FEBS 17677 FEBS Letters 396 (1996) 243 247 Specific photoaffinity labelling of a ferripyoverdin outer membrane receptor of Pseudomonas aeruginosa Aydin Ocaktan a, Isabelle Schalk a, Christophe Hennard a, Caroline Linget-Morice% Pavel Kyslik b, Anthony W. Smith c, Peter A. Lambert d, Mohamed A. Abdallah ~,* ~Laboratoire de Chimie Microbienne, Associb au C.N. R S., Facultb de Chimie, Universitd Louis Pasteur, F-67008-Strasbourg Cedex, France blnstitute of Microbiology, Academy of Sciences of the Czech Republic, Videnskd 1083, 142 20 Prague 4, Czech Republic CSchool of Pharmacy and Pharmacology, University of Bath, Bath BA2 7A Y, UK dDepartment of Pharmaceutical and Biological Sciences, Aston University, Birmingham, B4 7ET, UK Received 16 September 1996 Abstract In order to identify and characterize the receptors involved in pyoverdin-mediated iron transport in Pseudomonas aeruginosa ATCC 15692, a photoactivatable siderophore has been synthesized. In the dark, this probe is stable and is able to promote iron transport at the same rate as the native pyoverdin. Under irradiation at 312 nm, the molecule is photodecomposed and a clear inhibition of the iron transport is observed. With the radioactive form of this photoactivatable probe, we were able to visualize on a S D S - P A G E gel a labelled protein of approximately 90 kDa molecular mass, which is very likely the FpvA receptor or a yet unknown pyoverdin receptor. Key words: Iron transport; Pseudomonas aeruginosa; Outer membrane receptor; Photoaffinity labelling; Pyoverdin; Azidopyoverdin I. Introduction When grown under iron-limited conditions, many aerobic bacteria synthesize and secrete into the environment iron chelators termed siderophores [1]. Siderophores are low-molecular-mass molecules capable of chelating and delivering iron to bacterial cells via specific high-affinity transport systems [2]. Pseudomonas aeruginosa, an important opportunistic pathogen of humans [3], produces at least two known siderophores when grown in iron-deficient conditions: pyochelin [4] and pyoverdin [5]. Pyoverdin is an octapeptide bound to a fluorescent chromophore derived from 2,3-diamino-6,7dihydroxyquinoline [6] and pyochelin is a hydroxyphenylthiazolinylthiazolidine type of siderophore [7]. In addition, P. aeruginosa is able to utilize a number of heterologous siderophores, including pyoverdins produced by some other Pseudomonas [8], ferrioxamine B [9], aerobactin and enterobactin [10]. Two outer membrane receptors for ferripyochelin, of respectively 14 k D a [11] and 75 k D a [12] molecular mass, have been identified. The gene for the 75 k D a ferripyochelin receptor (fptA) has been cloned [13] and more recently sequenced [14]. Heinrichs et al. [12] suggested that the 14 k D a system operated in exponential phase cells, whereas the 75 *Corresponding author. Fax: (33) 88 60 44 07. E-mail: abdallah@chimie.u-strasbg.fr Abbreviations." PaA, pyoverdin PaA; PaA-NH2, aminopyoverdin; PaA-N3, azidopyoverdin; HOHAHA, homonuclear Hartmann Hahn spectroscopy; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum coherence k D a receptor transport was of greater importance in late exponential and early stationary phase. The receptor for ferripyoverdin has been identified as a 90 k D a outer membrane protein (FpvA) [15] and cloned [16]. A mutant deficient in expression of this protein still showed low uptake of ferripyoverdin, providing evidence for a second transport system [15]. As we mentioned above, P. aeruginosa is able to use heterologous siderophores like enterobactin [10]. Poole et al. [17] identified and cloned [18] a ferric-enterobactin receptor (PfeA) of 