Siderophores from Microorganisms and Plants (Structure and Bonding)

143 Pages·1985·6.5 MB·English

by  A. Chimiak, R.C. Hider, A. Liu, J.B. Neilands, K. Nomoto, Y. Sugiura

8 Structure
and Bonding
Editors:
M. J. Clarke, Chestnut Hill • J. B. Goodenough, Oxford
J. A. Ibers, Evanston • C. K. J¢rgensen, Gen6ve
D. M. P. Mingos, Oxford • J. B. Neilands, Berkeley
G. A. Palmer, Houston • D. Reinen, Marburg
P. J. Sadler, London • R. Weiss, Strasbourg
R. J. P. Williams, Oxford
Siderophores from
Microorganisms and Plants
With Contributions by
A. Chimiak R.C. Hider A. Liu J.B. Neilands
K. Nomoto Y. Sugiura
With 35 Figures and 16 Tables
Springer-Verlag
Berlin Heidelberg New York Tokyo 1984
Editorial Board
Professor Michael J. Clarke, Boston College, Department of Chemistry, Chestnut
Hill, Massachusetts 02167, U.S.A.
Professor John B. Goodenough, Inorganic Chemistry Laboratory, University of
Oxford, South Parks Road, Oxford OX1 3QR, Great Britain
Professor James A. Ibers, Department of Chemistry, Northwestern University, Evan-
ston, Illinois 60201, U.S.A.
Professor Christian K. Jcrgensen, D6pt. de Chimie Min6rale de l'Universit6, 30 quai
Ernest Ansermet, CH-1211 Gen6ve 4
Professor David Michael P. Mingos, University of Oxford, Inorganic Chemistry
Laboratory, South Parks Road, Oxford OX1 3QR, Great Britain
Professor Joe B. Neilands, Biochemistry Department, University of California, Ber-
keley, California 94720, U.S.A.
Professor Graham A. Palmer, Rice University, Department of Biochemistry, Wiess
School of Natural Sciences, P. O. Box 1892, Houston, Texas 77251, U.S.A.
Professor Dirk Reinen, Fachbereich Chemie der Philipps-Universit/it Marburg,
Hans-Meerwein-Stral3e, D-3550 Marburg
Professor Peter J. Sadler, Birkbeck College, Department of Chemistry, University of
London, London WC1E 7HX, Great Britain
Professor Raymond Weiss, Institut Le Bel, Laboratoire de Cristallochimie et de
Chimie Structurale, 4, rue Blaise Pascal, F-67070 Strasbourg Cedex
Professor Robert Joseph P. Williams, Wadham College, Inorganic Chemistry
Laboratory, Oxford OX1 3QR, Great Britain
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Foreword
This is the first volume of Structure and Bonding to be devoted entirely to
siderophores. These compounds have become a popular subject for study
by scientists whose interests range from synthetic organic chemistry to
molecular genetics. Annually a substantial number of papers on
siderophore systems are presented before such bodies as the American
Society for Microbiology. Siderophores were not always so much in fashion
even though three decades have passed since their general presence and
low-iron induction in microbes was demonstrated.
Volume 1 of Structure and Bonding carried a review on the compounds
now classed as "siderophores" (Gr. "iron bearers") under the somewhat
cumbersome title: "Naturally Occurring Non-Porphyrin Iron com-
pounds") ) By the mid-1950's ferrichrome had been postulated on the basis
of its structure, properties and, especially, its induction at low iron growth,
to function biologically as an iron carrier. 2) Mycobactin and terregens fac-
tor, which initially had no connection with iron, could be related to ferric-
hrome on the basis of binding of Fe(III) and/or growth tests with Pilobolus
kleinii or Arthrobacter terregens.3 ) At the end of the decade of the 1950's
Swiss workers produced a large number of ferrioxamines from Actinomyces
sp. and coined the word siderochromes for all "red-brown" iron-containing
compounds of this type: ) This was subsequently extended to the second
major chemical class of microbial products with equivalent function, the
phenolates and catechols. 5) As all of these ligands yield with Fe(III) charge
transfer bands in the visible, the designation siderochrome seemed approp-
riate. However, Lankford6 ) pointed out that some of the ligands may not
afford colored complexes and as a consequence he suggested the now gener-
ally accepted term of siderophore. His suggestion was timely since com-
pounds with siderophore activity are now turning up with amino, imino and
carboxylate functions only. These ligands do not generate with ferric ion
much if any color. 7) By definition, therefore, a siderophore is a low molecu-
lar weight, virtually ferric ion specific ligand induced by low iron growth of a
microorganism.
