Vol. 158, No. 2JOURNAL OF BACTERIOLOGY, May 1984, p. 496-5020021-9193/84/050496-07$02.00/0Copyright C 1984, American Society for Microbiology

Derepression of the Azotobacter vinelandii Siderophore System,Using Iron-Containing Minerals to Limit Iron Repletion

WILLIAM J. PAGE* AND MARIANNE HUYER

Department of Microbiology, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Received 21 December 1983/Accepted 11 February 1984

Azotobacter vinelandii solubilized iron from certain minerals using only dihydroxybenzoic acid, whichappeared to be produced constitutively. Solubilization of iron from other minerals required dihydroxyben-zoic acid and the siderophore N,N'-bis-(2,3-dihydroxybenzoyl)-L-ly'sine (azotochelin) or these chelatorsplus the yellow-green fluorescent siderophore azotobactin. In addition to this sequential production ofsiderophores, cells also demonstrated partial to hyperproduction relative to the iron-limited control. Theiron sources which caused partial derepression of the siderophores caused derepression of all the high-molecular-weight iron-repressible outer membrane proteins except a 77,000-molecular-weight protein,which appeared to be coordinated with azotobactin production. Increased siderophore production correlat-ed with increased production of outer membrane proteins with molecular weights of 93,000, 85,000, and77,000, but an 81,000-molecular-weight iron-repressible protein appeared at a constant level despite thedegree of derepression. When iron was readily available, it appeared to complex with a 60,000-molecular-weight protein believed to form a surface layer on the A. vinelandii cell.

Azotobacter vinelandii is a gram-negative nitrogen-fixingmicrobe which is found in the soil (33). This obligate aerobehas an absolute requirement for iron to support respirationwhich in turn serves to protect the oxygen-labile nitrogenasesystem (29). Iron is plentiful in the soil, but oxidized iron atneutral pH is very insoluble. It is not surprising that thisorganism has evolved a siderophore system (0. Knosp andW. J. Page, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983,K174, p. 206) analogous to those described for other bacteriafor the chelation and transport of iron (18, 19). A. vinelandiiproduces three iron-binding compounds under iron-limitedconditions (Fig. 1), but only N,N'-bis-(2,3,-dihydroxyben-zoyl)-L-lysine (trivial name, azotochelin; Fig. 1A) and theyellow-green fluorescent peptide (trivial name, azotobactin;Fig. 1C) appear to function as siderophores in high-affinityactive transport of iron (0. Knosp, submitted for' publica-tion). Iron-limited A. vinelandii also derepresses high-molec-ular-weight outer membrane proteins which may function assiderophore receptors or facilitate iron uptake (25).A common approach to studying siderophore system

derepression is to impose iron limitation on the culture, butthis condition of extreme "iron stress" is likely to betransient in a natural environment, where the induced sider-ophores are able to scavenge iron. Iron repletion will belimited by the rate of solubilization of iron from the naturaliron source, be it inorganic minerals or organic iron-contain-ing compounds, which in turn will be affected by thesiderophore(s) employed. The returning ferrisiderophoremay also repress part or all of the siderophore system (13,26). In this study, the addition of insoluble iron sources toiron-limited A. vinelandii was used to observe the effects oflimited iron repletion on the regulation of siderophore pro-duction. The results provide evidence for sequential dere-pression of siderophores and suggest the operation of anextracellular low-affinity iron accumulation system in A.vinelandii.

* Corresponding author.

MATERIALS AND METHODSBacterial strains and growth conditions. The capsule-nega-

tive strain UW ofA. vinelandii OP and a spontaneous variantof strain UW which failed to produce azotobactin (strainUA1) were used. These strains were maintained on slants ofBurk medium, pH 7.2 (24), containing 1% glucose, 1.1 g ofammonium acetate per liter, and 1.8% agar, incubated at30°C. Burk buffer was the same as Burk medium withoutadded glucose, ammonium, or agar. Iron-limited Burk medi-um contained no added iron. The inoculum for each studywas pregrown for 48 h on slants of Burk medium, by whichtime the cells were Fe limited and fluorescent green (3). Cellswere washed from slants with 1.7' ml of Fe-limited Burkbuffer and added as a 1% (vol/vol) inoculum to 100 ml of Fe-limited Burk medium in a 500-ml Erlenmyer flask. Minerals(50 mg) were autoclaved dry and then added at the time ofinoculation. Soluble iron sources FeSO4, ferric sodiumEDTA (FeEDTA), humic acid, and fulvic acid were auto-claved with the medium. The flasks were incubated for 20 hat 30°C with shaking at 270 rpm in a New'Brunswick modelG-76 gyratory water bath shaker.Chemical analysis of cells and minerals. The cells were

