1958] PROPERTIES OF PYOVERDINE 241 metabolites. The Chemical Society, Special Publication Roussos, G. G. AND VINING, L. C. 1956 Isolation and prop- No. 5, London, England. erties of pure actinomycins. J. Chem. Soc., 2469-2474. KOFFLER, H., EmERSON, R. L., PERLMAN, D., AND BURRIS, SAUNDERS, A. AND SYLVESTER, J. C. 1947 Synthetic media R. H. 1945 Chemical changes in submerged penicillin for the production of streptomycin. Abstracts of Papers, fermentations. J. Bacteriol., 60, 517-548. 112th meeting, Am. Chem. Soc., 9A-1OA. O'BRIEN, E., WAGMAN, G. H., AND PERLMAN, D. 1952 Syn- SCHMIDT-KASTNER, G. 1956 Actinomycin E und Actino- thetic media for growth and streptomycin production by mycin F, zwei neue biosynthetische Actinomycin gemische. Streptomyces griseus. Bacteriol. Proc., p. 25. Naturwissenschaften, 43, 131-132. PUGH, L., KATZ, E., AND WAKSMAN, S. A. 1956 Antibiotic WAKSMAN, S. A. AND WOODRUFF, H. B. 1940 Bacteriostatic and cytostatic properties of the actinomycins. J. Bac- and bactericidal substances produced by a soil actino- teriol., 72, 660-665. myces. Proc. Soc. Exptl. Biol. Med., 45, 609-614. Some Properties of Pyoverdine, the Water-soluble Fluorescent Pigment of the Pseudomonads R. P. ELLIOTT Food and Drug Administration, U. S. Department of Health, Education, and Welfare, Federal Office Building, San Francisco, California Received for publication December 3, 1957 The fluorescent water-soluble pigment complex pro- were probably determined on mixtures, and therefore duced by pseudomonads has been variously called not valid. Giral (1936), Chodat (1951), and Naves "bacterial fluorescein" or "fluorescin." These terms (1955) all claim to have separated blue and yellow frac- are unfortunate for they are synonymous with re- tions by chromatographic means. Giral (1936) con- sorcinolphthalein and resorcinolphthalin, respectively sidered pyoverdine to be a lyochrome or a pterine. (Merck Index, 1952). The bacterial pigment is not re- Birkhofer and Birkhofer (1948) reported that it was lated to either of these compounds. However, the term composed in part of riboflavin. "pyoverdine," suggested by Turfreijer (1941), spe- Giral (1936) found pyoverdine to be soluble in water, cifically identifies the bacterial pigment complex and formic acid, aqueous alcohol, aqueous acetone, and 90 will be used here. per cent pyridine. Turfitt (1937) found it was also Pyoverdine has interested bacteriologists for years, soluble in phenol and acetic acid. Giral (1936), Turfitt but few intensive studies have been made on it. Some (1937), and Turfreijer (1941) have presented absorp- investigators have studied media for maximum pig- tion spectra of pyoverdine in various reagents. Meader ment production. This work was adequately reviewed et al. (1925) described the indicator characteristics of by Seleen and Stark (1943) except for the important pyoverdine, and Giral (1936) described the effect of a contribution of Sullivan (1905), who was the first to large number of reagents on its appearance. He stated describe a simple synthetic medium composed of that air favored its production, but that light had the asparagine, magnesium sulfate, and potassium hydrogen opposite effect. phosphate. King et al. (1948), Baghdiantz (1952), and The object of the present study was to obtain enough Totterand Moseley (1953) studiedthe stimulation of pig- information about the properties of pyoverdine to form ment production by added minerals. King et al. (1954) a basis for determining it in frozen whole egg. The described simple solid media. resultant method is described in the second paper of No one has yet determined the exact chemical nature this series (Elliott, 1958). of pyoverdine or its fractions, nor has anyone yet suc- ceeded in crystallizing it. Various authors have used EXPERIMENTAL activated carbon to remove it from aqueous solution. Equipment and materials. The Pseudomonas ovalis When thus adsorbed it can be eluted with aqueous and Pseudomonas fluorescens cultures used in the ex- acetone or alcohol (Giral, 1936; Turfitt, 1937; Tur- periments had been isolated earlier from fluorescent freijer, 1941). Turfreijer succeeded in separating four eggs (Elliott, 1954) and identified by the method of fractions based on solubilities of phosphotungstic acid Haynes (1953). These cultures have been deposited at precipitates, and described many properties of these the Northern Utilization Research Branch, Agricul- fractions. Empirical formulae suggested by Turfitt tural Research Service, U. S. Department of Agricul- (1937), Turfreijer et al. (1938), and Bonde et al. (1957) ture, Peoria, Illinois, and have been numbered B1595 242 R. P. ELLIOTT [VOL. 6 and B1613, respectively. Asparagine broth for the organism was there any evidence of antibacterial production of