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FEM.9Microbiology Letters 132 (1995) 9-l 5 High-molecular-mass, iron-repressed cytoplasmic proteiris in fluorescent Pseudomonas: potential peptide-synthetases for pyoverdine biosynthesis Claude Georges, Jean-Marie Meyer * Luboratoire de Microbiologic. Unite’de Recherche Associk au Centre National de la Recherche Scientifique No. 1481, 28 rue Goethe, Uniuersite’ Louis-Pasteur, 67ooO Strasbourg, France Received 24 April 1995; revised 1 June 1995; accepted 3 June 1995 Abstract High molecular-mass cytoplasmic proteins were detected in iron-starved, pyoverdine-producing Pseudomonas aeruginosa, P. chlororaphis, P. fluorescens, P. pufih, P. apt&a and P. tofuasii.They appeared to be specifically located in the cytoplasm and thus were termed ‘IRCPs’, for iron-repressed cytoplasmic proteins. A strain-dependent gel electrophoresis pattern with multiple bands of M, values ranging from 180 to 600 kDa was usually observed for these proteins. Strains synthesizing pyoverdines differing in their peptide part presented different IRCP gel electrophoresis profiles, whereas strains synthesizing identical pyoverdines had identical IRCP gel electrophoresis profiles. Some mutants affected in pyoverdine biosynthesis presented a perturbed IRCP pattern, and no IRCPs were detected in non-fluorescent Pseudomonasstrains either unable to synthesize siderophores or synthesizing non-peptidic siderophores. The data strongly suggest that the IRCPs could be related to peptide synthetases involved in the biosynthesis of the peptidic part of pyoverdine-type siderophores. Keywords: Pseudomonas spp.; Iron metabolism; Fyoverdine biosynthesis; Peptide synthetases 1. Introduction Pyoverdines, the fluorescent pigments and main siderophores of fluorescent Pseudomonas species [l], share the presence of a peptidic chain in their structures. More than 20 pyoverdines produced by different strains of Pseudomonas have been purified and the structures of most have been determined. With the exception of two type strains, P. jluorescens ATCC 13525 and P. chlororaphis ATCC * Corresponding author. Tel.: +33 88 24 41 50; Fax: 88 35 84 84. 0378-1097/95/$09.50 0 SSDl 0378-1097(95)00250-2 1995 9446, which produce a structurally identical pyoverdine, each fluorescent Pseudomonas type strain studied so far synthesizes a specific pyoverdine: these differ from one another in the amino acid composition of their peptidic parts [2]. This diversity in structure is even found within a single species, as structurally different pyoverdines have been recognized among P. aeruginosa or P. jluorescens strains [2-41. The peptide part of pyoverdine is linked to a quinoleinic chromophore which is apparently always identical, whatever the producing strain [2]. Depending on the pyoverdine, the length of the peptide chain may vary from 6 to 12 aminoacyl residues [2], with some of them participating in the complexing of Federation of European MicrobiologicalSocieties. All rights reserved IO C. Georges. J.-M. Meyer / FEMS Microbiology iron, the others being presumably involved in the specific recognition of the ferripyoverdine receptor [4]. Another special feature of the peptide chain is the presence of o-amino acids together with L-amino acids. These amino acids usually form linear chains with possible internal loops, e.g. the pyoverdines of P. ueruginosa ATCC 15692 or P. fluorescens strain 12 [2]. The peculiarities found in the peptide chain of pyoverdine raises questions about its biosynthesis. Because of their short length, unusual amino acid composition and possible internal cyclic structures, these peptides appear to be structurally closely related to peptidic antibiotics of microbial origin such