removed from the growth medium by centrifugation andresuspended in 10 ml of 8 mM Tris-hydrochloride buffer, pH7.8. The cell suspension (2 ml) was extracted with 0.1 NNaOH for 1 h at 90°C before protein determination (16). Toseparate the cells from minerals, 2 ml of cell suspension wasoverlaid on a 3-ml Percoll-0.15 M NaCl (2:1 [vol/vol])cushion in a conical centrifuge tube and centrifuged for 5 minin an International clinical centrifuge at top speed. Therewas no trace of mineral in the cell pellet formed when thecells from the top of the Percoll cushion were centrifugedagain. The cell pellet was suspended in 2.5 ml of 7%perchloric acid and extracted overnight at room temperatureand for 4 h at 90°C before iron determination using a,a-bipyridyl (21). All glassware for iron analysis was acidwashed.

Freely exchangeable iron in each mineral was determinedby shaking 100 mg of mineral in 20 ml of 1 M ammonium

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AZOTOBACTER SIDEROPHORE DEREPRESSION 497

C H2-CH2-CH2-C H2-CH-COOH(A) NH NH

O=C C=OI IgJ OH HO

OH HO

OHOH

OH(B) COOH

H

N OH

O= ~ ~

(C) L.C peptide,,

0FIG. 1. Iron-binding compounds produced by A. vinelandii. (A)

White-green fluorescent (Amax310) N,N'-bis-(2,3-dihydroxybenzoyl)-L-lysine was isolated by Corbin and Bulen (6), and its affinity forFe2 , Fe3+, MoO42, V03 and W042 has been described

previously (25). (B) The blue-white fluorescent (Amax3i0) DHBA alsowas described by Corbin and Bulen (6). (C) Yellow-green fluores-cent siderophore (Amax410, pH 7.0; Amax380, pH 1.8) has beendescribed previously (3, 10). The peptide has been given thesequence chromophore-aspartyl-homoseryl-seryl-homoseryl-citrul-linyl-seryl-glycyl-hydroxyaspartate (10).

acetate, pH 7.2, under the conditions used for cultureincubation (20). Soluble iron was determined after reducing15 ml of extract to dryness and suspending the residue in 1ml of 0.35 N HCI.

Siderophore analysis. After the removal of cells by centrif-ugation, the culture supernatant fluid was acidified to pH 1.8with HCl and passed through a Millipore filter (0.8-,um poresize). The acidified culture fluid was scanned with a Perkin-Elmer Lambda 3 spectrophotometer, and absorbance at 310nm was used to estimate total catechol using purified dihy-droxybenzoic acid (DHBA) as a standard. Absorbance at380 nm was used to estimate azotobactin concentration withthe extinction coefficient previously published (10).

Catechols were extracted from 100 ml of acidified culturesupernatant fluid with ethyl acetate as described previously(6). Catechols were separated by thin-layer chromatographyon silica gel G developed with butanol-acetic acid-water(120:30:30). DHBA (Rf = 0.77) and azotochelin (Rf = 0.36)were detected by spraying chromatograms with FeCl3-bipyr-idyl or FeCl3-potassium ferricyanide reagents (14).

