ABSThACT OP THE THESE OP

Gerald Q. Still ror the Maeter oZ Science degree in Cheiiistry.

Dat. thesis le presented ----T -'í?'3.

Titi. TH OCCURRENCE OP THE N-DOUD1OFF PATHWAY IN ATìCT] VINELíNDII

Abstract Approved.

The c*tàcUrn of glucose in proltterating cille of Azotobactsi has been examined with r.epsst to the nature end Uts aticn of the individuai pathways. The radiorespi5lMtric method, as wefl as incorporation end degradation experita, were used in this investigation.

The radiorespirometric data has been interpreted to indicate that the &itner-Doudorot'f and Pentose Phosphate pathwa7s are playing inortent roles in glucose catabolien. The thden-yerhof-Parnas pathway, if' present, does not contribute xmich to the overall utilization of glucose, Incorporation rnd deadation experimente support the findings ob3erved in the radioreaplrnetric expernte and revealed the operation of the Hexose Cycle in these proliferating cuiture3. lt is estinted that the operation of the Pentosa Phornhat. pathy in this organism should be not greater than ZQ% o1 the totaUr eatabolized glucose. Therefore, the Etner-Do*ioroft pathway represents the major glucoac utilizing pathway in opbacter e14I,

THE OCCURRENCE OF THE ENTNER-DOtJDOROFF PATHWAY IN AZOTOBACTER VINELANDII

by

GERALD G. STILL

A THESIS

submitted to

OREGON STATE UNIVERSITY

in partial fulfillment of the requirements for the

degree of

MASTER OF SCIENCE

June 1963

APPROVED:

ernistry

In Charge of Major

rman of Department of Chemistry

Date thesis is presented 1zLLí,3T /3 Typed by Kay J. Stili

THIS THESIS IS DEDICATED TO "MY THREE STILLS" WHO WERE INSTRUMENTAL IN THE CONDENSATION AND COMPLETION OF THIS STUDY.

ACKNOWLEDGEMEiTS

The author wishes to acknowledge Dr. A. C. Zagallo

who introduced this unique microbe to this laboratory.

To Mrs. Julie Krackov who nurtured and studied this

nitrogen-fixing bacterium with sufficient finesse to

yield the basic patterns upon which this thesis was

built, I extend my appreciation.

My deepest gratitude to my major professor

and friend, Dr. Chih H. Wang, for his patience and

unfaltering confidence.

Financial assistance was provided by Federal

grants from the Atomic Energy Commission and. the

National Institute of Health.

TABLE OF CONTENTS

Page

INTRODUCTION. . . . . , . . . . . . . . . . . . . i

MATERIALS AND METHODS , . . . . . . . . . . . . . . 3

Culture Conditions . . . . . . . . e . s i s e 3 Radiochemical Substrates . . . . . . . . . . . :3

Radlorespirometric Experiments . . . . . . . .

Incorporation Experiments . . . . . e . e 14.

Degradation Procedures . . . . . . . s e e e e 6 Determination of Radioactivity . . . . . . . s 7

RESULTS. . . . . . . . . . . . . . . . . . . . . 9

DISCUSSION. . . . . . . . . . . . . . . . . . . .

BIBLIOGRAPHY . . . . . . . . . e e s 22

LIST OF FIGURES

Figure 1. Radiorespirometric Pattern. Azotobacter vinelan&1i metabolizing specifically labeled glucose . . . . . . 10

Figure 2. Rad.iorespirometrlo Pattern. Azptpbpcter vinelandli metabolizing specifically labeled gluconate . . . . . li

LIST OF TABLES

Page

Table I, Utilization of Labeled Substrate by ptobacter vinelandli . . . . . . 12

Table II. Incorporation of G1ucose-C NaHCO3 into Azotobacte vinelandii . . . . . 13

Table III. The Isotopic Distribution Pattcr of Cel1ulr Alanine , , 19

THE OCCURRENCE OF THE ENTNER-DOUDOROFF PATHWAY IN AZOTOBACTER VINELANDII

INTRODUCT ION

Glucose catabolism in &zptpbgcter vinelandl.i has

been of interest to a number of research workers for some

time. These investigations have been stimulated pri-

manly by the ability of this organism to assimilate

molecular nitrogen and to oxidize extensively carbona-

ceous substrates.

