Proc. Nail. Acad. Sci. USA Vol. 88, pp. 1878-1882, March 1991 Biochemistry

Mutasynthesis of siderophore analogues by Pseudomonas aeruginosa (pyochelin/iron transport/mutasynthesis)

ROBERT G. ANKENBAUER*t, ANDREW L. STALEYt§, KENNETH L. RINEHARTt,

AND

CHARLES D. Cox*¶

*Department of Microbiology, University of Iowa, Iowa City, IA 52242; and tRoger Adams Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL 61801

Communicated by I. C. Gunsalus, December 3, 1990

acid for pyochelin biosynthesis (Sal- phenotype) were isolated and shown to incorporate [14C]salicylate into [14C]pyochelin (12). The mutasynthetic technique (13-16) of mutating a microorganism so that it can no longer produce an unusual subunit of a secondary metabolite has been used to produce various synthetic antibiotics, some with improved antibacterial properties. We report here the production, isolation, and structure assignment by NMR and MS of three analogues ofpyochelin, produced through mutasynthetic incorporation of salicylic acid analogues into pyochelin by using the Sal- mutant ofP. aeruginosa. The precursors incorporated were 5-fluorosalicylic acid, 4-methylsalicylic acid, and 3-hydroxypicolinic acid. Each of the mutasynthetic analogues, 5-fluoro-, 4-methyl-, and 6-azapyochelin, had iron transport activities different from that of pyochelin.

The Gram-negative bacterium Pseudomonas ABSTRACT aeruginosa produces the phenolic siderophore pyochelin. Salicylic acid is an intermediate in the pyochelin biosynthetic pathway, and mutants blocked in salicylic acid biosynthesis (Sal-) are able to incorporate exogenously supplied salicylic acid into pyochelin. A P. aeruginosa SalP mutant was incubated with 13 salicylic acid analogues and was found to incorporate three (5-fluorosalicylic acid, 4-methylsalicylic acid, and 3-hydroxypicolinic acid) into pyochelin analogues, trivially designated as 5-fluoropyochelin, 4-methylpyochelin, and 6-azapyochelin. The structures of the mutasynthetic products were confirmed by 1H and 13C NMR and high-resolution fast atom bombardment mass spectrometry as being identical to pyochelin except for the expected changes in the aromatic ring. The biological activity of the three pyochelin analogues was determined in iron transport assays. In comparison to pyochelin, 4-methylpyochelin was more active in the assays whereas the activities of 5-fluoropyochelin and 6-azapyochelin were markedly decreased. In coincubation assays, 5-fluoropyochelin substantially inhibited iron transport by pyochelin; 4-methylpyochelin and 6-azapyochelin did not demonstrate this inhibitory effect.

MATERIALS AND METHODS Bacterial Strains and Medium. P. aeruginosa PAO1 and IA602 have been described (8, 12) and were grown in CAA medium (8). Iron Transport Assays. P. aeruginosa PAO1 was grown in CAA medium for 20 hr at 37°C and the bacteria were harvested and washed with water by centrifugation. Bacteria were resuspended in CAA/Mops/EGTA medium (1% CAA/5 mM Mops, pH 7.4/1 mM MgCl2/1 mM EGTA) prior to assays. The transport ability of pyochelin and the analogues was determined by combining "FeCl3 with the compounds and then adding it to the bacterial suspension to yield 0.038 ,Ci (4.2 pmol; 1 Ci = 37 GBq) of "FeCl3 and 10 pg of the analyzed compound per ml. Bacteria were separated from the reaction mixture by filtration through cellulose acetate filters of 0.45-pm pore size (OE-67; Schleicher & Schuell), which were washed with water to remove unreacted "Fe. Filters were dried and immersed in scintillation fluid and the radioactivity was determined by scintillation counting. Spectral Characterization. 1H NMR spectra (C2HC13) were recorded on a Nicolet NT 360 spectrometer at 360 MHz, with a spectral width of 2380 Hz and 32,768 data points zero-filled to 65,536 points. 13C NMR spectra were recorded in 100-150 pul of C2HC13 on a General Electric GN-500 spectrometer at 125 MHz, with a spectral width of 13,513 Hz and 16,384 data points zero-filled to 32,768 points. All chemical shifts are reported relative to (Me)4Si; for 13C, the center peak of the C2HC13 resonance at 77.0 ppm was used as standard. Fast atom bombardment (FAB) mass spectra were recorded on either a VG ZAB-SE spectrometer (low resolution) or a VG 70-SE4F four-sector instrument (high resolution). In either