Preparation of outer membranes and electrophoresis. Cellsfrom 100-ml cultures were concentrated by centrifugationand suspended in 10 ml of 8 mM Tris-hydrochloride buffer,pH 7.8. The cells were broken by sonication (Branson celldisrupter; model 350), and the broken cell suspension wastreated with RNase and DNase (0.5 mg each) for 30 min atroom temperature. Cell debris was removed by centrifuga-tion at 2,000 x g in a Sorval RC5B centrifuge, and theprotein content of the supernatant was determined. Sodiumlauroyl sarcosine (sarcosyl) was added in a 6:1 (wt/wt) ratiowith the protein present, and the solutions were shaken for30 min at room temperature. The sarcosyl extracts were

centrifuged at 46,000 x g to pellet outer membrane material(8). The outer membrane pellet was washed once with 20 mlof Tris buffer, followed by centrifugation at 46,000 x g for 60min, and then resuspended in 1 ml of Tris buffer. Samples (10,ug of protein) were boiled in sample buffer containing

sodium dodecyl sulfate (SDS) and P-mercaptoethanol andloaded onto SDS-polyacrylamide gels (15) with the condi-tions (SDS-polyacrylamide gel electrophoresis [SDS-PAGE]) and apparatus described previously (25). The pro-tein bands were stained with Coomassie brilliant blue R inisopropanol and acetic acid (7) and scanned with a Hoefermodel GS300 scanning densitometer. The molecular weightsof the proteins were determined by comparison with authen-tic standards (25).

Surface protein precipitation. Filter-sterilized culture su-pernatant fluid from cells grown in calcium- and iron-limitedBurk medium was used as a source of surface protein (23).This protein was precipitated from culture fluid by theaddition of 20 mM cation salts as described previously (23).The protein content of the precipitate was determined withthe Bio-Rad protein assay (Bio-Rad Laboratories, Rich-mond, Calif.) after dissolution in 0.5 M EDTA, pH 7.0.Samples were subjected to SDS-PAGE to confirm that the60,000-molecular-weight (60K) surface protein was the ma-jor protein in these precipitates (23). The iron content of theprecipitate also was determined after digestion in 7% per-chloric acid.

Chemicals and minerals. All fine chemicals including FeS,Fe3O4, iron metal (40 mesh), and FeEDTA were reagentgrade and obtained from Fisher Scientific Co. (Edmonton,Alberta, Canada). Enzymes, sarcosyl, SDS, Percoll, andDHBA were obtained from Sigma Chemical Co. (St. Louis,Mo.). Acrylamide and N,N'-methylene bisacrylamide werefrom Eastman Kodak Co. (Rochester, N.Y.). Other electro-phoresis chemicals were obtained from Bio-Rad Labora-tories (Mississauga, Ontario, Canada). Minerals were ob-tained as authenticated samples from the Department ofGeology Museum (University of Alberta) or purchased fromWards Natural Science Establishment, Inc., (Rochester,N.Y.). All minerals were ground to approximately 200 meshbefore use.Humic acid and fulvic acid were extracted from commer-

cial potting soil by a modified procedure of Cheshire et al.(4). The soil (200 g) was washed with 2 liters of 0.1 M HCI for15 h at room temperature, and then the residue was extract-ed with 2 liters of 0.2 M NaOH for 2 days. The suspensionwas centrifuged, the supernatant was filtered through What-man no. 1 paper and then acidified to pH 2.0 with 6 M HCI.The precipitate that formed was humic acid, which wascentrifuged out and freeze-dried. The remaining supernatantwas neutralized to pH 7.0 with NaOH pellets, and a precipi-tate of aluminum and iron hydroxides formed which wasremoved. The remaining solution was concentrated to 80 mlby using an Amicon hollow fiber ultrafiltration unit (<10,000molecular weight pore size), dialyzed against distilled water,and precipitated with 3 parts 95% ethanol at -20°C. Thefulvic acid precipitate was collected by centrifugation andfreeze-dried.

RESULTSRegulation of siderophore production. All the minerals

used in this study contained very little freely exchangeableiron (Table 1), and minerals preincubated overnight in Fe-limited growth medium did not release sufficient iron torepress the A. vinelandii siderophore system. Growth ofstrain UW with these minerals, however, resulted in theextraction and cellular accumulation of at least 450 to 6,000times more iron than was freely exchangeable. This mineralsolubilization was not due to acid production per se becausethe culture fluid remained at pH 6.8 to 7.2 throughout theincubation period.

