Tricarboxylic Acid Cycle Intermediates - Journal of Bacteriology

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Jan 9, 1973 - Isolation of a phosphotransferase system from Esche- richia coli. ... Romano, A. H., S. J. Eberhard, S. L. Dingle, and T. D. McDowell. 1970.
JOURNAL OF BACTERIOLOGY, OCt. 1973, p. 271-278 Copyright 0 1973 American Society for Microbiology

Vol. 116, No. 1 Printed in U.S.A.

Abolition of Crypticity of Arthrobacter pyridinolis Toward Glucose and a-Glucosides by Tricarboxylic Acid Cycle Intermediates MARK E. SOBEL, ELLEN B. WOLFSON,

AND

TERRY A. KRULWICH

Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York, New York 10029

Received for publication 9 January 1973

Arthrobacter pyridinolis cannot grow on glucose as sole carbon source, although the cells possess catabolic enzymes of the Embden-Meyerhof and pentose phosphate pathways as well as a complete tricarboxylic acid cycle. Crypticity toward glucose is abolished by a period of growth in a medium containing malate, succinate, citrate, or fumarate in addition to glucose. Other carbon sources, which support as rapid growth as does-malate (e.g. asparagine), do not enable the cells to use glucose. Malate, succinate, citrate, and fumarate abolish crypticity toward glucose only in the second phase of diauxic growth after the tricarboxylic acid cycle intermediate has been depleted. This sequence of events, first observed in growth curves, has been verified by experiments in which the incorporation of radioactive substrates into trichloroacetic acid-insoluble cellular material was followed. The tricarboxylic acid cycle intermediates which confer the ability to utilize glucose also enhance the utilization of the a-glucosides sucrose and maltose. The mechanism whereby growth on certain tricarboxylic acid cycle intermediates confers the subsequent ability to grow on glucose is related to a transport system for glucose and a-glucosides. This transport system has been assayed by measuring the uptake of [1-_4C]-2-deoxyglucose. Cells grown for varying periods of time in asparagine, asparagine plus glucose, or malate do not transport 2-deoxyglucose. Cells from malate-glucose cultures that are in the exponential phase of growth on glucose can transport 2-deoxyglucose. Transport of 2-deoxyglucose shows Michaelis-Menten kinetics with a Km of 2.9 x 10-4 M. It is competitively inhibited by glucose, a-methylglucopyranoside, and maltose. The transport of 2-deoxyglucose is inhibited by cyanide, dinitrophenol, azide, and N-ethylmaleimide, but not by malonate or fluoride. No phosphoenolpyruvate: D-glucose phosphotransferase activity has been detected, and the 2-deoxyglucose transported into the cell is not phosphorylated.

Through studies of whole cells and isolated membrane preparations, investigators of sugar transport in bacteria have elucidated seve,ral major mechanisms for these processes. Thus, it is clear that in many bacteria a spectrum of sugars enter the cell with concomitant phosphorylation via the Roseman phosphoenolpyruvate (PEP):hexose phosphotransferase system (13, 14, 18, 20, 22). Certain other sugars, e.g., lactose, are transported without phosphorylation by using energy derived from membranebound dehydrogenase systems (2, 6, 9-11, 19). A role for binding proteins has also been indicated in the transport of some sugars (3, 5).

We have been studying several species of Arthrobacter which are capable of growth on a wide variety of organic and amino acids but grow, and often poorly, on only a few sugars. The subject of the present study, A. pyridinolis, can use D-fructose or L-rhamnose as sole sources of carbon but cannot grow on over 10 other carbohydrates tested. D-Fructose is metabolized in this organism by conversion to D-fructose 1-phosphate by a PEP: D-fructose phosphotransferase followed by a second phosphorylation to form D-fructose 1, 6-diphosphate (21). The pathway for L-rhamnose uptake and metabolism is unknown. 271

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SOBEL, WOLFSON, AND KRULWICH