Siderophores are low molecular weight iron chelators produced in response to iron deprivation by microorganisms, in which they mediate high-affinity iron uptake mechanisms (1). Siderophore-mediated iron uptake systems have been implicated in the virulence of a number of pathogenic bacteria (2, 3). The chemical structures of these compounds have been intensively investigated (4), and a number have been chemically synthesized (5). Pyochelin (structure 1) is a phenolic siderophore produced

~2

OHCH

2-0>

4,,C02H

42 N

S 5'

S

5

1 (PYOCHELIN)

by Pseudomonas aeruginosa (6) and has been assigned the structure

2-[2-(2-hydroxyphenyl)-2-thiazolin-4-yl]-3-methyl-

4-thiazolidinecarboxylic acid (7) on the basis of 'H and 13C NMR spectroscopy and high-resolution (HR) mass spectrometry (MS). The assignment has been confirmed by total synthesis (8) and further spectral analyses (9). The pseudomonads Pseudomonas cepacia and Pseudomonasfluorescens have also been reported to produce pyochelin (10, 11). Recently, mutants of P. aeruginosa requiring salicylic

Abbreviations: HR, high resolution; MS, mass spectrometry; FAB, fast atom bombardment. tPresent address: Rocky Mountain Laboratories, National Institutes of Health, Hamilton, MT 59840. §Present address: Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853. $To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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case, dithiothreitol-dithioerythritol matrix ("magic bullet") was used in the positive ion mode with an Ion Tech fast atom gun and xenon atoms (8 keV; 1 ev = 1.602 x 10-19 J). IR spectra were recorded as thin films on NaCl plates on an IBM IR/30S Fourier transform IR (FTIR) spectrophotometer, and UV spectra were recorded on a Perkin-Elmer Lambda-3 spectrophotometer. Optical rotations were recorded on a JASCO DIP-360 digital polarimeter with a sodium lamp source. Production of Mutasynthetic Pyochelin Analogues. The salicylic acid analogues tested as mutasynthetic precursors were 3-chloro, 3-hydroxy-, 3,5-dichloro-, 3-methyl-, 4-amino-, 4-methyl-, 5-fluoro-, 5-hydroxy-, and 6-hydroxysalicylic acids, 2-hydroxynicotinic acid, 3-hydroxypicolinic acid, picolinic acid N-oxide, and 2-mercaptobenzoic acid. All compounds were purchased from Aldrich and used without further purification. The analogues were dissolved in 95% ethanol, with the exception of the nitrogen-containing acids, which were dissolved as their sodium salts in water and filter-sterilized before addition to the cultures. Strain IA602 was inoculated into CAA medium at 1 x 104 colony-forming units per ml and was incubated at 37°C with rapid shaking. At 4 hr after inoculation, the salicylic acid analogues were aseptically added to the cultures, and the cultures were incubated for a further 48 hr. Two liters of culture broth was used for each mutasynthetic precursor. Purification of Mutasynthetic Analogues. The analogues isolated from the cultures by vigorous extraction with two volumes of dichloromethane/acetic acid, 10:1 (vol/vol), were purified by silica gel chromatography as described for pyochelin (8). All of the mutasynthetic products were isolated as mixtures of two rapidly interconverting isomers, designated as isomers I and II. The partially purified carboxylic acids (each at 40-60 mg) were then dissolved in methanol (3 ml) and treated with ethereal diazomethane. The methyl esters were purified by medium-pressure liquid chromatography on silica gel (Merck Kieselgel PF254, Darmstadt, 42-60 mm mesh, 20 mm x 13 cm column), with carbon tetrachloride/ethyl acetate mixtures as eluant, 20:1 to 1:1 (vol/vol) step gradients (flow rate, 4 ml/min). Methyl 2-[2-(5-fluoro-2-hydroxyphenyl)-2-thiazolin-4-yl]-3methyl-4-thiazolidinecarboxylate (5b). The two isomeric forms (I and II) of the methyl ester were cleanly separated by medium-pressure liquid chromatography to provide 13.6 mg of 5-fluoropyochelin I, [a]' + 30.40 (c 0.4, CHC13), and 7.6 mg of 5-fluoropyochelin II, [a/]25 -34.2° (c 0.2, CHC13): IR (thin

