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Xylose metabolism in genetic variants of Salmonella typhosa
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSlCS
66,
128-139
(1967)
Xylose Metabolism in Genetic Variants of Salmonella typhosa E. S. Kline1 and L. S. Baron From the Division of Immunology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C.
Received May 2, 1956 INTRODUCTION It has been observed in enteric bacteria that the ability to utilize various pentoses could readily be transferred to bacterial strains previously unable to metabolize them (1, 2). This phenomenon, termed genetic transduction, involves the addition of hereditary determinants from one strain to another through the intervention of bacteriophage. Thus, it has been assumed that a bacterial strain incapable of using xylose may, by means of transduction, gain the ability to synthesize an enzyme or enzymes which would then allow this pentose to be metabolized. Confirmation of this hypothesis is dependent on the elucidation of the metabolic route of pentose utilization in the organism prior to and after transduction. It has been postulated that this synthetic ability is controlled by heritable factors transferred by the bacteriophage to the recipient bacterial strain. As part of a study of the metabolism of pentoses in enteric bacteria, strains of Salmonella typhosa that are positive for various pentoses were obtained by this method of genetic transfer. Aided by such transductions, one is provided with substrains which can facilitate comparative studies in metabolism. Recent studies on pentose metabolism have served to clarify much of the controversy regarding the mechanism by which xylose and other pentoses are utilized. Phosphorylation studies made by Lampen and Peterjohn (3) led these workers to believe that ribose 5phosphate was pentosus. They an intermediate of xylose degradation in Lactobacillus 1 Taken from a thesis submitted by E. S. Kline quirements for the degree of Master of Science, George Washington University. 128
in partial fulfillment of the reDepartment of Biochemistry,
XTLOSE METABOLISM
129
could find no evidence to indicate that either xylose 5-phosphate or arabinose 5-phosphate could be metabolized by xylose-grown cells. Cohen has demonstrated the existence of a pentose isomerase which catalyzes the conversion of D-arabinose to n-ribulose (4). Papers by Lampen and co-workers (5, 6) showed that extracts of L. pentosus could isomerize n-xylose to n-xylulose. Lampen also post’ulated that a xylulose kinase was present by studying the reaction of this sugar with adenosine triphosphate (XTP) (7). Hochster and Watson then demonstrated that xylose-grown cells of Pseudomonas hyclrophilia possessed an isomerase which converted n-xylose to n-xylulose (8). Evidence was also obtained for the formation of the phosphate esters of xylulose, ribulose, ribose, and sedoheptulose (9). Ashwell and Hickman (10) have demonstrated the conversion of ribose 5-phosphate to xylulose 5-phosphate in spleen extracts and have indicated that a 3-ketopentose may be involved in this system (11). The isolation of xylulose 5-phosphate from a bacterial source was first accomplished by Stumpf and Horecker (12). This report is concerned with cerbain aspects of xylose metabolism in strains of S. typhosa which by virtue of their genet,ic structure offer an opportunity for comparative study. The strains were chosen to study both the nature of the enzymatic changes incurred following transduction and the route of xylose degradation in enteric bacteria. RlaTE~1a~s
AND METHODS
Materials Two strains of 8. lyphosa were examined: strain 643, a xylose-negative strain, and a xylose-positive substrain (643T) isolated from a phage-treated suspension of strain 643 following a transduction experiment. Xylose, arabinose, ribose, and other aldol sugars were commercial preparations. Where necessary, they were further purified by recrystallization from ethanol. ATP (barium or disodium) was obtained from blann Research Laboratories, Inc. or Nutritional Biochemicals, Inc. The barium salt was converted to the disodium salt prior to use. In most cases, the pH of the ATP was adjusted to pH 6.8 with KOH. It was stored in the frozen condition, but not longer than 24 hours. n-Xylulose was prepared from D-XylOSe by the method of Schmidt and Treiber (13). This preparation was not free of unepimerized n-xylose and other pentoses. Where required, this preparat,ion was treated nith bromine at room temperature for 30 min. in order to oxidize t,he contaminating aldopentoses. Solid barium carbonate was added to maintain a pH of 7.0. Excess barium carbonate was removed by centrifugation; much of the soluble ions were removed by further precipitations. The products of the bromine oxidation were eliminated by passage through anion-exchange resins, either IRA-400 (HCO,- form) or Dowex 1 (HCO,form). The material which was obtained following this treatment was essentially
130
E. S. KLINE AND L. S. BARON
free of ddopentoses. Pure n-xylulose was also kindly supplied by Dr. A. Weissbach and by Dr. G. Ashwell, National Institutes of Health. n-Ribulose was prepared by the procedure of Glatthaar and Reichstein (14). Media for culturing the bacteria included Difco Nutrient Agar or broth, and, when applicable, xylose or xylulose was added in concentrations of 0.2-0.5oJ,. Incubations were carried out at 37°C. for 24-48 hr. Acetone powders were prepared from 24-hr. cultures grown in 6-gal. carboys. Cells were separated by centrifugation in a refrigerated Sharples centrifuge and washed with physiological saline. The cell paste was homogenized in a Waring blendor for 1 min. with several volumes of cold acetone, filtered through a Biichner funnel, and washed with additional cold acetone and finally with cold absolute ether to dry the powders. The entire procedure was carried out in a cold room. The acetone powders were further dried in an evacuated desiccator and stored at 4°C. Before use, the preparations were ground in a mortar and taken up in appropriate diluent. These powders were employed primarily for manometric procedures. Fresh extracts were prepared from cultures harvested by centrifugation and washed with saline. The paste was ground vigorously for several minutes in a cold mortar with No. 303 Alumina in a quantity of approximately 2.5 times the weight of the cell sample. The paste was taken up in suitable buffer, the cellular debris and alumina were removed by centrifugation, and the resulting cloudy supernatant was kept in the cold until used. Resting cells were obtained by washing organisms with saline and suspending them in appropriate buffers. The suspensions were standardized to the desired cell concentration by means of a Klett-Summerson photoelectric calorimeter.
