APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1991, p. 2246-2250 0099-2240191/082246-05$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 57, No. 8
Production of the Siderophore Aerobactin by a Halophilic Pseudomonad JEFFREY S. BUYER,'* VICTOR DE LORENZO,'t AND J. B. NEILANDS2 Soil Microbial Systems Laboratory, Building 318 BARC-East, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705,1 and Department of Biochemistry, University of California, Berkeley, California 947202 Received 22 January 1991/Accepted 14 May 1991
A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-linmiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pCoIV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.
Iron is an essential element for all living organisms, with the possible exception of certain lactobacilli. However, the solubility product of ferric hydroxide, 10-38, limits the concentration of dissolved iron to less than 10-17 M under aerobic conditions at biological pH. In order to solubilize and transport iron, bacteria and fungi synthesize, in response to low-iron stress, extracellular iron chelators called siderophores (29). Enteric bacteria commonly produce the tricatechol siderophore enterobactin. In addition, the dihydroxamate siderophore aerobactin (Fig. 1) is produced by certain strains of Shigella (21, 31), Escherichia coli (16, 41), Enterobacter (40), Salmonella (25), and Yersinia (39), all of which are members of the family Enterobacteriaceae. The siderophore aerobactin has received considerable attention in the past few years since the finding that its production could constitute a major virulence factor (9, 12). The aerobactin system encoded by the enteric plasmid pColV-K30 has been extensively studied in our laboratory (11, 13) and by other independent groups (17, 18, 33), and many details are known about its genetics and regulation (1, 5, 14). To our knowledge, aerobactin production has never been reported for species outside the family Enterobacteriaceae. During the course of a study on siderophore production and utilization by cyanobacteria, we observed the production of an iron-chelating agent by a Pseudomonas sp. isolated from a culture of the cyanobacterium Spirulina platensis. In this report, we demonstrate that the siderophore produced is aerobactin, although the genetic determinants for its production differ substantially from those in E. coli harboring the pColV-K30 plasmid.
*
Corresponding author.
t Present address: Consejo Superior de Investigaciones Cientificas, Centro de Investigaciones Biol6gicas, Velazquez 144, 28006 Madrid, Spain. 2246
MATERIALS AND METHODS and media. The cyanobacterium S. platensis Organisms 2340 was obtained from the UTEX culture collection (Department of Botany, The University of Texas at Austin, Austin, Tex.) and maintained in Spirulina medium (SM) (35). Bacterial strain X40 was isolated from the S. platensis culture by streaking the culture on Spirulina medium-yeast extract plates (SMYE; SM enriched with 5 g of Difco yeast extract per liter and 10 g of glucose per liter) and incubating them at 30°C. A pure culture was obtained by repetitive streaking of single colonies on SMYE plates. E. coli HB101 (23), HB101 ent fep (11), and BN3040 Nalr (4) were described previously. Plasmid ColV-K30 is the original source of the aerobactin system (4). Plasmid pVLN1 (13) contains the complete determinants for aerobactin production and transport from pColV-K30 cloned in a multicopy vector. pABN6 (13) contains the iutA gene from pColV-K30, which encodes the ferric aerobactin outer membrane receptor
protein. Identification of bacteria. Strain X40 was identified by microscopic examination, Gram stain, oxidase test (37), glucose fermentation, growth on single carbon sources, and fatty acid analysis. To test for salt tolerance, basal medium (2) supplemented with 1 g of Casamino Acids (Difco) per liter was prepared without NaCl, and various amounts of NaCl were added. Unless otherwise indicated, strain X40 was grown at 30°C throughout this study. Siderophore assay. Single colonies were inoculated into 2 ml of SMYE and shaken overnight. A total of 20 [uL was inoculated into 10 ml of Pseudomonas medium (PM) and into 10 ml of PM containing 2 ILM FeSO4. PM was derived from SM by eliminating the ferric chloride, EDTA, and vitamins and supplementing it with 10 g of glycerol per liter and 1 g of Casamino Acids (Difco) per liter. Stock solutions of salt and Casamino Acids were deferrated as described previously (3). The cultures were shaken at room temperature and assayed every 24 h for 3 days with Chrome Azurol S (CAS), as described previously (36), by using 0.5 ml of culture supernatant and 0.5 ml of CAS solution without shuttle. Production and isolation of aerobactin. A single colony of
AEROBACTIN PRODUCTION IN A PSEUDOMONAS SP.
VOL. 57, 1991
H
oH OHN
N
HNx
COWr
FIG. 1. Structure of aerobactin.
strain X40 was inoculated into 2 ml of SMYE and shaken overnight. A total of 20 was inoculated into 10 ml of PM, which was shaken overnight at room temperature and then inoculated into 1 liter of PM. After 2 days of shaking at room temperature, the cells were spun down, the pH was adjusted to 6 with HCl, and the supernatant was passed through a column (160 by 25 mm [inner diameter]) containing the strong anion-exchange resin Dowex AG-1X2 equilibrated with 0.4 M NH4Cl. The column was washed with 0.4 M NH4CI, and then a gradient (total volume, 400 ml) was run to 1.0 M NH4Cl. Fractions were assayed with CAS. The CAS-reactive band was acidified to pH 2, saturated with ammonium sulfate, and extracted with benzyl alcohol. The organic layer was filtered through grade 2S hydrophobic filter paper (Micro Filtration Systems, Dublin, Calif.), diluted four times (vol/vol) with diethyl ether, and extracted three times with H20. The aqueous extracts were combined and washed twice with diethyl ether, rotary evaporated, and lyophilized. The product was dissolved in 0.2 M acetic acid-pyridine buffer (pH 4.8) and loaded onto a Bio-Gel P-2 column (450 by 15 mm [inner diameter]) equilibrated in the same buffer. Fractions were eluted in the same buffer and assayed with CAS, and the reactive band was rotary evaporated and lyophilized. The trisodium salt was prepared by passing an aqueous solution of the siderophore through a short column of the weak cation-exchange gel Sephadex CM-C25, sodium form, in H20. Ferric aerobactin. Ferric hydroxide was prepared by dissolving ferric chloride in 0.1 M HCl and neutralizing the solution with 0.1 M NaOH. The resulting precipitate was collected by centrifugation, resuspended in distilled water, and centrifuged again. The ferric hydroxide was suspended and centrifuged twice more to remove any remaining salts. Ferric aerobactin was formed by overnight reaction with freshly prepared ferric hydroxide followed by chromatography on Bio-Gel P-2 (410 by 16 mm [inner diameter]) in 0.2 M pyridine-acetic acid buffer (pH 4.8). The brown band from the column was lyophilized, dissolved in H20, and run through a short column of Sephadex CM-C25, sodium form, in H,O. Bioassays. Nutrient broth (Difco) plates containing 200 p.M dipyridyl were overlaid with soft agar inoculated with E. coli HB101(pABN6) ent fep. Plates were spotted with 10 of 100 p.M authentic aerobactin (30) and pseudomonad aerobactin and stabbed with E. coli HB101 and HB101(pVLN1) entJep. SM plates enriched with 1% tryptone-1% glycerol-3 mM purified (34) ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDA) were inoculated with strain X40. Paper disks containing 10 pl. of FeCI3, aerobactin, and ferric aerobactin,
