INTERMEDIATE METABOLISM OF AEROBIC SPORES ... - Europe PMC

INTERMEDIATE METABOLISM OF AEROBIC SPORES III. THE MECHANISM OF GLUCOSE AND HEXOSE PHOSPHATE OXIDATION IN EXTRACTS OF Bacillus cereus SPORES' ROY DOI, HARLYN HALVORSON, AND BROOKS CHURCH Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, and Warner-Lambert Research Institute, Warner-Lambert Pharmaceutical Company, Morris Plains, New Jersey

Received for publication July 1, 1958

When dormant spores are sufficiently activated by heat (Church and Halvorson, 1955, 1957) or germinating agents (Murrell, 1955; Murty and Halvorson, 1957), an active glucose oxidation ensues in the absence of a loss of heat resistance. The oxidation of glucose, gluconate, 2KG2 and pyruvate by activated spores of Bacillus cereus var. terminalis (Church, 1955) suggested that the oxidation proceeded by a direct non-phosphorylated oxidation of glucose (Entner and Duodoroff, 1952). A similar conclusion was reached by Hachisuka et al. (1956) who observed that the oxidation of glucose to gluconate by germinated spores of B. cereus was phosphate independent and unaffected by the presence of inhibitors of glycolysis. Previous studies in our laboratory have shown that extracts of B. cereus var. terminalis spores rapidly oxidize glucose, gluconate, G6P, and 6PG (Church and Halvorson, 1955; Halvorson, 1958). ' This research was supported in part by an

Since these findings suggested that spores contain a system of carbohydrate metabolism involving phosphorylated intermediates, an examination of the mechanism of glucose utilization was undertaken. The present results demonstrate that glucose and gluconate are oxidized to 2KG which is phosphorylated to 2K6PG. The latter is either converted to pyruvate by an unknown pathway or reduced to 6PG which is metabolized by a hexose monophosphate oxidative system. MATERIALS AND METHODS

Preparation of extracts. The spores used in this study were prepared from Bacillus cereus var. terminalis. The methods employed for harvesting and washing the spores (Church et al., 1954) and for the preparation of sonic spore extracts (Halvorson and Church, 1957) have been previously described. Enzyme fractionation. The sonic extract from 6 g of spores was clarified by high speed centrifugation (140,000 X G for 1 hr). To the supernatant (41 ml), 3 ml of 2 per cent protamine sulfate, pH 5.0, were added and the precipitate which formed was immediately removed by centrifugation. The supernatant was dialyzed 15 hr against 10-2 M Tris buffer, pH 7.6. The precipitate which formed was removed by centrifugation and discarded. The ratio of absorption at 280 and 260 mu was approximately 0.85. The supernatant (43 ml) was fractionated with solid ammonium sulfate at 1 C. The precipitate fractions (0 to 0.6, 0.6 to 0.7, 0.7 to 0.9 saturated) were removed by centrifugation, resuspended in 10 ml of 0.1 M Tris buffer, pH 7.6, and dialyzed for 15 hr against 10-2 M Tris buffer, pH 7.6. The 0 to 0.6 fraction was further fractionated with solid ammonium sulfate. The precipitates were resuspended in 5 ml 0.1 M Tris buffer, pH 7.6, dialyzed as previously, and stored at -20 C. In some cases, DEAE cellulose chromatog-

Institutional research grant from the American Cancer Society and a grant-in-aid from the Wisconsin Alumni Research Foundation. An account of this research was presented at the 58th Annual Meeting of the Society of American Bacteriologists, Chicago, Illinois, May, 1958. 2 The following abbreviations will be used: 2KG, 2-keto-gluconate; G6P, D-glucose-6-phosphate; 6PG, D-gluconate-6-phosphate; R5P,

D-ribose-5-phosphate; 2K6PG, 2-keto-D-gluconate-6-phosphate; 2K3D6PG, 2-keto-3-deoxy-Dgluconate-6-phosphate; F6P, D-fructose-6-phosphate; G3P, D-glyceraldehyde-3-phosphate; Ru5P, D-ribulose-5-phosphate; Xu5P, D-xylulose-5-phosphate; HDP, hexose diphosphate; GIP, D-glucose1-phosphate; DPN, diphosphopyridine nucleotide; TPN, triphosphopyridine nucleotide; ATP, adenosine triphosphate; TCA, trichloracetic acid; GSH, glutathione; DEAE, diethylaminoethyl; Tris, tris(hydroxymethyl)aminomethane; Versene, disodium salt of ethylenediaminetetraacetic acid. 43

