Vol. 140, No. 3
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 1129-1132 0021-9193/79/12-1129/04$02.00/0
Siderophore Synthesis in Klebsiella pneumoniae and Shigella sonnei During Iron Deficiencyt R. D. PERRY::* AND C. L. SAN CLEMENTE Department ofMicrobiology and Public Health, Michigan State University, East Lansing, Michigan 48824
Received for publication 8 August 1979
Klebsiellapneumoniae 298/53 and Shigella sonnei 43-GG9 exhibited restricted growth and enterochelin synthesis only under iron-deficient conditions. S. sonnei also produced an unidentified secondary hydroxamate siderophore.
When ionic iron is present in relatively high concentrations, a low-affinity membrane-bound iron transport system in Escherichia coli is capable of satisfying its iron requirements. At lower iron concentrations, this system is unable to accumulate sufficient iron to meet microbial needs (5, 18); under these conditions many microorganisms synthesize and secrete small organic compounds (siderophores) which solubilize exogenous iron, making it available for transport into the cells (9). Two classes of compounds, phenolics (2,3-dihydroxybenzoate amino acid conjugates) and secondary hydroxamates, have been shown to be effective siderophores. The most thoroughly studied phenolate siderophore, enterochelin (or enterobactin, a cyclic trimer of 2,3-dihydroxybenzoylserine) is produced by iron-deficient cultures of E. coli, Enterobacter aerogenes, Enterobacter cloacae, and Salmonella typhimurium (11, 16, 20). Only a few bacterial species secrete hydroxamate siderophores during iron deprivation: Bacillus megaterium (schizokinen), E. aerogenes strain 62-1 (aerobactin), Arthrobacter pascens (arthrobactin or terregens factor), and Pseudomonas fluorescens (ferribactin) (3, 6, 9). In this study, we report on the synthesis of enterochelin by both Klebsiella pneumoniae 298/53 and Shigella sonnei 43-GG9 and the additional synthesis of a secondary hydroxamate siderophore by the S. sonnei strain. The iron-starved cells of K. pneumoniae secreted phenolate compounds and failed to attain the level of growth exhibited by the iron-sufficient cells (Fig. 1). Growth of the iron-deficient culture was reduced by 38%. In the iron-deficient culture, the secretion of extracellular phenolate compounds paralleled growth. Secretion of phenolate compounds by iron-sufficient cells was below detectable levels (less than 1 yg of 2,3-dihydroxybenzoic acid equivalents per ml). t Journal article no. 8610 from the Michigan Agricultural Experiment Station. t Present address: Department of Biology, Washington University, St. Louis, MO 63130.
Hydroxamate compounds were not observed in supernatant fluids of either culture (less than 10 ,ug of schizokinen equivalents per ml). When compared with iron-sufficient S. sonnei cells, iron-restricted cells exhibited an increased generation time and a reduced growth yield (Fig. 1). Iron restriction caused a 38% reduction in growth and nearly a twofold increase in generation time (1.3 h:0.75 h). Iron-starved cells synthesized both phenolate and hydroxamate compounds; secretion of these compounds paralleled growth. Enterochelin, its degradation products, and 2,3-dihydroxybenzoic acid have previously been separated on paper chromatograms run in 5.0% (wt/vol) ammonium formate in 0.5% (vol/vol) aqueous formic acid (11, 15, 17). In this study, purified phenolate isolates from each organism were separated into five iron-reactive spots [i.e., enterochelin, linear (2,3-dihydroxybenzoylserine)3, (2,3-dihydroxybenzoylserine)2, 2,3-dihydroxybenzoylserine, and 2,3-dihydroxybenzoic acid] by the formate solvent (Table 1). Although the formate solvent system has previously been reported to separate phenolate isolates from E. coli into six (17) and four (15) compounds, differences in growth conditions and isolation procedures may account for these discrepancies. The experimental Rf values for phenolate compounds from E. coli K-12 (Table 1) correlate reasonably well with previously published values of analogous compounds (15, 17). Phenolate compounds from E. coli yielded spots with Rf values which were nearly identical with analogous spots from phenolate isolates of K. pneumoniae and S. sonnei (Table 1). The slowest moving compounds from all three organisms turned a shade of mauve when sprayed with 0.5% ferric chloride; these results are in agreement with those previously reported by O'Brien et al. (11), who identified this compound as enterochelin. Thus, under iron-deficient conditions, both K. pneumoniae 298/53 and S. sonnei 43-GG9 syn-