2247
each at 0.1, 1, and 10 mM, were placed on the plates, and disks containing 10 ,ul of H20 were used as controls. All plates were incubated at 37°C overnight. Paper electrophoresis. Culture supernatants, aerobactin and ferric aerobactin isolated from Pseudomonas strain X40, and authentic aerobactin and ferric aerobactin were analyzed by paper electrophoresis on Whatman 3MM paper. Samples were run in pyridine-acetic acid buffer (pH 5.2) at 40 V/cm. Aerobactin was detected by spraying the dried paper with CAS assay solution or with FeCl3 in ethanol, while ferric aerobactin was detected by the brown color of the ferric complex. Acid hydrolysis. One micromole each of pseudomonad aerobactin and authentic aerobactin was hydrolyzed in 0.5 ml of hydriodic acid (47%) overnight at 110°C in vacuo. The digests were dried in vacuo and dissolved in H20. Each hydrolysate was subjected to preparative paper electrophoresis at 20 V/cm on Whatman 3MM paper by using a pyridine-acetic acid buffer at pH 5.6. A single cationic, ninhydrin-reactive band with mobility identical to that of lysine was observed for each hydrolysate. The bands were eluted with water and dried by rotary evaporation. An aliquot of each band was reacted with L-lysine decarboxylase (type VIII; Sigma) at 37°C for 1 h. Controls included L-lysine and DL-lysine. Each reaction mixture was analyzed by paper electrophoresis as described above. Spectroscopy. The visible absorption spectrum of ferric aerobactin from strain X40 at various pH values was recorded on a Beckman 25 spectrophotometer. The 1H nuclear magnetic resonance spectrum of the free ligand was taken on a 200-MHz Nicolet spectrometer in the Fourier transform mode. The fast-atom-bombardment mass spectrum was measured in the anionic detection mode. Outer membrane proteins. Pseudomonas sp. strain X40 was grown overnight in 10 ml of PM and 10 ml of PM containing 2 ,uM Fe. E. coli BN3040(pColV-K30) gyrA was grown overnight at 37°C in LB and LB containing 200 p.M dipyridyl. Protein preparations enriched in outer membrane proteins were obtained as Triton X-100-insoluble cellular fractions, as described elsewhere (32). DNA analyses. Plasmid preparations from E. coli BN3040(pColV-K30) gyrA and strain X40 were obtained by an alkaline-sodium dodecyl sulfate procedure (23). The resulting DNA was digested with restriction endonucleases BglII, HpaI, and SmaI under standard conditions (23). Digested and undigested DNAs were analyzed on 1% agarose-TAE gels (23) and stained with ethidium bromide. The DNA was transferred to nitrocellulose paper (23), and the resulting blot was probed with 32P-nick-translated (23) plasmid pVLN1 by standard hybridization techniques. Final washings of the blot were made in conditions of moderate to high stringency. The positions of DNA fragments with homology to the probe were revealed by autoradiography of the dried blot.
RESULTS A bacterium designated strain X40, isolated from a cyanobacterial culture, was identified as a Pseudomonas species by the following characteristics. The bacterium consisted of gram-negative motile rods. The organism tested positive for catalase, oxidase, and nitrate reduction but tested negative for glucose fermentation. The organism was negative for DNase activity, starch hydrolysis, gelatin hydrolysis, and growth on MacConkey agar, lysine, arginine, ornithine, citrate, urea, and indole. A fatty acid profile, determined by
2248
APPL. ENVIRON. MICROBIOL.
BUYER ET AL.
TABLE 1. Proton nuclear magnetic resonance chemical shift data for aerobactin in deuterium oxide from Aerobacter aerogenes and the Pseudomonas sp. 8 (multiplicityb)
Assignment
Lys a Lys E Citrate CH2 Acetyl CH3 Lys l, 8 Lys y
A. aerogenes
Pseudomonas sp.
4.37 (t) 3.62 (t) 2.85 (2 d) 2.12 (s) 1.0-2.0 (m) 1.0-2.0 (m)
4.46 (m) 3.78 (t) 2.99 (d of q) 2.32 (s) 1.75-2.1 (m) 1.56 (m)
a Chemical shift data for A. aerogenes were presented previously (14), whereas those for the Pseudomonas sp. are from this study. b s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
Myron Sasser of Microbial ID, Inc. (Newark, Del.), while not identical to any in the data base of Microbial ID, was most similar to that of Pseudomonas vesicularis. The strain was moderately halophilic, with maximum growth occurring at NaCl concentrations greater than 0.5 M and with no growth occurring below 0.2 M. No growth occurred below pH 8, maximum growth was at pH 9 to 10, and slower growth was observed at pH 12. The organism grew at 20 to 45°C, but incubation at 50°C was bactericidal. Supernatants of Pseudomonas strain X40 low-iron (PM) cultures were CAS reactive when compared with those of high-iron (PM plus 2 ,uM Fe) cultures. Paper electrophoresis of supernatants revealed a single spot. The purified material had paper electrophoretic mobility identical to that of authentic aerobactin, while the ferric siderophore had electrophoretic mobility identical to that of authentic ferric aerobactin. The variation in the visible absorbance spectrum with pH (data not shown) was virtually identical to that published for ferric aerobactin (19). Proton nuclear magnetic resonance spectra of the siderophore (Table 1) were virtually identical to published spectra (15). The mass spectrum confirmed the identity of the siderophore to be aerobactin. Peaks at 585, 607, and 629 atomic mass units corresponded to (NaHAerobactin)-7 (Na2Aerobactin)-, and (Na3[Aerobactin-H])-, respectively, where Na3Aerobactin is the neutral trisodium salt. Purified pseudomonad aerobactin and ferric pseudomonad aerobactin stimulated the growth of strain X40 on EDDA iron-limited plates, as is expected for a siderophore (28, 34). Pseudomonas aerobactin, authentic aerobactin, and E. coli HB101 (pVLN1) entfep, a strain which produces aerobactin but not enterobactin, reversed dipyridyl-induced iron starvation for E. coli HB1O1(pABN6) ent fep, a strain which cannot produce enterobactin, aerobactin, or the outer membrane receptor for enterobactin, but which can utilize aerobactin for iron transport. E. coli HB101, which produces enterobactin but not aerobactin, did not promote growth in this assay. Therefore, pseudomonad aerobactin and authentic aerobactin had similar biological activities for E. coli. In order to identify the stereochemistry of the lysine residue, both aerobactin from the Pseudomonas sp. and authentic aerobactin were hydrolyzed with hydriodic acid, producing lysine. After reaction with L-lysine decarboxylase, each aerobactin gave a single spot on paper electrophoresis with the same mobility as that of 1,5-pentane diamine. L-Lysine gave identical results, while DL-lysine, when it was reacted with L-lysine decarboxylase, produced two spots corresponding to lysine and 1,5-pentane diamine. Therefore,
FIG. 2. Outer membrane proteins of E. coli and the Pseudomo6, molecular weight markers (in thousands); lanes 2 and 3, E. coli grown under conditions of high and low iron availability, respectively; lanes 4 and 5, the Pseudomonas sp. grown under conditions of high and low iron availability, respectively.