as the gramicidins or tyrocidine produced by Bacillus spp. [5], actinomycin from Streptomyces spp. [6], or syringomycin and syringotoxin produced by strains of P. syringae [7,8]. Thus, the pyoverdine peptides may be synthesized through a multi-enzyme thiotemplate mechanism involving peptide synthetases as demonstrated for the antibiotics [5], rather than through a classical ribosomal pathway. Moreover, a multi-enzyme system catalysing the biosynthesis of the cyclic hexapeptide ferrichrome, a fungal siderophore, has already been characterized [9], as have some amino acid-activating enzymatic activities in Azotobacter, which suggest the involvement of a non-ribosomal pathway for the biosynthesis of the pyoverdine-like azotobactin siderophore [IO]. Finally, the recently published nucleotide sequence of gene pcdD involved in pyoverdine biosynthesis in P. ueruginosu demonstrated clear similarities with a range of bacterial and fungal antibiotic peptide synthetase genes [ 1 I], again strongly suggesting a nonribosomal way of biosynthesis for pyoverdine. In this paper, we report on the search for such pyoverdinerelated peptide-synthetases in fluorescent Pseudomonas spp., undertaken by looking for high M, soluble proteins, a general feature of antibiotic-related peptide synthetases [5]. 2. Materials and methods 2. I. Bacterial strains and growth conditions The fluorescent Pseudomonas strains used throughout this work were mainly collection strains Leirers 132 (1995) 9- 15 as P. ueruginosu ATCC 15692, P. ATCC 27853, P. putidu ATCC 12633, P. fluorescens ATCC 13525, P. fluorescens ATCC 17400, P. fluorescens CCM 2798, P. chlororaphis ATCC 9446, and P. tolaasii NCPPB 2192. P. fluorescens strain 12, P. f7uorescens strain ii, and P. aptata strain 4a were from the laboratory collection of H. Budzikiewicz (University of Kiiln, Germany) and 10 P. aeruginosa clinical isolates (strains Pa3 to Pal2), with pyoverdines previously characterized [3], were from G. Wauters (St. Luc Hospital, Brussels, Pseudomonas included Belgium). Non-fluorescent P. fragi ATCC 4973, P. stutzeri ATCC 17588 and P. diminuta CIP 7129. The P. ueruginosu pyoverdine-deficient mutants PA06601, PA06606, PA06609, PA06622, PA06624 and their parent strain PA06049, a methionine-auxotroph of P. ueruginosu ATCC 15692 (PA01 strain), have been described elsewhere [ 121. Iron-starved cells were obtained from 40-h cultures grown in succinate medium [3] at 25°C with shaking (200 rpm). Iron-replete cells were obtained from cultures grown under identical conditions in succinate medium supplemented, after sterilization, with 100 PM FeCl,. The PA0 mutants were grown in 1 mM methionine-supplemented succinate media as required [ 121. referenced ueruginosa 2.2. Detection of the IRCPs Bacterial protein extracts from stationary phase cells were prepared and partially purified by using a procedure described for the purification of antibiotic peptide synthetases [6], consisting of disruption of the cells using a French press or by sonication in 0.1 M phosphate buffer (pH 7.0). Partial purification of the soluble proteins was then obtained by a treatment with polymine P (O.l%, final concentration), followed by an overnight 50% saturation ammonium sulfate precipitation at 4°C. The resulting protein precipitate was solubilized in 0.125 M Tris HCl buffer (pH 6.8) and, after protein quantification by the Lowry method [13], subjected to SDS-PAGE according to Laemmli [14] with 5% polyacrylamide gels. Detection of the protein bands was performed with the conventional Coomassie brilliant blue R-250 reagent or by more sensitive procedures, i.e. a silver staining [15] or a modified Coomassie brilliant blue staining [ 161. A selective purification of the periplas- C. Georges, J.