Vol. 53, No. 7APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1987, p. 1418-14240099-2240/87/071418-07$02.00/0

Iron-Dependent Production of Hydroxamate by Sodium-DependentAzotobacter chroococcum

WILLIAM J. PAGE

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

Received 17 November 1986/Accepted 14 April 1987

The sodium-dependent strain 184 ofAzotobacter chroococcum was unable to grow significantly in iron-limitedmedium, but did produce iron-repressible outer membrane proteins. Siderophores were not produced underthese conditions. Citric acid was excreted, but not in response to iron limitation. This strain, however, was ableto grow in insoluble mineral iron sources, and under these conditions the cells produced a hydroxamate.Growth on minerals and hydroxamate production was dependent on a low level of freely exchangeable iron.Optimal hydroxamate production was observed with 0.75 ,iM ferric citrate, and hydroxamate production wasrepressed by >5 ,uM iron. Despite this iron requirement, hyroxamate was only formed during internal ironlimitation of the cells. Iron-containing cells were able to grow in iron-limited medium but only producedhydroxamate when their iron-per-cellular-protein content was low. These results, the spectral changesobserved upon Fe3+ addition, and iron-uptake coincident with hydroxamate production suggested that thehydroxamate was a siderophore.

Iron is a micronutrient required by almost all cells. This isespecially true of members of the Azotobacteraceae, afamily of obligate aerobic nitrogen-fixing bacteria. Iron isessential, not only for nitrogenase activity, but also forelectron transport reactions and respiratory protection of theoxygen-labile nitrogenase (28). The iron accumulation sys-tems of only a few members of this family have beenexamined. Azotobacter vinelandii grows in iron-limited me-dium and produces two high-affinity siderophores, thecatechol azotochelin and the fluorescent compoundazotobactin, which scavenge iron and transport it into theiron-limited A. vinelandii cell (16). When iron is moreabundant the siderophores are repressed, but 2,3-dihy-droxybenzoic acid continues to be formed and possibly actsas a low-affinity iron-chelating agent (23). Similarly, Azo-monas macrocytogenes grows in iron-limited medium andproduces a fluorescent iron-binding compound that appearsto act as a siderophore (S. K. Collinson and W. J. Page,Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, 065, p. 246).When iron is more available, the fluorescent compound isrepressed and only 3,4-dihydroxybenzoic acid is present inculture fluids (6, 34). This catechol has been shown tomobilize iron from some insoluble minerals, thereby promot-ing the growth of A. macrocytogenes (6). Azotobacterpaspali growing in iron-limited medium also produces afluorescent compound (32) and forms only 3,4-dihydroxy-benzoic acid when iron is more available (6). It wouldappear, even from this limited sampling, that members of thefamily Azotobacteraceae have developed similar strategiesfor iron accumulation whereby even the low-affinity uptakeof this essential ion may be chelator mediated.Azotobacter chroococcum is the member of this family

which is most frequently isolated from soils worldwide (32).Recently, sodium-dependent A. chroococcum strains havebeen described (21). These strains are both academicallyinteresting and have the potential for development as plantinoculants (4, 5) for saline soils. In bacterium-plant interac-tions, siderophores not only promote the growth of theproducer strain but may also have phytopathogen-suppressive properties or may facilitate iron transport into

the plant itself (15, 26, 27, 29, 31). Initial work with these A.chroococcum strains indicated, however, that they wereunable to grow in iron-limited medium and apparently didnot form siderophores. The present study was thereforeinitiated to explain how these strains could be successful andactive in an environment where iron would be very insoluble(3) and not readily available for their use.

MATERIALS AND METHODSBacterial strain and growth conditions. The sodium-

dependent, capsule-deficient strain 184 of A. chroococcumwas used (21). This culture was maintained on slants of Burkmedium (pH 7.2) (21), containing 1% glucose, 1.1 g ofammonium acetate per liter, and 1.8% agar, incubated at30°C. Sodium contamination of the agar provided sufficientNa+ for growth on solid medium. Iron-limited Burk mediumcontained no added iron, all glassware was acid washed, anddeionized Milli-Q water (Millipore Corp., Bedford, Mass.)was used in medium preparation to minimize iron contami-nation. Iron-sufficient medium contained 18 FM ferroussulfate. The inoculum for each study was pregrown for 72 hon slants of iron-sufficient Burk medium, by which time theiron content of the cells had decreased to a constant 0.35 ±0.05 p.g of Fe3+ per mg of protein. These cells were consid-ered to be iron starved.

