The Oxidative Dissimilation of Mannitol and Sorbitol by Pseudomonas Fluorescens Oldrich K
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THE OXIDATIVE DISSIMILATION OF MANNITOL AND SORBITOL BY PSEUDOMONAS FLUORESCENS OLDRICH K. SEBEK' AND CHESTER I. RANDLES Department of Bacteriology, Ohio State University, Columbus, Ohio Received for publication October 29, 1951 Bacteria belonging to the genus Pseudomonas are endowed with the property of oxidizing many organic substances which, depending upon the strains used and the prevailing conditions, may result in completely or partially oxidized compounds. Several investigators (Lockwood and Nelson, 1946; Lockwood et al., 1941; Stanier, 1947a; Vaughn, 1942) emphasize that in some morphological and physiological characteristics the pseudomonads closely resemble the genus Aceto- bacter, the members of which are recognized as producers of industrially valuable compounds through the oxidation of polyhydric alcohols to their homologous sugars (Fulmer et al., 1939; Wells et al., 1939) or sugars to acids (Stubbs et al., 1940). In view of this similarity, it was deemed of inlterest to investigate whether the reactions brought about by Acetobacter can also be demonstrated in Pseudo- monas. Accordingly, a study was undertaken to elucidate the manner in which mannitol and sorbitol are oxidized and the pathways through which the result- ing intermediates are further dissimilated (Sebek and Randles, 1951). It was believed that the data obtained might provide further information on the rela- tionship between the genera Acetobacter and Pseudomonas and on the oxidative mechanisms possessed by strict aerobes. EXPERIMENTAL METHODS AND RESULTS In a preliminary survey the ability of seven strains of Pseudomonas, including P. fluorescens, P. aeruginosa, P. fragi, and P. graveolens, to utilize alcohols for growth and cell synthesis was examined. The cultures were streaked on mineral agar (0.15 per cent K2HPO4 -5H20, 0.1 per cent KH2PO4, 0.1 per cent NH4Cl, 2 per cent agar) supplemented with 1 per cent of the alcohol under study. The plates were incubated at room temperature (25-27 C), and the results were recorded after four days. The results are summarized in table 1. Similar results were obtained both after 7 days and 5 weeks when the cultures were inoculated in mineral salt solutions of pH 7.0 (composition as before, no agar added) with 1 per cent of the alcohols present. For further study P. fluo- rescens, strain B-10 NRRL, was selected since, unlike the other strains, it grew on both mannitol and sorbitol. This strain has also been reported to oxidize glucose to 2-ketogluconic acid (Lockwood et al., 1941). The suspensions were adjusted with distilled water to the same density on the Klett-Summerson col- orimeter. The 0.5 ml used corresponded to 3.2 to 3.7 mg of dry cells. Two , moles of substrate were used throughout. 1 Mary S. Muellhaupt Postdoctoral Research Fellow. 693 694 OLDRICII K. SEBEK AND CHESTER I. RANDLES [VOL. 63 Whenl grown in nutrient broth or inorganiic salt solutioni with 1 per cent sodium lactate, the cells showed nio significanit activ-ity oIn either mainnitol or sorbitol. However, wheni the organiism had beeni growvn in the presence of these compounids, the washed cell suspenisioni immediately and rapidly oxidized both alcohols to about 80 per cenit completion. At the same time, these adapted cells oxidized other substances at a more rapid rate than did cells grown in lnutrient brioth oIr lactate. The adaptive niature of these enzymes thus provided a conlvell- ient tool for following the couIrse of oxidatioin of mannitol aind sorbitol (Karlsson, 1947; Randles anid Birkelaind, 1947; Stanier, 1947b). Oni the basis of structural relationships the oxidation product of D-mannitol could be either D-mannose if the terminal primary alcoholic groups were attacked, or D-fructose should secondary alcoholic groups be oxidized in the 2 or 5 positioins. When mannitol-grown cells were allowved to dissimilate these sugars, mannose TABLE 1 Utilization of polyhydric alcohols by different species of Pseudornonas P. FLUO- P. FLUO- P. FLUO- P. FRAGI, P. GRAVE- P. AERU- P. AERU- SUBSTRATE RESCENS, RESCFNS, RESCENS, 25 OLENS, 14 GINOSA, GINOSA, NRRL NBR-R 64 OSU NRRL* NRRL* 439 OSU 274 OSU D-Sorbitol .0 + 0 0 0 0 0 D-Mannlitol ..... + + + 0 + + + D-DUlCitol .................. 