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498 PAGE AND HUYER

TABLE 1. Derepression of siderophores and mineral iron solubilizationa

Exchange- Cellular Siderophore production (>M) Siderophore derepression'Mineral Ideal formula able ironb ironc (pg) Catechol Azoto- Catechol Azoto-

(ng) UW UA1 uw UA1 bactind UW UA1 bactind

None 0 11 9 250 246 37 1.00 1.00 1.00FeS FeS 120 232 416 48f 38f 0 0.07f 0.07f 0Marcasite FeS2 162 121 174 28f 3lf 0 0.03f 0.05f 0Vivianite Fe3(PO4)2 * 8H20 58 64 95 54 62 0 0.07 0.11 0Olivine (Mg,Fe)2SiO4 62 51 53 204 229 0 0.28 0.45 0Fe3O4 Fe3O4 8 60 50 149 125 0 0.22 0.24 0Hematite Fe2O3 20 55 44 334 333 13 0.34 0.63 0.11Siderite FeCO3 10 48 31 631 733 24 0.73 1.69 0.22Pyrite FeS2 28 42 17 697 419 41 0.90 1.59 0.43Goethite FeO(OH) 18 44 12 652 492 47 0.81 1.50 0.46Illite See footnote g 12 37 14 567 485 83 0.80 1.84 0.94Ilmenite FeTiO3 16 29 14 598 371 121 1.03 1.41 1.68Micaceous Unknown 28 25 10 582 322 107 1.06 1.63 1.56

hematitea All values are means of at least two duplicates.b Iron extracted from 50 mg of mineral with 1 M ammonium acetate, pH 7.2.c Cell-associated iron per 100 ml of culture after incubation for 20 h with 50 mg of mineral.d Strain UW only.e Siderophore derepression relative to the respective Fe-limited control normalized for cellular protein content.f DHBA only; all other figures are mixtures of DHBA and azotochelin.g Illite-bearing shale; ideal formula, 2K20 * 3(Mg,Fe)O * 8(Al,Fe)203 * 24SiO2 * 12H20.

The Fe-limited control culture grew to approximately 3 x

108 cells per ml after 20 h of incubation. These cells were

relatively small, with a protein content of 190 ,ug/ml and an

iron content of 114 ng/ml of culture. Repeated transfer onslants of Fe-limited medium depleted this iron backgroundbut prevented subsequent growth in liquid culture. There-fore, the control cells were not iron free, but the iron contentof these cells was low enough not to interfere with theinterpretation of these data.The minerals fell into four major classes in terms of their

effect on siderophore repression. (i) Marcasite and FeS were

strong repressors of siderophore production. The culturesupernatant fluid contained ethylacetate-extractable cate-chol, however, and this was entirely DHBA (Table 1).Similarly, readily available iron sources such as FeSO4 or

FeEDTA (5 ,ug of Fe per ml) and humic acid or fulvic acid(0.5 mg/ml; pH 7.0) resulted in repression of all the sidero-phores except 20 to 50 ,uM DHBA. Iron metal (0.5 mg/ml)released sufficient freely exchangeable iron into Fe-limitedculture to give at least 1 ,uM soluble iron plus a large amountof insoluble iron salts. Only DHBA was detected in thesupernatant fluid of cultures grown with iron metal. Itappeared, therefore, that DHBA was not stringently con-

trolled by iron availability and may be formed constitutively.These iron sources caused a twofold increase in viable

cells per milliliter, and the cells were larger and contained 7-to 10-fold more iron and 4-fold more protein per milliliter ofculture relative to the Fe-limited control. The viable numberper milliliter of these mineral-containing cultures continuedto increase relative to the Fe-limited control with furtherincubation. The control cell pellet was white, whereas theiron-sufficient cell pellet was pink, which suggested a differ-ent disposition of iron in these cells (i.e., an increased hemecontent [35] could account for the pink coloration and theincreased respiratory activity of these cells [22]). Iron reple-tion appeared to increase the biosynthetic activity of theculture, which in turn was conveniently measured as proteincontent.

(ii) Vivianite, olivine, and Fe3O4 caused repression ofazotobactin but derepressed azotochelin (Table 1). In thecase of olivine, the combination of partial derepression ofazotochelin coupled with increased biosynthetic activity(3.8-fold increased protein content) resulted in a concentra-tion of catechol in the culture supernatant that was onlyslightly less than that found in the fully derepressed Fe-limited control.