Siderophores appear to be so uniformly present in aerobic and faculta-
rive anaerobic microbial life that failure to detect them prompts a research
paper on the fact. Clearly, tests for hydroxamate or catechol functions will
miss the amino/imino carboxylates. Demonstration that the low iron grown
supernatant has the power to reverse iron starvation imposed by a non-
utilized chelator is a more inclusive test. Still, failure to detect a siderophore
may be a consequence of the specific cultural conditions. Furthermore, it is
VI Foreword
evident that enteric, and probably other species, possess uptake systems for
exogenous siderophores. Finally, siderophores may dwell in the lipid phase
of the microbial membrane, s)
Notwithstanding these qualifications, failure of a microorganism to
extract iron from a polymer when the macromolecule and cells are sepa-
rated by a membrane has been taken as evidence for absence of
siderophore. 9) Here one would have to be certain that the carrier iron does
not bind to the membrane.
Do siderophores occur in plants and animals? The paper by Sugiura and
Nomoto in this volume suggests that higher plants do indeed synthesize a
line of compounds which they designate as phytosiderophores. In plants, in
contrast to microbes, genetic confirmation of function is less easily
obtained. Animal tissue has yet to yield a bona fide siderophore but mutants
of SV40 transfomred BALB/3 T 3 cell line adapted to growth in picolinic
acid produce "siderophore-like" growth factors.I°)
What are the prospects for practical applications of siderophores? The
use of deferdferrioxamine B mesylate (Desferal) for deferration of siderotic
patients is well known. Unfortunately, the drug is inactive via the oral route
and must be injected; otherwise, it is relatively effective and non-toxic. The
observation that siderophores may complex loosely bound, errant iron
atoms and so eliminate or diminish the toxicity of partially reduced oxygen
species, such as superoxide anion and peroxide, suggests that compounds of
this type may have possibilities for treatment of rheumatoid arthritis and
other degenerative diseasesJ 1) The chemotherapeutic potential of
siderophores remains unexploited. Antibioses related to iron have been
relegated to three classes.12) In Type I there is a simple deprivation of iron,
as provoked by transferdn or deferdferdchrome A. In Type II, as exem-
plified by albomycin and ferrimycin, the lethal moiety is smuggled into the
cell on a siderophore transport system. In Type III a compound such as iron
bleomycin binds to the DNA and effects strand scission through generation
of oxygen radicals. The seemingly disparate fields of infection and neo-
plasia, and the role therein of iron and siderophores, is discussed in a recent
review by Weinberg. 13) The identifications of the tumor antigen of trans-
formed cells as the transferrin receptor1 4) and the very recent report 15) that
ras proteins in human carcinoma cells complex with the receptor underline
the significance of iron assimilation in the neoplastic state. Although phy-
topathogenic microorganisms in general synthesize siderophores, ~6) no cor-
relation between iron assimilation and virulence has yet been made in plants
as has been possible in human and animal subjectsJ 7)
It is hoped that the present volume of Structure and Bonding will stimu-
late the interest of colleagues in both pure and applied branches of science
to investigate further the properties and behaviour of the unique family of
compounds grouped generically under the term siderophores.
J. B. NEILANDS
Berkeley, California, May 1984
Foreword VII
References
1. Neilands, J. B.: Structure and Bonding I, 59 (1966)
2. Neilands, J. B.: Bactedol. Rev. 21, 101 (1957)
3. Burton, M. O., Sowden, F. J., Lochhead, A. G.: Can. J. Biochem. and Physiol.
32, 400 (1954)
4. Prelog, V.: Pure Appl. Chem. 6, 327 (1963)
5. Neilands, J. B.: In Inorganic Biochemistry (G. L. Eichhom Ed.), Elsevier, p. 167
(1973)