Methods Ketopentose determinations were carried out by means of the cysteine-carbazole method of Dische and Borenfreund (15). This method was found to be very sensitive for materials containing as little as 1 pg. of xylulose. The Klett-Summerson photoelectric calorimeter was employed for all routine analysis, and the Beckman DU spectrophotometer was used for absorption spectra. Optical densit.y was determined at 540 rnlr. Phosphorylation was studied by means of the manometric technique of Colowick and Kalckar (16). Respiration studies were carried out in the conventional Warburg apparatus. For aerobic experiments, 2Q’%KOH was added to the center well of the vessels; for anaerobic experiments, the vessels were flushed for at least 10 min. with a 95% N1-5% CO* gas mixture. All incubations were carried out at a temperature of 37°C. RESULTS
n-Xylose Utilization
in S. typhosa
This experiment illustrates the fact that n-xylose can be metabolized by the transduced strain of S. typhosa (strain 643T) and not by the
XYLOSE
131
METABOLISM
parent strain (strain 643). This utilization was found to be of an adaptive nature since prior exposure to the substrate was necessary to distinguish between the positive and negative strains (Fig. 1). n-Xylose Ismnerasein S. typhosa Table I shows the results obtained by incubating n-xylose with resting cells of strain 643T and 643 of S. typhosa grown both with and without xylose. After 24 hr. growth, the cells were harvested with saline, mashed, and resuspendedin 0.4 M borate buffer, pH 8.0. The suspension was standardized on the Klett calorimeter to approximately log organisms/ml. The incubation mixture consisted of 20 ml. of cell suspension
TIME, MINUTES STRAIN 6,s3,T I STRAIN 643
I - XYLOSE GROWN 2- NON XYLOSE GROWN 3- XYLOSE GROWN 4 - NON XYLOSE GROWN
I
FIG. 1. Net oxygen uptake in the presence of
D-xylose
by S. typhoaa.
132
E.
S. KLINE
AND
L.
S. BARON
TABLE I Ketopentose Formation in Zntact Cells of S. typhosa Time of incubation min.
0 20 40
Ketopentose concentration in intact cells, fig. Strain 643T strain 643 Xylose-grown Nouxylos~grown Xylose-grown Nouxylose-grown
131 >400 >400
40 36 43
40 42 34
36 36
containing 4 mg. xylose/ml. and was carried out aerobically at 37°C. At the specified times, 4-ml. aliquots were removed and the cells were packed by centrifugation, the supernatant was decanted, and 0.9 ml. of 0.1 iV HCl was then added to the pellet of packed cells. The cells were resuspended and allowed to stand for 10 min. Appropriate aliquots of this suspension were then used to carry out the cysteine-carbazole test. Color was measured after 1 hr. at 540 mp. This procedure is similar to that used by Cohen and Barner (17) for the detection of pentose isomerase activity in the intact cell. Acetone powder or alumina extracts prepared from S. Eyphosa have repeatedly failed to exhibit the xylose isomerase activity that has been demonstrated in the intact cell. In similar studies with Escherichia COG,extracts prepared in this laboratory by these procedures have invariably yielded xylose isomerase activity.’ The basis for this difference is as yet unexplained. It can be seen in this organism that only the strain which utilizes n-xylose possesses isomerase activity as demonstrated by the cysteine-carbazole reaction. Figure 2 shows the absorption spectra of n-xylose and n-xylulose in the cysteine-carbazole test. This determination was carried out in the Beckman DU spectrophotometer, and the color was read after 1 hr. It can be seen that xylose produces but little interference in the cysteinecarbazole test under the conditions employed. At the wavelength employed, xylulose produces approximately 100 times as much absorption as does xylose. Xylulose
Utilization
in S. typhosa
Using both xylose-grown and nonxylose-grown cells of strain 643T and 643 of S. typhosa, oxygen uptake was measured manometrically in the presence of n-xylose and n-xylulose. Each double-armed vessel contained approximately 2 X lo8 washed cells in 0.067 M (M/15) phosphate ’ Kline, E. S., and Baron, L. S., manuscript in preparation.
XYLOSE
133
METABOLISM
buffer pH 7.2 in a final volume of 2.5 ml. The center well contained 0.2 ml. of 20 % KOH. After a 20-min. period of equilibration, the substrates were tipped into the main compartments of the flask. Table II shows the net oxygen consumption of the two strains when incubated with xylose or xylulose after 30 min. These results shorn xylulose oxidation to be independent of the presence of xylose isomerase and that organisms which are incapable of metabolizing xylose can readily utilize xylulose oxidatively without prior growt,h in the presence of added xylulose. Xylulose disappearance as measured by the cysteine-carbazole test was noted during the course of these experiments. 2.0
I 0
16
I. 4
.4
.2
0
0
440
FIG. 2. Absorption
400
520
560
600
640
60Om~
WAVE LENGTH spectra of D-xylose and D-XyhdOSe in the cysteine-carbazole reaction. (7 = hg. = micrograms.)