(iii) Hematite, siderite, pyrite, and goethite caused partialderepression of azotobactin and partial to full derepressionof azotochelin (Table 1). The increased biosynthetic activity(2.8-fold increased protein content) coupled with derepres-sion of azotochelin resulted in micromolar catechol concen-trations up to 2.8-fold greater than the Fe-limited control.Similarly, the concentration of azotobactin produced bycells growing with pyrite or goethite exceeded that obtainedwith the fully derepressed control.

(iv) Illite, ilmenite, and micaceous hematite caused fullderepression of azotochelin and full to hyperproduction ofazotobactin (Table 1). The increased biosynthetic activity(1.8-fold increased protein content) of these cells coupledwith derepression of the siderophores resulted in 2.3-foldgreater catechol and 2.2- to 3.3-fold greater azotobactinconcentrations than found in the Fe-limited control culturesupernatant fluid.Although the cultures grown with minerals in groups (ii),

(iii), and (iv) accumulated more total iron than the Fe-limitedcontrol (Table 1), the relative content of iron/protein of allthese cultures including the control was 0.7 + 0.1 ng of Feper mg of protein. Clearly, all of these cultures were ironlimited, and the iron content of the cells per se did notprovide insight into the control of siderophore derepression.Although the viable number per milliliter obtained with all ofthese minerals was twofold greater than the Fe-limitedcontrol, further incubation did not significantly increase cellnumbers. This suggested that all of these cultures were in thestationary phase of growth.Mineral solubilization with DHBA. The results in Table 1

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AZOTOBACTER SIDEROPHORE DEREPRESSION 499

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5 10 15 20MINUTES

0 1 2 3 4 5HOURS

FIG. 2. Solubilization of mineral iron by DHBA. The ability of10 mM DHBA in 1 M ammonium acetate, pH 7.2, with 270 rpmshaking at 30°C to release soluble iron from 5 mg of the minerals (A)marcasite (A), FeS (A), iron metal (0), or vivianite (O) per mlwithin minutes of DHBA addition and (B) vivianite (L), olivine (0),illite (A), or Fe3O4 (U) per ml within hours of DHBA addition was

measured. The solubilization of iron metal in 1 M ammoniumacetate, pH 7.2, without DHBA (0) is also plotted. The otherminerals released nanogram amounts of iron in ammonium acetatecontrol flasks.

suggested that iron release from the minerals was achievedwith catechol alone in at least five cases. When mineralswere suspended in 10 mM DHBA buffered with 1 M ammo-nium acetate, pH 7.2, the release of iron from the mineralswas evident by the formation of the purple Amax52O ferri-DHBA. Although the absorbance of ferri-DHBA was direct-ly proportional to iron concentration, the release of yellow-colored compounds from the minerals necessitated an

independent iron assay.Iron was rapidly solubilized from the strong repressors

marcasite, FeS, and iron metal (Fig. 2A). Only iron metalreleased detectable soluble iron into 1 M ammonium acetate,pH 7.2, alone (Fig. 2). Iron was released more slowly fromvivianite, olivine, and Fe3O4 (Fig. 2B). After 20 h of incuba-tion, siderite released 0.38 ,ug of Fe per ml compared with0.61, 5.4, and 18.5 p,g of Fe per ml from Fe3O4, olivine, andvivianite, respectively. No detectable iron was released fromhematite, pyrite, goethite, ilmenite, or micaceous hematiteafter 20 h of incubation. The release of iron from illite-bearing shale was observed and was probably an artifact,analogous to that described previously (1), arising from thehigh concentration of ammonium acetate and DHBA em-ployed.

Mineral solubilization by strain UA1. The role of catecholsin mineral solubilization was confirmed by the use of strainUA1, which produces only DHBA and azotochelin. Thisstrain grew as well as the wild type on FeS and marcasite.Growth, iron accumulation, and siderophore production alsowere similar to the wild type in vivianite, olivine, and Fe3O4.The iron accumulated from hematite and siderite was lessthan that of the wild type, and in these cases, where strainUW derepressed azotobactin synthesis, strain UA1 in-creased or hyperproduced catechol (Table 1). Hyperproduc-tion of catechol coupled with increased biosynthetic activity(3.7-fold increased protein content) on siderite resulted inthe maximum yield of catechol observed. Hyperproductionof catechol was dependent on iron repletion and increasedbiosynthetic activity because catechol production by strains

UA1 and UW was identical in Fe-limited medium. Growth ofstrain UA1 on pyrite, goethite, illite, ilmenite, and mica-ceous hematite was characterized by very low iron accumu-lation despite hyperproduction of catechol and increasedbiosynthetic activity (1.9-fold increased protein content)relative to the control.

