Vol. 130, No. 1 Printed in U.S.A.
JOURNAL OF BACTZRIOLOGY, Apr. 1977, p. 173-180 Copyright X 1977 American Society for Microbiology
Specificity of Siderophore Receptors in Membrane Vesicles of Bacillus megaterium J. E. ASWELL,' A. H. HAYDON, H. R. TURNER, C. A. DAWKINS, J. E. L. ARCENEAUX, AND B. R. BYERS* Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216 Received for publication 19 July 1976
Membrane vesicles of Bacillus megaterium strains SK11 and Ardl bound the ferrischizokinen and ferriferrioxamine B siderophores (iron transport cofactors). An approximately equimolar uptake of both labels of [3H, 59Fe]ferrischizokinen indicated binding of the intact chelate. Binding reached equilibrium in 2 to 5 min, was temperature independent, and was unaltered by the addition of several energy sources. A 91% dissociation of bound [59Fe]ferrischizokinen was achieved in 60 s by the addition of excess ferrischizokinen. Ferriaerobactin, a siderophore which is structurally related to ferrischizokinen, caused no detectable release of bound [59Fe]ferrischizokinen. Of several other ferrihydroxamates tested, only ferriferrichrome A achieved the release (11%) of [59Fe]ferrischizokinen. Rapid dissociation (92%) of bound [59Fe]ferriferrioxamine B by the addition of ferriferrioxamine B was observed, and a 67% release of [59Fe]ferriferrioxamine B was caused by ferriA22765, its structural relative. Ferrischizokinen, ferriferrichrome A, and ferrirhodotorulic acid produced a 6, 25, and 29% dissociation, respectively, of [59Fe]ferriferrioxamine B; ferriaerobactin caused no dissociation. [59Felferriaerobactin was bound by the membranes, but its dissociation was not effected by unlabeled ferriaerobactin, suggesting no specific receptors for this chelate. The respective binding affinity constants and maximal binding capacities of membrane vesicles of strain SK1l were 2 x 107 M-' and 280 pmol per mg of protein for ferrischizokinen and 7 x 107 M-1 and 37 pmol per mg of protein for ferriferrioxamine B. These values in strain Ardl were, respectively, 1.4 x 107 M-' and 186 pmol per mg of protein for ferrischizokinen and 11 x 107 M-' and 23 pmol per mg of protein for ferriferrioxamine B. Separate, specific binding sites (receptors) for ferrischizokinen and ferriferrioxamine B exist on the vesicles. The ferrischizokinen receptors have a lower affinity but a higher binding capacity (eightfold) than that shown by the ferriferrioxamine B receptor. These receptors may be components of independent transport systems. ferrischizokinen is transported across the cell membrane to form a pool of available iron within the cytoplasm (2, 3). Ferriaerobactin, another member of the citrate-hydroxamate family (Fig. 1), does not function as a siderophore in B. megaterium (3, 17); however, ferriferrioxamine B, a ferrioxamine-type siderophore (Fig. 1), does supply iron to B. megaterium (4, 7, 8). An examination of ferrischizokinen and ferriferrioxamine B uptake suggested independent transport systems (3, 17). The specificity noted in ferrihydroxamate utilization might reside in membrane receptors, in the transport process, or in the factors responsible for release of iron from the chelates. Chelate transport can be separated from iron assimilation because B. megaterium Ardl transported ' Present address: Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, VA ferriferrioxamine B but could not release iron from the chelate for metabolic use (2). (The 24060.
The efficient uptake of iron by many microorganisms requires the participation of siderophores (6, 19, 24), which are defined as ironchelating agents functioning in the transport of iron to the cell and/or into the cell (19). The natural secondary hydroxamic acids comprise a major chemical category of siderophores, and several structurally different families of hydroxamate siderophores have been identified in various microorganisms (24). Bacillus megaterium ATCC 19213 (American Type Culture Collection) excretes deferrischizokinen, a member of the citrate-hydroxamate family of siderophores (Fig. 1). Studies of B. megaterium SK11 (a strain that is unable to produce deferrischizokinen) showed that externally formed
173
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ASWELL ET AL.
O II
0 II
H
/ NHNI
(CH2)2-N-C-CHs ii
CH2
HO-C-COOH
C/2 \
C H O
J. BACTERIOL. / \HNC \(CH2)4-N-C-CH3 CH,2H
HO-C-COzH
OHO NHs IN CH ACH,)a-N-C .CH2N-C H3 H
C/2
OH 0 \c,NHN (CH2)4-N-C-CH3 H II 0 CO2H
DEFERRISCHIZOKINEN
DEFERRIAEROBACTIN
COHN
H2-N
CO,H
CONH
(CHz)5 (CH2)2 (CH2)5 (CH2)2 (CH2)5 CH3 N-C II HOO
N-C 11 HO O
N-C 11 HO O
DEFERRIFERRIOXAMINE 8
FIG. 1. Structures of ferrischizokinen, ferriaerobactin, and ferriferrioxamine B. From reference 24.
ferric chelates and the iron-free forms of the siderophores are indicated by the prefixes ferriand deferri-, respectively.) The present paper will present evidence for separate receptors for ferrischizokinen and ferriferrioxamine B (possibly initial components of independent transport systems) in membrane vesicles of B. megaterium strains SK11 and Ardl. The ferrischizokinen receptors do not accept the structurally related siderophore ferriaerobactin, and no specific receptors for ferriaerobactin were detected. MATERIALS AND METHODS Bacterial strains and culture medium. Bacillus megaterium strains SK1l and Ardl were used in this study. Strain SK1l is a mutant that was isolated from B. megaterium ATCC 19213 by Arceneaux and Lankford (4); this strain is unable to synthesize deferrischizokinen, the secondary hydroxamate. Strain Ardl is a spontaneous mutant of strain SK11; unlike its parent, strain Ardl is resistant to the hydroxamate antibiotic ferriA22765 (8). Strain Ardl also has lost the capacity to utilize ferriferrioxamine B. Both strains were maintained in the lyophilized state and on brain heart infusion agar slants. The preparation of the sucrose-mineral salts medium and methods for treatment of this medium with Chelex-100 to reduce trace metal contamination have been described (9). Water used for the preparation of medium and reagents and for cleaning glassware was first purified by passage through a reverse osmosis-demineralizing unit (Culligan model RDS1) and then distilled in an all-glass water still. All plasticware and glassware were soaked for 6 or more h in 0.025 M ethylenediaminetetraacetic acid. The ethylenediaminetetraacetic acid was removed by extensive rinsing with the purified water. Preparation of membrane vesicles. Membrane vesicles were prepared by modification of previously used methods (18). B. megaterium strains SK11 and Ardl were harvested from brain heart infusion agar slants that had been previously incubated at 37°C for 6 to 8 h. A 100-ml portion of Chelex-treated,
sucrose-mineral salts medium (9) containing 0.2 ,ug of Fe per ml was inoculated at an initial cell density of 106 colony-forming units per ml. The culture was shaken at 37°C (Gyrotory Shaker, New Brunswick) until the population reached 108 colony-forming units per ml. The cells were harvested and washed three times by centrifugation in 10 ml of Chelextreated medium containing no trace metal supplements. These cells were then used to inoculate an initial level of 106 colony-forming units per ml of medium containing 0.005 ,ug of Fe2+ per ml ("lowiron conditions") or 2 ,ug of Fe2+ per ml ("high-iron conditions"). This culture was incubated with shaking at 37°C until the population reached about 5 x 107 colony-forming units per ml. The cells then were harvested by centrifugation, and the cells obtained from 1 liter were equally divided into three Erlenmeyer flasks (500 ml), each of which contained 100 ml of 0.5 M Chelex-treated sucrose in 0.05 M Chelextreated potassium phosphate buffer (pH 7) with 20 ,ug of Mg2+ per ml, 2 ,ug of Mn2+ per ml, 48 ,ug of Na+ (Na2SO4) per ml, and 200 ug of lysozyme (Sigma Chemical Co.) per ml. The flasks were shaken gently in a water bath at 37°C for 15 min. Conversion to protoplasts was monitored by light microscopy. When conversion was greater than 90%, the suspension of protoplasts was poured into centrifuge cups, chilled in ice, and centrifuged at 14,500 x g for 20 min at 5°C. The resulting pellet of protoplasts was lysed with cold 0.05 M potassium phosphate buffer containing 20 ,ug of Mg2+ per ml, 5 ,ug of deoxyribonuclease (Sigma) per ml, and 10 ,ug of ribonuclease (Sigma) per ml. After centrifugation at 121 x g for 10 min at 5°C to remove whole cells, the vesiclecontaining supernatant was centrifuged at 34,800 x g for 30 min at 5°C, and the pellet was resuspended in 0.05 M Chelex-treated potassium phosphate buffer (pH 7) containing 20 ,ug of Mg2+ per ml. These vesicles were either stored in packed ice or frozen at -70°C until used. An average of 12 to 18 mg of protein, as determined by the method of Lowry et al. (21), was obtained from 1 liter ofculture as described above. Electron microscopy of these vesicles (fixed with 1% glutaraldehyde and stained with uranylacetate and lead citrate) revealed bilayer membrane sacs of varying diameter. Diaminopimelic acid assays for cell wall contamination (14) indicated only about 5 ,ug of diaminopimelic acid per mg of membrane protein. On the day of preparation, membrane vesicles displayed reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3) activity (10) with a usual specific activity of about 97 nmol of reduced nicotinamide adenine dinucleotide oxidized per min per mg of protein; however, by day 6 of storage at 0WC, this activity declined to 70 nmol of reduced nicotinamide adenine dinucleotide oxidized per min per mg of protein. Immediately after preparation, membrane vesicles showed an adenosine triphosphatase (EC 3.6.1.3) activity (1) of about 0.7 nmol per min per mg of protein, but this activity was reduced by 60% on day 3 of storage at 0°C. Succinate dehydrogenase (EC 1.3.99.1) activity (10) reduced 73 nmol of dichlorophenolindophenol per mg of membrane protein on the day of preparation.
