Cleveland, Ohio. "adaptive." A similar phenomenon was observed in E. coli. Here the enzymes

Transcription

1 STUDIES ON THE SPECIFICITY OF THE FERMENTATION OF PENTOSES BY LACTOBACILLUS PENTOSUS' J. 0. LAMPEN AND H. R. PETERJOHN2 Department of Microbiology, School of Medicine, Western Reserve Univer8ity, Cleveland, Ohio Received for publication May 10, 1951 Lactobacillus pentosus, strain 124-2, was isolated from sauerkraut by Fred, Peterson, and coworkers (1919, 1921) during their studies on the fermentation of pentoses. They observed that the organism is a homofermentative lactic acid bacterium and under the proper conditions performs essentially the following conversions: Glucose -* 2 lactic acid D-xylose or L-arabinose -+ 1 acetic acid + 1 lactic acid These results were obtained when the organism was growing in a yeast-water medium containing glucose, xylose, or L-arabinose as the carbohydrate. The specificity of pentose fermentation by washed, resting cells was studied first by Karstrom (1938) who used the organisms Betacoccus arabinosaceus (Leuconostoc mesenteroides), Lactobacillus pentoaceticus, and Escherichia coli. His procedure was to grow cells on a medium containing only a single sugar, wash these cells, and subsequently test their ability to ferment various sugars. With B. arabinosaceus he observed that all cells, irrespective of the sugar on which they had been grown, were able to ferment glucose. Only cells raised on L-arabinose fermented L-arabinose. He considered the enzymes involved in the fermentation of glucose to be "constitutive," those for arabinose he termed "adaptive." A similar phenomenon was observed in E. coli. Here the enzymes fermenting xylose and arabinose were "adaptive," those fermenting glucose again "constitutive." Cohen and Raff (1951) have recently reported that the ribose-fermenting enzymes of E. coli are adaptive as well. Karstrom observed that the reverse situation occurred in L. pentoaceticus, i.e., the enzymes acting on xylose and arabinose were constitutive, whereas those acting on glucose were adaptive. It is of interest to note with all of these organisms that growth on a given pentose did not result in cells able to ferment the other pentoses tested. Ribose has been tested only with E. coli, however. The mechanism of the fermentation of _-Cl4-D-xylose by L. pentosus has been under investigation in this laboratory (Lampen, Gest, and Sowden, 1951). 1 This paper is based upon work sponsored in part by the Biological Department, Chemical Corps, Camp Detrick, Maryland, under Contract No. W CM-241 with Western Reserve University. 2 Crile Fellow in Microbiology. 281

2 282 J. 0. LAMPEN AND H. R. PETERJOHN [VOL. 62 During these studies it was observed that washed resting cells grown in a medium containing glucose as the carbohydrate did not form acid from xylose. Cells raised in a xylose medium attacked xylose rapidly. In the present communication we are reporting detailed data on the fermentative specificity of cells raised on various sugars. This study was undertaken to define conditions for raising cells of desired fermentative types. Data are also offered indicating an interrelation of the degradative pathways of the various pentoses. METHODS The culture of Lactobacillus pentosus, strain 124-2, used in these experiments was obtained from Dr. Elizabeth McCoy of the Department of Bacteriology, University of Wisconsin. Stock cultures were maintained as stabs in the medium described here with 2 per cent agar added. These were stored at 4 C and transferred monthly. The medium contained the following (per 100 ml): 0.4 g Difco yeast extract, 1.0 g Difco Nutrient Broth, 1.0 g sodium acetate, 1.0 g of the proper sugar, 0.02 g MgS4O7H20, g NaCl, g FeS04*7H20, and g MnSO4-4H20. In order to prevent charring of the medium the sugars were autoclaved separately as 10 per cent solutions and added aseptically to the sterilized medium. All incubations were at 37 C. To prepare cell suspensions for the various experiments, transfers were made from the stab culture to the glucose medium and then to one containing the desired sugar. Five ml of the latter culture were added to 100 ml of the identical medium, and after 24 hours this entire culture was used to inoculate 1 liter of medium. With glucose as the carbohydrate the yield of cells was about 5 g (wet weight) per liter in 24 hours, with xylose the yield was about 2 g per liter in 48 hours, and with ribose and L-arabinose about 1.5 g in 48 hours. The cells were harvested at 4 C, washed with 0.02 M NaHCO3, and suspended in either 0.01 M or 0.02 M NaHCO3. The experiments presented in figures 1, 2, and 4 were run in Warburg flasks with a volume of approximately 15 ml. The small yield of cells obtained with the pentoses made it necessary to use flasks with a volume of 9 ml for the other experiments in order to conserve material. The cell suspension was placed in the main compartment of the Warburg flask and the substrate solution in one side arm. After equilibration with 5 per cent C02-95 per cent N2 at 30 C, the substrate was tipped into the main compartment and readings taken at intervals. The second side arm received 0.1 ml of 5 N H2S04 which was tipped into the main compartment at the end of the experiment if an analysis for residual bicarbonate was desired. The endogenous acid production was negligible (e.g., 18,l in 6 hours for the cells used in the series shown in figure 1); however, the data have been corrected for these changes. Cell-free extracts were prepared by grinding with alumina and by sonic treatment. The alumina procedure3 was that of McIlwain (1948). The ground cell paste was extracted with 5 ml of 0.05 M phosphate buffer (ph 7.0) per gram of original wet cells. For the sonic treatment 1 ml of buffer was added per gram of 3Alumina A-303, supplied by the Aluminum Company of America, Pittsburgh, Pennsylvania, was used.