nas sp. Lanes 1 and
aerobactin isolated from both E. coli and Pseudomonas strain X40 contains only L-lysine. Polyacrylamide gel electrophoresis of outer membrane proteins is shown in Fig. 2. Under iron-starvation conditions, at least two outer membrane proteins were induced. The E. coli aerobactin receptor encoded by pColV-K30 is a 74-kDa protein (4) and is one of the induced proteins in lane 3 of Fig. 2. The Pseudomonas aerobactin receptor protein, presumed to be one of the induced proteins in lane 5 of Fig. 2, is similar in size but not identical to the E. coli aerobactin receptor protein. DNA preparations from Pseudomonas strain X40 were obtained by a sodium dodecyl sulfate-alkaline procedure in which the presence of a large plasmid (pVl) could be detected (Fig. 3) migrating more slowly than the bulk of chromosomal DNA. This plasmid, however, was substantially smaller than the control plasmid pColV-K30 (Fig. 3). To determine whether aerobactin determinants in Pseudomonas strain X40 were plasmid-borne and whether they were similar to those in pColV-K30, a DNA hybridization assay was carried out. Figure 4 shows the autoradiograph of a Southern blot obtained from DNA preparations from Pseudomonas strain X40 and E. coli BN3040(pColV-K30) gyrA either undigested or digested with some restriction endonucleases. Under conditions of moderate to high stringency, no hybridization could be detected between the probe pVLN1 (carrying the aerobactin determinants from pColVK30) and Pseudomonas strain X40 DNA.
212
pYl
pCoIV4(3W
FIG. 3. DNA preparation. Lane 1, Pseudomonas sp.; lane 2, E.
coli(pColV-K30).
AEROBACTIN PRODUCTION IN A PSEUDOMONAS SP.
VOL. 57, 1991
FIG. 4. Southern blot hybridization with 32P-nick-translated pVLN1. Lane 1, molecular weight markers; lane 2, Pseudomonas DNA preparation; lane 3, E. coli(pColV-K30) DNA preparation; lanes 4, 5, and 6, pColV-K30 DNA preparation cut with BglII, HpaI, and SmaI, respectively; lanes 7, 8, and 9, Pseudomonas DNA preparation cut with BglII, Hpal, and SmaI, respectively; lanes 10, 11, and 12, pVLN1 cut with BglII, HpaI, and SmaI, respectively.
DISCUSSION Pseudomonas strain X40 appears to be well adapted to growth in Spirulina culture. Like the cyanobacterium (7), Pseudomonas strain X40 grows vigorously under highly alkaline conditions at high Na concentrations. This suggests that Pseudomonas strain X40 may be a natural inhabitant of Spirulina ecosystems rather than a culture contaminant. When stressed for iron, Pseudomonas strain X40 produced a single siderophore which was identical to the aerobactin isolated from E. coli, as demonstrated by both biological and chemical tests. It seems somewhat surprising to find that aerobactin, previously found only among the enteric bacteria (24), is also utilized by a Pseudomonas sp. under conditions extremely different from those of the normal enteric environment.
A number of siderophores from the family Pseudomonadaceae have been identified. Pseudomonas fluorescens and Pseudomonas putida produce the fluorescent siderophores termed pseudobactins and pyoverdins (22). Pyochelin has been found in Pseudomonas aeruginosa and Pseudomonas cepacia (8, 38), while desferrioxamine E is produced by Pseudomonas stutzeri (26). Cepabactin was recently identified as the siderophore of P. cepacia and Pseudomonas alcaligenes (27). The pseudomonads are clearly very diverse in siderophore production. The system leading to aerobactin production in Pseudomonas strain X40 appears to be different in at least some respects from that in E. coli. Two outer membrane proteins induced by low iron were produced by Pseudomonas strain X40. One of these proteins is reasonably assumed to be the aerobactin receptor. Neither protein had electrophoretic mobility identical to that of the E. coli aerobactin outer membrane receptor protein of pColV-K30. No homology was observed between Pseudomonas strain X40 DNA and the aerobactin system of pColV-K30 cloned in pVLN1. Iron regulation in Pseudomonas strain X40 may also be different from that in E. coli, since no homology to the gene sequence of the Fur protein of E. coli could be detected by hybridization in Pseudomonas strain X40 DNA (1Sa). Preliminary data indicate that the N-OH-lysine acetylase of Pseudomonas strain X40 is different from the analogous E. coli protein (8a).
2249
Similar results have been reported previously. Certain clinical isolates of E. coli and Enterobacter cloacae which produce aerobactin show no homology to the aerobactin genome from the ColV plasmid (6, 10). Variations in the sizes of aerobactin receptors from different sources have been observed previously (20), even when large similarities in the other genes of the operon are present. The genes encoding the aerobactin biosynthetic enzymes of an aerobactin-producing strain of Aerobacter aerogenes show no homology to the analogous pColV genes of E. coli, while the outer membrane receptors were immunologically cross-reactive and of the same size, and the genes encoding them were homologous (42). While the lack of homology between Pseudomonas strain X40 DNA and E. coli pVLN1 could be due to variations in the third base of the codons, this would not explain the differences seen between the outer membrane receptor proteins and the acetylase enzymes. In this respect, Pseudomonas strain X40 may prove to be useful in providing comparative biochemical and genetic information on the aerobactin system. ACKNOWLEDGMENTS We thank Kenneth Nealson, University of Wisconsin, and Myron Sasser, Microbial ID, Inc., for assistance in identifying the organism. This work was supported by National Research Service Award ES05361-02 to J.S.B. V.D.L. was a postdoctoral fellow (1F05TWO3577-01-B1-5) of the John Fogarty NIH Center. Additional support was derived from Public Health Service grant A104156 and from grants PCM 78-12198 and CRCR-1-1633 from the National Science Foundation and the U.S. Department of Agriculture, respectively. REFERENCES 1. Bagg, A., and J. B. Neilands. 1987. Ferric uptake regulation protein acts as a repressor, employing Fe(II) as cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry 25:5471-5477. 2. Baumann, P., and R. H. W. Schubert. 1984. Family II. Vibrionaceae Veron 1965, 5245AL, p. 51-538. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 3. Bell, S. J., S. A. Friedman, and J. Leong. 1979. Antibiotic action 4.
5.
6.
7.
8.
of N-methylthioformohydroxamate metal complexes. Antimicrob. Agents Chemother. 15:384-391. Bindereif, A., and J. B. Neilands. 1983. Cloning of the aerobactin-mediated iron assimilation system of plasmid ColV. J. Bacteriol. 153:1111-1113. Bindereif, A., and J. B. Neilands. 1985. Promoter mapping and transcriptional regulation of the iron assimilation system of plasmid ColV-K30 in Escherichia coli K-12. J. Bacteriol. 162: 1039-1046. Carbonetti, N. H., S. Boonchai, S. H. Parry, V. Vaisanen-Rhen, T. K. Korhonen, and P. H. Williams. 1986. Aerobactin-mediated iron uptake by Escherichia coli isolates from human extraintestinal infections. Infect. Immun. 51:966-968. Ciferri, O., and 0. Tiboni. 1985. The biochemistry and industrial potential of Spirulina. Annu. Rev. Microbiol. 39:503-526. Cox, C. D., and R. Graham. 1979. Isolation of an iron-binding compound from Pseudomonas aeruginosa. J. Bacteriol. 137:
357-364. 8a.Coy, M. Personal communication. 9. Crosa, J. H. 1984. The relationship of plasmid-mediated iron transport and bacterial virulence. Annu. Rev. Microbiol. 38:6989. 10. Crosa, L. M., M. K. Wolf, L. A. Actis, J. Sanders-Loehr, and J. H. Crosa. 1988. New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae. J. Bacteriol. 170:5539-5544.