During the work on hexose utilization by A. pyridinolis, it became apparent that, whereas D-glucose could not be used when present as sole carbon source, this hexose could be used when certain other carbon sources were also present in the medium. We now describe these observations and relate them to the characteristics of the transport system for D-glucose in A. pyridinolis. This transport system appears to be inducible, and does not involve phosphorylation of the substrate. MATERIALS AND METHODS Bacteria and growth conditions. Arthrobacter pyridinolis was used for all studies. The growth conditions and mineral medium (MS) have been described previously (25). Carbon sources were added to MS from separate sterile solutions. For routine maintenance, cells were grown in PYE medium containing 0.2% peptone, 0.1% yeast extract, and 0.02% MgSO4 .7H2O. Growth studies were conducted by using 300-ml side-arm flasks as previously described (25). The turbidity of the cultures was determined at intervals by using a Klett-Summerson colorimeter with a no. 42 filter. Chemicals. "C-asparagine (U), [2,3-14C]-succinate, and a-[methyl-"4C]-glucopyranoside (U) were purchased from Amersham-Searle, and "4C-glucose (U) and [1-"C]-2-deoxyglucose were purchased from New England Nuclear Corp. Lysozyme and glucose 6-phosphate dehydrogenase were obtained from Boehringer-Mannheim Corp. Nicotinamide adenine dinucleotide phosphate and dichlorophenolindophenol were purchased from Sigma Chemical Co. All other chemicals were obtained commercially at the highest purity available. The D-isomers of all sugars were

used.

2-Deoxyglucose and a-methylglucopyranoside uptake assays. Cells were washed and resuspended in MS. The cell concentration was adjusted to 100 Klett units. Cells were then incubated at 30 C for 30 min with 40 ,ug of chloramphenicol per ml. Then, at room temperature, [1_-4C]-2-deoxyglucose or a[methyl-"C]-glucopyranoside (U) was added to the desired concentration. Uptake was observed only when aerobic conditions were maintained; therefore, the incubations were all carried out with shaking of the flasks at 200 rpm. At intervals, 1-ml samples were filtered through filters (0.45 um; Matheson-Higgins Co.). Filters were immediately washed with 10 ml of cold MS and dried. The radioactivity was measured by scintillation counting in Bray solution (4). Enzyme assays. Crude extracts were prepared by sonic disruption of washed cells as previously described (25). Before being used for enzyme assays, the extracts were dialyzed against the buffer to be used for the assay. Glucokinase (EC 2.7.1.2) was assayed by the methods of Patni and Alexander (17). Glucose dehydrogenase (EC 1.1.47) was determined by the method of Hauge (8). The spectrophotometric assays were conducted by using a Gilford model 240 recording

J. BACTERIOL.

spectrophotometer at 26 C. PEP:glucose phosphotransferase activity was assayed in extracts of malateglucose-grown cells by the method of Tanaka, Lerner, and Lin (23) by using 2 mM glucose. Samples from the reaction mixture were spotted on diethylaminoethyl disks (16), which were washed exhaustively with water, dried, and counted by scintillation counting. Quantitative recovery of "C-fructose 1-phosphate was obtained under the experimental conditions used (21). Protein was determined by the method of Lowry et al. (15) by using lysozyme as a standard. Substrate incorporation studies. Cells grown in PYE medium were inoculated into MS medium containing radioactively labeled glucose, succinate, or asparagine in various combinations with nonradioactive substrates. The concentration of glucose when present was 0.045 M; the concentration of succinate or asparagine when present was 0.015 M. The specific radioactivity of all labeled substrates was 2 x 10-3 Ci/mol. At intervals, 0.1-ml samples were filtered through filters (Matheson-Higgins Co.). The filters were washed with 10 ml of cold 10% trichloroacetic acid and dried. The radioactivity was measured as described above. RESULTS A. pyridinolis did not grow when glucose was the sole source of carbon for growth (Fig. 1). The organism grows readily on fructose, however, and glucokinase as well as other enzymes of the Embden-Meyerhof and pentose phosphate pathways are detectable in cell extracts (21). A. pyridinolis also has a complete tricarboxylic acid cycle; it is able to grow on citrate, glutamate, or succinate as sole source of carbon. Various compounds were screened for their ability to facilitate utilization of glucose. Comparisons were made between the turbidities attained in 30 h by cultures grown on the compound alone and cultures grown on the compound plus glucose. Glucose utilization seemed to occur in cultures containing citrate, succinate, fumarate, or malate in addition to glucose (Table 1). Oxaloacetate had a much smaller effect, and other carbon sources, such as asparagine, which support as rapid growth as does malate, did not confer the ability to utilize glucose. Fructose and L-rhamnose, which are the only hexoses able to support growth of A. pyridinolis when present as sole carbon source, also did not abolish crypticity toward glucose. The pattern of growth of A. pyridinolis on limiting (0.15 M) malate plus 0.05 M glucose is shown in Fig. 1. Initially, the culture grew at a rate that was characteristic of that observed on malate alone. Then, at the time when growth of cells on 0.015 M malate alone ceased, the cells on malate plus glucose exhibited a lag of 4 to 5 h. After the lag, a second phase of exponential growth occurred. Extracts of cells from the two