6. Lankford, C. E.: Crit. Rev. Microbiol. 2, 273 (1973)
7. Smith, M. J., Neilands, J. B.: J. Plant Nutr., in press
8. Ratledge, C., Marshall, B. V.: Biochim. Biophys. Acta 279, 58 (1972)
9. Simonson, C., Trivett, T., DeVoe, I. W.: Infect. Immun. 31, 547 (1981)
10. Fernandez-Pol, J. A.: In Microbiology 1983. (D. Schlessinger Ed.), Am. Soc.
Microbiol., Washington, D. C., p. 313
11. Hoe, S., Rowley, D. A., Halliwell, B.: Chem.-Biol. Interactions 41, 75 (1982)
12. Neilands, J. B., Valenta, J. R.: In Metal Ions in Biological Systems, Vol. 19, (H.
Sigel Ed.), Marcel-Dekker, New York, in press
13. Weinberg, E. D.: Physiol. Rev. 64, 65 (1984)
14. Trowbridge, I. S., Omary, M. B.: Proc. Natl. Acad. Sci. USA 78, 3039 (1981)
15. Finkel, T., Cooper, G. M.: Cell 136, 1115 (1984)
16. Leong, S. A., Neilands, J. B.: Arch. Biochem. Biophys. 218, 351 (1983)
17. Weinberg, E. D.: Microbiol. Rev. 42, 45 (1978)
Table of Contents
Methodology of Siderophores
J. B. Neilands . . . . . . . . . . . . . . . . . . . . . . . .
Siderophore Mediated Absorption of Iron
R. C. Hider . . . . . . . . . . . . . . . . . . . . . . . . . 25
Lysine Analogues of Siderophores
A. Chimiak, J. B. Neilands . . . . . . . . . . . . . . . . . 89
Mutational Analysis of Rhodotorulic Acid Synthesis in
Rhodotorula pilimanae
A. Liu, J. B. Neilands . . . . . . . . . . . . . . . . . . . . 97
Phytosiderophores - Structures and Properties of Mugineic
Acids and Their Metal Complexes
Y. Sugiura, K. Nomoto . . . . . . . . . . . . . . . . . . . 107
Author Index Volumes 1-58 . . . . . . . . . . . . . . . . . 137
Methodology of Siderophores
J.B. Neilands
Department of Biochemistry, University of California, Berkeley, CA 94720, USA
Siderophores, defined as iron(Ill) specific transport compounds, are widely distributed in aerobic
and facultative anaerobic microbial species. The list of microbes known to form siderophores
includes various enteric bacteria; human, animal and plant pathogenic bacteria and fungi; soil
microorganisms; Gram positive and negative species, blue green alga (Cyanobacteria) and higher
algae; nitrogen fixing bacteria; and many types of fungi including all species of Penicillia, Neuros-
pora, basidiomycetes and certain types of yeast. As natural products, siderophores are classed
conveniently as hydroxamates, catechols and "miscellaneous", the latter possibly structurally
related to the phytosiderophores of plants. A variety of standard methods has evolved for detection,
enhanced production, isolation, characterization and synthesis of the siderophores. The cloning of
the enterobactin and aerobactin systems of Escherichia coli has been reported. A very large number
of siderophores remain to he characterized as either known compounds or new products. The
opportunities for technical exploitation of the substantial reservoir of basic research knowledge of
siderophores abound in fields as diverse as clinical medicine and plant pathology.
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
II. Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
A. Detection and Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1. Chemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2. Biological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
a. Siderophore Auxotrophes . . . . . . . . . . . . . . . . . . . . . . . . . . 7
b. Wild-Type CeUs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
B. Enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1. Minimal Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2. Carbon Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3. Deferration Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
C. Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
D. Structure and Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
E. Chemical Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
F. Molecular Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
III. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
IV. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Structure and Bonding 58
© SpringeroVerlagB erlin Heidelberg 1984
2 J. B. Neilands
I . I n t r o d u c t i o n
Siderophores, defined as low molecular weight, virtually Fe(III) specific ligands, are
formed generally by aerobic and facultative anaerobic bacteria and by fungi. According
to present knowledge they do not appear to be present in strictly anaerobic bacteria, such
as the Clostridia, probably because the low potential of the growth media of these species
maintains the iron, which they require for synthesis of their iron-sulfur and other pro-
teins, in a more soluble Fe(II) state. Siderophores may not occur in strict lactic acid
bacteria since these lack cytochromes of all types, hydroperoxidases, and substitute the
cobalt-containing ribonucleotide reductase for the more usual iron-containing form of
this enzyme. Indeed, Lactobacillus plantarum, when grown in low iron media, has been
shown 1) to contain only 1.7 atoms of iron per bacterial cell and hence it is possible that
this species has zero requirement for this usually biologically precious metal. A species of
Legionella 2) does not appear to form siderophores of any kind when cultured in laborat-
ory media and it is not known how the pathogen acquires iron in the host. No
siderophore has thus far been reported from Saccharomyces spp., but other fungi com-
monly make siderophores of the ferrichrome type. So, apart from the few exceptions just
noted, siderophores seem to be almost universally present in the microbial world.
It is assumed that iron was firmly established as a bio-essential element during the
anaerobic phase of life on planet Earth 3). In view of the substantial solubility of Fe(II) at
biological pH, it is further speculated that specific ligands were not required and that iron
was assimi!atedby pathways analogous to those followed by other divalent metal ions.
