ANALYTICAL BIOCHEMISTRY 160,47-56 (1987)

Universal Chemical Assay for the Detection and

Determination of Siderophores’

BERNHARDSCHWYNANDJ. B. NEILANDS
Department of Biochemistry, University of California. Berkeley, California 94720

Received May 12, 1986

A universal method to detect and determine siderophores was developed by using their high
affinity for iron(ll1). The ternary complex chrome azurol S/iron(lII)/hexadecyltrimethylam-
monium bromide, with an extinction coefficient of approximately 100,000 M-’ cm-’ at 630
nm, servesas an indicator. When a strong chelator removes the iron from the dye, its color turns
from blue to orange. Because of the high sensitivity, determination of siderophores in solution
and their characterization by paper electrophoresis chromatography can be performed directly
on supematants of culture fluids. The method is also applicable to agar plates. Grange halos
around the colonies on blue agar are indicative of siderophore excretion. It was demonstrated
with Escherichia coli strains that biosynthetic, transport, and regulatory mutations in the
enterobactin system are clearly distinguishable. The method was successfully used to screen
mutants in the iron uptake system of two Rhizobium meliloti strains, DM5 and 1021. o 1987
Academic Press, Inc.
KEY WORDS:paper electrophoresis; chemical assay; bacterial genetic techniques; sidero-
phores; iron chelaton, iron uptake.

In an aerobic environment iron is virtually cently, mugineic acid, a phytosiderophore

insoluble at biological pH, where it exists in
the trivalent state as oxyhydroxide (1). In
blood serum it is not readily available owing
to its tight bond to transferrin (2). In re-
sponse, many microorganisms use a high af-
finity pathway to take up this essential ele-
ment. The transport system involves a low-
molecular-weight, Fe(III)-specific ligand,
termed siderophore (3). Previously the ma-
jority of these molecules fell into either the
hydroxamate or the catechol classes (4). Both
functional groups are chemically detectable
by calorimetric assays: catechols via Arnow’s
method (5) and hydroxamates via either the
Csaky test (6), the absorbance of the iron
complexes (7), or the competition for ferric
ions of a colored iron(II1) complex (8). Re-
’ This work was supported in part by Public Health
Service Grants AM 17 146 and AI04 156 from the Na-
tional Institutes of Health, and by National Science
Foundation Grant PCM78-12 198.
(9), and rhizobactin (lo), a structurally novel
type of siderophore, have been the focus of
interest. Using amine, carboxylate, and alco-
holate groups as binding sites for iron(III),
such chelators lack specific moieties for
chemical assay and their complexes are only
weakly colored in the visible region of the
spectrum. In these cases only biological
assays, which are sensitive but tedious, are
possible (I 1). This report describes a highly
sensitive chemical method for the detection
of siderophores, which is based on their af-
finity for iron(II1) and is therefore indepen-
dent of the structure.
The following chemical equation explains
the principle:
FeDye3-’ + L”- * FeL3-” + Dye’-.
A strong ligand L (e.g., a siderophore) is
added to a highly colored iron dye complex.
When the iron ligand complex is formed, the
release of the free dye is accompanied by a

47 0003-2697187 $3.00
Copyigbt 0 1987 by Academic Ras, Inc.
All rights of reproduction in any form reserved.
48 SCHWYN AND NEILANDS

