The Oxidative Dissimilation of Mannitol and Sorbitol by Pseudomonas Fluorescens Oldrich K

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 resulting 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. fluorescens, 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

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ISO0 160 FRUCoTOSE n

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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 fluorescens 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

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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. the oxidation of mannitol to fructose but can act in much the same way upon sorbitol, yielding sorbose. This would be expected if the enzyme possesses the specificity indicated by Bertrand's rule for Acetobacter. No evidence for phosphorylation of mannitol or sorbitol was obtained since adenosinetriphosphate (Na salt, Armour, 0.02 m final concentration per cup) incubated anaerobically (5 parts Of CO2 and 95 per cent of N2) with these sub- strates did not result in CO2 evolution from bicarbonate buffer (0.028 m, pH 7.2). The dissimilation of both alcohols as well as glucose and fructose was essentially the same both in the rate and extent of oxidation, whether phosphate or borate buffers (pH 7.2) were involved. Fructose-1, 6-phosphate was not dissimilated to an appreciable extent, 8 AtL02 being taken up per 2 Au moles of substrate in 180 minutes. NaF (0.05 to 0.1 m), which inhibits the transformation of phospho- glyceric into phosphopyruvic acids, did not influence either the rate or the total amount Of 02 taken up with these compounds. Under anaerobic conditions, as described before, no CO2 evolution occurred from the bicarbonate buffer. When 1952] DISSIMILATION OF MANNITOL AND SORBITOL 697 adenosinetriphosphate (0.02 M final concentration per vessel) was added to the cell suspensions under conditions such as these, no CO2 evolution was observed with either fructose or glucose. These results indicate that glucose and fructose may not be phosphorylated prior to further dissimilation and that the initial stages of glycolysis, at least, do not take place in the strain under study. In an attempt to determine what the initial stages in the oxidation of these sugars by P. fluorescens might be, further investigation was undertaken. Cells of P. fluorescens were grown in media containing 0.016 per cent nutrient broth to which fructose, glucose, gluconic and 2-ketogluconic acids were sys-

A B C D E 90 Go 80 G G G

w 60 F ~50 /GA

~20 F // 7 2-KGA / .. 10 __F_ 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 MIN UT E S Figure S. Oxidation of glucose (G), fructose (F), gluconic acid (GA), and 2-ketogluconic acid (2-KGA) by the cells of Pseudomonas fluorescens grown in 0.016 per cent nutrient broth (A), and in 0.016 per cent nutrient broth plus glucose (B), fructose (C), gluconic acid (D), and 2-ketogluconic acid (E); 2 u moles of the substrate; 0.067 M phosphate buffer (pH 7.2); endogenous respiration subtracted. tematically added. The washed young harvested cells were handled as described before. The rates of 02 uptake by these cells (figure 3) demonstrated that fructose- grown cells were adapted also to glucose and gluconic but not to 2-ketogluconic acid. Cells grown in glucose failed to adapt to fructose and 2-ketogluconic acid while the dissimilation of gluconic acid started immediately and was rapid. Should the conclusions of simultaneous adaptation be applied, then glucose and gluconic acid would be normal oxidation products of fructose, but 2-ketogluconic acid would not be the substance through which the main dissimilation pathway would proceed. However, when gluconic acid was partially oxidized by gluconic acid-grown cells, 2-ketogluconic acid was identified in the concentrate by its positive reaction with conventional alkaline oxidizing reagents and by paper 698 OLDRICH K. SEBEK AND CHESTER I. RANDLES [VOL. 63 partition chromatography, usinlg a methaniol-ethanol mixture as solvent and an ammoniacal solutioni of AgNO3 as developer (Norris anid Campbell, 1949). Experimenits designied to demonistrate the formationi of phosphorylated compounds of gluconic and 2-ketogluconic acids with adenosinetriphosphate unlder anaerobic conditions as described before were negative. DISCUSSION The oxidationi of manniiitol aiid sorbitol by P. fltuorescens may be tentatively pictured by the followinig scheme: Mannitol Fructose Sorbitol Sorbose CH20H CH.OH CH20H CH,OH HOCH C=0 -2H HC0H HCOH HOCH HOCH HOCH HOCH H1OH1 HCOH HCOH H0OH HCOH HCOH HC0H C-=O CH.00 CH,OH 1CH,OH0 CH.,OH