However, once a prokaryotic species, probably Cyanobacteria (blue-green algae),
achieved the capacity to generate O2 gas, the surface iron oxidized and precipitated as the
oxyhydroxide polymer, the analytical composition of which is FeOOH. The relevant
reactions leading to a trimer are
Fe3 + + 3 (OH-) = Fe(OH)3
3Fe(OH)3 = HO-Fe--[O-Fe-]O-Fe-OH + 2H20
I f I
OH OH OH
Taking 10 -38 M as an approximation of the solubility product constant (Ksp) for Fe 3+, the
maximum concentration of soluble ferric ion at pH 7 is given by
K,p = (Fe3+)(OH-)3 = 10-3SM
(Fe 3+) ---- 10-38/10-21 = 10-17 M
This very small number would be diminished even further by taking 7.4 as a more
reasonable value for biological pH. Besides hydroxyl ion, specific iron (Ill) binding
proteins such as transferrin, ovotransferrin, lactotransferrin and ferritin will serve to
decrease even further the level of free ferric ions. It is clear from this semi-quantitative
analysis that the appearance of siderophores concomitantly with the switch from a reduc-
ing to an oxidizing atmosphere makes good evolutionary sense.
The versatile role of iron as an electron transfer catalyst - the redox potentials from
the ferredoxins to cytochrome c oxidase span more or less 1000 m y - and the requirement
MethodologyofSiderophores 3
of the metal ion in such crucial biological reactions as fixation of dinitrogen, photosyn-
thesis, deoxyribotide synthesis4 ) and oxygen metabolism underscore the need for an
effective assimilation system. But precious though iron may be in its physiological roles,
it can also be extremely toxic5 ). Its capacity to generate OH" greatly exacerbates the
toxicity of H202 and O~. Thus in the Fenton reaction
H 2 0 2 + F e 2+ = HO" + HO- + Fe3 +
iron acts as a catalyst to produce the highly reactive OH" radical. For this reason the
uptake of iron is carefully regulated at the membrane level. This appears to be true in
bacteria, fungi, plants and animals. In the last named species it is well established that
iron is recycled internally and this may be the case for lower organisms as well.
Although living species may be expected - in view of the physiological importance of
the element - to have diverse pathways for the uptake of iron, it is the high affinity
siderophore-mediated process in microbial cells which is the most amenable to study.
This is because the system is comprised of two parts, namely, the biosynthetic pathway
leading to the siderophore and the transport system for the chelated iron. It is thus
possible to insert independent genetic blocks in both parts of the siderophore system.
The microbe for which we have the greatest depth of knowledge vis-a-vis its genetics is
Escherichia coli, and hence considerable progress has been made in unraveling the vari-
ous iron assimilation systems in this microorganism. No less than four such systems have
thus far been described in E. coli, namely, (i) enterobactin, (ii) ferrichrome and other
hydoxamate siderophores, (iii) citrate, and (iv) plasmid encoded systems such as the
aerobactin system found on pColV. The molecular mechanism whereby iron regulates at
least one of these systems will soon be known in detail and it is an article of faith among
the microbial iron mongers that these data may suggest how the corresponding controls
operate in fungi, plants and animals. Simple iron deficiency anemia is the single most
important nutritional deficiency in much of the world and basic research with micro-
organisms may indicate how this condition can be corrected in the human, where it seems
to be at root an inability to use efficiently the dietary iron.
Genetic analysis of E. coli shows beyond doubt that the role of siderophores is in iron
metabolism. Thus genetic blocks in either biosynthesis or transport of enterobactin can
be overcome by administration of massive doses of iron salts or by trace levels of a metal-
free siderophore that is utilized. This demonstrates that the role of the ligand is to make
iron available to the cell.
Iron assimilation can be correlated with public health in many other ways. The tumor
antigen has now been established with confidence to be the transferrin receptor6 ). Is is
assumed that the enhanced iron requirement associated with the neoplastic state can be
assigned to the higher energy requirement associated with unbridled protein synthesis,
higher levels of ribotide reductase, and other demands for iron.
The transfusion induced siderosis consequent to the particular therapy for thalasem-
mia (Cooley's anemia), aplastic anemia and, to some extent, sickle cell anemia, is now
treated with deferriferrioxamine B, a siderophore from Streptomyces pilosus. Although a
number of other siderophores and a wide range of synthetic compounds have been
screened, the S. pilosus siderophore, which is marketed as the mesylate salt by Ciba-
Geigy as Desferal, remains the drug of choice7 ). There is still a need for an orally
effective deferration drug. The theory of using the siderophores as the reservoir in which