RCOOH 2, 3, 4

film) 1740, 1570, 1489, 1207, 1170, 1024, 989, 862, 821, 785, and 684 cm-' for both isomers; UV Amax (MeOH) 321 (E 3360), 245 (5150), 220 (25,100) for the mixture of isomers. Anal. Calcd for C15H18FN2O3S2 (M + H): 357.0743. Found: 357.0717 (M + H, HRFABMS), measured on the mixture of isomers I and II. Methyl 2-[2-(2-hydroxy-4-methylphenyl)-2-thiazolin-4-yl]-3methyl-4-thiazolidinecarboxylate (6b). A clean chromatographic separation provided 9.6 mg of 4-methylpyochelin I, [a]25 + 36.20 (c 0.2, CHC13), and 4.5 mg of 4-methylpyochelin II, [a]D -46.6° (c 0.1, CHC13); IR (thin film) 1742, 1586, 1437, 1379, 1221, 1199, 1176, 1037, 968, 920, and 800 cm-' for both isomers; UV Ama, (MeOH) 318 (E 4330), 262 (7990), 209 (17,800) for the mixture of isomers. Anal. Calcd for C16H21N203S2 (M + H): 353.1016. Found: 353.1005 (M + H, HRFABMS), measured on the mixture of isomers I and II. Methyl 2-[2-(3-hydroxy-2-pyridyl)-2-thiazolin-4-yl]-3-methyl-4-thiazolidinecarboxylate (7b). Only the methyl ester of 6-azapyochelin I (6.8 mg) was obtained in pure form after medium-pressure liquid chromatography of the mixture: IR (thin film) 1740, 1589, 1448, 1298, 1182, 1026, 970, 806, and 763 cm-'; UV Ama. (MeOH) 312 (E 11,600), 205 (27,800). The pure isomer rapidly isomerized to the mixture of isomers I and II, so optical rotations were not obtained. The 1H and '3C NMR data reported in Tables 1 and 2 were obtained from a mixture of isomers. Anal. Calcd for C14Hj8N3O3S2 (M + H): 340.0798. Found: 340.0794 (M + H, HRFABMS), measured on the mixture of isomers I and II.

RESULTS Screening for Mutasynthetic Pyochelin Analogues. The 13 salicylic acid analogues listed above were administered to P. aeruginosa Sal- IA602, and the cultures were screened for the production of mutasynthetic pyochelin analogues by extraction and TLC. Three mutasynthetic pyochelin analogues (Scheme I), derived from 5-fluorosalicylic acid (2), 4-methylsalicylic acid (3), and 3-hydroxypicolinic acid (4), were assigned chemical structures as 2-[2-(5-fluoro-2-hydroxyphenyl)-2-thiazolin-4-yl]-3-methyl-4-thiazolidinecarboxylic acid (5a), 2-[2-(2-hydroxy-4-methylphenyl)-2-thiazolin-4-yl]-3methyl-4-thiazolidinecarboxylic acid (6a), and 2-[2-(3hydroxy-2-pyridyl)-2-thiazolin-4-yl]-3-methyl-4-thiazolidinecarboxylic acid (7a), respectively. The analogues also received CH3

P. aeruginosa

CH2N2

strain IA602

I a

Isomer I (4'R, 2"R, 4"R)

5a: 5-FLUOROPYOCHELIN 6a: 4-METHYLPYOCHELIN 7a: 6-AZAPYOCHELIN (R' = H)

R = F5hIIILIISS 2; 5a,b

Methyl Esters (R! = CH3) 5b, 6b, 7b

CH3

2

1879

OH

OH N6

3; 6a,b

Scheme I

4; 7a,b

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Biochemistry: Ankenbauer et al.