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z

GLUCOSE CRYPTICITY

dioactive glucose alone incorporated very little Ilabel. Cells incubated in 0.015 M succinate plus 0.045 M radioactive glucose incorporated only 1.Csmall amounts of label until 15 h after inoculattion. Incorporation of glucose was then oband continued at the rate shown until 1.served, C Iapproximately 40,000 counts per min per ml Iwas incorporated. Thus significant amounts of Ilabel from glucose are incorporated into trichloIroacetic acid-insoluble cell material only after the rate of incorporation of succinate has slowed and a lag in growth has occurred. The same incorporation experiment was conducted with the substitution of asparagine for succinate. Label from asparagine was incorporated for about 10 h either in the presence or absence of glucose; after this time the amount of label in trichloroacetic acid-insoluble material began to decrease. As expected from the growth data, no significant incorporation of label from radioactive glucose occurred, even after utilization of asparagine. Many carbohydrates other than glucose fail to support growth of A. pyridinolis when present as sole carbon source. The possibility that tricarboxylic acid cycle intermediates might

MALATE

1 00-

90 -

soW

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ABOLITION IN A. PYRIDINOLIS

70

60_ < soJ 50

40 30

20

0.05 M GLUCOSE

1~~~~~~~~ 20 10

0

30

40

HOURS FIG. 1. Growth of A. pyridinolis on glucose, malate, and malate plus glucose. Cells were inoculated from PYE cultures into mineral medium containing the indicated carbon sources.

exponential phases contained the same specific activities of glucokinase; these observations are consistent with previous data showing that glucokinase is constitutive in A. pyridinolis. Neither extract contained detectable glucose dehydrogenase activity. Growth curves of A. pyridinolis in 0.015 M citrate, succinate, or

TABLE 1. Ability of various additional carbon sources to enhance the utilization of D-glucose for growth by

Arthrobacter pyridinolisa Klett units after 30 h in

Supplement

D-Glucose Supplement AKlett units plus

alone

supplement

None

Asparagine Aspartate Citrate D-Fructose Fumarate Glutamate

21

185 80 338 370 378 144 16 383 300 390

16 167 38 220

375 204

5 18 42 118 -a 174 62

82 fumarate plus 0.05 M glucose show pattern Glycerol -1 17 similar to that shown in Fig. 1 for malate plus Malate 170 213 80 glucose. 220 Oxaloacetate 50 340 To determine whether, in fact, glucose is used L-Rhamnose 147 238 385 during the second period of the diauxic growth Succinate curve, and is used only during that period, an a Cells of A. pyridinolis were inoculated from PYE experiment was conducted with radioactive cultures into MS plus the indicated supplements (at into of label Incorporation substrates. growth and those supplements (0.015 M) plus 0.05 M) 0.015 was cold trichloroacetic acid-insoluble material After 30 h, the turbidities of the cultures M D-glucose. followed as described in Materials and were determined. The values in the last column are Methods. Cells grown on 0.015 M radioactive the Klett units for the glucose-containing cultures succinate, either with or without 0.045 M glu- minus the Klett units for the cultures containing the cose, incorporated label for 9 to 10 h (Fig. 2A). corresponding supplement alone. The first line reprecontaining gluIncorporation of label from succinate then sents the difference between cultures markedly decreased. Cells incubated with ra- cose and those containing no carbon source. a

