Siderophores from Microorganisms and Plants (Structure and Bonding)

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, Evanston, 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, Berkeley, 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

ISBN 3-540-13649-5 Springer-Verlag Berlin Heidelberg New York Tokyo ISBN 0-387-13649-5 Springer Verlag New York Heidelberg Berlin Tokyo

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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 compounds") ) 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 factor, which initially had no connection with iron, could be related to ferrichrome 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 t y p e : ) 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 appropriate. However, Lankford 6) pointed out that some of the ligands may not afford colored complexes and as a consequence he suggested the now generally accepted term of siderophore. His suggestion was timely since compounds 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 molecular weight, virtually ferric ion specific ligand induced by low iron growth of a microorganism. Siderophores appear to be so uniformly present in aerobic and facultarive 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 nonutilized 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 separated 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 exemplified 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 neoplasia, and the role therein of iron and siderophores, is discussed in a recent review by Weinberg. 13) The identifications of the tumor antigen of transformed cells as the transferrin receptor 14) 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 phytopathogenic microorganisms in general synthesize siderophores, ~6)no correlation 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 stimulate 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, Neurospora, 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Detection and Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Chemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Biological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Siderophore Auxotrophes . . . . . . . . . . . . . . . . . . . . . . . . . . b. Wild-Type CeUs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Minimal Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Carbon Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Deferration Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Structure and Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Chemical Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Molecular Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 5 5 7 7 11 12 12 13 13 14 14 16 18 18

III. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20

IV. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23

Structure and Bonding 58

© SpringeroVerlagBerlin Heidelberg 1984

2

J. B. Neilands

I. Introduction 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 proteins, 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 Legionella2) does not appear to form siderophores of any kind when cultured in laboratory 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 commonly 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

OH

f

OH

I

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

= 10-17M

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 reducing 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, photosynthesis, 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 various 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 microorganisms 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 metalfree 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 thalasemmia (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 CibaGeigy 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

4

J.B. Neilands

to search for such a nostrum is based primarily on the biological origin of these compounds and their specificity for Fe(III). It has been shown8) recently that a substantial fraction of clinical isolates of E. coli synthesizes an hydroxamate type siderophore, aerobactin, as well as the usual catechol type siderophore, enterobactin, the latter commonly encountered among Gram negative bacteria. In E. coli the aerobactin system is encoded on the ColV ("virulence") plasmid, but in other enterics the evidence suggests that the genetic determinants for the siderophore system may, like enterobactin, be on the chromosome9). Bacteria with the capacity to transport siderophores are frequently susceptible to attack by a variety of naturally occurring lethal agents. These outer membrane receptor systems 1°) might be exploited in chemotherapy as they provide specific sites of attachment for siderophore analogues. The high affinity of the receptor for the drug imparts specificity and sensitivity. While resistance to antibiotics, such as albomycin, has been cited as a serious defect to the "illicit transport" approach to drug design, this may not be a serious drawback in the siderophore series. An organism which underwent a genetic variation to no longer use the high affinity iron assimilation pathway would grow well in laboratory media but would be at a serious competitive disadvantage inside living cells where the iron is much less readily available. There is convincing evidence that one of the biological values of fever is interference with the iron assimilation system in the invading pathogen TM12). Exactly how elevated temperatures selectively block the iron gathering pathways of microorganisms remains to be determined but apparently the siderophore system is involved. It has been known for many years that there is a close correlation between availability of iron and pathogenicity in both bacteria and fungi attacking man t3). The list of microorganisms includes, but is not limited to, Yersinia pestis, Pasteurella multocida, Clostridium perfringens, Pseudomonas aeruginosa, E. coli, Listeria monocytogenes, Vibrio cholerae, Neisseria meningitidis, Neisseria gonorrhoeae, Salmonella typhimurium, Klebsiella pneumoniae, Clostridium septicum, and Erysipelothrix rhusiopathiae. In addition, the toxin of Corynebacterium diphtheriae and other organisms is only produced at low iron. It has been shown that the complete high affinity transport system of E. coli is expressed in infections in experimental animals. Moving out of the arena of public health, we see that the struggle for iron among all living species has an impact on biological selection. The fact that siderophore outer membrane receptors in enteric bacteria are "parasitized" by lethal agents constitutes the debit side of the high affinity iron transport system. Siderophore mediated antibiosis based on iron starvation appears to be one mechanism underlying selection and blooming in mixed populations of alga 14). A marine dinoflagellate, Prorocentrum minimum, produces a trihydroxamate type siderophore 15), but the subject of high affinity iron assimilation in marine and estuarine environments is in urgent need of further development. Iron is a limiting nutrient for growth in the blue water of the deep ocean. Similarly, plant growth promoting rhizobacteria are believed to excrete siderophores which render iron unavailable to noxious soil organisms 16). Mycorrhizal fungi, which have long been implicated in phosphorus nutrition in the host plant, may also produce siderophores which assist the plant to acquire iron tT). Soil Pseudomonads produce siderophores which display antifungal activity based on iron complexation, thus holding out the hope for development of novel pesticides ~s).

Methodologyof Siderophores

5

Magnetotactic bacteria have been shown to possess strings of cuboid particles rich in magnetite, Fe304, and it is believed that the microorganisms use this device for orientation to food sources19). The magnetic bacteria contain orders of magnitude more iron than non-magnetic species, but it has not been ascertained if they use the high affinity iron assimilation which is the subject of this review. The discovery of magnetic material in microbes, algae and animals makes the subject of general interest. In sum, the profound biological importance of iron coupled with its quantitative insolubility in aerobic environments at biological pH are the twin factors first eliciting interest in the siderophores. Secondarily, interest has centered on the practical applications of siderophores, as represented by the use of Desferal for treatment of siderosis. This review has been prepared in the hope that it will make available to future workers the expertise which has been accumulated gradually over three decades of siderophore research. It is safe to say that a very large number of siderophores remain to be isolated and characterized. Some of these compounds, such as the factor produced by plasmid bearing strains of Vibrio anguillarum2°), will have both theoretical and practical importance.

H. Methodology A. Detection and Determination 1. Chemical Methods We now recognize three general classes or types of siderophores, namely, hydroxamates, catechols and "miscellaneous"2t). In some instances, such as in the mycobactins, hydroxamate and phenolate ligands are present in the same molecule. The alpha hydroxycarboxylate function occurs rather frequently in the bacterial siderophores, but it is doubtful that this bidentate ligand would stand alone without assistance from the more powerful ligands, hydroxamate and catechol, present in the same molecule. The hydroxamates are, in some respect, the classical variety of siderophore since ferrichrome is the first member of the series to be recognized as an iron transporting natural product from microbial sourcesn). As hydroxamic acids are acylated organic hydroxylamines, digestion of the sample in sulfuric acid and oxidation with iodine to nitrous acid, a modification of the Blom test introduced by Csaky for detection of bound hydroxylamine, is generally employed. The test is sensitive since the nitrous acid is diazotized and coupled to 1-naphthylamine to yield a highly conjugated, intensely colored dye. The reaction depends on the release of hydroxylamine and since all hydroxamate siderophores are of the secondary variety, i.e., the hydroxylamino N is bound to a carbon chain, it is a little mysterious as to how this occurs. The presence of an alpha amino group and a chain length favorable for its cyclization to a potential aldehyde function enhances the yieldsn~. Nonetheless, the Csaky test can only be regarded as semi-

6

J.B. Neilands

quantitative in nature and it further suffers from the fact that it is quite ceremonial and requires several steps using noxious reagents. As an alternative to the Csaky test, reaction with ferric perchlorate in dilute acid, as introduced by Atkin et al. z4), is frequently employed. Hydroxamic acids form di, tetra and sexadentate derivatives with Fe(III) according to the following reactionsZS): F e II1

/

xl-3 O O-

I1 I

Fe3+ + 1-3 R-CO==-N(OH) - R' : R-C--N-R' + 1-3 H + The 1 : I complex is produced in acid solution and is purplish in color with a maximum of 500-520 rim. The 1 : 3 complex is generated in neutral pH and is orange in color with a maximum at ca. 420-450 rim. The intensity of absorption is about 1000 per ring so that the a,,M of ferrichrome, for example, is 2.9 at 425 rim. The perchlorate is used as counterion since its large size precludes coordination with Fe 3÷ and generation of the familiar yellow color of ferric chloride. The ferric ion reaction of the hydroxamic acids can be made to reveal certain intimate details of the structure of siderophores. A trihydroxamate such as ferrichrome remains orange colored even at pH 2. At still lower pH, in mineral acid, the color fades through a fleeting purple and the solution becomes colorless. In contrast, a ferric monohydroxamic acid, such as aceto- or benzo-hydroxamic acid, is purplish already at pH 5; at neutral pH these simple hydroxamic acids have absorption spectra as their ferric derivatives which are quantitatively indistinguishable from that of ferrichrome. A dihydroxamic such as rhodotorulic acid displays intermediate behavior. It is thus apparent that a careful analysis of the effect of the pH on the intensity and position of the absorption peak of the ferric hydroxamate can reveal much about the number of R-CO-N(OH)R' groups per mole and their disposition vis-a.vis the iron atom. In one study of eight different opportunistic and systemic fungal pathogens, the presence of hydroxamate siderophore in the cell-free supernatant was measured by the generation of the sharp peak at 264 nm following periodate oxidation (see below)26). All catechol containing sideroph0res display fluorescence in the ultraviolet that is reminiscent of that observed with 2,3-dihydroxybenzoic acid. The 2,3-dihydroxybenzoyl moiety has three absorption bands in the ultraviolet, one at about 320 rim, one at around 250 nm and the third and most intense in the stepwise sequence, at about 210 nm. The intensity of the band in the near ultraviolet is such that the extinction is 3.2 per millimole. All catechol type siderophores yield wine colored Fe(III) complexes at neutral pH. The very high pKa's of the ring hydroxyls mean that these are not good chelating agents for Fe(III) at slightly acid pH and, in addition, there is a tendency for the metal ion to oxidize the ligand. This has been suggested as one mode of biological unloading of the ironr0; it is also the basis of one test for catechol where the product measured is the ferrous ion28). It is possible to determine hydroxamic acid type siderophores in the presence of the catechol type with the perchlorate reagent since the pH in this medium is low enough to block binding to the aromatic ligand. Oxidation products of the catechol may, however, interfere with quantitation of the reaction. Thus a safer procedure may be first to lower the pH of the cell free supernatant, extract the catechols with ethyl acetate and then apply the ferric perchlorate reagent to the aqueous phase. This avoids dealing

Methodology of Siderophores

7

with any insoluble neutral iron complexes that may be generated in the course of decreasing the pH of the assay solution. The third category of siderophores, designated above as "miscellaneous", probably are amino or imino carboxylic acids. The Fe(III) complexes of these compounds lack the charge transfer bands of ferric hydroxamates and ferric catechols in the visible and are hence not highly colored. In this respect they resemble the ferric derivatives of EDTA and nitrilotriacetate. Although these chelates display considerable absorbancy in the ultraviolet, this is more difficult to quantitate. Thus we are here left with some type of bioassay as the only practical method of detection. There is a need for a universal method for detection of siderophore ligands in cell-free culture supernatants. One possibility is to add excess iron, check the pH to 7, spin out the insoluble excess iron as Fe(OH)3 and measure the dissolved iron by ferrozine (after reduction) or directly by atomic absorption. Attempts in the author's laboratory to perfect this test have been frustrated by refusal of the excess iron to precipitate quantitatively. In a few rare cases the siderophore ligand bears a chromophore. Thus certain of the siderophores produced by the Pseudomonads and variously referred to as pseudobactins 10 and pyoverdines29) are yellow-green fluorescent ligands and may be detected directly by their light absorption in the visible, and by their fluorescence. Fekete et al. a°) described a novel method for detection of siderophore iigands excreted into agar surfaces. The colony is grown up on purified agar prepared from low iron media and after growth a segment adjacent to the colony is cut out and placed on filter paper. After electrophoretic separation, the paper is viewed in ultraviolet light for detection of catechols and sprayed with iron solution for detection of hydroxamate siderophores. Using a number of different species known to form defined siderophores, the expected compounds were detected together with some spots of unknown substances.

2. Biological Methods a. Siderophore A u x o t r o p h e s Bioassays for siderophores are many orders of magnitude more sensitive than the best chemical methods. In essence, we have a choice of use of either a "natural" or artificial (mutant) auxotrophe on the one hand, vs. a wild-type cell grown under iron stress on the other hand. The organism most commonly used for assay of hydroxamate siderophores is the soil isolate Arthrobacter flavescens JG-9 ATCC 29 091. Lochhead 31) and co-workers showed that certain strains of Arthrobacter produced a factor required by other strains of Arthrobacter. Subsequently, a soil enrichment culture growing on the antibiotic puromycin as sole source of carbon yielded an Arthrobacter species named JG-9. It was shown to require hydroxamate type siderophores. Although it does not respond to catechol type siderophores, it is quite omnivorous as regards the hydroxamate type and even uses, at the proper level, synthetic chelating agents. Some caution must hence be used in the interpretation of bioassays based solely on the use of this tester strain.

