Eur. J . Biochem. YY, 39-47 (1979)
Iron Supply of Escherichia coli with Polymer-Bound Ferricrocin James W. COULTON, Hans-Ulrich NAEGELI, and Volkmar BRAUN Lehrbereich Mikrobiologie, Universitat Tiibingen (Received March 14, 1979)
Uptake of ferric iron from ferricrocin was studied in Escherichia coli using a polymer-coupled ferricrocin that was unable to penetrate into the cell. Ferricrocinyl polyethylene glycol succinate ( M , 7000-8500) promoted growth of E. coli K-12 AB2847 uroB under iron-limiting conditions. In iron-starved cells, uptake of "Fe could be demonstrated; the amount of iron accumulated amounted to 10% of that observed with free ferricrocin. The iron supply by ferricrocin bound to polyethylene glycol was strictly dependent upon the functions expressed by the tonA and the tonB genes, as was the iron uptake promoted by free ferricrocin. Polymer-bound ferricrocin protected cells against colicin M and phage T5 by competition for the common tonA-coded outer membrane receptor protein. In addition, the rate of iron transport via the negatively charged ferricrocinyl succinate was as fast as via the neutral ferricrocin molecule. No ligand was found associated with the cells. Penetration of chelator beyond receptor is not necessary for siderophoremediated iron uptake. It is concluded that sufficient amounts of iron can be released from the polymer complex to satisfy growth requirements.
In Escherichia coli three high affinity systems for the uptake of ferric iron have been studied [1,2]. They involve chelators (ferrichrome, enterochelin and citrate), receptor proteins in the outer membrane, and additional functions, most likely permeases in the cytoplasmic membrane, each specific for a single iron complex. Common to all three iron uptake systems is the requirement for the tonB function which is also essential for other translocation processes across the outer membrane and cytoplasmic membrane. The tonB gene product has not yet been characterized biochemically. This report concerns one aspect of the uptake of iron mediated by the ferrichrome complex. During the time that iron is being rapidly taken up into the cell, the amount of ligand associated with the cell is low and does not appear to accumulate within the cell [3]. Conversely, a lag phase of about 10 min was reported before a slow increase in incorporation of tritiated ferrichrome [4]. Ferrichrome binds first to a protein of the outer membrane, the product of the tonA gene. Mutants of tonA are unable to bind or to take up iron from ferrichrome. The further pathway of ferrichrome or of iron after its release from the ligand across the outer membrane and cytoplasmic membrane is unknown. To determine whether uptake of the whole complex into the cell is essential for iron uptake or whether iron can be released close to the cell surface, a high molecular weight derivative of
ferrichrome carrying a bulky pendant side chain was synthesized. Ferricrocin is a close structural analogue of ferrichrome and mediates iron uptake with the same rate as ferrichrome by using the tonA, tonBcoded uptake system. One of the glycine residues of the cyclic hexapeptide (glycine)3-(N-hydroxy-N-acetylornithine)3 of ferrichrome is replaced by a serine residue in ferricrocin (Fig. 1A). The hydroxyl group of serine was used to couple a ferricrocin derivative to polyethylene glycol ( M ,6000 - 7500). The biological response of E. coli to this complex was determined with regard to donation of iron, stimulation of growth, and binding specificity to the tonA-coded protein. MATERIALS AND METHODS Synthesis of Ferricrocinyl Succinate Ferricrocin was coupled with succinic anhydride to yield the succinyl monoester. Ferricrocin (200 mg, 0.259 mmol) was combined with 260 mg (2.6 mmol) succinic anhydride in 4 m l absolute pyridine and stirred for 3 h at 60 "C. The reaction mixture was then dried in vucuo and the residue dissolved and chromatographed on a Sephadex LH-20 column in chloroform/methanol (l/l, v/v). An orange-brown powder was recovered (180 mg, 0.207 mmol) with an RF value of 0.22 on silica gel 60 F245 thin-layer plates (Merck, Darmstadt) developed with CHCI3/CH30H/HzO (65/ 25/4, v/v/v); Amax (CHC13) 430 nm; E = 1980 M - '
40
cm-'; infrared spectrum (KBr) with peaks at 1730 cm-', 1680 cm-', 1640 cm-', 1580 cm-'. The deferri form of ferricrocinyl succinate was prepared by extraction of iron with 8-hydroxyquinoline [ 5 ] to give a colourless powder with an RF value of 0.1 on the same type of plates as above developed with CHC13/ CH30H/H20 (65/25/4, v/v/v) ; 6 (dimethylsulphoxided6): 1.62 (broad, 12H); 2.02 (singlet, 9H); 2.41 (singlet, 4H); 3.1 -4.8 (complex, 16H); 8.0-9.0 (complex, 6H); 9.44 (broad, 3H). Synthesis of Ferricrocinyl Polyethylene Glycolyl Succinate
The esterification of ferricrocinyl succinate with poly(ethy1ene glycol) was carried out in a single reaction vessel using a modification of the method of Stadler [6]. An activated dimethyl formamide imidechloride/ferricrocinyl succinate adduct was prepared for reaction with poly(ethy1ene glycol) at room temperature. Dimethyl formamide (24 y1,0.311 mmol) was dissolved in 1 ml absolute CHC13.Then 10 pl(0.113 mmol) oxalyl chloride (Merck, 97% pure) was added with vigorous stirring, followed after 5 min by a solution of 20 mg (23 pmol) ferricrocinyl succinate in 400 pl absolute CHC13 plus three drops of dimethyl formamide. After strong stirring for 3 min, 70 mg poly(ethy1ene glycol) ( M , 6000 - 7500; Serva, Heidelberg) and 20 p1 (0.25 mmol) absolute pyridine were added. The reaction was complete in a few minutes. The reaction mixture showed a diffuse orange spot as major component with an RF value above 0.9 on silica gel thin-layer chromatogram developed with CHC13/CH30H/H20 (65/25/4, v/v/v). There were several unidentified impurities at RF from 0 to 0.2. The reaction mixture was evaporated to dryness, dissolved in water and the crude product ferricrocinpolymer extracted twice with CHC13. Column Chromatography and Analyses
Separation of the products from the reaction mixture utilized the following gel filtration columns : BioGel P-6 (Bio-Rad Laboratories, Richmond, Calif., U.S.A.) run with distilled water, 38 x 1.8-cm column; and Sephadex LH-20 (Pharmacia, Uppsala) eluted with CHC13/CH30H ( l / l , v/v), 36 x 1.8-cm column. Fractions of 1 ml were collected at l-min intervals at room temperature. The eluate was scanned for absorption at 436nm on a Gilford250 spectrophotometer, and all fractions were tested for poly(ethylene glycol) by using the quantitative assay of Ingham and Ling [7] with the following modification to avoid interference with solvent: 50-yl samples of column fractions were mixed with 1 ml of isopropanol/ acetic acidlwater (4/1/1, v/v/v). Then 0.5 ml of alkaline Nessler's reagent (2 parts Merck reagent: 1 part of
Iron Supply of Escherichia coli
4 M KOH) were added with vigorous stirring, and the turbidity measured after incubation at room temperature for 15 min. A standard curve for poly(ethy1ene glycol) alone showed a linear relationship of the absorbance at 578 nm versus poly(ethy1ene glycol) concentration in the region of 5 - 50 yg/ml; ferricrocin did not disturb the test. High-pressure Liquid Chromatography
High-pressure liquid chromatography was performed on a LiChrosorb RP-8 (Merck) column of 7.5-mm internal diameter as described by Fiedler and Sauerbier [13]. The column was first calibrated with 10 pmol free ferricrocin. A linear gradient of water/ acetonitrile from 0 to 100% acetonitrile was used to elute all of the material at a flow rate of 5%/min, corresponding to 4 ml/min. Twenty fractions were collected at l-min intervals. Radioisotopes and Labelling of Sideramine Derivatives 55Fe. To radioactively label ferricrocin and ferricrocinyl succinate, the deferri form of the sideramine was prepared by extraction of iron with 8-hydroxyquinoline. The lyophilized powder was then resuspended in distilled water, and an aliquot incubated with equimolar amounts of 55FeC13(Amersham Buchler ; 1.01 mCi/ml, 150 pg Fe/ml) for 1 h at room temperature. Because iron in the ferricrocin-polymer was not readily extractable by 8-hydroxyquinoline, a solution of carrier-free iron (Amersham Buchler ; 0.43 mCi/ml, 2.1 yg/ml) was incubated with sideramine-polymer. The half-time of exchange of labelled for unlabelled iron is about 8 min, and the above reaction mixture was allowed to stand for 1 h. Excess 55FeC13that was not exchanged was separated on a Sephadex LH-20 column in CHC13/CH30H ( l / l , v/v), or in some instances was scavanged by inclusion of 100 yM nitrilotriacetate (C6H9NO6) pH 7.8, in the bacterial suspension. The iron nitrilotriacetate complex is not taken up by E. coli. 14C.Lyophilized ferricrocin (10 pmol) was labelled with 100 pmol [l ,4-'4C]succinic anhydride (New England Nuclear), specific activity 50 yCi/700 pg. The tenfold excess of succinic anhydride over ferricrocin was required to force the reaction to completion. Excess reagents were separated on a Sephadex LH-20 column in CHC13/CH30H ( l / l , v/v). The deferri derivative was prepared by extraction with 8-hydroxyquinoline as described above. Bacterial Strains and Culture Conditions