274

SOBEL, WOLFSON, AND KRULWICH

facilitate the utilization of these carbohydrates was investigated. Maltose and sucrose could be utilized when malate but not asparagine was also present in the medium (Table 2). Growth curves of A. pyridinolis in 0.015 M malate plus 0.05 M maltose or sucrose were identical to that shown in Fig. 1 for glucose. Utilization of a variety of other carbohydrates tested was not enhanced by malate. The possibility was examined that the crypticity of A. pyridinolis toward glucose and the abolition of this crypticity are related to transport of the hexose. The uptake of 2-deoxyglucose, which is nonmetabolizable in A. pyridinolis, was used to monitor the transport system for glucose. Uptake of radioactive 2-deoxyglucose was measured in cells grown in asparagine, asparagine plus glucose, malate, and malate plus glucose for 9, 13, or 20 h. Only malate-glucose-grown cells which had been growing for 20 h and were in the phase of exponential growth on glucose took up 2-deoxyglucose appreciably. The data for the 20-h cultures on each of the media are shown in Fig. 3. An experiment was conducted to determine whether the failure of the 9- and 13-h cultures in malate-glucose to take up 2-deoxyglucose might be due to an insufficiently long period of exposure to glucose. Cells were incubated in 0.015 M malate for 12 h. Glucose was then added to a final concentration of 0.05 M, and the cells were

12

8

24 0

r8) 0

8

4

TABLE 2. Enhanced utilization of various growth substrates in the presence of additional compoundsa Growth substrate

OL,

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Growth increment (AKIett units) supported by Malate Asparagine

D-Glucose (21) 18 149 D-Galactose (12) 5 35 D-Ribose (16) -8 1 D-Maltose (26) 40 215 25 224 FIG. 2. Incorporation of label from radioactive Sucrose (34) -24 8 growth substrates into trichloroacetic acid-insoluble D-Mannitol (19) -33 cell material. A: Cells were grown in mineral medium Lactose (27) -21 plus 0.015 M 12,9-I4C1-succinate (0); 0.015 M [2,3'4CJ-succinate plus 0.045 M glucose (0); 0.045 M aAsparagine and malate (0.015 M) were tested for "C-glucose (U) (x); and 0.015 M succinate plus 0.045 ability to enhance the growth of A. pyridinolis on the M 'IC-glucose (U) (A). B: Cells were grown in mineral carbon sources listed in the first column. The values medium plus 0.015 M "4C-asparagine (U) (0); 0.015 are the AKlett units between cultures containing 0.05 M '4C-asparagine (U) plus 0.045 Mglucose (0); 0.045 M of the carbohydrate growth substrate plus 0.015 M M "4C-glucose (U) (x); and 0.015 M asparagine plus of the supplement and the cultures containing 0.015 0.045 M "4C-glucose (U) (A). The specific radioactiv- M supplement alone. The numbers in parentheses ity of all labeled substrates was 2 x 10-1 Ci/mol. next to the growth substrates indicate the Klett units Samples (0.1 ml) were taken at intervals, treated with attained in 30 h in cultures with that substrate cold trichloroacetic acid and counted as described in present alone at 0.05 M. The experiment was conMaterials and Methods. ducted as described in Table 1. 0