8

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Rhodotorulic Acid (picomol) Fig. 1. Growth response of Arthrobacterflavescens JG-9 ATCC 29091 to rhodotorulic acid. Small volumes (1 to 20 p.l) of an aqueous solution of rhodotorulic acid were pipetted onto 6 mm paper discs. American Type Culture Collection (ATCC) Medium No. 424 consisting of 1 g Bacto Peptone, 1 g Difco Yeast Extract, 0.2 g K2HPO4 and 1.5 g Bacto Agar were dissolved in 100 ml of distilled water, cooled to 40 °C and seeded with two drops of an overnight culture of A. flavescens growing in Medium 424 supplemented with 2 Ixg of rhodotorulic acid per 100 ml. After impregnation with the siderophore solution, the paper discs were placed on the surface of the seeded agar plates and the latter incubated at 30 *C. The diameter of the halo of exhibition of growth was measured. Other hydroxamate siderophores, such as ferrichrome and deferriferrioxamine B are comparably potent in their capacity to stimulate the growth of A. flavescens which, however, does not respond to eatechol type siderophores

The assay can be performed on agar surfaces containing ATCC medium or in solution 32) using a formula of defined medium. Somewhat more precise assays can be achieved via the latter route. However, in this instance the sample should be filter sterilized since hydroxamates, although reasonably stable, will suffer some degradation in the autoclave. Figure I shows the growth response of Arthrobacterflavescens JG 9 to rhodotorulic acid. As an alternative to the use of Arthrobacter flavescens JG 9, a mutant enteric bacterium can be employed for the assay of hydroxamate siderophores. Although it does not synthesize any detectable siderophore other than the catechol, enterobactin, Salmonella typhimurium LT-2 strains use a wide variety of siderophores of this type 33). Mutants

Methodology of Siderophores

9

blocked in the biosynthesis of enterobactin have been placed in two major Classes, I and I134). The latter are blocked between chorismate and 2,3-dihydroxybenzoate while Class I mutants are defective in the conversion of this catechol to enterobactin. Mutants enb-1 and enb-7 are typical of Class I and II, respectively. The inability of these strains to synthesize enterobactin means that, under certain conditions, a siderophore will be required for growth. These conditions are easily achieved in S. typhimurium since, although it uses citrate as a carbon source, it cannot transport ferric citrate. [In E. coli the converse is true - it does not use citrate as an energy source but has an inducible outer membrane receptor for ferric citrate.] Thus the use of high levels of citrate in the assay medium ties up the adventitious iron in a form unavailable to the enb mutants and, at the same time, provides a metabolizeable substrate for the microorganism. It must be stressed that the enb mutants will respond to enterobactin as well as to the range of hydroxamates tested by Luckey et al. 33). In addition, Class II mutants such as enb-7 will respond to enterobactin precursors at the level of 2,3-dihydroxybenzoic acid since these can be converted to the finished siderophore. In the test of Luckey et al. 33) the siderophores are pipetted onto 6 mm paper discs which are then placed on the surface of the agar medium. While the point does not seem to have been researched systematically, it is likely that the enteric bacteria are somewhat more fastidious than Arthrobacter spp. in their requirements for chelated iron. For some siderophores a more specialized bioassay must be adopted. This is the case for the aerobactin formed by ColV plasmids of E. coli. Fortunately, mutant plasmids are available 35), designated iuc, which are blocked in the biosynthesis of aerobactin but which retain full capacity to use the siderophore. The test organism bearing the iuc plasmid can be seeded into warm agar and the sample applied on the 6 mm paper disc or in wells scooped out of the agar. In addition, a quantity of cells can be patched onto the surface of the seeded agar and the plate then examined for a halo of exhibition of growth. No general assay organism has been described for either the catechols or the "miscellaneous" group of siderophores. In these cases resort must be made to the source organism. As mutants are readily acquired in the enteric bacteria as a class, this is the solution of choice in, for example, the enterobactin produced by E. coli. Here we have two useful mutants 36), RW 193 and RWB 18, both derivatives of E. coli K 12. These have now been desposited with the ATCC and bear the numbers ATCC 33 475 and 33 476, respectively. Both are entA and are hence blocked between chorismate and 2,3-dihydroxybenzoate; neither can achieve the synthesis of enterobactin. The simultaneous use of both tester strains in an assay is recommended in order to confirm that the activity measured is, in fact, enterobactin. Strain ATCC 33 475 forms the 81,000 molecular weight outer membrane receptor for ferric enterobactin and responds to low levels of this siderophore. Strain ATCC 33 476, on the other hand, lacks this outer membrane protein and is not stimulated by low levels of enterobactin, although very high levels may give some activity. Both of these tester strains use ferrichrome and a range of other hydroxamate siderophores comparable to that seen with the enb mutants of S. typhimurium. Figure 2 shows the results of a bioassay for enterobactin as performed in this laboratory. The test depends on the ~use of a chelating agent incapable of supplying iron to E. coli. Our favorite compound of this type is deferriferrichrome A; it is available in this laboratory and its antibiosis against E. coli appears to be based entirely on iron complexation. Ferrichrome A 37) is isolated from low iron grown Ustilago sphaerogena and the

10

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E n t e r o b a c t i n ( p i c o tool) Fig. 2. Growth response of Escherichia coli RW 193 ATCC 33475 to enterobactin. The assay was performed essentially as described in the legend to Fig. 1 except that the medium consisted of 0.8 g Bacto Nutrient Broth, 1.5 g of Bacto Agar and 1 ml of 10 mM deferriferrichrome A in 100 ml of distilled water. Inoculum was two drops of an overnight culture of E. coli RW 193 growing in nutrient broth. The incubation was at 30 *C. In practice it is useful to set up a companion plate seeded with E. coli RWB 18 ATCC 33476 which, owing to the absence of the outer membrane receptor for ferric enterobactin, is not stimulated by low levels of this siderophore. As an additional control, 10 ~1 of 10 IxM ferrichrome can be applied to discs set on all test plates, where the response should be ca. 15 mm for both strains

traces of ferrichrome removed by extraction of a neutral solution with benzyl alcohol. The pure ferrichrome A is treated with cyanide and sodium hydrosulfite in the usual way to remove the iron atom 3a). A 0.1 mM solution of deferriferrichrome A, which retains some yellow color despite the apparent absence of iron, is stored in the refrigerator. In a 100 ml erlenmeyer flask are placed 50 ml of water and 0.4 g of Difco nutrient broth powder and 0.75 g agar. The flask is heated to dissolve the agar; completely aseptic conditions are not required. To the still hot solution is added 0.5 ml of the stock 0.1 mM deferriferrichrome A and the resulting solution divided into two 25 ml portions and placed in the 40 °C water bath. After temperature equilibration, one drop of an overnight culture of A T C C 33 475 and A T C C 33 476 are added to individual flasks, the flasks are swirled and poured into separate petri plates. When the agar has solidified, 6 mm paper discs impregnated with siderophore solution are deftly placed on the surface. Five or six discs can be placed on each plate. The sterile paper discs can imbibe up to about 20 Ixl of solution in a single application. If the siderophore is applied in an organic fluid, such as one of the lower alcohols, the solvent should be allowed to evaporate before the discs are placed on the agar surface. The initial working range, which can subsequently be raised or lowered, is around 10 ~tl of 10 ~tM

Methodology of Siderophores

11

siderophore solution. After the discs have been placed, the plates are inverted and incubated at 37 °C overnight. The readout, which may be visible after a half day of incubation, is a halo of exhibition of growth. Although the diameter of the halo varies monotonically with siderophore concentration, the test is not very precise in quantitative terms. In the absence of deferriferrichrome A, bipyridyl or ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA or EDDHA) 39) may be substituted as the deferration agent applied in situ. As the last named agent is certain to show an appreciable affinity for divalent metal ions in general, it can be expected and, indeed, does have substantial toxicity. One has simply to experiment with a range of concentrations from 0.1 mg up to a few mg/ml. The EDDA should be purified by the methods of Rogers39a). The substantial level of iron in nutrient broth absolutely requires that some such deferration agent be added to suppress low affinity iron uptake and overgrowth in these E. coli strains. This service is performed by citrate in the test of Luckey et al. 33) based on S. typhimurium. The E. coli tester strains require several amino acids, which can be supplied at 40 ~tg/ml for growth in minimal medium. These additions are not required in nutrient broth agar. Yet another deferration agent often employed is one of the transferrins. Some four decades ago4°) it was discovered that the bacteriostatic effect of serum could be reversed with iron - the effect was therefore attributed to binding of iron by transferrin, which is normally only 30-35% saturated. The particular transferrin to use is the ovotransferrin or conalbumin of egg white since this is commercially available at modest cost. It is usually prepared in 5 mM sodium bicarbonate, filter sterilized and diluted into the medium. For short periods of growth, such as overnight growth with relatively heavily seeded plates, it may not be necessary to work under totally asceptic conditions.

b. Wild-Type Cells In the absence of a suitable siderophore auxotrophe, either natural or laboratory acquired, the sole option for the bioassay is the wild-type cell. Negative activity for A. flavescens and E. coli and negative chemical tests for hydroxamate and catechol will require such examination of the wild-type strain before it can be concluded that siderophores are absent. By all accounts the most commonly used deferration agent is EDDA, again used at a concentration that can only be determined by trial and error experimentation. In working with the wild-type cell it must be borne in mind that such cells probably synthesize some level of siderophore regardless of the level of iron in the culture medium. In order to avoid excessive cross feeding and overgrowth of the test plates, it is hence essential to use a light seedling, ca. 103 colony forming units/ml. The zone or halo of exhibition of growth around the paper disc will not be confluent growth, as seen above for the 1 drop of nutrient broth culture of E. coli seeded into 25 ml agar, but a constellation of small, individual colonies. A good illustration of the use of the EDDA method is given by Vandenberg et al. 18~, who employed it for detection of siderophores from Pseudomonas spp. believed to exert anti-fungal activity based on iron starvation. Some of the compounds they detected appear to be superior to known siderophores in their ability to complex iron. Mutants blocked in production of siderophore were, as expected, the most sentitive to EDDA. In all of these biological tests it seems immaterial whether the siderophore is applied as ligand or as iron complex. There will be enough environmental iron available for

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J.B. Neilands

reconstitution of the complex in any growth medium. It is little wonder that most workers have opted for the paper disc assay introduced by Luckey et al. 33) over the more elaborate solution assay. Some of the siderophores, such as the catechols, only dissolve readily in organic solvents. This presents no problem with the paper discs since the solvent can simply be allowed to evaporate before the discs are placed on the agar.

B. Enhancement 1. Minimal Media It has been known for almost three decades that iron starvation elicits overproduction of both hydroxamate and catechol type siderophores. The focus of this discussion will hence be on the various techniques used for derepression of the siderophore synthesizing gene complexes of microorganisms. While a completely generalized relationship between iron concentration and siderophore production cannot be made, it is apparent that many systems are optimally derepressed at ca. 0.1 IxM iron. A certain variation can be expected depending on the

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Time (days) Fig. 3. Generalized relationship between microbial growth and siderophore production. Siderophore appearance awaits depletion of the iron concentration of the medium and generally reaches a maximum in 2-7 days. Vigorous aeration enhances the yield. Most hydroxamates remain stable in the medium (an exception may be prorocentrin - see reference No. 15), but catechols are rapidly oxidized and polymerized. Iron is the most important nutrient affecting production, its regulatory effect generally being evidenced at a concentration of about 1.0 I~M

Methodologyof Siderophores

13

particular species under investigation. Obviously, some cell growth will be required in order to obtain any metabolic products from the microorganism. An iron concentration of 10 ~M is considered to he "high" in iron and generally results in excellent cell mass with only modest yields of siderophore. The relationship between cell growth and siderophore yield is further illustrated in Fig. 3. Obviously the culture must become iron starved before the gene products (enzymes) responsible for siderophore production are optimally expressed. It is apparent that the first task in dealing with a new siderophore system is to devise a purely synthetic or defined (minimal) medium for growth of the cells. This is the most convenient means, not always easy with pathogenic microorganisms or other species with complex nutritional requirements, of achieving a medium low in iron. Regardless of what type of medium is adopted, it is necessary initially to demonstrate enhanced growth in the presence of graded levels of iron salts. This is the only way to be certain that the medium has been made poor in iron.

2. Carbon Source It is often possible to offer the aerobic and facultative anaerobic microorganisms that synthesize siderophores a choice of energy substrate. Glucose, while generally the preferred substrate, may not be optimum for siderophore production. This is well illustrated for aerobactin formation in enteric bacteria such as Aerobacter aerogenes8). Growth of this organism on glucose affords very low and erratic yields of aerobactin. Growth on succinate, on the other hand, gives, quite reproducibly, higher yields. Apparently this is because the succinate can only be metabolized via an aerobic metabolic pathway requiring iron-containing enzymes. As a consequence, the cells rapidly become depleted in iron. A secondary advantage in using a substrate such as succinate or citrate is that these can be added to the culture medium prior to sterilization. The potential aldehyde group of glucose can attach a nitrogen atom in Schiff base linkage and in this way initiate the well known "chemical browning" reactions. Thus glucose should not be added to any culture medium containing nitrogen prior to sterilization; sucrose, or other metabolizeable saccharides without the potential aldehyde function, present no problems of this type.