-M. Meyer/ FEMS Microbiology Letters 132 (1995) 9-15 mic proteins of P. aeruginosa ATCC 15692 was done according to [17]. 3. Results and discussion 3.1. Detection of high M,, iron-regulated cytoplasmic proteins in P. aeruginosa ATCC 15692 When the soluble proteins of the pyoverdine producer P. aeruginosa ATCC 15692 (PA01 strain) were analysed by SDS-PAGE (5% polyacrylamide), high M, proteins were observed (Fig. 1). Depending on the bacterial growth conditions and on the amount of protein loaded on the gel, five protein bands ranging from 185 to 550 kDa were stained by the Coomassie brilliant blue reagent. These bands were not visible when gels were loaded with less than 100 pg protein. They were detectable and increased in intensity at higher protein concentrations, appearing PA0 PA0 P.chl. P.chl. P. n. P.rl. 15692 15692 9446 9446 13525 13525 P.SWw.. P.SlUlz. 17588 17588 kDa II as pronounced bands when gels were loaded with 400 Fg protein per lane. As shown in Fig. 1, these bands were observed in bacteria grown under iron starvation (succinate medium), but were never visible in cell extracts obtained from bacteria grown in a 100 PM iron-enriched succinate medium. For intermediate iron concentrations in growth medium, the high M, proteins were visible at roughly the same intensity over the range of O-4 PM added iron and not seen at higher iron concentrations. It should be emphasized that the O-4 PM iron concentration range in succinate growth medium allows the production, by P. aeruginosa ATCC 15692, of pyoverdine and the synthesis of a bacterial outer membrane protein acting as ferri-pyoverdine receptor [18]. For higher iron concentrations, all these biosyntheses (high molecular mass proteins, siderophore, outer membrane receptor) were severely repressed. Thus, the soluble high molecular mass proteins appeared to be under the same iron control as the siderophore and the ferri-siderophore receptor. Analysis of the periplasmic proteins of P. aeruginosa ATCC 15692 did not reveal any high M, proteins (data not shown). Thus, the large proteins should be located in the cytoplasm only. For convenience, we propose to name these high M, proteins ‘IRCPs’, for iron-repressed cytoplasmic proteins, by analogy with the term ‘IROMPs’ which is commonly used to designate iron-regulated outer membrane proteins expected to act as ferri-siderophore receptors. 3.2. lRCPs in other Pseudomonas strains - w Fig. 1. Iron-repressed cytoplasmic proteins (IRCPs) in Pseudomonas. Bacterial extracts (400 pg protein) of P. aeruginosa ATCC 15692 (PA0 15692). P. chlororuphis ATCC 9446 (P. chlor. 9446). P. fruorescens ATCC 13525 (P. fl. 13525) and P. stutzeri ATCC 17588 (P. stutz. 17588) grown in succinate medium ( -1, or in 100 PM FeCl,-supplemented succinate medium ( + ), were analysed by SDS-PAGE (5% polyactylamide) according to Laemmli [14]. The electrophomtic mobility of high molecular protein markers (Pharmacia) with size in kDa am indicated on the right side. Electrophoresis conditions were maintained during a supplementary hour after the dye had reached the bottom of the gel. Initially determined for P. aeruginosa ATCC 15692, the 400 pg protein load for SDS-PAGE was convenient to observe well formed IRCP bands for a majority of other pyoverdine producer Pseudomonas strains, as illustrated in Fig. 1 for P. chlororuphis ATCC 9446 (lane 3) and P. jluorescens ATCC 13525 (lane 51, and also seen for P. aptata 4a, P. j7uorescens ATCC 17400, P. jluorescens CCM 2798, P. jluorescens 12, P. jluorescens ii (data not shown). In some cases, however, as in P. aeruginosu ATCC 27853 and P. putida ATCC 12633, these proteins appeared as diffuse or very faint bands, even with a 400 pg protein load. Higher amounts of proteins did not result in a better visualization of these bands and disturbed the electrophoresis pattern. The use of 12 C. Geoqes, J.