3 1951] SPECIFICITY OF FERMENTATION OF PENTOSES 283 wet cells and the suspension placed in a magnetostriction sonic oscillator (Type R-22-3 of the Raytheon Manufacturing Company). Rupture of the cells was almost complete in 1 hour. All extracts were centrifuged at 4,OOOX g for 10 minutes to remove cell debris. Pentose was determined by the method of Mejbaurm (1939). The hypoxanthine desoxyriboside was provided by Dr. L. A. Manson, and the D-xylose-5-phosphate and D-arabinose-5-phosphate were from the Levene Collection of the Rockefeller Institute for Medical Research, New York. Inosine was prepared by the method 0 CD MINUTES Figure 1. Acid production by cells grown on glucose. Each vessel contained 2.0 ml of the cell suspension (12 mg dry weight per ml) in 0.01 M NaHCO3 and 0.2 ml of substrate solution. 0, 0.1 M D-glucose; 0, 0.3 M D-xylose; A, 0.3 M D-ribose; 0, 0.3 M L-arabinose. of Levene and Tipson (1935) and ribose-5-phosphate according to Albaum and Umbreit (1947). The other substrates were commercial samples. RESULTS Cells grown on glucose. Figure 1 shows the acid production with various substrates. Glucose was rapidly utilized whereas xylose and L-arabinose were almost completely inert. Acid production occurred at a low but significant rate with free ribose and with two ribose nucleosides, inosine and uridine (cf. Cells grown on ribose).

4 284 J. 0. LAMPEN AND H. R. PETERJOHN [VOL. 62 It seemed possible that the ability of these cells to attack ribose was due to the small amounts of ribose derivatives present in the yeast extract of the medium. To test this, a quantity of cells was raised on a synthetic medium free of ribose-containing compounds.4 Such cells showed the same moderate activity with ribose, inosine, and uridine as did cells grown on the medium containing yeast extract. Therefore, the ability of these cells to ferment ribose does not depend on the presence of added ribose in the medium. It may be noted from figure 1 that acid production from xylose was insignificant even after 6 hours; that is, the organism did not "adapt." It was thought O l60 lwtnutes Figure 2. Acid production by cells grown on xylose. Each vessel contained 2.0 ml of the cell suspension (10 mg dry weight per ml) in 0.02 M NaHCOs and 0.2 ml of substrate solution. 0, 0.1 M glucose; 0, 0.3 M xylose; A, 0.3 M ribose; *, 0.3 M L-arabinose. that a source of energy might be required for adaptation. This might be provided by the fermentation of a small amount of glucose. To test this, 3 ;um of glucose were added to one Warburg vessel, 30,uM of xylose to a second vessel, and 3 pm of glucose and 30,4M of xylose to a third. The other experimental conditions were those given in figure 3. In the first cup, 116 pl of C02 were produced within 10 minutes, 5,ul in the second, and 114,1 in the third. No further acid production (<5 IAI) occurred in any cup within 2 hours. Thus the cells did not acquire the ability to degrade xylose under these conditions. 4Medium B of Krueger and Peterson (1948) to which guanine and uracil had been added at concentrations of 10 p&g per ml.