3. Deferration Methods Should the organism under investigation have complex nutritional requirements, it is then only possible to deferrate the medium in some way. Lankford4t) has published a number of ways in which this can be achieved, dating from the classic work of Waring and Werkman42), who extracted contaminating iron with 8-hydroxyquinoline. A 1% solution of the synthetic chelator is prepared in chloroform and the medium extracted until the gray-green color of the ferric :complex is no longer visible in the solvent layer. The medium is then extracted with pure chloroform in order to remove any residue of the chelator. Since 8-hydroxyquinoline binds a large number of metal ions, it may be necessary to add back some of those essential for growth.

14

J.B. Neilands

Filtration through Chelex, a polyaminocarboxylate resin related in structure of EDTA, has proven to be an effective means of iron removal. Complexation to ovotransferrin followed by ultrafiltration through a protein-proof filter has proven satisfactory for small scale deferration 43).

4. Additives The addition of metal ions such as AI3+ or Cr 3+ to culture media sometimes results in overproduction of siderophores. Both of these ions would be exptected to compete for iron sites although Cr 3+, being subsitution inert, would only slowly enter into complexation in the absence of an externally supplied energy source. It is believed to co-polymerize with iron and in this way to tie up the nutritious metal ion in a form unavailable to the cell. One of the common ways to render enteric and other Gram negative bacteria iron deficient is to employ media containing up to 0.1 M trihydroxymethylaminomethane (Tris). The pKa of this organic base, about 8.1, maintains the pH in the neutral range even with glucose added as energy source. The neutral pH helps to keep iron in an insoluble state and, in addition, the "Iris may have a direct effect on the cell which enhances its iron deficiency.

C. Isolation Siderophores may be recovered from the spent culture fluid either as the free ligand or as the iron complex. In general, it is found that charged species do not extract and crystallize well as their iron complexes and hence such compounds, e.g., aerobactin, are best recovered as the uncomplexed ligand. In any case, it will be necessary to remove the iron before any NMR measurements can be made since the paramagnetism of Fe 3+ will eliminate all signals via line broadening. If desired, AI3÷ or Ga 3+, both diamagnetic ions, can be substituted for the paramagnetic Fe 3+. Gallium is particularly useful since both its charge and radius are closely parallel to those of Fe z+ 44). Some success has attended the direct adsorption of neutral ferric hydroxamates to XAD resins 15), from which they may be eluted with an organic solvent such as methanol. This provides a convenient means of rapid concentration of the siderophore from a large volume of culture fluid. Detailed descriptions have been given elsewhere 4s) for the current methods used in this laboratory for the isolation of ferrichrome and enterobactin, prototypical representatives of the hydroxamate and catechol type siderophores, respectively. A few additional examples will be given here. Rhodotorulic acid. Rhodotorula pilimanae or one of the related basidiomycetous yeasts producing the siderophore is grown in the same low iron medium used for production of the ferrichromes, except with double the sugar and ammonium ion concentration 46). The medium contains,~per liter: K2SO4, 1 g; K2HPO4, 3 g; ammonium acetate, 6 g; sucrose, 40 g; citric acid, 1 g; thiamine, 2 mg; Cu 2+, 5 ~tg; Mn 2+, 35 ttg; Zn z+, 2 mg und Mg2+, 80 rag. The metal ions were supplied as their sulfates. The pH was adjusted to 6.8 by addition of about 0.7 ml of concentrated ammonium hydroxide and the medium

Methodology of Siderophores

15

dispensed in 200 ml volumes contained in I flasks. Growth was carried out with vigorous aeration for about one week. The cells were removed and the supernatant flash evaporated to ca. 0.1 volume of the original culture. After standing overnight, crude rhodotornlic acid precipitated. Recrystallization was achieved from hot water. The yield was about 1-2 g/1. As an alternative means of isolation, the rhodotorulic acid can be extracted into 1 : 1 :: CHCI3 : phenol and then returned to water as described for fen'ichrome. The maximum solubility in water is about 3 mM; most hydroxamic acids are substantially more water soluble.

Aerobactin. A. aerogenes 62-I or another enteric strain producing aerobactin is cultured in "Iris47) medium containing 1% sodium succinate as carbon source, pH 7.4. If A. aerogenes 62-I is used, the aromatic amino acids phenylalanine, tyrosine and tryptophane must be added at a level of 40 mg each per liter. A trace of added iron, 0.5 to 1.0 0M may improve cell yield and hence aerobactin synthesis; iron levels of 10 ~tM or higher will result in repression and should be avoided. After about two days at 37 °C with vigorous aeration the supernatant solution is freed from cells and passed through a column of anion exchanger, such as AG 1-X2, of sufficient capacity to retain all of the siderophore. The aerobactin is eluted with 1 N NH4CI, the effluent fractions giving a ferric chloride positive reaction on the spot plate are pooled and passed through a column of cation exchanger, A G 50 W-X 2. The already acidic solution was adjusted to pH 2, the solution saturated with (NH4)2SO 4 and extracted with benzyl alcohol to remove all hydroxamate from the aqueous phase. Three extractions are generally sufficient to remove all of the aerobactin. The pooled benzyl alcohol extracts are centrifuged to clarity and then filtered through a double layer of filter paper. The clear filtrate is diluted with ten volumes of diethyl ether and the aerobactin extracted into a small volume of water. The aqueous extract containing the aerobactin is swirled with ether to remove traces of benzyl alcohol and then lyophilized. The yield of aerobactin varies from 20-60 mg/J and consists of a slightly tan colored, hygroscopic and deliquescent solid. Aerobactin moves as a trivalent anion on paper electrophoresis at neutral pH. On field desorption mass spectrometry the major line for M + 1 occurs at 565 mass units. Some cultures producing aerobactin have been found to form the side chain, N ~hydroxy-N~-acetyl-L-lysine9). As this product is neutral it comes directly through the ion exchange resins. It is extracted into chloroform: phenol from saturated ammonium sulfate solution at pH 6.5 as the ferric complex. It was deferrated with 8-hydroxyquinoline and crystallized from absolute ethanol to yield prisms with mp 210-211 °C, with evolution of gas. The recovery of this compound suggests that aerobactin arises by oxidation of lysine on the N% followed by the acetylation of the resulting hydroxylamine and, finally, condensation of the a-amino groups with the distal carboxyl groups of citrate. Quite a fair number of enteric bacteria make both aerobactin and enterobactin 9), and Vibrio vulnificus has just been shown to make hydroxamate and catechol, neither yet identified43). The method given above for isolation of aerobactin differs from the original procedure of Gibson and Magrath 4s) essentially by inclusion of a solvent extraction step. In our hands this step has proven indispensable for obtaining hydroxamate type siderophores completely free from salts and other impurities. In lieu of benzyl alcohol, a 1 : 1 :: phenol : chloroform solution may be used as the organic extraction fluid. In this case the organic phase may be the lower one, even in the presence of a relatively high salt

16

J.B. Neflands

concentration. The phenol:chloroform mixture probably has greater solvation power than benzyl alcohol but the latter, which is relatively expensive, can be recovered by distillation. Vibriobactin. In addition to enterobactin, the catechol type siderophores known at this date include agrobactin, parabactin and vibriobactin49). All of these are isolated as the ligands by extraction into ethyl acetate, followed by precipitation with hexane or benzene. The preparation of vibriobactin is illustrative of the general procedures involved. Vibrio cholerae Lou 15 is maintained on 3.7% Difco Brain Heart Infusion agar. This strain is only weakly pathogenic and it is believed that an infective dose would require ingestion of about 1 ml of fully grown culture fluid. A broth culture of V. cholerae Lou 15 is transferred into 6 x 40 ml of "Iris medium containing, as carbon source, 0.4% each of the sodium salts of lactic and succinic acids. After overnight growth at 37 °C with vigorous shaking, the culture was transferred into 6 x 1 1of Tris medium, pH 6.8. During the course of growth the pH drifts upward owing to the consumption of the organic acid. The pH is not allowed to exceed 8.0 since catechols are sensitive to air oxidation, especially at alkaline pH, with consequent formation of quinones and brown polymers. The catechol concentration is monitored by the Arnow reaction which, after 1.5 to 2 days of growth, will reach a maximum value. The pH of the cell free supernatant solution is reduced to about 6.0 by addition of HC1 and then extracted with 3 x 1 1 portions of ethyl acetate. The combined ethyl acetate extract is concentrated to about 100-200 ml on the rotary evaporator, washed with 0.1 M citrate buffer pH 5.5, water and dried over anhydrous MgSO4. The dried ethyl acetate is concentrated to a few ml and the vibriobactin precipitated with excess n-hexane. The crude product is dissolved in the least volume of methanol and again precipitated with hexane. To remove traces of bound ethyl acetate, which NMR spectroscopy shows cling tenaciously to catechol type siderophores, the product is dissolved in the smallest volume of methanol and precipitated with water. After lyophilization the vibriobactin is obtained as a white, amorphous powder in a yield of ca. 20 mg per 6 1. An unusual feature of vibriobactin is the presence of norspermidine rather than spermidine, as in agrobactin and parabactin, as the polyamine backbone. A survey5°) of a large number of Vibrio spp. indicates that norspermidine is very common in these bacteria and its presence there has been suggested to have some taxonomic value.

D. Structure and Characterization The usual methods of structural analysis in the natural product series may be applied to the siderophores. Such methods include the use of standard instrumental techniques such as mass spectrometry and NMR, guided by, in the first instance, knowledge of the C, H and N composition of the sample. A simple flat bed paper electrophoresis is most convenient for study of both the intact siderophores and any hydrolysis products released by digestion in 6 N HCI. Many siderophores, for example, all of those elaborated from citrate as the skeletal backbone, will carry charges and will move on paper electrophoresis with or without the iron. The rate of migration will depend on, primarily, charge, and secondarily, molecular weight. The frictional coefficient has a cube root dependency on the molecular weight, while mobility is directly dependent on charge.

Methodology of Siderophores

17

Thus by use of known standards it is a relatively simple matter to arrive at a postulation regarding size and charge of the unknown. Hydrolysis of hydroxamic acids will yield an acyl moiety and an organic hydroxylamine, both of which will move nicely in an electric field. Similarly, amide and ester groups are present in catechol type siderophores. The bidentate metal-coordinating functional groups are held together by subunits which are themselves condensed together and hence labile to cleavage. The technique of periodate oxidation is most useful for the hydroxamic acids since it cleaves the -CO--N(OH)- bond while leaving intact the amide bonds, which are invariably present elsewhere in the molecule. Total hydrolysis should not be performed when iron is present in the siderophore since this will lead to extensive decomposition of the hydroxylamino moiety, which is sensitive to oxidoreduction. The iron can be removed by treatment with excess 8-hydroxyquinoline in methanol, evaporation of the solvent and extraction of the excess reagent with chloroform. This lability of the -N(OH)H group has prompted de novo use of a variety of methods for its reduction to the amide level. While the hydroxamic acid bond is notoriously difficult to reduce, Raney nickel at 50 lbs pressure will give satisfactory results if the target bond is not too hindered. Treatment with hot 50% HI will both cleave and reduce the -CON(OH)-function. Recently, Akers 51) has suggested the use of TiCI3 as reductant. The recently improved technology of mass spectrometry enables direct examination of a range of siderophores as their iron complexes. If the complex crystallizes, the preferred structural analysis is via X-ray diffraction. A special virtue of the X-ray diffraction method of structural analysis lies in the fact that it reveals at once the sites of attachment of the iron. In determining the nature of the complex ion, the first step is to measure the proton count and then to calculate the overall charge on the complex. The iron must be linked to O, N or S and the base "hardness" of the first of these atoms means that it will be the preferred site of transition metal binding in siderophores. The stability constants are measured by the standard methods of inorganic and coordination chemistry. For biological studies it is sometimes desirable to insert a kinetically inert ion such as Cr 3÷. In the case of ferrichrome, this was accomplished at reflux temperature in methanol 52). The pale green needles of the chromic complex are virtually indistinguishable from the natural product, apart from the change in color. A good illustration of the identification of iron binding sites in a siderophore is the work of Plowman et al. 53) on ferric schizokinen. They showed by a variety of techniques that a deprotonated hydroxyl and carboxyl group of the citrate moiety are linked directly to the iron. The siderophore ligand will enter into complex formation with the-actinides 54), and the binding to AI3÷ and Ga 3÷ have just been cited. The latter element is rare and the aluminum, while abundant, is orders of magnitude weaker than Ga 3+ or Fe 3+ in its affinity for the usual oxygen ligands in siderophores. Thus we are left with the high spin d5 octahedral Fe(III) complex as essentially the only form in which the siderophore ligand can be expected to be encountered in nature. A special feature of siderophore ligands containing optically active centers is the capacity to form A or A coordination isomers. These are in addition to the geometrical isomers, c/s and trans, which are also possible, although c/s is the most usual because of constraints imposed by length of the ligand arms. The enantio isomers of both enterobactin 55) and ferrichrome56) have been synthesized. In E. coli the synthetic A, c/s ferric enterobactin is totally inactive and the ligand, which is prepared from D-serine, com-

18

J.B. Neilands

pletely denies iron to the bacteria. The linear catechols agrobactin and parabactin apparently form isomers which are, as expected, inactive in E. coli. However, on hydrolysis of the oxazoline ring to afford the "A" analogues, which still contain L-threonine, the complex appears to be of the A variety and, interestingly, now acquires some activity for E. coli 56a). This specificity may reside in the siderophore attachment sites in the outer membrane receptor proteins. Siderophore ligands which are 6-coordinate hydroxamates or catechols are often accompanied in the culture fluid by bidentate ligands which appear to be the building stones of the complete ligand. Thus 2,3-dihydroxybenzoic acid is nearly always found in the cell free supernatant of bacteria forming the catechol type siderophores. It has been suggested that these simpler ligands may be involved in interllgand exchange as a device for unloading siderophore iron 57). Low though the redox potentials5s) of ferric siderophores may be, microbial cells do appear to have the capacity for their reduction59).