Derepression of high-molecular-weight outer membraneproteins. The production of 93K, 85K, 81K, and 77K pro-teins in response to iron limitation of A. vinelandii has beenshown previously using outer membranes prepared by su-crose gradient fractionation (25). Outer membranes used inthis study were prepared by sarcosyl extraction to facilitatethe handling of a large number of samples. Outer membranesprepared by these two methods were identical except thatthe 60K surface protein (W. H. Bingle, J. L. Doran, andW. J. Page, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983,K155, p. 202) was lost in most samples and a 50K proteinwas present in all membrane samples prepared by sarcosylextraction (Fig. 3). The latter protein has been shown to bepresent in outer membrane fragments banding at a densityintermediate between pure outer membrane and cytoplasmicmembrane on sucrose gradients (J. L. Doran, Ph.D. thesis,University of Alberta, 1983).Minerals and iron sources which caused total repression of

siderophore synthesis also repressed the high-molecular-weight outer membrane proteins. A trace of the 85K proteinwas detected in all of these samples except the FeS-growncells (Fig. 3 and 4). Even the partial derepression of thesiderophore system caused by vivianite, olivine, or Fe3O4was sufficient to derepress these outer membrane proteins(SDS-PAGE patterns were all identical to the olivine sampleshown). Increased siderophore production correlated withincreased production of the 93K, 85K, and 77K proteins(Fig. 3 and 4). The production of the 77K protein wasdirectly proportional to the appearance of azotobactin andwas absent from cells grown with olivine (Fig. 4), vivianite,or Fe3O4. The 81K protein appeared at a constant leveldespite the degree of derepression, as did minor Fe-repress-ible proteins, not previously reported, of 86K, 75K, and 74K(Fig. 4).

93K 1 2 3 4 5 6 7 8 91011

85K- __

7 K - _ m ._6__K77K Z~jw mS 50 5K*~~~:iw _ 0

__ - 3~~~~36

_in______m~tm-1 7K

FIG. 3. SDS-PAGE of outer membranes prepared by sarcosylextraction. Strain UW was grown as described in the text in iron-limited Burk medium (lane 1) containing 5 pLg of Fe per ml asFeEDTA (lane 2) and FeSO4 (lane 3) or 0.5 mg of the mineralsolivine (lane 4), hematite (lane 5), illite (lane 6), micaceous hematite(lane 7), siderite (lane 8), goethite (lane 9), iron metal (lane 10), andFeS (lane 11) per ml. The molecular weights indicated were estab-lished by using standards previously described (25). Major iron-repressible polypeptides are indicated by the molecular weights atthe left.

z0CC

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500 PAGE AND HUYER

< -llSideritecc

< tHem~Heatite

,Oliv~~iine

93K 77K, _Ferrous

85K84K SulfateFIG. 4. Densitormetric scans of iron-repressible outer mem-

brane proteins. The high-molecular-weight proteins present in theouter membranes (from Fig. 3) prepared from cells grown for 20 hwith the minerals indicated were scanned to show the relativeabundance of each protein. Total protein applied to each well wasconstant (10 jig). Molecular weights of the proteins are indicated.

Binding of iron to the cell surface. When A. vinelandii wasgrown with FeSO4, iron metal, humic acid, fulvic acid,marcasite, FeS, or vivianite, the 60K surface protein waswell preserved in the sarcosyl membrane preparations(FeSO4, iron metal, and FeS are shown in Fig. 3). The acidicsurface protein has a well-established affinity for cations andcan be precipitated from Burk medium with 20 mM CaCl2(23). Precipitates also formed with unbuffered 20 mMFeSO4, SrCl2, or FeCl3 and contained 140, 90 or 33% of theprotein precipitated by CaCl2. A precipitate containing sur-face protein (SDS-PAGE results not shown) also formedwith 20 mM (Al)2(SO4)3, but this metal interfered withprotein determination. No precipitate formed with 20 mMGaCl3 or CrCl3. Similarly, no precipitate formed with 20 mMFeEDTA, pH 7.0, and the surface protein was poorlypreserved in this membrane preparation (Fig. 3).FeSO4 (20 mM) formed a visible precipitate in Burk buffer