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B. MEGATERIUM SIDEROPHORE RECEPTOR SPECIFICITY
Siderophore accumulation by membrane vesicles. The uptake of [59Felferrihydroxamates and doubly labeled [3H, 59Fe]ferrischizokinen by membrane vesicles was determined according to the following procedures. The chelates were formed by mixing 59FeCI3 (5 to 20 mCi per mg of Fe, New England Nuclear, Boston, Mass.) and deferrischizokinen, [3Hldeferrischizokinen (52 mCi/mmol), or Desferal (the methane sulfonate of deferriferrioxamine B) at a molar ratio of 2:1 (hydroxamate-iron) and allowing 1 to 2 h for chelate formation at room temperature. Membrane vesicles (either used immediately after preparation or rapidly thawed from -70°C storage) were diluted to 1 mg of membrane protein per ml in 0.05 M potassium phosphate buffer (pH 7.0) containing 20 ug of Mg per ml. One milliliter of the vesicle suspension was then added to an Erlenmeyer flask (125 ml), and the volume was brought to 4.9 ml with the above buffer. The final concentration of membrane protein was 200 ,lg/ml. The flasks were placed in a water bath at 37°C and gently shaken. After temperature equilibration, the assays were initiated by adding the desired concentration of labeled ferrihydroxamate in 0.1 ml to the vesicle suspension. At timed intervals 0.2-ml samples were removed and rapidly filtered (0.45-,um pore size filters; Millipore Corp., Bedford, Mass.). The filters were washed with 10 ml of 0.05 M phosphate buffer and, after drying, assayed for 59Fe and/ or 3H content by liquid scintillation spectrophotometry using Aquasol (New England Nuclear) in a Packard scintillation spectrometer (model 3003). Appropriate controls containing no membrane vesicles were included to assay for the nonspecific retention of label by the filters. The number of picomoles of ferric hydroxamate accumulated by the vesicles were calculated from the specific activity of the radioactive iron or from the specific activity of the
transport system in strain Ardl is functional because this chelate appears in the cytoplasmic fraction of protoplasts exposed to this chelate (2); however, strain Ardl is not capable of separating iron from ferriferrioxamine B for metabolic use. It was of interest to compare the uptake of the two chelates, ferrischizokinen and ferriferrioxamine B, by membrane vesicles prepared from both strains by osmotic rupture of protoplasts. Initial observations showed that membrane vesicles of B. megaterium strains Ardl and SK11 (prepared from protoplasts of cells grown at low-iron conditions; see Materials and Methods) accumulated both [59Fe]ferrischizokinen and [59Fe]ferriferrioxamine B. Their uptake by the vesicles was rapid, usually reaching equilibrium between 2 and 5 min of the assay (for example, Fig. 2). Vesicles prepared from either strain accumulated more [59Fe]ferrischizokinen than [59Fe]ferriferrioxamine B (described in detail below).
30-
z
w
z
25-
N
[3H]deferrischizokinen.
Sources of siderophores. Deferrischizokinen and
175
w
E 15-;
[3Hldeferrischizokinen were prepared by previously
described methods (3, 7). Desferal was a gift of CibaGeigy Pharmaceutical Co. The hydroxamate antibiotic ferriA22765 was supplied by F. Knusel, CibaGeigy Pharmaceutical Company, Basel, Switzerland. Deferriferrichrome A, deferrirhodotorulic acid, and deferriaerobactin were purified from culture filtrates of Ustilago sphaerogena, Rhodotorula utilis, and Aerobacter aerogenes strain 62-1, respectively, by previously determined methods (5, 12, 13).
0:
2
RESULTS Binding of ferrihydroxamates by membrane vesicles. Neither of the two strains of B. megaterium (SK1l and Ardl) used in this study synthesizes deferrischizokinen, the hydroxamate produced by B. megaterium ATCC 19213, and iron uptake by both strains is stimulated by ferrischizokinen (2, 8). Unlike strain SKil, strain Ardl cannot utilize the hydroxamate ferriferrioxamine B. The ferriferrioxamine B
4
6
8
10
MIN UTES FIG. 2. Binding of [59Fe]ferrischizokinen by membrane vesicles of B. megaterium strain Ardl and dissociation of bound [59Fe]ferrischizokinen by the addition of non-radioactive ferrischizokinen. Assay contained initially 102 pmol of [59Fe]ferrischizokinen per ml and 200 pg of membrane vesicle protein per ml in 0.05 M Chelex-treated potassium phosphate buffer (pH 7). At 5 min, 10,200 pmol of non-radioactive ferrischizokinen per ml was added to 50% of the assay sample (dashed line). The control (solid line) received equal volume of buffer.
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ASWELL ET AL.
The accumulation of radioiron added as the ferrihydroxamate represented the uptake of the chelate as an intact unit. A small quantity of [3H]deferrischizokinen was available for testing, and the simultaneous uptake of a doubly labeled chelate, [3H, 59Fe]ferrischizokinen, was followed in vesicles of strain SK11. A rapid uptake of both labels occurred; maximal levels were reached within 2 min. The picomole quantities of ligand and metal that had accumulated were approximately equivalent, suggesting accumulation of the intact chelate. As measurement of 59Fe uptake was indicative of ferrihydroxamate binding, the remainder of the work reported here utilized only [59Fe]ferrihydroxamates. Competition between ferrischizokinen and deferrischizokinen for the transport system in whole cells has been reported (17). In the present assays, the addition of deferrischizokinen at a deferrihydroxamate:59Fe ratio of 15:1 caused a reduction in radioiron accumulation by membrane vesicles of both strains Ardl and SK11 to less than 50% of that seen at lower deferrischizokinen:59Fe ratios. Thus, the accumulation of ferrischizokinen by membrane vesicles resembled ferrischizokinen transport by whole cells in that the ferri and deferri forms of schizokinen appear to compete. Similar additions of excess deferriferrioxamine B reduced the accumulation of [59Fe]ferriferrioxamine B by membrane vesicles of both strains. Because deferriferrioxamine B also impedes the transport of its ferric chelate in whole cells (17), it is suggested that [59Fe]ferrihydroxamate binding by membrane vesicles was accomplished by components of the transport processes. The assays described above were conducted in Chelex-treated potassium phosphate buffer (0.05 M, pH 7). Identical results were obtained after the addition of the following energy sources: 15 mM sucrose, 3.9 mM adenosine 5'triphosphate, 9 mM 1)-lactate, 0.14 mM reduced nicotinamide adenine dinucleotide or 20 mM succinate plus 20 mM ascorbate with 0.1 mM phenazine methosulfate. All of these energy sources failed to stimulate uptake of the radioiron-labeled chelates by membrane vesicles. Moreover, the addition of 0.1 M sodium azide to assays containing the energy sources had no measurable effect on accumulation of the [59Fe]ferrihydroxamates. Identical uptake curves were obtained at 0, 25, and 37°C. These results appear to exclude active transport as the system for [59Felferrihydroxamate accumulation by membrane vesicles of both strains. Although such uptake appeared independent of temperature, the possibility that a facilitated diffusion-type process might be operating was
J. BACTERIOL.
considered. It was found that a fourfold dilution of membrane vesicles previously exposed to [59Fe]ferrischizokinen or [59Fe]ferriferrioxamine B did not cause a rapid release of the label, suggesting that the chelates were not subjet to immediate efflux as might be predicted with a facilitated diffusion-type system. These data indicate that accumulation of the two ferrihydroxamates by membrane vesicles of the two strains represented binding without transport. Dissociation of bound ferrihydroxamates. Evidence for specific receptors. If binding of ferrihydroxamates by the membrane vesicles represents an association between the chelates and specific receptor sites, then the addition of excess non-radioactive ferrihydroxamate (after equilibration between [59Felferrihydroxamate and the membrane vesicles) would result in a rapid exchange of the non-radioactive and radioiron-labeled chelates at the specific receptor. On the other hand, if the chelates are held by relatively nonspecific binding sites, rapid exchange should not occur. It has been shown in a number of membrane systems that excess nonradioactive ligand will both block the binding of radioactive ligand to a specific receptor and cause a rapid release of radioactive ligand that is bound to a specific receptor (for example, see reference 20). When the binding of [59Fe]ferrischizokinen by membrane vesicles of strain Ardl was allowed to reach equilibrium (5 min) and nonradioactive ferrischizokinen was added at 100 times the initial concentration of this chelate, more than 90% of the bound label was released within 60 s after the addition of non-radioactive ferrischizokinen (Fig. 2). The capacity of various structurally different ferrihydroxamates to cause the dissociation of [59Fe]ferrischizokinen from membrane vesicles should represent a measure of the specificity of the binding site. The dissociation of bound [59Fe]ferrischizokinen from vesicles of strain Ardl by ferriferrioxamine B, ferriferrichrome A, ferriaerobactin, ferrirhodotorulic acid, and ferriA22765 was tested (Table 1). Of these ferrihydroxamates, only ferriferrichrome A effected some release of [59Fe]ferrischizokinen, showing 11% dissociation of the label by 2 min. It was interesting that the structurally related siderophore ferriaerobactin caused no dissociation. The ferrischizokinen binding component(s) present on the membranes displayed a marked specificity for ferrischizokinen. Similar to the ferrischizokinen binding system, 92% of bound [59Fe]ferriferrioxamine B could be dissociated rapidly by the addition of non-radioactive ferriferrioxamine B (Table 1).