c,Q
l I soti from laboratory stock. All other materials
were of the highest purity available.
“.ay-Or CH3 FeW CH3(CH2),5N+(C~)3 Glassware was cleaned with 6 M HCl.
Double distilled water was used without ex-
0-
coo- coo- ception.
Strains (relevant genotype is given in
Chrome Azurol S HDTMA brackets). Escherichia coli: AN 194 [proC,
FIG. 1. The indicator used, a complex of unknown leu, trp, thi], AN93 [as AN194, entE],
structure comprised of chrome azurol S, iron(III), and RW193 (=ATCC 33475) [as AN194, entA],
HDTMA, has an extinction coefficient of approximately BN0203 [as AN194,fepB], and 294(pColV-
100,000 M-’ cm-’ at 630 nm and pH 5.6. K30) [thi] were from the laboratory collec-
tion. AB401 [his, arg, leu, thr, thi] and
Ab4020 [his, arg, thr, thi, fur] were con-
color change. With EDTA as ligand L the structed by A. Bagg in this laboratory. Rhi-
color change occurs near the endpoint of a zobium meliloti: 102 1 was provided by Dr. S.
complexometric titration ( 12). Hence indi- Long (Stanford University). DM5 is a spon-
cators used for the determination of iron(II1) taneous, streptomycin-resistant mutant, de-
by this well known analytical method should rived from DM4 (15), and was provided by
be suitable for our purpose. Chrome azurol S Dr. P. Gill in this laboratory. 102 F28, the
(Fig. 1) was chosen, because a similar com- parent strain of DM4, and 102 F34 were ob-
pound (chrome azurol B) was used, together tained from Dr. K. T. Shanmugam (Univer-
with HDTMA,2 to determine serum iron and sity of California, Davis). Rhodotorula pi-
was reported to have an extremely high sensi- limanae UCD 67-64 was from the laboratory
tivity (13). collection.
CAS assay solutions. A 6-ml volume of 10
MATERIALS AND METHODS mM HDTMA solution was placed in a lOO-
Reagents. CAS and HDTMA were pur- ml volumetric flask and diluted with water.
chased from Fluka Chemical corporation. A mixture of 1.5 ml iron(II1) solution (1 mrvr
Anhydrous piperazine and 5-sulfosalicylic FeCl, * 6H2O, 10 mM HCl) and 7.5 ml 2 mM
acid were procured from Sigma Chemical aqueous CAS solution was slowly added
Company. Pipes (free acid) was obtained under stirring. A 4.307-g quantity of anhy-
from U.S. Biochemical Corporation. Stock drous piperazine was dissolved in water and
solutions of neutralized succinic acid and 6.25 ml of 12 M hydrochloric acid was care-
casamino acids (Difco Laboratories, acid-hy- fully added, This buffer solution (pH = p&
drolyzed) were deferrated by extraction with = 5.6) was rinsed into the volumetric flask
3% (w/w) 8-hydroxyquinoline in chloroform which was then filled with water to afford
solution. MM9 growth medium was derived 100 ml of CAS assay solution. The CAS
from M9 (14) by reduction of the phosphate shuttle solution was obtained by adding 5-
to 0.03% KH2P04 and was supplemented sulfosalicylic acid to the above solution at a
with an adequate buffer. Deferriferrioxamine concentration of 4 IIIM. The solutions were
B was procured from Ciba-Geigy Corpora- stored in the dark. A reversible crystalliza-
tion. The other siderophores were taken tion took place in the CAS shuttle solution
below 25°C. After months of storage an in-
crease in the absorbance was observed owing
* Abbreviations used: CAS, chrome azurol S; DHBA, to pickup of trace metals in glass flasks pre-
2,3-dihydroxybenzoic acid; EDDA, ethylenediamine-
NJ”-bis(2-hydroxyphenylacetic acid); HDTMA, hexa- viously cleaned with 6 M HCl. Hence, storage
decyltrimethylammonium bromide; Pipes, 1,4-pipera- in polyethylene bottles is recommended.
zinediethanesulfonic acid. Determination of siderophores in solution.
 ASSAY FOR DETERMINATION
A 0.5-ml aliquot of siderophore solution or
supematant containing less than 7.5 nmol of
the iron chelator was mixed with 0.5 ml CAS
assay solution. A reference was prepared
using exactly the same components except
the siderophore (e.g., the uninoculated me-
dium used for culture of the bacteria or
fungi). After reaching equilibrium the absor-
bance was measured at 630 nm. The CAS
shuttle solution was used when the iron ex-
change was slow. In this case the absorbance
at 630 nm was measured within 6 h because
the blue dye precipitated during longer
storage.
Paper electrophoresis. The ionophoric
mobilities of siderophores were determined
with a flat-bed device using a volatile buffer
at pH 5.6 (5.7 ml glacial acetic acid, 24.3 ml
pyridine per liter). Up to 0.1 ml supematant,
adjusted to pH 5.6, or siderophore solution
was spotted on Whatman No. 3 paper. The
electrophoresis was run at approximately 30
V/cm for l-2 h. The paper was then dried
carefully to remove all traces of pyridine and
acetic acid. It was then sprayed on both sides
with CAS assay solution. (The CAS shuttle
solution could not be used because the blue
color was not stable.) After some minutes
pink spots appeared on a light blue back-
ground. Phosphate was also detectable and
was used as an internal standard.
Siderophore production in liquid culture. A
IO-ml volume of an adequate low iron me-
dium, not interfering with the CAS assay (see
below), was inoculated with a bacterial or
fungal culture and incubated at a proper
temperature. After sufficient growth 0.1 ml
of this culture, demonstrably starved for
iron, was transferred into 10 ml of the same
medium. As a control a flask with 2 PM
added iron (freshly prepared, filter sterilized
FeS04 * 7H20 stock solution) was also inoc-
ulated. During incubation the cell density
was monitored at 600 nm to verify iron star-
vation, which was manifested by a slower
growth rate and a lower final optical density.
When stationary phase was reached samples
were taken, the cells were spun down, and
OF SIDEROPHORES 49
the supematant was assayed for siderophores
as described above. E. coli strains were
grown in MM9, Tris buffer, casamino acids
(0.3%), thiamine - HCl (2 ppm), and succi-
nate (0.2%) at 37°C; Rhizobium meliloti