pyoverdine consisted of asparagine, 0.1 action. per cent; MgSO4.7H20, 0.05 per cent; and K2HPO4, Absorption spectra. The absorption spectrum offered 0.05 per cent in distilled water. The filtrates of fluores- a possible means of identification and measurement of cent cultures used in several experiments were prepared the pigment. Five mg of crude pyoverdine, prepared by inoculating asparagine broth with one of the above as described above, when dissolved in 25 ml of water organisms, incubating it in the dark for 1 to 3 weeks presented the absorption curve shown in figure 1. at room temperature, and filtering it through a Seitz Similar spectra were obtained on the fluorescent fil- pad or a Mandler filter, previously washed free of trates of cultures (figures 2-5) and on this same pyo- soluble fluorescent materials. verdine preparation dissolved in 0.5 per cent AlCl3. Absorption spectra were determined on a Becknman' The curves obtained agreed well with those presented model DU spectrophotometer, and pH measurements by Giral (1936), Turfitt (1937), and Turfreijer (1941). on a Beckman' model H2 pH meter. Fluorescence emis- Fluorescence emission spectra and the effect of pH sion spectra were obtained using the equipment de- change. Fluorescence emission spectra were determined scribed by French (1955). All emission spectra were at three pH values on a fluorescent culture filtrate of corrected for the instrument response to a standard Pseudomonas ovalis and on pure riboflavin solutions. light source. Fluorescence measurements were made on Absorption spectra then were run on the same solutions. a Coleman2 model 12B photofluorometer. Visual fluores- Results are plotted in figure 2. The pH shift in absorp- cence of materials was determined in subdued lighting tion maxima in pyoverdine solutions as described by or in the dark using a Vogelite3 model 6W101 hand Turfitt (1937) is evident here. Also, when the pH is fixture previously described (Elliott, 1954). A Vogel either raised or lowered, the emission maximum shifts blue to green fluorescence color comparison chart was toward the red. Total emission is lessened by such pH used for recording small changes in black-light fluores- changes, the greater decrease occurring in alkali. The cent colors (Benson and Vogel, 1955). Reagents and same pH variations cause nearly complete quenching glassware were tested frequently for the presence of of fluorescence emission of riboflavin, though little foreign fluorescent materials. In general, chemically change in its absorption spectrum. pure reagents were nonfluorescent, whereas technical That pyoverdine is a sensitive pH indicator was grades had to be purified. Darco G604 was the activated shown by adjusting the pH values of an asparagine carbon used for adsorption of pyoverdine. broth culture of P. ovalis using HC1 and NaOH. The Solvents. As a means of separating pyoverdine from colors noted are shown in table 1. All color changes were frozen whole egg magma, a pyoverdine solvent im- reversible. miscible in water would be ideal. A large number of Riboflavin content. If riboflavin were a free component such solvents was tried, but no suitable one was found of pyoverdine, as described by Birkhofer and Birk- since pyoverdine has a strong affinity for the aqueous hofer (1948), the identification and measurement of phase in any extraction system. Water and aqueous the pigment could be accomplished easily by established alcohol were found most practical for the present work. methods. Riboflavin was found to be absent when a Attempts at purification. A crude preparation of P. ovalis culture was analyzed by the official method pyoverdine was obtained by adsorption on activated (AOAC, 1955). Furthermore, the absorption spectrum carbon from a culture filtrate of P. ovalis, followed by elution with aqueous alcohol. This material dried to a brown amorphous residue but did not crystallize. On heating, it began to soften at 230 C, but even at 252 C it did not melt completely. Although this indicates .8001 that pyoverdine is a mixture, it will be considered a / i single entity in further discussion here. Unsuccessful .6001 attempts were made to fractionate this material by paper and column chromatography. In z3 .400 Antibiotic properties. Brief experiments using Micro- a coccus pyogenes var. aureus and Escherichia coli with -J the agar-cup-plate method showed a concentrated a. .200 aqueous solution of crude pyoverdine to diffuse rapidly 0 ./ into the medium surrounding the cup. With neither 250 300 350 400 450 Soo 1 Beckman Instruments, Inc., Fullerton, California. 2 Coleman Instruments, Inc., Maywood, Illinois. WAVELENGTH my 3 Ultra-Violet Products, Inc., San Gabriel, California. Figure 1. Absorption spectrum of crude pyoverdine, 0.2 mg 4Atlas