44

DOI, HALVORSON, AND CHURCH

raphy was employed for enzyme purification. The crude extract was dialyzed for 24 hr against 0.01 M Tris-0.005 M magnesium acetate buffer, pH 7.5. A 25-cm long DEAE cellulose column in a 1-cm diameter glass column was prepared using commercial DEAE cellulose according to the method of Sober et al. (1956). The dialyzed fraction was added to the column and an increasing NaCl gradient applied by means of two reservoirs: 200 ml of the above buffer and 200 ml of 1.5 M NaCl. Fractions of 1.5 ml were collected at 3 C and analyzed for protein and enzyme activity. Analytical methods. Dehydrogenase determinations were carried out at 30 C at 340 m,u in a Beckman spectrophotometer in 3.0-ml capacity cells having a 1-cm light path. Spectrum measurements were made with a recording Beckman DK2 spectrophotometer. Protein was determined by the method of Lowry et al. (1951) using serum albumin as a standard. Glucose was measured by the arseno-molybdate method of Nelson (1944), pyruvate by the procedure of Friedemann and Haugen (1943), gluconate by the method of Hestrin (1949), 2-KG by the method of Lanning and Cohen (1951), total hexose by the cysteineH2SO4 method of Dische et al. (1949), keto sugars by the cysteine-carbazole method of Dische and Borenfreund (1951), and pentose and sedoheptulose by the orcinol method of Brown (1946) with a 40-min heating period and calculated by the method of Horecker et al. (1953). Chemicals. Crystalline barium 6-phosphogluconate (6PG), barium glucose-6-phosphate heptahydrate (G6P), barium fructose-1, 6-diphosphate (F-1,6-P), diphosphopyridine nucleotide "90" (DPN), triphosphopyridine nucleotide "90" (TPN), and disodium adenosine triphosphate (ATP) were obtained from the Sigma Chemical Company. Barium ribose-5-phosphate (R5P) was obtained from the Schwarz Laboratories. Barium 2-keto-3-deoxy-D-gluconate-6phosphate (2K3D6PG) and barium 2-keto-6phosphogluconate were kindly furnished by Dr. W. A. Wood, University of Illinois. Calcium 2-ketogluconate (2KG) was kindly furnished by Dr. H. Blumenthal, University of Michigan. Neutralized 6-gluconolactone (Nutritional Biochemicals) served as a source of gluconate. Dowex 1 was obtained from Microchemical Specialities Co. and DEAE cellulose type 40 from the Brown Company.

[VOL. 77

EXPERIMENTAL RESULTS

Glucose oxidation. Previous studies (Halvorson, 1958) have indicated that sonic extracts of B. cereus var. terminalis spores oxidize glucose, gluconate, 2KG, and 2K6PG in the presence of DPN or TPN. Gluconate was found to be the primary end product of glucose oxidation with traces of 2KG, 2K6PG, and pyruvate (Church, 1955; Halvorson, 1958). To further characterize the reactions involved, several enzymes were purified and the stoichiometry of the reactions determined. The active glucose dehydrogenase of these spore extracts was purified 33-fold by protamine treatment and ammonium sulfate fractionation as shown in table 1. The 0.7 to 0.9 fraction was devoid of side reactions on glucose and was used for further studies on characterization of the enzyme. The enzyme has a pH optimum at 7.8. DPNH formation was found to be linear with time and enzyme concentration. The effect of substrate concentrations upon the initial reaction velocity is shown in figure 1. The Michaelis constants calculated from plots according to Lineweaver and Burk (1934) were 6.7 X 10-3 M for glucose and 9.1 X 10-5 M for DPN. Enzyme activity was unaffected by the addition of 0.1 M phosphate, 0.01 M Ca++, or by dialysis against 10-3 M Versene. Although DPNH formation was stimulated by glutathione, this activity was attributed to a contamination of the fraction by a glutathione reductase which reacted with both DPN and TPN. The substrate specificity of glucose dehydrogenase is shown in figure 2. The enzyme TABLE 1 Purification of glucose dehydrogenase from spore extracts Specific Activity Units* Protein Units (mg) per ml per ml per mng

Step No.

Protein

3

23,000 820 Sonic extract High speed centrifu- 18,400 398 gation Protamine superna- 6,900 151

4

tant 0.7-0.9 Saturated

1 2

2,130

28.1 46 45.6

2.26 940

(NH4)2SO4 ppt * One unit = 0.001 change in optical density (340 m,u) per 100 sec.

INTERMEDIATE METABOLISM OF AEROBIC SPORES

1959]

45

S(M x03)

S (M)

Figuire 1. Substrate saturation of glucose dehydrogencase. In the left-hand section the 3-ml reaction mixture contained DPN, 0.7 ,Lmole; extract, 0.10 ml; Tris buffer, pH 7.6, 300 /imoles; and various conlAmoles of glucose were added centrations of glucose. The right-hand section was the same except 10 and the DPN concentrations varied. SPECIFICITY OF GLUCOSE DEHYDROGENASE

70-90 %(NH4)O4 FRACTION 300

_____

/ GLUCOSE+ DPN ______ -~______ ____

E

IT2Co

_/ /

_

x

z 1OO w

L

}

* DPN

/

_J

/ O 1, o 2

4 MINUTES

_c,_o

NOE GLUCOSE+ TPN

SEA I-L PN

6

Figucre 2. Specificity of glucose deh drogenase. The 3-ml reaction mixture contained extract 0.10 ml; DPN or TPN, 0.7 usmole; Tris btuffer, pH 7.6, 300 umoles; and 10lOmoles of substrates where in(licatedl.