The strains of Escherichia coli K-12 used in this study were as follows: AB2847, aroB, thi, Ar, as wild type-strain; P8, a tonA derivative of AB2847; BR158,
41
J. W. Coulton, H.-U. Naegeli, and V. Braun
a tonB derivative of AB2847. Bacteria were grown in the following media : tryptone-yeast medium consisting of Difco tryptone (8 g/l), Difco yeast extract ( 5 g/l), NaCl ( 5 g/l); nutrient broth, Difco, (8 g/l); minimal medium (M9 salts, 0.4% glucose, plus required growth supplements [3] and 1 mM citrate as iron chelator; dihydroxybenzoate at 20 pM was added for growth of strain BR158); a low iron M9 salt solution was prepared by passage over a Chelex-100 column which resulted in about 0.1 pM residual iron concentration. Glassware was cleaned of iron by washing with 10 mM ethylenediamine tetraacetate and double-distilled water. Uptake of Radioactively Labelled Iron Transport Assay. Cells grown overnight in minimal medium were resuspended in low iron medium and grown for 4 h at 37 "C. Cells were harvested by centrifugation, washed three times in iron-uptake medium (10 mM Tris-HCl, pH 7.2, 0.5% glucose, 1 mM MgS04), and resuspended in cold iron-uptake medium plus 100 pM nitrilotriacetate to an absorbance of 0.40 at 578 nm, corresponding to 0.38 mg dry weight/ml. 10 ml of cells were equilibrated at 37 "C for 5 min, and uptake started by adding an equimolar solution of iron and sideramine. Samples were removed at intervals, rapidly collected onto cellulose nitrate filters (Schleicher and Schiill, 0.45 pm), washed twice with 4 ml of 0.1 M LiCl, dried, and counted in a Nuclear Chicago Mark I1 liquid scintillation counter. When the ferricrocin-polymer was used in transport assays, it was necessary to avoid non-specific binding of poly(ethylene glycol) to cellulose nitrate filters. Control experiments demonstrated that when cells were collected on glass filters (Whatman GF/C) and twice washed with CHC13/CH30H ( l / l , v/v), there was equivalent incorporation of "Fe as with cellulose nitrate filters/LiCl wash. Poly(ethy1ene glycol) did not bind to glass filters and could be quantitatively washed through with CHCl&H30H. Growing Cultures. Bacteria grown to stationary phase in M9 medium were resuspended in 10ml of low iron medium. Radioactively labelled sideramine was added either immediately or added after growing cells for 4 h at 37°C. The methods of sampling and counting were as described above. Test,for Growth Promotion Screening of substances which promote iron uptake in E. coli involved a plate test: a 0.1 ml portion from an overnight culture of AB2847 cells grown in nutrient broth was mixed with 0.1 ml of 20 mM dipyridyl solution in distilled water, plated with 2.5 ml soft agar overlay on 20ml nutrient broth plates, and allowed to dry for 15 min. Since E. coli cells do not
take up the iron-pyridyl complex, they grow very slowly. Compounds to be tested as growth supporting iron chelators were spotted onto the plates in 5-pl volumes, and those which were active gave a growth halo of a diameter proportional to the number of pmoles applied. The optimal incubation time of the plates was 6 h at 37 "C.
Test for Protection against Colicin M Killing The test is based on the assumption that ferricrocin and the synthesized derivative would compete with colicin M or, as described later, with phage T5, for binding to the common tonA receptor. Logarithmically growing E. coli AB2847 (2 x lo8 cells/O.l ml) and 100 pl of a colicin M solution sufficient to kill all cells were spread with 2.5 ml of 0.6% agar in tryptone-yeast medium over tryptone-yeast plates and allowed to dry for 20 min. Then 5 p1 of various concentrations of ferricrocin or the ferricrocin-polymer were spotted on the plates which were incubated at 37 "C for 6 h. Growth of cells under points of application was measured. Application of water alone resulted in no cell growth. Inhibition of T5 Adsorption
E. coli AB2847 grown for 6 h in tryptone-yeast medium were washed twice with M9 salts and suspended in M9 salts plus 10 mM MgS04 and 0.5 mM CaC12to a concentration of 2 x lo9 cells/ml. T5 phage (lo9 plaque-forming units/ml) was pre-incubated with a suspension (2.0 mg/ml) of lipopolysaccharide in M9 salts at 23 "C for 2 h. Materials were added in the following order: 5 pl ferricrocin solution, 40 p1 of cell suspension, 5 pl bacteriophage. The number of phages added per cell was 0.04-0.06; higher ratios gave erratic results. Following incubation at 37 "C for 13 min, adsorption was terminated by 90-fold dilution with M9 salts. After centrifugation to pellet the bacteria (15 min, 5000 x g ) , phages remaining in the supernatant were titered on E. coli AB2847. Inactivation of tonA Protein Samples which contain ligand would be expected to bind the tonA protein and render it unavailable for inactivation of T5 phages. Hence, 95 pl of phage buffer (0.10 M Tris-HC1, pH 8.0,0.5 M NaCl, 1.0 mM CaC12, 0.01 % gelatin) were mixed with 100 pl of an enriched tonA protein solution which after sodium dodecylsulphate electrophoresis showed one major band and three faint contaminating components. 5 p1 were removed from each of the high-pressure liquid chromatography fractions, added to tonA protein in phage buffer, and the tubes incubated for 60 min at 37°C. Between 600-800 T5 phages in 100 p1 were
42
Iron Supply of Escherichiu coli
then pipetted into the samples, and further incubated for 120 min at 37°C. The entire mixture was then plated on E. coli K-12 AB2847. Plates were scored for plaque-forming units.
RESULTS
HO-CH2-tH
Synthesis of a High-Molecular- Weight Derivative of Ferricrocin In order to obtain a high-molecular-weight derivative of a ferrichrome-like sideramine, ferricrocin was bound to poly(ethy1ene glycol) ( M , 6000 - 7500). An esterification reaction offered a relatively easy method of coupling the terminal OH-group of poly(ethylene glycol) to a sideramine. Since no naturally occurring ferrichrome-like sideramine possesses a free carboxyl group, an activation reaction was carried out between ferricrocin's free serine OH-group (Fig. 1A) and succinic anhydride to yield the monoester, ferricrocinyl succinate (Fig. 1 B). The free carboxyl group of the monoester had to be activated such that the sensitive sideramine moiety was not destroyed. Hence, the elegant method of Stadler was employed: the activation was successfully achieved by producing a dimethyl formamide-imidechloride adduct. In order to have a relatively short activation time, the reaction was carried out in absolute CHC13 at room temperature. The reverse order of reaction, that is first coupling poly(ethy1ene glycol) with succinic anhydride and then esterification with ferricrocin, was abandoned since separation of free poly(ethylene glycol) from derivatized poly(ethy1ene glycol) was difficult. Furthermore, preparation of radioactively labelled ferricrocin-polymer was much easier in the reaction sequence shown in Fig. 1 C, since the intermediary compounds could be purified and their biological activity tested. Purification of the Ferricrocin-Polymer Separation of the high-molecular-weight ferricrocin-polymer complex from any free ferricrocinyl succinate was imperative to interpret information on uptake of iron from sideramine-polymer. Molecular sieving on columns of Bio-Gel P6 in distilled water and on Sephadex LH-20 in CHC13/CH30H was used to purify the ferricrocin-polymer. In both systems, the ferricrocin-polymer eluted in the void volume, and was detected by assay for poly(ethylene glycol), for absorbance maximum of ferricrocin at 430 nm, and for s5Fe radioactivity. The elution profiles coincided for all these three criteria (data not shown). A trailing peak of uncovalently bound fericrocinyl succinate was free of poly(ethy1ene glycol). Rechromatography of the ferricrocin-polymer-containingfraction on LH-20 showed only a single symmetrical peak.