10 HOURS

20

VOL. 116, 19,73 17 n

GLUCOSIE CRYPTICITY ABOLITION IN A. PYRIDINOLIS XNT TT----

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tially identical to the uptake of 2-deoxyglucose observed under the same conditions. y )_ / The effect of 2-deoxyglucose concentration on -c the initial rate of its uptake is shown in Fig. 4. The Km for uptake was 2.9 x 10-4 M. This Km 0-i;00 3 _ / value as well as the rates of uptake observed are in the same range as the values found for glucose uptake in Arthrobacter crystallopoietes OD0(12) and are consistent with the doubling times > E of cultures growing on glucose. The uptake of 2-deoxyglucose is competitively inhibited by 0.5 x E _ mM glucose, a-methylglucopyranoside, and X W maltose (Fig. 5). 9Oc;jcm / 0 o No PEP-dependent phosphorylation of glu4.O _ cose or 2-deoxyglucose could be detected in extracts from cells grown in 0.015 M malate plus )< E| 0.05 1 M glucose for 20 h. To ascertain whether accumulated as a phosphoryl2-deoxyglucose 3 0 6 9 12 ated derivative, iswashed cells were incubated in MINUTES MS containing 1 mM radioactive 2-deoxyFIG. 3. Ulptake of 2-deoxyglucose by A. pyridinolis. glucose for 10 min as described for uptake The cells we;re grown for 20 h in 0.015 M malate (0); experiments in Materials and Methods. The 0.015 M mailate plus 0.05 M glucose (0); 0.015 M cells were then harvested by centrifugation and asparagine ((A); 0.015 M asparagine plus 0.05 M resuspended in 0.05 M tris(hydroxymethyl)glucose (A). Uptake was determined as described in aminomethane buffer, pH 7.0. Extracts were Materials arid Methods by using 1 mM [1-14C]-2- prepared and were found to contain 6,160 deoxyglucose (0.025 Ci/mol). counts per min per ml. Approximately 4% of this radioactivity was retained by cationic DE incubated f.or an additional 8 h. These cells took 81 filter disks after the disks were washed with up 2-deox)yglucose as well as did the cells water. A sample of the extract was adjusted to incubated in 0.015 M malate plus 0.05 M pH 9 and incubated with alkaline phosphatase glucose for the entire 20 h. for 10 min at 37 C. After neutralization, the An exper,iment was then conducted to deter- treated extract was spotted on DE 81 disks. mine whet}her protein synthesis is required for Again, 4% of the total counts was retained by developmernt of the capacity to transport glu- the disks after the disks had been washed with cose. Two cultures were grown in 0.015 M water. The same results were obtained when malate for 14 h. Chloramphenicol was added to one of the cultures (to a final concentration of 40 Ag/ml). After 15 min glucose (0.05 M) was added to both cultures. The cells were incubated for an additional 5 h, after which they were washed and assayed for viability and for the capacity to transport 2-deoxyglucose. Incubation with chloramphenicol caused no loss of viability, but prevented development of transport activity. Cells grown for 21 h in 0.015 M malate plus 01 2 4 6 8 10 4I 0.05 M maltose took up 2-deoxyglucose as well [SIS] as did cells grown for the same period of time in I malate plus glucose. Cells grown for 21 h in 0 2.0 4.0 6.0 8.0 10.0 0.015 M asparagine plus 0.05 M maltose, how2-DEOXYGLUCOSE CONCENTRATION (mM) ever, did not take up 2-deoxyglucose. 4. Effect of 2-deoxyglucose concentration on Cells grown for 21 h in 0.015 M malate or theFIG.initial rate of its uptake into malate-glucoseasparagine, with and without 0.05 M glucose or grown cells. Uptake was determined as described in maltose, were also tested for uptake of a- Materials and Methods by using cells grown for 25 h [methyl- "C]-glucopyranoside (U). In all cases, on 0.015 M malate plus 0.05 M glucose. Inset: uptake of a-methylglucopyranoside was essen- Lineweaver-Burk plot of the data. l

w

X

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SOBEL, WOLFSON, AND KRULWICH

J. BACTERIOL.

It thus appeared that, after a period of growth certain tricarboxylic acid cycle intermediates, a transport system for glucose and a-glucosides could be induced and that this transport system did not involve phosphorylation of the substrate but was sensitive to uncouplers and inhibitors of electron transport. In order to determine whether the presence of the tricarboxylic acid cycle intermediate was still required after the transport system had been induced, cells which had been grown in malate (0.015 M) plus glucose (0.05 M) for 21 h were washed with MS and inoculated into medium containing 0.05 M glucose alone, 0.05 M glucose plus 0.015 M malate, or 0.015 M malate alone. Only the cells inoculated into medium containing both glucose and malate grew on the glucose; these cells grew to a much greater density than did cells inoculated into 0.015 M malate alone. on

22.5 0

2.0

2

E

wc

1.0

c'J E~0.5

0

2

4

6

[2-DEOXYGLUCOSEI

8

10

(mM)

DISCUSSION

FIG. 5. Inhibition of 2-deoxyglucose uptake by A. pyridinolis is cryptic toward glucose, malmaltose, glucose, and a-methylglucopyranoside. The cells and procedure used for uptake were as described tose, and sucrose, and this crypticity is abolin Fig. 4. Maltose (x), glucose (A), or a-methyl- ished by tricarboxylic acid cycle intermediates. glucopyranoside (0) were added at 0.5 mM; the The organism grows on fructose or rhamnose, kinetics of uptake in the absence of additives other however, without any special prior conditions of than substrate is designated by closed circles. growth. Therefore, A. pyridinolis is not simply

radioactive a-methylglucopyranoside was substituted for 2-deoxyglucose in the experiment. The possibility remained that phosphatase activity in the cell rendered PEP-dependent phosphorylation of glucose or 2-deoxyglucose undetectable. If this were true, however, mutants lacking enzyme I of the Roseman phosphotransferase system would not be expected to transport and use glucose as well as the wildtype strain. Such mutants have been isolated as fructose-negative strains and have been characterized (E. B. Wolfson and T. A. Krulwich, unpublished data); when tested for growth on malate plus glucose, they exhibit a growth pattern that is identical to that of the wild type. The effects of several inhibitors on 2-deoxyglucose uptake were determined. N-ethylmaleimide inhibited uptake almost totally (Table 3). Both cyanide and 2, 4-dinitrophenol also inhibited strongly. Azide caused less inhibition, and fluoride and malonate had only a slightly inhibitory effect. The possibility that malate might inhibit or stimulate 2-deoxyglucose uptake was also tested. Concentrations of malate ranging from 0.5 to 50 mM stimulated 2-deoxyglucose uptake up to 50% over that observed in the control.

