF23 appeared only after a lag of about one division time, and the receptors remained functional for B(12) uptake throughout. Therefore, possession of receptor is insufficient for colicin sensitivity, and some interaction of receptor with subsequent uptake components is indicated. Another example of physiological alteration of colicin sensitivity is the protection against many of the tonB-dependent colicins afforded by provision of iron-supplying siderophores. The rate of acquisition of this nonspecific protection was found to be consistent with the repression of receptor synthesis, rather than through direct and immediate effects on the tonB product or other components of colicin uptake or action.     

Mechanisms of siderophore iron transport in enteric bacteria.

Uptake of 55Fe- and 3H-labeled siderophores and their chronic analogues have been studied in Salmonella typhimurium LT-2 and Escherichia coli K-12. In S. typhimurium LT-2, at least two different mechanisms for siderophore iron transport may be operative. Uptake of 55Fe- and 3H-labeled ferrichrome and kinetically inert lambda-cis-chromic [3H]deferriferrichrome by the S. typhimurium LT-2 enb7 mutant, which is defective in the production of its native siderophore, enterobactin, appears to occur by two concurrent mechanisms. The first mechanism is postulated to involve either rapid uptake of iron released from the ferric complex by cellular reduction without penetration of the complex or ligand or dissociation of the complex and simultaneous uptake of both ligand and iron coupled with simultaneous expulsion of the ligand. The second mechanism appears to consist of slower uptake of the intact ferric complex.     

Enterochelin System of Iron Transport in Escherichia coli: Mutations Affecting Ferric-Enterochelin Esterase

Three mutant strains of Escherichia coli have been isolated which are lacking ferric-enterochelin esterase activity. This enzyme catalyzes the hydrolysis of the enterochelin moiety of ferric-enterochelin to yield ultimately three molecules of N-2,3-dihydroxybenzoylserine. The mutants (designated fes(-)) were shown to be unaffected in enterochelin biosynthesis, capable of enterochelin-mediated iron uptake, and able to utilize ferric-dihydroxybenzoylserine complexes normally. When grown under iron-deficient conditions, however, they showed an absolute requirement for added iron or citrate, a phenotype characteristic of mutants defective in some part of the enterochelin system of iron uptake. These results support the theory that iron, taken up by the cell as ferric-enterochelin is only available for general cell metabolism after hydrolysis of the ligand by enterochelin esterase. The three fes(-) strains were shown to be affected in the B component of enterochelin esterase. The fesB gene which is probably the structural gene coding for component B of the esterase, was shown to be located at about minute 14 on the E. coli chromosome together with seven other genes involved in the enterochelin system of iron transport.     

Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775.

The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.     

Ferrous iron uptake by a magnesium transport system is toxic for Escherichia coli and Salmonella typhimurium.

At low magnesium concentrations, Escherichia coli and Salmonella typhimurium LT2 accumulate ferrous iron independent of the ferrous iron transport system feo. Mutant strains with mutations in the magnesium transport gene corA accumulated less ferrous iron than the parent strains. corA+ and corA strains also differed in their sensitivity to ferrous iron under oxic conditions. corA mutants were more resistant to ferrous iron than their parent corA+ strains. Part of the ferrous iron accumulated can be chased by the addition of magnesium. Much less iron was chased when ferric iron was taken up by the siderophore ferrichrome. These results may indicate that the intracellular metabolism of the iron taken up by these systems differs and that it depends on the uptake route of the iron.