134
E. S. KLINE
AND L. S. BARON
TABLE II D-Xylulose Oxidation by S. typhosa Microliters 02 uptake strain 643 Strain643T Strain 6MT Substrate
Xylose Xylulose
(Xylosegmwn)
(Nonxylose-grown)
(Xylose-grown)
strain 643 (Nondose-grown)
312 361
24 243
25 281
30 306
Phmphorylation
by Xylose-Grown Cells of S. typhosa
Alumina extracts of S. typhusa were prepared, and 0.6 ml. of extract was added to each double-armed vessel. One side arm contained 8 pmoles of ATP pH 6.8 plus 0.1 ml. of 0.01 M MgS04 in 0.01 M sodium bicarbonate. The other side arm contained either 20 mg. of n-xylose or 15 mg. of n-xylulose, and 0.01 M sodium bicarbonate was added to give a final volume of 2.5 ml. Incubations were carried out in an atmosphere of 95 % N2-5 % CO* at a temperature of 37°C. The low endogenous values and the values obtained after addition of substrate to the main compartment of the vessels were subtracted. ATP was tipped into the main compartment at zero time. Control vessels contained ATP, extract, and bicarbonate buffer. Figure 3 shows the relative rates of phosphorylation for both of these strains. A comparison of the results obtained with the two strains of S. typhosa illustrates the following differences: curve 5 in Fig. 3 (strain 643T xylose-grown, incubated with xylulose) shows phosphorylation with ATP, while curve 4 (strain 643T, xylose-grown, incubated wit’h xylose) fails to indicate any evidence of phosphorylation. This is consistent with the previous finding that xylose isomerase activity is lost when extracts of 8. typhosa are prepared. This fact also serves to eliminate xylose as the substrate of the phosphorylation in this organism. When xylulose was added to the preparation which showed no phosphorylation of xylose (curve 4), immediate phosphorylation was observed. This strain shows no phosphorylation when grown in the absence of xylose, while the genetically negative strain fails to show phosphorylation regardless of the presence of xylose in the medium during growth. Phosphorylation
by Xylulose-Grown
Cells of S. typhosa
The following experiment was undertaken in order to whether the negative strain could be induced to form the growth in the presence of xylulose, since previous results xylose cannot serve as the inducer of the kinase in this strain.
determine kinase by show that A xylose-
XYLOSE
135
METABOLISM
negative strain of Escherichia co2i was included in this experiment for comparative purposes since previous experiments have demonstrated that this strain, although unable to isomerize xylose, is nevertheless able to phosphorylate xylulose by means of a xylulose-induced kinase.? The cells were grown in the presence of n-xylulose for 24 hr. at 37°C. and were harvested and washed, and acetone powders mere prepared. The same general manometric procedure was employed as in previous phosphorylation studies with the following modifications: the vessels contained 40 mg. of acetone powders, 20 pmoles of ATP, 0.01 M MgCL , 0.01 211NaF plus substrate (either n-xylose or n-xylulose) in 0.01 11f NaHCOa . The xylulose control vessel lacked ATP and MgClz , and the
220 200 160 180 160 140 120 100 60 60 40 20 0
5
IO
15 20
26
TIME, Sm&&Jm
;‘-
RfN
30
STRAIN
643
40
XYYSE
3” 4 - XILOSE 56n 7 - N$N 6II
35
45
50
MINUTES OR~WN,INCU?ATED
” ; CONTROL 6ROaWN. INCU?ATED WITH 11
XYL$SE
I: 1 XYLOSE II’ ” 12”
WIJH
;;$;;S2
I
INCU?ATED
II
’ CONTROL GRO”WN , lN+ATED WITH I
ADDED mEA
~CONTROL GRtWN.
.
XYLOSEMYLULOSE XYLULOSE Wl,TH XYLDSE XYLULOSE XYLOSE XYLULOSE
; CONTROL
FIG. 3. Phosphorylation pattern with S. typhosa (alumina
xylose- and nonxylose-grown extracts).
25 MIN.
136
E.
8.
KLINE
AND
L.
S. BARON
ATP control vessel lacked substrate. Sodium fluoride, at the concentration used, reduced the ATPase activity of the extract about 50%, but it can be seen that a substantial amount of ATPase activity still remained. Figure 4 shows the pattern obtained wherein phosphorylation does not occur in the presence of either xylulose or xylose with the xylose-negative strain of S. typhosa. In experiments performed with the posifive strain of S. typhosa (643T) under similar conditions, the xylulokinase is induced by growth in the presence of xylulose. This was shown by testing acetone prepara-
flME.MINUTES s. STRAIN ----
FIG.