Liquid Burk medium also contained 50 [ig of NaCl per ml,and the Fe3+ content was varied by use of ferric citrate. Thismedium was inoculated with a 1% (vol/vol) inoculum ofiron-starved cells suspended in iron-limited Burk buffer(Burk medium lacking the C and N source) to give an initialdensity of approximately 5 x 105 cells per ml. The cultureswere incubated at 30°C with gyratory shaking at 225 rpm. Intime course sampling, a large batch of medium was inocu-lated and dispensed in 20-ml portions per 50-ml Erlenmeyerflask, and a flask was removed at each time point.Growth with mineral iron sources. Mineral iron sources

were obtained as massive minerals from Wards NaturalScience Establishment, Inc., Rochester, N.Y., and thencrushed and ground to approximately 200 mesh before use.Mineral (50 mg) was added either as a loose powder to 100 ml

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I-

E 100.,,,,,,80

lu 60 -

08

a: 40 _

10

14

0 8

0 02 468 012141618

HOURS

FIG. 1. Growth of strain 184 in iron-limited medium. Growth

activities reported include increased cell mass (0), glucose (U) and

acetate (A) consumption, and citric acid (A) excretion.

Of iron-limited Burk medium in a 500-ml Erlenmeyer flask or

was isolated from the cells by sealing the mineral, suspended

in 2 ml of iron-limited medium, inside a dialysis bag

(Spectrapor, 1-cm diameter, 12,000- to 14,000-molecular-

weight cutoff; Fisher Scientific Co., Edmonton, Alberta,

Canada). This bag was small enough to be completely

covered by the medium in the flask. Mineral free in the

medium (loose mineral) and mineral contained in dialysis

bags were sterilized by autoclaving. To determine the

growth supported by freely exchangeable iron released from

the mineral, a flask of iron-limited medium containing min-

eral in a dialysis bag was autoclaved, allowed to cool, and

incubated for 24 h at 30°C with shaking; the dialysis bag was

then removed, and the medium remaining in the flask was

inoculated with strain 184. This same procedure was modi-

fied by the addition of 10 F.M citric acid (pH 6.8) to promote

iron release from the mineral. One flask was used for each

time point in sequential sampling studies.

Chemical analysis of cells and culture fluids. At timed

intervals, cells from 5 ml of culture were collected by

centrifugation and digested in 2 ml of 0.1 M NaOH before the

determination of protein content as described previously

(23). Similarly, 15 ml of cell suspension was used as a source

of cells for digestion in 2 ml of 7%S perchloric acid before

determination of iron content (23). The glucose content of

the culture supernatant fluid was estimated colorimetrically

by a glucose oxidase assay (Statzyme; Worthington Diag-

nostics, Mississauga, Ontario, Canada). Citric acid and

acetate were determined by using enzyme reagent kits from

Boehringer Mannheim, Dorval, Quebec, Canada.

Organic acids in the culture supernatant fluids were first

detected by thin-layer chromatography (TLC) on cellulose

with a fluorescent indicator (no. 6065; Eastman Kodak Co.,

Rochester, N.Y.) developed in a phenol-water-formic acid

(75:25:1, vol/vol) solvent system (19). Amino compounds

were detected by spraying with ninhydrin (0.25 g 100 ml-' ofacetone).The culture supernatant fluid was fractionated further on

Dowex 1-Cl-, 8% cross-linked, 200/400 mesh resin. Theresin was pretreated as described by Cooper (7), convertedto the formic acid form, and used to fill a 2.2-by-25-cmcolumn to a height of 15 cm. Strain 184 culture supernatantfluid (30 ml) was loaded onto the column, washed with aninitial 30 to 40 ml of distilled water, and then eluted with a500-ml 0 to 5 N formic acid gradient. Fractions of 8 ml,decreasing to 6 ml at the end of the gradient, were collected.Each fraction was allowed to evaporate under an air streamand finally dried by heating at 35°C for 10 min. Samples wererehydrated in 2 ml of distilled water, and 25 ,ul of each wasexamined by cellulose TLC with an ether-formic acid-water(140:40:20, vol/vol) solvent system (19). Standards weremade to 0.1 M and spotted (3 ,ul) in parallel. Succinic acidwas quantitated by comparing the TLC spot size to gradedstandards. Citric acid was quantitated enzymatically.