It has been shown by Mortenson and Wilson (12, 13)

that glucose can be converted to 6-phosphogluconate

(6-PG) by extracts vinelandit. This

finding implies that either the Pentose Phosphate (P?)

pathway (i1i) or the Entner-Doudoroff (ED) pathway (1i), or

both, are operative in this organism. The operation

of the ED pathway was later suggested by W11on J,.,

(li) on the grounds that 6-PG was found to be cleaved by

coil-free extracts to g3yceraldehyde-3-phosphate (G_3-P)

and pynuvic acid. However, the two key enzymes associated

with the ED pathway, i.e., 6-phosphogluconic dehydrogenase

and 2-keto-3-deoxy-6-phosphogluconic-aldolase, have not

been detected in this organism. In addition, there has

been demonstrated the occurrence of 6-phosphogluconic dehy-

drogenase (13), transaldolase and transketolase (10) in

cell-free extracts of vinelandil which are the key steps

2

in the PP pathway. Al]. of the enzymes of the

Embden-Meyerhof-Parnas (Er'1P) pathway, excluding

phosphohexokinase, have been demonstrated to occur in

cell-free extracts of vinelandli. It was suggested.

by Wilson ., (13) that phosphohexokinase may have

been destroyed in preparation of the cell-free extract.

More recently Sobek and Clifton (7, 17) suggested that

in Azotobacter ai.lis, glucose may have been utilized

via the concurrent operation of the EMP and PP pathways.

The utilization of pyruvate, a key intermediate in

glucose catabolism, has been extensively studied in

vineandi (19, 20, 21). This includes the demon-

stration of the occurrence of the tricarboxylic acid

(TCA) cycle as evidenced by the observed oxidation of

TCA cycle intermediates with cell-free extracts and

the incorporation of acetate-l-C into cellular

constituents.

In the present study the catabolism of glucose

in proliferating cells of vinelandil has been examined

particularly with respect to the nature and the partici.-

pation of the individual pathways. Radiorespirometric

data (26) for the utilization of C specifically labeled

glucose and gluconate are presented along with the experi-

mental findings of the incorporation of specifically

labeled glucose and NaHCO3 into the cellular amino acids.

MATERIALS AND METHODS

Culture Cpndjtjpn

vineland.tl (ATCC 91014.) was maintained on

agar slants. Experimental cells were grown in a medium

containing: K2HPOj4., 0.8 g.; KH2POk, 0.2 g.;

MgSOz. 7H20, 0.2 g.; CaSO. . 2H20, 0.1 g.; NHj4.C1, 0.2 g.;

NHNO3, 0.3 g.; 1.0 cc of' Mo, Fe trace element solution

(No 03, 7.5 mg.; FeSOj., i 7H20, 299.0 mg.; water added to

two volumes of 50 cc.); glucose 12 g.; all dissolved in

one liter of water. Incubation was carried out aerobi-

cally on a rotary shaker at 28° C for 16 hours at which

time the culture was in the first third of the exponential

phase. The cells were harvested by centrifugation and

resuspended in growth medium to a prescribed concentration

prior to experimentation (excluding glucose).

Radiochemical Substrates

The radiochemical substrates used in the present

work were Glucose-1,-2, or 6-Cfl obtained from the National

Bureau of Standards. Glucose-3-C was kindly furnished

by William Sacks of Rockland State Hospital, Orangeburg,

New York.