-M. Meyer/ FEMS MicrobiologyLetters132 11995) 9-15 more sensitive methods for gel protein detection, i.e. a silver staining [15] or a modified Coomassie blue staining [16], likewise did not result in better visualization. Nonetheless, the general conclusion of these studies was that all the pyoverdine-producing strains analysed synthesized IRCPs. Usually they all responded to iron supplementation as P. aeruginosu ATCC 15692 did, with no high M, protein bands detectable for cells grown in presence of 100 PM added iron, as shown in Fig. 1 for P. aeruginosa ATCC 15692 (lane 2) and P. fluorescens ATCC 13525 (lane 6). P. chlororuphis ATCC 9446 was the only strain which showed a high M, protein, with a molecular mass different from the two major IRCPs detected in this strain (370 kDa and 530-480 kDa, respectively), specific for iron-fed cells (Fig. 1, lane 4). In this respect, P. chlororaphis behaved the same cytoas P. syringae, which produces iron-inducible plasmic proteins related to syringotoxin and syringomycin synthesis [7,8]. For all the pyoverdineproducing strains analysed, IRCP biosynthesis correlated well with pyoverdine biosynthesis and both syntheses appeared to be under the same iron regulation. Additional evidence for a relationship between IRCPs and pyoverdines came from analyses of the cytoplasmic protein content of several pyoverdinedeficient mutants of P. aeruginosa PA06049, a methionine auxotroph of P. aeruginosu ATCC 15692 (PA01 strain). The mutants analysed belonged to the two mutation groups recognized previously [ 121, harbouring mutations at the 23-min region of the bacterial chromosome (35 min in a former map, strains PA06601 and PA06622), or at the 47-49 min region (65-70 min) for strains PA06606, PA06609 and PA06624. As shown in Fig. 2, PA06601 presented an IRCP profile which lacked the two 550and 480-kDa upper bands and showed only a faint band at 250 kDa when compared to the parent strain PA06049; the two other IRCPs (290 and 185 kDa) were apparently not affected. However, PA06622, which belongs to the same mutation group as PA06601, presented the same IRCP profile as the parent strain (data not shown), suggesting for this strain a mutational event not related to the synthesis of the peptidic part of the pyoverdine molecule. Mutants of the second group presented a similar heterogeneity since PA06606 gave an identical IRCP PA0 PA0 PA0 PA0 PA0 PA0 PA0 PA0 fxiol - 6601 + 6ah - 6606 6609 - 6609 + 6019 - 601’) + + Fig. 2. Iron-repressed cytoplasmic proteins (IRCPs) in the pyoverdine-deficient mutants PAO6601, PA06606, PA06609 their pyoverdine producer parental strain PAO6049. and in Electrophore- sis, bacterial growth conditions. symbols and markers are the same as in the legend of Fig. I, excepted that electrophoresis was stopped when the blue dye had reached the bottom of the gel, and that the growth media were supplemented with 1 mM methionine according to Hohnadel et al. [ 121. profile as PA06049 (Fig. 2), whereas PA06609 (Fig. 2) and PA06624 (not shown) were characterized by an almost complete absence of the 250-kDa IRCP, the other IRCPs being unchanged. 3.3. Correlations between peptidic siderophore-reluted IRCPs and peptide synthetuses In order to determine if the IRCPs are directly related to pyoverdine biosynthesis and if they are specific to peptidic siderophore-producing bacteria, we extended the search of IRCPs to a strain known to produce a non-peptidic siderophore, i.e. the desferriferrioxamine E-producer P. stutzeri ATCC 17588 [ 191, and to wild-type strains which apparently do not produce siderophores under iron starvation, such as P. diminuta CIP 7 129 or P. frugi ATCC 4973 [l]. As shown in Fig. 1, lanes 7-8, no IRCPs were detectable in iron-starved P. stutzeri ATCC 17588 cell extracts, even at the maximal 400 kg protein load. The non-siderophore-producing strains reacted the same: cells of P. diminutu and P. frugi, which were iron-deficient as judged by their lower C. Georges, J.