rapi(lly catalyzes the reduction of DPN but not TPN in the presence of glucose. A slow rate of DPN reduction occurs in the presence of mannose. The stoichiometry of glucose oxidation by fraction 0.7-0.9 (see table 1) and of gluconate oxidation by crude extr acts is shown in table 2. Chromatographic analysis of the end products of glucose oxidation (methanol: ethanol: water, 4.5: 4.5:1) showed the presence of gluconate (Rf 0.43). Church (1955), with similai extracts, also observed traces of 2KG from glucose oxidlation. The primary product formed a purple color with o-phenvlendiamine and when eluted supported TPN reduction in the presence of ATP, gluconokinase, and 6PG dehydrogenase. In the absence of ATP, gluconate disappearance can be quantitatively accounted for as 2KG and agrees with the product previously identified from glucose oxidation (Halvoison, 1958). Attempts to detect gluconokinase have consistently been negative (Halvorson and Church, 1957). the addition of ATP, M and a gluconokinase

IUpon

partially purified from yeast by the method of Sable and Guarino (1952), rapid DPN reduction OCCUI'S.

The stoichiometry of 2KG phosphorylation by

46) 4)OI, HALVORSON, AND CHURCH TABLE 2 Stoichiomnetry of glucose oxidation by spore extracts

mnioles

Glucose. Gluconate....

Gluconate

Glucose

Substrate

.

60 12.5

2cKnetegl

A.moles (60 min.)

-44.3

+39.6 -1.70

+1.53

The reaction mixture for glucose oxidation (6.45 ml) contained glucose, 60 ,umoles; pyruvate, 60 ,moles; DPN, 7 ,moles; crystalline lactic dehydrogenase, 1 mg; fraction 0.7-0.9 1 ml (see table 1); and 64.5 ,moles of glycylglycine buffer, pH 7.3. The reaction mixture for gluconate oxidation (3 ml) contained gluconate, 12.5 jimoles; TPN, 2.1 ,jmoles; crude extract, 1.5 ml; and 85 ,umoles of glycylglycine buffer, pH 7.3. The reaction mixtures were incubated for 60 min at 30 C, deproteinized and analyzed for glucose by the method of Nelson (1944), gluconate by the method of Hestrin (1949), and 2KG by the method of Lanning and Cohen (1951). TABLE 3 Products of 2-keto-D-glutconate-6-phosphate (2K6PG) oxidation Substance

Rf

2K6PG O0 39 Pentose phosphate Hexose phosphate.. 0.21 Pv-ruvatel

Glyceraldehyde

Spectrum Peak 670 m,u 410 inmA

[VOL. 77

ATP and a magnesium requiring 2-ketogluconokinase has been previously reported (Halvorson, 1958). The product of this reaction, 2K6PG, is formed quantitatively from 2KG. Preliminary experiments indicated that 2K6PG was utilized by crude extracts. In order to further characterize the mechanism involved, an examination of end product formation from 2K6PG oxidation was undertaken (table 3). Assuming that G3P was formed in equimolar quantities to pyruvate, approximately 80 per cent of the 2K6PG utilized could be recovered as pentose phosphate, hexose phosphate, and pyruvate. These results suggest that 2K6PG is converted to pyruvate at least in part via intermediates of the hexose monophosphate cycle. The mechanism whereby 2K6PG enters the hexose monophosphate cycle has been clarified in Enterobacteriaceae (DeLey, 1955) and Pseudomonads (Wood et al., 1955) by the discovery of 2K6PG reductase which catalyzes the reduction of 2K6PG to 6PG in the presence of TPNH or DPNH. Several observations indicate that a similar enzyme is present in spore extracts. First,

lmoles ~soe

-4.54 +1.87 +1.25 +0.45 +

The 4-ml reaction mixture contained 2K6PG, 7.2/Amoles; arsenite, 100,umoles; DPNH, 2,uAmoles; ATP, 1 ,Amole; Mg++, 10 /Amoles; TPN, 0.23 /Amole; erude extract, 1.0 ml; and glycylglycine buffer, pH 7.3, 45 ;Amoles. After 60 min incubation at 30 C, the reaction mixture was deproteinized and tested for pyruvate by the method of Friedemann and Haugen (1943), glyceraldehyde by the method of Beck (1955), and 2K6PG- by the method of Lanning and Cohen (1951). The phosphate compounds in 2.0 ml of the deproteinized reaction mixture were precipitated by barium, washed, and converted to the free acid with Dowex 50 cation exchange resin in the H+ form. After concentrating to a small volume in vacuo, the sample, with appropriate controls, was chromatogrammed with the solvent system of Benson et al. (1950). The phosphate compounds were located with a molybdate spray, eluted and tested either for pentose by the orcinol method (Brown, 1946) or hexose by the cysteine-H2SO4 method of Dische et al. (1949).

MINUTES

Figure S. Stoichiometry of G6P and 6PG oxidation. The 3-ml reaction mixtures contained extract, 0.2 ml; (fraction 8, table 3); Mg++, 10 ,umoles; Tris buffer, pH 7.6, 300 ,umoles; and TPN or DPN, 0.7 ,umole. Where indicated, 0.2 /Amole of G6P or 6PG were added.