Siderophore assays. The presence of catechols was testedby the method of Arnow (2). Hydroxamates were tested byperiodate oxidation (10) and by the Csaky test (8). Theuniversal siderophore detection medium, described bySchwyn and Neilands (30), was used to show siderophoreproduction during the growth of strain 184. The presence ofsiderophores also was tested by the ability of filter-sterilizedculture supernatant fluids to reverse the inhibition of strain184 growth on Burk medium plates containing deferratedEDDA (ethylendiamine-di(o-hydroxyphenylacetic acid) asdescribed previously (22).

Outer membrane isolation and analysis. Outer membraneswere prepared by sarcosyl extraction as described previ-ously (23). Samples were prepared for sodium dodecylsulfate-polyacrylamide gel electrophoresis and run under theconditions previously described (23).

RESULTS

Iron-limited growth characteristics. A. chroococcum 184was unable to grow beyond a doubling of the initial inoculumin iron-limited medium (Fig. 1). The iron-limited culturesupernatant fluid from A. chroococcum 184 did not form anyfluorescent compounds, and the UV-visible spectrum of theculture supernatant fluid was uneventful and nonreactivewith Fe3". There were no iron-reactive catechols extractedwith ethyl acetate from the culture supernatant fluid or thecells at acidic or neutral pH. Similarly, there was no reactionin the Arnow assay for o-dihydric phenols, and hydroxam-ates were not detected by periodate oxidation or the Csakytest.The iron-limited A. chroococcum cells did, however,

continue to metabolize acetate and glucose (Fig. 1). Strain184 appeared to cometabolize acetate and glucose in contrastto the diauxic utilization of these C sources reported for A.vinelandii (13). By 14 h the cells had consumed 86% of theacetate and 20% of the glucose originally present. As strain184 was incubated in the iron-limited medium, the pH of theculture decreased from pH 7.2 to 6.2, which was unusual foran obligate aerobe but has been previously noted for A.chroococcum (21, 32). Analysis of the iron-limited culturesupernatant fluid by chromatography on Dowex-1, and TLCof the effluent fractions revealed the presence of a number ofninhydrin-reactive compounds, of which glutamic acid was amajor component. The culture supernatant fluids also con-tained approximately 6 mM succinic acid and 10 ,uM citricacid. Up to 20 ,uM citric acid was also excreted in iron-

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sufficient medium containing 18 to 90 ,uM FeSO4, whichindicated that its production was constitutive in relation toiron availability.

Analysis of the iron-limited cell outer membranes pre-pared by sarcosyl extraction revealed the production ofhigh-molecular-weight proteins in response to iron limitation(Fig. 2). These proteins with molecular weights rangingbetween 70,000 and 76,000 were not observed in membranesfrom cells grown with 90 ,uM Fe3+.Hydroxamate production with iron minerals. A. vinelandii

has been shown to obtain iron from a variety of insolublechemical and mineral iron sources via the production ofsiderophores (23). A. chroococcum 184 was similarly cul-tured in the presence of these minerals to determine whichwould promote growth in the absence of high-affinitysiderophores. The surprising result was that the mineralscontaining iron silicates, sulfides, oxides, and oxyhydrox-ides promoted the growth of strain 184 (Table 1). Thesupernatant fluids from the cultures which had enhancedgrowth did not react in the Arnow assay but did react in theCsaky assay. The positive Csaky reaction was dependent onhydrolysis, which indicated that the hydroxamate was in abound form and that the compound was a potential sidero-phore.

Culture fluids containing hydroxamate reversed the inhi-bition of growth caused by the iron chelator EDDA at 0.25mg ml-1', but not at 0.5 to 1.0 mg ml-' (data not shown).Similarly, these culture fluids gave a positive reaction in theuniversal siderophore assay described by Schwyn and Nei-lands (30), which confirmed its siderophore nature (data notshown).