E. Chemical Synthesis Most of the siderophores, including such relatively complicated structures as mycobactin6°), have been obtained by chemical synthesis. The list includes aerobactin61) and schizokinen 62), and various members of the ferrioxamine series 63). Among the catechols, enterobactin55) has been synthesized from both L- and D-serine. Much of this work has been inspired by the search for an orally effective substitute or replacement for Desferal. The classical chemical route to the hydroxamic acid bond is over the nitro compound, which can be reduced with zinc dust in ammonium chloride solution to the hydroxylamino stage. Acylation of the latter generally results in an N,O-diacyl product, but the O-acyl bond can be cleaved in dilute ammonium hydroxide leaving intact the substantially more stable hydroxamic acid function. Unfortunately, very few nitro compounds useful as siderophore synthons are commercially available. Oxidation of the amino group is difficult to arrest at the hydroxylamino stage. However, the special oxidation method introduced by Keller-Schiedein64) and his colleagues shows considerable promise. Here the amino group is first condensed in Schiff base linkage with benzaldehyde, the Schiff base is then oxidized and hydrolyzed to yield the organic hydroxylamine. These gentle series of reactions can be applied to optically active to-amino acids suitably blocked on the alpha amino group to afford good yields of products and retention of configuration. Some details of the chemical synthesis of specific catechol type siderophores are discussed elsewhere in this volume (Chimiak and Neilands, p. 89). The first member of the series, 2,3-diliydroxy-N-benzoylglycine, was prepared by direct condensation of 2,3dihydroxybenzoic acid with glycine in the presence of dicyclohexycarbodiimide. Generally, however, some type of reversible blocking on the ring hydroxyls will be required for good yields.

F. Molecular Cloning The simple expedient of lowering the iron level of the medium will generally suffice to give reasonable yields of siderophores. While iron starvation also, and apparently coordi-

Methodology of Siderophores

19

Table 1. Siderophore outer membrane receptors in Escherichia coli K 12

Receptor

MoleculaP weight

Ferrichrome

78,000

Ferric enterobactin Ferric aerobactin

81,000 74,000

Gene locus 2.5 rain

13.0 rain plasmid ColV

Lethal agents Albomycin; bacteriophages T 1, T5, 80 and UC-167); Colicin M Colicin B Cloacin

' Relative molecular weight as measured in sodiumdodecylsulfate-polyacrylamidegel electrophoresis

natelyr5, 66), induces overproduction of the transport system, it is still desirable to further enhance this segment of the siderophore system by molecular cloning. Indeed, a complete understanding of the mechanism of action of the high affinity system will require possession of all of the DNA and gene products involved in the system. Cloning in the siderophore series is greatly facilitated by the fact, already noted, that siderophore transport systems are often "parasitized" by specific lethal agents. Table 1 lists the outer membrane receptors in E. coli for three siderophores which are known to act as common binding sites for a variety of bacteriophages, bacteriocins and antibiotics. The enterobactin gene complex of E. coli has been cloned68'69) on bacteriophage Mu and shown to be organized into several transcriptional units across some 26 kb of DNA. The tonA 7°) protein or outer membrane receptor for ferrichrome type siderophores has been cloned in a number of laboratories. The cloning of the aerobactin system from ColV bearing, hospital isolates of E. coli illustrates the general principles 71). ColV DNA is separated by density gradient sedimentation and subjected to complete digestion with restriction enzyme HindIII. This afforded 11 fragments which ranged in size from 1.2 to 30 kb and which summed to about 90 kb, the known size of pColV. The fragments were mixed in 10 fold molar excess with the expression vector pPlac, opened with HindIII. Strain 294 of E. coli K 12 was then transformed with this mixture, after ligation with T 4 ligase. Since the vector carries ampicillin resistance, a primary selection screen was based on resistance to this antibiotic. In a second, negative selection, the ampicillin resistance strains were replicated to plates containing cloacin. This served as an indicator for the presence of the ferric aerobactin receptor, which would make the colonies sensitive to cioacin. From 500 penicillin-resistant transformants, one clone was isolated and sized to I8.3 kb. This plasmid, pABN 1, contained the entire aerobactin gene complex of pColV-K 30 inserted into the single Hind III restriction site of pPlac. A sub-clone of the plasmid yielded pABN5, which contained only the regulatory and biosynthetic genes of the complex on 8.7 kb of DNA, of which 1.9 kb represents the vector programming origin of replication of the plasmid and the 30,000 molecular weight 13-1actamase protein. In minicell preparations pABN 1 was shown to form five proteins 72), in addition to ~-lactamase, with molecular weights ranging from 27,000 to 74,000, the latter the outer membrane ferric aerobactin-cioacin receptor. A sys/,ematic deletion analysis of the largest plasmid revealed the gene order and some information about the function of each

20

J.B. Neilands

of the proteins encoded in the complex, which appears to be a single operon. The promoter73) region was detected by S 1 mapping and the transcription start site identified by sequence analysis of the transcript. The nucleotide base sequence through the promoter region indicates it to be unusually "strong" and closely related to the concensus sequence for E. coli promoters. Thus virulent and invasive strains of E. coli have become equipped with a very efficient mechanism for expression of aerobactin. Since the stability constant of this siderophore is many orders of magnitude below that observed for enterobactin, the question arises as to why the former is at all necessary. At least part of the answer seems to be that enterobactin, the catechol type siderophore, does not function well in a protein aceous environment 74'75). In fact, serum albumin appears to form a relatively stable 1:1 complex with enterobactin easily detected by either equilibrium dialysis or via shift of the near ultraviolet absorption band of the siderophore to longer wavelengths. According to this view, E. coli would maintain a chromosomally encoded siderophore, enterobactin, which it uses for survival in the environment and then resorts to the plasmid encoded aerobactin, non-aromatic in character and with no detectable affinity for serum proteins, for invasion of the host. There is increasing evidence that in enteric bacteria other than E. coli, the aerobactin system may exist side-by-side with the enterobactin system on the bacterial chromosome. It is hence conceivable that the former is on a transposon or other type of mobile genetic element9).

HI. Summary and Conclusions Although at the moment of writing many dozens of siderophores have been characterized, it is apparent that many more such compounds remain to be identified from microbial sources. Hydroxamate and catechol bidentate ligands, as found in ferrichrome and enterobactin, respectively, are the most usual functional groups encountered in siderophores. The preponderance of oxygen atoms at the metal ion binding center of the siderophore provides the specificity for iron(III) and, at the same time, affords a release mechanism via reduction of the bound metal to iron(II). That oxygen is not the exclusive atom linked to iron is illustrated by mycobactin, where coordination to an oxazoline nitrogen has been proven by electron diffraction structural analysis. This type of bonding also probably occurs in agrobactin, parabactin and vibriobactin, all siderophores from Gram negative bacteria. The a-hydroxycarboxylic center occurs in a number of siderophores derived from citrate, namely, aerobactin, arthrobactin (Terregens Factor) and schizokinen. The same grouping occurs in the pseudobactins, where it is supplemented by a brace of catechol and hydroxamate functions. There remain certain compounds with siderophore activity which contain neither catechol nor hydroxamate groups; these may be more closely related to the phytosiderophores described elsewhere in this volume (Sugiura and Nomoto, p. 107) or to the opines. The only clinical application of siderophores of note is the use of deferriferrioxamine B for treatment of transfusion induced siderosis, which is sold by Ciba-Geigy as the mesylate under the trade name of Desferal. Unfortunately, it is not effective via the oral route. There is thus still a requirement for development of a cheap, non-toxic orally active deferration drug. The identification of aerobactin as the siderophore of virulent

Methodology of Siderophores

21

E. coli opens up some interesting possibilities for illicit transport of chemotherapeutic drugs, the latter yet to be synthesized. Optimum production of siderophores will depend primarily on selection of the most propitious microorganism and, secondarily, on choice of a medium readily depleted in iron. The objective is to secure a reasonably robust growth of iron-starved cells. Hydroxamate type siderophores can usually be allowed to accumulate in the growth medium but catechols are much more readily destroyed by oxidation, especially at alkaline pH. Structural determination in the siderophore series is most efficiendy accomplished by X-ray diffraction of the crystals of the iron complex, if these are forthcoming. While most siderophores are now available by chemical synthesis, the biosynthetic method generally yields sufficient material for purposes of basic research. A variety of lethal agents gain access to the cell via outer membrane receptors designed for the transport of siderophores. There would thus seem to exist a unique opportunity for the pharmaceutical industry to design and synthesize an array of drugs which could be smuggled into the cell on this uptake pathway. Cloning of the siderophore systems of E. coli is relatively easily achieved, thanks to the existence of specific lethal agents, and it remains to apply such modern methods of molecular genetics to the fungi and yeasts. The biosynthetic enzymes for the siderophores will prove to be of considerable interest as this will provide another target for crippling the iron-gathering function of the pathogenic microorganism. Finally, the molecular mechanism whereby iron represse s the siderophore system of iron assimilation will soon be known in E. coli, and we can then await the rapid extension of this line of research to fungi, plants and animals. Table 2 gives a list of siderophores which have been characterized to date. Although there is a growing tendency to now isolate known siderophores from new species - thus dimerum acid turned up in Verticillium dahliae, schizokinen in Anabaena sp. andferrioxamine E in Pseudomonas stutzeri- the list given in Table 2 is certain to be elaborated and will include new compounds of clinical interest. It is hoped that this cursory review of the field will be of some assistance in this fascinating work.

Table

2. List of siderophores and related compounds~ Sourceb

L Hydroxamic Acids Ferrichrome Ferrichrome A Ferrichrome C Ferrichrysin Ferricrocin Ferrirubin Ferrirhodin Albomycins (gdsein)76) Ferribactin Sake colorant A Verticillins Pseudobactins16)

Penicillia spp. Ustilago sphaerogena Cryptococcus raelibiosum Aspergillus melleus Aspergillus fumigatus Paecilomyces varioti Aspergillus nidulans Actinomyces subtropicus Pseudornonas fluorescens Aspergillus oryzae Verticillium dahliae Pseudomonas fluorescens

J. B. Neilands

22 Table 2 (continued)

Sourceb Pyoverdines 29) Rhodotomlic acid Dimemm acid Coprogen Triornoicin ~ Isotriornicin TM Schizokinen Terregens Factor (Arthrobactin) Aerobactins) Fnsarinine (Fusigen) Fusarinine A, B, C Triacetyl fusarinine Ferrioxamine A1, A2, B, D1, D2, E, G, H ~) Ferrimycin AI Mycobactins Nocobactins Aspergillic acids (neo-, meta, hydroxy, neohydrory) Mycelianamide Pulcherrimic acid Hadacidin Actinonin

Thioformin (Fluopsin)

Pseudomonas fluorescens Rhodotorula pilimanae Fusarium dimerum Neurospora crassa Epicoccum purpurascens Epicoccum purpurascens Bacillus megaterium Arthrobacter terregens Escherichia coli ColV Fusarium roseum Fusarium roseum Fusarium roseum Streptomyces spp. Streptomyces spp. Mycobacterial spp. Nocardia spp. AspergiUus flavus Penicillium griseofulvum Candida pulcherimma Penicillium aurantioviolaceum Streptomyces sp. Pseudomonas fluorescens

II. Catechols Enterobactin 2,3-dihydroxy-N-benzoyl lysine 2,3-dihydroxy-N-benzoyl serine 2,3-dihydroxy-N-benzoyl glycine 2,3-dihydroxy-N-benzoyl threonlne 2,3-dihydroxybenzoic acid Compound II Agrobactin s°) parabactin sl) Vibriobactin 49) Pyochelinsz)