Proc. Natl. Acad Sci. USA 88 (1991)

Table 1. 'H NMR spectral data for the methyl esters of the mutasynthetic pyochelins: 8, m, and J Proton, H 5b-I 6b-I 6b-Il 5b-iI 3 4

7.04 dd (8.8, 2.9) 6.86 dd (9.0, 4.6)

S

-

6 4' 5'a 5'b 2" 4"

5"a 5"'b N-CH3 O-CH3

6.99 dd (8.0, 3.1) 5.01 dt (8.8, 5.1) 3.43 dd (11.3, 8.7) 3.35 dd (11.2, 9.0) 4.45 d (5.2) 3.58 dd (9.1, 6.4) 3.10 dd (10.7, 9.2) 3.05 dd (10.7, 6.4) 2.51 s 3.68 s

7.02 d (8.3) 6.87 dd (8.6, 4.1)

6.73 s

-

6.61 dd (8.0, 1.0) 7.20 d (8.0) 5.08 dt (8.8, 5.2) 3.38 dd (11.2, 8.5) 3.33 dd (11.2, 8.9) 4.44 d (5.1) 3.57 dd (9.1, 6.3) 3.10 dd (10.5, 9.2) 3.03 dd (10.6, 6.2) 2.51 s 3.68 s 2.29 s

7.00 m 4.86 q (7.9) 3.47 dd (11.2, 8.7) 3.34 dd (11.2, 8.3) 4.49 d (6.8) 4.01 t (6.0) 3.18 dd (10.7, 6.5) 3.10 dd (10.7, 5.5) 2.43 s 3.71 s

-

6.73 s 6.61 dd (7.9, 0.9) 7.19 d (7.9) 4.97 q (7.7) 3.43 dd (11.2, 8.6) 3.30 dd (11.2, 8.2) 4.48 d (6.9) 4.01 t (6.0) 3.17 dd (10.7, 6.5) 3.10 dd (10.7, 5.6) 2.41 s 3.70 s 2.26 s

7b-I

7b-I*

7.24 dd (8.4, 1.6) 7.22 dd (8.4, 4.3) 8.13 dd (4.3, 1.5)

7.3 m (t) 7.3 m (t) 8.21 dd (4.1, 1.1)

4.98 td (8.9, 5.3) 3.37 dd (11.4, 8.6) 3.31 dd (11.4, 9.3) 4.45 d (5.3) 3.59 dd (9.1, 6.2) 3.13 dd (9.2, 10.7) 3.05 dd (6.2, 10.7) 2.52 s 3.69 s

4.82 q (8.4) 3.3-3.5 m (t) 3.3-3.5 m (1) 4.48 d (7.1) 4.10 t (6.1) 3.1-3.2 m (t) 3.1-3.2 m (t) 2.43 s 3.72 s

CH3 Chemical shift (8) in ppm from (Me)4Si, multiplicity m (s, singlet; d, doublet; t, triplet; and m, multiplet), and coupling constant J (in Hz in parentheses) are shown. *6-Azapyochelin II methyl ester (7b-II) could not be isolated in pure form. tMultiplicity and coupling constant assignments were based on analysis of the mixture of isomers I and II, from which accurate values could not be obtained. The numbering system for 7b-I and 7b-Il does not accord with International Union of Pure and Applied Chemistry practice but instead follows the numbering scheme for pyochelin.

the respective trivial designations of 5-fluoropyochelin (5a), 4-methylpyochelin (6a), and 6-azapyochelinll (7a). Production and Isolation of Mutasynthetic Pyochelin Analogues. Optimal production of pyochelin analogues was obtained with 250 ILM 5-fluoro- and 4-methylsalicylic acids and 5 mM 3-hydroxypicolinic acid in CAA medium. Precursors 2 and 3 can be extracted into dichloromethane/acetic acid, but very little of these compounds was observed in the culture broth extracts. This indicates that the precursors either had been incorporated into the mutasynthetic products 5a and 6a, respectively, or had been metabolized to other unidentified products. Precursor 4 could not be extracted into dichloromethane/acetic acid, which facilitated the separation ofthe product 7a from the high levels of precursor necessary for incorporation. II

Numbering scheme for 6-azapyochelin in scheme I does not conform to International Union of Pure and Applied Chemistry (IUPAC) rules but instead is consistent with the numbering scheme for pyochelin and the other two mutasynthetic analogues.