5 1951] SPECIFICITY OF FERMENTATION OF PENTOSES 285 Cells grown on xylose. Several interesting features were noted with these cells. As shown in figure 2, the rate of fermentation of ribose was equal to that with glucose or xylose. The rate with L-arabinose was considerably less than with the other pentoses. The effect of substrate concentration on the rate of acid formation is illustrated by the experiment shown in figure 3. The initial rate was identical when 5, 10, or )M on XYLOSE 5pM RIBOSE MINUTES Figure 3. Effect of substrate concentration on the rate of acid production by cells grown on xylose. Each vessel contained 1.0 ml of the cell suspension (10 mg dry weight per ml) in 0.02 M NaHCO, and 0.1 ml of substrate solution containing the quantities shown on the graph. 30 Mm of ribose were present per 1.1 ml. The values obtained with 10 Mm of ribose yield a curve superimposable on that with 30 Mm. In contrast the initial rate with 5 um of xylose was two-fifths that found with 30 gm of xylose. The results with glucose were similar to those shown for ribose. It was observed that 9.4 Mm of acids were produced from 5 Mm of xylose and 9.5 um of acid from 5 Mm of ribose. These data agree well with the previously reported production of 1 mole each of acetic and lactic acids per mole of pentose.

6 286 J. 0. LAMPEN AND H. R. PETERJOHN [VOL. 62 The stability of the enzymic activity during storage at 4 C was studied. The cells were tested on glucose, xylose, and ribose at various intervals with the results shown in figure 4. After 24 hours' storage the rates with xylose or ribose had decreased slightly. With glucose, acid production occurred only after a lag period.' After 96 hours' storage the cells fermented all three substrates at reduced rates, but xylose was utilized at a rate definitely higher than that observed co 0 0 MINUTES Figure 4. Effect of storage at 4 C on acid production by cells grown on xylose. Experimental details as in figure 3. 0, 0.1 M glucose; 0, 0.3 M xylose; A, 0.3 m1 ribose. with ribose or glucose. This series of events has been noted in several tests. In one experiment the cells were almost completely inactive on ribose after 96 hours' storage, while retaining good activity on xylose. In contrast to these results with cells grown on xylose, cells from a glucose medium retained about 75 per cent of their original activity on glucose after storage at 4 C for 96 hours. X It should be noted that acid production stopped at about 1 mole per mole of sugar (19.7 pm C02 released per 20 Nm of glucose). This reduced yield of acid by stored cells has also been observed in experiments with pentoses. This phenomenon is under investigation. 180

7 1951] SPECIFICITY OF FERMENTATION OF PENTOSES 287 Cells grown on ribose. Glucose and ribose are attacked readily by such cells (figure 5). Acid production is slow with L-arabinose and almost absent on xylose. Acid production from equivalent concentrations of ribose and its nucleosides is shown in figure 6. The rate with ribose is about twice that observed with the nucleosides. Cells raised on glucose, xylose, or arabinose were also tested. In all instances the rate with ribose was greater than that with the nucleosides. It is of interest, however, that the nucleosides were attacked much more rapidly by cells raised on the pentoses than by cells raised on glucose. Thus the activities on ribose and on its nucleosides vary in a similar manner with the sugar present during growth. These supplementary data will not be presented in detail * 300/ O 180/ O30b , MINUTES Figure 6. Acid production by cells grown on ribose. Experimental details as in figure 3 (7 mg dry weight of cells per ml of suspension). 0, 0.1 M glucose; 0, 0.3 M xylose; A, 0.3 M ribose; *i 0.3 M L-arabinose. Cells grown on L-arabinose. These cells utilized glucose, ribose, or L-arabinose rapidly, xylose to a negligible extent. Representative data are shown in figure 7. Experiments with cell-free extracts. Extracts prepared by the alumia procedure from cells grown on glucose or xylose had only weak pentose-degrading activity. Free ribose and xylose-5-phosphate were not degraded. Ribose-5-phosphate was degraded slowly by an extract of xylose-grown cells (table 1). In similar experiments with extracts of glucose-grown cells the disappearance of pentose was within the experimental error of the pentose determination. Highly active pentose-degrading extracts were obtained by the sonic procedure. Typical data are presented in tables 1 and 2. Free ribose was not me-

8 0-140/ MINUTES Figure 6. Rate of acid production from ribose and ribose nucleosides by oells grown on ribose. Experimental details as in figure 5. *, 0.1 m ribose; X, 0.1 m inosine; A, 0.1 M undine o MINUTES Figure 7. Acid production by cells grown on L-arabinose. Experimental details as in figure 3. 0, 0.1 M glucose; 0, 0.3 M xylose; A, 0.3 M ribose; 0, 0.3 M L-arabinose. 288