Enteric bacteria Azotobacter vinelandii Escherichia coli Bacillus subtilis Klebsiella oxytoca Accompanies conjugates Paracoccus denitrificans Agrobacterium tumefaciens Paracoccus deni~flcans Vibrio cholerae Pseudomonas aeruginosa

Unless otherwise referenced, see Neilands, J. B., Ratledge, C. In Handbook of Microbiology, Vol. IV, 2nd ed., Laskin, A. I., Lechevalier, H. A. (eds.), CRC Press, Cleveland, p. 565 (1982), for structures and original literature citations b Not all of these compounds have been shown to meet the definition of a siderophore: low molecular weight, virtually iron(III) specific ligands induced by growth of microorganisms at suboptimal levels of iron. In addition, only a single source is listed. Thus ferrichrome is produced by Aspergillus niger, Ustilagosphaerogena, other fungi and all PeniciUia. Similarly, schizokinen is the product of various cyanobacteria as well as Bacillus megaterium

Methodology of Siderophores

23

IV. References Archibald, F.: FEMS Microbiol. Lettr. 19, 29 (1983) Reeves, M. W., Pine, L., Neilands, J. B., Balows, A.: J. Bacteriol. 154, 324 (1983) Neilands, J. B.: Structure and Bonding 11, 145 (1972) Lammers, M., FoUmann, H.: ibid. 54, 27 (1983) Flitter, W., Rowley, D. A., Halliwell, B.: FEBS Lettr. 158, 310 (1983) Trowbridge, I. S., Bishr Omary, M.: Proc. Natl. Acad. Sci. USA 78, 3039 (1981) Martell, A. E., Anderson, W. F., Badman, D. G. (eds.): Development of Iron Chelators for Clinical Use, Elsevier/North Holland, New York 1981 8. Warner, P. J., Williams, P. H., Bindereif, A., Neilands, J. B.: Infect. Immun. 33, 540 (1981) 9. Neilands, J. B.: Microbiology 1983 (Schlessinger, D., ed.), Am. Soc. Microbiol., Washington, D.C., p. 284 (1983) 10. Neilands, J. B.: Ann. Rev. Microbiol. 36, 285 (1982) 11. Garibaldi, J. A.: J. Bacteriol. 110, 262 (1972) 12. Kluger, M. J., Rothenburg, B. A.: Science 203, 374 (1978) 13. Bullen, J. J.: Rev. Infect. Dis. 3, 1127 (1981) 14. Murphy, T. P., Lean, D. R. S., Nalewajko, C.: Science 192, 900 (1976) 15. Trick, C. G., Andersen, R. J., Gillam, A., Harrison, P. J.: ibid. 219, 306 (1983) 16. Teintze, M., Hossain, M. B., Barnes, C. L., Leong, J., Vander Helm, D.: Biochemistry 20, 6446 (1982) 17. Powell, P. E., Szaniszlo, P. J., Cline, G. R., Reid, C. P. P.: J. Plant Nutr. 5, 653 (1982) 18. Vandenberg, P. A., Gonzalez, C. F., Wright, A. M., Kunka, B. S.: Appl. Environ. Microbioi. 46, 128 (1983) 19. Maugh, T. H.: Science 215, 1492 (1982) 20. Crosa, J. H.: Nature 284, 566 (1980) 21. Neilands, J. B.: Ann. Rev. Biochem. 50, 715 (1981) 22. Neilands, J. B.: Bacteriol. Rev. 21, 101 (1957) 23. Tomlinson, G., Cruickshank, W. H., Viswanatha, T.: Anal. Biochem. 44, 670 (1971) 24. Atkin, C. L., Phaff, H., Neilands, J. B.: J. Bacteriol. 103, 722 (1970) 25. Neilands, J. B.: Structure and Bonding 1, 59 (1965) 26. Holzberg, M., Artis, W. M.: Infect. Immun. 40, 1134 (1983) 27. Hider, R. C., Silver, J., Neilands, J. B., Morrison, I. E. G., Rees, L. V. C." FEBS Lettr. 102, 325 (1979) 28. Rioux, C., Jordan, D. C., Rattray, J. B. M.: Anal. Biochem., 133, 163 (1983) 29. Philson, S. B., Llinas, M.: J. Biol. Chem. 257, 808 (1982) 30. Fekete, F. A., Spence, J. T., Emery, T.: Anal. Biochem. 131, 516 (1983) 31. Lochhead, A. G., Burton, M. O., Thexton, R. H.: Nature 170, 282 (1952) 32. Morrison, N. E., Antoine, A. D., Dewbrey, E. E.: J. Bacteriol. 89, 1630 (1965) 33. Luckey, M., Pollack, J. R., Wayne, R., Ames, B. N., Neilands, J. B.: ibid. 111, 731 (1972) 34. Pollack, J. R., Ames, B. N., Neilands, J. B.: ibid. 104, 635 (1970) 35. Williams, P. H., Warner, P. J.: Infect. Immun. 29, 411 (1980) 36. Wayne, R., Frick, K., Neilands, J. B.: J. Bacteriol. 126, 7 (1976) 37. Garibaldi, J. A., Neilands, J. B.: J. Am. Chem. Soc. 77, 2429 (1955) 38. Emery, T., Neilands, J. B.: Org. Chem. 27, 1075 (1962) 39. Miles, A. A., Khimji, P. L.: J. Med. Microbiol. 8, 477 (1975) 39a. Rogers, H. J.: Infect. Immun. 7, 445 (1973) 40. Schade, A. L., Caroline, L.: Science 100, 14 (1944) 41. Lankford, C. E.: Critical Reviews of Microbiology 2, 273 (1973) 42. Waring, W. S., Werkman, C. H." Arch. Biochem. 1, 303 (1942) 43. Simpson, L. M., Oliver, J. D.: Infect. Immun. 41, 644 (1983) 44. Llinas, M., Wilson, D. M., Neilands, J. B.: Biochemistry 12, 3836 (1973) 45. Neilands, J. B.: In Development of Iron Chelators for Clinical Use (Martell, A. E., Anderson, W. F., Badman, D. G., eds.), Elsevier/North Holland, New York, p. 13 (1981) 46. Atkin, C. L., Neilands, J. B.: Biochemistry 7, 3734 (1968) 1. 2. 3. 4. 5. 6. 7.

24

J.B. Neilands

Simon, E. H., Tessman, I.: Proc. Natl. Acad. Sci. USA 50, 526 (1963) Gibson, F., Magrath, D. I.: Biochim. Biophys. Acta 192, 175 (1969) Griffiths, G., Sigel, S. P., Payne, S. M., Neilands, J. B.: J. Biol. Chem., 259, 383 (1984) Yamamoto, S., Shinoda, S., Kawaguchi, M., Wakamatsu, K., Makita, M.: Can. J. Microbiol. 29, 724 (1982) 51. Akers, H. A.: Appl. Environ. Microbiol. 45, 1704 (1983) 52. Leong, J., Raymond, K. N.: J. Am. Chem. Soc. 96, 6628 (1974) 53. Plowman, J. E., Loehr, T. M., Goldman, S. J., Sanders-Loehr, J.: J. Inorg. Biochem., 20, 183 (1984) 54. Bulman, R. A.: Structure and Bonding 34, 39 (1978) 55. Rastetter, W. H., Erickson, T. J., Venuti, M. C.: J. Org. Chem. 45, 5011 (1980) 56. Winkelmann, G.: FEBS Lettr. 97, 43 (1979) 564. Neilands, J. B., Peterson, T., Leong, S. A.: In ACS Symposium Series 140 (Martell, A. E., ed.) American Chemical Society, Washington, DC p 263 (1980) 57. Monzyk, B., Crumbliss, A. L.: J. Inorganic. Biochem. 19, 19 (1983) 58. Raymond, K. N., Carrano, C. J.: Acc. Chem. Res. 12, 183 (1979) 59. Lodge, J. S., Gaines, C. G., Arceneaux, J. E. L., Byers, B. R.: J. Bacteriol. 149, 771 (1982) 60. Manrer, P. J., Miller, M. J.: J. Am. Chem. Soc. 105, 240 (1983) 61. Manrer, P. J., Miller, M. J.: ibid. 104, 3096 (1982) 62. Lee, B. H., Miller, M. J.: J. Org. Chem. 48, 24 (1983) 63. Keller-Schierlein, W., Prelog, V., Zahner, H.: Prog. Chem. Org. Nat. Products 22, 279 (1964) 64. Naegeli, H.-U., Keller-Schierlein, W.: Helv. Chim. Acta 61, 2089 (1978) 65. Uemura, J., Mizushima, S.: Biochim. Biophys. Acta 413, 163 (1975) 66. Mclntosh, M. A., Earhart, C. F.: Biochem. Biophys. Res. Commun. 70, 315 (1976) 67. Lundrigan, M. D., Lancaster, J. H., Earhart, C. F.: J. Virol. 45, 700 (1983) 68. Laird, A. J., Ribbons, D. W., Woodrow, G. C., Young, I. G.: Gene 11, 347 (1980) 69. Laird, A. J., Young, I. G.: ibid. 11, 359 (1980) 70. Menichi, B., Buu, A.: J. Bacteriol. 154, 130 (1983) 71. Bindereif, A., Neilands, J. B.: ibid. 153, 1111 (1983) 72. Bindereif, A., Thorsness, P. E., Neilands. J. B.: Inorganica Chimica Acta 79, 78 (1983) 73. Bindereif, A., Neilands, J. B.: Unpubfished 74. Konopka, K., Bindereif, A., Neilands, J. B.: Biochemistry 21, 6503 (1982) 75. Konopka, K., Neilands, J. B.: ibid., 23, 2122 (1984) 76. Benz, G., Schr6der, T., Kurz, J., Wiinsche, C., Karl, W., Steffens, G., Pfitzner, J., Schmidt, D.: Angew. Chem. Suppl., 1322 (1982) 77. Frederick, C. B., Szaniszlo, P. J., Vickrey, P. E., Bentley, M. D., Shive, W.: Biochemistry 20, 2432 (1981) 78. Frederick, C. B., Bentley, M. D., Shive, W.: Biochem. Biophys. Res. Commun. 105, 133 (1982) 79. Adapa, S., Huber, P., KeHer-Schiedein, W.: Helv. Chim. Acta 65, 1818 (1982) 80. Ong, S. A., Peterson, T., Neilands, J. B.: J. Biol. Chem. 254, 1860 (1979) 81. Peterson, T., Neilands, J. B.: Tetrahedron Lettr. 50, 4805 (1979) 82. Cox, C. D., Rinehart, K. L., Moore, M. L., Cook, J. C.: Proc. Natl. Acad. Sci. USA 78, 4256 (1981) 47. 48. 49. 50.

Siderophore Mediated Absorption of Iron Robert C. Hider Department of Chemistry, Essex University, Wivenhoe Park, Colchester, Essex, UK

A brief presentation of iron chemistry is made with emphasis on those aspects relevant to siderophore biochemistry. Siderophore structure and biosynthesis is described. The underlying chemistry associated with, 1, the movement of iron(III) complexes across membranes and 2, the removal of iron from such complexes is discussed in detail. The ability of siderophores to interact with other metals is considered. Finally, the role of siderophores in infection and their clinical potential as iron scavenging molecules are reviewed.

Symbols and Abbreviations 1 Introduction

.................................

26

.......................................

27

2 Siderophore Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Bidentate Ligands Possessing High Affinity for Iron(III) . . . . . . . . . . . . . . 2.2 Hexadentate Siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Kinetic Lability of Iron(III) Complexes . . . . . . . . . . . . . . . . . . . . . . . 2.4 Redox Activity of Catechol Ligands . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Stereochemistry of Octahedral Hydroxamate and Catecholate Complexes . . . . .

34 34 38 40 42 46

3 Siderophore Structure and Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Siderophore Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Siderophore Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48 48 50

4 Iron Transport in Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Iron(III) Siderophore Uptake by Fungi . . . . . . . . . . . . . . . . . . . . . . . 4.1.1 Iron(III) Siderophore Uptake by Ustilago . . . . . . . . . . . . . . . . . . 4.1.2 Iron(III) Siderophore U p t a k e by Neurospora . . . . . . . . . . . . . . . . 4.1.3 Iron(III) Siderophore Uptake by Rhodotorula . . . . . . . . . . . . . . . . 4.1.4 Iron(III) Siderophore Uptake by Fusaria . . . . . . . . . . . . . . . . . . . 4.1.5 General Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Iron(III) Siderophore Uptake by Enteric Bacteria . . . . . . . . . . . . . . . . . 4.2.1 Iron(III) Siderophore Translocation of the Outer Membrane . . . . . . . . 4.2.1.1 Iron(III) enterobactin Receptor . . . . . . . . . . . . . . . . . . 4.2.1.2 Ferrichrome Receptor . . . . . . . . . . . . . . . . . . . . . . . 4.2.1.3 Iron(III) citrate Receptor . . . . . ................. 4.2.1.4 Iron(III) aerobactin Receptor . . . . . . . . . . . . . . . . . . . 4.2.1.5 Regulationof the Outer Membrane Transport Systems . . . . . . . 4.2.1.6 Outer Membrane Proteins of Enteric Bacteria General Comments . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1.7 Outer Membrane Proteins of Non Enteric Bacteria . . . . . . . . . 4.2.2 Iron(III) Siderophore Translocation of the Cytoplasmic Membrane . . . . .