0 0 0 0 0 0 meso-Inositol .............. + 0 0 0 0 0 Glycerol ........... + + + O = no growsth, + = growth. No growth occurre(l on control plates. * We are indelted to D)r. H. J. Koepsel of the -Northern Regional Research Laboratory, U. S. Departmenit of Agriculture, leoria, Illinois, for providing us with the cultures. was oxidized only slowly while fructose was oxidized much more rapidly than by cells grown in niutrient broth (figure 1). The rate of dissimilation of fiuctose was comparable to that of mannitol, thus inidicatiing that fructose was probably an oxidation product of this alcohol. Since the transieintly formed sugar was rapidly metabolized, several attempts to demonstrate it failed. Finally the use of resting cells was found satisfactory and enough material was isolated to permit the identificationi. Washed manrlitol- grown cells of a 20-hour culture were shakeni for 10 hours at room temperature in 150 ml of a 3 per cent a(ueous solution of mannitol containing 0.5 per cent CaCO3. At the end of this period the suspensioni was filtered, passed through cationic anid aniionic exchange resin columins ("amberlite" IR-120 and IRA-410), and evaporated under reduced pressure to a small volume. The resulting con- cenitrate reduced alkaline oxidizing- reageints and gave a positive Seliwanoff test showing that the unknowvni reducing sugar wvas a ketose. It was further evaporated to drynvess, redissolIved in a miniimum amounit of H20, anld diluted with 96 per cent ethaniol. It wvas separate(d from the initerferinlg residual mainnitol by repeated passages through a clay chromatographic column (Lew et al., 1946). The con- 1952] DISSIMILATION OF MANNITOL AND SORBITOL 695 centrated purified solution was tested by paper partition chromatography using phenol saturated with H20 as solvent and 3 , 5-dinitrosalicylic acid (0.5 g in 100 ml of 1 N NaOH) as developer. The RF value (0.51) of the unknown sugar was identical with that of fructose (Partridge, 1948). The melting point and the mixed mp of its phenylosazone were 205 C. Since no other sugars were detected by these methods, it was assumed that fructose was the only primary oxidation product of mannitol. It was concluded, therefore, that the oxidation of mannitol by the strain under study yielded fructose and that further oxidation proceeded through the fructose stage. The oxidation of D-sorbitol to the homologous sugar might be expected to yield D-glucose or L-gulose should primary alcoholic groups be attacked at the 220 220 200 200 180 A 180 B MANNITOL ISO0 160 FRUCoTOSE n 120 Yj 120 NO 02 UPTAKE w12l20/ I100 WITH MANNITOL a Dso 0 60 i 40 40 MANNOSE 20 FRUCTOSE 20 0 ~~~~~~~MANNOSE 10 2030 46 60 90 120 l0 20 30 46 60 90 120 MINUTES MINUTES Figure 1. Oxidation of mannitol, fructose, and mannose by cells of Pseudomonas fluores- cens grown in 0.016 per cent nutrient broth (A) and 0.016 per cent nutrient broth + 1 per cent mannitol (B); 2 , moles of the substrate; 0.067 M phosphate buffer (pH 7.2); endogenous respiration subtracted. 1 or 6 positions, respectively, or D-fructose or L-sorbose if the secondary alcoholic groups are oxidized at the 2 or 5 positions, respectively. These sugars were allowed to be acted upon by sorbitol-grown cells, and their ability to be dissimilated was determined. Gulose failed to be oxidized. Sorbose, surprisingly enough, also was not attacked and remained unaffected even by cells previously grown in the presence of sorbose. Consequently, both sugars were eliminated as potential intermediates in the oxidation of sorbitol. Fructose and glucose, however, were dissimilated at a rapid and essentially similar rate, suggesting that the oxidation of sorbitol may proceed through these sugars (figure 2). Further data showed that fructose-grown cells metabolized glucose at a rapid rate while glucose-grown cells did not produce an initial rapid oxidation of fructose (figure 3C). These results suggest that the enzyme normally oxidizing sorbitol may form fructose which could in turn be converted to glucose. 696 OLDRICH K. SEBEK AND CHESTER I. RANDLES [VOL. 63 In an attempt to determine the initial product of sorbitol oxidation, the technique used in the isolation and identification of fructose from mannitol was used. The substance obtained reduced alkaline oxidizing reagents and gave a positive Seliwanoff test. In view of the results obtained with the adaptation technique, it was expected that fructose would be isolated. The isolated sugar, however, was not dissimilated by sorbitol-grown cells and its RF value (0.41) corresponded to that of sorbose (Partridge, 1948). The melting point of its phenylosazone (169 to 171 C) also indicated that the unknown ketose was sorbose (mp 168 C). An identical substance was also obtained when sorbitol was oxidized by cells grown in mannitol. This phenomenon is in agreement with our findings (Randles and Sebek, unpublished) that sorbitol-grown cells adapt to mannitol as well as to sorbitol and that a mannitol oxidizing enzyme accounts not only for 220 20, 200 zo ISO AIgosFRCO 140 NO ON UPTAKE WITH ui140,T GC5. L&i SORSITOL, SORSOSE 4% 120 AND GULOSE I.20SOB.O 0. 0 00 )0 GLUCOSE AND ± / ~~~~SORSOSE SULOSE 0C4 FRUCTOSE 10 2020o 45 50 5 1 1 00 (0t- VR z MINUTES MINUTE S Figure 2. Oxidation of sorbitol, fructose, glucose, sorbose, and gulose by cells of Pseudo- monas fluorescens grown in 0.016 per cent nutrient broth (A) and 0.016 per ce&i nutrient broth + 1 per cent sorbitol (B); 2 g moles of the substrate; 0.067 m phosphate buffer (pH 7.2); endogenous respiration subtracted.