1129
1130 NOTES
J. BACTERIOL.
E
0
E
0
0
C
Q
'U
uJ
I.-
I-
4
I-
ui
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z 'U
z -j
0
4 lO
4
x
0
-d
4
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0
In
I
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HOURS OF INCUBATION FIG. 1. Growth and siderophore synthesis by K. pneumoniae and S. sonnei at 37°C. (A) Viability (in colonyforming units per milliliter) and (B) optical density (620 nm) of cultures: iron-sufficient cultures of K. pneumoniae (0) and S. sonnei (O); iron-deficient cultures of K. pneumoniae (0) and S. sonnei (U). Optical density values (540 nm) of the Csaky assay (4) for the secondary hydroxamate in the iron-deficient culture of S. sonnei (K, A). No detectable hydroxamateproduction occurred in the iron-sufficient cultures. (B) Equivalent pg of 2,3-dihydroxybenzoic acid (DHB) produced per ml by the iron-deficient cultures of K. pneumoniae (Q) and S. sonnei (i). No detectable production ofphenolate compounds occurred in the iron-sufficient cultures. Growth curves were performed in a chemically defined medium as previously described (13). Iron was extracted from this medium by the method of Waring and Werkman (19); iron-deficient medium contained 0.1 to 0.3 ,iM iron as determined by a modified bathophenanthroline iron assay (14). Iron-sufficient medium was prepared by supplementing the iron-deficient medium with ferric chloride to a final concentration of 20 ,iM. Phenolate compounds were detected by the method ofArnow (1).
TABLE 1. Rf values for the components of the phenolate isolates and 2,3-dihydroxybenzoic acid separated by descending paper chromatography in an ammonium formate solvent systema Phenolates
Rf in 5% (wt/vol) ammonium formate in 0.5% (vol/vol) aqueous formic acidb
C D E 0.19 0.32 0.56 0.67 0.76 From E. coli From K. pneumoniae 0.19 0.35 0.55 0.66 0.74 From S. sonnei 0.20 0.35 0.56 0.66 0.75 0.76 2,3-dihydroxybenzoic A
B
acid a Phenolate compounds were isolated by ethyl acetate extraction as described by O'Brien and Gibson (12). The phenolate isolates were diluted with redistilled ethanol such that each solution contained equivalent quantities of Arnow assay-reactive material. b A, Enterochelin; B, linear (2,3-dihydroxybenzoylserine)3; C, (2,3-dihydroxybenzoylserine)2; D, 2,3-dihydroxybenzoylserine; E, 2,3-dihydroxybenzoic acid. Chromatograms were developed by spraying with a 0.5% (wt/vol) ferric chloride solution.