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B. MEGATERIUM SIDEROPHORE RECEPTOR SPECIFICITY
The ferrischizokinen chelate caused the release of only 6% of bound [59Fe]ferriferrioxamine B. FerriA22765, which is thought to be structurally similar to ferriferrioxamine B, initiated an exchange with [59Fe]ferriferrioxamine B, showing a release of 67% of the label at 2 min. Both ferriferrichrome A and ferrirhodotorulic acid also showed some apparent exchange with [59Felferriferrioxamine B, causing a release of 25 and 29%, respectively, of the label. Ferriaerobactin caused no removal of [59Felferriferrioxamine B. These results suggest that ferriferrioxamine B binding sites are present on the membrane and that they may have an affinity for a number of other hydroxamates. Estimation of total numbers of receptor sites and affinity constants. Scatchard analysis is well suited for describing both the affinity and maximal binding capacity, at equilibrium, of protein receptors for small molecules (25). Accordingly, the specific binding of [59Fe]ferriferrioxamine B and [59Fe]ferrischizokinen to membrane vesicles of both strains was determined at chelate concentrations from 8 to 330
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pmol/ml. Specific binding of the chelates to the receptor sites was determined by subtracting the amount of nonspecifically associated chelate from the total chelate bound by the vesicles. Nonspecifically bound chelate (i.e., ferrihydroxamate associated with sites other than the receptor) is defined as the [59Fe]ferrihydroxamate that is membrane associated in the presence of a 100-fold concentration of non-radioactive ferrihydroxamate. It has been shown in other systems that some radioactive ligand binding cannot be blocked by additions of large excesses of unlabeled ligand; this binding is considered to be nonspecific (20). A typical Scatchard plot of data obtained in membrane vesicles of strain Ardl is shown in Fig. 3. Differences in both the affinity constants (Ka, calculated from the slopes of the lines) and
TABLE 1. Dissociation of bound
[59Fe]ferrihydroxamates from membrane vesicles of B. megaterium Ardl by non-radioactive
ferrihydroxamates % Dissociation of:°
Non-radioactive fernhydroxamnate addeda
[59Fe]fer- [59Felferririschizokinen
ferrioxamine B
Ferrischizokinen 91 6 Ferriferrioxamine B 0 92 Ferriferrichrome A 11 25 Ferriaerobactin 0 0 Ferrirhodotorulic acid 0 29 FerriA22765 0 67 a Added at 100 times the initial concentration (102 pmol/ml) of radioiron-labeled ferrihydroxamate. Assays contained 200 ,ug of protein per ml. b Dissociation of [59Felferrihydroxamate determined 120 s after the addition of non-radioactive ferrihydroxamate.
20 30 moles BOUND FIG. 3. Scatchard analyses of specific binding of [5"Fe]ferrischizokinen (open circle) and [59Felferriferrioxamine B (closed circle) by membrane vesicles of B. megaterium strain Ardl (200 pg of membrane protein per ml) at ferrihydroxamate concentrations from 8 to 330 pmol/ml. p
TABLE 2. Affinity constants and maximal binding capacities of membrane vesicles of B. megaterium strains for [59Fe]ferrischizokinen and [59Felferriferrioxamine B a
K.
Maximal binding (pmol/mg of protein)
Strain
[59Fe]ferrischizokinen
[59Fe]ferriferrioxamine B
[59Fe]ferrischi["Fe]ferriferzokinen rioxamine B
280 7.0 x 107 M-l 37 186 11.0 X 107 M-' 23 a Affinity constants (Ka) and maximal binding capacities were calculated from Scatchard plots of specific binding at 8 to 330 pmol of ferrihydroxamate per ml and 200 lAg of membrane vesicle protein per ml (prepared from cells grown in low-iron conditions).
SK11 Ardl
2.0 x 107 M-1 1.4 x 107 M-l
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ASWELL ET AL.
the maximal binding capacities (X intercepts) of the membrane vesicles for the two ferrihydroxamates were apparent. Such plots were used to produce the data summarized in Table 2. In vesicles from both strains, the [59Fe]- ferrischizokinen binding system had less affinity for. [59Fe]ferrischizokinen than the [59Fe]ferriferrioxamine B binding system showed for its chelate; however, the membrane sites binding [59Fe]ferrioxamine B were fewer in number than the [59Fe]ferrischizokinen accepting sites. Thus, the [59Fe]ferrischizokinen binding sites appeared to have a higher capacity, but lower affinity, when compared to those sites that bound [59Fe]ferriferrioxamine B. Effect of iron on synthesis of siderophore receptors. The above information was collected using membrane vesicles prepared from cells grown in "low-iron medium" (see Materials and Methods). Because elevated concentrations of iron in the growth medium will lower the production of deferrischizokinen in wild-type B. megaterium (7) and because cells that have been cultivated in "high-iron medium" show a reduced capacity to transport ferrihydroxamates (J. E. L. Arceneaux, unpublished observation), it was of interest to determine the effect of the iron concentration in the growth medium on the binding capacity of membrane vesicles. Scatchard analyses of the association of high-iron membrane vesicles of strain SK1l with [59Fe]ferrihydroxamates produced a K,, for [59Fe]ferrischizokinen of 2.0 x 107 M-1 and a maximal binding capacity of 198 pmol per mg of membrane protein. This K, is identical to that observed in low-iron membrane vesicles (Table 2), and the total binding capacity was lowered by only 30%. The K,, of high-iron membrane vesicles for [59Fe]ferriferrioxamine B was 6.7 x 107 M-1, and these vesicles bound 28 pmol of [59Fe]ferriferrioxamine B per mg of membrane protein. This Ka is essentially identical to that obtained with low-iron vesicles (Table 2), and the total binding capacity was lowered by 25%. These data reveal the presence of large numbers of binding sites for both ferrischizokinen and ferriferrioxamine B in membrane vesicles of cells that have been cultivated in a medium containing excess iron and suggest that synthesis of the ferrihydroxamate receptors may continue even when the cells are replete for iron. Binding of [59Felferriaerobactin. Lack of specific receptors. Although ferriaerobactin is structurally related to ferrischizokinen (Fig. 1), ferriaerobactin is not utilized by B. megaterium SK1l (17). Membrane vesicles of strain SK11 bound about 18 pmol of [59Fe]ferriaerobactin per ml (in an assay containing 102 pmol of [59Fe]ferriaerobactin and 200 ,ug of mem-
J. BACTERIOL.
brane protein per ml). However, subsequent addition of 10,200 pmol of non-radioactive ferriaerobactin per ml caused no detectable release of the label. Because ferriaerobactin also is unable to exchange with [59Fe]ferrischizokinen (indicating its probable inability to bind to the ferrischizokinen receptor) these results are interpreted to mean that the membrane vesicles lack specific receptors for ferriaerobactin. Transport of ferriaerobactin appears unlikely, explaining the lack of ferriaerobactin utilization by whole cells. DISCUSSION An interesting aspect of microbial iron transport is the number of substances that can serve as siderophores (iron transport cofactors). Many organisms have used the ferric chelating capacity of secondary hydroxamic acids; however, the natural hydroxamate siderophores can be divided into several structurally related families. Some organisms can utilize more than one ferrihydroxamate. The transport of ferrihydroxamates in B. megaterium probably involves an initial interaction between the ferrihydroxamate and specific receptors at the cell surface. The present work revealed separate receptors for ferrischizokinen and ferriferrioxamine B, implying independent transport systems for these siderophores. Membrane vesicles of both strains rapidly accumulated the radioiron-labeled chelates ferrischizokinen and ferriferrioxamine B by processes that appear to be temperature and energy independent. Such accumulation is thought to represent only binding of the chelates without transport. Limited studies with a doubly labeled chelate, [3H, 59Fe]ferrischizokinen, suggested that the chelate was bound as an intact unit. It is not clear why the membrane vesicles were unable to transport the ferrihydroxamates, because whole cells and protoplasts of the strains do transport the two siderophores (2, 3). It is possible that certain membraneassociated or cytoplasmic components, essential for such transport, are lost during the preparation of membrane vesicles. Binding studies in other systems (for example, see reference 20) have shown that membrane-bound radioactive ligand can be rapidly dissociated from its specific receptor by the addition of some chemically similar non-radioactive ligands. Therefore, the capacity of various structurally different ferrihydroxamates to cause the dissociation of bound [59Fe]ferrischizokinen and [59Fe]ferriferrioxamine B was used to establish the specificity of the binding sites. In the case of bound [59Fe]ferrischizo-
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B. MEGATERIUM SIDEROPHORE RECEPTOR SPECIFICITY