2250
BUYER ET AL.
11. DeLorenzo, V., A. Bindereif, B. H. Paw, and J. B. Neilands. 1986. Aerobactin biosynthesis and transport genes of plasmid CoIV-K30 in Escherichia coli K-12. J. Bacteriol. 165:570-578. 12. DeLorenzo, V., and J. L. Martinez. 1988. Aerobactin production as a virulence factor: a reevaluation. Eur. J. Clin. Microbiol. Infect. Dis. 7:621-629. 13. DeLorenzo, V., and J. B. Neilands. 1986. Characterization of genes iucA and iucC of the aerobactin system of pColV-K30 in E. coli K-12. J. Bacteriol. 167:350-355. 14. DeLorenzo, V., S. Wee, M. Herrero, and J. B. Neilands. 1987. Operator sequences of the aerobactin operon of plasmid ColVK30 binding the ferric uptake regulation (fur) repressor. J. Bacteriol. 169:2624-2630. 15. Gibson, G., and D. I. Magrath. 1969. The isolation and characterization of a hydroxamic acid (aerobactin) formed by Aerobacter aerogenes 62-I. Biochim. Biophys. Acta 192:175-184. 15a.Giovannini, F. Personal communication. 16. Griffiths, E., P. Stevenson, T. L. Hale, and S. B. Formal. 1985. Synthesis of aerobactin and a 76,000-dalton iron-regulated outer membrane protein by Escherichia coli K-12-Shigella flexneri hybrids and by enteroinvasive strains of Escherichia coli. Infect. Immun. 49:67-71. 17. Gross, R., F. Engelbrecht, and V. Braun. 1984. Genetic and biochemical characterization of the aerobactin synthesis operon on pColV. Mol. Gen. Genet. 196:74-80. 18. Gross, R., F. Engelbrecht, and V. Braun. 1985. Identification of the genes and their polypeptide products responsible for aerobactin synthesis by pColV plasmids. Mol. Gen. Genet. 201:204212. 19. Harris, W. R., C. J. Carrano, and K. N. Raymond. 1979. Coordination chemistry of microbial iron transport compounds. 16. Isolation, characterization, and formation constants of ferric aerobactin. J. Am. Chem. Soc. 101:2722-2727. 20. Krone, W. J., F. Stegehuis, G. Koningstein, C. vanDoorn, B. Roosendaal, F. K. deGraaf, and B. Oudega. 1985. Characterization of the pColV-K30 encoded cloacin DF13 aerobactin outer membrane receptor protein of Escherichia coli; isolation and purification of the protein and analysis of its nucleotide sequence and primary structure. FEMS Microbiol. Lett. 26:153161. 21. Lawlor, K. M., and S. M. Payne. 1984. Aerobactin genes in Shigella spp. J. Bacteriol. 160:266-272. 22. Leong, J. 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogens. Annu. Rev. Phytopathol. 24:187-209. 23. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Martinez, J. L., J. L. Cercenado, F. Baquero, J. C. Perez Diaz, and A. Delgado-Iribarren. 1987. Incidence of aerobactin production in gram-negative hospital isolates. FEMS Microbiol. Lett. 43:351-353. 25. McDougall, S., and J. B. Neilands. 1984. Plasmid- and chromosome-coded aerobactin synthesis in enteric bacteria: insertion
APPL. ENVIRON. MICROBIOL.
26. 27. 28. 29. 30.
31. 32.
33.
34. 35.
36. 37.
38. 39.
40.
41. 42.
sequences flank operon in plasmid-mediated systems. J. Bacteriol. 159:300-305. Meyer, J. M., and M. A. Abdallah. 1980. The siderochromes of non-fluorescent pseudomonads: production of nocardamine by Pseudomonas stutzeri. J. Gen. Microbiol. 118:125-129. Meyer, J. M., D. Hohnadel, and F. Halle. 1989. Cepabactin from Pseudomonas cepacia, a new type of siderophore. J. Gen. Microbiol. 135:1479-1487. Miles, A. A., and P. L. KhimJi. 1975. Enterobacterial chelators of iron: their occurrence, detection, and relation to pathogenicity. J. Med. Microbiol. 8:477-490. Neilands, J. B. 1981. Microbial iron compounds. Annu. Rev. Biochem. 50:715-731. Neilands, J. B. 1984. Methodology of siderophores. Struct. Bonding 58:1-24. Payne, S. M., D. W. Niesel, S. S. Peixotto, and K. M. Lawlor. 1983. Expression of hydroxamate and phenolate siderophores by Shigella flexneri. J. Bacteriol. 155:949-955. Pugsley, A. P., F. Moreno, and V. deLorenzo. 1986. MicrocinE492-insensitive mutants of E. coli K12. J. Gen. Microbiol. 132:3253-3259. Roberts, M., R. W. Leavitt, N. H. Carbonetti, S. Ford, R. A. Cooper, and P. H. Williams. 1986. RNA-DNA hybridization analysis of transcription of the plasmid ColV-K30 aerobactin gene cluster. J. Bacteriol. 167:467-472. Rogers, H. J. 1973. Iron-binding catechols and virulence in Escherichia coli. Infect. Immun. 7:445-456. Schlosser, U. G. 1982. Sammlung von Algenkulturen. Ber. Dtsch. Bot. Ges. 95:181-276. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47-56. Smibert, R. M., and N. R. Krieg. 1981. General characterization, method 1, p. 420. In P. Gerhardt (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. Sokol, P. A. 1986. Production and utilization of pyochelin by clinical isolates of Pseudomonas cepacia. J. Clin. Microbiol. 23:560-562. Stuart, S. J., J. K. Prpic, and R. M. Robins-Browne. 1986. Production of aerobactin by some species of the genus Yersinia. J. Bacteriol. 166:1131-1133. Van Tiel-Menkveld, G. J., J. M. Mentjox-Vervuurt, B. Oudega, and F. K. deGraaf. 1982. Siderophore production by Enterobacter cloacae and a common receptor protein for the uptake of aerobactin and cloacin DF13. J. Bacteriol. 150:490-497. Warner, P. J., P. H. Williams, A. Bindereif, and J. B. Neilands. 1981. ColV plasmid-specified aerobactin synthesis by invasive strains of Escherichia coli. Infect. Immun. 33:540-545. Waters, V. L., and J. H. Crosa. 1988. Divergence of the aerobactin iron uptake systems encoded by plasmids pColVK30 in Escherichia coli K-12 and pSMN1 in Aerobacter aerogenes 62-1. J. Bacteriol. 170:5153-5160.