thesize enterochelin. The quantities of enterochelin secreted by both organisms are approximately equivalent to that secreted by E. coli K12 under similar conditions (13). The synthesis of 2,3-dihydroxybenzoylthreonine in iron-deficient cultures of E. coli and some Klebsiella
species has been reported (7, 8). This compound was either not secreted under the conditions employed in this study or indistnguishable from enterochelin and its degradation products in the formate solvent system. The hydroxamate compound from S. sonnei has not been completely isolated and purified. This compound gave a positive reaction for the presence of hydroxylamine only after hydrolysis with 6 M sulfuric acid at 1000C, indicating that the compound is a secondary hydroxamate. The compound can be extracted into phenol when fully chelated with iron at pH 4.0, whereas the unchelated compound is not. Attempts to remove the hydroxamate from phenol have been unsuccessful. Ethyl acetate extraction at pH 1.0 did not remove this compound from culture supernatant fluids. Dowex-1 columns (4 by 7 cm) did not remove the hydroxamate from culture supematant fluids adjusted to pH's of 7.0 and 9.0. At pH 12.0, the hydroxamate was partially retained on a Dowex-1 column and appeared to be eluted with 0.1 M NH4Cl (data not shown). The compound was partially purified by passage of culture supernatant fluids at pH 7.0 through a Dowex-1 column. The eluant was adjusted to pH 9.0 with 1 M NaOH and passed through a second Dowex-1 column. The eluant was flash evaporated and applied to a waterequilibrated Sephadex G-10 column which ex-
NOTES
VOL. 140, 1979
cluded the hydroxamate compound. Positive fractions were pooled, concentrated by flash evaporation, and analyzed by descending paper chromatography in n-butanol-acetic acid-water (60:15:25, by vol). A chromatogram developed with 0.5% ferric chloride revealed a single red spot with an Rf of 0.63. In the same system, aerobactin, desferrioxamine B, and schizokinen gave reddish-purple spots with respective R,'s of 0.69, 0.86, and 0.73. Development of a second chromatogram with ammoniacal silver nitrate indicated that the hydroxamate sample from S. sonnei contained several other organic compounds as minor contaminants (data not shown). For growth and inhibition factor studies, S. typhimurium LT-2 enb-7 (a mutant blocked in the synthesis of 2,3-dihydroxybenzoic acid) was grown in iron-sufficient medium before inoculation onto petri plates. Petri plates contained either iron-sufficient solid medium (iron-deficient medium supplemented with 1% [wt/vol] Ionagar no. 2 and 50 ,uM ferric chloride) or citrate-supplemented solid medium E, which completely inhibits the growth of the S. typhimurium mutant (10, 15). Petri plates were seeded by overlayering with approximately 105 cells in the appropriate solid medium. Volumes of 10 pl of sterile siderophore and iron solutions (1 mM) were spotted onto the seeded plates. All phenolate isolates were diluted with water to 0.15 optical density units by the Arnow assay (1) before spotting 10-1l volumes onto the seeded plates. The same volume of the S. sonnei hydroxamate solution (0.58 optical density units by the CMky assay [4]) was applied to seeded plates. The hydroxamate solution was the eluant obtained by passing culture supernatant through a Dowex-1 column and was not contaminated with phenolate compounds since these compounds were irreversibly bound by Dowex-1 (Perry, unpublished data). The growtL of S. typhimurium LT2 enb-7 on citrate-supplemented medium E was stimulated by ferric chloride, ferrous chloride, 2,3-dihydroxybenzoic acid, desferrioxamine B, schizokinen, the S. sonnei hydroxamate, and the phenolate isolates from E. coli, K. pneumoniae, and S. sonnei. Aerobactin, hemin, and uninoculated iron-deficient medium did not stimulate growth. Only aerobactin inhibited the growth of the S. typhimurium mutant on iron-sufficient solid medium (data not shown). The secondary hydroxamate produced by S. sonnei is not aerobactin, desferrioxamine B, or schizokinen since its chemical or biological properties or both differ from the properties of these secondary hydroxamate siderophores. The synthesis of phenolate and hydroxamate siderophores by Shigella boydii M44 (Perry, unpub-
1131
lished data) and S. sonnei 43-GG9 suggests that the simnultaneous expression of both types of siderophores may be common within this genus. The simultaneous synthesis of both phenolate and hydroxamate compounds has been previously reported only in E. aerogenes 62-1 (enterochelin and aerobactin) and in Bacillus subtilis var. niger (a secondary hydroxamate and 2,3dihydroxybenzoylglycine) (2, 6, 18). The physiological necessity for synthesizing two separate siderophores is not readily apparent. This work was supported by the Michigan Agricultural Experiment Station. We thank B. R. Byers for providing schizokinen, aerobactin, and E. aerogenes 62-1; R. R. Brubaker for E. coli K-12, K. pneumoniae 298/53 (ATCC 13883), and S. sonnei 43-GG9 (ATCC 9290); F. Archibald for S. typhinurium LT-2 enb-7; and Carol Lee Luscombe for figure preparation.