9 1951] SPECIFICITY OF FERMENTATION OF PENTOSES 289 TABLE 1 Action of cell-free extracts of Lactobacillus pentosus on ribose and on pentose phosphates Each incubation mixture contained the substrate, 0.5 ml of the enzyme preparation (equivalent to 250 mg of wet cells), and 1.0 ml of 0.1 M NaF phosphate buffer, ph 7.0. Final volume = 4.0 ml. At the indicated times samples were deproteinized by adding an equal volume of 4 per cent perchloric acid. The protein-free filtrates were analyzed for pentose. All values have been corrected for the small changes observed in a parallel incubation without substrate. INC rodt TYPE OF EXTRACT SUBSTRATE #M OF PENTOSE 0 hr 0.5 hr 2 hr 5 hr Glucose Sonic Ribose Sonic Ribose-5-phosphate Xylose Alumina* Ribose-5-phosphate Sonic Ribose Sonic Ribose-5-phosphate Sonic Xylose-5-phosphate * 1.0 ml of enzyme preparation equivalent to 200 mg wet cells was used. TABLE 2 Degradation of ribose-5-phosphate by extracts of Lactobacillus pentosus Each incubation mixture contained 2.9 pm of ribose-5-phosphate, a volume of a sonic extract equivalent to 150 mg of wet cells, 0.5 ml of 0.1 m NaF phosphate buffer (ph 7.0), and 0.1 ml of 1 M NaF in a final volume of 2.5 ml. Other details as in table 1. #M PENTOSE CARBOHYDRATE IN MEDIUM_- 0 mm 30 min 2 hr 3 hr 5 hr Glucose Xylose Ribose Arabinose TABLE 3 Specificity of fermentation by Lactobacillus pentosus CARBOHYDRATE IN MEDIUM RATE OF SUBSEQUENT ACID PRODUCTION FROM D-glucose D-riboSe D-Xylose L-arabinose D-Glucose * + D-Ribose _- + D-Xylose L-Arabinose * Rates are graded from -, no fermentation, to ++++, maximum rate obtained with given cell type. tabolized by any of the extracts under the conditions used. Ribose-5-phosphate was degraded rapidly by the extract of xylose-grown cells, but xylose-5-phosphate

10 290 J. 0. LAMPEN AND H. R. PETERJOHN [VOL. 62 was not attacked. An extract prepared by 2 hours' treatment in the sonic oscillator had approximately the same activity as did those treated 1 hour. The enzyme complex degrading ribose-5-phosphate appears to be in solution since the activity remained in the supernatant after 30 minutes' centrifugation at 20,OOOX g. Extracts prepared from cells grown on glucose utilized ribose-5- phosphate only very slowly. This "activation" of the ribose-5-phosphate degrading system observed as a consequence of growth on xylose was found to occur during growth on ribose or arabinose as well. The data of table 2 demonstrate that extracts from cells grown on the various pentoses are of comparable activity and are all much more active than is the extract of cells grown on glucose. These extracts were also incubated with xylose-5-phosphate and arabinose-5- phosphate. The total pentose in the mixtures remained constant within experimental error. Slight increases in pentose were observed in parallel incubations without added substrates, hence degradation of approximately 10 per cent of the added pentose may have occurred in 5 hours. These results can be considered as essentially negative. It should be noted that the presence of the xylose or arabinose esters did not prevent the degradation of equimolar amounts of ribose- 5-phosphate by the extracts. DISCUSSION The data on the rates of fermentation are summarized in table 3. The constant presence of the enzymes for glucose fermentation and the ability to ferment the sugar present during growth have been observed numerous times in such studies (Karstrom, 1938). Activation of the ribose-fermenting system by growth in the presence of other pentoses has not to our knowledge been previously reported. Cells grown on each of the sugars were -also tested with D-arabinose and hypoxanthine desoxyriboside. None of the cell types formed acid from these substrates. The differences noted here could be the result of permeability changes during growth of the cells under the various conditions. More likely, we feel, is the possibility that they represent changes in the enzymic composition of the cells. The only direct evidence at present on this point is the fact that extracts of cells grown on pentoses degrade ribose-5-phosphate, whereas extracts of cells grown on glucose do not do so at a significant rate. It is planned to continue this study of the differences among cell-free extracts of these cells in an attempt to correlate these differences with the specificities observed with the whole cells. The data presented here can probably be explained most simply by assuming that xylose and L-arabinose are converted to ribose derivatives by L. pentosus before cleavage of the pentose chain occurs. This would explain the correlation between growth of the cells in pentose media and the ability of extracts prepared from these cells to degrade ribose-5-phosphate. Small amounts of free ribose might form during this process and bring about the "adaptation" to ribose. However, free ribose may not be an essential intermediate in these conversions since one can readily obtain cells whose rate of acid production from xylose is at least twice that from ribose (figure 4). If this assumption is correct, then adapta-