643,
I234-
INCUBATED WITH XYLOSE INCUBATED WITH XYLULOSE ATP CONTROL XYLULOSE CONTROL
(I6 7 S-
INCUBATED WITH XYLOSE INCUBATED WITH XYLULOSE ATP CONTROL XYLULOSE CONTROL
4. Phosphorylation pattern with xylulose-grown and E. coli (acetone powders).
S. typhosa
XYLOSE
METABOLISM
137
tions of the positive strain grown in the presenceof xylulose. In a typical experiment, vessels containing 25 mg. of acetone powder, 20 pmoles of ATP, 0.01 M MgCL , 0.04 M NaF plus xylulose as substrate yielded a total of 140 ~1.of CO:!as compared with control values of 28 ~1.of COZ . Thus, these data indicate that the xylose-negative strain of S. typh.osa lacks both isomerase and kinase activity, whereas the xylose-negative strain of E. coli lacks only isomeraseactivity. Substrate Specijicity of the Kinase
n-Ribose, D-arabinose, L-arabinose, o-ribulose, and L-xylose did not show phosphorylation when incubated with S. typhosa extracts which were capable of phosphorylating xylulose. DISCUSSION
The experimental results obtained in this comparative study illustrate the metabolic route of xylose utilization in genetic variants of S. typhosa. The comparison of the strain made positive by transduction and its negative parent strain has established the fact that the negative strain cannot isomerize xylose to xylulose, while the positive strain has now acquired the ability to synthesize the isomerase.A similar situation has been encountered in studies of a spontaneous mutation from xylose utilization to nonutilization in E. coli2 In both of these species, the negative strains have been found to lack the isomerase, while the positive strains are able to synthesize this enzyme following induction in the presence of xylose. Experimenm designed to study the phosphorylation patterns of S. typhosa have shown that when positive cells were grown without xylose, they lacked kinase activity. These cells, following adaptation to xylose, displayed kinase activity only in the presence of xylulose. This was attributed to the fact that the isomerase activity shown to be present in intact xylose-adapted cells is lost in the preparation of extracts made from these cells. The loss of isomeraseacbivity accompanied by the phosphorylat,ion of xylulose indicates that the kinase does n,ot act on xylose. Negative cells grown in the presence of xylose showed no induction of kinase activity. Furthermore, negative cells grown with xylulose were unable to form the xylulokinase, whereas positive cells grown with xylulose were able to produce this enzyme. In contrast, data obtained with the xylose negative isolate of E. coli, referred to above, indicated that this strain still possessedthe ability to
138
E.
S. KLINE
AND
L.
S. BARON
form the enzyme xylulokinase, which was inducible in the presence of xylulose. Thus, in the case of acquisition of xylose utilization by the negative strain of S. typhosa, the mechanism of transduction seems to involve the addition of the ability to synthesize at least two enzymes, xylose isomerase and xylulokinase. A gain of two enzymes by spontaneous mutation has been demonstrated recently by Englesberg (18) who studied rhamnose metabolism in Pasteurella pestis. The preceding discussion has served to establish the following metabolic sequenceof xylose utilization in the strains of S. typhosa examined: Positive cells xylose
xylose-induced isomerase
) xylulose
rylulose-induced kinase
Negative xylose
no lmzyme v xylulose A ’
) xylulose phosphate
cells no ensyme V ,, b xylulose phosphate
In addition, evidence has been presented showing that under conditions whereby these enzymes are not inducible, xylulose is rapidly oxidized by resting cells of both the negative and the positive strain. Thus, it appears that xylulose cab be metabolized by an alternate mechanism as well as direct phosphorylation. This observation has also been noted in the caseof free ribulose. Similar experiments with xylose and arabinose have revealed no such direct oxidation as occurs with the ketopentoses, xylulose and ribulose. Xylulose phosphate assumes added importance in view of the recent findings by several investigators (19, 12, 20) that xylulose phosphate rather than ribulose phosphate serves as the actual substrate for transketolase. The data by Srere et al. (19) showing that free xylulose is also acted upon by transketolase may explain the observation in this paper that xylulose is actively utilized in the absence of phosphorylation. Experiments designed to further elucidate the route of pentose metabolism in enteric bacteria are being continued. SUMMARY
Evidence has been presented which establishes steps in the route of xylose utilization in certain strains of S. typhosa. The failure of the negative strain to metabolize n-xylose stems from its inability to form the enzyme, xylose isomerase, and the enzyme, xylulokinase. The positive
STLOSE
METABOLISM
139
strain of S. typhma obtained by transduction has acquired the ability to synthesize both the isomerase and the kinase. Under conditions whereby these enzymes are not induced, xylulose is rapidly oxidized by resting cells of both the negative and positive strains. REFERENCES 1. ZINDER, N., AND LEDERBERQ, J., J. BacteCoZ. 64, 679 (1952). 2. BARON, L. S., FORNAL, S. B., AND SPILMAN, W., Proc. Sot. Exptl. Biol. &fed. 83, 292 (1953). 3. LAMPEN, J. O., AND PETERJOHN, H. R., J. Bacterial. 62,281 (1951). 3. COHEN, s. s., J. &Ok them. 201, 71 (1953). 5. MITSUHASHI, S., AND LAMPEN, J. O., J. Biol. Chem., 204, 1011 (1953). 6. L.~MPEN, J. O., in “Symposiumof Phosphorus Metabolism” (W. D. MC&BOY and B. GLASS, ads.), Vol. 2 p,. 363. Johns Hopkins Press, Baltimore, Md., 1952. 7. LAMPEN, J. O., J. Biol. Chem. 204, 999 (1953). 8. HOCHSTER, R. M., AND WATSON, R. W., J. Am. Chem. Sot. 76,3284 (1953). 9. HOCHSTER, R. M., Can. J. Microbial. 1, 346 (1955). 10. ASHWELL, G., AND HICKMAN, J., J. Am. Chem. Sot. 76,5SS9 (1954). 11. ASHWELL, G., AND HICKMAN, J., J. Am. Chem. Sot. 77, 1062 (1955). 12. STUMPF, P. K., AND HORECKEH, B. L., J. Biol. Chem. 218,753 (1956). 13. SCHMIDT, 0. TH., AND TREIBER, R., Ber. 66, 1765 (1933). 14. GLATTHAAR, C., AND REICHSTEIN, T., Helu. Chim. Acta 18, 80 (1935). 15. DISCHE, Z., AND BORENFREUND, B., J. Biol. Chem. XI!& 583 (1951). 16. COLOWICK, S. P., AND KALCKAR, H. M., J. Biol. Chem. 146, 117 (1943). 17. COHEN, S. S., AND BARNER, H., J. Bacterial. 69, 59 (1956). 18. ENQLESBERG, E., Federation Proc. 16, 586 (1956). 19. SRERE, P. A., COOPER, J. R., KLYBAS, V., AND RACKER, E., Arch. Bio&m. and Biophys. 69, 535 (1955). 20. HORECKER, B. L., AND SMYRNIOTIS, P. Z., Federation Proc. 16, 277 (1966).