78 k D a molecular mass. It is now clear that P. aeruginosa has multiple iron transport systems, but the contribution of these systems to the growth in vivo is still unclear. The way all these receptors are activated is not yet known [19]. However, the presence in F p v A and in PleA of sequences apparently conserved in TonB-dependent receptors [20] certainly suggests a TonB-like mechanism [16]. A method for studying the siderophore-receptor interactions during the iron transport and ultimately for isolating the protein receptors is photoaffinity labelling [21]. In this paper we describe the synthesis and the iron transport properties of a photoactivatable analogue of pyoverdin PaA, a peptidic siderophore of P. aeruginosa A T C C 15692. We also show how a photoactivatable radiolabelled pyoverdin analogue can be used to label specifically, when irradiated in the presence of whole cells of P. aeruginosa A T C C 15692, the outer membrane receptor involved in pyoverdin-mediated iron uptake. 2. Materials and methods 2.1. Growth condition P. aeruginosa strain ATCC 15692 was grown under aerobic conditions in a succinate medium as described by Demange et al. [6]. 2.2. Isolation and purification of the pyoverdins The pyoverdins have been isolated and purified as described earlier by Albrecht-Gary et al. [22]. The purity of pyoverdins was monitored by electrophoresis on cellulose acetate films using a SEBIA horizontal electrophoresis tank. The electrophoreses were run in 100 mM pyridine-acetic acid pH 5.0 buffer, at a constant voltage (300 V), for 30 min. 2.3. Synthesis" of aminopyoverdin-Fe( III) ( PaA-NH2-Fe( III) ) complex Pyoverdin PaA-Fe(III) complex (90 mg, 65 pmol) was dissolved in 500 pl of DMSO/H20 (7:1, v/v). 34 mg (177 gmol) of EDCI (1-(3dimethylaminopropyl)-3-ethycarbodiimide bydrochloride) (Aldrich Chemic, Steinheim, Germany) was added and the flask was stirred at room temperature for 3 h. 50 pl (615 pmol) of 1,2-diaminoethane was added and the solution stirred for 5 h at room temperature. The reaction was monitored by film electrophoresis on cellulose acetate. After evaporation of the solvent, the residue was dissolved in 500 pl 0014-5793/96/$12.00 © 1996 Federation of European Biochemical Societies. All rights reserved. PH S00 I 4 - 5 7 9 3 ( 9 6 ) 0 1 07 1 -X 244 pyridine-acetic acid 50 m M p H 5.0 buffer, and applied on a CMSephadex C-25 column (1.5 x 20 cm) eluted with a linear gradient of pyridine-acetic acid pH 5.0 buffer (5(~500 m M , 2 x 100 ml). 67 mg of aminopyoverdin-Fe(III) was obtained (74°/,0. Electrophoresis on cellulose acetate films: migration distance= 3.0 cm. UV-Vis: X,n~x= 400 nm, ~ = 19 000 M -1 x L; ~ , = 460 nm, e = 6200 M ~ x L; ~ h = 540 nm, ~= 2300 M ~ x L in pyridine-acetic acid pH 5.0 buffer. FAB-MS: m / z = 1429 m.u. (M+). 2.4. Decomplexation of aminopyoverdine-Fe (III) (PaA-NH2_ Fe (HI) ) The decomplexation of PaA-NH2-Fe(III) was performed as for pyoverdin-Fe(III) according to Albrecht-Gary et al. [22]. Electrophoresis on cellulose acetate films: migration d i s t a n c e = 8 cm. UV-Vis: 7,,,~×=380 nm, ~ = 1 6 5 0 0 M - 1 x L ; ;~...... =360 nm, E= 16000 M 1 X L in 50 m M pyridine-acetic acid pH 5.0 buffer. FABS-MS: m / z = 1376 m.u. (M+). 