Separation of the minerals into dialysis bags appeared tomake the mineral iron less available to strain 184 in the casesof hematite, goethite, and limonite (Table 1). These culturesdid not grow better than the iron-limited control and did notproduce hydroxamate. In cultures containing olivine, Fe3O4,pyrite, or glauconite in the dialysis bags, the growth of strain184 was less than or equal to that obtained with loose mineralbut greater amounts of hydroxamate were produced. Simi-larly, FeS and muck soil isolated in dialysis bags were nolonger repressive to hyroxamate production (Table 1).Hydroxamate was detected in both the culture superna-

tant fluid and the dialysis bag fluid in all cases. The fluid inthe dialysis bag had a yellow to yellow-orange color whichwas characteristic of this ferrihydroxamate. Addition of ironto hydroxamate-containing culture supernatant fluid resultedin a yellow-orange color at pH 7, which gave increased A460to A520 values without the formation of a distinct peak.Acidification resulted in an increased absorbance in the samerange with a color change to yellow. The ferrihydroxamatecould not be extracted into benzyl alcohol by standardprocedures (12) and thus appeared to be very hydrophilic.

Iron-dependent hydroxamate production. It was curiousthat iron-limited medium did not promote hydroxamateproduction but that the same medium containing insolubleiron sources did. There was a limited amount of freelyexchangeable iron in these minerals but this available irononly promoted growth of strain 184 slightly greater than thatof the iron-limited control (Table 1). Iron could also beextracted from the minerals with 10 FLM citric acid iniron-limited medium, pH 6.8. This mobilized iron also pro-moted somewhat greater growth than did the iron-limitedcontrol (50 to 85 pLg of protein ml-') and also promoted theformation of approximately 0.2 ,ug of hydroxamate-N ml-'.Significantly, the mineral from which the citrate-extractableiron had been removed did not promote the growth or

FIG. 2. Absence of high-molecular-weight outer membrane pro-teins in strain 184 grown in iron-sufficient medium (lane A) and theappearance of these proteins in outer membranes of cells fromiron-limited medium (lane B). Molecular weight standards areshown on the right margin (K = x 1,000).

hydroxamate production by strain 184 to levels greater thanthe iron-limited control.The preceding results suggested that a low level of soluble

iron was necessary to promote hydroxamate production bystrain 184. This was confirmed in cultures which containedlow levels of iron citrate (Fig. 3). The cell yield increased inan almost linear fashion with 0.25 to 2.0 FxM Fe3+. The cellyield increased to 480 pLg of protein ml-' at 5 ,uM Fe3+ anddid not increase further with iron concentrations to 20 FLM.The iron content of the cells from the cultures containing0.25 to 5.0 ,uM Fe3 indicated that all of the iron initiallyadded had become cell associated during the 22-h incuba-tion. Cells from cultures with 10, 15, and 20 ,uM Fe3+contained only 75 to 78% of the added iron after 22 h. Theiron content of the cells from the cultures containing 0.25 to2.5 ,uM Fe> was x = 0.36 + 0.04 pLg of Fe> per mg ofprotein, which was within the range of the iron-limitedcontrol. Hydroxamate was found in the supernatant fluids ofthese cultures but not in the cultures containing 10 to 20 ,uMFe3, which had Fe> values (microgram of Fe> per milli-gram of protein) in excess of the iron-limited control. Cellsfrom the culture containing 5 ,uM Fe> produced a smallamount of hydroxamate (0.5 ,ug of hydroxamate-N ml-') andcontained 0.59 ,ug of Fe> per mg of protein, which indicatedthey also were becoming iron limited. Hydroxamate produc-tion had a definite iron requirement and was optimal at 0.75,uM Fe> (Fig. 3).Analysis of the culture grown with 2 FLM Fe3+ over time

indicated a pattern similar to that observed with no addediron (Fig. 4). At 8 h the citrate level had reached 9.6 to 12FLM, acetate consumption was nearly complete, and thegrowth curve demonstrated a change in slope. At 10 h,citrate production increased to approximately 17 ,M, glu-cose consumption continued without significant acetatecometabolism, and hydroxamate was detectable in the cul-

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TABLE 1. Growth and hydroxamate production by strain 184 in iron-limited medium containing insoluble iron mineralsa

Loose mineralb Dialysatec Mineral in dialysisdIron source Ideal formula Protein Hydroxamate protein, Protein Hydroxamate

(,ug/ml) (,ug of N/ml) (pg/ml) (,g/ml) (pg of N/ml)