Vol. 57, No. 8
Production of the Siderophore Aerobactin by a Halophilic Pseudomonad JEFFREY S. BUYER,'* VICTOR DE LORENZO,'t AND J. B. NEILANDS2 Soil Microbial Systems Laboratory, Building 318 BARC-East, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705,1 and Department of Biochemistry, University of California, Berkeley, California 947202 Received 22 January 1991/Accepted 14 May 1991
A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-linmiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pCoIV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.
Iron is an essential element for all living organisms, with the possible exception of certain lactobacilli. However, the solubility product of ferric hydroxide, 10-38, limits the concentration of dissolved iron to less than 10-17 M under aerobic conditions at biological pH. In order to solubilize and transport iron, bacteria and fungi synthesize, in response to low-iron stress, extracellular iron chelators called siderophores (29). Enteric bacteria commonly produce the tricatechol siderophore enterobactin. In addition, the dihydroxamate siderophore aerobactin (Fig. 1) is produced by certain strains of Shigella (21, 31), Escherichia coli (16, 41), Enterobacter (40), Salmonella (25), and Yersinia (39), all of which are members of the family Enterobacteriaceae. The siderophore aerobactin has received considerable attention in the past few years since the finding that its production could constitute a major virulence factor (9, 12). The aerobactin system encoded by the enteric plasmid pColV-K30 has been extensively studied in our laboratory (11, 13) and by other independent groups (17, 18, 33), and many details are known about its genetics and regulation (1, 5, 14). To our knowledge, aerobactin production has never been reported for species outside the family Enterobacteriaceae. During the course of a study on siderophore production and utilization by cyanobacteria, we observed the production of an iron-chelating agent by a Pseudomonas sp. isolated from a culture of the cyanobacterium Spirulina platensis. In this report, we demonstrate that the siderophore produced is aerobactin, although the genetic determinants for its production differ substantially from those in E. coli harboring the pColV-K30 plasmid.
*
Corresponding author.
t Present address: Consejo Superior de Investigaciones Cientificas, Centro de Investigaciones Biol6gicas, Velazquez 144, 28006 Madrid, Spain. 2246
MATERIALS AND METHODS and media. The cyanobacterium S. platensis Organisms 2340 was obtained from the UTEX culture collection (Department of Botany, The University of Texas at Austin, Austin, Tex.) and maintained in Spirulina medium (SM) (35). Bacterial strain X40 was isolated from the S. platensis culture by streaking the culture on Spirulina medium-yeast extract plates (SMYE; SM enriched with 5 g of Difco yeast extract per liter and 10 g of glucose per liter) and incubating them at 30°C. A pure culture was obtained by repetitive streaking of single colonies on SMYE plates. E. coli HB101 (23), HB101 ent fep (11), and BN3040 Nalr (4) were described previously. Plasmid ColV-K30 is the original source of the aerobactin system (4). Plasmid pVLN1 (13) contains the complete determinants for aerobactin production and transport from pColV-K30 cloned in a multicopy vector. pABN6 (13) contains the iutA gene from pColV-K30, which encodes the ferric aerobactin outer membrane receptor
protein. Identification of bacteria. Strain X40 was identified by microscopic examination, Gram stain, oxidase test (37), glucose fermentation, growth on single carbon sources, and fatty acid analysis. To test for salt tolerance, basal medium (2) supplemented with 1 g of Casamino Acids (Difco) per liter was prepared without NaCl, and various amounts of NaCl were added. Unless otherwise indicated, strain X40 was grown at 30°C throughout this study. Siderophore assay. Single colonies were inoculated into 2 ml of SMYE and shaken overnight. A total of 20 [uL was inoculated into 10 ml of Pseudomonas medium (PM) and into 10 ml of PM containing 2 ILM FeSO4. PM was derived from SM by eliminating the ferric chloride, EDTA, and vitamins and supplementing it with 10 g of glycerol per liter and 1 g of Casamino Acids (Difco) per liter. Stock solutions of salt and Casamino Acids were deferrated as described previously (3). The cultures were shaken at room temperature and assayed every 24 h for 3 days with Chrome Azurol S (CAS), as described previously (36), by using 0.5 ml of culture supernatant and 0.5 ml of CAS solution without shuttle. Production and isolation of aerobactin. A single colony of
AEROBACTIN PRODUCTION IN A PSEUDOMONAS SP.
VOL. 57, 1991
H
oH OHN
N
HNx
COWr
FIG. 1. Structure of aerobactin.
strain X40 was inoculated into 2 ml of SMYE and shaken overnight. A total of 20 was inoculated into 10 ml of PM, which was shaken overnight at room temperature and then inoculated into 1 liter of PM. After 2 days of shaking at room temperature, the cells were spun down, the pH was adjusted to 6 with HCl, and the supernatant was passed through a column (160 by 25 mm [inner diameter]) containing the strong anion-exchange resin Dowex AG-1X2 equilibrated with 0.4 M NH4Cl. The column was washed with 0.4 M NH4CI, and then a gradient (total volume, 400 ml) was run to 1.0 M NH4Cl. Fractions were assayed with CAS. The CAS-reactive band was acidified to pH 2, saturated with ammonium sulfate, and extracted with benzyl alcohol. The organic layer was filtered through grade 2S hydrophobic filter paper (Micro Filtration Systems, Dublin, Calif.), diluted four times (vol/vol) with diethyl ether, and extracted three times with H20. The aqueous extracts were combined and washed twice with diethyl ether, rotary evaporated, and lyophilized. The product was dissolved in 0.2 M acetic acid-pyridine buffer (pH 4.8) and loaded onto a Bio-Gel P-2 column (450 by 15 mm [inner diameter]) equilibrated in the same buffer. Fractions were eluted in the same buffer and assayed with CAS, and the reactive band was rotary evaporated and lyophilized. The trisodium salt was prepared by passing an aqueous solution of the siderophore through a short column of the weak cation-exchange gel Sephadex CM-C25, sodium form, in H20. Ferric aerobactin. Ferric hydroxide was prepared by dissolving ferric chloride in 0.1 M HCl and neutralizing the solution with 0.1 M NaOH. The resulting precipitate was collected by centrifugation, resuspended in distilled water, and centrifuged again. The ferric hydroxide was suspended and centrifuged twice more to remove any remaining salts. Ferric aerobactin was formed by overnight reaction with freshly prepared ferric hydroxide followed by chromatography on Bio-Gel P-2 (410 by 16 mm [inner diameter]) in 0.2 M pyridine-acetic acid buffer (pH 4.8). The brown band from the column was lyophilized, dissolved in H20, and run through a short column of Sephadex CM-C25, sodium form, in H,O. Bioassays. Nutrient broth (Difco) plates containing 200 p.M dipyridyl were overlaid with soft agar inoculated with E. coli HB101(pABN6) ent fep. Plates were spotted with 10 of 100 p.M authentic aerobactin (30) and pseudomonad aerobactin and stabbed with E. coli HB101 and HB101(pVLN1) entJep. SM plates enriched with 1% tryptone-1% glycerol-3 mM purified (34) ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDA) were inoculated with strain X40. Paper disks containing 10 pl. of FeCI3, aerobactin, and ferric aerobactin,