INTERMEDIATE METABOLISMI OF AEROBIC SPORES

1959]

47

pyruvate formation from 2K6PG in crude ex- crude extracts invarably form pyruvate 2 to 4 tracts is increased 400 per cent by the addition of DPNH. Secondly, although 2K6PG utilization is largely lost during fractionation, a weak enzyme activity is sharpy distributed between fractions 70 to 73 of the experiment in figure 4 which oxidizes DPNH only in the presence of 2K6PG. The main end product of this reaction was hexose phosphate, probably due to the presence of G6P dehydrogenase in the fractions. The equilibrium of the 2K6PG reductase is strongly in favor of 6PG since attempts to demonstrate TPN or DPN reduction by 6PG have been negative. The above demonstration in spore extracts of a 2K6PG reductase and the subsequent reactions involved in 6PG metabolism readily explain the production of pyruvate from 2K6PG. If this mechanism represented the primary route of 2K6PG utilization, one would expect that the rate of pyruvate formation from 6PG would be equal or greater than that from 2K6PG. However,

0

20

times faster from 2K6PG than from 6PG. Foi example, in an experiment employing the extract and conditions described in table 3, 0.12,moles of pyruvate were formed from 10 Mumoles of 6PG after 60 min incubation. The simplest explanation of these observations is that spore extracts containl an alternate route of pyruvate formation from 2K6PG not involving 6PG as an interine(liate. Wood (personal communication) has alternatively suggested that 2K6PG may be re(luced to 6PG which in turn is cleaved to glyceraldehyde phosphate. The oxidation of the latter would provide the supply of hydrogen atoms subsequently used in the reduction of a second molecule of 2K6PG. The increased production of pyruvate by such a cycle over 6PG oxidation could be ieadily understood if either the system were devoid of hydrogen acceptors for the oxidation of glyceraldehyde phosphate or if pyruvate itself served as the hydrogen acceptor.

40 FRACTION NUMBER

60

80

Figure 4. Chromatography on DEAE cellulose columns. A dialyzed extract (2.0 ml) was applied to a DEAE cellulose column as described in Materials and Methods. Each fraction, 1.5 ml, was tested for protein and dehydrogenases by following spectophotometrically the formation of I)PNH or TPNH in presence of various substrates. Fraction A is glucose dehydrogenase, fraction B, 6PG dehydrogenase, and fraction C, G6P dehydrogenase.

48 8DOI, HALVORSON, AND CHURCH

[VOL. 77

fractions 60 to 70. In all three cases, the activities applied to the column were quantitatively recovered in the three fractions described in figure 4. The properties of the G6P dehydrogenase from the cellulose DEAE column were determined by measuring spectrophotometrically the initial rate of TPN reduction. The rate of the enzyme reaction was proportional both to time and enzyme concentration and had a pH optimum of 7.6. The effect of substrate concentrations on the initial reaction rates are shown in figure 5. The enzyme has a KM of 6.6 X 10-5 M for G6P and 5.6 X 10-5 M for TPN. The activity of dialyzed preparations of G6P dehydrogenase was increased 56 per cent by 3 X 10-3 M Mg++ ions. The substrate specificity of the 50 to 60 per cent fraction (see table 4) of G6P dehydrogenase is shown in figure 6. The enzyme preparation is active only in the presence of TPN and is contaminated with phosphohexoisomerase. Analysis of the end products by 6PG dehydrogenase and DPN showed that for each mole of G6P oxidized, 1 mole of 6PG is formed. TPN is not reduced in the presence of hexoses, HDP, GlP, or R5P. These findings indicate that G6P dehydrogenase from spores is essentially the same as that occurring in Escherichia coli and in Aerobacter (Gunsalus et al., 1955). 6PG dehydrogenase (fraction B, figure 4) is TABLE 4 for 6PG and DPN. The reduction of specific Purification of glucose-6-phosphate (G6P) TPN 6PG oxidation in crude extracts can during dehydrogenase and 6-phosphogluconate (6PG) to the activity of pyridine nuclebe attributed dehydrogenase from spore extracts otide transhydrogenase since (a) the activity of G6P Dehy6PG Dehythe crude extract can be accounted for in fraction drogenase drogenase B, (b) no evidence for a TPN enzyme could be Step Unlits Units No. Total per mg Total per mg detected, and (c) pyridine nucleotide transhyunits* pro- units prodrogenase activity was detected in tubes 45 to 60. tein tein The 6PG dehydrogenase was very labile; after 2 1 Sonic extract 27,000 33 6,010 7.5 days at 4 C over 50 per cent of the activity was 2 High speed centrif- 26,400 46 5,900 11.5 lost. G6P dehydrogenase preparations could ugation easily be prepared free of 6PG dehydrogenase by 3 Protamine superna- 27,000 188 5,000 32.0 aging the 0.4 to 0.5 ammonium sulfate fractions tant 4 0-0.60 Saturated 32,000 275 4,500 40.0 (table 4). To measure the end products of the oxidation (NH4)2 S04 ppt of 6PG, the following experiment was performed. Subfractions of 4: The reaction mixture contained 200 ,umoles of 5 0.3-0.4 Saturated 15 2 710 98 (NH4)2 S04 ppt Tris buffer, 1.5 ml of 6PG dehydrogenase (frac6 0.4-0.5 62 4.7 tion B), 5 ,umoles of DPN, and 1.65 ,umoles of 2,080 119 7 0.5-0.6 7,350 650 425 11.3 6PG. After 60 min incubation anaerobically at 8 0.6-0.7 12,700 727 1,700 228 30 C, at which period CO2 evolution had ceased, * One unit = 0.001 change in optical density 0.2 ml 2 N H2S04 was added from the side arm. The reaction mixture was diluted to 5 ml with (340 m,u) per 100 sec.