FeS FeS 320 0 53 310 1.3Muck soil Fe-humate 350 0 90 365 2.1Glauconite e 385 0.5 44 410 3.9Pyrite FeS2 325 0.5 62 300 3.3Fe3O4 Fe3O4 295 1.0 39 245 1.5Olivine (Fe, Mg)2SiO4 265 0.6 57 150 2.2Goethite a-FeO(OH) 220 2.1 42 45 0Limonite FeO(OH)n, H20 175 1.5 45 40 0Hematite Fe2O3 50 0.6 45 35 0None 42 0 45 40 0

a All values are means of at least two duplicates.b Iron-limited medium containing 50 mg of loose mineral per 100 ml of culture.c Estimate of freely exchangeable iron: iron-limited medium was preincubated for 24 h with 50 mg of mineral sealed in a dialysis bag, the bag was removed

aseptically, and the medium remaining in the flask (dialysate) was inoculated with strain 184.d Iron-limited medium contained 50 mg of mineral sealed in a dialysis bag.eGlauconite ideal formula: K2(Mg, Fe)2Al6(Si4010)3(OH)12.

ture supernatant fluid. At 10 h the iron content of the cellswas 0.63 ,ug of Fe3+ per mg of protein, and this declined to0.40 ± 0.03 ,ug of Fe3+ per mg of protein from 12 h onwards.Thus hydroxamate production was coincident with bothacetate depletion and iron deficiency in the cells.

This analysis was repeated with cultures containing 1 puMFe3+ and with glauconite sealed in dialysis bags, and essen-tially the same results were obtained. In the case of theculture containing 1 p.M Fe3+, hydroxamate was first de-tected at 14 h, which coincided with a cellular iron content of0.53 p.g of Fe3+ per mg of protein and with acetate depletion(data not shown). Similarly, hydroxamate was first detectedat 16 h in the cells grown with glauconite, which followedacetate depletion at 12 h and coincided with a cellular ironcontent of 0.45 jig of Fe3+ per mg of protein (Fig. 5). In thisculture, however, the level of solubilized iron started toincrease coincident with hydroxamate production. The sta-tionary phase started at 36 h when glucose was depleted andhydroxamate did not increase further. The cells continued toaccumulate iron, however, consistent with the continued

Ecm)I-J

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00r

a-

1.0 1.5 2.0iiM IRON

100D

8 > .5

6 "2 a

4 < EfO Z2ra

_ Ir

FIG. 3. Iron-dependent hydroxamate formation by strain 184.Culture growth (0) after 24 h of incubation and the yield ofhydroxamate (0) are shown.

activity of the hydroxamate siderophore in the stationaryphase.

Iron shift-down and hydroxamate production. Iron-limitedcultures very occasionally produced trace amounts ofhydroxamate. This appeared to occur when the cells werenot rigorously iron starved before inoculation into the iron-limited medium. To test this further, iron-sufficient strain 184cells were pregrown in media containing 5, 10, 15, or 20 ,uMFe3+ for 22 h, harvested, and used to inoculate iron-limitedmedium at a constant cell density of 1 mg of protein per 20 mlof medium. After 12 h of growth in iron-limited medium, allof the cell types were iron limited (0.30 + 0.05 ,ug of Fe3+ permg of protein) and hydroxamate production was induced.

Ez

3wu-:

0

x

D . a I i I0 2 4 6 8 10 12 14 16 18 20

HOURS

a

5-0

FIG. 4. Growth of strain 184 in medium containing 2 ,uM ferriccitrate. Growth activities reported include increased cell mass (0),glucose (E) and acetate (A) consumption, iron accumulation (0),and citric acid (A) and hydroxamate (Li) excretion.

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The amount of hydroxamate formed by 18 h was dependenton the initial iron content of the cells, with the greatestamount being formed by the iron-rich cells (Fig. 6). Theproduction of hydroxamate was not simply an amplificationdue to increased cell mass, but was due to increased synthe-sis (Fig. 6), presumably as a consequence of the initial ironcontent of the cells.The acetate in these cultures was depleted by 12 to 14 h.

Hydroxamate was detectable at a low level (0.5 ,ug ofhydroxamate-N ml-1) in all of the cultures at 12 h. Thisinitial low level of hydroxamate was characteristic of thatproduced after shift-down of iron-containing cells into iron-limited medium containing 30 mM acetate as the sole Csource (data not shown). Thus acetate was not a repressor ofhydroxamate production, but was a very poor C source forhydroxamate production.