alone, but much greater flocs were formed when the surfaceprotein was present. The precipitates formed at pH 5, 6, 7, 8,and 9 had the same protein content (1.7 mg/ml) but contained0.4, 2,4, 4.2, 4.9, and 4.7 mg of Fe per mg of protein,respectively, in excess of that precipitated by Burk bufferalone at each pH.When strain UW was grown for 20 h with 0.25 to 2.5 jig of

Fe per ml using FeSO4, there was no detectable soluble ironin the culture supernatant fluid, and throughout the range of0.25 to 10 jig of Fe per ml approximately 50% of the ironadded initially was cell bound (Fig. 5). Catechol synthesiswas derepressed at 1 to 2.5 ,ug of Fe per ml, similar to the Fe-limited control, and at 0.25 jig of Fe per ml catechol washyperproduced. When iron was supplied as FeEDTA theculture grew the same as with FeSO4, but the cell-associated

iron was much less. Catechol synthesis was repressed bysoluble iron in all except the lowest initial iron concentration(Fig. 5).Washing the cells pregrown in 5 ,ug of Fe per ml of FeSO4

six times with iron-limited Burk buffer removed 53% of thecell-bound iron. Similar washing with 10 ,uM EDTA or 10mM sodium citrate, pH 7.0, removed 78% of the cell-boundiron and left the same amount of cell-associated iron asfound in the cells grown on 5 jig of Fe per ml of FeEDTA.Significantly, washing the cells pregrown in FeSO4 withdistilled water at 42°C, a treatment known to release thesurface protein (30), also removed 78% of the cell-boundiron.

DISCUSSIONA. vinelandii was able to obtain iron from all of the mineral

and chemical iron sources that were tested. Certain of theseminerals, hematite, goethite, and olivine, are the mostcommon iron sources in soil (32). Siderite and vivianite maybe abundant in the aerobic, neutral to alkaline pH soils richin calcium, phosphate, and carbonate preferred by A. vine-landii (33). Pyrite, FeS, and marcasite, however, are morecommon in acidic anaerobic conditions, and their degrada-tion is usually associated with acidophilic iron-oxidizingbacteria (31). Marcasite and FeS proved to be excellent ironsources for A. vinelandii. Even the most recalcitrant miner-als, illite, ilmenite, and micacious hematite, resulted inlimited iron repletion and increased cellular protein com-pared with the Fe-limited control.

Solubilization of FeS, marcasite, and iron metal or vivian-ite, olivine, and Fe3O4 was achieved with DHBA only orDHBA plus azotochelin, respectively, at neutral pH. DHBAacid alone at neutral pH was very effective in solubilizingthese minerals in vitro, and the ease of iron release byDHBA paralleled the in vivo repressive effect of eachmineral on the siderophore system. The mechanism of iron

4

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0.25 1 2.5 5 10jug IRON / ml

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60-

FIG. 5. Comparison of cell-bound iron using FeSO4 or FeEDTAas the iron source. Strain UW was grown with FeSO4 (A) orFeEDTA (0) added in the original iron concentrations indicated,and the amount of the cell-associated iron per milliliter of culturewas determined. The cell pellets were washed twice with 10 F.MEDTA, pH 7.0, to remove loosely associated iron before perchloricacid digestion and iron assay. Total catechol in the FeSO4 (A) andFeEDTA (0) culture supernatant fluid also was determined.