kinen, ferriaerobactin, ferrirhodotorulic acid, ferriferrioxamine B, and ferriA22765 caused no measurable release of the chelate. On the other hand, non-radioactive ferrischizokinen was rapidly exchanged (91%) with membrane-associated [59Fe]ferrischizokinen. Low-level (11%) release of [59Fe]ferrischizokinen was effected by ferriferrichrome A, but the reason for this is uncertain. It is clear that the ferrischizokinen receptor has marked specificity for this ferrihydroxamate. Similar studies of the association of [59Fe]ferriferrioxamine B with membrane vesicles revealed a rapid exchange (92%) of this labeled chelate with ferriferrioxamine B and, to a lesser extent (67%), ferriA22765, a structural relative of ferriferrioxamine B. It is interesting that both ferrirhodotorulic acid and ferriferrichrome A caused significant release (29 and 25%) of bound [59Fe]ferriferrioxamine B. This suggests binding of these two chelates by the ferriferrioxamine B receptor. Whether or not these two ferrihydroxamates might be normally transported by the ferriferrioxamine B system is unknown; however, the membrane receptor sites with specificity for ferriferrioxamine B appear independent from the system which binds ferrischizokinen. Dissociation attempts with ferriaerobactin yielded interesting results. Previously, it was shown that ferriaerobactin (a structural relative of ferrischizokinen) inhibited the growth of B. megaterium strain SK11 (17). A low-level uptake of [3H, 59Fe]ferriaerobactin by whole cells of strain SKil was noted, although it was suggested that this uptake might represent only binding of the chelate (3). In the present work, membrane vesicles of strain SK11 were capable of binding [59Fe]ferriaerobactin; however, subsequent addition of non-radioactive ferriaerobactin caused no detectable release of the label. This suggests only nonspecific binding of ferriaerobactin and implies that B. megaterium lacks a transport system for ferriaerobactin, explaining previous results obtained in whole cells. The possibility that the dissociation of the iron label from the membrane vesicles was due to the exchange of iron between the bound radioiron-labeled chelates and free non-radioactive ferrihydroxamates was considered. Such a possibility was discounted, partly because of the rapidity of dissociation and by the fact that not all hydroxmates were capable of causing dissociation of bound chelate. Scatchard analyses of [59Fe]ferrihydroxamate binding at several concentrations of chelate also indicated that [59Fe]ferrischizokinen and [59Fe]ferriferrioxamine B were bound by
179
independent sites. The [59Fe]ferrischizokinen receptors appeared to constitute a higher capacity, but lower affinity, binding system when compared to the binding of [59Fe]ferriferrioxamine B by membrane vesicles of both strains of B. megaterium. The accumulation of the two ferrihydroxamates by membrane vesicles is consistent with the physiological mechanism operative in intact cells. Earlier studies in whole cells have shown that the addition of excess deferrihydroxamate will impede the uptake of ferrihy-
droxamate (17); the addition of excess deferrihydroxamate also reduced the binding of [59Fe]ferrihydroxamates by membrane vesicles. It is known that, at high concentrations of ferrihydroxamate, the rate of uptake of ferrischizokinen exceeds the rate of uptake of ferriferrioxamine B in whole cells (17). This may be explained by current findings that the ferrischizokinen receptors are present on the membranes in greater numbers (about eightfold) and that more rapid rates of ferrischizokinen uptake could be predicted at higher concentrations. Additional evidence supporting a physiological role for the receptors may come from the finding that the non-utilizable ferriaerobactin may have no specific receptors on the B. megaterium membrane. Finally, both temperature- and energy-independent binding of ferrischizokinen and ferriferrioxamine B has been noted in whole cells, and these two siderophores do not compete for uptake by protoplasts of strain Ardi (2). Cultivation in elevated iron medium reduced the total number of siderophore receptors by only about 25 to 30%. Even in the presence of high iron concentrations in the growth medium, and in the absence of either ferrischizokinen or ferriferrioxamine B, the two strains continued the synthesis of receptor sites for these ferrihydroxamates. The critical need for continued influx of iron during rapid growth appears to have made it imperative that the cells maintain transport capabilities for both ferrischizokinen and for other ferrihydroxamates which might be present in a natural environment, even when the cells are temporarily satisfied for iron. These studies imply independent transport systems for at least two of the ferrihydroxamates utilized by B. megaterium and suggest the possibility of additional ferrihydroxamate receptors on the membranes. At what point in the iron assimilation process these multiple transport systems might converge (if they do) is not known. Recently, siderophore binding sites were discovered on the outer membrane of Escherichia
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coli and Salmonella typhimurium (11, 15, 16, 22, 23, 26, 27). Some of these sites are involved with, or constitute part of, receptors for certain bacteriophages and colicins. It has been suggested that outer-membrane receptors also may be part of siderophore transport systems. ACKNOWLEDGMENTS This research was supported by Public Health Service research career development award GM29366 (to B.R.B.) from the National Institute of General Medical Science and by Public Health Service research grant CA11886 from the National Cancer Institute. Ferriaerobactin and ferrirhodotorulic acid were prepared by C. G. Gaines and C. V. Sciortino under support from research contract NO1-AM-42226 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. We thank J. B. Neilands for ferriferrichrome A, F. Knusel for ferriA22765, and Ciba-Geigy Pharmaceutical Company for Desferal. Electron micrographs were taken by N. Mansfield. The technical assistance of L. Eubanks is acknowledged, and D. Milner is thanked for typing the manuscript. 1.
2.
3.
4.
5.
6.
7.
LITERATURE CITED Abrams, A. 1965. The release of bound adenosine triphosphate from isolated bacterial membranes and the properties of the solubilized enzyme. J. Biol. Chem: 240:3675-3681. Arceneaux, J. E. L., and B. R. Byers. 1976. Ferric hydroxamate transport without subsequent iron utilization in Bacillus megaterium. J. Bacteriol. 127:1324-1330. Arceneaux, J. E. L., W. B. Davis, D. N. Downer, A. H. Haydon, and B. R. Byers. 1973. Fate of labeled hydroxamates during iron transport from hydroxamateiron chelates. J. Bacteriol. 115:919-927. Arceneaux, J. L., and C. E. Lankford. 1966. A schizokinen (siderochrome) auxotroph ofBacillus megaterium induced with N-methyl-N'-nitro-N-nitrosoguanidine. Biochem. Biophys. Res. Commun. 24:370-375. Atkin, C. L., and J. B. Neilands. 1968. Rhodotorulic acid, a diketopiperazine dihydroxamic acid with growth factor activity. I. Isolation and characterization. Biochemistry 7:3734-3739. Byers, B. R. 1974. Iron transport in gram positive and acid fast bacilli, p. 83-105. In J. B. Neilands (ed.), Microbiol iron metabolism. Academic Press Inc., New York. Byers, B. R., M. V. Powell, and C. E. Lankford. 1967. Iron-chelating hydroxamic acid active in initiation of cell division in Bacillus megaterium. J. Bacteriol.
J. BACTERIOL.
11.
12.
13.
14.
15.
15. 17.
18.
19.
20.
21. 22.
23.
24.
93:286-294. 8. Davis, W. B., and B. R. Byers. 1971. Active transport of iron in Bacillus megaterium: role of secondary hy-
droxamic acids. J. Bacteriol. 107:491-498.
25.
9. Davis, W. B., M. J. McCauley, and B. R. Byers. 1971.
Iron requirements and aluminum sensitivity of an hydroxamic acid-requiring strain of Bacillus megaterium. J. Bacteriol. 105:589-594. 10. Ferrandes, B., C. Frehel, and P. Chaix. 1970. Fractionement et purification des systemes membranaires cytoplasmiques et mesosomiques de Bacillus subtilis. Etude de quelques-unes de leurs proprietes oxydo-
26. 27.
reductrices associees a la chaine respiratorie. Biochim. Biophys. Acta 223:292-308. Frost, G. E., and H. Rosenberg. 1975. Relationship between the tonB locus and iron transport in Escherichia coli. J. Bacteriol. 124:704-712. Garibaldi, J. A., and J. B. Neilands. 1955. Isolation and properties of ferrichrome A. J. Am. Chem. Soc. 77:2429-2430. Gibson, F., and D. I. Magrath. 1969. The isolation and characterization of a hydroxamic acid (aerobactin) produced by Aerobacter aerogenes 62-1. Biochim. Biophys. Acta 192:175-184. Gilvarg, C. 1962. Diaminopimelic acid synthesis in Escherichia coli, p. 848-849. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 5. Academic Press Inc., New York. Hancock, R. E. W., K. Hantke, and V. Braun. 1976. Iron transport in Escherichia coli: involvement of the colicin B receptor and of a citrate-inducible protein. J. Bacteriol. 127:1370-1375. Hantke, K., and V. Braun. 1975. Membrane receptor dependent iron transport in Escherichia coli. FEBS Lett. 49:301-305. Haydon, A. H., W. B. Davis, J. E. L. Arceneaux, and B. R. Byers. 1973. Hydroxamate recognition during iron transport from hydroxamate-iron chelates. J. Bacteriol. 115:912-918. Kaback, H. R. 1971. Bacterial membranes, p. 99-120. In W. B. Jacoby (ed.), Methods in enzymology, vol. 22. Academic Press Inc., New York. Lankford, C. E. 1973. Bacterial assimilation of iron. Crit. Rev. Microbiol. 2:273-331. Lefkowitz, R. J., M. C. Caron, C. Mukherjee, J. Mickey, and R. Tate. 1976. Beta-adrenergic receptors: direct identification and physiological regulation, p. 49-87. In R. J. Beers, Jr., and E. G. Bassett (ed.), Cell membrane receptors for viruses, antigens and antibodies, polypeptide hormones, and small molecules. Raven Press, New York. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Luckey, M., and J. B. Neilands. 1976. Iron transport in Salmonella typhimurium LT-2: prevention, by ferrichrome A, of adsorption of bacteriophages ES18 and ES18.hl to a common cell envelope receptor. J. Bacteriol. 127:1036-1037. Luckey, M., R. Wayne, and J. B. Neilands. 1975. In vitro competition between ferrichrome and phage for the outer membrane T5 receptor complex of Escherichia coli. Biochem. Biophys. Res. Commun. 64:687693. Neilands, J. B. 1973. Microbial iron transport compounds (siderochromes), p. 167-202. In G. I. Eichhorn (ed.), Inorganic biochemistry. Elsevier Press, Amsterdam. Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660-672. Wayne, R., K. Frick, and J. B. Neilands. 1976. Siderophore protection against colicins M, B, V, and Ia in Escherichia coli. J. Bacteriol. 126:7-12. Wayne, R., and J. B. Neilands. 1975. Evidence for common binding sites for ferrichrome compounds and bacteriophage qb80 in the cell envelope ofEscherichia coli. J. Bacteriol. 121:497-503.