DISCUSSIONThe sodium-dependent strain 184 of A. chroococcum has

an iron-uptake system that is basically similar to thoseexpressed by other members of the Azotobacteraceae. Thecells produce a low level of citric acid constitutively and thismay promote low-affinity uptake of Fe3" and possibly act asa primitive siderophore (14, 19). Furthermore, localizedacidification of the soil may render iron complexes moresoluble, provided this acidification does not exceed pH 6.2,the lower limit for A. chroococcum growth (21). When the A.chroococcum cells become iron limited they produce ahydroxamate siderophore. Although this compound has notbeen purified, its siderophore character is extremely likely.It appears coincident with growth on insoluble iron sources,demonstrates spectral changes upon Fe3+ addition, appearsto promote Fe3+ uptake into cells grown on glauconite, andis repressed by >5 ,uM Fe3". Iron limitation also causes thecells to produce four iron-repressible outer membrane pro-teins in the 70,000- to 76,000-molecular-weight range.The hydroxamate formed by these cells does not appear to

be a typical hydroxamate, as evidenced by its failure to forma red Fe3` complex and its failure to partition into benzylalcohol. Similarly, its ability to reverse EDDA inhibition islimited, suggesting a low affinity for Fe3+. Culture fluids

400-40 -4

z30 E

20 - 222403

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E m 80 a 0.8 -.06 N

cmG3 5. Gw o stai 1 in m

0- cr~~~~~~~~~~~~~~~-3r o

0 <~~~~~

020 ~~~~~~~~0.20 -.02ccI

0 12 24 36HOURS

FIG. 5. Growth of strain 184 in iron-limited medium containingglauconite sealed in dialysis tubing. Growth activities reportedinclude increased cell mass (0), iron solubilized per milliliter ofculture (O), cellular iron (0), and hydroxamate excreted (c).

3

za3 2

w

x .10cr0

I

C._

0)

Ez

3

0.5 1.0 1.5 2.0 2.5iig CELLULAR IRON ADDED

FIG. 6. Influence of the initial iron content of inoculant cells onhydroxamate produced in iron-limited medium after 18 h of incuba-tion. Hydroxmate per milliliter of culture (0) and hydroxamate yieldper cell mass (0) are shown.

containing this hydroxamate also will not promote thegrowth of Arthrobacter sp. strain JG-9 in the standardhydroxamate bioassay (1), although other growth-inhibitorycompounds could also be present in these fluids (unpub-lished data). This description of the production of ahydroxamate-containing siderophore by an Azotobacter sp.is not unique. Demange et al. (9) have reported that thefluorescent siderophore azotobactin formed by A. vinelandiiD (CCM289) contains N6-hydroxyornithine. Similarly, A.chroococcum B-8 produces a hydroxamate-containing com-pound likely to be a siderophore (P. Lapp, L. A. Profenno,M. Toder, G. A. Mabbott, and F. A. Fekete, Abstr. Annu.Meet. Am. Soc. Microbiol. 1986, K134, p. 215), but itschemical properties and induction characteristics appear tobe completely different from those of strain 184.

Conditions leading to the production of the hydroxamateof strain 184 differ from other members of theAzotobacteraceae and most other siderophore-producingbacteria (20). Strain 184 is unable to grow significantly iniron-limited medium. Metabolism continues, however, andiron-repressible proteins are inserted into the outer mem-brane. This response is similar to that observed with Neis-seria gonorrhoeae, Yersinia spp., and Rhodopseudomonassphaeroides, which derepress siderophore receptors in theirouter membranes without the production of siderophores(18, 25, 33). Strain 184, however, is able to form itshydroxamate siderophore when a small amount of iron isprovided.