CH3-
6= O
Fe3+E-N-(CH2)3-dH
0-
NH
0
N
CH NH
Ferrlcrocin
B
0
0
II
II
HOC -CH2-O$-
CO
F e r r i c r o c i n y l succinate
+
I
Fe rricrociny I succinat e (RCOOH)
+
IPolyethylene glycol
RCOOR'+ 2 PymHCI +dimethylformamide=
Ferricrocinyl polyethylene glycolyl succinate
Fig. 1. Structurulformulae cv substances used in the synthesis of the hi~h-moleculur-weightderivative of a ,frrrichrome-like sideramine. ( A ) Ferricrocin, M , 770; (B) ferricrocinyl succinate, M , 870; (C) reaction of dimethylformarnide imidcchloride with ferricrocinyl succinate and polyethylene glycol to yield the covalent complex, ferricrocinyl polyethylene glycolyl succinate. Py, pyridine
43
J. W. Coulton, H.-U. Naegeli, and V. Braun
0.7
i
.. c
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w c c
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06
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200 -
9
100
I \
/-J \
0.1
I
0 0
0
1
10
20
30
120
Time (min)
Fraction number
Fig. 2. Elution profile of'(ssFe]ferricrocin-polymer f r o m high-pressure liquid chromatography on an RP-8 column, using a O-lOO% gradient of acetonitrile in water. Samples were collected at 1-min intervals and each fraction analyzed for inactivation of tonA protein (A)and for radioactivity from 55Fe in a 5-pl aliquot).(
The best method for separation of the ferricrocinpolymer from ferricrocin was high-pressure liquid chromatography. Collected fractions were analyzed for absorbance at 278 nm; for "Fe radioactivity; and for inactivation of tonA protein. The major material was the ferricrocin-polymer (Fig. 2) and although no radioactivity or absorption was found elsewhere in the gradient, a minor peak of tonA protein-inactivation capacity was eluted at the position corresponding to free ferricrocinyl succinate (fraction 5). Fractions containing sideramine-polymer were pooled, rotary evaporated to dryness, and dissolved in 3 .O ml CHCl3. Determining the amount of poly(ethy1ene glycol) by gravimetry or by turbidity at 578 nm [7] and using the molar absorption coefficient of ferricrocinyl succinate (log E = 3.3, 430 nm), a stoichiometry of 1 mol FCo bound for every 9 mol of poly(ethy1ene glycol) was demonstrated. The infrared spectrum (KBr) for the ferricrocin-polymer showed peaks at 2900 cm - , 1330 cm-', and and 1110 cm-' which were characteristic of poly(ethy1ene glycol); A,, (CHC13) at 430 nm was typical of the absorbance of ferricrocin. The ester-specific band at 1730 cm-' was clearly seen, but was weak due to the surplus of unmodified poly(ethy1ene glycol).
'
BIOLOGICAL EFFECTS OF T H E FERRICROCIN-POLYMER O N STRAINS O F E. coli K-12
Testfor Growth Promotion on Nutrient Agar Plates. Compounds which provide iron to bacteria which have been starved of iron (by addition of dipyridyl) yield a growth zone where the iron compound has been spotted. The ferrichrome-promoted iron uptake system was the only high affinity iron uptake system operative
Fig. 3. Transport curves for uptake of [55Fe]ferricrocin and /s5Fe/ferricrocin-polymer into E. coli K-12 strain AB2847 and its tonA derivative, P8. Cells were starved for iron for 4 h by growth in low iron M9 medium, washed and tested for uptake of radioacetively labelled substrates. Uptake of f'5F]ferricrocin into strain AB2847 (M); of [55Fe]ferricrocin into strain P8 (0); of [55Fe]ferricrocinpolymer into strain AB2847 ( 0 ) ;and of [55Fe]ferricrocin-polymer into strain P8 (0)
in aroB strains in the absence of added enterochelin or citrate. Even 1-2 pmol of ferricrocin provided detectable growth. Higher levels showed more dense growth spots, and below this amount there was no growth promotion. Neither mutant P8 (ton.4) nor BR 158 (tonB) gave growth responses to ferricrocin or ferricrocinyl succinate. A comparable effect of growth promotion was displayed by ferricrocinyl polyethylene glycolyl succinate, and the tonA and tonB dependance was identical to ferricrocin and to ferricrocinyl succinate. All amounts of the ferricrocin-polymer from 105 pmol down to 6.6 pmol (calculated with respect to ferricrocin) gave a growth zone, but 3.3 pmol failed to provide a growth response. Free ferricrocin was spotted as a positive control on the same plates; poly(ethy1ene glycol) and FeC12 gave no growth zone. Transport Assay for Uptake of "Fe from the Ferricrocin-Polymer. Iron 55Feuptake complexed to ferricrocin and the ferricrocin-polymer was measured with washed cells. Rapid incorporation into wild type cells of [55Fe]ferricrocinwas found (Fig. 3), but in the tonA strain only low levels of "Fe were detected which represent non-specific binding. The ferricrocin-polymer failed to readily donate "Fe in the same transport assay, even over an extended period of 120 min. There was a rather high level of bound ferricrocinpolymer which was about the same in wild type and the tonA mutant. Uptake of 55Fefrom Ferricrocin and from the Ferricrocin-Polymer by Growing Cells. Bacteria grown to stationary phase in iron-rich medium were inoculated into extracted M9 salts in which the sole iron source
Iron Supply of Escherichia coli
44 I
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g -
20.0 15.0 10.0
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Fig. 4. Stinzulation of growth and uptake of iron into E. coli K-I2 strain AB2847 after provision of ferricrocin as sole iron source. Bacteria were grown overnight to stationary phase in unextracted M9 medium and were resuspended in extracted minimal medium. ["Fe]Ferricrocin was added at time zero or after 4 h of iron starvation (arrow); final concentration of sideramine was 1.0 pM. (A) Samples were taken to determine the amount of 5sFe from ferricrocin which was incorporated into the cells. (0)Iron uptake following addition of [5sFe]ferricrocin at time zero; and (B) iron uptake after addition of [55Fe]ferricrocin at 4 h. (B) The increase in absorbance of the growing cells was followed at 578 nm. (A) No added iron source; ( 0 ) ferricrocin added to the culture at zero time; (A) ferricrocin added after 4 h growth in extracted medium
Fig. 5. Stimulation of grotcth and uptake oJ iron by strain AB2847 following addition ofpolymer-boundferricrocin.Cultures were grown to stationary phase in M9 medium and then resuspended in extracted (low iron) medium. [55Fe]Ferricrocin-polymerwas added either immediately or after an interval of 4 h growth without iron supplement (arrow). (A) The amount of 55Fe incorporated into the cells from siderophore-polymer was determined. (0)Uptake of iron after addition of [s5Fe]ferricrocin-polymerat time zero; (W) uptake of iron after addition of [55Fe]ferricrocin-polymerat 4 h. (B) The absorbance of growing cultures was followed at 578 nm. (A) No iron addition to the culture; (0)polymer-bound ferricrocin added upon resuspension of cells in extracted medium ; (A) stimulation of growth after pipetting the ferricrocin-polymer into the culture following 4 h iron starvation
was either [55Fe]ferricrocin or [55Fe]ferricrocin-polymer. Upon addition of [55Fe]ferricrocin, there was immediate uptake of iron even though the cells were iron loaded (Fig. 4). In contrast, providing [55Fe]ferricrocin-polymer did not result in immediate uptake of 55Fe;only after a 2 h lag during which the endogenous iron supply might be depleted did the cells begin to take up iron from the polymer complex (Fig.5). Under the test conditions, both ferricrocin and the ferricrocin-polymer enhanced growth in liquid culture as seen by comparison with the growth curve of a control culture in which no exogenous source of iron was provided (Fig. 4,5). A markedly increased rate of uptake of 55Fefrom either ferricrocin or the ferricrocin-polymer could be demonstrated if the cells were iron-starved for 4 h before adding the iron source. As demonstrated in Fig. 4, radioactivity was incorporated into growing cells of E. coli AB2847 immediately upon addition of [55Fe]ferricrocin and rapidly reached a plateau. En-
hancement of growth was also demonstrable as shown by the increase of absorbance at 578 nm over a control culture without any added iron chelator. In parallel with this situation, 55Feuptake from the ferricrocinpolymer was observed over 90 min after addition of sideramine-polymer, a plateau in incorporation was achieved, and growth was stimulated (Fig. 5). The amount of iron incorporation was tenfold lower from the ferricrocin-polymer as compared to ferricrocin (20 pmol versus 200 pmol). Iron-starved cells of tonA and tonB mutants did not take up iron from ferricrocin or from the ferricrocin-polymer. Protection against Killing by Colicin M . After plates had been seeded with sufficient colicin to prevent growth of sensitive cells, various amounts of ferricrocin or the ferricrocin-polymer were applied and observation made of the zone of protection conferred by sideramine. Between 5000 pmol and 50 pmol, ferricrocin gave 9- 11 mm wide growth zones; 5 pmol failed to protect against colicin M killing. The ferri-
J. W. Coulton, H.-U. Naegeli, and V. Braun
45
0.4
02
I
c
3 % L
U
0.001 0.00 0.10 1.0 10.0 Ferricrocin or ferricrocinyl polyethylene glycolyl succinate ( p M )
0.0094 0.094 0 9 4
9.4
94.0
Polyethylene glycol ( p M )
Fig. 6. Inhibition of bacteriophage T5 adsorption to strain AB2847. Increasing concentrations of ferricrocin (m) or of the ferricrocinpolymer (0)or of poly(ethy1ene glycol) alone (A) were mixed with a sample of bacteria. To the mixtures was added a sample of T5 bacteriophage previously incubated with lipopolysaccharide (2.0 mg/ml solution in M9 salts) to dissociate the phages. After 13 min incubation at 37 "C, the samples were diluted 90-fold to terminate adsorption, centrifuged to pellet the bacteria with adsorbed phage, and the supernatant titered on the indicator strain AB2847
crocin-polymer at 127 pmol ferricrocin bound to polymer showed an 8-mm diameter growth zone, but 68 pmol were ineffective. Poly(ethy1ene glycol) in the amounts used in the test did not protect against colicin M killing. Inhibition of T5 Infection by Ferricrocin and the Ferricrocin-Polymer. Ferrichrome has been shown to protect energy-starved cells against infection by T5 phage [8]. These experiments were repeated with increasing concentrations of the ferricrocin-polymer ; as a control, poly(ethy1ene glycol) was tested for its inhibition and found to have no effect (Fig. 6). Phages were first dissociated with lipopolysaccharide; failure to pre-incubate with lipopolysaccharide gave spurious results due to the capacity of poly(ethy1ene glycol) to disaggregate the phages. Low levels of ferricrocin and the ferricrocin-polymer (up to 0.01 pM) gave no inhibition of T5 adsorption (Fig. 6); 0.1 pM ferricrocin gave a maximum adsorption inhibition such that no further phage inhibition could be shown by increasing the concentration further. The ferricrocinpolymer was an even more effective inhibitor in that inhibition of phage adsorption went twice as high as that reached by ferricrocin at concentrations of 1.0 and 10 pM. It is conceivable that the ferricrocin-tonA protein complex has a higher dissociation rate than the ferricrocin-polymer which is bound to the receptor ; the ferricrocin-polymer would block the receptor site and thus be a more effective inhibitor of phage adsorption. Transport of Ferricrocinyl Succinate. Cells were tested for their ability to take up 55Fe from the
. -F 0
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H
.