1.
2.
3.
4.
LITERATURE CITED Arnow, L. E. 1937. Colorimetric determination of the components of 3,4-dihydroxyphenylalanine-tyrosine mixtures. J. Biol. Chem. 118:531-537. Byers, B. R. 1974. Iron transport in gram-positive and acid-fast bacilli, p. 83-105. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Preas Inc., New York. Byers, B. R., M. V. Powell, and C. E. Lankford. 1967. Iron-chelating hydroxamic acid (schizokinen) active in initiation of cell division in Bacillus megaterium. J. Bacteriol. 93:286-294. Csaiky, T. Z. 1948. On the estimation of bound hydroxylamine in biological materials. Acta Chem. Scand. 2:
450-454. 5. Frost, G. E., and H. Rosenberg. 1973. The inducible citrate-dependent iron transport system in Escherichia coli K-12. Biochim. Biophys. Acta 330:90-101. 6. Gibson, F., and D. I. Magrath. 1969. The isolation and characterization of a hydroxamic acid (aerobactin) formed by Aerobacter aerogenes 62-1. Biochim. Biophys. Acta 192:175-184. 7. Korth, H. 1970. Ober das Vorkommen von 2,3-Dihydroxybenzoesaure und ihrer Aminosaurederivate in Kulturmedien von Klebsiella oxytoca. Arch. Mikrobiol. 70: 297-302. 8. Korth, H., C. S. Spieckermann, and G. Pulverer. 1971. Ober die Ablagerung von 2,3-Dihydroxybenzoesaure und deren Aminosaurederivate bei Klebsiellen und Escherichia coli. Med. Microbiol. Immunol. 157:52-57. 9. Lankford, C. E. 1973. Bacterial assimilation of iron. Crit. Rev. Microbiol. 2:273-331. 10. Luckey, M., J. R. Pollack, R. Wayne, B. N. Ames, and J. B. Neilands. 1972. Iron uptake in Salmonella typhimurium: utilization of exogenous siderochromes as iron carriers. J. Bacteriol. 111:731-738. 11. O'Brien, I. G., G. B. Cox, and F. Gibson. 1970. Biologically active compounds containing 2,3-dihydroxybenzoic acid and serine formed by Escherichia coli. Biochim. Biophys. Acta 201:453-460. 12. O'Brien, L. G., and F. Gibson. 1970. The structure of enterochelin and related 2,3-dihydroxy-N-benzoylserine conjugates from Escherichia coli. Biochim. Biophys. Acta 215:393-402. 13. Perry, R. D., and R. R. Brubaker. 1979. Accumulation of iron by yersiniae. J. Bacteriol. 137:1290-1298. 14. Perry, R. D., and C. L. San Clemente. 1977. Determination of iron with bathophenanthroline following an improved procedure for reduction of iron (III) ions. Analyst (London) 102:114-119.
1132
NOTES
15. Pollack, J. R., B. N. Ames, and J. B. Neilands. 1970. Iron transport in Salmonella typhimurium: mutants blocked in the biosynthesis of enterobactin. J. Bacteriol. 104:635-639. 16. Pollack, J. R., and J. B. Neilands. 1970. Enterobactin, an iron transport compound from Salmonella typhimurium. Biochem. Biophys. Res. Commun. 38:989-992. 17. Rogers, H. J. 1973. Iron-binding catechols and virulence in Escherichia coli. Infect. Immun. 7:445456. 18. Rosenberg, H., and L G. Young. 1974. Iron transport in
J. BACTERIOL. the enteric bacteria, p. 67-82. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Press Inc., New York. 19. Waring, W. S., and C. H. Werkman. 1942. Growth of bacteria in an iron-free medium. Arch. Biochem. 1:303310. 20. Wawszkiewicz, E. J., H. A. Schneider, B. Starcher, J. Poilack, and J. B. Neilands. 1971. Salmoneilosis pacifarin activity of enterobactin. Proc. Natl. Acad. Sci. U.S.A. 68:2870-2873.