were grown in MM9, Tris buffer, casamino
acids (0.3%), L-glutamic acid (0.05%), (+)-
biotin (0.5 ppm), and sucrose (0.2%) at 30°C;
and Rhodotorula pilimanae were grown in
Wickerham’s medium ( 16) with sucrose (2%)
at 30°C.
CAS agar plates. To prepare 1 liter of blue
agar, 60.5 mg CAS was dissolved in 50 ml
water and mixed with 10 ml iron(II1) solu-
tion (1 mM FeCl, * 6H20, 10 mM HCl).
Under stirring this solution was slowly added
to 72.9 mg HDTMA dissolved in 40 ml
water. The resultant dark blue liquid was au-
toclaved. Also autoclaved was a mixture of
750 ml H20, 100 ml 10X MM9 salts, 15 g
agar, 30.24 g Pipes, and 12.00 g of a 50%
(w/w) NaOH solution to raise the pH to the
pK, of Pipes (6.8). After cooling to 50°C 30
ml casamino acids (lo%), the carbon source,
and other required supplements like vita-
mins and antibiotics were added as sterile
solutions. The dye solution was finally added
along the glasswall, with enough agitation to
achieve mixing without generation of foam.
Each plate received 30 ml of blue agar. E.
coli specific ingredients: 10 ml glucose (20%)
as a carbon source, 1 ml thiamine. HCl
(0.2%), and 3 ml L-tryptophan (l%), if re-
quired. R. meliloti specific ingredients: 10 ml
glucose or sucrose (20%) as a carbon source,
5 ml L-glutamic acid (lo%, neutralized), and
2.5 ml (+)-biotin (0.02%).
RESULTS
Determination of siderophores in solution.
For the Fe-CAS-HDTMA complex shown
in Fig. 1 an extinction coefficient of approxi-
mately 100,000 M-’ cm-’ was found at 630
nm in solutions buffered at pH 5.6. At this
wavelength the orange-colored, iron-free sys-
tem has essentially no absorption. The re-
sulting sensitivity is remarkably higher than
50 SCHWYN AND
that of procedures which use charge transfer
bands of iron(II1) complexes with extinction
coefficients of a few thousand. The structure
of the blue complex and its stability are un-
known, although the constants for the pure
iron-CAS complexes have been measured
(17). Above pH 7 the blue color changes to
yellow, possibly owing to formation of iron
hydroxide, and is no longer distinguishable
from the iron-free system. Consequently, in
the presence of HDTMA, CAS is competi-
tive in chelating the metal below neutral pH,
while ferric hydroxide seems to have a higher
stability at pH values above 7.
Chelators in the growth media interfere
and have to be avoided. For ligands with
lower iron affinity, like phosphate, citrate, or
DHBA, this interference is observed only at
higher concentrations. Thus, phosphate as a
0.1 M buffer had to be replaced by Tris or
Pipes in MM9 medium, but a 20 mM con-
centration added as phosphorus source did
not interfere. Rich media, like LB, interfered
in an irreproducible manner probably be-
cause of varying compositions of the compo-
nents.
To demonstrate the universality of the
assay, purified representatives of the differ-
ent types of siderophores were tested. The
exchange rate for the iron from CAS into the
siderophore was found to differ widely, de-
pending on structure (for siderophore struc-
tures see Refs. (4) and (10)). Using the cate-
chols enterobactin and vibriobactin the color
changed from blue to orange within minutes.
The polyamino carboxylic acids rhizobactin
and aerobactin, the latter using hydroxa-
mates and an cu-hydroxycarboxylic acid as
iron ligands, reacted within hours. With the
pure hydroxamates rhodotorulic acid and
deferriferrioxamine B equilibrium was
reached only within days. Although knowl-
edge of the reaction rate is useful for a pre-
liminary distinction between different types
of siderophores, acceleration of the rate was
deemed essential for analytical purposes. To
speed the iron exchange, 5-sulfosalicylic acid
was used as a shuttle. It chelates iron quickly,
NEILANDS
but since the stability of the complex is low,
the metal is immediately handed over to the
siderophore. Although the visible spectrum
of the blue iron dye complex is slightly
changed (data not shown), 5-sulfosalicylic
acid does not diminish the sensitivity of the
method since without shuttle an extinction
coefficient of approximately 100,000 M-’
cm-’ is observed at 630 nm and pH 5.6.
All the tested siderophores exhibit a more
or less linear dependence of the absorbance
at 630 nm versus concentration of the chela-
tor (Fig. 2). The varied behavior of entero-
bactin, vibriobactin, aerobactin, and deferri-
ferrioxamine B is probably due to different
purities of the siderophores since in slight
excess (20 PM; the assay solution originally
contains 15 PM Fe) these strong chelators
reach the same absorbance. Although the sta-
bility of the iron complex with rhodotorulic
acid and rhizobactin might be insufficient to
allow complete exchange of the iron, the de-
termination of chelators in solution is
usually possible with moderate accuracy.
In supernatant solutions probably the
most important application is the qualitative
check for the presence of siderophores by
simply watching the color change from blue
to orange. The strains described in Table 1
gave positive results except for 102 F34,
which seems to produce no siderophore
under these conditions. The hrst new sidero-
phore discovered by the method is excreted
by R. meliloti 102 1 (Fig. 3E). The structure is
under investigation in this laboratory.
The quantitative use of the method al-
lowed us to study the dependence of sidero-
phore excretion on the concentration of iron
and other nutrients like amino acids. How-
ever preliminary experience with yeast and
certain Pseudomonas strains suggests cau-
tion. Occasionally microorganisms under
stress excrete metabolic chelators, like citric
acid, not directly involved in the uptake of
iron. Chelators with a relatively low iron af-
finity have to be present in high concentra-
tion to give a positive test; furthermore, they
lack the iron regulation of siderophores.
 ASSAY FOR DETERMINATION OF SIDEROPHORES 51