0;
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1
3
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10
Fig. 7. Transport of radioactively labelled substrates, ferricrocin and ferricrocinyl succinate, by E . coli K-12 strains. Cells were starved of iron by growth for 4 h in low iron M9 medium, washed, and tested for uptake of radioactivity from the sideramines which were added at a concentration of 1.0 pM. Uptake of ["Fe]ferricrocin intowild-type strain AB2847 (W); of [''Fe]ferricrocinyl succinate into strdin-2847 (A);of [55Fe]ferricrocinylsuccinate into strain P8 (A); and of ferricrocinyl ['4C]succinate into strain AB2847 (0).If the ferricrocinyl succinate were doubly labelled as ['5Fc]fcrricrocinyl['4C]succinate, the uptakes of iron label and chelator label were the same as shown for strain AB2847, i.e. (A,.) combined
modified siderophore, ferricrocinyl succinate, M , 870. This chelator was equally effective as ferricrocin in donating iron to wild-type cells, since the same rates of uptake of 55Fewere observed (Fig. 7). Mutants of tonA and tonB strains showed no uptake from the esterified chelator. Transport of the chelator was also determined by following the 14C label of the succinate moiety. With a final concentration of 1.0 pM ferri~rocinyl['~C]succinate, no radioactivity above the level of binding was taken up by strain AB2847 or by its tonA derivative P8.
DISCUSSION Cells of E. coli contain two permeability barriers for the uptake of substances, the outer membrane and the cytoplasmic membrane. The outer membrane is permeable to hydrophilic substances up to a M , of about 600 [9]. For uptake of substrates above this exclusion limit, highly specific translocation systems exist across the outer membrane [l]. The systems for
Iron Supply of Escherichia coli
46
uptake of ferric iron complexes belong to this latter category because their M , values are above 700. For ferrichrome, it is questionable whether the whole iron complex has to pass into the cytoplasm or whether iron is mobilized within the membrane system, presumably by reduction to the ferrous form. In this paper, it is shown that iron donated from ferricrocin can be taken up in sufficient amounts into the cell to satisfy growth requirements without penetration of the complex into the cytoplasm. This was achieved by covalent attachment of ferricrocin to poly(ethy1ene glycol) to yield the ferricrocin-polymer; due to its high M , of 7000-8500, this complex is excluded from the cell interior. Nikaido and coworkers have shown that poly(ethylene glycol) with a M , of 1540 is unable to pass across the outer membrane, whereas poly(ethy1ene glycol) with M , 600 flows through water-filled channels formed by the major proteins [9]. In addition, the same group has shown that cyanogen bromide bound to dextran ( M , 10000) only reacts with amino groups of components localized at the cell surface [lo, 111. Total uptake of iron from ferricrocinyl polyethylene glycolyl succinate was ten times lower as compared with that from free ferricrocin. It needed to be guaranteed that the polymer-bound ferricrocin was not contaminated by trace amounts of free ferricrocin, and that no ferricrocin was released during incubation in aqueous medium. Three chromatographic separations on columns of Bio-Gel P-6, on Sephadex LH-20 and on LiChrosorb RP-8 yielded a pure sample. To account for the growth-promoting effect, the ferricrocin-polymer would have to be contaminated with ferricrocin by 15% which can be excluded. Re-examination after incubation in buffer using thin-layer chromatography and the most sensitive high-pressure liquid chromatography gave no indication of breakdown products. To guarantee that ferricrocin was not released by an enzyme, we synthesized ferricrocin linked to Sepharose 6B by an ether linkage. Again we obtained tonA and tonB dependent growth promotion on plates and in liquid culture. It is therefore concluded that ferricrocin need not be taken up into the cell to act as a growthpromoting factor. This does not exclude the suggestion by Leong and Neilands [4] that ferrichrome is transported into the cytoplasm and rapidly expelled as free ligand. That argument was based upon the irreversible transport of the kinetically inert chromium analogue into the cell. It is not surprising that the ferricrocin-polymer effectively serves as iron donor despite the fact that iron is transported at a low rate. There are a large number of well documented systems which behave in the same way. For example, measurement of growth promotion of appropriate mutants by vitamin BIZ is much more sensitive than the uptake assay using
radioactive substrate [12]. The transport capacity of many systems is often far beyond the growth requirement and is often only required under starvation conditions. Questions remain open as to how far the bound ferricrocin penetrates into the membrane, how the iron is released, and how it is passed into the cell. A significant observation is that the strict requirement for the tonA and the tonB gene functions is retained by the ferricrocin-polymer. This was shown by the following results : the ferricrocin-polymer did not serve as iron donor in tonA or tonB mutants; killing of cells by colicin M via the common tonA receptor protein could be prevented by adding the ferricrocin-polymer ; polymer-bound ferricrocin inhibited binding and inactivation of T5 phage by isolated tonA protein. It is also of interest that introduction of a negative charge into the uncharged ferricrocin by synthesis of the succinyl monoester yielded a product which donated iron to the cells with the same fast rate as ferricrocin (Fig.7). In addition, the amount of ligand associated with the cells is, as in the case of ferrichrome and ferricrocin, only at a basal level and not higher than that found in mutants defective in ferrichrome-promoted iron uptake. It appears that the extra negative charge on an otherwise uncharged carrier has no effect in this uptake system across the membranes. Again, the iron may be mobilized near the periphery of the cell while ferricrocinyl succinate is still bound to the tonA receptor protein. The low iron uptake rate observed with the polymer-bound ferricrocin could result from steric hindrance of ferricrocinyl residues bound to the large, randomly coiled poly(ethy1ene glycol). A considerable portion of the ferricrocin residues may be prevented from binding to the tonA receptor protein because they are situated within the poly(ethy1ene glycol) molecule and not sufficiently exposed at the surface of the complex. We thank Dr H. P. Fiedler for performing the high-pressure liquid chromatography analyses, H. Wolff and P. Burry for help in some of the experiments, J. A . Kashul for the figures, and Dr R. J. Kadner for a critical review of the manuscript. J. W. C. was a Fellow of the Medical Research Council of Canada and of the Alexander von Humboldt Stifung, Bonn. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 76)
REFERENCES 1. Braun, V. & Hantke, K. (1977) in Microbial Interactions (Receptors and Recognition, Series B, vol. 3) (Reissig, J. L., ed.), pp. 100-137, Chapman and Hall, London. 2. Neilands, J. B. (1977) Adv. Chem. Ser. 162,3-32. 3. Braun, V., Hancock, R. E. W., Hantke, K. & Hartmann, A. (1976) J . Supramol. Struct. 5 , 37-58. 4. Leong, J. & Neilands, J. B. (1976) J . Bacferiol. 126, 823-830. 5. Keller-Schierlein, W. & Dekr, A . (1963) Helv. Chim. Acta, 46, 1907-1920.
41
J. W. Coulton, H.-U. Naegeli, and V. Braun
6. Stadler, P. A. (1978) Helv. Chim.Acta, 61, 1675-3681. 7. Ingham, K. C. & Ling, R. C. (1978) Anal. Biochem. 85, 139145. 8. Hantke, K. & Brdun, V. (1978) J . Bacteriol. 135, 190- 197. 9. Nakae, T. (1976) J . Biol. Chem. 251, 2176-2178. 10. Kamio, Y. & Nikaido, H. (1976) Biochemislry, 15, 2561 -2570.
11. Kamio, Y. & Nikaido, H. (1977) Biochim. Biophys. Acta, 464, 489 - 601. 12. Kadner, R. J. (1978) in Bucteriul Transport (Rosen, B. P., ed.) pp. 463-493, Marcel Dekker, New York. 13. Fiedler, H. P. & Sauerbier, J. (1978) Eur. J . Appl. Microhiol. Biotechnol. 5, 51 -57.
J. W. Coulton, Department of Microbiology and Immunology, McCill University, 3775 University Street, Montreal, Quebec, Canada, H3A 2B4 H.-U. Naegeli and V. Braun*, Lehrbereich Mikrobiologie, Institut fur Biologie 11, Eberhard-Karls-Universitat, Tiibingen, Auf der Morgenstelle 28, D-7400 Tiibingen 1, Federal Republic of Germany
* To whom correspondence should be addressed.