53 53 53 57 57 57 58 59 59 60 61 61 61 62 63 63 63

St~cture and Bonding58 © Springer-VerlagBerlinHeidelberg1984

26

R.C. Hider 4.2.2.1 Iron(III) enterobactin Permease . . . . . . . . . . . . . . . . . . 4.2.2.2 Ferrichrome Permease . . . . . . . . . . . . . . . . . . . . . . . 4.2.3 Iron(III) Uptake by M y c o b a c t e r i a c e a e . . . . . . . . . . . . . . . . . . . . 4.3 Antibiotics Based on Siderophore Structure . . . . . . . . . . . . . . . . . . . . . 4.3.1 The Ferrimycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 The Albomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

64 67 68 68 68 69

5 Removal of Iron From Siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Removal of Iron From Hydroxamate Siderophores . . . . . . . . . . . . . . . . . 5.2 Removal of Iron From Phenolate Siderophores . . . . . . . . . . . . . . . . . . . 5.3 Utilisation of Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 70 71 74

6 The Interaction of Siderophores and Siderophore-like Molecules with Metals Other Than Iron(III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Copper(II) Siderophore Complexes . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Molybdenium(VI) Siderophore Complexes . . . . . . . . . . . . . . . . . . . . .

75 75 76

7 The Role of Siderophores in Infection

77

..........................

8 Clinical Applications of Siderophores and Their Analogues

...............

9 Conclusions and Suggestions for Further Work . . . . . . . . . . . . . . . . . . . . . .

79 80

10 Notes Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

80

11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81

Symbols and Abbreviations DNP CCCP NEM I~ I I~

2,4-dinitrophenol (Proton unconpier) Carbonylcyanide phenylhydrazone (Proton uncoupler) N-ethylmaleimide Solubility product59~ Formation constant59)

I(1

First equilibrium constant of a multistep association of more than one ligand molecule with the same cation59)

1~

Cumulative equilibrium constant of a multistep association of more than one ligand molecule with the same cation 59)

Siderophore Mediated Absorption of Iron

27

1 Introduction Siderophores are compounds produced by microorganisms for scavanging iron from the environment 1). They have been defined as low molecular weight compounds (500-100 daltons) possessing a high affinity for iron(III) (Ke > 103°), the biosynthesis of which is regulated by iron levels and the function of which is to supply iron to the cell2). During the period 1949-52, four different siderophores were isolated and identified as growth factors. Mycobactin and arthrobactin (Terregens factor) were isolated as free ligands 3, 4), while ferrichrome5) and coprogen6) were isolated as iron complexes. Snow, in two classical papers, largely characterised the structure of mycobactin, correctly identifying it's coordination groups as two hydroxamates and 2-hydroxyphenyloxazoline7, 8). He also demonstrated that mycobactin possesses a high affinity for AI3+, Cu2+ and Fe 3+. This seminal work was followed in 1960 by the discovery of the ferrioxamines9), which like the ferrichromes1°) possess hydroxamate ligands. A key observation concerning the mode of action of these growth factors was made by Garibaldi and Neilands in 19561~),when it was demonstrated that the production of ferrichrome A was enhanced by growing the organism Ustilago sphaerogena in medium deficient in iron. This finding was subsequently confirmed for mycobactin12) and many other siderophores1). The complete structures of the ferrioxaminet3) and ferrichrome14) classes were finally elucidated in 1962 and 1963 respectively. Thus by the early 1960's, three types of structurally diverse hydroxamate containing siderophores had been characterised as iron binding growth factors. Characterisation of the catecholato siderophores was initiated by the discovery, in 1958, that an iron(III) binding agent, the glycine conjugate of 2,3-dihydroxybenzoic acid, was secreted by Bacillus subtilis when grown under low iron conditions ~5). Analogous serine 16) and threonine 17)conjugates were subsequently isolated but it was not until 1970 that the first tricatechol siderophore was identified. O'Brien, Cox and Gibson reported evidence for condensed units of 2,3-dihydroxybenzoylserine in culture fluids of both Escherichia coli and Aerobacter aerogenes is) and in the same year enterobactin was independently isolated by Pollock and Neilands from Salmonella typhimurium 19) and O'Brien, and Gibson from Escherichia coli2°). The latter workers term this siderophore enterochelin. Since 1970 the number of well characterised siderophores has risen to over forty (Table 1). The majority of these molecules fall into either the hydroxamate or the phenolate classes and despite considerable structural variation, chelate iron in a hexadentate fashion. Siderophores, coordinated to iron, are accumulated by microorganisms by specific translocation mechanisms and the tightly bound iron is removed for utilisation by the cell1, 51, 52). Siderophore uptake varies between the different classes of molecule, some enter the cytoplasm, while others apparently donate iron at the cytoplasmic membrane surface. Indeed some siderophores may be secreted in order to deprive competing organisms of iron2°' 53) and as such will influence the ecology of the environment occupied by the secreting colony of microorganisms. An analysis of the experimental data concerning membrane permeation by siderophores is presented in this review and methods by which iron may be actively accumulated by organisms against considerable concentration gradients is discussed. Because of the high affinity of siderophores for iron(III) the mechanism of iron removal is of considerable chemical interest. Indeed, the manner in which iron is

6

7

8 (a) (b) (c) (d) (e) (f) (g) (h)

Pseudomonas fluorescensputida

Pseudomonas Bi0

Pseudomonas aeruginosa

Mycobacteria Mycobacterium phlei M. aurum M. fortuitum M. thermoresistible M. marinum M. terrae M. smegmatis M. tuberculosis

Pseudobactin u)

Pseudobactin A zs)

Pyoverdine ~)

Mycobactins~) Mycobactin P Mycobactin A Mycobactin F Mycobactin H Mycobactin M Mycobactin R Mycobactin S Mycobactin T

5

Pseudomonas aeruginosa

Pyochelin~)

6 (a)

4

Vibrio Cholerae

Vibriobactin ")

3

2(a) 2(c) 2(b) 2(d)

1

Azotobacter vinelandii

Paracoccus denitrificans

Agrobacterium tumefacie~

Aerobacter aerogenes, Escherichia coil Salmonella typhimurim

Ri F.A? F.A. F.A. F.A. CH3 F.A. F.A. F.A.

Structure

a, e-bis-2,3-dihydroxybenzoyllysine21)

-

Agrohactin49) Agrohactin A Parabactin49, 5o) - Parahactin A

Catechol type Enterobactin (enterochelin)tT. 1~)

Bacterium

1 a. Bacterial siderophores

Siderophore

Table

R2 CH3 CH3 H CH3 H H H H

R3 H H CH3 CH3 CH3 H H H

IL C2H5 CH3 CH3 CH3 F.A. C.zH5 CH3 CHa

R5 CH3 H H H CH3 CH3 H H

2-Hydroxyphenyloxazoline and hydroxamate

Catechol, hydroxamate and a-hydroxy acid Catechol and hydroxamate

Catechol, hydroxamate and a-hydroxy acid

Phenol and thiazoline

Catechol and 2-hydroxyphenyloxazoline

Catechol

Catechol and 2-hydroxyphenyloxazoline

Catechol and 2-hydroxyphenyloxazoline

Catechol

Ligands

to oo

B 33)

Nocardia, Micromonospom, Streptomyces, Acfinomyces

Bacillus megaterium, Anabaena sp. E. coli, Aerobacter aerogenes (now Klebsiella pneumoniae) Arthrobacter sp. R=H,n=2

CH = CH; n = 12, 14, 16

n = 5 R1 = H, R2 = CH3, n = 5 RI=CH3CO, R2=CH3, n=5 RI = H, R2 = CH2CH2CO2H, n = 5 R1=H, R2=CH3, n=4

n = 5

R=H,n=4

-= C H 3 • ( C H 2 ) n •

10(a) 10(b) ll(a) ll(b) 11(c) 11(d)

9(c)

9(b) R = C O 2 H , n = 4

9(a)

a F.A. = mixture of long chain fatty acids, for example Mycobactin P; F.A.

Desferrioxamine D134) Desferrioxamine G 35) Desferrioxamine A: 32)

Desferrioxamine

Desferrioxanune E 31) Desferrioxamine D232)

Ferrioxamines

Arthrobactin 3°) (Terregens factor)

Citrate-Hydroxamates Schizokinen2s) Aerobactin 29)

Hydroxamate

Hydroxamate and a-hydroxycarboxylate

o

O

O

>.

r~

~t 2o.

t~

fD

R. C. Hider

30 Table 1 b, Fungal siderophores (All hydroxamates) Siderophore

Fungi

Structure

Ferriehrome (name refers to iron complex)

Aspergillus, Neurospora Penicillium, Ustilago, Actinomyces, Streptomyces

12

R1 R2 Ferrichromet3, ~) Ferrichrome A ~s)

(a) H (b) H

R3 R H H CH3 H CH2OH CH2OH "C /

(c) H (d) H

CH3~'C02 H CH2OH CH2OH CH3 H CH2OH CH3

(e) H

CH2OH CH2OH

(f) H

CH2OH CH2OH

(g) H (h) H

CH3

Ustilago sphaerogena

Ferrichrysin37. 3s) Ferricrocin37, 3s) Ferrirubin3S. 39)

Aspergillus sp.

Ferrirhodin ~, 39) Ferrichrome C~) Malonichrome 41) Fusarinines (Fusigens) Triacetyi fusarine 42) (Triacetyl fusigen) Fusarinine A 43) } Fusarinine B43) Fusafinine C~) Rhodotorulic acid derivatives Rhodotorulic acid45)

Dimerumic acid~

Cryptococcus melibiosum Fusarium roseum

oc~H ]I CH~ "CH~OH H~C. jl. CH~ -CH2OH

H H

CH 3

CH3

•CH2CO2H

Fusaria, Aspergillus Penicillium, Gibberella Penicillium sp.

13

R = CH3CO

Fusarium roseum F. cubense

14(a) R = H , n = 2

(b) R = H , n = 3 13(b) R = H

Rhodotorula, Sporobolomyces Leucosporidium

15(a) R = CH3

Fusarium dimerum

15(b) R : C---~

H

?H2OH

CHa = cH

Coprogen47)

16(a) R=CHaCO; RI

--IO H CHa

PeniciUium sp. Neurospora sp.

(b) R=H, RI =~==~__.OH

Epicoccum purpurascens

(c) R = CHACO, R1 = CH3

CH3

Coprogen B 47)

(name refers to iron complex) Tfiornicin ~)

31

Siderophore Mediated Absorptionof Iron

HO

NH OH OH

O~

~..o.r..O o~o

l l / ~ y "OH

r~"...~/OH

~OH o~>--~,~o. 0 ~'H~

N"~ N H ~ O a.

,o,

X = OH

b, X = H

o

@ HO

0

Me

OH

~NH~0 C,

X = OH

d,

x

=

H

OH

OH OH

OH

N~ d£merumlcacid (ISb)

Coprogen(16a)

Siderophore Mediated Absorption of Iron

53

the siderophore complex before acting as a repressor or inhibitor124'12s).It is conceivable that in some organisms, siderophore biosynthesis is regulated indirectly by cellular oxidative metabolism7a, nO

4 Iron Transport in Microorganisms In view of the variety of siderophores that have been identified it is not surprising that a corresponding diversity of iron(III) transport mechanisms exists. Indeed there are several examples of more than one system operating in a single species, and in the presence of relatively high levels of iron (> 10-5 M) a low affinity iron translocation mechanism operates. Thus the kinetic interpretation of iron uptake can be complicated. This difficulty is compounded by the different levels and types of trace iron present in the incubation media. Lankford 1), Neilands 124), and Winkelmann 126) present clear discussion of these problems. Uptake sudies have been carded out with fungi (for instance Ustilago sphaerogena and Fusarium roseum) and bacteria (mainly enteric bacteria for instance Escherichia coli and Salmonella typhimurium). As the structures of these two classes of organism are quite different they will be considered separately.

4.1 Iron(Ill) Siderophore Uptake by Fungi Fungi are eukaryotic organisms and possess a single cytoplasmic membrane usually surrounded by a hyphal wall. This hyphal wall does not contain an integral lipid membrane and thus unlike enteric bacteria, the iron only has to permeate a single bilayer structure. Siderophores have been isolated from three different fungal classes: Basidiomycetes (Ustilago); Ascomycetes (Neurospora and Rhodotorula) and Deuteromycetes (Penicillium, Aspergillus, and Fusarium). A more complete list is provided by Winkelmann 126). Iron(III) siderophore accumulation is an active transport process, being severely inhibited in the presence of metabolic poisonsTM.