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 1129-1132 0021-9193/79/12-1129/04$02.00/0
Siderophore Synthesis in Klebsiella pneumoniae and Shigella sonnei During Iron Deficiencyt R. D. PERRY::* AND C. L. SAN CLEMENTE Department ofMicrobiology and Public Health, Michigan State University, East Lansing, Michigan 48824
Received for publication 8 August 1979
Klebsiellapneumoniae 298/53 and Shigella sonnei 43-GG9 exhibited restricted growth and enterochelin synthesis only under iron-deficient conditions. S. sonnei also produced an unidentified secondary hydroxamate siderophore.
When ionic iron is present in relatively high concentrations, a low-affinity membrane-bound iron transport system in Escherichia coli is capable of satisfying its iron requirements. At lower iron concentrations, this system is unable to accumulate sufficient iron to meet microbial needs (5, 18); under these conditions many microorganisms synthesize and secrete small organic compounds (siderophores) which solubilize exogenous iron, making it available for transport into the cells (9). Two classes of compounds, phenolics (2,3-dihydroxybenzoate amino acid conjugates) and secondary hydroxamates, have been shown to be effective siderophores. The most thoroughly studied phenolate siderophore, enterochelin (or enterobactin, a cyclic trimer of 2,3-dihydroxybenzoylserine) is produced by iron-deficient cultures of E. coli, Enterobacter aerogenes, Enterobacter cloacae, and Salmonella typhimurium (11, 16, 20). Only a few bacterial species secrete hydroxamate siderophores during iron deprivation: Bacillus megaterium (schizokinen), E. aerogenes strain 62-1 (aerobactin), Arthrobacter pascens (arthrobactin or terregens factor), and Pseudomonas fluorescens (ferribactin) (3, 6, 9). In this study, we report on the synthesis of enterochelin by both Klebsiella pneumoniae 298/53 and Shigella sonnei 43-GG9 and the additional synthesis of a secondary hydroxamate siderophore by the S. sonnei strain. The iron-starved cells of K. pneumoniae secreted phenolate compounds and failed to attain the level of growth exhibited by the iron-sufficient cells (Fig. 1). Growth of the iron-deficient culture was reduced by 38%. In the iron-deficient culture, the secretion of extracellular phenolate compounds paralleled growth. Secretion of phenolate compounds by iron-sufficient cells was below detectable levels (less than 1 yg of 2,3-dihydroxybenzoic acid equivalents per ml). t Journal article no. 8610 from the Michigan Agricultural Experiment Station. t Present address: Department of Biology, Washington University, St. Louis, MO 63130.
Hydroxamate compounds were not observed in supernatant fluids of either culture (less than 10 ,ug of schizokinen equivalents per ml). When compared with iron-sufficient S. sonnei cells, iron-restricted cells exhibited an increased generation time and a reduced growth yield (Fig. 1). Iron restriction caused a 38% reduction in growth and nearly a twofold increase in generation time (1.3 h:0.75 h). Iron-starved cells synthesized both phenolate and hydroxamate compounds; secretion of these compounds paralleled growth. Enterochelin, its degradation products, and 2,3-dihydroxybenzoic acid have previously been separated on paper chromatograms run in 5.0% (wt/vol) ammonium formate in 0.5% (vol/vol) aqueous formic acid (11, 15, 17). In this study, purified phenolate isolates from each organism were separated into five iron-reactive spots [i.e., enterochelin, linear (2,3-dihydroxybenzoylserine)3, (2,3-dihydroxybenzoylserine)2, 2,3-dihydroxybenzoylserine, and 2,3-dihydroxybenzoic acid] by the formate solvent (Table 1). Although the formate solvent system has previously been reported to separate phenolate isolates from E. coli into six (17) and four (15) compounds, differences in growth conditions and isolation procedures may account for these discrepancies. The experimental Rf values for phenolate compounds from E. coli K-12 (Table 1) correlate reasonably well with previously published values of analogous compounds (15, 17). Phenolate compounds from E. coli yielded spots with Rf values which were nearly identical with analogous spots from phenolate isolates of K. pneumoniae and S. sonnei (Table 1). The slowest moving compounds from all three organisms turned a shade of mauve when sprayed with 0.5% ferric chloride; these results are in agreement with those previously reported by O'Brien et al. (11), who identified this compound as enterochelin. Thus, under iron-deficient conditions, both K. pneumoniae 298/53 and S. sonnei 43-GG9 syn-