The mutasynthetic analogues were partially purified by preparative TLC in a manner identical to that for pyochelin (8) to yield, under the conditions listed above, Sa at 63 mg/liter of culture, 6a at 54 mg/liter, and 7a at 54 mg/liter. All the analogues (as the free acids) behaved similarly to pyochelin in that they (i) yielded black spots when sprayed with ammoniacal silver nitrate spray reagent (8), thus indicating the presence of an N-methylthiazolidine ring; (it) yielded colored spots (red to orange-red) when sprayed with a 0.1 M FeCl3/HCl solution; (iii) had fluorescent and spectral properties similar to those of pyochelin; (iv) had Rf values (0.40 and 0.35, in an approximately 2:1 ratio of isomers) identical to those of the pyochelins when chromatographed on silica gel plates with chloroform/acetic acid/ethanol [19:1:1 (vol/vol)] as solvent; and (v) had the same behavior as pyochelin on silica gel plates, whereby either of the two spots at Rf 0.40 and 0.35, after isolation, interconverted rapidly to produce the original mixture of two isomers. In contrast to the acids, the methyl esters of the mutasynthetic compounds (5b, 6b, and 7b) proved to be relatively

Table 2. '3C NMR spectral data for the methyl esters of the mutasynthetic pyochelins, listed as chemical shift (8) from (Me)4Si, the center resonance of the C2HCl3 triplet being at 77.0 ppm Sb-I 6b-I 7b-I Carbon, C Sb-II 6b-II 7b-II * * 1 113.76t 134.21t 113.76t 134.22t 155.27 158.92 158.92 155.72 2 155.34 155.67 118.23t 117.22t 117.32t 3 118.12t 124.70 124.82 127.11 4 120.18§ 120.30§ 144.09 144.09 127.02 119.96 5 154.981 154.981 119.96 140.70t 140.70t 115.7611 115.6511 6 130.24 130.24 170.59 170.65 2' 171.33 170.55 170.62 170.62 80.50 79.11 80.01 4' 79.44 80.02 79.72 33.41 34.25 32.93 5' 32.89 32.83 33.96 75.74 2" 75.72 75.83 75.41 75.99 76.07 70.39 4" 72.27 70.32 72.31 70.38 72.39 31.73 31.91 31.95 5" 32.53 31.95 32.23 172.49 171.37 171.37 4"a 172.07 171.96 172.49 52.44 52.35 52.40 52.29 52.38 52.29 O-CH3 37.78 37.73 41.23 37.77 41.20 41.08 N-CH3 21.72 21.72 Ar-CH3 Spectra were obtained on mixtures of isomers I and II. Ar-CH3, aromatic CH3. *Resonance not observed. tResonances may be interchanged. $Doublet, 3JC-F = 6.9 Hz, 7.4 Hz for Sb-I and 5b-Il, respectively. §Doublet, 2JC-F = 23.1 Hz, 22.7 Hz for Sb-I and Sb-II, respectively. 9Doublet, VJcF = 273.3 Hz. "IDoublet, 2JC-F = 24.3 Hz, 23.8 Hz for Sb-I and 5b-II, respectively.