OF BIOCHEMISTRY
AND
BIOPHYSlCS
66,
128-139
(1967)
Xylose Metabolism in Genetic Variants of Salmonella typhosa E. S. Kline1 and L. S. Baron From the Division of Immunology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C.
Received May 2, 1956 INTRODUCTION It has been observed in enteric bacteria that the ability to utilize various pentoses could readily be transferred to bacterial strains previously unable to metabolize them (1, 2). This phenomenon, termed genetic transduction, involves the addition of hereditary determinants from one strain to another through the intervention of bacteriophage. Thus, it has been assumed that a bacterial strain incapable of using xylose may, by means of transduction, gain the ability to synthesize an enzyme or enzymes which would then allow this pentose to be metabolized. Confirmation of this hypothesis is dependent on the elucidation of the metabolic route of pentose utilization in the organism prior to and after transduction. It has been postulated that this synthetic ability is controlled by heritable factors transferred by the bacteriophage to the recipient bacterial strain. As part of a study of the metabolism of pentoses in enteric bacteria, strains of Salmonella typhosa that are positive for various pentoses were obtained by this method of genetic transfer. Aided by such transductions, one is provided with substrains which can facilitate comparative studies in metabolism. Recent studies on pentose metabolism have served to clarify much of the controversy regarding the mechanism by which xylose and other pentoses are utilized. Phosphorylation studies made by Lampen and Peterjohn (3) led these workers to believe that ribose 5phosphate was pentosus. They an intermediate of xylose degradation in Lactobacillus 1 Taken from a thesis submitted by E. S. Kline quirements for the degree of Master of Science, George Washington University. 128
in partial fulfillment of the reDepartment of Biochemistry,
XTLOSE METABOLISM
129
could find no evidence to indicate that either xylose 5-phosphate or arabinose 5-phosphate could be metabolized by xylose-grown cells. Cohen has demonstrated the existence of a pentose isomerase which catalyzes the conversion of D-arabinose to n-ribulose (4). Papers by Lampen and co-workers (5, 6) showed that extracts of L. pentosus could isomerize n-xylose to n-xylulose. Lampen also post’ulated that a xylulose kinase was present by studying the reaction of this sugar with adenosine triphosphate (XTP) (7). Hochster and Watson then demonstrated that xylose-grown cells of Pseudomonas hyclrophilia possessed an isomerase which converted n-xylose to n-xylulose (8). Evidence was also obtained for the formation of the phosphate esters of xylulose, ribulose, ribose, and sedoheptulose (9). Ashwell and Hickman (10) have demonstrated the conversion of ribose 5-phosphate to xylulose 5-phosphate in spleen extracts and have indicated that a 3-ketopentose may be involved in this system (11). The isolation of xylulose 5-phosphate from a bacterial source was first accomplished by Stumpf and Horecker (12). This report is concerned with cerbain aspects of xylose metabolism in strains of S. typhosa which by virtue of their genet,ic structure offer an opportunity for comparative study. The strains were chosen to study both the nature of the enzymatic changes incurred following transduction and the route of xylose degradation in enteric bacteria. RlaTE~1a~s
AND METHODS
Materials Two strains of 8. lyphosa were examined: strain 643, a xylose-negative strain, and a xylose-positive substrain (643T) isolated from a phage-treated suspension of strain 643 following a transduction experiment. Xylose, arabinose, ribose, and other aldol sugars were commercial preparations. Where necessary, they were further purified by recrystallization from ethanol. ATP (barium or disodium) was obtained from blann Research Laboratories, Inc. or Nutritional Biochemicals, Inc. The barium salt was converted to the disodium salt prior to use. In most cases, the pH of the ATP was adjusted to pH 6.8 with KOH. It was stored in the frozen condition, but not longer than 24 hours. n-Xylulose was prepared from D-XylOSe by the method of Schmidt and Treiber (13). This preparation was not free of unepimerized n-xylose and other pentoses. Where required, this preparat,ion was treated nith bromine at room temperature for 30 min. in order to oxidize t,he contaminating aldopentoses. Solid barium carbonate was added to maintain a pH of 7.0. Excess barium carbonate was removed by centrifugation; much of the soluble ions were removed by further precipitations. The products of the bromine oxidation were eliminated by passage through anion-exchange resins, either IRA-400 (HCO,- form) or Dowex 1 (HCO,form). The material which was obtained following this treatment was essentially
130
E. S. KLINE AND L. S. BARON
free of ddopentoses. Pure n-xylulose was also kindly supplied by Dr. A. Weissbach and by Dr. G. Ashwell, National Institutes of Health. n-Ribulose was prepared by the procedure of Glatthaar and Reichstein (14). Media for culturing the bacteria included Difco Nutrient Agar or broth, and, when applicable, xylose or xylulose was added in concentrations of 0.2-0.5oJ,. Incubations were carried out at 37°C. for 24-48 hr. Acetone powders were prepared from 24-hr. cultures grown in 6-gal. carboys. Cells were separated by centrifugation in a refrigerated Sharples centrifuge and washed with physiological saline. The cell paste was homogenized in a Waring blendor for 1 min. with several volumes of cold acetone, filtered through a Biichner funnel, and washed with additional cold acetone and finally with cold absolute ether to dry the powders. The entire procedure was carried out in a cold room. The acetone powders were further dried in an evacuated desiccator and stored at 4°C. Before use, the preparations were ground in a mortar and taken up in appropriate diluent. These powders were employed primarily for manometric procedures. Fresh extracts were prepared from cultures harvested by centrifugation and washed with saline. The paste was ground vigorously for several minutes in a cold mortar with No. 303 Alumina in a quantity of approximately 2.5 times the weight of the cell sample. The paste was taken up in suitable buffer, the cellular debris and alumina were removed by centrifugation, and the resulting cloudy supernatant was kept in the cold until used. Resting cells were obtained by washing organisms with saline and suspending them in appropriate buffers. The suspensions were standardized to the desired cell concentration by means of a Klett-Summerson photoelectric calorimeter.