GI itcose

Gluconic acid

2-Ketogluconic acid The initial stages by which Pseadomonas oxidizes manniiitol to fructose and sorbitol to sorbose point to the mechanism kniowni to operate in Acetobacter. The elaborationi of the identical enizyme system may serve as aniother example of the close similarity of both genera. Since it has beeni demonstrated that not onily sorbitol but also mainnitol-growni cells oxidize sorbitol to the same compound (sorbose), it seems probable that the production of sorbose is due to the action of an enzyme normally oxidizing manniiitol to fructose and that this enizyme possesses a specificity corresponiding to that of Bertrand's rule for Acetobacter. Since the sorbose formed proved to be physiologically inactive, it was evident that P. fltorescens uses aiiother mechaniism through which the oxidation of sorbitol is niormally directed. Adaptationi studies suggested fructose as the most probable compound wvhich would in turn be converted to glucose before further oxidation takes place. Neither sugar, however, was isolated and identified. Gluconic acid is unquestioniably the product resulting from oxidation of glucose and is further oxidized to 2-ketogluconic acid (Norris and Campbell, 1949; Stokes and Campbell, 1951). The 2-ketogluconic aci(l may accumulate or be slowly further dissimilated. An identical finidinig w-as also reported by Enitner 1952] DISSIMILATION OF MANNITOL AND SORBITOL 699 and Stanier (1951) who reasoned that not 2-ketogluconic acid itself but rather its derivative is a member of the main pathway or that two divergent pathways exist for glucose dissimilation, one of which proceeds via 2-ketogluconic acid. Our experiments failed to reveal the formation of phosphorylated compounds in the initial stages of dissimilation of mannitol and sorbitol. These results are in agreement with those of Barker and Lipmann (1950) who worked with Propionibacterium pentosaceum. Absence of phosphorylation prior to oxidation of the sugars was reported for P. aeruginosa by Norris and Campbell (1949). Our studies as well as those of Norris and Campbell were conducted with whole cells. Under these conditions, it is possible that adenosinetriphosphate could not enter the cells, which fact might account for the recorded absence of phosphorylated compounds in initial stages of oxidation. Stokes and Campbell (1951), however, failed to demonstrate phosphorylation even with dried cells of P. aeruginosa which would be expected to have greater permeability than living cells and thus allow adenosinetriphosphate to react.

SUMMARY By application of the techniques of simultaneous adaptation, paper chromatography, and chemical determination, it has been demonstrated that mannitol and sorbitol are normally oxidized by different adaptive enzymes of Pseudomonas fluorescens to fructose. The enzyme attacking mannitol apparently is not specific for mannitol and can oxidize sorbitol to sorbose which is not further dissimilated. The further oxidation of fructose probably proceeds through glucose and gluconic acid to 2-ketogluconic acid. No evidence has been found for the formation or participation of phosphorylated derivatives of any of these compounds. REFERENCES BARKER, H. A., AND LIPMANN, F. 1950 The role of phosphate in the metabolism of Pro- pionibacterium pentosaceum. J. Biol. Chem., 179, 247-257. ENTNER, N., AND STANIER, R. Y. 1951 Studies on the oxidation of glucose by Pseudo- monas flutorescens. J. Bact., 62, 181-186. FULMER, E. I., DUNNING, J. W., AND UNDERKOFLER, L. A. 1939 The effect of concentration of mannitol upon the production of levulose by the action of Acetobacter suboxy- datns. Iowa State Coll. J. Sci., 13, 279-281. KARLSSON, J. L. 1947 The use of induced physiological mutants to study the energy metabolism of Azotobacter agilis. Dissertation, Univ. of California. LEW, B. M., WOLFROM, M. L., AND GOEPP, R. M., JR. 1946 Chromatography of sugars and related polyhydroxy compounds. J. Am. Chem. Soc., 68, 1449-1453. LOCKWOOD, L. B., AND NELSON, G. E. NI. 1946 The oxidation of pentoses by Pscudcnionas. J. Bact., 52, 581-586. LOCKWOOD, L. B., TABENKIN, B., AND WARD, G. E. 1941 The production of gluconic acid and 2-ketogluconic acid from glucose by species of Pseudomonas and Phytomonas. J. Bact., 42, 51-61. NORRIS, F. C., AND CAMPBELL, J. J. R. 1949 The intermediate metabolism of Pseudo- monas aeruginosa III. The application of paper chromatography to the identification of gluconic and 2-ketogluconic acids, intermediates in glucose oxidation. Can. J. Research, C, 27, 253-261. 700 OLDRICH K. SEBEK AND CHESTER I. RANDLES [VOL. 63

I'ARTRIDGE, S. 'M. 1948 Filter-paper partitioIn chromatography of sugIars. Bioclhenm. J., 42, 238-250. RANDLES, C. I., AND13IRKELAND), J. M\. 1947 The effects of mialic and maloniic ticids oII methvlene blue reduction bv bacteria. J. Bact., 54, 275. SEBEK, 0. K., AND) RANDLES, C. I. 1951 The oxi(dation of polyhydric alcohols by PscI(lo- Moonas. Bact. Proc., 1951, 137-138. STANIER, R. Y. 1947a Acetic acid productioll from ethiainol by fluoreseniit pseudomona(ls. J. Bact., 54, 191-194. STANIER, R. Y. 1947b Simultaneous adaptation: a new technii(lue for the study of mIeta- bolic plathwvays. J. Bact., 54, 339-348. STOKES, F. C., AND CA-MPBELL, J. J. R. 1951 Thei oxidationi of glucose and gluconiciacid by dried cells of Pseuidoo0iontas aeriyqinosa. Arch. Biochem., 30, 121-125. STUBBS, J. J., LOCKWOOD, L. B., ItOE, E. T., TABENKIN, B., AND WARD, G. E. 1940 Keto- gluconic acids from glucose. Ind. EnIg. Cheii., 32, 1626-1631. VAUGHN, R. II. 1942 The acetic acid bacteria. Wallerstein Labs. Comm., 5, 5-26. WELLS, 1. A., LOCKWOOD, L. B., STIUBBS, J. J., Rot:, E. T., PORGE:S, N., A-ND GASTROCK, E. A. 1939 Sorbose from sorbitol. Semiplant-scale production by .4cetobacter solb- oxiydans. Ind. Eng. Chem., 31, 1518-1521.