- RB
__D RA
. . . . . . . . .. “0
. . . . . . . . .0 EB
-m OF
-A AB

0
0 10 20
C*( pM)
FIG. 2. Relative absorbance at 630 nm as a function of C*, the siderophore concentration, except for
rhodotorulic acid, where C* is 3 the concentration, because rhodotorulic acid forms 3:2 complexes with
iron(W). The values refer to the concentration before mixing with the assay solution. 0, enterobactin; 0,
vibriobactin; A, rhizobactin; A, aerobactin; q , rhodotorulic acid, n , defeniferrioxamine B. (---) CAS assay
solution used; (-) CAS shuttle solution used.

Often such systems can be successfully ana-
lyzed by various methods, including paper
electrophoresis, as described below.
Paper electrophoresis. Because of the high
sensitivity of the assay we were able to ana-
lyze supematants of bacterial cultures for
siderophores via paper electrophoresis. Table
1 shows the results for the tested strains. The
assignments were made by comparison with
the purified siderophores. The method was
successfully used for the analysis of the aero-
bactin biosynthetic precursors, distinguish-
ing between aerobactin, N’-acetyl-N’-hy-
droxylysine, and N”-citryl-N’-acetyl-N’-hy-
droxylysine ( 18).
Blue agar plates. The exceptionally high
sensitivity of the assay also enables its use on
agar plates. The 10 MM Fe(II1) tints the agar
with a rich blue color, while the concentra-
tion of siderophores excreted by iron-starved
miroorganisms generally exceeds this level.
This results in a complete change to orange.
Interaction of the dye with the agar seems to
extend the applicability of the method to
higher pH values, although a turn to green
was observed above pH 7. Considering the
color as well as bacterial growth, plates buf-
fered with Pipes at pH 6.8 gave satisfactory
results. The concentration of HDTMA was
found to be crucial; too low it led to precipi-
tation of the blue dye and too high it turned
out to be toxic even for gram-negative bacte-
ria. Because of this toxicity we are not yet
able to extend the method to plate cultures of
certain other microorganisms. While some
fungi grow poorly on the blue agar, gram-
positive bacteria seem to be especially sensi-
tive to the detergent used. However, an im-
provement was achieved either by neutraliz-
ing excessive HDTMA with perchlorate,
52 SCHWYN AND NEILANDS