1129
1130 NOTES
J. BACTERIOL.
E
0
E
0
0
C
Q
'U
uJ
I.-
I-
4
I-
ui
5;
z 'U
z -j
0
4 lO
4
x
0
-d
4
Id
0 uJ
0
In
I
0
HOURS OF INCUBATION FIG. 1. Growth and siderophore synthesis by K. pneumoniae and S. sonnei at 37°C. (A) Viability (in colonyforming units per milliliter) and (B) optical density (620 nm) of cultures: iron-sufficient cultures of K. pneumoniae (0) and S. sonnei (O); iron-deficient cultures of K. pneumoniae (0) and S. sonnei (U). Optical density values (540 nm) of the Csaky assay (4) for the secondary hydroxamate in the iron-deficient culture of S. sonnei (K, A). No detectable hydroxamateproduction occurred in the iron-sufficient cultures. (B) Equivalent pg of 2,3-dihydroxybenzoic acid (DHB) produced per ml by the iron-deficient cultures of K. pneumoniae (Q) and S. sonnei (i). No detectable production ofphenolate compounds occurred in the iron-sufficient cultures. Growth curves were performed in a chemically defined medium as previously described (13). Iron was extracted from this medium by the method of Waring and Werkman (19); iron-deficient medium contained 0.1 to 0.3 ,iM iron as determined by a modified bathophenanthroline iron assay (14). Iron-sufficient medium was prepared by supplementing the iron-deficient medium with ferric chloride to a final concentration of 20 ,iM. Phenolate compounds were detected by the method ofArnow (1).
TABLE 1. Rf values for the components of the phenolate isolates and 2,3-dihydroxybenzoic acid separated by descending paper chromatography in an ammonium formate solvent systema Phenolates
Rf in 5% (wt/vol) ammonium formate in 0.5% (vol/vol) aqueous formic acidb
C D E 0.19 0.32 0.56 0.67 0.76 From E. coli From K. pneumoniae 0.19 0.35 0.55 0.66 0.74 From S. sonnei 0.20 0.35 0.56 0.66 0.75 0.76 2,3-dihydroxybenzoic A
B
acid a Phenolate compounds were isolated by ethyl acetate extraction as described by O'Brien and Gibson (12). The phenolate isolates were diluted with redistilled ethanol such that each solution contained equivalent quantities of Arnow assay-reactive material. b A, Enterochelin; B, linear (2,3-dihydroxybenzoylserine)3; C, (2,3-dihydroxybenzoylserine)2; D, 2,3-dihydroxybenzoylserine; E, 2,3-dihydroxybenzoic acid. Chromatograms were developed by spraying with a 0.5% (wt/vol) ferric chloride solution.