devoid of PEP carboxylase and other anaplerotic enzymes whose absence would create a requirement for tricarboxylic acid cycle interTABLE 3. Effects of various inhibitors on

2-deoxyglucose uptake by Arthrobacter pyridinolisa 2-Deoxyglucose uptake Inhibitor

Amt of uptake

(x 102 Mmol/mg

Inhibition (%)

6.3 0.1 1.1 1.9 4.3 5.6 5.8

0 99 83 71 32 11 8

of protein)

None

N-ethylmaleimide Potassium cyanide Dinitrophenol

Sodium azide Sodium fluoride Malonate

aCells grown for 22 h (to the glucose logarithmic

phase) in 0.015 M malate plus 0.05 M glucose were washed with MS and incubated in MS plus chloramphenicol as described in Materials and Methods. The

inhibitor indicated was added to a final concentration of 10 mM. After 10 min of incubation with the inhibitor, [1-_4C]-2-deoxyglucose was added to a final concentration of 0.5 mM, 0.025 ACi/ml. The uptake of radioactive substrate was determined after 9 min as described in Materials and Methods.

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mediates during growth on hexoses. Were such a deficiency responsible for the lack of growth on glucose, not only would fructose be nonutilizable, but aspartate and oxaloacetate would be expected to allow growth on glucose as they do with a PEP carboxylase-deficient mutant of Salmonella typhimurium (24) but do not with A. pyridinolis. The crypticity of A. pyridinolis towards glucose and a-glucosides and abolition of the crypticity by certain tricarboxylic acid cycle intermediates are apparently related to an unusual property of the transport system for these carbohydrates. This transport system does not involve phosphorylation of the carbohydrates and is, therefore, distinct from the PEP:hexose phosphotransferase used for fructose metabolism in A. pyridinolis (21). Transport of glucose and a-glucosides is inducible, requiring a period of incubation with a substrate of the transport system. More interesting, however, is the requirement for the presence of certain tricarboxylic acid cycle intermediates for the activity and possibly the induction of the transport system. Perhaps, as in other bacterial transport systems (2, 6, 7, 9-11, 19) and as suggested by the inhibitor experiments, transport of glucose and a-glucosides in A. pyridinolis is coupled to respiration and may utilize a membrane-bound dehydrogenase and associated electron transport chain. In this organism, malate may be the required electron donor for a constitutive dehydrogenase and electron transport chain which are coupled to sugar transport, but the intracellular malate concentration does not reach a high enough level unless malate or a direct precursor is present in the medium. This possibility would be consistent with previous work on the anaplerotic routes in this species (25) and with the continued incorporation of some, albeit very small, amounts of label from the Krebs cycle intermediate during the phase of growth on glucose (Fig. 2A). It would also be consistent with the requirement for malate by cells in which the glucose transport system is already induced. In addition to its role as a substrate for the dehydrogenase, malate or some precursor thereof may be required to induce sufficient levels of the dehydrogenase. Barnes (1) has shown that glucose transport in membrane vesicles from Azotobacter vinelandii is coupled to malate oxidation by a membrane-bound malic dehydrogenase. We are presently preparing isolated membrane preparations of A. pyridinolis for use in determinations of possible electron donors whose oxidation is coupled to transport, and in studies of the transport chain.