JOURNAL OF BACTZRIOLOGY, Apr. 1977, p. 173-180 Copyright X 1977 American Society for Microbiology
Specificity of Siderophore Receptors in Membrane Vesicles of Bacillus megaterium J. E. ASWELL,' A. H. HAYDON, H. R. TURNER, C. A. DAWKINS, J. E. L. ARCENEAUX, AND B. R. BYERS* Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216 Received for publication 19 July 1976
Membrane vesicles of Bacillus megaterium strains SK11 and Ardl bound the ferrischizokinen and ferriferrioxamine B siderophores (iron transport cofactors). An approximately equimolar uptake of both labels of [3H, 59Fe]ferrischizokinen indicated binding of the intact chelate. Binding reached equilibrium in 2 to 5 min, was temperature independent, and was unaltered by the addition of several energy sources. A 91% dissociation of bound [59Fe]ferrischizokinen was achieved in 60 s by the addition of excess ferrischizokinen. Ferriaerobactin, a siderophore which is structurally related to ferrischizokinen, caused no detectable release of bound [59Fe]ferrischizokinen. Of several other ferrihydroxamates tested, only ferriferrichrome A achieved the release (11%) of [59Fe]ferrischizokinen. Rapid dissociation (92%) of bound [59Fe]ferriferrioxamine B by the addition of ferriferrioxamine B was observed, and a 67% release of [59Fe]ferriferrioxamine B was caused by ferriA22765, its structural relative. Ferrischizokinen, ferriferrichrome A, and ferrirhodotorulic acid produced a 6, 25, and 29% dissociation, respectively, of [59Fe]ferriferrioxamine B; ferriaerobactin caused no dissociation. [59Felferriaerobactin was bound by the membranes, but its dissociation was not effected by unlabeled ferriaerobactin, suggesting no specific receptors for this chelate. The respective binding affinity constants and maximal binding capacities of membrane vesicles of strain SK1l were 2 x 107 M-' and 280 pmol per mg of protein for ferrischizokinen and 7 x 107 M-1 and 37 pmol per mg of protein for ferriferrioxamine B. These values in strain Ardl were, respectively, 1.4 x 107 M-' and 186 pmol per mg of protein for ferrischizokinen and 11 x 107 M-' and 23 pmol per mg of protein for ferriferrioxamine B. Separate, specific binding sites (receptors) for ferrischizokinen and ferriferrioxamine B exist on the vesicles. The ferrischizokinen receptors have a lower affinity but a higher binding capacity (eightfold) than that shown by the ferriferrioxamine B receptor. These receptors may be components of independent transport systems. ferrischizokinen is transported across the cell membrane to form a pool of available iron within the cytoplasm (2, 3). Ferriaerobactin, another member of the citrate-hydroxamate family (Fig. 1), does not function as a siderophore in B. megaterium (3, 17); however, ferriferrioxamine B, a ferrioxamine-type siderophore (Fig. 1), does supply iron to B. megaterium (4, 7, 8). An examination of ferrischizokinen and ferriferrioxamine B uptake suggested independent transport systems (3, 17). The specificity noted in ferrihydroxamate utilization might reside in membrane receptors, in the transport process, or in the factors responsible for release of iron from the chelates. Chelate transport can be separated from iron assimilation because B. megaterium Ardl transported ' Present address: Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, VA ferriferrioxamine B but could not release iron from the chelate for metabolic use (2). (The 24060.
The efficient uptake of iron by many microorganisms requires the participation of siderophores (6, 19, 24), which are defined as ironchelating agents functioning in the transport of iron to the cell and/or into the cell (19). The natural secondary hydroxamic acids comprise a major chemical category of siderophores, and several structurally different families of hydroxamate siderophores have been identified in various microorganisms (24). Bacillus megaterium ATCC 19213 (American Type Culture Collection) excretes deferrischizokinen, a member of the citrate-hydroxamate family of siderophores (Fig. 1). Studies of B. megaterium SK11 (a strain that is unable to produce deferrischizokinen) showed that externally formed
173
174
ASWELL ET AL.
O II
0 II
H
/ NHNI
(CH2)2-N-C-CHs ii
CH2
HO-C-COOH
C/2 \
C H O
J. BACTERIOL. / \HNC \(CH2)4-N-C-CH3 CH,2H
HO-C-COzH
OHO NHs IN CH ACH,)a-N-C .CH2N-C H3 H
C/2
OH 0 \c,NHN (CH2)4-N-C-CH3 H II 0 CO2H
DEFERRISCHIZOKINEN
DEFERRIAEROBACTIN
COHN
H2-N
CO,H
CONH
(CHz)5 (CH2)2 (CH2)5 (CH2)2 (CH2)5 CH3 N-C II HOO
N-C 11 HO O
N-C 11 HO O
DEFERRIFERRIOXAMINE 8
FIG. 1. Structures of ferrischizokinen, ferriaerobactin, and ferriferrioxamine B. From reference 24.
ferric chelates and the iron-free forms of the siderophores are indicated by the prefixes ferriand deferri-, respectively.) The present paper will present evidence for separate receptors for ferrischizokinen and ferriferrioxamine B (possibly initial components of independent transport systems) in membrane vesicles of B. megaterium strains SK11 and Ardl. The ferrischizokinen receptors do not accept the structurally related siderophore ferriaerobactin, and no specific receptors for ferriaerobactin were detected. MATERIALS AND METHODS Bacterial strains and culture medium. Bacillus megaterium strains SK1l and Ardl were used in this study. Strain SK1l is a mutant that was isolated from B. megaterium ATCC 19213 by Arceneaux and Lankford (4); this strain is unable to synthesize deferrischizokinen, the secondary hydroxamate. Strain Ardl is a spontaneous mutant of strain SK11; unlike its parent, strain Ardl is resistant to the hydroxamate antibiotic ferriA22765 (8). Strain Ardl also has lost the capacity to utilize ferriferrioxamine B. Both strains were maintained in the lyophilized state and on brain heart infusion agar slants. The preparation of the sucrose-mineral salts medium and methods for treatment of this medium with Chelex-100 to reduce trace metal contamination have been described (9). Water used for the preparation of medium and reagents and for cleaning glassware was first purified by passage through a reverse osmosis-demineralizing unit (Culligan model RDS1) and then distilled in an all-glass water still. All plasticware and glassware were soaked for 6 or more h in 0.025 M ethylenediaminetetraacetic acid. The ethylenediaminetetraacetic acid was removed by extensive rinsing with the purified water. Preparation of membrane vesicles. Membrane vesicles were prepared by modification of previously used methods (18). B. megaterium strains SK11 and Ardl were harvested from brain heart infusion agar slants that had been previously incubated at 37°C for 6 to 8 h. A 100-ml portion of Chelex-treated,
sucrose-mineral salts medium (9) containing 0.2 ,ug of Fe per ml was inoculated at an initial cell density of 106 colony-forming units per ml. The culture was shaken at 37°C (Gyrotory Shaker, New Brunswick) until the population reached 108 colony-forming units per ml. The cells were harvested and washed three times by centrifugation in 10 ml of Chelextreated medium containing no trace metal supplements. These cells were then used to inoculate an initial level of 106 colony-forming units per ml of medium containing 0.005 ,ug of Fe2+ per ml ("lowiron conditions") or 2 ,ug of Fe2+ per ml ("high-iron conditions"). This culture was incubated with shaking at 37°C until the population reached about 5 x 107 colony-forming units per ml. The cells then were harvested by centrifugation, and the cells obtained from 1 liter were equally divided into three Erlenmeyer flasks (500 ml), each of which contained 100 ml of 0.5 M Chelex-treated sucrose in 0.05 M Chelextreated potassium phosphate buffer (pH 7) with 20 ,ug of Mg2+ per ml, 2 ,ug of Mn2+ per ml, 48 ,ug of Na+ (Na2SO4) per ml, and 200 ug of lysozyme (Sigma Chemical Co.) per ml. The flasks were shaken gently in a water bath at 37°C for 15 min. Conversion to protoplasts was monitored by light microscopy. When conversion was greater than 90%, the suspension of protoplasts was poured into centrifuge cups, chilled in ice, and centrifuged at 14,500 x g for 20 min at 5°C. The resulting pellet of protoplasts was lysed with cold 0.05 M potassium phosphate buffer containing 20 ,ug of Mg2+ per ml, 5 ,ug of deoxyribonuclease (Sigma) per ml, and 10 ,ug of ribonuclease (Sigma) per ml. After centrifugation at 121 x g for 10 min at 5°C to remove whole cells, the vesiclecontaining supernatant was centrifuged at 34,800 x g for 30 min at 5°C, and the pellet was resuspended in 0.05 M Chelex-treated potassium phosphate buffer (pH 7) containing 20 ,ug of Mg2+ per ml. These vesicles were either stored in packed ice or frozen at -70°C until used. An average of 12 to 18 mg of protein, as determined by the method of Lowry et al. (21), was obtained from 1 liter ofculture as described above. Electron microscopy of these vesicles (fixed with 1% glutaraldehyde and stained with uranylacetate and lead citrate) revealed bilayer membrane sacs of varying diameter. Diaminopimelic acid assays for cell wall contamination (14) indicated only about 5 ,ug of diaminopimelic acid per mg of membrane protein. On the day of preparation, membrane vesicles displayed reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3) activity (10) with a usual specific activity of about 97 nmol of reduced nicotinamide adenine dinucleotide oxidized per min per mg of protein; however, by day 6 of storage at 0WC, this activity declined to 70 nmol of reduced nicotinamide adenine dinucleotide oxidized per min per mg of protein. Immediately after preparation, membrane vesicles showed an adenosine triphosphatase (EC 3.6.1.3) activity (1) of about 0.7 nmol per min per mg of protein, but this activity was reduced by 60% on day 3 of storage at 0°C. Succinate dehydrogenase (EC 1.3.99.1) activity (10) reduced 73 nmol of dichlorophenolindophenol per mg of membrane protein on the day of preparation.