Iron Supply of Escherichia coli with Polymer-Bound Ferricrocin James W. COULTON, Hans-Ulrich NAEGELI, and Volkmar BRAUN Lehrbereich Mikrobiologie, Universitat Tiibingen (Received March 14, 1979)
Uptake of ferric iron from ferricrocin was studied in Escherichia coli using a polymer-coupled ferricrocin that was unable to penetrate into the cell. Ferricrocinyl polyethylene glycol succinate ( M , 7000-8500) promoted growth of E. coli K-12 AB2847 uroB under iron-limiting conditions. In iron-starved cells, uptake of "Fe could be demonstrated; the amount of iron accumulated amounted to 10% of that observed with free ferricrocin. The iron supply by ferricrocin bound to polyethylene glycol was strictly dependent upon the functions expressed by the tonA and the tonB genes, as was the iron uptake promoted by free ferricrocin. Polymer-bound ferricrocin protected cells against colicin M and phage T5 by competition for the common tonA-coded outer membrane receptor protein. In addition, the rate of iron transport via the negatively charged ferricrocinyl succinate was as fast as via the neutral ferricrocin molecule. No ligand was found associated with the cells. Penetration of chelator beyond receptor is not necessary for siderophoremediated iron uptake. It is concluded that sufficient amounts of iron can be released from the polymer complex to satisfy growth requirements.
In Escherichia coli three high affinity systems for the uptake of ferric iron have been studied [1,2]. They involve chelators (ferrichrome, enterochelin and citrate), receptor proteins in the outer membrane, and additional functions, most likely permeases in the cytoplasmic membrane, each specific for a single iron complex. Common to all three iron uptake systems is the requirement for the tonB function which is also essential for other translocation processes across the outer membrane and cytoplasmic membrane. The tonB gene product has not yet been characterized biochemically. This report concerns one aspect of the uptake of iron mediated by the ferrichrome complex. During the time that iron is being rapidly taken up into the cell, the amount of ligand associated with the cell is low and does not appear to accumulate within the cell [3]. Conversely, a lag phase of about 10 min was reported before a slow increase in incorporation of tritiated ferrichrome [4]. Ferrichrome binds first to a protein of the outer membrane, the product of the tonA gene. Mutants of tonA are unable to bind or to take up iron from ferrichrome. The further pathway of ferrichrome or of iron after its release from the ligand across the outer membrane and cytoplasmic membrane is unknown. To determine whether uptake of the whole complex into the cell is essential for iron uptake or whether iron can be released close to the cell surface, a high molecular weight derivative of
ferrichrome carrying a bulky pendant side chain was synthesized. Ferricrocin is a close structural analogue of ferrichrome and mediates iron uptake with the same rate as ferrichrome by using the tonA, tonBcoded uptake system. One of the glycine residues of the cyclic hexapeptide (glycine)3-(N-hydroxy-N-acetylornithine)3 of ferrichrome is replaced by a serine residue in ferricrocin (Fig. 1A). The hydroxyl group of serine was used to couple a ferricrocin derivative to polyethylene glycol ( M ,6000 - 7500). The biological response of E. coli to this complex was determined with regard to donation of iron, stimulation of growth, and binding specificity to the tonA-coded protein. MATERIALS AND METHODS Synthesis of Ferricrocinyl Succinate Ferricrocin was coupled with succinic anhydride to yield the succinyl monoester. Ferricrocin (200 mg, 0.259 mmol) was combined with 260 mg (2.6 mmol) succinic anhydride in 4 m l absolute pyridine and stirred for 3 h at 60 "C. The reaction mixture was then dried in vucuo and the residue dissolved and chromatographed on a Sephadex LH-20 column in chloroform/methanol (l/l, v/v). An orange-brown powder was recovered (180 mg, 0.207 mmol) with an RF value of 0.22 on silica gel 60 F245 thin-layer plates (Merck, Darmstadt) developed with CHCI3/CH30H/HzO (65/ 25/4, v/v/v); Amax (CHC13) 430 nm; E = 1980 M - '
40
cm-'; infrared spectrum (KBr) with peaks at 1730 cm-', 1680 cm-', 1640 cm-', 1580 cm-'. The deferri form of ferricrocinyl succinate was prepared by extraction of iron with 8-hydroxyquinoline [ 5 ] to give a colourless powder with an RF value of 0.1 on the same type of plates as above developed with CHC13/ CH30H/H20 (65/25/4, v/v/v) ; 6 (dimethylsulphoxided6): 1.62 (broad, 12H); 2.02 (singlet, 9H); 2.41 (singlet, 4H); 3.1 -4.8 (complex, 16H); 8.0-9.0 (complex, 6H); 9.44 (broad, 3H). Synthesis of Ferricrocinyl Polyethylene Glycolyl Succinate
The esterification of ferricrocinyl succinate with poly(ethy1ene glycol) was carried out in a single reaction vessel using a modification of the method of Stadler [6]. An activated dimethyl formamide imidechloride/ferricrocinyl succinate adduct was prepared for reaction with poly(ethy1ene glycol) at room temperature. Dimethyl formamide (24 y1,0.311 mmol) was dissolved in 1 ml absolute CHC13.Then 10 pl(0.113 mmol) oxalyl chloride (Merck, 97% pure) was added with vigorous stirring, followed after 5 min by a solution of 20 mg (23 pmol) ferricrocinyl succinate in 400 pl absolute CHC13 plus three drops of dimethyl formamide. After strong stirring for 3 min, 70 mg poly(ethy1ene glycol) ( M , 6000 - 7500; Serva, Heidelberg) and 20 p1 (0.25 mmol) absolute pyridine were added. The reaction was complete in a few minutes. The reaction mixture showed a diffuse orange spot as major component with an RF value above 0.9 on silica gel thin-layer chromatogram developed with CHC13/CH30H/H20 (65/25/4, v/v/v). There were several unidentified impurities at RF from 0 to 0.2. The reaction mixture was evaporated to dryness, dissolved in water and the crude product ferricrocinpolymer extracted twice with CHC13. Column Chromatography and Analyses
Separation of the products from the reaction mixture utilized the following gel filtration columns : BioGel P-6 (Bio-Rad Laboratories, Richmond, Calif., U.S.A.) run with distilled water, 38 x 1.8-cm column; and Sephadex LH-20 (Pharmacia, Uppsala) eluted with CHC13/CH30H ( l / l , v/v), 36 x 1.8-cm column. Fractions of 1 ml were collected at l-min intervals at room temperature. The eluate was scanned for absorption at 436nm on a Gilford250 spectrophotometer, and all fractions were tested for poly(ethylene glycol) by using the quantitative assay of Ingham and Ling [7] with the following modification to avoid interference with solvent: 50-yl samples of column fractions were mixed with 1 ml of isopropanol/ acetic acidlwater (4/1/1, v/v/v). Then 0.5 ml of alkaline Nessler's reagent (2 parts Merck reagent: 1 part of
Iron Supply of Escherichia coli
4 M KOH) were added with vigorous stirring, and the turbidity measured after incubation at room temperature for 15 min. A standard curve for poly(ethy1ene glycol) alone showed a linear relationship of the absorbance at 578 nm versus poly(ethy1ene glycol) concentration in the region of 5 - 50 yg/ml; ferricrocin did not disturb the test. High-pressure Liquid Chromatography
High-pressure liquid chromatography was performed on a LiChrosorb RP-8 (Merck) column of 7.5-mm internal diameter as described by Fiedler and Sauerbier [13]. The column was first calibrated with 10 pmol free ferricrocin. A linear gradient of water/ acetonitrile from 0 to 100% acetonitrile was used to elute all of the material at a flow rate of 5%/min, corresponding to 4 ml/min. Twenty fractions were collected at l-min intervals. Radioisotopes and Labelling of Sideramine Derivatives 55Fe. To radioactively label ferricrocin and ferricrocinyl succinate, the deferri form of the sideramine was prepared by extraction of iron with 8-hydroxyquinoline. The lyophilized powder was then resuspended in distilled water, and an aliquot incubated with equimolar amounts of 55FeC13(Amersham Buchler ; 1.01 mCi/ml, 150 pg Fe/ml) for 1 h at room temperature. Because iron in the ferricrocin-polymer was not readily extractable by 8-hydroxyquinoline, a solution of carrier-free iron (Amersham Buchler ; 0.43 mCi/ml, 2.1 yg/ml) was incubated with sideramine-polymer. The half-time of exchange of labelled for unlabelled iron is about 8 min, and the above reaction mixture was allowed to stand for 1 h. Excess 55FeC13that was not exchanged was separated on a Sephadex LH-20 column in CHC13/CH30H ( l / l , v/v), or in some instances was scavanged by inclusion of 100 yM nitrilotriacetate (C6H9NO6) pH 7.8, in the bacterial suspension. The iron nitrilotriacetate complex is not taken up by E. coli. 14C.Lyophilized ferricrocin (10 pmol) was labelled with 100 pmol [l ,4-'4C]succinic anhydride (New England Nuclear), specific activity 50 yCi/700 pg. The tenfold excess of succinic anhydride over ferricrocin was required to force the reaction to completion. Excess reagents were separated on a Sephadex LH-20 column in CHC13/CH30H ( l / l , v/v). The deferri derivative was prepared by extraction with 8-hydroxyquinoline as described above. Bacterial Strains and Culture Conditions