277

By using membrane preparations, we will examine the possible differences between the transport chain of asparagine- and malategrown cells, as well as the characteristics of glucose transport in membranes from malateglucose-grown cells. It is clear that the presence of certain tricarboxylic acid cycle intermediates is required for function of the glucose transport system. It is also possible that whereas the tricarboxylic acid cycle intermediate is present in high concentrations, formation of the glucose transport system is repressed. Thus, the capacity to transport glucose appears only after the tricarboxylic acid cycle intermediate has been depleted to levels which are not repressive but are sufficient to supply substrate for a transport-linked dehydrogenase and a lag has occurred. In another species of Arthrobacter, A. crystallopoietes, succinate has been shown to repress glucose transport (12). In that species, however, succinate also inhibits glucose transport (12), whereas in A. pyridinolis malate does not inhibit the glucose transport system but instead seems required for its operation. Future experiments will be directed at the possible repression of the glucose transport system in A. pyridinolis by high concentrations of the same tricarboxylic acid cycle intermediates required for function of the system. ACKNOWLEDGMENTS This work was supported by research grant GB-20481 from the National Science Foundation, Public Health Service grant AM-1466301 from the National Institute of Arthritis and Metabolic Diseases, and a grant from the Research Foundation of the American Diabetes Association. LITERATURE CITED 1. Barnes, E. M., Jr. 1972. Respiration-coupled glucose transport in membrane vesicles from Azotobacter vinelandii. Arch. Biochem. Biophys. 152:795-799. 2. Barnes, E. M., Jr., and H. R. Kaback. 1972. Mechanisms of active transport in isolated membrane vesicles. I. The site of energy coupling between D-lactic dehydrogenase and ,-galactoside transport in Escherichia coli membrane vesicles. J. Biol. Chem. 246:5518-5522. 3. Boos, W., and A. S. Gordon. 1971. Transport properties of the galactose-binding protein of Escherichia coli. J. Biol. -Chem. 246:621-628. 4. Bray, G. E. 1960. A simple efficient liquid scintillation for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:279-285. 5. Fox, C. F., J. R. Carter, and E. P. Kennedy. 1967. Genetic control of the membrane protein component of the lactose transport system of Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 57:698-705. 6. Harold, F. M. 1972. Conservation and transformation of energy by bacterial membranes. Bacteriol. Rev. 36:172-230. 7. Harold, F. M., and J. R. Baarda. 1968. Inhibition of membrane transport in Streptococcus faecalis by un-

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8. 9.

10.

11.

12.

13.

14.

15. 16.

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couplers of oxidative phosphorylation and its relationship to proton conduction. J. Bacterlol. 96:2025-2034. Hauge, J. G. 1966. Glucose dehydrogenases-particulate, p. 92-98. In W. A. Wood (ed.), Methods in enzymology, vol. 9. Academic Press Inc., New York. Kaback, H. R., and E. M. Barnes, Jr. 1971. Mechanisms of active transport in isolated membrane vesicles. II. The mechanism of energy coupling between D-lactic dehydrogenase and ,8-galactoside transport in membrane preparations from Escherichia coli. J. Biol. Chem. 246:5523-5531. Kerwar, G. K., A. S. Gordon, and H. R. Kaback. 1972. Mechanisms of active transport in isolated membrane vesicles. IV. Galactose transport by isolated membrane vesicles from Escherichia coli. J. Biol. Chem. 247:291297. Konings, W. N., E. M. Barnes, Jr., and H. R. Kaback. 1971. Mechanisms of active transport in isolated membrane vesicles. III. The coupling of reduced phenazine methosulfate to the concentrative uptake of ,8-galactosides and amino acids. J. Biol. Chem. 246:5857-5861. Krulwich, T. A., and J. C. Ensign. 1967. Alteration of glucose metabolism of Arthrobacter crystallopoietes by compounds which induce sphere to rod morphogenesis. J. Bacteriol. 97:526-534. Kundig, W., F. D. Kundig, B. Anderson, and S. Roseman. 1966. Restoration of active transport of glycosides in Escherichia coli by a component of a phosphotransferase system. J. Biol. Chem. 241:3243-3246. Kundig, W., and S. Roseman. 1971. Sugar transport. I. Isolation of a phosphotransferase system from Escherichia coli. J. Biol. Chem. 246:1393-1406. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Newsholme, E. A., J. Robinson, and K. Taylor. 1967. A radiochemical enzymatic assay for glycerol kinase and hexokinase. Biochim. Biophys. Acta 132:338-346.

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17. Patni, N. J., and J. K. Alexander. 1971. Utilization of glucose by Clostridium thermocellum: presence of glucokinase and other glycolytic enzymes in cell extracts. J. Bacteriol. 105:220-225. 18. Romano, A. H., S. J. Eberhard, S. L. Dingle, and T. D. McDowell. 1970. Distribution of the phosphoenolpyruvate: glucose phosphotransferase system in bacteria. J.

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