2247
each at 0.1, 1, and 10 mM, were placed on the plates, and disks containing 10 ,ul of H20 were used as controls. All plates were incubated at 37°C overnight. Paper electrophoresis. Culture supernatants, aerobactin and ferric aerobactin isolated from Pseudomonas strain X40, and authentic aerobactin and ferric aerobactin were analyzed by paper electrophoresis on Whatman 3MM paper. Samples were run in pyridine-acetic acid buffer (pH 5.2) at 40 V/cm. Aerobactin was detected by spraying the dried paper with CAS assay solution or with FeCl3 in ethanol, while ferric aerobactin was detected by the brown color of the ferric complex. Acid hydrolysis. One micromole each of pseudomonad aerobactin and authentic aerobactin was hydrolyzed in 0.5 ml of hydriodic acid (47%) overnight at 110°C in vacuo. The digests were dried in vacuo and dissolved in H20. Each hydrolysate was subjected to preparative paper electrophoresis at 20 V/cm on Whatman 3MM paper by using a pyridine-acetic acid buffer at pH 5.6. A single cationic, ninhydrin-reactive band with mobility identical to that of lysine was observed for each hydrolysate. The bands were eluted with water and dried by rotary evaporation. An aliquot of each band was reacted with L-lysine decarboxylase (type VIII; Sigma) at 37°C for 1 h. Controls included L-lysine and DL-lysine. Each reaction mixture was analyzed by paper electrophoresis as described above. Spectroscopy. The visible absorption spectrum of ferric aerobactin from strain X40 at various pH values was recorded on a Beckman 25 spectrophotometer. The 1H nuclear magnetic resonance spectrum of the free ligand was taken on a 200-MHz Nicolet spectrometer in the Fourier transform mode. The fast-atom-bombardment mass spectrum was measured in the anionic detection mode. Outer membrane proteins. Pseudomonas sp. strain X40 was grown overnight in 10 ml of PM and 10 ml of PM containing 2 ,uM Fe. E. coli BN3040(pColV-K30) gyrA was grown overnight at 37°C in LB and LB containing 200 p.M dipyridyl. Protein preparations enriched in outer membrane proteins were obtained as Triton X-100-insoluble cellular fractions, as described elsewhere (32). DNA analyses. Plasmid preparations from E. coli BN3040(pColV-K30) gyrA and strain X40 were obtained by an alkaline-sodium dodecyl sulfate procedure (23). The resulting DNA was digested with restriction endonucleases BglII, HpaI, and SmaI under standard conditions (23). Digested and undigested DNAs were analyzed on 1% agarose-TAE gels (23) and stained with ethidium bromide. The DNA was transferred to nitrocellulose paper (23), and the resulting blot was probed with 32P-nick-translated (23) plasmid pVLN1 by standard hybridization techniques. Final washings of the blot were made in conditions of moderate to high stringency. The positions of DNA fragments with homology to the probe were revealed by autoradiography of the dried blot.
RESULTS A bacterium designated strain X40, isolated from a cyanobacterial culture, was identified as a Pseudomonas species by the following characteristics. The bacterium consisted of gram-negative motile rods. The organism tested positive for catalase, oxidase, and nitrate reduction but tested negative for glucose fermentation. The organism was negative for DNase activity, starch hydrolysis, gelatin hydrolysis, and growth on MacConkey agar, lysine, arginine, ornithine, citrate, urea, and indole. A fatty acid profile, determined by
2248
APPL. ENVIRON. MICROBIOL.
BUYER ET AL.
TABLE 1. Proton nuclear magnetic resonance chemical shift data for aerobactin in deuterium oxide from Aerobacter aerogenes and the Pseudomonas sp. 8 (multiplicityb)
Assignment
Lys a Lys E Citrate CH2 Acetyl CH3 Lys l, 8 Lys y
A. aerogenes
Pseudomonas sp.
4.37 (t) 3.62 (t) 2.85 (2 d) 2.12 (s) 1.0-2.0 (m) 1.0-2.0 (m)
4.46 (m) 3.78 (t) 2.99 (d of q) 2.32 (s) 1.75-2.1 (m) 1.56 (m)
a Chemical shift data for A. aerogenes were presented previously (14), whereas those for the Pseudomonas sp. are from this study. b s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
Myron Sasser of Microbial ID, Inc. (Newark, Del.), while not identical to any in the data base of Microbial ID, was most similar to that of Pseudomonas vesicularis. The strain was moderately halophilic, with maximum growth occurring at NaCl concentrations greater than 0.5 M and with no growth occurring below 0.2 M. No growth occurred below pH 8, maximum growth was at pH 9 to 10, and slower growth was observed at pH 12. The organism grew at 20 to 45°C, but incubation at 50°C was bactericidal. Supernatants of Pseudomonas strain X40 low-iron (PM) cultures were CAS reactive when compared with those of high-iron (PM plus 2 ,uM Fe) cultures. Paper electrophoresis of supernatants revealed a single spot. The purified material had paper electrophoretic mobility identical to that of authentic aerobactin, while the ferric siderophore had electrophoretic mobility identical to that of authentic ferric aerobactin. The variation in the visible absorbance spectrum with pH (data not shown) was virtually identical to that published for ferric aerobactin (19). Proton nuclear magnetic resonance spectra of the siderophore (Table 1) were virtually identical to published spectra (15). The mass spectrum confirmed the identity of the siderophore to be aerobactin. Peaks at 585, 607, and 629 atomic mass units corresponded to (NaHAerobactin)-7 (Na2Aerobactin)-, and (Na3[Aerobactin-H])-, respectively, where Na3Aerobactin is the neutral trisodium salt. Purified pseudomonad aerobactin and ferric pseudomonad aerobactin stimulated the growth of strain X40 on EDDA iron-limited plates, as is expected for a siderophore (28, 34). Pseudomonas aerobactin, authentic aerobactin, and E. coli HB101 (pVLN1) entfep, a strain which produces aerobactin but not enterobactin, reversed dipyridyl-induced iron starvation for E. coli HB1O1(pABN6) ent fep, a strain which cannot produce enterobactin, aerobactin, or the outer membrane receptor for enterobactin, but which can utilize aerobactin for iron transport. E. coli HB101, which produces enterobactin but not aerobactin, did not promote growth in this assay. Therefore, pseudomonad aerobactin and authentic aerobactin had similar biological activities for E. coli. In order to identify the stereochemistry of the lysine residue, both aerobactin from the Pseudomonas sp. and authentic aerobactin were hydrolyzed with hydriodic acid, producing lysine. After reaction with L-lysine decarboxylase, each aerobactin gave a single spot on paper electrophoresis with the same mobility as that of 1,5-pentane diamine. L-Lysine gave identical results, while DL-lysine, when it was reacted with L-lysine decarboxylase, produced two spots corresponding to lysine and 1,5-pentane diamine. Therefore,
FIG. 2. Outer membrane proteins of E. coli and the Pseudomo6, molecular weight markers (in thousands); lanes 2 and 3, E. coli grown under conditions of high and low iron availability, respectively; lanes 4 and 5, the Pseudomonas sp. grown under conditions of high and low iron availability, respectively.