Glucose-3,k-C was

from rats injected, with Na:

of Wood, Libson and Lorber

6-Ca was prepared in this

Li..

prepared from liver glycogen

according to the method

(28). Gluconate-2,-3,-3,Li or

laboratory from the corre-

spondingly labeled glucose samples according to the

method Oi Moore and Lirk (9). Gluconate-l-C was

obtained from Nuclear Chicago Corporation.

Rpdjprejrpnietrje Exneriments

The radiorespirometric studies on the utilization

of glucose and gluconate by proliferating cells were

carried out according to the method of Wang and Krackov

(26). At the termination of each of the experiments,

the cells and incubation media were separated by centri-

fugation and processed for the analysis of radioactivity

therein.

IncprlDpration ExDerjmpnt$

These experiments were designed to trace the

individual carbon atoms of glucose in biosynthetic

functions. To 62 cc of a coU suspension, containing

50 mg (dry wt.) of cells, were added a given amount of

specifically labeled glucose (Table II). The culture was

incubated at 28° C in the manner similar to that used under

5

radioresplron!etric experiments. Utilization of the

respective substrates was followed by examining the

iLl. rate of respiratory C 02 production. Upon termina-

tion of each of the experiments the cells were

harvested, washed, and dried over P205 j vacuo.

In a separate experiment the incorporation of

metabolic CO2 into cellular constituents was examined

by the use of NaHCO3 (2OO''c; specific activity

233 /(c/mg) as co-substrate to unlabeled glucose in an

enclosed system. After four hours of incubation the

flask was chilled to 00C, the p11 of the media adjusted

to 5 and the residual CO2 recovered from the system. Thereafter, the cells were processed as described under

Radiorespirometric Experiments.

The cell samples obtained from the incorporation

experiments were hydrolyzed individually with 20% 11Cl

(in sealed tubes) at C for 19 hours. The hydroly-

sates were evaporated to dryness j and taken up

in a defined amount of water. These hydrolysate

solutions were then chromatograhed on large sheets of

Schleicher and Schuell 14.70-A chromatograph paper employing

several solvent systems.

6

The solvent, 80% phuo1-water, was used for the

separctio. of alauinc, aspartic acid and glutamlo acid

(is). TI-ic a1mine fraction which wai slightly contami-

nated waE; purified by paper ohroruatography using

2butano1-3 &q, (15). The aspartic acid arid gluta-

mie acid fractions were individually purified with

butariol:acetic acid:water, O:1O :50 paper chromatography.

Specific activities of' each of the amino acids were

calculated from the radioactivity counting data using

liquid scintillation counting and the chemical analysis

according to the method of lemm and cocking (29).

For degradation studies each of the amino acids

was diluted with carrier to a suitable quantity.

Dezradation Procedures

lnthe:

The distribution of C in the samples of cellular

alanine was determined by means of the following chemical

operations (A) The total radiochemical assay of alanine

was carried out according to the combustion method

described by Schöniger (8); (B) The ninhydrin decarboxy-

latiori reaction (23) was used to convert C-1 of alanine to

CO2 for radiochemical assay; (C) C-2 and C-3 of alanine

7

were trapped j. HS03 as acetaldohyde, the product of

the nirthydrin reaction, and further degraded by means

of the iodoforni reaction (16) forming CHI from C-3,

CHI3 was combusted. to CO2 by the Van S1ykeFo1ch wet

combustion as described by Calvin (2); (D) The

radioactivity of C-2 was calculated by difference.

Determination Rpdjopctvjty

The respiratory CO2 obtained in radiorespiro-

metric experiments was absorbed in 10 cc of ethanolic

0.25 ?4 hyamne hydroxide [(p-diisobutylcresoxyethyl)

dimethylbenzyl ammonium hydroxide] and placed lU a

radiorespirometric CO2 trap (26). The trap solution

was replaced hourly and its radioactivity assayed by

liquid scintillation counting. Usually 5 cc aliquote

of the trapped solution were mixed with 10 cc of

toluene containing terphenyl (3g/l.) and l,14-bis-2

(5-phenyloxazolyl)-benzene (30 mg./l), in a 20 cc glass

counting vial. Countings were carried out with a

tri-carb liquid scintillation counter in the manner

described by Wang (26). The radioactivity of the cell

samples was also counted by means of liquid scintillation

counting as described by White and Helf (27). The

efficiency of radioactivity detectim for each type of

[;:1

counting sample was determined by the use of internal

standards.