Hexose phosphate metabolism. The presence of at least a portion of the hexose monophosphate pathway in erude extracts was evident by the demonstration of phosphoglucomutase, phosphohexoisomerase, and by the reduction of TPN by G6P and 6PG and DPN by 6PG (Halvorson and Church, 1957; Halvorson 1958). The suggestion that G6P is oxidized via 6PG is indicated by the stoichiometry of figure 3. Two moles of TPN are reduced per mole of G6P oxidized whereas only 1 mole of TPN or DPN is reduced per mole of 6PG oxidized. In order to further characterize these reactions, the sonic extract was fractionated as indicated in table 4, and assayed for G6P and 6PG utilization. Although G6P dehydrogenase was purified over 22-fold and the 6PG oxidizing system over 30-fold, it was not possible to separate the activities by ammonium sulfate fractionation. The separation of glucose, G6P, and 6PG dehydrogenases was achieved by means of DEAE cellulose columns (figure 4). Glucose dehydrogenase (curve A) was eluted first from the column as a sharp peak. This fraction was closely followed by a second sharp peak which was identified as 6PG dehydrogenase. G6P dehydrogenase was cleanly separated from the other two dehydrogenases as a broad peak between


1959]

>

G6P

w

TPN

Z

E

o: 0

49

6/

O

f

I

/I

1 S/VV 4

l 0/

0

0

0

0

-Q 0~ w

-5

-5

~

~

~

SF,eXIO5)

~

~

~

0

1

StMX lO0)

Figure 5. Substrate saturation of G6P dehydrogenase. In the left-hand section the 3-ml reaction mixture contained TPN, 0.7,umoles; G6P dehydrogenase (fraction C, figure 4), 0.1 ml Tris buffer, pH 7.6, 300 ,moles; and various concentrations of G6P. The right-hand section was the same except 10 ,umoles of G6P were added and the TPN concentrations varied.

water, centrifuged, passed through norite to remove DPN and analyzed for pentose by the oreinol method. From the initial 1.65 ,umoles of 6PG, theoretical recoveries of CO2 (1.8 ,umoles) and pentose (1.6 ,umoles) were observed. The pentose was further identified by running a larger scale (10 X) experiment. After the rieaction was complete, the mixture was deproteinized, dlesalted, adsorbed with norite, concentrated, and introduced to a Dowex 1 formate column (Horecker et al., 1953). The column (1.3 by 11 cm) wAas eluted for 0.75 L with 0.26 N formic acid and 0.03 M sodium formate; 0.65 L with 0.4 N formic and 0.06 M sodium formate; 0.25 L with 1 N formate andl 0.15 NI sodium formate. Five-ml fractions were recovered and analyzed for pentose. Only 1 peak was recovered (tubes 312 to 319) which agreed with the elution pattern of R5P. The pro(luct on chromatography had the same Rf as R5P, and was identified by the phloroglucinol spray as aldopentose and the molybdate spray as pentose phosphate. The orcinol spectrum MINUTES of the eluate from either the Dowex column or the chromatogram was identical to that of R5P. Figuire 6. Specificity of G6P dehydrogenase Pentose metabolism. Although the oxidation of preparation. The 3-ml reaction mixtuire conitained R5P has been demonstrated in sonic extracts of extract, 0.10 ml; TPN or DPN, 0.7 ,mole; 1\g++, spores (Halvorson and Church, 1957), the prod- 10 MAmoles; Tris buiffer, pH 7.6, 300 ,umoles; and ucts of the reaction wAere not identified. The 10 l,moles of stubstrates where indicate(l.

5,0


PENTOSE

[V'OL. 77

UTILIZATION

Pentose

E

5

_

Py ruvate @ t" -At AJ5>o

Sedoheptulose

O0

60

........

120 Minutes

ISO

a

240

Figure 7. Products of R5P utilization. The 10-ml reaction mixture contained R5P, 25,umoles; Mg++, 200,4moles; NaF, 190 ,umoles; crude extract, 5.0 ml; and glycylglycine buffer, pH 7.5, 225 ,umoles. Oneml aliquots w-ere taken out at indicated times, deproteinized, and tested for various products as indicated in Mlethods.

kinetic analysis of the end products formed from R5P is shown in figure 7. Similar results were observed during 6PG oxidation. The hexose and ketohexose formed were identified as G6P and F6P by a chromatographic analysis of the sugar phosphates after deproteinization (figure 8) and enzymatically with G6P dehydrogenase and a coupled phosphohexoisomerase and G6P dehvdrogenase system. The initial rapid utilization of R5P is paralleled by an increase in sedoheptulose, G6P and F6P. The presence of phosphohexoisomerase in these preparations has previously been established (Halvorson and Church, 1957). The secondary decrease in G6P and F6P after 30 min and the later increase in R5P and pyruvate suggests the operation in these extracts of a functional hexose monophosphate cycle.