TABLE 1
ELEC~ROPHORE~CMOBIL~~OFIRONCHELATINGCOMPOUNDSINSUPERNATANTS
OFLOWIRONMICROBIALCULTURES

Mobility” relative
Strain to HSPGT at pH 5.6 Assignment

Escherichia coli
294 (pColV-K30) 0.74 (main spot) Aerobactin
-0.12 Enterobactin and N’-acetyl-W-hydroxylysine
Rhizobium meliloti
102 F28 0.39 Rhizobactin
DM5 0.39 Rhizobactin
102 F34 - No siderophore detected
1021 0.20 (main spot) Unknown anionic siderophore(s)
0.69
Rhodotorula pilimanae
UCD 67-64 -0.13 Rhodotorulic acid

r?Mobilities are not corrected for endosmosis, thus neutral molecules give slightly negative values.

which forms strong ion pairs, or by topping
the plates with agar containing cation ex-
change resin, which adsorbs the free deter-
gent.
To ascertain the behavior of different types
of mutants, the blue plates were applied to
the well known enterobactin system of Es&
erichia co/i. Figure 3A shows a wild-type
strain. To get the iron out of the dye, the
bacterium has to express the high affinity
uptake system but only to a level that satis-
fies its requirements for the metal. This re-
sults in relatively small halos around the col-
onies. Probably due to polymerization of the
blue dye, diffision seems to be minimal and
the zones are astonishingly well focused.
A mutation in the ferric uptake regulation
(fur) gene is known to result in constitutive
derepression of the high affinity uptake sys-
tem (19). In Fig, 3B a strain carrying this
mutation (AB4020) is compared to the wild
type; the different level of enterobactin ex-
cretion is clearly demonstrated.
All four strains shown in Fig. 3C are re-
lated to each other. This is important, be-
cause wild-type E. co/i strains with different
background obviously vary in their behavior
on blue plates (AB40 1 in Fig. 3A and AN 194
in Fig. 3C). BN0203 carries a mutation in
one of the transport genes (&II)). Because
this bacterium is unable to take up ferric en-
terobactin, it deprives itself of iron by its own
siderophore. Consequently the synthesis and
excretion of enterobactin is fully turned on,

FTC. 3. (A-D) E. coli: (A) enterobactin producing wild type; (B) a constitutively deregulated mutant
(fur) is compared to the wild type; (C) transport mutant (fipB) and two synthesis mutants (enti and entE)
of the enterobactin system are compared to the wild type; (D) complementation of the two synthesis
mutations. (E-F) R. meliloti 102 1: (E) assayed supematant solutions of bacterial cultures grown with and
without added iron; (F) screening of mutants for altered siderophore production: the mutant in the second
row is an auxotroph not connected with the iron uptake system, the mutant in the fifth row is obviously an
overproducer. These mutants were generated by Dr. Paul Gill of this laboratory and will be described in a
subsequent publication.
ASSAY FOR DETERMINATION OF SIDEROPHORES 53