4.1.1 Iron(Ill) Siderophore Uptake by Ustilago Emery and his coworkers have made a major contribution to our understanding of ferrichrome uptake by Ustilago sphaerogenaTM. Washed cells of this organism rapidly accumulate ferrichrome from the medium which can be monitored by the loss of extracellular colour and the formation of highly pigmented cells127). When [14C]- and [59Fe]ferrichrome are utilised in such experiments both isotopes are initially accumulated at the same rate but as the experiment progresses [14C]-deferriferdchrome is released from the cell and thus is capable of scavenging more iron (Fig. 7) 127). That ferrichrome is accumulated independently of the removal of iron from the siderophore is confirmed by the finding that gallium(III) ferrichrome is also actively accumulated by U. sphaerogena128).This property is most readily explained by the exist-

54

R.C. Hider I

100

I

I

I

[s9 Fe ] Ferrichrome 80 \

@1

\

60

/

_

e-s

20

[ 16C ]

.......

0 0

Desferriferrichrorne 4-. . . . .

"t"-. . . . .

I

1

2

3 houFs

I

Fig. 7. Uptake of ferrichrome by Us. tilago sphaerogena IzO. Percentage radioactivity (59Feor 14C)taken up by a cell suspension upon addition of [i:C]ferrichrome, [SgFe]ferrichromeor [ C]deferriferrichrome. Siderophore concentration, 36 ~M; incubation carried out at 30 °C and pH 7.0. After 2 h the medium iron(HI) level was increased to 6.8 mM in the samples containing [l~C]ferrichrome. Additional uptake of 14C was observed ( . . . )

ance of a shuttle mechanism for iron as originally suggested by Neilands 129). Ferrichrome is accumulated against a concentration gradient 127)and therefore it is to be expected that anaerobic conditions and the presence of low concentrations of azide and cyanide will inhibit the transport process. Ferrichrome uptake shows saturation kinetics and thus is likely to be mediated by a protein carrier. The specificity of the process confirms this view (Table 6). Both molecular features of the ferrichrome molecule, namely the metal centre and the cyclic hexapeptide appear to be involved in receptor interaction. When the metal centre is blocked by branched side chains poor uptake rates result. Furthermore, when the complex adopts a net charge of 1-, as for instance with the hexadentate Cobalt(II)deferriferrichrome, no uptake is observed. Slight modification of the hexapep-

Table 6. Relative Rates of Uptake of Iron Chelates by Ustilago 1~' 1~o,131) Compound

Relative rate of uptake (%)

Ferrichrome deferriferrichrome Ferrichrome A Ferrichrome A trimethylester Ferrichrysin Ferrirhodin Ferrirubin Tripropionylferdchrome Tributylferrichrome Ferrioxamine B Fusarine Rhodotorulic acid Ahminium(IH)deferriferrichrome Gallium(III)deferriferrichrome Chromium(II)deferriferrichrome Cobalt (III)deferriferrichrome

100 0 8 2 55 very low very low approx. 50 220 very low very low very low 74 70 90 0

Siderophore Mediated Absorption of Iron

55

tide ring decreases the uptake rate as demonstrated by ferrichrysin and siderophores lacking the cyclic structure have little or no affinity for the uptake mechanism (Table 6). The hexapeptide ring of ferrichrome is capable of coordinating the alkaline earth metals Mg 2+ and Ca 2÷ 132)and it has been suggested that by doing so, ferrichrome uptake would become sensitive to membrane potential. The cell membrane of fungal cells supports an electrical potential which in yeast is generated by a proton pump 133' 134). In yeasts, nutrients are accumulated by symport mechanisms, for instance aminoacids135, 136), phosphateZ37) and sugars 137). In principle a similar mechanism could operate for iron(III) siderophore uptake. However, Emery has demonstrated that 2,4dinitrophenol, (DNP) at the relatively high concentration of 10-3M, only decreased ferrichrome uptake to 75% that of normal. Clearly then, proton symport cannot be involved. However, with magnesium symport (Fig. 8) DNP would not be predicted to completely inhibit ferrichrome uptake. Although its presence would discharge the membrane potential, it would not directly influence the trans-membrane distribution of Mg 2+ ions. If the intracellular uncomplexed magnesium levels are lower than those on the outside, a reasonable supposition as indicated in Fig. 8, ferrichrome accumulation could still occur. Such a symport process is in agreement with the finding that the uptake system does not exhibit exchange diffusion properties. Furthermore, the observation that it is possible to stimulate the release of accumulated ferrichrome by the addition of alkaline phosphate solutions 127), can be explained in terms of this mechanism. The extracellular magnesium would be decreased to low levels (10 -6 M) by alkaline-induced precipitation of magnesium phosphate x3s), thus stimulating the symport mechanism to run in reverse. A likely structure for the magnesium ferrichrome complex involves coordination by 3 amide oxygen atoms (32). Such a structure offers an explanation for the conserved nature of glycine-1 in the entire series of ferrichromes (Table 1 b). Glycine in this position of the

in

out

+

AT p " ~ ' ~ _ Mg2+(H20)n AOP+P l~Keq=106 M g 2+(H2p

Mg2+(HzO)n

-

~

I~ Keq= 102"s ~

Mg 2+

Hg2+(H2PO~)(H20)n-1

Ferrichrome CeLL

membrane Fig. 8. Possible symport mechanism for ferrichrome uptake by Ustilago. By analogy with yeast cells the cytoplasmic membrane potential is probably generated by an electrogenic proton pump. An asymmetric distribution of non complexed magnesium ions will be maintained by the differential affinities of magnesium for orthophosphate and pyrophosphate (e.g. ADP). Under the incubation conditions used by Emery 127) the extracellular [Mg2+]totalis approximately 3 mM. When a magnesium ion enters the cell via the symport carrier it will move down an electrochemical gradient and therefore can "pull" the ferrichrome molecule with it. The advantage of utilising a divalent anion in a symport process is that a small membrane potential is able to support a large distribution ratio. In the above system a membrane potential of 100 mV would support a ferrichrome concentration gradient of approximately l&

56

R.C. Hider

@ cyclohexapeptide favours the ~-turn to adopt a Type II conformation which is ideal for cation coordination 132). The binding of alkaline earth cations to synthetic cyclohexapeptides has been reported previously 139' 140). U. sphaerogena synthesises deferriferrichrome A as well as deferriferdchrome, indeed under low iron conditions the secretion of ferrichrome A predominates over ferrichrome. When grown in culture, their synthetic rates are initially similar, however, as the culture progresses and becomes more iron deficient, deferriferrichrome A is preferentially synthesised 141). In contrast at higher iron levels, whereas deferriferrichrome A synthesis is completely repressed at 10-SM iron, deferriferrichrome can be isolated from cultures grown in 5 x 10-SM iron 141). Ferrichrome A, by virtue of the conjugated nature of its hydroxamate functions, possesses a higher affinity for iron than ferrichrome (1030 vs 1029)141). This, together with its acidic nature renders deferriferrichrome A more efficient than deferriferrichrome at solubilising ferric oxides74). Thus the highly efficient scavenging properties of defferriferrichrome A are only called upon when the organism senses severe stress due to low iron levels. The uptake of ferrichrome A is much slower than that of ferrichrome (Table 6) and yet iron associated with ferrichrome A enters cells rapidly and as demonstrated by ESR spectroscopy, is released as iron(II) 142). Originally it was thought that ferrichrome A donated iron(III) to deferriferrichrome and indeed this does occur to some extent, but at an insufficient rate to account for the accumulation of iron from ferrichrome A 143). From a number of lines of evidence it has become apparent that ferrichrome A supplies iron to the cell by directly interacting with a specific protein in the cytoplasmic membrane. This protein reduces the siderophore bound iron, which dissociates into the cytoplasm and the deferrifenichrome A is returned to the extracellular fluid. If the metal cannot be released by reduction, the complex dissociates from the receptor. This additional uptake system for iron presents U. sphaerogena with a strong competitive advantage over other organisms. In more favourable environments ([Fe] > 1 ~tM) the cells are able to utilise deferriferrichrome as an iron scavenger. Ferrichrome has an advantage over ferdchrome A in that it may also be stored intracellularly 142). Thus whereas there are conditions where deferriferrichrome A is not synthesised by the cell, the presence of deferriferrichrome is always required. This pattern of iron uptake may well exist for other fungal species.

Siderophore Mediated Absorption of Iron

57

4.1.2 lron(III) Siderophore Uptake by Neurospora The first uptake studies with Neurospora crassa were reported by Padmanaban and Sarma in 1965144)when they demonstrated that 59Fe coordinated to the organism's major siderophore, coprogen, is taken up much more quickly than [59Fe] citrate and that the iron is efficiently utilised for haeme synthesis. Ferrichrysin (12 c) and ferricrocin (12 d) are also efficiently transported by the cells, but rather surprisingly ferrichrome is not 145). With the aid of Mtssbauer spectroscopy it has been shown that iron(III) coprogen enters the cell where it is reduced. The resulting iron(II) dissociates and the coprogen becomes available for recycling146). A large proportion of iron(III) coprogen is not reduced and remains unchanged in the cytoplasm, possibly as a storage form of iron 146). Thus the uptake mechanism would appear to be similar to that operating in Ustilago for ferrichrome. Presumeably ferricrocin, a minor siderophore of N. crassa147)and ferrichrysin are transported in a similar manner. As with the ferrichrome receptor in Ustilago, ferrichrome A (12b) is not effectively transported by Neurospora and likewise ferrirubin (12e). Rather surprisingly, ferrirubin is a powerful competitive inhibitor of iron(III) coprogen (Ki = 5 × 10-7M). That there is such a difference between the uptake rates of ferrichrome and ferricrotin indicates that the cytoplasmic membrane receptor interacts with the peptide backbone. However, the metal centre must also be involved in the binding process since the hexapeptide sections of ferrirubin and ferrichrysin are identical. Although enantioferrichrome possesses a markedly lower rate of uptake than ferrichrome in N. crassa148),it is not clear whether this difference results from the different stereochemistry of the iron, the peptide or both.

4.1.3 Iron(III) Siderophore Uptake by Rhodotorula Rhodotorula pilimanae is capable of producing enormous quantities of rhodotorulic acid when grown under low iron conditions. The cell membrane of this yeast is able to transport iron, presented by rhodotorulic acid, the process being inhibited by respiratory poisons 149).The uptake of the ligand itself is very slow and thus the iron(III) rhodotulate receptor appears to be similar in mechanism to that of the ferrichrome A receptor of Ustilago143).Ferrichrome A and ferrioxamine B are unable to donate iron to this receptor, in contrast to iron(III) citrate which is equally efficient as iron(III) rhodotulate 149). Significantly neither of the active ligands are hexadentate and therefore will be kinetically more labile than hexadentate siderophores.

4.1.4 Iron(Ill) Siderophore Uptake by Fusaria As with Ustilago, Fusariurn roseum secretes two major siderophore types, the family of fusarinines (13 and 14) and malonichrome (12 h) 41). The relative production of these two classes varies with the age of the culture (Fig. 9). It is tempting to suggest that these two siderophore types have different functions, namely the fusarinines act as true siderophores and possibly form intracellular iron stores while malonichrome acts as a powerful iron scavenger. Certainly the highly negatively charged nature of deferrimalonichrome,

58

R.C. Hider

/rnnes l

E

I

I

6

o c

, , / / /

0 5

10

days

15

Fig. 9. The relative production of fusarinines and malonichrome by Fusarium roseum as a function of the culture age41)

endows the molecule with potent iron solubilising properties. Iron(III) fusarinine C by virtue of its net positive charge would be accumulated by the cell as a result of sensing the membrane potential; symport would not be necessary (Fig. 10). At present experiments which test this possibility have not been reported. Malonichrome is able to donate iron to cells of F. roseum but not in an efficient manner 40. In contrast ferrichrome, a siderophore not produced by this organism, is very much more efficient 4x). In related Aspergillus sp. two siderophores are also synthesised, namely triacetylfusarinine C and ferricrocin 151). 4.1.5 G e n e r a l C o m m e n t s

The overall picture emerging from this limited study of fungal iron transport is that fungi secrete a single potent iron scavanger into the environment. This scavanger is able to donate iron to the external face of the cytoplasmic membrane. However, in addition to in

m

our

+

2

H

ATP ~ ~ _ _ AOP+P

~

+ - ++

[iro~fusarinine]3+ Celt

membrane

Fig. 10. Possible uptake mechanism for fusarinine in Fusaria. A facilitated transport system for the positively charged iron(Ill) fusarinine will lead to accumulation of the complex. A symport mechanism is not required. Similar uptake mechanisms are known for basic aminoacids 15°)

Siderophore Mediated Absorption of Iron

59

this iron assimulation mechanism, some fungi possess additional independent uptake systems which are centred on true siderophore activity. Such molecules are capable of coordinating extracellular iron, permeating the cell membrane and of either donating the coordinated iron for biosynthetic requirements or retaining it as an intracellular store.