The hexose monophosphate cycle of various organisms (DeLey, 1955) involves the conversion of R5P to Ru5P and Xu5P by the action of isomerase and ketopentoepimerase. Ru5P may then be converted to F6P by the action of transaldolase and transketolase. The operation of this cycle is supported not only by the above evidence but also by the shift in the maximal absorption in the cysteine-carbazole test for keto sugars during 6PG and R5P utilization. In an experiment similar to that described in figure 7, R5P decreased very rapidly. During the first 10 min, a ketopentose (max 545 m,u) was formed, presumably Ru5P, which disappeared again rapidly. After 25 to 30 min ketohexose (max 565 m,u), identified above as F6P, was formed and slowly disappeared.

1959]


51

Figure 8. Paper chromatography of products from R5P utilization. Two ml of reaction mixture (figure 7) were deproteinized, concentrated, and applied to Whatman no. 1 paper. The developing solvent of Benson et al. (1950) and the spray reagent and ultraviolet irradiation of Bandurski and Axelrod (1952) were used to identify the sugar phosphates.

Alternate route of 6PG utilization. Extracts of Pseudomonads degrade 6PG to pyruvate and triose phosphate by reactions not involving R5P or CO2 (Entner and Doudoroff, 1952). Kovachevich and Wood (1955) have shown that the mechanism involves the reduction of 6PG by a Fe++ and GSH requiring 6-phosphogluconate dehydrase to 2K3D6PG and its subsequent cleavage by 2-keto-3-deoxy-6-phosphogluconate aldolase to pyruvate and D-glyceraldehyde-3phosphate. The oxidation of the intermediate 2K3D6PG by spore extracts (Halvorson and Church, 1957) has led us to examine this system in these extracts. Dialyzed sonic extracts (1.0 ml) were incubated in the presence of Tris buffer, 200 ,umoles; 6PG; 4 ,umoles, Fe++; 12 /moles, and GSH, 6 ,umoles,

for various periods of time, the reaction stopped by boiling and analyzed for 6PG utilization by purified 6PG dehydrogenase and DPN. Our results have invariably been negative. Since the 6PG dehydrase is generally labile, the enzyme may have been inactivated during preparation of the sonic extract. The presence of 2K3D6PG aldolase was tested by following the rate of pyruvate formation from 2K3D6PG. The reaction mixture (2.0 ml) contained spore extracts, 1.0 ml; Tris buffer, 200 ,umoles; arsenite, 50 ,umoles; and 2K3D6PG, 4 j,moles. Following deproteinization with 5 per cent TCA, the samples were analyzed for pyruvate. Pyruvate was formed at a rate of 0.4 ,umoles per hr per ml of extract. The addition of ATP, 1 umole; DPN, 1.4 ,umoles; Mg++, 10

52

~


,umoles; and thiamin pyrophosphate, 0.5 ,moles, did not enhance the aldolase activity. DISCUSSION

Although hexose metabolism has previously been reported in spore extracts (Church and Halvorson, 1957), the role of these systems in glucose metabolism by spores remained obscure. The absence of hexokinase and glucokinase (Halvorson and Church, 1957) was consistent with the observations that glucose is solely utilized by a direct oxidative route through gluconate and 2KG. The demonstration that spores contain 2KG kinase and 2K6PG reductase of the type described by DeLey (1955) provided at least one route of pyruvate formation via 6PG. In view of the fact that pyruvate is formed more rapidly from 2K6PG than 6PG, it seems likely that another route of 2K6PG utilization may exist in these extracts. Wood et al. (1955), however, have demonstrated that the conversion of 2KG to pyruvate in Pseudomonas fluorescens involves 2K6PG, 6PG, and 2K3D6PG as intermediates. An analysis of the metabolism of phosphosugars, summarized in figure 9, shows that spores contain the pathway analogous if not identical to the HTMIP oxidative route which has been extensively studied in yeast, bacteria, animal, and plant systems. G6P dehydrogenase is TPN specific but 6PG dehydrogenase in contrast to the enzyme from yeast and bacteria (DeLey, 1955) is DPN specific. The sequential formation of R5P, ketopentoses (presumably Ru5P and Xu5P), sedoheptulose, F6P, and G6P provide a cyclic system for G3P formation. These extracts contain not only D-glyceraldehyde-3-phosphate