The samples obtained in the degradation

experiments were converted into BaCO and mounted on

aluminum planchets by means of the centrifugation

technique (6). Determination of radioactivity was

carried out on a Geiger-Muller counter. Corrections

of counting data for background and self-absorption

were applied in the usual manner. Ali counting operations

were carried out to a relative standard deviation no

greater than 2%.

RESt'LTS

The radiorespirometric data on the utilization of

specifically labeled glucose and gluconate by

vinelandil are given in Figuren I and 2 respectively.

The experimental conditions as well as the distribution

of the substrate activity in cells, i.ncubation media and

respiratory CO2 at the end of the respective radio-

respirometric experiment are given in Table I.

In Table II is given the essential findings

obtained in the incorporation experiment including the

isotope distribution of cellular alanine samples derived

from the respective labeled substrates.

H z w C)

w ;5o N

o

4o

w > o o

3o -J

> n: w H z

Elli

lo

Fjure 1.

__80L . AZOTOBACTER VNELANDII 60 . //

4O- /ì'Ï

.

»\ - 7 \ TIME (HRS)

f 2

I iì

.

/i: 6 //

/ì2

/1/ II

Ì 5

TIME (HRS)

Radlorespi.rometric Pattern. Azotobacter vthelandii. specifically labeled glucose.

Glucosel-C _________ -2..0 _____ ____ 3-C11 _ _ - -, .J4-C1.

, _6_Cl'.

I- z w o w Q-

('J

o

IL o >-

w > o o w

-J

w H z

u

FIgure 2,

>-

w -

60 AZOTOBACTER VNELANDII

50 -

_\ 02:

' I t

I/::::::N\ o 2 4 6 8

40 - 1/e'

6 \\\\ TIME (HRS)

II //i:

30-

/,y.:

20-

/ Io- ii

ooJI TIME (HRS)

Radlorespirornetric Pattern. Aztpbacer vne].adl1 netabo11zing specifically labeled gluconate.

G1uconato_1_C14 ________ _2_C14 ____ (I1l h r1 ¿ n14 - _ - .-, --r-%.. I S I L)'.l $

12

Table I

Utilization of C- Labeled Substrate by Azotobacter viaelandii

Subsi.rate % Recovçry of C1

Amount mg. CO2 Cells Mediwn

G1ucose-1-C 16 0.25 93 5 3

G1ucose_2_C14 16 O.21 80 18 6

Giucose-3-C 16 9.8x103 69 22 8

Glucose _3,L4,_C1 16 0.063 71 21 12

Glucose-4-C1 -- --- 73 20 16

Glucose 6_C1k 16 0.27 59 2

Gluconate_l_C1 16 0.23 77 1 10

Giuconate_2_Chl 16 0.15 73 13 8

G1uoonate_3_Chi 16 0.055 72 23 10

Giuconate_3,L_C1 16 0.081 63 19 12

G1uconate4_C1* -- --- 5 1l,

G1uconate_6_C1 16 0.17 6i 24 15

*Clculated values.