2.5. Synthesis of the azido derivative (PaA-N3) 15 m g (10 [amol) of aminopyoverdin was dissolved in 75 tll of a mixture of dimethylformamide-water (7:1, v/v) and 18 [al of a 10% solution of triethylamine in dimethylformamide was added. After stirring the mixture for 5 min at room temperature, 3.5 m g (13 [amol) of 4-azidobenzoic acid N-hydroxysuccinimide ester dissolved in 200 [al of dimethylformamide was added. After 1 h at room temperature, 1 ml of ethyl acetate was added. The mixture was centrifuged and the pellet dissolved in 200 I11 water. Excess reagent was removed by two successive extractions with 100 ~1 ethyl acetate. Electrophoresis on cellulose acetate film: migration distance 1.0 cm. UV-Vis: ;~.... =258 nm, E = 4 1 5 0 0 M 1 x L ; ;~..... =360 nm, e = 1 6 0 0 0 M 1 X L ; X~,~x=380 nm, E = 1 6 5 0 0 M - 1 x L in 50 m M acetic acid/sodium acetate pH 5.0 buffer. 2.6. Synthesis of the tritiated azido derivative-Fe(lll) complex ( ['JH ] PaA-N3-Fe( III) ) 50 [ag (35 nmol, determined spectrophotometrically) of PaA-NH2Fe(III) were dissolved in 5 [al of dimethylformamide. 1 [al of a solution of triethylamine in dimethylformamide (diluted 50 times) and 5 lal (5 [aCi) of a solution of 4-azido-3,5[3H]benzoic acid N-hydroxysuccinimide ester (Dupont N E N , specific activity 45.7 Ci/mmol) in dimethylformamide were added and the tube was vigorously stirred at room temperature. The reaction was monitored by electrophoresis on cellulose acetate film. An aliquot of the reaction medium was removed, mixed with a concentrated solution of unlabelled PaA-N3-Fe(III) and electrophoresed for 12 min. The film was then sliced into 5 m m wide sections and each slice was counted in 4 ml of AqualumaPlus scintillation liquid ( L U M A , L.S.C., France). The counting was perfonned after 2 h. After 1 h, 50 [al of distilled water was added and the aqueous phase was extracted with ethyl acetate (3 X 50 t11). The yield of the coupling reaction was 32% (calculated from the radioactivity counted) with a specific activity of 45.7 Ci/mmol. A. Ocaktan et al./FEBS Letters 396 (1996) 243~47 2.9. r'SFe uptake in P. aeruginosa in the presence or absence of irradiation 10 ml of bacterial culture harvested at the end of the exponential phase of growth was centrifuged 10 min at 1 2 0 0 0 x g . After having been washed with 50 m M MOPS pH 7.0 buffer, the pellet was suspended again in MOPS buffer and the optical density of the suspension at 600 n m was adjusted to 0.5. The cells were incubated for 15 min at 29°C before the beginning of the transport experiments. The solutions of the siderophores complexed to 55Fe (from 55FEC13, NEN, specific activity of 3 Ci/g) were prepared using a 1 m M solution of PaA or PaA-N3 as a free ligand in 50 m M MOPS pH 7.0 buffer. To 200 [al of this solution was added 5 [al of a solution of 55FEC13 (1 [aCi/ [al) obtained by dilution of the stock solution, plus 800 [al of 50 m M MOPS pH 7.0 buffer. 500 [al of the radioactive complex solution thus formed were then added to 4.5 ml of the bacterial suspension. The mixture was stirred at 29°C in the dark or under irradiation (bench lamp maintained at 12 cm above the surface of the liquid). 500 [al aliquots were removed at different times and filtered on 0.45 [am porosity filters (Micronsep, France), pre-soaked in a 0.3% solution of polyethyleneimine. Each filter was rapidly washed twice with 2 ml 50 m M MOPS buffer at pH 7.0, and its radioactivity measured in 3 ml of scintillation liquid (AqualumaPlus) after 4 h incubation. 2.10. Analysis' of the labelled outer membrane proteins The bacteria were prepared as above in 50 m M MOPS pH 7.0 buffer. 