Methods Ketopentose determinations were carried out by means of the cysteine-carbazole method of Dische and Borenfreund (15). This method was found to be very sensitive for materials containing as little as 1 pg. of xylulose. The Klett-Summerson photoelectric calorimeter was employed for all routine analysis, and the Beckman DU spectrophotometer was used for absorption spectra. Optical densit.y was determined at 540 rnlr. Phosphorylation was studied by means of the manometric technique of Colowick and Kalckar (16). Respiration studies were carried out in the conventional Warburg apparatus. For aerobic experiments, 2Q’%KOH was added to the center well of the vessels; for anaerobic experiments, the vessels were flushed for at least 10 min. with a 95% N1-5% CO* gas mixture. All incubations were carried out at a temperature of 37°C. RESULTS
n-Xylose Utilization
in S. typhosa
This experiment illustrates the fact that n-xylose can be metabolized by the transduced strain of S. typhosa (strain 643T) and not by the
XYLOSE
131
METABOLISM
parent strain (strain 643). This utilization was found to be of an adaptive nature since prior exposure to the substrate was necessary to distinguish between the positive and negative strains (Fig. 1). n-Xylose Ismnerasein S. typhosa Table I shows the results obtained by incubating n-xylose with resting cells of strain 643T and 643 of S. typhosa grown both with and without xylose. After 24 hr. growth, the cells were harvested with saline, mashed, and resuspendedin 0.4 M borate buffer, pH 8.0. The suspension was standardized on the Klett calorimeter to approximately log organisms/ml. The incubation mixture consisted of 20 ml. of cell suspension
TIME, MINUTES STRAIN 6,s3,T I STRAIN 643
I - XYLOSE GROWN 2- NON XYLOSE GROWN 3- XYLOSE GROWN 4 - NON XYLOSE GROWN
I
FIG. 1. Net oxygen uptake in the presence of
D-xylose
by S. typhoaa.
132
E.
S. KLINE
AND
L.
S. BARON
TABLE I Ketopentose Formation in Zntact Cells of S. typhosa Time of incubation min.
0 20 40
Ketopentose concentration in intact cells, fig. Strain 643T strain 643 Xylose-grown Nouxylos~grown Xylose-grown Nouxylose-grown
131 >400 >400
40 36 43
40 42 34
36 36
containing 4 mg. xylose/ml. and was carried out aerobically at 37°C. At the specified times, 4-ml. aliquots were removed and the cells were packed by centrifugation, the supernatant was decanted, and 0.9 ml. of 0.1 iV HCl was then added to the pellet of packed cells. The cells were resuspended and allowed to stand for 10 min. Appropriate aliquots of this suspension were then used to carry out the cysteine-carbazole test. Color was measured after 1 hr. at 540 mp. This procedure is similar to that used by Cohen and Barner (17) for the detection of pentose isomerase activity in the intact cell. Acetone powder or alumina extracts prepared from S. Eyphosa have repeatedly failed to exhibit the xylose isomerase activity that has been demonstrated in the intact cell. In similar studies with Escherichia COG,extracts prepared in this laboratory by these procedures have invariably yielded xylose isomerase activity.’ The basis for this difference is as yet unexplained. It can be seen in this organism that only the strain which utilizes n-xylose possesses isomerase activity as demonstrated by the cysteine-carbazole reaction. Figure 2 shows the absorption spectra of n-xylose and n-xylulose in the cysteine-carbazole test. This determination was carried out in the Beckman DU spectrophotometer, and the color was read after 1 hr. It can be seen that xylose produces but little interference in the cysteinecarbazole test under the conditions employed. At the wavelength employed, xylulose produces approximately 100 times as much absorption as does xylose. Xylulose
Utilization
in S. typhosa
Using both xylose-grown and nonxylose-grown cells of strain 643T and 643 of S. typhosa, oxygen uptake was measured manometrically in the presence of n-xylose and n-xylulose. Each double-armed vessel contained approximately 2 X lo8 washed cells in 0.067 M (M/15) phosphate ’ Kline, E. S., and Baron, L. S., manuscript in preparation.
XYLOSE
133
METABOLISM
buffer pH 7.2 in a final volume of 2.5 ml. The center well contained 0.2 ml. of 20 % KOH. After a 20-min. period of equilibration, the substrates were tipped into the main compartments of the flask. Table II shows the net oxygen consumption of the two strains when incubated with xylose or xylulose after 30 min. These results shorn xylulose oxidation to be independent of the presence of xylose isomerase and that organisms which are incapable of metabolizing xylose can readily utilize xylulose oxidatively without prior growt,h in the presence of added xylulose. Xylulose disappearance as measured by the cysteine-carbazole test was noted during the course of these experiments. 2.0
I 0
16
I. 4
.4
.2
0
0
440
FIG. 2. Absorption
400
520
560
600
640
60Om~
WAVE LENGTH spectra of D-xylose and D-XyhdOSe in the cysteine-carbazole reaction. (7 = hg. = micrograms.)