thesize enterochelin. The quantities of enterochelin secreted by both organisms are approximately equivalent to that secreted by E. coli K12 under similar conditions (13). The synthesis of 2,3-dihydroxybenzoylthreonine in iron-deficient cultures of E. coli and some Klebsiella
species has been reported (7, 8). This compound was either not secreted under the conditions employed in this study or indistnguishable from enterochelin and its degradation products in the formate solvent system. The hydroxamate compound from S. sonnei has not been completely isolated and purified. This compound gave a positive reaction for the presence of hydroxylamine only after hydrolysis with 6 M sulfuric acid at 1000C, indicating that the compound is a secondary hydroxamate. The compound can be extracted into phenol when fully chelated with iron at pH 4.0, whereas the unchelated compound is not. Attempts to remove the hydroxamate from phenol have been unsuccessful. Ethyl acetate extraction at pH 1.0 did not remove this compound from culture supernatant fluids. Dowex-1 columns (4 by 7 cm) did not remove the hydroxamate from culture supematant fluids adjusted to pH's of 7.0 and 9.0. At pH 12.0, the hydroxamate was partially retained on a Dowex-1 column and appeared to be eluted with 0.1 M NH4Cl (data not shown). The compound was partially purified by passage of culture supernatant fluids at pH 7.0 through a Dowex-1 column. The eluant was adjusted to pH 9.0 with 1 M NaOH and passed through a second Dowex-1 column. The eluant was flash evaporated and applied to a waterequilibrated Sephadex G-10 column which ex-
NOTES
VOL. 140, 1979
cluded the hydroxamate compound. Positive fractions were pooled, concentrated by flash evaporation, and analyzed by descending paper chromatography in n-butanol-acetic acid-water (60:15:25, by vol). A chromatogram developed with 0.5% ferric chloride revealed a single red spot with an Rf of 0.63. In the same system, aerobactin, desferrioxamine B, and schizokinen gave reddish-purple spots with respective R,'s of 0.69, 0.86, and 0.73. Development of a second chromatogram with ammoniacal silver nitrate indicated that the hydroxamate sample from S. sonnei contained several other organic compounds as minor contaminants (data not shown). For growth and inhibition factor studies, S. typhimurium LT-2 enb-7 (a mutant blocked in the synthesis of 2,3-dihydroxybenzoic acid) was grown in iron-sufficient medium before inoculation onto petri plates. Petri plates contained either iron-sufficient solid medium (iron-deficient medium supplemented with 1% [wt/vol] Ionagar no. 2 and 50 ,uM ferric chloride) or citrate-supplemented solid medium E, which completely inhibits the growth of the S. typhimurium mutant (10, 15). Petri plates were seeded by overlayering with approximately 105 cells in the appropriate solid medium. Volumes of 10 pl of sterile siderophore and iron solutions (1 mM) were spotted onto the seeded plates. All phenolate isolates were diluted with water to 0.15 optical density units by the Arnow assay (1) before spotting 10-1l volumes onto the seeded plates. The same volume of the S. sonnei hydroxamate solution (0.58 optical density units by the CMky assay [4]) was applied to seeded plates. The hydroxamate solution was the eluant obtained by passing culture supernatant through a Dowex-1 column and was not contaminated with phenolate compounds since these compounds were irreversibly bound by Dowex-1 (Perry, unpublished data). The growtL of S. typhimurium LT2 enb-7 on citrate-supplemented medium E was stimulated by ferric chloride, ferrous chloride, 2,3-dihydroxybenzoic acid, desferrioxamine B, schizokinen, the S. sonnei hydroxamate, and the phenolate isolates from E. coli, K. pneumoniae, and S. sonnei. Aerobactin, hemin, and uninoculated iron-deficient medium did not stimulate growth. Only aerobactin inhibited the growth of the S. typhimurium mutant on iron-sufficient solid medium (data not shown). The secondary hydroxamate produced by S. sonnei is not aerobactin, desferrioxamine B, or schizokinen since its chemical or biological properties or both differ from the properties of these secondary hydroxamate siderophores. The synthesis of phenolate and hydroxamate siderophores by Shigella boydii M44 (Perry, unpub-
1131
lished data) and S. sonnei 43-GG9 suggests that the simnultaneous expression of both types of siderophores may be common within this genus. The simultaneous synthesis of both phenolate and hydroxamate compounds has been previously reported only in E. aerogenes 62-1 (enterochelin and aerobactin) and in Bacillus subtilis var. niger (a secondary hydroxamate and 2,3dihydroxybenzoylglycine) (2, 6, 18). The physiological necessity for synthesizing two separate siderophores is not readily apparent. This work was supported by the Michigan Agricultural Experiment Station. We thank B. R. Byers for providing schizokinen, aerobactin, and E. aerogenes 62-1; R. R. Brubaker for E. coli K-12, K. pneumoniae 298/53 (ATCC 13883), and S. sonnei 43-GG9 (ATCC 9290); F. Archibald for S. typhinurium LT-2 enb-7; and Carol Lee Luscombe for figure preparation.