VOL. 130, 1977
B. MEGATERIUM SIDEROPHORE RECEPTOR SPECIFICITY
Siderophore accumulation by membrane vesicles. The uptake of [59Felferrihydroxamates and doubly labeled [3H, 59Fe]ferrischizokinen by membrane vesicles was determined according to the following procedures. The chelates were formed by mixing 59FeCI3 (5 to 20 mCi per mg of Fe, New England Nuclear, Boston, Mass.) and deferrischizokinen, [3Hldeferrischizokinen (52 mCi/mmol), or Desferal (the methane sulfonate of deferriferrioxamine B) at a molar ratio of 2:1 (hydroxamate-iron) and allowing 1 to 2 h for chelate formation at room temperature. Membrane vesicles (either used immediately after preparation or rapidly thawed from -70°C storage) were diluted to 1 mg of membrane protein per ml in 0.05 M potassium phosphate buffer (pH 7.0) containing 20 ug of Mg per ml. One milliliter of the vesicle suspension was then added to an Erlenmeyer flask (125 ml), and the volume was brought to 4.9 ml with the above buffer. The final concentration of membrane protein was 200 ,lg/ml. The flasks were placed in a water bath at 37°C and gently shaken. After temperature equilibration, the assays were initiated by adding the desired concentration of labeled ferrihydroxamate in 0.1 ml to the vesicle suspension. At timed intervals 0.2-ml samples were removed and rapidly filtered (0.45-,um pore size filters; Millipore Corp., Bedford, Mass.). The filters were washed with 10 ml of 0.05 M phosphate buffer and, after drying, assayed for 59Fe and/ or 3H content by liquid scintillation spectrophotometry using Aquasol (New England Nuclear) in a Packard scintillation spectrometer (model 3003). Appropriate controls containing no membrane vesicles were included to assay for the nonspecific retention of label by the filters. The number of picomoles of ferric hydroxamate accumulated by the vesicles were calculated from the specific activity of the radioactive iron or from the specific activity of the
transport system in strain Ardl is functional because this chelate appears in the cytoplasmic fraction of protoplasts exposed to this chelate (2); however, strain Ardl is not capable of separating iron from ferriferrioxamine B for metabolic use. It was of interest to compare the uptake of the two chelates, ferrischizokinen and ferriferrioxamine B, by membrane vesicles prepared from both strains by osmotic rupture of protoplasts. Initial observations showed that membrane vesicles of B. megaterium strains Ardl and SK11 (prepared from protoplasts of cells grown at low-iron conditions; see Materials and Methods) accumulated both [59Fe]ferrischizokinen and [59Fe]ferriferrioxamine B. Their uptake by the vesicles was rapid, usually reaching equilibrium between 2 and 5 min of the assay (for example, Fig. 2). Vesicles prepared from either strain accumulated more [59Fe]ferrischizokinen than [59Fe]ferriferrioxamine B (described in detail below).
30-
z
w
z
25-
N
[3H]deferrischizokinen.
Sources of siderophores. Deferrischizokinen and
175
w
E 15-;
[3Hldeferrischizokinen were prepared by previously
described methods (3, 7). Desferal was a gift of CibaGeigy Pharmaceutical Co. The hydroxamate antibiotic ferriA22765 was supplied by F. Knusel, CibaGeigy Pharmaceutical Company, Basel, Switzerland. Deferriferrichrome A, deferrirhodotorulic acid, and deferriaerobactin were purified from culture filtrates of Ustilago sphaerogena, Rhodotorula utilis, and Aerobacter aerogenes strain 62-1, respectively, by previously determined methods (5, 12, 13).
0:
2
RESULTS Binding of ferrihydroxamates by membrane vesicles. Neither of the two strains of B. megaterium (SK1l and Ardl) used in this study synthesizes deferrischizokinen, the hydroxamate produced by B. megaterium ATCC 19213, and iron uptake by both strains is stimulated by ferrischizokinen (2, 8). Unlike strain SKil, strain Ardl cannot utilize the hydroxamate ferriferrioxamine B. The ferriferrioxamine B
4
6
8
10
MIN UTES FIG. 2. Binding of [59Fe]ferrischizokinen by membrane vesicles of B. megaterium strain Ardl and dissociation of bound [59Fe]ferrischizokinen by the addition of non-radioactive ferrischizokinen. Assay contained initially 102 pmol of [59Fe]ferrischizokinen per ml and 200 pg of membrane vesicle protein per ml in 0.05 M Chelex-treated potassium phosphate buffer (pH 7). At 5 min, 10,200 pmol of non-radioactive ferrischizokinen per ml was added to 50% of the assay sample (dashed line). The control (solid line) received equal volume of buffer.
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ASWELL ET AL.
The accumulation of radioiron added as the ferrihydroxamate represented the uptake of the chelate as an intact unit. A small quantity of [3H]deferrischizokinen was available for testing, and the simultaneous uptake of a doubly labeled chelate, [3H, 59Fe]ferrischizokinen, was followed in vesicles of strain SK11. A rapid uptake of both labels occurred; maximal levels were reached within 2 min. The picomole quantities of ligand and metal that had accumulated were approximately equivalent, suggesting accumulation of the intact chelate. As measurement of 59Fe uptake was indicative of ferrihydroxamate binding, the remainder of the work reported here utilized only [59Fe]ferrihydroxamates. Competition between ferrischizokinen and deferrischizokinen for the transport system in whole cells has been reported (17). In the present assays, the addition of deferrischizokinen at a deferrihydroxamate:59Fe ratio of 15:1 caused a reduction in radioiron accumulation by membrane vesicles of both strains Ardl and SK11 to less than 50% of that seen at lower deferrischizokinen:59Fe ratios. Thus, the accumulation of ferrischizokinen by membrane vesicles resembled ferrischizokinen transport by whole cells in that the ferri and deferri forms of schizokinen appear to compete. Similar additions of excess deferriferrioxamine B reduced the accumulation of [59Fe]ferriferrioxamine B by membrane vesicles of both strains. Because deferriferrioxamine B also impedes the transport of its ferric chelate in whole cells (17), it is suggested that [59Fe]ferrihydroxamate binding by membrane vesicles was accomplished by components of the transport processes. The assays described above were conducted in Chelex-treated potassium phosphate buffer (0.05 M, pH 7). Identical results were obtained after the addition of the following energy sources: 15 mM sucrose, 3.9 mM adenosine 5'triphosphate, 9 mM 1)-lactate, 0.14 mM reduced nicotinamide adenine dinucleotide or 20 mM succinate plus 20 mM ascorbate with 0.1 mM phenazine methosulfate. All of these energy sources failed to stimulate uptake of the radioiron-labeled chelates by membrane vesicles. Moreover, the addition of 0.1 M sodium azide to assays containing the energy sources had no measurable effect on accumulation of the [59Fe]ferrihydroxamates. Identical uptake curves were obtained at 0, 25, and 37°C. These results appear to exclude active transport as the system for [59Felferrihydroxamate accumulation by membrane vesicles of both strains. Although such uptake appeared independent of temperature, the possibility that a facilitated diffusion-type process might be operating was
J. BACTERIOL.
considered. It was found that a fourfold dilution of membrane vesicles previously exposed to [59Fe]ferrischizokinen or [59Fe]ferriferrioxamine B did not cause a rapid release of the label, suggesting that the chelates were not subjet to immediate efflux as might be predicted with a facilitated diffusion-type system. These data indicate that accumulation of the two ferrihydroxamates by membrane vesicles of the two strains represented binding without transport. Dissociation of bound ferrihydroxamates. Evidence for specific receptors. If binding of ferrihydroxamates by the membrane vesicles represents an association between the chelates and specific receptor sites, then the addition of excess non-radioactive ferrihydroxamate (after equilibration between [59Felferrihydroxamate and the membrane vesicles) would result in a rapid exchange of the non-radioactive and radioiron-labeled chelates at the specific receptor. On the other hand, if the chelates are held by relatively nonspecific binding sites, rapid exchange should not occur. It has been shown in a number of membrane systems that excess nonradioactive ligand will both block the binding of radioactive ligand to a specific receptor and cause a rapid release of radioactive ligand that is bound to a specific receptor (for example, see reference 20). When the binding of [59Fe]ferrischizokinen by membrane vesicles of strain Ardl was allowed to reach equilibrium (5 min) and nonradioactive ferrischizokinen was added at 100 times the initial concentration of this chelate, more than 90% of the bound label was released within 60 s after the addition of non-radioactive ferrischizokinen (Fig. 2). The capacity of various structurally different ferrihydroxamates to cause the dissociation of [59Fe]ferrischizokinen from membrane vesicles should represent a measure of the specificity of the binding site. The dissociation of bound [59Fe]ferrischizokinen from vesicles of strain Ardl by ferriferrioxamine B, ferriferrichrome A, ferriaerobactin, ferrirhodotorulic acid, and ferriA22765 was tested (Table 1). Of these ferrihydroxamates, only ferriferrichrome A effected some release of [59Fe]ferrischizokinen, showing 11% dissociation of the label by 2 min. It was interesting that the structurally related siderophore ferriaerobactin caused no dissociation. The ferrischizokinen binding component(s) present on the membranes displayed a marked specificity for ferrischizokinen. Similar to the ferrischizokinen binding system, 92% of bound [59Fe]ferriferrioxamine B could be dissociated rapidly by the addition of non-radioactive ferriferrioxamine B (Table 1).