nas sp. Lanes 1 and
aerobactin isolated from both E. coli and Pseudomonas strain X40 contains only L-lysine. Polyacrylamide gel electrophoresis of outer membrane proteins is shown in Fig. 2. Under iron-starvation conditions, at least two outer membrane proteins were induced. The E. coli aerobactin receptor encoded by pColV-K30 is a 74-kDa protein (4) and is one of the induced proteins in lane 3 of Fig. 2. The Pseudomonas aerobactin receptor protein, presumed to be one of the induced proteins in lane 5 of Fig. 2, is similar in size but not identical to the E. coli aerobactin receptor protein. DNA preparations from Pseudomonas strain X40 were obtained by a sodium dodecyl sulfate-alkaline procedure in which the presence of a large plasmid (pVl) could be detected (Fig. 3) migrating more slowly than the bulk of chromosomal DNA. This plasmid, however, was substantially smaller than the control plasmid pColV-K30 (Fig. 3). To determine whether aerobactin determinants in Pseudomonas strain X40 were plasmid-borne and whether they were similar to those in pColV-K30, a DNA hybridization assay was carried out. Figure 4 shows the autoradiograph of a Southern blot obtained from DNA preparations from Pseudomonas strain X40 and E. coli BN3040(pColV-K30) gyrA either undigested or digested with some restriction endonucleases. Under conditions of moderate to high stringency, no hybridization could be detected between the probe pVLN1 (carrying the aerobactin determinants from pColVK30) and Pseudomonas strain X40 DNA.
212
pYl
pCoIV4(3W
FIG. 3. DNA preparation. Lane 1, Pseudomonas sp.; lane 2, E.
coli(pColV-K30).
AEROBACTIN PRODUCTION IN A PSEUDOMONAS SP.
VOL. 57, 1991
FIG. 4. Southern blot hybridization with 32P-nick-translated pVLN1. Lane 1, molecular weight markers; lane 2, Pseudomonas DNA preparation; lane 3, E. coli(pColV-K30) DNA preparation; lanes 4, 5, and 6, pColV-K30 DNA preparation cut with BglII, HpaI, and SmaI, respectively; lanes 7, 8, and 9, Pseudomonas DNA preparation cut with BglII, Hpal, and SmaI, respectively; lanes 10, 11, and 12, pVLN1 cut with BglII, HpaI, and SmaI, respectively.
DISCUSSION Pseudomonas strain X40 appears to be well adapted to growth in Spirulina culture. Like the cyanobacterium (7), Pseudomonas strain X40 grows vigorously under highly alkaline conditions at high Na concentrations. This suggests that Pseudomonas strain X40 may be a natural inhabitant of Spirulina ecosystems rather than a culture contaminant. When stressed for iron, Pseudomonas strain X40 produced a single siderophore which was identical to the aerobactin isolated from E. coli, as demonstrated by both biological and chemical tests. It seems somewhat surprising to find that aerobactin, previously found only among the enteric bacteria (24), is also utilized by a Pseudomonas sp. under conditions extremely different from those of the normal enteric environment.
A number of siderophores from the family Pseudomonadaceae have been identified. Pseudomonas fluorescens and Pseudomonas putida produce the fluorescent siderophores termed pseudobactins and pyoverdins (22). Pyochelin has been found in Pseudomonas aeruginosa and Pseudomonas cepacia (8, 38), while desferrioxamine E is produced by Pseudomonas stutzeri (26). Cepabactin was recently identified as the siderophore of P. cepacia and Pseudomonas alcaligenes (27). The pseudomonads are clearly very diverse in siderophore production. The system leading to aerobactin production in Pseudomonas strain X40 appears to be different in at least some respects from that in E. coli. Two outer membrane proteins induced by low iron were produced by Pseudomonas strain X40. One of these proteins is reasonably assumed to be the aerobactin receptor. Neither protein had electrophoretic mobility identical to that of the E. coli aerobactin outer membrane receptor protein of pColV-K30. No homology was observed between Pseudomonas strain X40 DNA and the aerobactin system of pColV-K30 cloned in pVLN1. Iron regulation in Pseudomonas strain X40 may also be different from that in E. coli, since no homology to the gene sequence of the Fur protein of E. coli could be detected by hybridization in Pseudomonas strain X40 DNA (1Sa). Preliminary data indicate that the N-OH-lysine acetylase of Pseudomonas strain X40 is different from the analogous E. coli protein (8a).
2249
Similar results have been reported previously. Certain clinical isolates of E. coli and Enterobacter cloacae which produce aerobactin show no homology to the aerobactin genome from the ColV plasmid (6, 10). Variations in the sizes of aerobactin receptors from different sources have been observed previously (20), even when large similarities in the other genes of the operon are present. The genes encoding the aerobactin biosynthetic enzymes of an aerobactin-producing strain of Aerobacter aerogenes show no homology to the analogous pColV genes of E. coli, while the outer membrane receptors were immunologically cross-reactive and of the same size, and the genes encoding them were homologous (42). While the lack of homology between Pseudomonas strain X40 DNA and E. coli pVLN1 could be due to variations in the third base of the codons, this would not explain the differences seen between the outer membrane receptor proteins and the acetylase enzymes. In this respect, Pseudomonas strain X40 may prove to be useful in providing comparative biochemical and genetic information on the aerobactin system. ACKNOWLEDGMENTS We thank Kenneth Nealson, University of Wisconsin, and Myron Sasser, Microbial ID, Inc., for assistance in identifying the organism. This work was supported by National Research Service Award ES05361-02 to J.S.B. V.D.L. was a postdoctoral fellow (1F05TWO3577-01-B1-5) of the John Fogarty NIH Center. Additional support was derived from Public Health Service grant A104156 and from grants PCM 78-12198 and CRCR-1-1633 from the National Science Foundation and the U.S. Department of Agriculture, respectively. REFERENCES 1. Bagg, A., and J. B. Neilands. 1987. Ferric uptake regulation protein acts as a repressor, employing Fe(II) as cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry 25:5471-5477. 2. Baumann, P., and R. H. W. Schubert. 1984. Family II. Vibrionaceae Veron 1965, 5245AL, p. 51-538. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 3. Bell, S. J., S. A. Friedman, and J. Leong. 1979. Antibiotic action 4.
5.
6.
7.
8.
of N-methylthioformohydroxamate metal complexes. Antimicrob. Agents Chemother. 15:384-391. Bindereif, A., and J. B. Neilands. 1983. Cloning of the aerobactin-mediated iron assimilation system of plasmid ColV. J. Bacteriol. 153:1111-1113. Bindereif, A., and J. B. Neilands. 1985. Promoter mapping and transcriptional regulation of the iron assimilation system of plasmid ColV-K30 in Escherichia coli K-12. J. Bacteriol. 162: 1039-1046. Carbonetti, N. H., S. Boonchai, S. H. Parry, V. Vaisanen-Rhen, T. K. Korhonen, and P. H. Williams. 1986. Aerobactin-mediated iron uptake by Escherichia coli isolates from human extraintestinal infections. Infect. Immun. 51:966-968. Ciferri, O., and 0. Tiboni. 1985. The biochemistry and industrial potential of Spirulina. Annu. Rev. Microbiol. 39:503-526. Cox, C. D., and R. Graham. 1979. Isolation of an iron-binding compound from Pseudomonas aeruginosa. J. Bacteriol. 137:
357-364. 8a.Coy, M. Personal communication. 9. Crosa, J. H. 1984. The relationship of plasmid-mediated iron transport and bacterial virulence. Annu. Rev. Microbiol. 38:6989. 10. Crosa, L. M., M. K. Wolf, L. A. Actis, J. Sanders-Loehr, and J. H. Crosa. 1988. New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae. J. Bacteriol. 170:5539-5544.