134
E. S. KLINE
AND L. S. BARON
TABLE II D-Xylulose Oxidation by S. typhosa Microliters 02 uptake strain 643 Strain643T Strain 6MT Substrate
Xylose Xylulose
(Xylosegmwn)
(Nonxylose-grown)
(Xylose-grown)
strain 643 (Nondose-grown)
312 361
24 243
25 281
30 306
Phmphorylation
by Xylose-Grown Cells of S. typhosa
Alumina extracts of S. typhusa were prepared, and 0.6 ml. of extract was added to each double-armed vessel. One side arm contained 8 pmoles of ATP pH 6.8 plus 0.1 ml. of 0.01 M MgS04 in 0.01 M sodium bicarbonate. The other side arm contained either 20 mg. of n-xylose or 15 mg. of n-xylulose, and 0.01 M sodium bicarbonate was added to give a final volume of 2.5 ml. Incubations were carried out in an atmosphere of 95 % N2-5 % CO* at a temperature of 37°C. The low endogenous values and the values obtained after addition of substrate to the main compartment of the vessels were subtracted. ATP was tipped into the main compartment at zero time. Control vessels contained ATP, extract, and bicarbonate buffer. Figure 3 shows the relative rates of phosphorylation for both of these strains. A comparison of the results obtained with the two strains of S. typhosa illustrates the following differences: curve 5 in Fig. 3 (strain 643T xylose-grown, incubated with xylulose) shows phosphorylation with ATP, while curve 4 (strain 643T, xylose-grown, incubated wit’h xylose) fails to indicate any evidence of phosphorylation. This is consistent with the previous finding that xylose isomerase activity is lost when extracts of 8. typhosa are prepared. This fact also serves to eliminate xylose as the substrate of the phosphorylation in this organism. When xylulose was added to the preparation which showed no phosphorylation of xylose (curve 4), immediate phosphorylation was observed. This strain shows no phosphorylation when grown in the absence of xylose, while the genetically negative strain fails to show phosphorylation regardless of the presence of xylose in the medium during growth. Phosphorylation
by Xylulose-Grown
Cells of S. typhosa
The following experiment was undertaken in order to whether the negative strain could be induced to form the growth in the presence of xylulose, since previous results xylose cannot serve as the inducer of the kinase in this strain.
determine kinase by show that A xylose-
XYLOSE
135
METABOLISM
negative strain of Escherichia co2i was included in this experiment for comparative purposes since previous experiments have demonstrated that this strain, although unable to isomerize xylose, is nevertheless able to phosphorylate xylulose by means of a xylulose-induced kinase.? The cells were grown in the presence of n-xylulose for 24 hr. at 37°C. and were harvested and washed, and acetone powders mere prepared. The same general manometric procedure was employed as in previous phosphorylation studies with the following modifications: the vessels contained 40 mg. of acetone powders, 20 pmoles of ATP, 0.01 M MgCL , 0.01 211NaF plus substrate (either n-xylose or n-xylulose) in 0.01 11f NaHCOa . The xylulose control vessel lacked ATP and MgClz , and the
220 200 160 180 160 140 120 100 60 60 40 20 0
5
IO
15 20
26
TIME, Sm&&Jm
;‘-
RfN
30
STRAIN
643
40
XYYSE
3” 4 - XILOSE 56n 7 - N$N 6II
35
45
50
MINUTES OR~WN,INCU?ATED
” ; CONTROL 6ROaWN. INCU?ATED WITH 11
XYL$SE
I: 1 XYLOSE II’ ” 12”
WIJH
;;$;;S2
I
INCU?ATED
II
’ CONTROL GRO”WN , lN+ATED WITH I
ADDED mEA
~CONTROL GRtWN.
.
XYLOSEMYLULOSE XYLULOSE Wl,TH XYLDSE XYLULOSE XYLOSE XYLULOSE
; CONTROL
FIG. 3. Phosphorylation pattern with S. typhosa (alumina
xylose- and nonxylose-grown extracts).
25 MIN.
136
E.
8.
KLINE
AND
L.
S. BARON
ATP control vessel lacked substrate. Sodium fluoride, at the concentration used, reduced the ATPase activity of the extract about 50%, but it can be seen that a substantial amount of ATPase activity still remained. Figure 4 shows the pattern obtained wherein phosphorylation does not occur in the presence of either xylulose or xylose with the xylose-negative strain of S. typhosa. In experiments performed with the posifive strain of S. typhosa (643T) under similar conditions, the xylulokinase is induced by growth in the presence of xylulose. This was shown by testing acetone prepara-
flME.MINUTES s. STRAIN ----
FIG.