VOL. 130, 1977
B. MEGATERIUM SIDEROPHORE RECEPTOR SPECIFICITY
The ferrischizokinen chelate caused the release of only 6% of bound [59Fe]ferriferrioxamine B. FerriA22765, which is thought to be structurally similar to ferriferrioxamine B, initiated an exchange with [59Fe]ferriferrioxamine B, showing a release of 67% of the label at 2 min. Both ferriferrichrome A and ferrirhodotorulic acid also showed some apparent exchange with [59Felferriferrioxamine B, causing a release of 25 and 29%, respectively, of the label. Ferriaerobactin caused no removal of [59Felferriferrioxamine B. These results suggest that ferriferrioxamine B binding sites are present on the membrane and that they may have an affinity for a number of other hydroxamates. Estimation of total numbers of receptor sites and affinity constants. Scatchard analysis is well suited for describing both the affinity and maximal binding capacity, at equilibrium, of protein receptors for small molecules (25). Accordingly, the specific binding of [59Fe]ferriferrioxamine B and [59Fe]ferrischizokinen to membrane vesicles of both strains was determined at chelate concentrations from 8 to 330
177
pmol/ml. Specific binding of the chelates to the receptor sites was determined by subtracting the amount of nonspecifically associated chelate from the total chelate bound by the vesicles. Nonspecifically bound chelate (i.e., ferrihydroxamate associated with sites other than the receptor) is defined as the [59Fe]ferrihydroxamate that is membrane associated in the presence of a 100-fold concentration of non-radioactive ferrihydroxamate. It has been shown in other systems that some radioactive ligand binding cannot be blocked by additions of large excesses of unlabeled ligand; this binding is considered to be nonspecific (20). A typical Scatchard plot of data obtained in membrane vesicles of strain Ardl is shown in Fig. 3. Differences in both the affinity constants (Ka, calculated from the slopes of the lines) and
TABLE 1. Dissociation of bound
[59Fe]ferrihydroxamates from membrane vesicles of B. megaterium Ardl by non-radioactive
ferrihydroxamates % Dissociation of:°
Non-radioactive fernhydroxamnate addeda
[59Fe]fer- [59Felferririschizokinen
ferrioxamine B
Ferrischizokinen 91 6 Ferriferrioxamine B 0 92 Ferriferrichrome A 11 25 Ferriaerobactin 0 0 Ferrirhodotorulic acid 0 29 FerriA22765 0 67 a Added at 100 times the initial concentration (102 pmol/ml) of radioiron-labeled ferrihydroxamate. Assays contained 200 ,ug of protein per ml. b Dissociation of [59Felferrihydroxamate determined 120 s after the addition of non-radioactive ferrihydroxamate.
20 30 moles BOUND FIG. 3. Scatchard analyses of specific binding of [5"Fe]ferrischizokinen (open circle) and [59Felferriferrioxamine B (closed circle) by membrane vesicles of B. megaterium strain Ardl (200 pg of membrane protein per ml) at ferrihydroxamate concentrations from 8 to 330 pmol/ml. p
TABLE 2. Affinity constants and maximal binding capacities of membrane vesicles of B. megaterium strains for [59Fe]ferrischizokinen and [59Felferriferrioxamine B a
K.
Maximal binding (pmol/mg of protein)
Strain
[59Fe]ferrischizokinen
[59Fe]ferriferrioxamine B
[59Fe]ferrischi["Fe]ferriferzokinen rioxamine B
280 7.0 x 107 M-l 37 186 11.0 X 107 M-' 23 a Affinity constants (Ka) and maximal binding capacities were calculated from Scatchard plots of specific binding at 8 to 330 pmol of ferrihydroxamate per ml and 200 lAg of membrane vesicle protein per ml (prepared from cells grown in low-iron conditions).
SK11 Ardl
2.0 x 107 M-1 1.4 x 107 M-l
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ASWELL ET AL.
the maximal binding capacities (X intercepts) of the membrane vesicles for the two ferrihydroxamates were apparent. Such plots were used to produce the data summarized in Table 2. In vesicles from both strains, the [59Fe]- ferrischizokinen binding system had less affinity for. [59Fe]ferrischizokinen than the [59Fe]ferriferrioxamine B binding system showed for its chelate; however, the membrane sites binding [59Fe]ferrioxamine B were fewer in number than the [59Fe]ferrischizokinen accepting sites. Thus, the [59Fe]ferrischizokinen binding sites appeared to have a higher capacity, but lower affinity, when compared to those sites that bound [59Fe]ferriferrioxamine B. Effect of iron on synthesis of siderophore receptors. The above information was collected using membrane vesicles prepared from cells grown in "low-iron medium" (see Materials and Methods). Because elevated concentrations of iron in the growth medium will lower the production of deferrischizokinen in wild-type B. megaterium (7) and because cells that have been cultivated in "high-iron medium" show a reduced capacity to transport ferrihydroxamates (J. E. L. Arceneaux, unpublished observation), it was of interest to determine the effect of the iron concentration in the growth medium on the binding capacity of membrane vesicles. Scatchard analyses of the association of high-iron membrane vesicles of strain SK1l with [59Fe]ferrihydroxamates produced a K,, for [59Fe]ferrischizokinen of 2.0 x 107 M-1 and a maximal binding capacity of 198 pmol per mg of membrane protein. This K, is identical to that observed in low-iron membrane vesicles (Table 2), and the total binding capacity was lowered by only 30%. The K,, of high-iron membrane vesicles for [59Fe]ferriferrioxamine B was 6.7 x 107 M-1, and these vesicles bound 28 pmol of [59Fe]ferriferrioxamine B per mg of membrane protein. This Ka is essentially identical to that obtained with low-iron vesicles (Table 2), and the total binding capacity was lowered by 25%. These data reveal the presence of large numbers of binding sites for both ferrischizokinen and ferriferrioxamine B in membrane vesicles of cells that have been cultivated in a medium containing excess iron and suggest that synthesis of the ferrihydroxamate receptors may continue even when the cells are replete for iron. Binding of [59Felferriaerobactin. Lack of specific receptors. Although ferriaerobactin is structurally related to ferrischizokinen (Fig. 1), ferriaerobactin is not utilized by B. megaterium SK1l (17). Membrane vesicles of strain SK11 bound about 18 pmol of [59Fe]ferriaerobactin per ml (in an assay containing 102 pmol of [59Fe]ferriaerobactin and 200 ,ug of mem-
J. BACTERIOL.
brane protein per ml). However, subsequent addition of 10,200 pmol of non-radioactive ferriaerobactin per ml caused no detectable release of the label. Because ferriaerobactin also is unable to exchange with [59Fe]ferrischizokinen (indicating its probable inability to bind to the ferrischizokinen receptor) these results are interpreted to mean that the membrane vesicles lack specific receptors for ferriaerobactin. Transport of ferriaerobactin appears unlikely, explaining the lack of ferriaerobactin utilization by whole cells. DISCUSSION An interesting aspect of microbial iron transport is the number of substances that can serve as siderophores (iron transport cofactors). Many organisms have used the ferric chelating capacity of secondary hydroxamic acids; however, the natural hydroxamate siderophores can be divided into several structurally related families. Some organisms can utilize more than one ferrihydroxamate. The transport of ferrihydroxamates in B. megaterium probably involves an initial interaction between the ferrihydroxamate and specific receptors at the cell surface. The present work revealed separate receptors for ferrischizokinen and ferriferrioxamine B, implying independent transport systems for these siderophores. Membrane vesicles of both strains rapidly accumulated the radioiron-labeled chelates ferrischizokinen and ferriferrioxamine B by processes that appear to be temperature and energy independent. Such accumulation is thought to represent only binding of the chelates without transport. Limited studies with a doubly labeled chelate, [3H, 59Fe]ferrischizokinen, suggested that the chelate was bound as an intact unit. It is not clear why the membrane vesicles were unable to transport the ferrihydroxamates, because whole cells and protoplasts of the strains do transport the two siderophores (2, 3). It is possible that certain membraneassociated or cytoplasmic components, essential for such transport, are lost during the preparation of membrane vesicles. Binding studies in other systems (for example, see reference 20) have shown that membrane-bound radioactive ligand can be rapidly dissociated from its specific receptor by the addition of some chemically similar non-radioactive ligands. Therefore, the capacity of various structurally different ferrihydroxamates to cause the dissociation of bound [59Fe]ferrischizokinen and [59Fe]ferriferrioxamine B was used to establish the specificity of the binding sites. In the case of bound [59Fe]ferrischizo-
VOL. 130, 1977
B. MEGATERIUM SIDEROPHORE RECEPTOR SPECIFICITY