[VOL. 77

dehydrogenase (Halvorson and Church, 1957) but also the complete system for its conversion to pyruvate. The existence of an active pentose metabolism in these spores was evident from the studies of Lawrence (1955) who observed that stoichiometric amounts of ribose could not be recovered from adenosine following its hydrolysis by the nucleoside ribosidase of spores. The reactions involved have recently been demonstrated by Krask (personal communication) to involve RIP formation from adenosine by nucleoside phosphorylase and its conversion to R5P by phosphoribo-mutase. Alternatively R5P is directly formed from ribose, Mg++, and ATP by a ribokinase present in these extracts. The presence of 2K3D6PG aldolase in these extracts poses the possibility that an alternative route of 6PG oxidation of the Pseudomonas type (Kovachevich and Wood, 1955) may occur. In these organisms 2K3D6PG, formed from 6PG by the action of 6PG dehydrase, is hydrolyzed by 2K3D6PG aldolase to pyruvate and G3P. Although 6PG dehydrase may be functional in activated spores, attempts to demonstrate its presence in sonic extract were negative. The presence of a functional hexose monophosphate oxidative pathway in sonic extracts of activated spores is perhaps not unexpected since its presence has been demonstrated in vegetative cells of this genus (DeLey, 1955; Kunita and Fukumaru, 1956). Spores and vegetative cells of the genus Bacillus contain in addition to the systems for a direct oxidation of glucose an operative tricarboxylic acid cycle (DeLey, 1955; Gunsalus et al., 1955). The multiplicity of the reactions thus far demonstrated in activated spores strongly indicates that the differences in

GLUCOSE

|2H

GLUCONATE

2H

o

2-KETOGLUCONATE

I~~~~~~~~~~ + ATP

-2H

0-6PPt4-GLUCONATEQ* 2 H4 2-KETO-6-P%-GL1UCONATE

GLUCOSE-6-PO4

-2H -C( RIBOSE-5-PO0

s

KETOPENTOSE--PO4

SEDOHE

FRUC TOSE-6-PO4

2-KETO-3-DEOXY6-PO -GLUCONATE

LSE-7-PO4

[GLYCERALDEHYDE-O3PO4-

-- PYRUVATE

Figture 9. Pathways of glucose oxidation in spore extracts

1959]

INTERMEDIATE MIETABOLISM OF AEROBIC SPORES

53

enzymatic patterns of spores and vegetative cells by gradient elution from diethylaminoethyl may- be considerably less than originally expected. cellulose columns. Some of the properties of each Although vegetative cells of the Bacillaceae contain a complete glycolytic system (Dedonder, 1952; Keynam et al., 1954), it is not clear whether the absence of these systems in spores represents a fundamental difference between the two forms. Since sporulation is characterized by an increasing emphasis on oxidative metabolism (H. Orin Halvoison, 1957), one might expect the metabolic activity of spores to more closely resemble that of the sporulating vegetative cell. An examination of the metabolic systems present in vegetative cells during sporulation and in newly formed vegetative cells during germination under aerobic conditions would greatly aid in our understanding of the physiology and biosynthesis of spores.

of these three enzymes are described. REFERENCES BANDtuRSKI, R. S. AND AXELROD, B. 1952 The

chromatographic identification of some biologically important phosphate esters. J. Biol. Chem., 193, 405-410. BECK, W. S. 1955 Determination of those phosphates and proposed modifications in the aldolase method of Sibley and Lehninger. J. Biol. Chem., 212, 847-857. BENSON, A. A., BASSHAM, J. A., CALVIN, MI., GOODALE, T. C., HAAS, V. A., AND STEPKA, W. 1950 The path of carbon in photosynthesis. V. Paper chromatography and radioautography of the products. J. Am. Chem. Soc., 72, 1710-1718. BROWN, A. H. 1946 Determination of pentose ACKNOWLEDGMENT in the presence of large quantities of glucose. The authors wish to express their appreciation Arch. Biochem., 11, 269-278. for valuable assistance to Mr. Ray Epstein, and, CHURCH, B. D. 1955 The role of L-alanine and to Dr. WV. A. Wood, University of Illinois, for glucose on dormancy in spores of aerobic his advice and encouragement throughout bacilli. Ph.D. Thesis, University of Michigan. CHURCH, B. D. AND HALVORSON, H. 1957 The this work. activation of glucose oxidation in spores of B. SUMMARY cereus var. terminalis. J. Bacteriol., 73, 470476. Cell free extracts of spores of Bacillus cereus B. D. AND HALVORSON, H. 1955 Gluvar. terminalis contain glucose dehydrogenase, a CHURCH, cose metabolism by resting spores of aerobic system for the direct oxidation of gluconate to bacilli. Bacteriol. Proc., 1955, 41. 2-keto-gluconate, 2-keto-gluconate kinase, 2- CHURCH, B. D., HALVORSON, H., AND HALVORSON, keto-D-gluconate-6-phosphate reductase, a comH. 0. 1954 Studies on spore germination: plete hexose monophosphate shunt system, its independence from alanine racemase activity. J. Bacteriol., 68, 393-399. 2-keto-3-deoxy-D-gluconate-6-phosphate aldolase, and a system for pyruvate formation from DEDONDER, R. 1952 Glycolysis by Bacillus subtilis and Bacillus megatheriulm. Congr. D-glyceraldehyde-3-phosphate. D-Ribose-5-phosintern. biochim., 2 Congr. Paris, p. 77. phate, ketopentophosphates, sedoheptulose, J. 1955 The hexose monophosphate D-fructose-6-phosphate, and D-glucose-6-phos- DELEY, route in microorganisms. Proc. oxidative phate were identified as products of either Intern. Congr. Biochem., 3rd Congr. Brussels, D-gluconate-6-phosphate or D-ribose-5-phosphate pp. 182-184. oxidation. A kinetic study of pyruvate formation DISCHE, Z. AND BORENFREUND, E. 1951 A new from 2-keto-D-gluconate-6-phosphate and Dspectrophotometric method for the detection and determination of keto sugars and trioses. gluconate-6-phosphate suggested that an J. Biol. Chem., 192, 583-587. alternate pathway for 2-keto-D-gluconate-6phosphate metabolism may be present not DISCHE, Z., SHETTLES, L. B., AND OSNOS, M. 1949 New specific color reactions of hexoses involving D-gluconate-6-phosphate as an interand spectrophotometric micromethods for mediate. their determination. Arch. Biochem., 22, A diphosphopyridine nucleotide linked glucose 169-184. dlehydrogenase was purified by ammonium sulfate ENTNER, N. AND DOUDOROFF, M. 1952 Glucose fractionation and a triphosphopyridine nucleotide and gluconic acid oxidation of Pseudomonas linked D-glucose-6-phosphate dehydrogenase and saccharophilia. J. Biol. Chem., 196, 853-862. a diphosphopyridine nueleotide linked D- FRIEDEMANN, T. E. AND HAUGEN, G. E. 1943 Pvruvic acid. II. The determination of keto gluconate-6-phosphate dehydrogenase puirified