643,
I234-
INCUBATED WITH XYLOSE INCUBATED WITH XYLULOSE ATP CONTROL XYLULOSE CONTROL
(I6 7 S-
INCUBATED WITH XYLOSE INCUBATED WITH XYLULOSE ATP CONTROL XYLULOSE CONTROL
4. Phosphorylation pattern with xylulose-grown and E. coli (acetone powders).
S. typhosa
XYLOSE
METABOLISM
137
tions of the positive strain grown in the presenceof xylulose. In a typical experiment, vessels containing 25 mg. of acetone powder, 20 pmoles of ATP, 0.01 M MgCL , 0.04 M NaF plus xylulose as substrate yielded a total of 140 ~1.of CO:!as compared with control values of 28 ~1.of COZ . Thus, these data indicate that the xylose-negative strain of S. typh.osa lacks both isomerase and kinase activity, whereas the xylose-negative strain of E. coli lacks only isomeraseactivity. Substrate Specijicity of the Kinase
n-Ribose, D-arabinose, L-arabinose, o-ribulose, and L-xylose did not show phosphorylation when incubated with S. typhosa extracts which were capable of phosphorylating xylulose. DISCUSSION
The experimental results obtained in this comparative study illustrate the metabolic route of xylose utilization in genetic variants of S. typhosa. The comparison of the strain made positive by transduction and its negative parent strain has established the fact that the negative strain cannot isomerize xylose to xylulose, while the positive strain has now acquired the ability to synthesize the isomerase.A similar situation has been encountered in studies of a spontaneous mutation from xylose utilization to nonutilization in E. coli2 In both of these species, the negative strains have been found to lack the isomerase, while the positive strains are able to synthesize this enzyme following induction in the presence of xylose. Experimenm designed to study the phosphorylation patterns of S. typhosa have shown that when positive cells were grown without xylose, they lacked kinase activity. These cells, following adaptation to xylose, displayed kinase activity only in the presence of xylulose. This was attributed to the fact that the isomerase activity shown to be present in intact xylose-adapted cells is lost in the preparation of extracts made from these cells. The loss of isomeraseacbivity accompanied by the phosphorylat,ion of xylulose indicates that the kinase does n,ot act on xylose. Negative cells grown in the presence of xylose showed no induction of kinase activity. Furthermore, negative cells grown with xylulose were unable to form the xylulokinase, whereas positive cells grown with xylulose were able to produce this enzyme. In contrast, data obtained with the xylose negative isolate of E. coli, referred to above, indicated that this strain still possessedthe ability to
138
E.
S. KLINE
AND
L.
S. BARON
form the enzyme xylulokinase, which was inducible in the presence of xylulose. Thus, in the case of acquisition of xylose utilization by the negative strain of S. typhosa, the mechanism of transduction seems to involve the addition of the ability to synthesize at least two enzymes, xylose isomerase and xylulokinase. A gain of two enzymes by spontaneous mutation has been demonstrated recently by Englesberg (18) who studied rhamnose metabolism in Pasteurella pestis. The preceding discussion has served to establish the following metabolic sequenceof xylose utilization in the strains of S. typhosa examined: Positive cells xylose
xylose-induced isomerase
) xylulose
rylulose-induced kinase
Negative xylose
no lmzyme v xylulose A ’
) xylulose phosphate
cells no ensyme V ,, b xylulose phosphate
In addition, evidence has been presented showing that under conditions whereby these enzymes are not inducible, xylulose is rapidly oxidized by resting cells of both the negative and the positive strain. Thus, it appears that xylulose cab be metabolized by an alternate mechanism as well as direct phosphorylation. This observation has also been noted in the caseof free ribulose. Similar experiments with xylose and arabinose have revealed no such direct oxidation as occurs with the ketopentoses, xylulose and ribulose. Xylulose phosphate assumes added importance in view of the recent findings by several investigators (19, 12, 20) that xylulose phosphate rather than ribulose phosphate serves as the actual substrate for transketolase. The data by Srere et al. (19) showing that free xylulose is also acted upon by transketolase may explain the observation in this paper that xylulose is actively utilized in the absence of phosphorylation. Experiments designed to further elucidate the route of pentose metabolism in enteric bacteria are being continued. SUMMARY
Evidence has been presented which establishes steps in the route of xylose utilization in certain strains of S. typhosa. The failure of the negative strain to metabolize n-xylose stems from its inability to form the enzyme, xylose isomerase, and the enzyme, xylulokinase. The positive
STLOSE
METABOLISM
139
strain of S. typhma obtained by transduction has acquired the ability to synthesize both the isomerase and the kinase. Under conditions whereby these enzymes are not induced, xylulose is rapidly oxidized by resting cells of both the negative and positive strains. REFERENCES 1. ZINDER, N., AND LEDERBERQ, J., J. BacteCoZ. 64, 679 (1952). 2. BARON, L. S., FORNAL, S. B., AND SPILMAN, W., Proc. Sot. Exptl. Biol. &fed. 83, 292 (1953). 3. LAMPEN, J. O., AND PETERJOHN, H. R., J. Bacterial. 62,281 (1951). 3. COHEN, s. s., J. &Ok them. 201, 71 (1953). 5. MITSUHASHI, S., AND LAMPEN, J. O., J. Biol. Chem., 204, 1011 (1953). 6. L.~MPEN, J. O., in “Symposiumof Phosphorus Metabolism” (W. D. MC&BOY and B. GLASS, ads.), Vol. 2 p,. 363. Johns Hopkins Press, Baltimore, Md., 1952. 7. LAMPEN, J. O., J. Biol. Chem. 204, 999 (1953). 8. HOCHSTER, R. M., AND WATSON, R. W., J. Am. Chem. Sot. 76,3284 (1953). 9. HOCHSTER, R. M., Can. J. Microbial. 1, 346 (1955). 10. ASHWELL, G., AND HICKMAN, J., J. Am. Chem. Sot. 76,5SS9 (1954). 11. ASHWELL, G., AND HICKMAN, J., J. Am. Chem. Sot. 77, 1062 (1955). 12. STUMPF, P. K., AND HORECKEH, B. L., J. Biol. Chem. 218,753 (1956). 13. SCHMIDT, 0. TH., AND TREIBER, R., Ber. 66, 1765 (1933). 14. GLATTHAAR, C., AND REICHSTEIN, T., Helu. Chim. Acta 18, 80 (1935). 15. DISCHE, Z., AND BORENFREUND, B., J. Biol. Chem. XI!& 583 (1951). 16. COLOWICK, S. P., AND KALCKAR, H. M., J. Biol. Chem. 146, 117 (1943). 17. COHEN, S. S., AND BARNER, H., J. Bacterial. 69, 59 (1956). 18. ENQLESBERG, E., Federation Proc. 16, 586 (1956). 19. SRERE, P. A., COOPER, J. R., KLYBAS, V., AND RACKER, E., Arch. Bio&m. and Biophys. 69, 535 (1955). 20. HORECKER, B. L., AND SMYRNIOTIS, P. Z., Federation Proc. 16, 277 (1966).