kinen, ferriaerobactin, ferrirhodotorulic acid, ferriferrioxamine B, and ferriA22765 caused no measurable release of the chelate. On the other hand, non-radioactive ferrischizokinen was rapidly exchanged (91%) with membrane-associated [59Fe]ferrischizokinen. Low-level (11%) release of [59Fe]ferrischizokinen was effected by ferriferrichrome A, but the reason for this is uncertain. It is clear that the ferrischizokinen receptor has marked specificity for this ferrihydroxamate. Similar studies of the association of [59Fe]ferriferrioxamine B with membrane vesicles revealed a rapid exchange (92%) of this labeled chelate with ferriferrioxamine B and, to a lesser extent (67%), ferriA22765, a structural relative of ferriferrioxamine B. It is interesting that both ferrirhodotorulic acid and ferriferrichrome A caused significant release (29 and 25%) of bound [59Fe]ferriferrioxamine B. This suggests binding of these two chelates by the ferriferrioxamine B receptor. Whether or not these two ferrihydroxamates might be normally transported by the ferriferrioxamine B system is unknown; however, the membrane receptor sites with specificity for ferriferrioxamine B appear independent from the system which binds ferrischizokinen. Dissociation attempts with ferriaerobactin yielded interesting results. Previously, it was shown that ferriaerobactin (a structural relative of ferrischizokinen) inhibited the growth of B. megaterium strain SK11 (17). A low-level uptake of [3H, 59Fe]ferriaerobactin by whole cells of strain SKil was noted, although it was suggested that this uptake might represent only binding of the chelate (3). In the present work, membrane vesicles of strain SK11 were capable of binding [59Fe]ferriaerobactin; however, subsequent addition of non-radioactive ferriaerobactin caused no detectable release of the label. This suggests only nonspecific binding of ferriaerobactin and implies that B. megaterium lacks a transport system for ferriaerobactin, explaining previous results obtained in whole cells. The possibility that the dissociation of the iron label from the membrane vesicles was due to the exchange of iron between the bound radioiron-labeled chelates and free non-radioactive ferrihydroxamates was considered. Such a possibility was discounted, partly because of the rapidity of dissociation and by the fact that not all hydroxmates were capable of causing dissociation of bound chelate. Scatchard analyses of [59Fe]ferrihydroxamate binding at several concentrations of chelate also indicated that [59Fe]ferrischizokinen and [59Fe]ferriferrioxamine B were bound by
179
independent sites. The [59Fe]ferrischizokinen receptors appeared to constitute a higher capacity, but lower affinity, binding system when compared to the binding of [59Fe]ferriferrioxamine B by membrane vesicles of both strains of B. megaterium. The accumulation of the two ferrihydroxamates by membrane vesicles is consistent with the physiological mechanism operative in intact cells. Earlier studies in whole cells have shown that the addition of excess deferrihydroxamate will impede the uptake of ferrihy-
droxamate (17); the addition of excess deferrihydroxamate also reduced the binding of [59Fe]ferrihydroxamates by membrane vesicles. It is known that, at high concentrations of ferrihydroxamate, the rate of uptake of ferrischizokinen exceeds the rate of uptake of ferriferrioxamine B in whole cells (17). This may be explained by current findings that the ferrischizokinen receptors are present on the membranes in greater numbers (about eightfold) and that more rapid rates of ferrischizokinen uptake could be predicted at higher concentrations. Additional evidence supporting a physiological role for the receptors may come from the finding that the non-utilizable ferriaerobactin may have no specific receptors on the B. megaterium membrane. Finally, both temperature- and energy-independent binding of ferrischizokinen and ferriferrioxamine B has been noted in whole cells, and these two siderophores do not compete for uptake by protoplasts of strain Ardi (2). Cultivation in elevated iron medium reduced the total number of siderophore receptors by only about 25 to 30%. Even in the presence of high iron concentrations in the growth medium, and in the absence of either ferrischizokinen or ferriferrioxamine B, the two strains continued the synthesis of receptor sites for these ferrihydroxamates. The critical need for continued influx of iron during rapid growth appears to have made it imperative that the cells maintain transport capabilities for both ferrischizokinen and for other ferrihydroxamates which might be present in a natural environment, even when the cells are temporarily satisfied for iron. These studies imply independent transport systems for at least two of the ferrihydroxamates utilized by B. megaterium and suggest the possibility of additional ferrihydroxamate receptors on the membranes. At what point in the iron assimilation process these multiple transport systems might converge (if they do) is not known. Recently, siderophore binding sites were discovered on the outer membrane of Escherichia
180
ASWELL ET AL.
coli and Salmonella typhimurium (11, 15, 16, 22, 23, 26, 27). Some of these sites are involved with, or constitute part of, receptors for certain bacteriophages and colicins. It has been suggested that outer-membrane receptors also may be part of siderophore transport systems. ACKNOWLEDGMENTS This research was supported by Public Health Service research career development award GM29366 (to B.R.B.) from the National Institute of General Medical Science and by Public Health Service research grant CA11886 from the National Cancer Institute. Ferriaerobactin and ferrirhodotorulic acid were prepared by C. G. Gaines and C. V. Sciortino under support from research contract NO1-AM-42226 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. We thank J. B. Neilands for ferriferrichrome A, F. Knusel for ferriA22765, and Ciba-Geigy Pharmaceutical Company for Desferal. Electron micrographs were taken by N. Mansfield. The technical assistance of L. Eubanks is acknowledged, and D. Milner is thanked for typing the manuscript. 1.
2.
3.
4.
5.
6.
7.
LITERATURE CITED Abrams, A. 1965. The release of bound adenosine triphosphate from isolated bacterial membranes and the properties of the solubilized enzyme. J. Biol. Chem: 240:3675-3681. Arceneaux, J. E. L., and B. R. Byers. 1976. Ferric hydroxamate transport without subsequent iron utilization in Bacillus megaterium. J. Bacteriol. 127:1324-1330. Arceneaux, J. E. L., W. B. Davis, D. N. Downer, A. H. Haydon, and B. R. Byers. 1973. Fate of labeled hydroxamates during iron transport from hydroxamateiron chelates. J. Bacteriol. 115:919-927. Arceneaux, J. L., and C. E. Lankford. 1966. A schizokinen (siderochrome) auxotroph ofBacillus megaterium induced with N-methyl-N'-nitro-N-nitrosoguanidine. Biochem. Biophys. Res. Commun. 24:370-375. Atkin, C. L., and J. B. Neilands. 1968. Rhodotorulic acid, a diketopiperazine dihydroxamic acid with growth factor activity. I. Isolation and characterization. Biochemistry 7:3734-3739. Byers, B. R. 1974. Iron transport in gram positive and acid fast bacilli, p. 83-105. In J. B. Neilands (ed.), Microbiol iron metabolism. Academic Press Inc., New York. Byers, B. R., M. V. Powell, and C. E. Lankford. 1967. Iron-chelating hydroxamic acid active in initiation of cell division in Bacillus megaterium. J. Bacteriol.
J. BACTERIOL.
11.
12.
13.
14.
15.
15. 17.
18.
19.
20.
21. 22.
23.
24.
93:286-294. 8. Davis, W. B., and B. R. Byers. 1971. Active transport of iron in Bacillus megaterium: role of secondary hy-
droxamic acids. J. Bacteriol. 107:491-498.
25.
9. Davis, W. B., M. J. McCauley, and B. R. Byers. 1971.
Iron requirements and aluminum sensitivity of an hydroxamic acid-requiring strain of Bacillus megaterium. J. Bacteriol. 105:589-594. 10. Ferrandes, B., C. Frehel, and P. Chaix. 1970. Fractionement et purification des systemes membranaires cytoplasmiques et mesosomiques de Bacillus subtilis. Etude de quelques-unes de leurs proprietes oxydo-
26. 27.
reductrices associees a la chaine respiratorie. Biochim. Biophys. Acta 223:292-308. Frost, G. E., and H. Rosenberg. 1975. Relationship between the tonB locus and iron transport in Escherichia coli. J. Bacteriol. 124:704-712. Garibaldi, J. A., and J. B. Neilands. 1955. Isolation and properties of ferrichrome A. J. Am. Chem. Soc. 77:2429-2430. Gibson, F., and D. I. Magrath. 1969. The isolation and characterization of a hydroxamic acid (aerobactin) produced by Aerobacter aerogenes 62-1. Biochim. Biophys. Acta 192:175-184. Gilvarg, C. 1962. Diaminopimelic acid synthesis in Escherichia coli, p. 848-849. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 5. Academic Press Inc., New York. Hancock, R. E. W., K. Hantke, and V. Braun. 1976. Iron transport in Escherichia coli: involvement of the colicin B receptor and of a citrate-inducible protein. J. Bacteriol. 127:1370-1375. Hantke, K., and V. Braun. 1975. Membrane receptor dependent iron transport in Escherichia coli. FEBS Lett. 49:301-305. Haydon, A. H., W. B. Davis, J. E. L. Arceneaux, and B. R. Byers. 1973. Hydroxamate recognition during iron transport from hydroxamate-iron chelates. J. Bacteriol. 115:912-918. Kaback, H. R. 1971. Bacterial membranes, p. 99-120. In W. B. Jacoby (ed.), Methods in enzymology, vol. 22. Academic Press Inc., New York. Lankford, C. E. 1973. Bacterial assimilation of iron. Crit. Rev. Microbiol. 2:273-331. Lefkowitz, R. J., M. C. Caron, C. Mukherjee, J. Mickey, and R. Tate. 1976. Beta-adrenergic receptors: direct identification and physiological regulation, p. 49-87. In R. J. Beers, Jr., and E. G. Bassett (ed.), Cell membrane receptors for viruses, antigens and antibodies, polypeptide hormones, and small molecules. Raven Press, New York. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Luckey, M., and J. B. Neilands. 1976. Iron transport in Salmonella typhimurium LT-2: prevention, by ferrichrome A, of adsorption of bacteriophages ES18 and ES18.hl to a common cell envelope receptor. J. Bacteriol. 127:1036-1037. Luckey, M., R. Wayne, and J. B. Neilands. 1975. In vitro competition between ferrichrome and phage for the outer membrane T5 receptor complex of Escherichia coli. Biochem. Biophys. Res. Commun. 64:687693. Neilands, J. B. 1973. Microbial iron transport compounds (siderochromes), p. 167-202. In G. I. Eichhorn (ed.), Inorganic biochemistry. Elsevier Press, Amsterdam. Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660-672. Wayne, R., K. Frick, and J. B. Neilands. 1976. Siderophore protection against colicins M, B, V, and Ia in Escherichia coli. J. Bacteriol. 126:7-12. Wayne, R., and J. B. Neilands. 1975. Evidence for common binding sites for ferrichrome compounds and bacteriophage qb80 in the cell envelope ofEscherichia coli. J. Bacteriol. 121:497-503.