Table II

Incorporation of Glucose-Ca NaHC'O3 into Azotobacter vinelandil

Substrate G1ucose-l-C- Glucose_2Cl Glucose-6Cl C1Ll.02

Glucose mg 125 125 125 125 G1ucoseA4- e lo 10 10 NaECO31'+'&c 200 Initial cell

(dry) wt. mg 50 50 50 50 % increase of cell mass 23 17 19 32

Specific Activity*

Fies, CO2 (DPM x i7) 1,93 1.53 1.Li5 32,5 Alanine DPM x 1O1'/miM 1.21 1.81 1.i6 1.81 Aspartic Acid DPN x

106/mM 0.27 1,79 1.12 5.91 Gluttnjo Acid DPM x

106/mN 0.12 1.91 0.66 5.09

Alanjue Carbon Distribution (1) COOM 98 11 32 57 (2) CFINH2 1 85 16 31 (3) CH3 1 52 12 *The amino acid specific activity represents the label in isolated cellular amino acid.

I-J

A)

DIS CUSS ION

Prevous1y, enzymological findings wore presented

by Wilson ., (19, 20, 21) to demonstrate the

operation of the TCA cycle in cell-free extracts of

vinelandii. These extrct oxUized pyruvic acid,

acetic acid. and the acids of the TCA cycle. Upon

addition of acetate_1_C1, radio carbon was rapidly

incorporated into the cellular TCA cycle intermediates.

Radiorespirometric experiments with proliferating cells

of vinelandti fed specifically labeled pyruvate and

acetate have added further proof that these substrates

are routed through the TCA cycle to CO2. With respect

to this information lt is desirable to elucidate the

pathways involved in the production of pyruvate from

substrate glucose. Previous work with cell-free extracts

by Wilson L l) suggested. strongly the

occurrence of the ED pathway and the PP pathway. The

operation of the EMP pathway was uncertain since these

workers failed to detect the presence of phosphohexo-

kinase. However, recent work of Sobek and Clifton (17)

with intact cells tends to support the concurrent

operation of the E1P and the PP pathways for glucose

utilization. Inasmuch as glucose can be catabolizec3. by

15

either the ED the EMP pathway giving rise to

un.ts, and the fact that the concurrent operation of

these two pathways in a given organism has never been

demonstrated, one is therefore interested in which

one of these two pathways is operating in vinelandli. Much is learned by an examination of the radiorespiro-

metric data for the utilization of glucose (Figure 1 and

Table I) by vin,elandii. The C1Z4O2 production patterns

for individual carbon atoms of glucose are basically

similar to those observed with Pseudotnonas fluorescens

KBl (18). There is strong evidence that the latter

organism relies on the concurrent operation of the PP

and ED pathways for the utilization of glucose and

gluconate. In comparing the findings with these two

organisris, a noticeable difference is that with

L2 vinelandU the conversion of C-Lf of glucose to CO2

is significantly less than that of C-2. Nevertheless,

the radiorespirometric data (Figure 1) can be interpreted

to indicate that: (A) the ED and PP pathways are playing

important roles in glucose catabolism; (B) the EM? pathway,

if present, does not contribute much to the overall utili-

zation of glucose. The latter conclusion is drawn from

the fact that a C_3 and Ck of glucose were converted to

CO2 to a baser extent as compared to that of C-2 and

16

particularly C-1. This observation is not in line with

the degradation of glucose via the EMP-TCA sequence

which calls for the preferential oxidation of C3 and C13 of glucose to CO2.

The foregoing analysis is further and more

clearly supported by the radiorespirornetric data on

gluconate utilization (Figure 2, Table I). Here again, the conversion of either C3 or C-11 of gluconate to CO2

is lower in rate and extent as compared to that of C-].

arid C2. A striking observation in the gluconate

experiments is the extraordinary low yield of CO2 from

C-J4 of gluconate even in comparison with C-6. This fact

cannot be explained on the basis of the PP scheme or the

EDTCA route. Some other mechanism must be operative to

account for this finding which may involve either a

preferential combustion of C-6 to CO2 or the retarded

combustion of C-k to CO2.