11 1951] SPECIFICITY OF FERMENTATION OF PENTOSES 291 tion to free ribose is not an essential feature of the over-all mechanism. The data obtained with E. coli (Cohen and Raff, 1951) could therefore still be consistent with this idea. The exact nature of the genetic and biochemical changes occurring during these "adaptations" is not known. There is no evidence at present to indicate whether all the cells can gain the ability to ferment a given sugar, or only a fraction of the original population is selected by these procedures. It would be desirable to study this question by plating cell suspensions on media containing the individual sugars. This has not yet been done since L. pentosus will not grow with xylose as the carbon source on a synthetic medium (Krueger and Peterson, 1948) satisfactory for growth with glucose.6 The existence of a "xylose factor" makes it necessary to add a supplement such as yeast extract to the medium when pentoses are used. Traces of sugars present in such a supplement might well invalidate the results of a plating experiment. The nature of this special requirement for growth on xylose is under investigation. ACKNOWLEDGMENTS The authors are indebted to Miss Barbara J. Danielson and Mr. George 0. Carrington for technical assistance. SUMMARY Lactobacillus pentosus was grown with various sugars as its energy source. Rates of acid production from glucose, pentose sugars, and inosine and uridine have been determined with washed, resting cells. All cells formed acid rapidly from glucose and from the sugar present in the growth medium. Cells raised on xylose or L-arabinose fermented ribose rapidly; cells raised on ribose utilized the other pentoses poorly if at all. All cells studied utilized free ribose more rapidly than either inosine or uridine. None of the types of cells fonned acid from D-arabinose or hypoxanthine desoxyriboside. Ribose-5-phosphate was degraded rapidly by sonic extracts of cells grown on pentoses, only slowly by extracts of cells from a glucose medium. None of the extracts degraded xylose-5-phosphate, arabinose-5-phosphate, or free ribose. The mechanism of pentose degradation by L. pentosus is discussed. It is suggested that the fermentation of D-xylose and L-arabinose occurs via ribose phosphates. REFERENCES ALBAIUM, H. G., AND UMBREIT, W. W Differentiation between ribose-3-phosphate and ribose-5-phosphate by means of the orcinol-pentose reaction. J. Biol. Chem., 167, COHEN, S. S., AND RAFF, R Adaptive enzymes in the estimation of gluconate, D-arabinose, and D-ribose. J. Biol. Chem., 188, FRED, E. B., PETERSON, W. H., AND DAVENPORT, A Acid fermentation of xylose. J. Biol. Chem., 39, Lampen, J. O., unpublished observations.

12 292 J. 0. LAMPEN AND H. R. PETERJOHN [VOL. 62 FRED, E. B., PETERSON, W. H., AND ANDERSON, J. A The characteristics of certain pentose-destroying bacteria, especially as concerns their action on arabinose and xylose. J. Biol. Chem., 48, KARSTROM, H Enzymatische Adaptation bei Mikroorganism. Ergeb. Enzymforsch., 7, KRUEGER, K. K., AND PETERSON, W. H The nutritional requirements of Lactobacillus pentosus J. Bact., 55, LAMPEN, J. O., GEST, H., AND SOWDEN, J. C Observations on the mechanism of fermentation of 1-C'4-D-xylose by Lactobacillus pentosus. J. Bact., 61, LEVENE, P. A., AND TIPSON, R. S The partial synthesis of ribose nucleosides. II. Muscle inosinic acid. J. Biol. Chem., 111, McILwAIN, H Preparation of cell-free bacterial extracts with powdered alumina. J. Gen. Microbiol., 2, MEJBAUM, W Uber die Bestimmung kleiner Pentosemengen, insbesondere in Derivaten der Adenylsaure. Z. physiol. Chem., 258, Downloaded from on November 11, 2018 by guest