AB 401 AB 4020
 ASSAY FOR DETERMINATION
but the growth is very slow, which results in
large halos around small colonies. The other
two mutants are blocked at different points
in the biosynthetic pathway: RW 193 (entA)
is deficient in the production of the interme-
diate 2,3-dihydroxybenzoic acid, while
AN93 (entE) cannot use it for synthesis of
enterobactin (20). Both strains do not show
any halo, although they are able to grow and
must obtain iron via a low affinity pathway
(3). On blue plates containing 250 pM
DHBA, RW 193 reverts to the phenotype of
AN194 (data not shown). As expected,
RW193 is complemented by AN93, which
delivers the necessary DHBA (Fig. 3D).
The blue agar was used by Dr. P. R. Gill in
this laboratory for genetic investigations of
the iron uptake in two R. meliloti strains:
DM5 (like DM4) produces rhizobactin (10)
while 102 1 forms a siderophore of unknown
structure, as mentioned earlier. Well-defined
chromosomal insertion mutants were
screened for altered siderophore production.
Bacteria in colonies without zones (data not
shown) are obviously defective in the synthe-
sis or excretion of the siderophore since they
do not grow on agar containing iron chela-
tors like 2,2’-bipyridine or EDDA. An exam-
ple of a colony with an unusually large halo
is shown in Fig. 3F. The nature of these mu-
tations is not yet fully understood. Details of
this study will be reported elsewhere.
DlSCUSSlON
For the study of iron uptake systems the
detection and determination of siderophores
are absolutely crucial. For this purpose a va-
riety of methods, recently reviewed (1 I), has
evolved. However, no universal assay was
known. This gap is now closed with the
highly sensitive method herein described.
The method should facilitate the discovery of
new siderophores of possible biomedical and
agricultural interest, and should enable a
more penetrating analysis of the biology of
known siderophore systems.
OF SIDEROPHORES 55
To be of any use to the microorganism, the
iron affinity of an excreted siderophore
should be high enough to be competitive
with iron hydroxide. At neutral pH the esti-
mated stability of the blue complex in Fig. 1
seems to be exactly in this range, which
makes it an ideal indicator. Paper electro-
phoretic analysis (in special cases the detec-
tion method might be extended to paper and
thin-layer chromatography) and the fact that
the iron exchange rate depends on the struo
ture of the chelator allow a preliminary char-
acterization of the siderophores. Certainly
the method complements existing tests for
specific chemical functionalities such as hy-
droxamate and catechol. When both ligand
types are absent, as in rhizobactin and in the
siderophore of R. meliloti strain 1021, the
new assay is an indispensable tool to track
the compounds during isolation.
Since the assay is based on the competitive
exchange of iron(III), potential chelators are
detectable corresponding to their affinity for
the metal; i.e., strong chelators like sidero-
phores react in a 1: 1 ratio, while weaker ones
need to be present in an excess. Hence, it is
conceivable that, at least at higher concen-
trations, transition metal binding metabo-
lites, especially antitumor antibiotics such as
bleomycin, adriamycin, and streptonigrin,
could also be detected.
The blue agar may prove to be especially
useful for investigation of the molecular ge-
netics of siderophore systems. Its use as a
screening technique for mutations in the
high affinity uptake system of E. coli and R.
meliloti and the complementation experi-
ment with E. coli demonstrate the potency of
the method. Applications can be imagined
wherever connections with iron metabolism
exist. The following remarks, based on our
experience, may facilitate extension of the
method to other bacterial strains. The agar
contains 10 PM iron, which is primarily
available to the cells without mediation of a
siderophore. Because iron uptake systems
are very well regulated, siderophore excre-
56 SCHWYN
tion and therefore halo formation occur only
when the rate of iron supply limits the
growth. As a rule slowly growing cultures
make smaller zones than faster growing ones.
Hence the nutrient composition of the agar
plate has to be adapted to each bacterial
strain to secure a growth rate sufficiently
rapid to result in deprivation of iron. In other
words, the halo size can be controlled via
growth conditions.
Concerning identification of mutants we
can say that, as a first approximation, bio-
synthetic mutants should give no halo, trans-
port mutants a large iron-repressible halo,
and regulatory mutants a large, non-iron-re-
pressible halo under conditions which lead to
the formation of a small halo by wild type
colonies.
Since E. coli as well as R. meliloti grow on
blue agar, one can conclude that the direct
plating mode is probably applicable to all
gram-negative bacteria. It is desirable to ex-
tend the direct plating technique to other mi-
croorganisms like fungi and gram-positive
bacteria, the siderophores of which respond
to the test. However, this will depend on the
identification of an additive other than
HDTMA or the development of procedures
which separate this compound from the
growing cells.
Note added in proof Recently Jason Cheung of this
laboratory has observed that the zwitterionic detergent
3-(dimethyldodecylammonium) propanesulfonate
(Fluka) may be used as a relatively non-toxic additive for
single cells of the basidiomycetous yeast Rhodotorula
pilimanae propagated on agar surfaces.
AND NEILANDS
ACKNOWLEDGMENTS
We thank Dr. P. R. Gill for providing us with the
preliminary results of the R. meliloti study. We also
thank him and Dr. J. S. Buyer for helpful discussions.
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