To elucidate further the mechanism of glucose and

gluconate assimilation in proliferating cells of

vinelandil, the results obtained in the incorporation experiment are helpful. These experiments were designed

to trace the fate of the individual carbon atoms of

glucose in biosynthetic functions, particularly those

responsible for the formation of the carbon skeleton of

'7

key amino acids such as alanine, aspartic acid and

glutamlo acid. An understanding of the or1gin of the

carbon skeletons of these amino acids, which are closely

related to the TCA intermediates, should also provide

information on the nature of the primary pathways of

carbohydrate breakdown (25). This is particularly

true when studying the metabolism of microorganisms

which do not excrete detectable amounts of carbohydrate

intermediates into the incubation medium. To account

for the res-entry of metabolic CO2 into the skeleton of

these amino acids, a separate experiment was carried

out in which unlabeled glucose and NaHCO3 were used

as co-substrates. Insofar as the specific activities

of these amino acidsare concerned, one finds that the

extent of labeling from C-1, C-2, or C-6 of glucose,

in alanine is approximately the same in magnitude.

However, preferential incorporation of C2 and C-6 of

glucose over that of C-1 into either aspartic acid or

glutamic acid was noted. This latter fact is not in

line with the operation of the EP pathway but is

compatible with the operation of the ED scheme for glucose

catabolism. This is true since the precursor of alanine,

pyruvic acid, is one of the key intermediates of glucose

degradation. Prior to the analysis of these patterns,

it was recognized that proliferating cells are capable

of incorporating metabolic CO2 into cellular const-

tuents. This was evidenced by the observed labeling

of amino acids from adnilnistered NaHC14O3 (Table II).

Consequently, the isotopic distribution pattern of

cellular alanine samples, corrected for the re-entry

of metabolic CO2 given in Table III, is much more

meaningful. The corrections were made from the iso-

topic distribution pattern of cellular alanine observed

In the NaHCO3 experiment as described by Wang and

:eaa (25).

The exclusive role played by the ED pathway in

the conversion of glucose to pyruvate is Indicated by

analysis of these isotopic distribution patterns. Thus,

C-1 of glucose was found to be exclusively incorporated

into C-1 of alanine and C-2 of glucose was heavily

incorporated into C-2 of alanlne. The anticipated isotopic

distribution pattern of alanine, or pyruvate, from a

number of possible pathways is summarized in Table III.

The minor labeling of C-1 and C-3 of alanine from C-2

of glucose may reflect the randomization of the glucose

skeleton via the operation of the PP pathway (i, 3).

19 Table III

The Isotopic Distribution Pattern of Cellular Alanine

Pathway Followed by the Expected and Observed Labeled Carbon Atoms Isotopic Distribution

Pattern %

G1ucose-l-C COOH CH(NH2) CH3

Via ED route lOO 0 0

Via EMP route O O loo

Via PP rQute O O O Observed* loo O O

Glucose_2_C14

Via ED route o loo O

Via EMP route 0 lOO O

VIa PP and ED routes (a) 66 o 33 Via PP and EMP routes (a) 33 O 66 Observed* lo 86

Gluco s_6_C

Via ED route o O 100 Via EMP route o O 100 Via liC and ED routes (b) 50 0 50

Via BC and ENP routes (b) O O 100 Observed* 31 16 53

'J 02

Via CO2 fixation (C3 plus C1) lOO 0 0

Observed 57 31 12

a) PP pathway cycles to reform hexose phosphate which is again cleaved to yield triose (i, 26).

b) Triose may be supplied from ED, PP, or EMP pathway. The Hexose Cycle (HC) wifl result in the formation of hoxose phosphate which may then be cleaved as shown.

* Corrected for the re-entry of metabolic C1L102.

ç'

The observed isotopic dlstrlbuti.on. of alanine in the

g1uoose_6-C1' experiment provides additional lnfor-.

matlon on the glucose catabolism of this organism.