5 [aCi of [3H]PaA-N3-Fe(III) was added to the suspension, and after 2 min incubation, the tubes were irradiated for 8 min at 312 nm. In parallel the same experiment was performed in the presence of 1 [aCi of [~H]PaA-N3-Fe(III) plus 5 [aM PaA-Fe(III). Both suspensions were centrifuged 5 min at 1 2 0 0 0 x g , and the pellet washed with 50 m M MOPS pH 7.0 buffer. The analysis of the labelled outer membrane proteins was performed according to Mizuno and K a g e y a m a [23] and Hancock and Nikaido [24] with some modifications. The two bacterial pellets were suspended in a 25 m M Tris-HC1 pH 6.8 in the presence of 0.5 m g lysozyme. The cells, maintained in an ice bath were then sonicated (Bransonic 12, Branson Co., Shelton, CT, USA) for 6 x 3 0 s, with 30 s cooling between each sonication. Addition of sodium N-lauryl sarcosinate (sarkosyl) at a final concentration of 2% (v/v), solubilized the cytoplasmic membranes leaving the outer membranes intact. After incubation at 37°C for 10 min, 200 [al of a solution containing 1 mg DNase and 1 m g R N a s e per ml was added. The mixtures were reincubated for 5 min at 37°C. Centrifugation for 5 min at 5000Xg removed the unbroken cells, and the supernatant was then centrifuged 60 min at 2 0 0 0 0 x g . The pellets were washed with 50 m M Tris-HC1 pH 6.8 and centrifuged as before. The membranes were loaded on a 12% SDS-PAGE gel. After staining, the gel was sliced in 2.5 m m wide bands, each band being then placed in a counting vial and digested by hydrogen peroxide (300 [al) for 48 h at room temperature. 100 I11 o f S D S l%/urea 8 M were added to each counting vial and the radioactivity counted after 12 h in 4.5 ml of scintillation liquid (AqualumaPlus). 3. Results and discussion 2. 7. Photolysis of PaA-N3-Fe(Ill) Photolysis was performed in 50 m M MOPS pH 7.0 buffer at 29°C with a bench lamp emitting at 312 n m (VL-M, 6W, Bioblock, France). The samples were kept in a quartz cell (Hellma, 454 X 12.5 X 9.5 mm), and irradiated with the light source maintained at a distance of 12 cm. A UV-visible spectrum of this solution was determined every 15 s. 2.8. P. aeruginosa growth in the presence of PaA-N.~ 5 ml of a culture of P. aeruginosa A T C C 15692 harvested at the end of the exponential phase of growth was centrifuged (10 min at 12000Xg) under sterile conditions and the pellet was washed twice with a sterile succinate medium. After the last centrifugation the bacteria were suspended again in the culture medium at a concentration of 1.3X 109 cfu/ml (OD470 = 1.3; OD600 =0.81). 30 I11 of this suspension was used to inoculate 3 ml of succinate medium and a solution of pyoverdin or its photoactivatable analogues was added at a final concentration of 25 [aM. The cuvettes were stirred at 37°C in the dark, and the optical densities at 470 n m and 600 n m were measured as a function of time. 3.1. Properties o f the photoactivatable pyoverdin analogue A r y l a z i d e s a r e k n o w n to be u s e f u l p h o t o r e a c t i v e m o i e t i e s o f p h o t o a f f i n i t y p r o b e s [21]. T h e y a r e c h e m i c a l l y inert, b u t w h e n p h o t o l y s e d , t h e y p r o d u c e aryl n i t r e n e s , e x t r e m e l y reactive species f o r m i n g s t a b l e b o n d s w i t h p r a c t i c a l l y all k i n d s o f c o m p o u n d s [25,26]. N i t r e n e s a r e sufficiently r e a c t i v e to i n s e r t e v e n into C-H bonds. T h e site c h o s e n f o r t h e b i n d i n g o f t h e p h o t o a c t i v a t a b l e g r o u p to p y o v e r d i n ( P a A ) w a s t h e s u c c i n y l m o i e t y l i n k e d to t h e c a r b o n C-3 o f t h e c h r o m o p h o r e . T h e first step w a s t h e introduction of a nucleophilic amino group on this spacer a r m . F o r this p u r p o s e P a A - F e ( I I I ) w a s c o u p l e d w i t h a l a r g e e x c e s s o f 1 , 2 - d i a m i n o e t h a n e , in t h e p r e s e n c e o f E D C I . T h e r e a c t i o n , m o n i t o r e d b y film e l e c t r o p h o r e s i s , g a v e o n e m a j o r d e r i v a t i v