2250
BUYER ET AL.
11. DeLorenzo, V., A. Bindereif, B. H. Paw, and J. B. Neilands. 1986. Aerobactin biosynthesis and transport genes of plasmid CoIV-K30 in Escherichia coli K-12. J. Bacteriol. 165:570-578. 12. DeLorenzo, V., and J. L. Martinez. 1988. Aerobactin production as a virulence factor: a reevaluation. Eur. J. Clin. Microbiol. Infect. Dis. 7:621-629. 13. DeLorenzo, V., and J. B. Neilands. 1986. Characterization of genes iucA and iucC of the aerobactin system of pColV-K30 in E. coli K-12. J. Bacteriol. 167:350-355. 14. DeLorenzo, V., S. Wee, M. Herrero, and J. B. Neilands. 1987. Operator sequences of the aerobactin operon of plasmid ColVK30 binding the ferric uptake regulation (fur) repressor. J. Bacteriol. 169:2624-2630. 15. Gibson, G., and D. I. Magrath. 1969. The isolation and characterization of a hydroxamic acid (aerobactin) formed by Aerobacter aerogenes 62-I. Biochim. Biophys. Acta 192:175-184. 15a.Giovannini, F. Personal communication. 16. Griffiths, E., P. Stevenson, T. L. Hale, and S. B. Formal. 1985. Synthesis of aerobactin and a 76,000-dalton iron-regulated outer membrane protein by Escherichia coli K-12-Shigella flexneri hybrids and by enteroinvasive strains of Escherichia coli. Infect. Immun. 49:67-71. 17. Gross, R., F. Engelbrecht, and V. Braun. 1984. Genetic and biochemical characterization of the aerobactin synthesis operon on pColV. Mol. Gen. Genet. 196:74-80. 18. Gross, R., F. Engelbrecht, and V. Braun. 1985. Identification of the genes and their polypeptide products responsible for aerobactin synthesis by pColV plasmids. Mol. Gen. Genet. 201:204212. 19. Harris, W. R., C. J. Carrano, and K. N. Raymond. 1979. Coordination chemistry of microbial iron transport compounds. 16. Isolation, characterization, and formation constants of ferric aerobactin. J. Am. Chem. Soc. 101:2722-2727. 20. Krone, W. J., F. Stegehuis, G. Koningstein, C. vanDoorn, B. Roosendaal, F. K. deGraaf, and B. Oudega. 1985. Characterization of the pColV-K30 encoded cloacin DF13 aerobactin outer membrane receptor protein of Escherichia coli; isolation and purification of the protein and analysis of its nucleotide sequence and primary structure. FEMS Microbiol. Lett. 26:153161. 21. Lawlor, K. M., and S. M. Payne. 1984. Aerobactin genes in Shigella spp. J. Bacteriol. 160:266-272. 22. Leong, J. 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogens. Annu. Rev. Phytopathol. 24:187-209. 23. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Martinez, J. L., J. L. Cercenado, F. Baquero, J. C. Perez Diaz, and A. Delgado-Iribarren. 1987. Incidence of aerobactin production in gram-negative hospital isolates. FEMS Microbiol. Lett. 43:351-353. 25. McDougall, S., and J. B. Neilands. 1984. Plasmid- and chromosome-coded aerobactin synthesis in enteric bacteria: insertion
APPL. ENVIRON. MICROBIOL.
26. 27. 28. 29. 30.
31. 32.
33.
34. 35.
36. 37.
38. 39.
40.
41. 42.
sequences flank operon in plasmid-mediated systems. J. Bacteriol. 159:300-305. Meyer, J. M., and M. A. Abdallah. 1980. The siderochromes of non-fluorescent pseudomonads: production of nocardamine by Pseudomonas stutzeri. J. Gen. Microbiol. 118:125-129. Meyer, J. M., D. Hohnadel, and F. Halle. 1989. Cepabactin from Pseudomonas cepacia, a new type of siderophore. J. Gen. Microbiol. 135:1479-1487. Miles, A. A., and P. L. KhimJi. 1975. Enterobacterial chelators of iron: their occurrence, detection, and relation to pathogenicity. J. Med. Microbiol. 8:477-490. Neilands, J. B. 1981. Microbial iron compounds. Annu. Rev. Biochem. 50:715-731. Neilands, J. B. 1984. Methodology of siderophores. Struct. Bonding 58:1-24. Payne, S. M., D. W. Niesel, S. S. Peixotto, and K. M. Lawlor. 1983. Expression of hydroxamate and phenolate siderophores by Shigella flexneri. J. Bacteriol. 155:949-955. Pugsley, A. P., F. Moreno, and V. deLorenzo. 1986. MicrocinE492-insensitive mutants of E. coli K12. J. Gen. Microbiol. 132:3253-3259. Roberts, M., R. W. Leavitt, N. H. Carbonetti, S. Ford, R. A. Cooper, and P. H. Williams. 1986. RNA-DNA hybridization analysis of transcription of the plasmid ColV-K30 aerobactin gene cluster. J. Bacteriol. 167:467-472. Rogers, H. J. 1973. Iron-binding catechols and virulence in Escherichia coli. Infect. Immun. 7:445-456. Schlosser, U. G. 1982. Sammlung von Algenkulturen. Ber. Dtsch. Bot. Ges. 95:181-276. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47-56. Smibert, R. M., and N. R. Krieg. 1981. General characterization, method 1, p. 420. In P. Gerhardt (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. Sokol, P. A. 1986. Production and utilization of pyochelin by clinical isolates of Pseudomonas cepacia. J. Clin. Microbiol. 23:560-562. Stuart, S. J., J. K. Prpic, and R. M. Robins-Browne. 1986. Production of aerobactin by some species of the genus Yersinia. J. Bacteriol. 166:1131-1133. Van Tiel-Menkveld, G. J., J. M. Mentjox-Vervuurt, B. Oudega, and F. K. deGraaf. 1982. Siderophore production by Enterobacter cloacae and a common receptor protein for the uptake of aerobactin and cloacin DF13. J. Bacteriol. 150:490-497. Warner, P. J., P. H. Williams, A. Bindereif, and J. B. Neilands. 1981. ColV plasmid-specified aerobactin synthesis by invasive strains of Escherichia coli. Infect. Immun. 33:540-545. Waters, V. L., and J. H. Crosa. 1988. Divergence of the aerobactin iron uptake systems encoded by plasmids pColVK30 in Escherichia coli K-12 and pSMN1 in Aerobacter aerogenes 62-1. J. Bacteriol. 170:5153-5160.