-M. Meyer/ FEMS Microbiology Letters 132 (1995) 9-15 growth yield in succinate medium compared to growth in iron-supplemented medium, were devoid of IRCPs, just like P. stutzeri (not shown). A comparison of the IRCP profiles of pyoverdine-producing Pseudomonas strains as a function of the structure of the pyoverdine they produce disclosed siderophore-specific profiles of IRCPs. As shown in Fig. 1, strains which are known to produce pyoverdines, differing by their peptide chain, presented a different number of IRCPs: four major IRCP bands were detectable for P. aenrginosa ATCC 15692, whereas P. C~~OFOFC-@~S ATCC 9446 only showed two major IRCPs. Other examples are found in Table 1, where both the number of IRCPs 13 recognized for each strain and the peptidic structures of the respective pyoverdines are given. Conversely, two strains producing the same pyoverdine presented identical IRCP profiles. This is illustrated in Fig. 1 for P. chlororaphis ATCC 9446 and P. jluorescens ATCC 13525. The same conclusion held when comparing IRCP profiles of several P. aeruginosa isolates belonging to an identical pyoverdine group: strains Pa5, Pa& Pa9, Pal0 and Pall, which have been shown to elaborate identical pyoverdines and pyoverdine uptake systems as P. aeruginosa ATCC 15692 [3], presented exactly the same IRCP profile as their type-strain (data not shown), whereas strains Pa3, Pa4 and Pa7, belonging to the pyoverdine group Table 1 Pyoverdines and IRCPs in various fluorescent pseudomonas species Stitl Numberof Number Apparentmolecularmass a.a.resfdues OflRBs OffRCPs &Da+ . _ sequenceof thepyoveldinepepddea P. aer~@~sa Pa6 (Chr)-Srt-Dab_FooHom-Gln-GlprFoOHaCp-CIy c 6 4 520,xlo. 465.330 P. optatasmin 4a (Chr)-dir-L~Tbr_SIl-AcOHOm-cOHOHOm 6 2 430.400 P. uemginosa ATCC 27853 (au)Scc_FooHorn-OrGl~~-~r~OHOm 7 3 550,470.3od P. ch&nw&&ATCC 9446 (~~-L~-Cly-FoOfIOnr-L~~HOrnSerC 7 3 530.480.180 P. jluonwcenr ATCC! 13525 (Cln)-gCr-LJlcly-F-L~~HOm~J 7 3 SM. 480.180 P. wngines~ ATCC 15692 (au)Srr-Arlr-SrC-OHOm-~~OHOm-Thr-Thrl a 5 550.480,290.250,185f 9 4 520,510,4MJ380 P.j7uorescensATCC17400 (Chr)-Ala-Lys-GIy-GIy-O~pGln-D~er-~8-~HOm 8 P. jhwesrenr CCM 2798 (Chr)Scr-~~Gly~r~~A~-Gly-~-Gly~OHOm 9 4 530.sclo.330,240 P. tohsii NCPPB 2192 (~)_Src-Lurser-Src_Tar-~-~HOm-~r~~~ 10 5 550.510.460.400.360 P.j78lOrescenssuain 12 (chr>src-Lys-Cb_FoOHOm~~-Gl~-LfbF 11 4 480.440.380.310 P.jlmescens (Chr)-~-~~lyly-cly-O~~-~-A~-~-~-~~Orn 12 5 600,470.440,230,210 strainii a See [2,20] for sources. BAmino acids are underlined. Abbreviations:Chr, chtomophore; OHOm, 6N-hydroxy Om; cOHOm, cycle-OHOm; OHAsp, threo-Shydroxy Asp; aThr, alloThr; FdAc,Bu)OHOm, 6N-formyl (ace@, Phydroxy butyryl) OHOm, Dab, diaminobutyric acid. h Average values obtained from two to ten independent experiments, depending on the strain (see also footnote 0. ’ Structure determined for the pyoverdine of strain P. aeruginosa R [2]. The pyoverdine of strain Pa6 has an identical structure (R. Tappe, personal communication). d Values obtained for strains P. aeruginosa Pa3, Pa4 and Pa7 which produce the same pyoverdine as P. aeruginosa ATCC 27853 [3]. e H. Budzikiewicz and K. Taraz, personal communication. ‘Average values with standard deviations (10 independent experiments) were 550 f 37. 