Optimal production of strain 184 hydroxamate (9.5 ,ug ofNper mg of protein) occurs with 0.75 ,uM ferric citrate in themedium. This iron-enhanced production of siderophore isvery similar to that observed for nocardamine production byPseudomonas stutzeri (17). Optimal production of thishydroxamate occurs at 0.9 ,uM Fe3+, but approximately 80%of the optimal level is also produced in iron-limited medium.Aquaspirillum magnetotacticum offers an extreme exampleof iron-dependent siderophore production in which morehydroxamate is formed in the presence of 20 ,uM ferricquinate than in the presence of 5 F.M ferric quinate (24). A.vinelandii has been reported to produce optimal levels of its

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siderophores in the presence of 2.5 ,uM FeSO4 (11). Theamount of iron actually available to these cells may be muchlower because the iron added to the highly aerated mediumused in this study would quickly become insoluble (3, 6, 16,23). Similarly, low levels of iron obtained from insolubleiron-containing minerals enhance siderophore production byA. vinelandii (23). The growth of A. chroococcum on mineraliron sources is, however, dependent on the low levels ofsoluble iron released from the minerals. Direct contactbetween A. chroococcum and Fe2O3 or the oxyhydroxides,goethite and limonite, is necessary for hydroxamate produc-tion and growth promotion, although Fe2O3 is a very pooriron source for these bacteria. Less recalcitrant minerals(23), having iron that is perhaps more easily released fromtheir fracture faces by simple diffusion or by the low levels ofcitric acid produced by strain 184, promote hydroxamateproduction and growth even when the mineral is separatedfrom the cells.The reason for this iron requirement is unknown. Iron may

be a cofactor in hydroxamate biosynthesis or may be re-quired for a more general aspect of cell metabolism. Forexample, iron-limited Escherichia coli strains have signifi-cantly decreased heme content and catalase activity (G.Morris and S. M. Hammond, XIV Int. Congr. Microbiol.1986, P.G4-75, p. 257), and a similar failure to protect A.chroococcum cells from oxygen or toxic oxygen productswould probably have a general growth-inhibitory effect.Alternatively, low levels of iron may be required for thecitrate-mediated uptake of iron, a situation analogous to thatobserved in E. coli (14). These possibilities demand furtherexperimental work.Once the cells have been exposed to even limiting

amounts of iron they are capable of growth in iron-limitedmedium, and when the iron content of the cells is low (<0.5jig of Fe31 per mg of protein) hydroxamate production isderepressed. A. chroococcum 184 also produces hydroxam-ate after a shift-down from iron-excess to iron-limited con-ditions. The amount of hydroxamate formed is directlyproportional to the original iron content of the cells.Hydroxamate, however, is formed in response to internaliron limitation, confirming that the derepression of thiscompound is like that of other bacterial siderophores (20).Attempts to purify and characterize this hydroxamate are

under way (S. Shivprasad, unpublished data). Consideringthe recognized ability of hydroxamates to persist in the soil,to bind Fe3+ over a wide range of pH, and to promote Fe3+uptake into plants (26, 27), the previously noted beneficialeffects of A. chroococcum inoculation of various plantspecies (4, 5) may be more related to hydroxamate produc-tion than to nitrogen-fixing ability.

ACKNOWLEDGMENTS

I thank Margaret von Tigerstrom and Linda Jackson for excellenttechnical assistance. Laboratory assistance also was provided byDavid Engelberg. Special thanks are extended to J. B. Neilands forsharing unpublished data.

This study was supported by grants from the Natural Sciences andEngineering Research Council of Canada.

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Specificity and improved methods for mycobactin bioassay byArthrobacter terregens. J. Bacterol. 88:1672-1677.

2. Arnow, L. E. 1937. Colorimetric determination of the compo-nents of 3,4-dihydroxyphenylalanine-tyrosine mixtures. J. Biol.Chem. 118:531-537.

3. Biedermann, G., and P. Schindler. 1957. On the solubilityproduct of precipitate iron hydroxide (III). Acta Chem. Scand.11:731-740.

4. Brown, M. E. 1974. Seed and root bacterization. Annu. Rev.Phytopathol. 12:181-197.

5. Brown, M. E. 1982. Nitrogen fixation by free-living bacteriaassociated with plants: fact or fiction?, p. 25-41. In M. E.Rhodes-Roberts and F. A. Skinner (ed.), Bacteria and plants.Academic Press, Inc., New York.

6. Collinson, S. K., J. L. Doran, and W. J. Page. 1987. Productionof 3,4-dihydroxybenzoic acid by Azomonas macrocytogenesand Azotobacter paspali. Can. J. Microbiol. 33:169-175.

7. Cooper, T. G. 1977. Tools of biochemistry. John Wiley & Sons,Inc., New York.

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