The strains of Escherichia coli K-12 used in this study were as follows: AB2847, aroB, thi, Ar, as wild type-strain; P8, a tonA derivative of AB2847; BR158,
41
J. W. Coulton, H.-U. Naegeli, and V. Braun
a tonB derivative of AB2847. Bacteria were grown in the following media : tryptone-yeast medium consisting of Difco tryptone (8 g/l), Difco yeast extract ( 5 g/l), NaCl ( 5 g/l); nutrient broth, Difco, (8 g/l); minimal medium (M9 salts, 0.4% glucose, plus required growth supplements [3] and 1 mM citrate as iron chelator; dihydroxybenzoate at 20 pM was added for growth of strain BR158); a low iron M9 salt solution was prepared by passage over a Chelex-100 column which resulted in about 0.1 pM residual iron concentration. Glassware was cleaned of iron by washing with 10 mM ethylenediamine tetraacetate and double-distilled water. Uptake of Radioactively Labelled Iron Transport Assay. Cells grown overnight in minimal medium were resuspended in low iron medium and grown for 4 h at 37 "C. Cells were harvested by centrifugation, washed three times in iron-uptake medium (10 mM Tris-HCl, pH 7.2, 0.5% glucose, 1 mM MgS04), and resuspended in cold iron-uptake medium plus 100 pM nitrilotriacetate to an absorbance of 0.40 at 578 nm, corresponding to 0.38 mg dry weight/ml. 10 ml of cells were equilibrated at 37 "C for 5 min, and uptake started by adding an equimolar solution of iron and sideramine. Samples were removed at intervals, rapidly collected onto cellulose nitrate filters (Schleicher and Schiill, 0.45 pm), washed twice with 4 ml of 0.1 M LiCl, dried, and counted in a Nuclear Chicago Mark I1 liquid scintillation counter. When the ferricrocin-polymer was used in transport assays, it was necessary to avoid non-specific binding of poly(ethylene glycol) to cellulose nitrate filters. Control experiments demonstrated that when cells were collected on glass filters (Whatman GF/C) and twice washed with CHC13/CH30H ( l / l , v/v), there was equivalent incorporation of "Fe as with cellulose nitrate filters/LiCl wash. Poly(ethy1ene glycol) did not bind to glass filters and could be quantitatively washed through with CHCl&H30H. Growing Cultures. Bacteria grown to stationary phase in M9 medium were resuspended in 10ml of low iron medium. Radioactively labelled sideramine was added either immediately or added after growing cells for 4 h at 37°C. The methods of sampling and counting were as described above. Test,for Growth Promotion Screening of substances which promote iron uptake in E. coli involved a plate test: a 0.1 ml portion from an overnight culture of AB2847 cells grown in nutrient broth was mixed with 0.1 ml of 20 mM dipyridyl solution in distilled water, plated with 2.5 ml soft agar overlay on 20ml nutrient broth plates, and allowed to dry for 15 min. Since E. coli cells do not
take up the iron-pyridyl complex, they grow very slowly. Compounds to be tested as growth supporting iron chelators were spotted onto the plates in 5-pl volumes, and those which were active gave a growth halo of a diameter proportional to the number of pmoles applied. The optimal incubation time of the plates was 6 h at 37 "C.
Test for Protection against Colicin M Killing The test is based on the assumption that ferricrocin and the synthesized derivative would compete with colicin M or, as described later, with phage T5, for binding to the common tonA receptor. Logarithmically growing E. coli AB2847 (2 x lo8 cells/O.l ml) and 100 pl of a colicin M solution sufficient to kill all cells were spread with 2.5 ml of 0.6% agar in tryptone-yeast medium over tryptone-yeast plates and allowed to dry for 20 min. Then 5 p1 of various concentrations of ferricrocin or the ferricrocin-polymer were spotted on the plates which were incubated at 37 "C for 6 h. Growth of cells under points of application was measured. Application of water alone resulted in no cell growth. Inhibition of T5 Adsorption
E. coli AB2847 grown for 6 h in tryptone-yeast medium were washed twice with M9 salts and suspended in M9 salts plus 10 mM MgS04 and 0.5 mM CaC12to a concentration of 2 x lo9 cells/ml. T5 phage (lo9 plaque-forming units/ml) was pre-incubated with a suspension (2.0 mg/ml) of lipopolysaccharide in M9 salts at 23 "C for 2 h. Materials were added in the following order: 5 pl ferricrocin solution, 40 p1 of cell suspension, 5 pl bacteriophage. The number of phages added per cell was 0.04-0.06; higher ratios gave erratic results. Following incubation at 37 "C for 13 min, adsorption was terminated by 90-fold dilution with M9 salts. After centrifugation to pellet the bacteria (15 min, 5000 x g ) , phages remaining in the supernatant were titered on E. coli AB2847. Inactivation of tonA Protein Samples which contain ligand would be expected to bind the tonA protein and render it unavailable for inactivation of T5 phages. Hence, 95 pl of phage buffer (0.10 M Tris-HC1, pH 8.0,0.5 M NaCl, 1.0 mM CaC12, 0.01 % gelatin) were mixed with 100 pl of an enriched tonA protein solution which after sodium dodecylsulphate electrophoresis showed one major band and three faint contaminating components. 5 p1 were removed from each of the high-pressure liquid chromatography fractions, added to tonA protein in phage buffer, and the tubes incubated for 60 min at 37°C. Between 600-800 T5 phages in 100 p1 were
42
Iron Supply of Escherichiu coli
then pipetted into the samples, and further incubated for 120 min at 37°C. The entire mixture was then plated on E. coli K-12 AB2847. Plates were scored for plaque-forming units.
RESULTS
HO-CH2-tH
Synthesis of a High-Molecular- Weight Derivative of Ferricrocin In order to obtain a high-molecular-weight derivative of a ferrichrome-like sideramine, ferricrocin was bound to poly(ethy1ene glycol) ( M , 6000 - 7500). An esterification reaction offered a relatively easy method of coupling the terminal OH-group of poly(ethylene glycol) to a sideramine. Since no naturally occurring ferrichrome-like sideramine possesses a free carboxyl group, an activation reaction was carried out between ferricrocin's free serine OH-group (Fig. 1A) and succinic anhydride to yield the monoester, ferricrocinyl succinate (Fig. 1 B). The free carboxyl group of the monoester had to be activated such that the sensitive sideramine moiety was not destroyed. Hence, the elegant method of Stadler was employed: the activation was successfully achieved by producing a dimethyl formamide-imidechloride adduct. In order to have a relatively short activation time, the reaction was carried out in absolute CHC13 at room temperature. The reverse order of reaction, that is first coupling poly(ethy1ene glycol) with succinic anhydride and then esterification with ferricrocin, was abandoned since separation of free poly(ethylene glycol) from derivatized poly(ethy1ene glycol) was difficult. Furthermore, preparation of radioactively labelled ferricrocin-polymer was much easier in the reaction sequence shown in Fig. 1 C, since the intermediary compounds could be purified and their biological activity tested. Purification of the Ferricrocin-Polymer Separation of the high-molecular-weight ferricrocin-polymer complex from any free ferricrocinyl succinate was imperative to interpret information on uptake of iron from sideramine-polymer. Molecular sieving on columns of Bio-Gel P6 in distilled water and on Sephadex LH-20 in CHC13/CH30H was used to purify the ferricrocin-polymer. In both systems, the ferricrocin-polymer eluted in the void volume, and was detected by assay for poly(ethylene glycol), for absorbance maximum of ferricrocin at 430 nm, and for s5Fe radioactivity. The elution profiles coincided for all these three criteria (data not shown). A trailing peak of uncovalently bound fericrocinyl succinate was free of poly(ethy1ene glycol). Rechromatography of the ferricrocin-polymer-containingfraction on LH-20 showed only a single symmetrical peak.