e . T h e yield a f t e r p u r i f i c a t i o n w a s 74%. U s i n g F A B - 245 A. Ocaktan et al./FEBS Letters 396 (1996) 243~47 0.9 0.8 0.7 0.6 0.5 0.4 © 0.3 0.2 0.1 0 240 340 440 540 640 Wavelength (nm) Fig. 1. Photodecomposition of PaA-Na-Fe(III) under irradiation at 312 nm. PaA-Na-Fe(III) was dissolved in 50 mM MOPS pH 7.0 buffer at 29°C at a concentration of 22 p.M and irradiated at 312 nm. A UV spectrum of this solution was determined every 15 s. MS techniques and 2D N M R correlations ( H O H A H A , 1HlaC correlations, H M Q C and H M B C , all performed on the free ligand with a 500 M H z Bruker spectrometer) we could assign all the signals corresponding to the protons and the carbon atoms (data not shown) and obtain the proofs that the 1,2-diaminoethane moiety is bound to the succinyl group (3j correlation between the C H 2 C O carbonyl of the succinate moiety at 177.2 ppm and the N H C H 2 protons of the 1,2diaminoethane group at 3.60 ppm, and 2 j correlation between the N H C H 2 proton of the 1,2-diaminoethane group at 8.28 ppm and the C H 2 C O carbonyl of the succinyl group at 177.2 ppm). All the other resonances were found to be very similar to those reported for pyoverdin PaA [6]. The introduction of the arylazido groups on the amino derivative of PaA was performed by treatment with a slight excess of 4-azidobenzoic acid N-hydroxysuccinimide ester, in the presence of triethylamine. One major product was observed by electrophoresis on cellulose acetate films. Due to its fairly low stability as a free ligand, PaA-N:~ was used without further purification in the experiments described in this paper. Further characterization of the products is under investigation. The UV-visible absorption spectra of the ferric complex of PaA-Na (Fig. 1) shows two maxima, at 258 nm and at 400 nm corresponding respectively to the absorption of the arylazido group and of the complexed chromophore. The photodecomposition of PaA-N3-Fe(III) in 50 m M M O P S p H 7.0 buffer under irradiation at 312 nm is characterized by the decrease of the absorption band of the arylazido group at 258 nm and by the unaltered m a x i m u m absorption of the chromophore. The half-life of the c o m p o u n d deduced from this photodecomposition is about 1 min, but is expected to be longer in the presence of bacteria, due to light scattering. 3.2. Iron transport properties of PaA-N3 ~SFe transport measurements, mediated by pyoverdin PaA or PaA-N3, have been performed according to Knosp et al. [27], with slight modifications. The rates of uptake of 55Fe presented in Fig. 2A show that PaA-Nz behaves exactly like pyoverdin P a A at the same concentration. PaA-N3 has still the properties required for the recognition of a pyoverdin by its receptors and for the transport of iron in P. aeruginosa A T C C 15692. The photodecomposition product obtained after pre-irradiation of a solution of PaA-Nz at 312 nm, and its addition to the uptake medium, gave a ~ F e uptake rate similar to that of PaA (Fig. 2A). This proves that the photolyzed analogue can still be recognized by the receptors. Before measuring the inactivation of the 5~Fe uptake by irradiation in the presence of the photoactivatable pyoverdin analogue during transport, it was essential to determine the effect of U V light itself on the bacterial iron transport. In two separate experiments the bacteria were or were not subjected to irradiation at 312 nm. 55Fe complexed to pyoverdin PaA was then added and its uptake measured. N o difference in uptake was seen (results not shown), indicating that irradiation at 312 nm has no effect on pyoverdin-mediated iron uptake. A ~"~ 40" (3 v30 O E 20 ¢-~ z 10" tl tO tO 0 0 2 4 6 8 10 12 14 time (min) B v,- '~ 40" 30" o E "~ 20" ¢-~ I1) kt.. tO to 10 0 i 0 10 i 20 time (min) 30 40 Fig. 2. ~Fe uptake by P. aeruginosa. P. aeruginosa ATCC 15692 at a concentration of 1.3× 109 cfu/ml was incubated 15 min in 4.5 ml 50 mM MOPS pH 7.0 buffer at 29°C before the beginning of the transport assays. A: After this incubation, 500 gl PaA-5~Fe(III) ( - ) , PaA-N3-55Fe(III) (O) or pre-irradiated PaA-N3-55Fe(III) (©) was added to the cells at a final concentration of 10 /aM. 500 gl aliquots were removed at different times, filtered and counted. B: After this incubation, 500 gl PaA-N3-S~Fe(III) (10 gM) was added and 500 gl aliquots were removed at different times, in the dark (e) or under continuous irradiation at 312 nm (©). 246 A. Ocaktan et al./FEBS Letters 396 (1996) 243-247 A kDa mately 90 k D a molecular mass. A characteristic feature of the photoaffinity labelling process is that the natural substrate, in our case pyoverdin PaA, should protect against irreversible labelling [21]. The competition experiment, irradiation in the presence of [aH]PaA-Na-Fe(III) plus an excess of PaA, shows that the radioactive peak is abolished in the presence of PaA during irradiation. These data indicate that the labelled protein is a specific receptor of PaA of approximately 90 kDa involved in the transport of iron via the pyoverdin system. These data are consistent with binding of the azidopyoverdin to the F p v A receptor, although we cannot preclude that a yet unknown pyoverdin-specific receptor different from F p v A lies in the same molecular mass range. Further experiments using blocking antibodies would be required to identify the receptor unambiguously. Attempts to determine the Nterminal sequence of P. aeruginosa high molecular mass iron regulated outer membrane proteins have not been successful (A.W. Smith, unpublished data). Gel slice IROMPs s 10 15 20 25 30 B 800 4. Conclusion eo "-~ 600 > 400 ¢) .~ 200 0 10 20 30 40 Gel slice Fig. 3. Analysis of the labelled outer membrane proteins by SDSPAGE. P. aeruginosa ATCC 15692 at a concentration of 1.3× l0 ~ cfu/ml in 50 mM MOPS pH 7.0 buffer at 29°C was irradiated at 312 nm in the presence of [aH]PaA-Na-Fe(III) (22 nM) and in the presence or absence of PaA-Fe(III) (5 ~M). The outer membrane proteins of this labelled bacteria were loaded on a SDS-PAGE gel. A: The Coomassie-stained gel; B: the corresponding radioactive tracing of the sliced gel (l slice corresponds to 2.5 ram), for the bacteria irradiated in presence of [aH]PaA-Na-Fe(III) (©) or in the presence of [aH]PaA-Na-Fe(III) plus PaA-Fe(III) (e). The results presented here indicate that PaA-Na is a successful photoaffinity probe for the pyoverdin-mediated iron transport system in P. aeruginosa A T C C 15692. In the dark this molecule behaves like PaA, but under irradiation it inhibits efficiently, irreversibly and specifically the pyoverdinmediated iron transport. A photolabelled outer membrane receptor, which is very likely F p v A or a yet unknown pyoverdin receptor, has been visualized on S D S - P A G E gel. PaA-Na should be a good probe to localize and identify some other proteins involved in the transport of PaA-Fe(III) in the other cell compartments of P. aeruginosa. Acknowledgements." We wish to thank Professor Maurice Goeldner for his kind help in providing us the possibility of performing some of the radiolabelling experiments in his laboratory, and for his very stimulating discussions as well. We also wish to thank Mr. Bachir Machi and Mr. Rui Ventura for their expert technical assistance. References The rates of '~SFe