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AZOTOBACTER SIDEROPHORE DEREPRESSION 501

release from these minerals is most likely related to thereductive properties of catechol rather than chelation ability.Parallel mechanisms of iron solubilization have been report-ed for plants and soil itself. Iron-efficient plants producephenolic "reductants" which solubilize mineral iron andmake it available for chelation and transport (2, 32). Thephenolics in peat cause abiotic conversion of insolublemineral iron into more available humic iron complexes (11).Similarly, the estimation of "iron oxides" in soil involvesextraction with a strong reductant (dithionite) and simplechelator (citrate) at neutral pH (20). Reduction of Fe(III)held in organic complexes by plant root "reductants" resultsin destabilization of the complex and allows a chelator with alower binding coefficient to remove the Fe(II) (2), a situationanalogous to the release of Fe(III) from siderophores at thebacterial cell surface (5). Reductive destabilization byDHBA may have been used by A. vinelandii to remove ironfrom humic and fulvic acids or FeEDTA without the induc-tion of a superior chelator.When excess iron becomes available it appears to complex

with the regularly arranged surface layer of A. vinelandii,thereby being concentrated at the cell surface. Complexescontain maximum iron content when formed at neutral toalkaline pH. Acidic ion solutions (e.g., FeCl3, GaCl3, orCrCl3) failed to complex well with the acidic surface protein(23). The continued association of surface protein with outermembrane after sarcosyl extraction of high available ironcells versus the loss of this protein in low available iron cellsis a matter of speculation. Magnesium and calcium ions arerequired for the organization of the surface protein layer (W.Bingle, unpublished data), and the magnesium ion-bindingactivity of sarcosyl (34) probably caused the solubilization ofthis protein during membrane preparation. Magnesium,however, may be less available for sarcosyl binding whenthe surface protein is complexed with iron (and phosphate).The iron complexed on the cell surface may be resolubi-

lized by DHBA and carried through outer membrane poresfor transport across the cytoplasmic membrane. Similarly,DHBA carries extracellular iron to a cytoplasmic membranetransport protein in enterobactin-deficient strains of Esche-richia coli (9, 12), and DHBA makes iron available toBacillus sp. but does not participate in transport per se (28).The presence of a chelator, even citrate, will mask oreliminate this activity (9, 12, 27; Knosp et al., submitted).This low-affinity iron uptake system of A. vinelandii is quitesimilar to that described for Neurospora crassa (36). Bothorganisms bind iron complexes to the outer surface of thecell in a pH-dependent manner. The surface iron is solubi-lized by chelators and apparently used by the cell as an ironreserve, being mobilized with citric or malic acid by N.crassa or DHBA by A. vinelandii.As iron becomes more difficult to obtain, A. vinelandii

combines chelation with reductive solubilization. Derepres-sion of the siderophores appears to be ordered as azotoche-lin first, followed by azotobactin production. Derepressionof azotobactin without DHBA and azotochelin has not beenobserved. Partial derepression and hyperproduction of ei-ther siderophore is possible. The azotochelin is only hyper-produced, however, when azotobactin cannot be formed. Asa result of iron repletion and increased biosynthetic activity,the cells growing with ferriminerals accumulate greaterconcentrations of siderophores in the growth supernatantthan found in the Fe-limited control. Although the effect ofthis on iron transport remains to be determined, this methodto increase yield should be exploited in siderophore purifica-tion schemes.

The performance of mutant UA1 suggests that azotobactinis a superior chelator compared with azotochelin, and hyper-production of azotochelin cannot substitute for azotobactin.Therefore, the derepression of A. vinelanii siderophoresfollows a logical strategy of reductant production to solubi-lize/destabilize iron and siderophores of increasing bindingaffinity for iron transport. This sequence also may endow alevel of biosynthetic and nitrogen economy on nitrogen-fixing A. vinelandii. As suggested by Neilands (19), ironassimilation is governed by a range of intracellular controls,and the feedback repression by ferrisiderophore provides arapid and fine control of the high-affinity system (13). Thus,fem-DHBA and ferriazotochelin could be involved in feed-back regulation of azotochelin and azotobactin plus the 77Kouter membrane protein synthesis, respectively. The func-tion of the 77K protein as a receptor for azotobactin remainsto be determined. It also will be important to challenge otherbacteria capable of producing multiple siderophores withsuitable iron sources to determine whether siderophorederepression is indeed coordinate (17) or sequential.

ACKNOWLEDGMENTSWe thank Scott Reed for supplying mineral samples and Ashish

Sanon for the preparation of fulvic and humic acid. Wade Bingle andRobert Carmichael also assisted with some laboratory procedures.

This study was funded through grants from the Natural Sciencesand Engineering Research Council of Canada and the AgriculturalResearch Council of Alberta Farming for the Future Program.

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