CH3-
6= O
Fe3+E-N-(CH2)3-dH
0-
NH
0
N
CH NH
Ferrlcrocin
B
0
0
II
II
HOC -CH2-O$-
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F e r r i c r o c i n y l succinate
+
I
Fe rricrociny I succinat e (RCOOH)
+
IPolyethylene glycol
RCOOR'+ 2 PymHCI +dimethylformamide=
Ferricrocinyl polyethylene glycolyl succinate
Fig. 1. Structurulformulae cv substances used in the synthesis of the hi~h-moleculur-weightderivative of a ,frrrichrome-like sideramine. ( A ) Ferricrocin, M , 770; (B) ferricrocinyl succinate, M , 870; (C) reaction of dimethylformarnide imidcchloride with ferricrocinyl succinate and polyethylene glycol to yield the covalent complex, ferricrocinyl polyethylene glycolyl succinate. Py, pyridine
43
J. W. Coulton, H.-U. Naegeli, and V. Braun
0.7
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9
100
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/-J \
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10
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30
120
Time (min)
Fraction number
Fig. 2. Elution profile of'(ssFe]ferricrocin-polymer f r o m high-pressure liquid chromatography on an RP-8 column, using a O-lOO% gradient of acetonitrile in water. Samples were collected at 1-min intervals and each fraction analyzed for inactivation of tonA protein (A)and for radioactivity from 55Fe in a 5-pl aliquot).(
The best method for separation of the ferricrocinpolymer from ferricrocin was high-pressure liquid chromatography. Collected fractions were analyzed for absorbance at 278 nm; for "Fe radioactivity; and for inactivation of tonA protein. The major material was the ferricrocin-polymer (Fig. 2) and although no radioactivity or absorption was found elsewhere in the gradient, a minor peak of tonA protein-inactivation capacity was eluted at the position corresponding to free ferricrocinyl succinate (fraction 5). Fractions containing sideramine-polymer were pooled, rotary evaporated to dryness, and dissolved in 3 .O ml CHCl3. Determining the amount of poly(ethy1ene glycol) by gravimetry or by turbidity at 578 nm [7] and using the molar absorption coefficient of ferricrocinyl succinate (log E = 3.3, 430 nm), a stoichiometry of 1 mol FCo bound for every 9 mol of poly(ethy1ene glycol) was demonstrated. The infrared spectrum (KBr) for the ferricrocin-polymer showed peaks at 2900 cm - , 1330 cm-', and and 1110 cm-' which were characteristic of poly(ethy1ene glycol); A,, (CHC13) at 430 nm was typical of the absorbance of ferricrocin. The ester-specific band at 1730 cm-' was clearly seen, but was weak due to the surplus of unmodified poly(ethy1ene glycol).
'
BIOLOGICAL EFFECTS OF T H E FERRICROCIN-POLYMER O N STRAINS O F E. coli K-12
Testfor Growth Promotion on Nutrient Agar Plates. Compounds which provide iron to bacteria which have been starved of iron (by addition of dipyridyl) yield a growth zone where the iron compound has been spotted. The ferrichrome-promoted iron uptake system was the only high affinity iron uptake system operative
Fig. 3. Transport curves for uptake of [55Fe]ferricrocin and /s5Fe/ferricrocin-polymer into E. coli K-12 strain AB2847 and its tonA derivative, P8. Cells were starved for iron for 4 h by growth in low iron M9 medium, washed and tested for uptake of radioacetively labelled substrates. Uptake of f'5F]ferricrocin into strain AB2847 (M); of [55Fe]ferricrocin into strain P8 (0); of [55Fe]ferricrocinpolymer into strain AB2847 ( 0 ) ;and of [55Fe]ferricrocin-polymer into strain P8 (0)
in aroB strains in the absence of added enterochelin or citrate. Even 1-2 pmol of ferricrocin provided detectable growth. Higher levels showed more dense growth spots, and below this amount there was no growth promotion. Neither mutant P8 (ton.4) nor BR 158 (tonB) gave growth responses to ferricrocin or ferricrocinyl succinate. A comparable effect of growth promotion was displayed by ferricrocinyl polyethylene glycolyl succinate, and the tonA and tonB dependance was identical to ferricrocin and to ferricrocinyl succinate. All amounts of the ferricrocin-polymer from 105 pmol down to 6.6 pmol (calculated with respect to ferricrocin) gave a growth zone, but 3.3 pmol failed to provide a growth response. Free ferricrocin was spotted as a positive control on the same plates; poly(ethy1ene glycol) and FeC12 gave no growth zone. Transport Assay for Uptake of "Fe from the Ferricrocin-Polymer. Iron 55Feuptake complexed to ferricrocin and the ferricrocin-polymer was measured with washed cells. Rapid incorporation into wild type cells of [55Fe]ferricrocinwas found (Fig. 3), but in the tonA strain only low levels of "Fe were detected which represent non-specific binding. The ferricrocin-polymer failed to readily donate "Fe in the same transport assay, even over an extended period of 120 min. There was a rather high level of bound ferricrocinpolymer which was about the same in wild type and the tonA mutant. Uptake of 55Fefrom Ferricrocin and from the Ferricrocin-Polymer by Growing Cells. Bacteria grown to stationary phase in iron-rich medium were inoculated into extracted M9 salts in which the sole iron source
Iron Supply of Escherichia coli
44 I
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Time (h)
Time (h)
Fig. 4. Stinzulation of growth and uptake of iron into E. coli K-I2 strain AB2847 after provision of ferricrocin as sole iron source. Bacteria were grown overnight to stationary phase in unextracted M9 medium and were resuspended in extracted minimal medium. ["Fe]Ferricrocin was added at time zero or after 4 h of iron starvation (arrow); final concentration of sideramine was 1.0 pM. (A) Samples were taken to determine the amount of 5sFe from ferricrocin which was incorporated into the cells. (0)Iron uptake following addition of [5sFe]ferricrocin at time zero; and (B) iron uptake after addition of [55Fe]ferricrocin at 4 h. (B) The increase in absorbance of the growing cells was followed at 578 nm. (A) No added iron source; ( 0 ) ferricrocin added to the culture at zero time; (A) ferricrocin added after 4 h growth in extracted medium
Fig. 5. Stimulation of grotcth and uptake oJ iron by strain AB2847 following addition ofpolymer-boundferricrocin.Cultures were grown to stationary phase in M9 medium and then resuspended in extracted (low iron) medium. [55Fe]Ferricrocin-polymerwas added either immediately or after an interval of 4 h growth without iron supplement (arrow). (A) The amount of 55Fe incorporated into the cells from siderophore-polymer was determined. (0)Uptake of iron after addition of [s5Fe]ferricrocin-polymerat time zero; (W) uptake of iron after addition of [55Fe]ferricrocin-polymerat 4 h. (B) The absorbance of growing cultures was followed at 578 nm. (A) No iron addition to the culture; (0)polymer-bound ferricrocin added upon resuspension of cells in extracted medium ; (A) stimulation of growth after pipetting the ferricrocin-polymer into the culture following 4 h iron starvation
was either [55Fe]ferricrocin or [55Fe]ferricrocin-polymer. Upon addition of [55Fe]ferricrocin, there was immediate uptake of iron even though the cells were iron loaded (Fig. 4). In contrast, providing [55Fe]ferricrocin-polymer did not result in immediate uptake of 55Fe;only after a 2 h lag during which the endogenous iron supply might be depleted did the cells begin to take up iron from the polymer complex (Fig.5). Under the test conditions, both ferricrocin and the ferricrocin-polymer enhanced growth in liquid culture as seen by comparison with the growth curve of a control culture in which no exogenous source of iron was provided (Fig. 4,5). A markedly increased rate of uptake of 55Fefrom either ferricrocin or the ferricrocin-polymer could be demonstrated if the cells were iron-starved for 4 h before adding the iron source. As demonstrated in Fig. 4, radioactivity was incorporated into growing cells of E. coli AB2847 immediately upon addition of [55Fe]ferricrocin and rapidly reached a plateau. En-
hancement of growth was also demonstrable as shown by the increase of absorbance at 578 nm over a control culture without any added iron chelator. In parallel with this situation, 55Feuptake from the ferricrocinpolymer was observed over 90 min after addition of sideramine-polymer, a plateau in incorporation was achieved, and growth was stimulated (Fig. 5). The amount of iron incorporation was tenfold lower from the ferricrocin-polymer as compared to ferricrocin (20 pmol versus 200 pmol). Iron-starved cells of tonA and tonB mutants did not take up iron from ferricrocin or from the ferricrocin-polymer. Protection against Killing by Colicin M . After plates had been seeded with sufficient colicin to prevent growth of sensitive cells, various amounts of ferricrocin or the ferricrocin-polymer were applied and observation made of the zone of protection conferred by sideramine. Between 5000 pmol and 50 pmol, ferricrocin gave 9- 11 mm wide growth zones; 5 pmol failed to protect against colicin M killing. The ferri-
J. W. Coulton, H.-U. Naegeli, and V. Braun
45
0.4
02
I
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3 % L
U
0.001 0.00 0.10 1.0 10.0 Ferricrocin or ferricrocinyl polyethylene glycolyl succinate ( p M )
0.0094 0.094 0 9 4
9.4
94.0
Polyethylene glycol ( p M )
Fig. 6. Inhibition of bacteriophage T5 adsorption to strain AB2847. Increasing concentrations of ferricrocin (m) or of the ferricrocinpolymer (0)or of poly(ethy1ene glycol) alone (A) were mixed with a sample of bacteria. To the mixtures was added a sample of T5 bacteriophage previously incubated with lipopolysaccharide (2.0 mg/ml solution in M9 salts) to dissociate the phages. After 13 min incubation at 37 "C, the samples were diluted 90-fold to terminate adsorption, centrifuged to pellet the bacteria with adsorbed phage, and the supernatant titered on the indicator strain AB2847
crocin-polymer at 127 pmol ferricrocin bound to polymer showed an 8-mm diameter growth zone, but 68 pmol were ineffective. Poly(ethy1ene glycol) in the amounts used in the test did not protect against colicin M killing. Inhibition of T5 Infection by Ferricrocin and the Ferricrocin-Polymer. Ferrichrome has been shown to protect energy-starved cells against infection by T5 phage [8]. These experiments were repeated with increasing concentrations of the ferricrocin-polymer ; as a control, poly(ethy1ene glycol) was tested for its inhibition and found to have no effect (Fig. 6). Phages were first dissociated with lipopolysaccharide; failure to pre-incubate with lipopolysaccharide gave spurious results due to the capacity of poly(ethy1ene glycol) to disaggregate the phages. Low levels of ferricrocin and the ferricrocin-polymer (up to 0.01 pM) gave no inhibition of T5 adsorption (Fig. 6); 0.1 pM ferricrocin gave a maximum adsorption inhibition such that no further phage inhibition could be shown by increasing the concentration further. The ferricrocinpolymer was an even more effective inhibitor in that inhibition of phage adsorption went twice as high as that reached by ferricrocin at concentrations of 1.0 and 10 pM. It is conceivable that the ferricrocin-tonA protein complex has a higher dissociation rate than the ferricrocin-polymer which is bound to the receptor ; the ferricrocin-polymer would block the receptor site and thus be a more effective inhibitor of phage adsorption. Transport of Ferricrocinyl Succinate. Cells were tested for their ability to take up 55Fe from the
. -F 0
-g 0.2 c
e
H
.