uptake in the presence of PaA-N3 in the dark and under continuous irradiation at 312 nm are represented in Fig. 2B. An inhibition of 15% is observed between both curves. Since the pre-irradiated photolyzed analogues can promote the uptake of SSFe with the same rate as natural PaA or PaA-Na, the effect of inhibition can be assigned exclusively to the inactivation of some receptor(s) involved in the iron transport, and not to the modification of the affinity of the analogue after photolysis. Therefore, this inhibition would be due to the photolabelling of the proteins occurring in the transport, by the nitrene generated by photoactivation. 3.3. Analysis o f the labelled outer membrane In order to identify on the wild strain which proteins on the outer membrane are the receptors of pyoverdin, photoaffinity labelling experiments have been performed on bacteria in the presence of tritiated PaA-Na-Fe(III) or in the presence of [aH]PaA-Na-Fe(III) plus a large excess of PaA. The outer membrane proteins of these labelled bacteria were analyzed by S D S - P A G E . The Coomassie-stained gel and the corresponding radioactive trace are shown in Fig. 3. The position of the labelled band corresponds to a protein of approxi- [1] Neilands, J.B. (1981) Annu. Rev. Biochem. 50, 715 731. [2] Neilands, J.B. (1982) Annu. Rev. Microbiol. 36, 285 309. [3] Botzenhart, K. and Ruden, H. (1986) in: Basic Research and Clinical Aspect of Pseudomonas aeruginosa (D6ring, G., Holder, I.A. and Botzenhart, K., Eds.), pp. 15 24, Karger, Basel. [4] Cox, C.D. (1980) J. Bacteriol. 142, 581-587. [5] Cox, C.D. and Adams, P. (1985) Infect. Immun. 48, 13~138. [6] Demange, P., Wendenbaum, S., Linget, C., Mertz, C., Cung, M.T., Dell, A. and Abdallah, M.A. (1990) Biol. Metals 3, 155 170. [7] Cox, C.D., Rinehart Jr., K.L., Moore, M.L. and Cook Jr., J.C. (1981) Proc. Natl. Acad. Sci. USA 78, 4256-4260. [8] Hohnadel, D. and Meyer, J.-M. (1988) J. Bacteriol. 170, 4865 4873. [9] Cornelis, P., Maguilevsky, N., Jacques, J.F. and Masson, P. (1986) in: Basic Research and Clinical Aspect of Pseudomonas aeruginosa (D6ring, G., Holder, I.A. and Botzenhart, K., Eds.), pp. 290 306, Karger, Basel. [10] Liu, P.V. and Shokrani, F. (1978) Infect. Immun. 22, 878-890. [1l] Sokol, P.A. and Woods, D.E. (1983) Infect. Immun. 40,665669. [12] Heinrichs, D.E., Young, L. and Poole, K. (1991) Infect. Immun. 59, 3680-3684. [13] Ankenbauer, R.G. (1992) J. Bacteriol. 174, 44014409. [14] Ankenbauer, R.G. and Quan, H.N. (1994) J. Bacteriol. 176, 307319. A. Ocaktan et aI./FEBS Letters 396 (1996) 243-247 [15] Poole, K., Neshat, S. and Heinrichs, D. (1991) FEMS Microbiol. Lett. 78, 1-5. [16] Poole, K., Neshat, S., Krebes, K. and Heinrichs, D.E. (1993) J. Bacteriol. 175, 4597-4604. [17] Poole, K., Young, L. and Neshat, S. (1990) J. Bacteriol. 172, 6991-6996. [18] Dean, C.R. and Poole, K. (1993) J. Bacteriol. 175, 317-324. [19] Gensberg, K., Hughes, K. and Smith, A.W. (1992) J. Gen. Microbiol. 138, 2381-2387. [20] Braun, V. (1995) FEMS Microbiol. Lett. 16, 295-307. [21] Bayley, H. and Knowles, J.R. (1977) Methods Enzymol. 46, 69114. 247 [22] Albrecht-Gary, A.-M., Blanc, S., Rochel, N., Ocaktan, A.Z. and Abdallah, M.A. (1994) Inorg. Chem. 33, 6391 6402. [23] Mizuno, T. and Kageyama, M. (1978) J. Biochem. 84, 179-191. [24] Hancock, R.E.W. and Nikaido, H. (1978) J. Bacteriol. 136, 381390. [25] Knowles, J.R. (1972) Acc. Chem. Res. 5, 155 160. [26] Bayley, H., Staros, J.V., (1984) in: Azides and Nitrenes. Reactivity and Utility (Scriven, E.F.V., Ed.), Chapter 9, Academic Press, New York. [27] Knosp, O., Von Tigerstrom, M. and Page, W.J. (1984) J. Bacteriol. 159, 341 347.