54


acids in blood and urine. J. Biol. Chem., 147, 415-442. GUNSALUS, I. C., HORECKER, B. L., AND WOOD, W. A. 1955 Pathways of carbohydrate metabolism in microorganisms. Bacteriol. Revs., 19, 79-128. HACHISUKA, Y., ASANO, N., KANEKO, M., AND KANBE, T. 1956 Evolution of respiratory enzyme system during germination of Bacillus sutbtilis. Science, 124, 174-175. HALVORSON, H. 0. 1958 Oxidative enzymes of bacterial spore extracts. In Spores. Edited by H. 0. Halvorson, pp. 144-160. Am. Inst. Biol. Sci., Washington, D. C. HALVORSON, H. AND CHURCH, B. D. 1957 Intermediate metabolism of aerobic spores. II. The relationship between oxidative metabolism and germination. J. Appl. Bacteriol., 20, 359-372. HALVORSON, H. ORIN 1957 Rapid and simultaneous sporulation. J. Appl. Bacteriol., 20, 305-314. HESTRIN, S. 1949 The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application. J. Biol. Chem., 180, 249-261. HORECKER, B. L., SMYRNIOTIS, P. Z., AND KLENOW, H. 1953 The formation of sedoheptulose phosphate from pentose phosphate. J. Biol. Chem., 205, 661-682. KEYNAM, A., STRECKER, H. J., AND WALSCH, H. 1954 Glutamine, glutamic acid, and glycolysis in Bacillus subtilis. J. Biol. Chem., 211, 883-891. KoVACHEVICH, R. AND WOOD, W. A. 1955 Carbohydrate metabolism by Pseudomonas fluorescens. III. Purification and properties of a 6-phosphogluconate dehydrase. IV. Purification and properties of a 2-keto-3-deoxy-6phosphogluconate aldolase. J. Biol. Chem., 213, 745-767.

KUNITA, N.

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AND FUKUMARU, T. 1956 Carbohydrate oxidation by Bacillus subtilis. 1. Mechanism of glucose oxidation. Med. J. Osaka Univ., 6, 955-967. LANNING, M. C. AND COHEN, S. S. 1951 The detection and estimation of 2-ketohexonic acids. J. Biol. Chem., 189, 109-114. LAWRENCE, N. L. 1955 The cleavage of adenosine by spores of Bacilluts cereuts. J. Bacteriol., 70, 577-582. LINEWEAVER, H. AND BURK, D. 1934 The determination of enzyme dissociation constants. J. Am. Chem. Soc., 56, 658-666. LOWRY, 0. H., ROSEBROUGH, N. J., FARE, A. L., AND RANDALL, R. J. 1951 Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265-275. MIURRELL, W. G. 1955 The bacterial endospore. Monograph published by the University of Sydney, Australia. MURTY, G. G. K. AND HALVORSON, H. 0. 1957 Effect of duration of heating, L-alanine and spore concentration on the oxidation of glucose by spores of B. cereus var. terminalis J. Bacteriol., 73, 235-240. NELSON, N. 1944 A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153, 375-380. SABLE, H. F. AND GUARINO, A. J. 1952 Phosphorylation of gluconate in yeast extracts. J. Biol. Chem., 196, 395-402. SOBER, H. A., GUTTER, F. J., WYCKOFF, M. M., AND PETERSON, E. A. 1956 Chromatography of proteins. II. Fractionation of serum protein on anion-exchange cellulose. J. Am. Chem. Soc., 78, 756-763. WOOD, W. A., NARROD, S. A., AND HERTLEIN, B. C. 1955 The conversion of 2-ketogluco nate to pyruvate by enzymes from Pseudomonas fluorescens. Proc. Intern. Congr. Biochem., 3rd Congr., Brussels.