1.
2.
3.
4.
LITERATURE CITED Arnow, L. E. 1937. Colorimetric determination of the components of 3,4-dihydroxyphenylalanine-tyrosine mixtures. J. Biol. Chem. 118:531-537. Byers, B. R. 1974. Iron transport in gram-positive and acid-fast bacilli, p. 83-105. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Preas Inc., New York. Byers, B. R., M. V. Powell, and C. E. Lankford. 1967. Iron-chelating hydroxamic acid (schizokinen) active in initiation of cell division in Bacillus megaterium. J. Bacteriol. 93:286-294. Csaiky, T. Z. 1948. On the estimation of bound hydroxylamine in biological materials. Acta Chem. Scand. 2:
450-454. 5. Frost, G. E., and H. Rosenberg. 1973. The inducible citrate-dependent iron transport system in Escherichia coli K-12. Biochim. Biophys. Acta 330:90-101. 6. Gibson, F., and D. I. Magrath. 1969. The isolation and characterization of a hydroxamic acid (aerobactin) formed by Aerobacter aerogenes 62-1. Biochim. Biophys. Acta 192:175-184. 7. Korth, H. 1970. Ober das Vorkommen von 2,3-Dihydroxybenzoesaure und ihrer Aminosaurederivate in Kulturmedien von Klebsiella oxytoca. Arch. Mikrobiol. 70: 297-302. 8. Korth, H., C. S. Spieckermann, and G. Pulverer. 1971. Ober die Ablagerung von 2,3-Dihydroxybenzoesaure und deren Aminosaurederivate bei Klebsiellen und Escherichia coli. Med. Microbiol. Immunol. 157:52-57. 9. Lankford, C. E. 1973. Bacterial assimilation of iron. Crit. Rev. Microbiol. 2:273-331. 10. Luckey, M., J. R. Pollack, R. Wayne, B. N. Ames, and J. B. Neilands. 1972. Iron uptake in Salmonella typhimurium: utilization of exogenous siderochromes as iron carriers. J. Bacteriol. 111:731-738. 11. O'Brien, I. G., G. B. Cox, and F. Gibson. 1970. Biologically active compounds containing 2,3-dihydroxybenzoic acid and serine formed by Escherichia coli. Biochim. Biophys. Acta 201:453-460. 12. O'Brien, L. G., and F. Gibson. 1970. The structure of enterochelin and related 2,3-dihydroxy-N-benzoylserine conjugates from Escherichia coli. Biochim. Biophys. Acta 215:393-402. 13. Perry, R. D., and R. R. Brubaker. 1979. Accumulation of iron by yersiniae. J. Bacteriol. 137:1290-1298. 14. Perry, R. D., and C. L. San Clemente. 1977. Determination of iron with bathophenanthroline following an improved procedure for reduction of iron (III) ions. Analyst (London) 102:114-119.
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NOTES
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J. BACTERIOL. the enteric bacteria, p. 67-82. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Press Inc., New York. 19. Waring, W. S., and C. H. Werkman. 1942. Growth of bacteria in an iron-free medium. Arch. Biochem. 1:303310. 20. Wawszkiewicz, E. J., H. A. Schneider, B. Starcher, J. Poilack, and J. B. Neilands. 1971. Salmoneilosis pacifarin activity of enterobactin. Proc. Natl. Acad. Sci. U.S.A. 68:2870-2873.