4.2 Iron(Ill) Siderophore Uptake by Enteric Bacteria A large proportion of bacterial siderophore uptake studies has been centred on enteric bacteria as typified by E. coli and S. typhimurium. The relative ease of mutant production and study has greatly facilitated progress in this area. These bacteria possess a cell wall consisting of both an outer membrane layer and a peptidoglycan layer152)(Fig. i1). The former acts as a molecular sieve-type barrier and the latter confers mechanical stability. Thus the metabolically active cytoplasmic membrane is protected against bile salts and hydrolytic enzymes found in mammalian gastrointestinal tract. The outer membrane basically consists of a lipid bilayer, rich in lipopolysaccharide which contains approximately 50 proteins of which only 3-5 are major species. Three of these form pore-fike structures called porins which render the outer membrane freely permeable towards hydrophilic molecules ( 300 °C, and [a]D + 16.4 °, showed positive color with ninhydrin reagent. The IR spectrum gave the absorption bands at 3450 (NH and OH), 1710 (COOH), 1580 and 1395 (COO-) and 2850 cm -1 (> +NH2). The 1H and 13C NMR data of 2 revealed the presence

Phytosiderophores

117

of three -CHz--CHz--CH- systems and three carbonyl carbons in the molecule (see Tables 2 and 3). The oxidation of 2 with KMnO4 yielded aspartic acid, homoserine, and malic acid. The CD curves of all the products obtained by the KMnOa-oxidation exhibited a positive Cotton effect (aspartic acid: [0]201nm= + 13 370, homoserine: [0h97nm = + 8384, and malic acid: [01206m = + 10 340), indicating that the absolute configurations of all asymmetric centers in 2 are S15). Thus, the structure of avenic acid A is 2(S)•3•(S)•y'(S)-N-[3-(3-hydr•xy-3-carb•xypr•py•amin•)-3-carb•xypr•py•]-h•m•serine as shown in the formula 2.

3. 2'-Deoxymugineic Acid (3) 2'-Deoxymugineic acid(3) isolated from wheat, C~2H20OTN2 (El-MS; m/z 286 (M ÷ - H20), mp 198.4--200.5°, and [a]D -- 70.5 °, was positive for ninhydrin reaction. The IR spectrum showed the absorption bands at 3450 (NH and OH), 1715 (COOH), and 1610 and 1395 cm -1 (CO0-). The 1H and t3c NMR spectral data indicated the presence of three -CH2-CH2-CH- systems and three carbonyl carbons in the molecule (see Tables 2 and 3). Detailed comparison of the NMR information among 3, 1, and nicotianamine(ll) afforded the evidence for the presence of azetidine-2-carboxylic acid(C-I-C-4), 3-carboxypropyl(C-l'-C-4'), and 3-hydroxy-3-carboxypropylamino (C1"-C-4") moieties in 3. The oxidation of 3 with KMnO4 gave L-azetidine-2-carboxylicacid ([aiD -- 129.3°), a slight amount of L-aspartic acid (positive CD Cotton effect at 205 nm), and 2(S),3'(S)-N-[(3-amino-3-carboxypropyl)]azetidine-2-carboxylicacid ([a]o -72.5°). Therefore, it is concluded that 2'-deoxymugineic acid is 2(S),3'(S),Y'(S)-N-[(3-carboxy3-hydroxypropylamino)-3-carboxypropyl]azetidine-2-carboxylic acid.

4. 3-Hydroxymugineic Acid (4) 3-Hydroxymugineic acid(4) isolated from rye, C12H2009N2(FD-MS; 337 (M + Na)+), mp 205-213 °C (decomp), and [a]D -- 52.4°, showed positive color with ninhydrin reagent. The IR spectrum exhibited the absorption bands at 3400, 3230, and 3050 (NH and OH) and 1620 cm -1 (COO-). The 1H and 13C NMR spectral data of 4 were similar to those of 1, except for the proton and carbon signals of C-1-C-4 in the azetidine-2carboxylic acid moiety. The methylene signal at 2.57 ppm due to C-3 in 1 is absent in 4, and instead the methine signal at 4.80 ppm is observed. The methine signal attributed to C-2 in 4 was detected as doublet (J = 6.5 Hz) at 4.98 ppm. Furthermore, the splitting pattern of the methylene signal to C-4 is simplified, compared with the corresponding signal of 1 (see Table 2). In the comparative t3C NMR spectra of 4 and 1, conspicuous differences were the presence of a methylene carbon signal at 23.8 ppm due to C-3 in 1 and of a methine carbon signal at 65.3 ppm in 4. The downfield shifts of the carbon signals at a-position (C-2 and C-4) and the slight upfield shift of the carbon signal at r position (C-1) are also observed in 4 as compared with 1. These findings indicate that an hydroxyl group is situated at C-3. The configurations at C-2', C-3', and C-3" in 4 were the same as those in 1, since the chemical shifts and the splitting patterns of C-2', C-3', and C-3" proton signals of 4 coincide with those of the corresponding signals of 1 (see Table 2). The CD curve of y-amino-a-hydroxybutyric acid obtained by 6 N HC1 hydro-

118

Y. Sugiuraand K. Nomoto

lysis of 4 gave a positive Cotton effect (Ae216,m = + 0.083), suggesting that the chiral center at C-3" in 4 is S configuration18). Thus, the structure including the streochemistry of C-2', C-3', and C-3" of 3-hydroxymugineic acid is represented by the formula 4.

5. Distichonic Acid A (5) Distichonic acid A(5) isolated from beer barley, C10HasOsN2 (FD-MS; m/z 259 (M + 1 - 2 H20)+), mp 220-221 °C (decomp), and Ae207~ = + 3.25, was positive for ninhydrin reaction. The IR spectrum exhibited the absorption bands at 3450, 3350, 3170 (NH and OH), 1720 (COOH), and 1605 and 1380 cm -1 (CO0-). In the 1H NMR spectrum of 5, all the proton signals were assigned thoroughly with the aid of the double resonance technique. Table 2 shows the presence of one -CH2--CH2--CH-, one --CH2--CH--CH-, and one -CH2- system in the molecule 5. The 13C NMR data also revealed the signals due to three carbonyl carbons, four methylene carbons, and three methine carbons (see Table 3). Since this compound contains no unsaturation rings, the formula 5 is concluded to be the structure for 5.

6. Other Similar Amino Acids Nicotianamine, 2(S),3'(S),3"(S)-N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]-azetidine-2-carboxylic acid(//), has been isolated from the leaves of Nicotiana tobacum L. (Solanaceae)2°' 21) and seeds of Fagus silvatica L. (Fagaceae)22,23). Another amino acid, 2(S),3'(S)-N-(3-amino-3-carboxypropyl)-azetidine-2-carboxylicacid(12), has also been obtained from the latter. Further, azetidine-2-carboxylic acid(13) which is the constituent of mugineic acid(I) has been found in various species of Liliaceae24-27), Agaraceae 19), Chenopodiaceae 28), and Leguminosae 29), and this compound has been proposed as a precursor of mugineic acid and nicotianamine23). Recently, Budensinsky suggested that nicotianamine(ll) is a possible phytosiderophore which functions in iron transport and/or metabolism in the vascular plants 3°). Of special interest is the fact that mugineic acid(/) and nicotianamine(ll) have remarkably similar structures (see Charts 1 and 4).

IV. Chemical Synthesis of Mugineic Acids

1. Synthesis of 2'-Deoxymugineic Acid (3) 31) The synthesis of 2'-deoxymugineic acid(3) was achieved through reductive coupling of three optically active units, L-a-hydroxy-y-butyrolactone(14), L-homoserine(6), and the unique L-azetidine-2-carboxylic acid(13) as shown in Scheme 1. L-a-hydroxy-y-butyrolactone(14)32) was prepared from L-malic acid in three steps. The compound(14) was converted into a diasteromeric mixture of tetrahydropyranylated derivatives(15). The diasteroisomers were separated by chromatography. The hydrolysis of 15 with 2.5%

119

Phytosiderophores

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Scheme 1

59%

C02"VSu 24

100%

120

Y. Sugiuraand K. Nomoto

KOH-tetrahydrofuran followed by benzylation afforded y-hydroxybenzylester(16). The oxidation of 16 with pyridinium chlorochromate (PPC) yielded L-malic halfaldehyde(17). The coupling of 17 and the homosefine moiety(18) was achieved via reducfive amination33) by using of NaBHaCN, and the procedure gave the desired lactone amine(19). The protection of 19 with di-tert-butyldicarbonate yielded the tert-butoxycarbonyl derivative(20) and the subsequent benzylation gave rise to the dibenzylester(21). Azetidine-2-carboxylic acid(13) was transformed into the trifluoroacetic acid salt of 2benzyloxycarboxylazetidine(22) in three steps. The PPC oxidation of 21 afforded the aldehyde(23), which upon reductive amination with 22 yielded the protected 2'-deoxymugineic acid(24). Removal of all the protecting groups under mild acidic condition followed by chromatographic separation on Dowex 50W X4 and Sephadex G-10 afforded optically pure 2'-deoxymugineic acid(3) as white crystals, mp 196-199 °C and [a]D - 66.6°.

2. Synthesis of Avenic Acid A(2) 34) The synthesis of avenic acid A(2) was performed by reductive coupling of the protected aldehyde(23), an intermediate employed in the synthesis of 2'-deoxymugineic acid(3), and L-homoserine lactone trifluoroacetic acid(18) (see Scheme 2). Thus, L-tert-butoxyearbonyl-N-(3-O-tetrahydropyranyl-3-carboxybenzylpropyl)homoserine aldehyde benzylester(23) and homoserine lactone trifluoroacetic acid(18) were treated with NaBH3CN to yield the protected avenic acid A lactone(25). The hydrogenation with H2/Pd-C, followed by treatment with trifluoroacetic acid gave the lactone trifluoroacetate(26) in quantitative yield. The hydrolysis of 26 with 2.5% KOH afforded the potassium salt of 2, which upon treatment with Dowex 50W X4 resin (H + form), and subsequently Sephadex G-10, gave optically pure avenic acid A(2), mp > 300 °C and Aezo~m = + 2.60.

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121

Phytosiderophores

V. Is Mugineic Acid a Phytosiderophore? In price plants, the effect of mugineic acid on the 59Fe-uptake and the synthesis of chlorophyll was investigated35). Figure 4 shows significant stimulation of mugineic acid for both the iron-uptake and the chlorophyll synthesis. In contrast, the effects of nicotianamine, EDTA, desferrioxamine, and citric acid were remarkably small and comparable to the control. 2'-Deoxymugineic acid and HEDTA gave an appreciably positive effect, although their abilities were lower than that of mugineic acid. It is of interest to note that mugineic acid and HEDTA are structurally analogous. Indeed, the formation constants of the HEDTA-Fe(III) complex (log K~L = 19.8)36) and the HEDTA-Fe(II) complex (log K~th = 11.6) are close to those (18.1 and 8.1) 37) of the corresponding mugineic acid-iron complexes, and the HEDTA ligand has the same kind of hydroxyl coordination for Fe(III). The structural similarity of 2'-deoxymugineic acid and HEDTA to mugineic acid probably contributes to the positive effect on the 59Fe-uptake and the chlorophyll synthesis. Several iron-solubilizing amino acids isolated from other gramineous plants all have similar structures with six coordination groups (see Chart 1). The large difference in the biological activity between 2'-deoxymugineic acid and nicotianamine strongly suggests the important contribution of the terminal alcoholic

Chlorophyll contents, %(wet wt.)

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?

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citric acid mugineic acid(-Fe)

o

•

°

59Iron uptake(~g/ a plant)

Fig. 4. Effect of some iron-chelatorson iron-uptake and chlorophyllsynthesisin water-culturedrice plant

122

Y. Sugiuraand K. Nomoto

hydroxyl group in the mugineic acid ligands for the iron-uptake. In spite of the high solubilizing ability of desferrioxamine, this compound did not stimulate iron-uptake by the rice plant. Presumably, the phenomenon is due to little permeability of the desferrioxamine-Fe(III) complex into membranes of the root. The experimental results of the 59Fe-uptake and the chlorophyll synthesis demonstrate that mugineic acid is really a phytosiderophore in rice plant.

VI. Some Properties of Mugineic Acid-Iron(IH) Complexes37) The electronic absorption spectrum of the golden yellow-colored mugineic acid-Fe(III) complex exhibited peaks at 350(e 1520), 250(8350), and 204 nm(35 000). In the CD spectrum, three Cotton peaks were also detected at 380(he -2.39), 270(+2.22), and 235 nm(+ 9.11). The visible absorption band of 350 nm must be due to a ligand - , metal charge-transfer transition, because high-spin ferric ion has no spin-allowed d-d transition. Figure 5 shows the Mfssbauer spectra of the mugineic acidJTFe(III) complex under the ,

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