0;
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5 7 Time (min)
10
Fig. 7. Transport of radioactively labelled substrates, ferricrocin and ferricrocinyl succinate, by E . coli K-12 strains. Cells were starved of iron by growth for 4 h in low iron M9 medium, washed, and tested for uptake of radioactivity from the sideramines which were added at a concentration of 1.0 pM. Uptake of ["Fe]ferricrocin intowild-type strain AB2847 (W); of [''Fe]ferricrocinyl succinate into strdin-2847 (A);of [55Fe]ferricrocinylsuccinate into strain P8 (A); and of ferricrocinyl ['4C]succinate into strain AB2847 (0).If the ferricrocinyl succinate were doubly labelled as ['5Fc]fcrricrocinyl['4C]succinate, the uptakes of iron label and chelator label were the same as shown for strain AB2847, i.e. (A,.) combined
modified siderophore, ferricrocinyl succinate, M , 870. This chelator was equally effective as ferricrocin in donating iron to wild-type cells, since the same rates of uptake of 55Fewere observed (Fig. 7). Mutants of tonA and tonB strains showed no uptake from the esterified chelator. Transport of the chelator was also determined by following the 14C label of the succinate moiety. With a final concentration of 1.0 pM ferri~rocinyl['~C]succinate, no radioactivity above the level of binding was taken up by strain AB2847 or by its tonA derivative P8.
DISCUSSION Cells of E. coli contain two permeability barriers for the uptake of substances, the outer membrane and the cytoplasmic membrane. The outer membrane is permeable to hydrophilic substances up to a M , of about 600 [9]. For uptake of substrates above this exclusion limit, highly specific translocation systems exist across the outer membrane [l]. The systems for
Iron Supply of Escherichia coli
46
uptake of ferric iron complexes belong to this latter category because their M , values are above 700. For ferrichrome, it is questionable whether the whole iron complex has to pass into the cytoplasm or whether iron is mobilized within the membrane system, presumably by reduction to the ferrous form. In this paper, it is shown that iron donated from ferricrocin can be taken up in sufficient amounts into the cell to satisfy growth requirements without penetration of the complex into the cytoplasm. This was achieved by covalent attachment of ferricrocin to poly(ethy1ene glycol) to yield the ferricrocin-polymer; due to its high M , of 7000-8500, this complex is excluded from the cell interior. Nikaido and coworkers have shown that poly(ethylene glycol) with a M , of 1540 is unable to pass across the outer membrane, whereas poly(ethy1ene glycol) with M , 600 flows through water-filled channels formed by the major proteins [9]. In addition, the same group has shown that cyanogen bromide bound to dextran ( M , 10000) only reacts with amino groups of components localized at the cell surface [lo, 111. Total uptake of iron from ferricrocinyl polyethylene glycolyl succinate was ten times lower as compared with that from free ferricrocin. It needed to be guaranteed that the polymer-bound ferricrocin was not contaminated by trace amounts of free ferricrocin, and that no ferricrocin was released during incubation in aqueous medium. Three chromatographic separations on columns of Bio-Gel P-6, on Sephadex LH-20 and on LiChrosorb RP-8 yielded a pure sample. To account for the growth-promoting effect, the ferricrocin-polymer would have to be contaminated with ferricrocin by 15% which can be excluded. Re-examination after incubation in buffer using thin-layer chromatography and the most sensitive high-pressure liquid chromatography gave no indication of breakdown products. To guarantee that ferricrocin was not released by an enzyme, we synthesized ferricrocin linked to Sepharose 6B by an ether linkage. Again we obtained tonA and tonB dependent growth promotion on plates and in liquid culture. It is therefore concluded that ferricrocin need not be taken up into the cell to act as a growthpromoting factor. This does not exclude the suggestion by Leong and Neilands [4] that ferrichrome is transported into the cytoplasm and rapidly expelled as free ligand. That argument was based upon the irreversible transport of the kinetically inert chromium analogue into the cell. It is not surprising that the ferricrocin-polymer effectively serves as iron donor despite the fact that iron is transported at a low rate. There are a large number of well documented systems which behave in the same way. For example, measurement of growth promotion of appropriate mutants by vitamin BIZ is much more sensitive than the uptake assay using
radioactive substrate [12]. The transport capacity of many systems is often far beyond the growth requirement and is often only required under starvation conditions. Questions remain open as to how far the bound ferricrocin penetrates into the membrane, how the iron is released, and how it is passed into the cell. A significant observation is that the strict requirement for the tonA and the tonB gene functions is retained by the ferricrocin-polymer. This was shown by the following results : the ferricrocin-polymer did not serve as iron donor in tonA or tonB mutants; killing of cells by colicin M via the common tonA receptor protein could be prevented by adding the ferricrocin-polymer ; polymer-bound ferricrocin inhibited binding and inactivation of T5 phage by isolated tonA protein. It is also of interest that introduction of a negative charge into the uncharged ferricrocin by synthesis of the succinyl monoester yielded a product which donated iron to the cells with the same fast rate as ferricrocin (Fig.7). In addition, the amount of ligand associated with the cells is, as in the case of ferrichrome and ferricrocin, only at a basal level and not higher than that found in mutants defective in ferrichrome-promoted iron uptake. It appears that the extra negative charge on an otherwise uncharged carrier has no effect in this uptake system across the membranes. Again, the iron may be mobilized near the periphery of the cell while ferricrocinyl succinate is still bound to the tonA receptor protein. The low iron uptake rate observed with the polymer-bound ferricrocin could result from steric hindrance of ferricrocinyl residues bound to the large, randomly coiled poly(ethy1ene glycol). A considerable portion of the ferricrocin residues may be prevented from binding to the tonA receptor protein because they are situated within the poly(ethy1ene glycol) molecule and not sufficiently exposed at the surface of the complex. We thank Dr H. P. Fiedler for performing the high-pressure liquid chromatography analyses, H. Wolff and P. Burry for help in some of the experiments, J. A . Kashul for the figures, and Dr R. J. Kadner for a critical review of the manuscript. J. W. C. was a Fellow of the Medical Research Council of Canada and of the Alexander von Humboldt Stifung, Bonn. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 76)
REFERENCES 1. Braun, V. & Hantke, K. (1977) in Microbial Interactions (Receptors and Recognition, Series B, vol. 3) (Reissig, J. L., ed.), pp. 100-137, Chapman and Hall, London. 2. Neilands, J. B. (1977) Adv. Chem. Ser. 162,3-32. 3. Braun, V., Hancock, R. E. W., Hantke, K. & Hartmann, A. (1976) J . Supramol. Struct. 5 , 37-58. 4. Leong, J. & Neilands, J. B. (1976) J . Bacferiol. 126, 823-830. 5. Keller-Schierlein, W. & Dekr, A . (1963) Helv. Chim. Acta, 46, 1907-1920.
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6. Stadler, P. A. (1978) Helv. Chim.Acta, 61, 1675-3681. 7. Ingham, K. C. & Ling, R. C. (1978) Anal. Biochem. 85, 139145. 8. Hantke, K. & Brdun, V. (1978) J . Bacteriol. 135, 190- 197. 9. Nakae, T. (1976) J . Biol. Chem. 251, 2176-2178. 10. Kamio, Y. & Nikaido, H. (1976) Biochemislry, 15, 2561 -2570.
11. Kamio, Y. & Nikaido, H. (1977) Biochim. Biophys. Acta, 464, 489 - 601. 12. Kadner, R. J. (1978) in Bucteriul Transport (Rosen, B. P., ed.) pp. 463-493, Marcel Dekker, New York. 13. Fiedler, H. P. & Sauerbier, J. (1978) Eur. J . Appl. Microhiol. Biotechnol. 5, 51 -57.
J. W. Coulton, Department of Microbiology and Immunology, McCill University, 3775 University Street, Montreal, Quebec, Canada, H3A 2B4 H.-U. Naegeli and V. Braun*, Lehrbereich Mikrobiologie, Institut fur Biologie 11, Eberhard-Karls-Universitat, Tiibingen, Auf der Morgenstelle 28, D-7400 Tiibingen 1, Federal Republic of Germany
* To whom correspondence should be addressed.