Biochemistry: Ankenbauer et al. stable and were easily purified by silica gel chromatography. The crude methyl esters each showed two diastereomeric products on TLC using hexane/ethyl acetate [1:1 (vol/vol)] as solvent. The two stereoisomers of 5b (Rf, 0.46 and 0.36) and 6b (Rf, 0.60 and 0.52) were isolated in pure form. However, the slower moving diastereomer of 7b (Rf, 0.31 and 0.10) apparently was too unstable to be separated in pure form. The isomers of each product were trivially named as "Is' and "II" on the basis of their elution order from the silica columns. The yields of purified methyl esters were 13.6 mg and 7.6 mg of 5b-I and 5b-H, respectively; 9.6 mg and 4.5 mg of 6b-I and 6b-IH, respectively; and 6.8 mg of 7b-I. Spectral Characterization. The 1H and '3C NMR spectral data for the three mutasynthetic products are given in Tables 1 and 2, respectively. The methyl esters 5b and 6b each provided two diastereomeric products that were isolated in an approximate molar ratio of 2:1 (isomer I/isomer II). Except for the expected changes in the aromatic regions of the 1H NMR spectra of the compounds, the spectra of the products are virtually indistinguishable from those of the natural pyochelins. For each diastereomer (I or II), the resonances for the H4', H-2", H4", and N-CH3 protons show nearly identical chemical shifts and coupling patterns with respect to the other mutasynthetic products (I or II). This indicates (as expected) that the only change in the mutasynthetic products relative to natural pyochelin is in the aromatic region, ultimately derived from the salicylic acid analogue precursors. Low- and high-resolution FABMS provided molecular ions (M + H) at the predicted values of m/z 357 (C15H18FN203S2), m/z 353 (C16H21N203S2), and m/z 340 (Cj4Hj8N303S2) for 5b, 6b, and 7b, respectively. The IR spectra of all the compounds showed similar absorption patterns, with intense absorptions at 1740-1743 (C=O), 1570-1590 (C==N), 1440-1490 (CH2), and 1170-1180 cm-' (C-O). In contrast, the aromatic fingerprint regions were distinctly different for the three mutasynthetic products (989, 862, 821, and 684 cm-' for 5b; 960, 920, and 800 cm' for 6b; and 970, 806, and 763 cm' for 7b), as expected from the change in the aromatic chromophore. Iron Transport Activity of Mutasynthetic Pyochelin Analogues. The mutasynthetic pyochelin analogues were compared to pyochelin in their ability to transport iron in P. aeruginosa (Table 3). Of the three analogues, only 4-methylpyochelin mediated iron transport as well as pyochelin. In fact, 4-methylpyochelin was more active than pyochelin by a factor of -1.4. In contrast, 5-fluoropyochelin and 6-azapyochelin were markedly less active than pyochelin with both analogues transporting iron at 17% the level of pyochelin and the transport levels only 3-4 times background. In coincubation assays with pyochelin and each analogue present at 10 ,g/ml, 4-methylpyochelin again demonstrated enhanced transport when compared to pyochelin alone. The transport activity of pyochelin was unchanged in the presTable 3. Iron transport activities of pyochelin and mutasynthetic analogues Iron uptake, pmol of "5Fe per Substrate 109 bacteria per ml Control 0.04 Pyochelin 0.84 4-Methylpyochelin 1.21 5-Fluoropyochelin 0.15 6-Azapyochelin 0.15 Pyochelin + 4-methylpyochelin 1.05 Pyochelin + 5-fluoropyochelin 0.14 0.91 Pyochelin + 6-azapyochelin All assays contained 0.038 ,tCi (4.2 pmol) of "5FeCl3 per ml. Substrate compounds were present at 10 ,g/ml.

Proc. Natl. Acad. Sci. USA 88 (1991)

1881

ence of 6-azapyochelin, indicating no effect on ferripyochelin transport. However, the addition of 5-fluoropyochelin decreased the ferripyochelin transport to the level exhibited by 5-fluoropyochelin alone in transport assays. The addition of 5-fluoropyochelin inhibited the transport activity of pyochelin by 85%.

DISCUSSION Several siderophore analogues have been chemically synthesized (17-20), and investigations utilizing them have revealed significant structural requirements for siderophore-mediated iron transport (21). To our knowledge, this is the first report of siderophore analogues produced by biological methods. We employed the methodology of mutasynthesis (13-16), in which a metabolite-producing organism is mutated to a nonproducing strain with a presumed block at some point in

the biosynthetic pathway. Subsequent incorporation of an exogenously supplied unnatural moiety (a mutasynthon) yields a modified metabolite. In this report, P. aeruginosa mutants that are blocked in salicylic acid biosynthesis incorporated salicylic acid analogues, thus yielding mutasynthetic pyochelins. The initial report on the isolation of Sal- mutants of P. aeruginosa (12) demonstrated rapid incorporation of exogenously supplied salicylic acid into pyochelin, and the present