480 f 22, 290 f 17, 250 f 22 and 185 f 18, respectively. Strains P. aeruginosa Pa5, Pa8, Pa9, Pal0 and Pal 1, which produce the same pyoverdine as P. aeruginosa ATCC 15692 [3], displayed the same IRCP profile. g Stereochemistry partially determined or unknown. 14 C. George& J.-M. Meyer/ FEMS Microbiology P. aeruginusa ATCC 27853 [3]) presented all the same IRCP gel pattern, with bands at 550, 470 and 300 kDa. Another strain belonging to the same group (Pa12) and the type strain itself gave too faint and diffuse bands on SDS gels to accurately estimate their apparent molecular mass. They appeared, however, at positions corresponding to the cited mass values. Attempts to correlate the number of IRCPs detected in one strain with the peptide length of its pyoverdine roughly suggested that the longer peptide chain, the greater the number of IRCPs: strains with pyoverdines having 6 or 7 aminoacyl residues presented the lowest number of IRCPs (middle average of 3), whereas strains with pyoverdines exceeding 7 amino acids had 4 or 5 IRCPs. Finally, a last correlation between IRCPs, peptidic siderophores and peptide synthetases concerned the unusual high IV, observed for both kinds of proteins. Antibiotic-related peptide synthetases are characterized by a M, higher than 100 kDa, often reaching the 200-500-kDa range [5]. Examples close to our work concern the production of the antibiotics syringomycin and syringotoxin by the pyoverdine-producing P. syringae pv. syringae. Five proteins ranging from 130 to 470 kDa are involved in syringomycin production [8], whereas two proteins of M, 470 and 435 kDa were related to syringotoxin production [7]. Moreover, at least four other large proteins in the range of 200-400 kDa were described in that strain and tentatively related to pyoverdine synthesis since, unlike the antibiotic-related proteins, they were visible only in iron-deficient cells [7]. The IRCP IV, values, as estimated in the present work, appeared within the same order of magnitude, ranging from 180 to 600 kDa (Table 1). Interestingly, a molecular size of 1100 kDa has been determined by gel filtration for ferrichrome synthetase, the only multi-enzyme complex involved in a non-ribosomal synthesis so far characterized for a peptidic siderophore [9]. In conclusion, the presence of high A4, iron-repressed cytoplasmic proteins in all the pyoverdineproducing Pseudomonas strains so far analysed, together with their absence in strains devoid of pyoverdine production, as well as the multiple correlations between these proteins and pyoverdine structures and regulation, strongly suggest that IRCPs are peptide II (type strain Letters 132 (1995) 9-15 synthetases which are specifically involved in the biosynthesis of the peptidic chain of pyoverdines. It should be emphasized that during the completion of this work the gene pvdD involved in the biosynthesis of pyoverdine in P. aeruginosa OTll (a strain related to P. aeruginosa ATCC 15692; I.L. Lamont, personal communication) has been fully sequenced [ 111. Although the gene product and its enzymatic function remain unknown, the multiple similarities of this gene with peptide synthetase genes strongly suggests that pvdD indeed codes for a peptide synthetase. The length of the gene (7374 bp) allows a deduced M, of 273 kDa for the PvdD protein, a size which is within the range of the IRCP h4, values and close to the estimated 290 5 17 and 250 + 22 kDa IRCPs (Table I) found in P. aeruginosa ATCC 15692. Acknowledgements We are indebted to H. Kleinkauf and H. von Dohren for valuable discussions and encouragements and to I.L. Lamont for providing us with the pvdD sequence before publication. C.F. Earhart is particularly acknowledged for advice and manuscript improvement, and H. 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