The heavy labeling of C-3 of alanine (52% of the total

ratio in the alanine) is in line with the operation of

the ED mechanism. It is surprising to note that signi-

ficant labeling also ocoured. at C-2 and particularly

C-1 of this atnino acia. As shown in Table III, this

finding may have been the consequence of the operation

of the Hexose Cycle (FTC) pathway. Such a pathway

involves the isomerization of G3P to dihydroxyacetone phosphate, condensation of these trioses to fructose

l,6-.diphosphate, followed by dephosphorylization to

fructose 6-P and isornerization to glucose 6-P. This

has been demonstrated by Hochster and Katznelson

(28, -29). It is recalled that earlier Wilson

failed to detect the presence of phosphohexokinase in

this organism. This may account for the observed

operation of the HC in this organism (28). The findings

given in the present work indicate strongly that glucose

in vinelandii is primarily metabolized via the ED

pathway. The extent of participation of the ED pathway

in overall glucose catabolism cannot be quantitatively

21

ascertained without additional information. The method

of estimation previously given in connection with the

studies on Pseudornonas cannot be directly applied to

the present case. This is true since the occurrence

of the NC pathway results in the shifting of a fraction

of the C-4 of glucose to the C-3 position, thereby

reducing the extent of conversion of this carbon atom to

co2. This consideration implies that at the end of the

radiorespirometric experiment the fraction of C..Lf of

glucose detected in CO2 should be greater than that

given in Table I (i.e., 73% of the total radioactivity

in the glucose molecule). Therefore, it can be

concluded that the operation of the PP pathway of this

organism should not exceed 20% of the total catabolized

glucose, I.e., yielded in the glucose_1_C1U1

experiment minus CO2 yield in the g1ucose_1_C1 experiment (calculated) (25).

, ' '-

B IBLIOGRAPHY

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:3. Dawos, E. A, and W. H, Ha13. On the Quantitative evaluation of routes of glucose metabolism by the use of radioactive glucose. Biochimica et Biophysica Acta 21:1711._175, 1960.

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8. Kelly, R. C., E, A, Peets, S. Gordon and D, A. Buyske. Determination of Cl'4 and 113 in biological samples by Schonl.ger combustion and liquid scintillation techniques. Analytical Biochemistry 2:267-273. 1961.

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23

10. Nortenson, L. E. and P, W. Wilson. Metabolism of r1bose-5-phophate by Azotobgoter vine1andi.. Journal of Biological Chemistry 213:713-721. 1955.

li. Nortenson, L. E., P. B. Hamilton and P. W. Wilson. Dissimilation of 6-phosphogluconate by Azotobacter v1rxelruili. Biochimica et Biophysica Acta 16: 238-24h. 1955.

12. Mortenson, L. E. and P. W, Wilson, Initial steps in breakdown of glucose by the Azotobacter. Bacteriological Proceedings of the 514th General Neeting of the Society of American Bacteriologists 54:108. 195L$.

13. Mortenson, L, E, and P. W. Wilson. Initial stages Iti the breakdown of carbohydrates by the Azotobacter vinelandlì. Archives of Biochemistry and Biophysics 53:)+25-1435, l95.

Racker, Efrain, Alternate pathways of glucose and fructose metabolism. Advances in Enzymology 15: 141-178. l95.

15. Roland, J. F. Jr., and A. N. Gross. Quantitative determination of amino acids using monodiniensional paper chromatography. Analytical Chemistry 26: 502-505. l95.

16. Seelye, R. N. and T. A, Turney. The lodoform reaction. Journal of Chemical Education 36:572_5714., 1959.

17. Sobek, J. N, nd C, F, Clifton. Oxidative assirni- lation and 1+ distribution in Azotobacter agilts. Proceedings of the Society for Experimental Biology and Medicine 109:k09-kfl. 1962,

18. Stern, Ivan J., C, H. Wang and C. M, Gilmour, Comparative catabolism of carbohydrates in Pseudomonas species. Journal of Bacteriology 79:601-611. 1960.

19. Stone, R, W, and P, W. Wilson, The effect of oxalacetate on the oxidation of suocinate by Azotobacter extracts. Journal of Bacteriology 63: 619-622. 1952.

21

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