results indicate that P. aeruginosa has an efficient transport mechanism for the uptake of salicylic acid and at least some of its structural analogues. Transport of salicylic acid by P. aeruginosa is unusual because the organism is unable to catabolize salicylic acid (22). Of the 13 salicylic acid analogues tested, only 5-fluorosalicylic acid, 4-methylsalicylic acid, and 3-hydroxypicolinic acid were observed to be transformed into mutasynthetic pyochelins, the first 2 being as efficiently incorporated as salicylic acid and the last -25% as efficiently. These three salicylate analogues are apparently recognized by both the salicylic acid transport system and the pyochelin biosynthetic enzymes, whereas the other 10 were presumably not recognized by one or both of these systems. The substitution pattern of the nonincorporated precursors suggests that steric considerations are most important for efficient incorporation of the precursors. For instance, 5-fluorosalicylic acid and 3-hydroxypicolinic acid are nearly isosteric with salicylic acid, and 4-methylsalicylic acid has the bulky substituent placed far from the chelating region. The inability to incorporate 3-chloro-, 3,5-dichloro-, 3-hydroxy-, 6-hydroxy-, and 3-methylsalicylic acids, in which the bulky substituents are ortho to either the carboxyl or the phenolic hydroxyl, appears to agree with a steric argument. Electronic factors, however, must be invoked to explain the nonincorporation of 2-hydroxynicotinic acid, picolinic acid N-oxide, 4-amino- and 5-hydroxysalicylic acids, and 2-mercaptobenzoic acid. The two diastereomeric forms of each modified metabolite isolated showed distinctive 'H NMR spectra. All of the "I" isomers (including natural pyochelin) exhibited the same chemical shift and coupling patterns for the H-4', H-2", H-4", and N-CH3 protons, and the "II" isomers also gave consistent patterns. The 13C NMR spectra provided a similar result in that the resonances for the C-4', C-2", and C-4" methine carbons for isomers I and II were found in the narrow regions of 79-82, 75-76, and 72-73 ppm. These results are reasonable as the only structural change to be found in the mutasynthetic products is in the aromatic ring, far removed from the diastereotopic centers in the heterocyclic rings. In addition, all of the spectroscopically similar isomers I eluted from silica gel before isomers II. The structures of the mutasynthetic and natural pyochelin isomers described above are corroborated by a crystallographic study of the methyl ester of 4-methylpyochelin 1,

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Proc. Natl. Acad. Sci. USA 88 (1991)

from which the assignment of the absolute stereochemistry of the two diastereomeric (and interconvertible) natural products follows as (4'R,2"R,4"R) for isomers in series I and as (4'R,2"S,4"R) for isomers in series II (23). This study (23) and the syntheses and spectral analyses (unpublished results) of four of the eight possible diastereomers of pyochelin itself will be the subject of a separate paper. The mutasynthetic analogue 6-azapyochelin bears structural similarities with ferrithiocin (8), a Streptomyces sp. OH

N

do

CO2H

N

S

H / C3

8 (FERRITHIOCIN)

metabolite (24). The structural features shared by pyochelin, ferrithiocin, and anguibactin (9), a siderophore produced by OH K Z

OH

S

N OH

N H

9 (ANGUIBACTIN) Vibrio anguillarum (25), suggest similar biosynthetic steps in their production. The presence of a 3-hydroxysalicylic acid unit in anguibactin poses an interesting biosynthetic question in terms of the nonincorporation of 3-hydroxysalicylic acid into pyochelin and may argue against a steric explanation for substrate specificity in P. aeruginosa. However, the two organisms are sufficiently different that biogenetic comparisons are tenuous at best. Each of the three mutasynthetic analogues demonstrated a different biological activity in iron transport assays. 4-Methylpyochelin, the only analogue with strong siderophore activity, was more efficient than pyochelin in the transport of iron into P. aeruginosa. 6-Azapyochelin and 5-fluoropyochelin had little siderophore activity; the low activity measured might result from nonspecific adsorption of the hydrophobic chelates to the membrane. However, the observation that 5-fluoropyochelin is capable of inhibiting fempyochelin transport would suggest that 5-fluoropyochelin is specifically recognized by the ferripyochelin transport receptor but that this analogue and its ferric chelate cannot be correctly processed by the transport system. Unlike 5-fluoropyochelin, 6-azapyochelin did not inhibit ferripyochelin transport.

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