/Ae,

ENDOGENOUS NUCLEOTIDE POOLS IN GROWING

CELLS OF AZOTOBACTER VINELANDII

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Yick-Shun Lee, B.S.

Denton, Texas

August, 1987

Lee, Yick-Shun, Endogenous Nucleotide Pools in Growing

Cells of Azotobacter vinelandii. Master of Science

(Biology), August, 1987, 44 pp., 5 tables, 8 illustrations,

bibliography, 35 titles.

The objective of this investigation was to examine the

changes in the nucleotide pools of Azotobacter vinelandii

during the growth cycle. Endogenous ribonucleotides were

extracted from A._vinelandii using trichloroacetic acid

(TCA; 12% w/v). The 5' mono-, di- and triphosphates of

adenine, guanine, uracil and cytosine were separated and

quantified by anion-exchange high performance liquid

chromatography. Results indicated that the adenylate energy

charge of A. vinelandii paralleled the growth rate during

exponential phase and that it declined rapidly as the

stationary phase was reached. In addition, the amount of

each nucleotide in A. vinelandii tended to increase in the

logarithmic phase and decrease in the stationary phase in a

similar manner to the energy charge.

TABLE OF CONTENTS

Page

LIST OF TABLES. .0.. ...... .*...... . iv

LIST OF ILLUSTRATIONS *. . . . . .. . . . . ... . v

Chapter

I. INTRODUCTION . . . . . . . . . . . . . . . 1

II. MATERIALS AND METHODS . . . . . . . . . . 7

Chemicals and ReagentsBacterial StrainMedia and CulturesExtraction of NucleotidesChromatographic ApparatusChromatographic ConditionsIdentification and Quantitation

of Nucleotides

III. RESULTS... .. . . .. . .. . . . 13

IV. DISCUSSION . . ..... ... . . . . . . 35

BIBLIOGRAPHY . ....0 .....0 .... 41

iii

LIST OF TABLES

Table Page

I. Identification of the Twelve NucleotideStandards Used in this Work . . . . . . . . 30

II. Distribution of Adenosine Nucleotides,and the Energy Charge of A. vinelandiiGrown in Soil Medium Supplemented with0.5% Glucose . . . . . . . . . . . . . . . 31

III. Distribution of Guanosine Nucleotides, andthe Guanylate Energy Charge of A. vinelandiiGrown in Soil Medium Supplemented with 0.5%Glucose . . . . . . . . . . . . . . . . . . 32

IV. Distribution of Uridine Nucleotides of A.vinelandii Grown in Soil Medium Supplementedwith 0.5% Glucose . . . . . . . . . . . . . 33

V. Distribution of Cytidine Nucleotides of A.vinelandii Grown in Soil Medium Supplementedwith 0.5% Glucose . .. .. ... . . ....... 34

iv

LIST OF ILLUSTRATIONS

Figure

1. Schematic diagram representing theextraction procedure for ribonucleotidesfrom A. vinelandii cultures . . . . . ..

2. Chromatograph of the 12 ribonucleotidestandards . . . . . . . . . . . . . . .

3. Elution profile of ribonucleotidesextracted with 12% (w/v) trichloroaceticacid from A. vinelandii . . . . . . .

4. Growth (e)1, and energy charge (0) of A.vinelandii in soil medium containing 0.5%(w/v) glucose . . . . . . . . . . .*.*..*

17

19

21

23

5. Growth rate (o), concentrations of nucleosidetriphosphates per 10 cells, ATP (C),GTP (®), and UTP ($) of A. vinelandiiin soil medium containing 0.5% (w/v)glucose . **- . . . . . . . . . . . . . . .

6. Growth rate (o), concentrations of nucleosidediphosphates per 10 cells, ADP (O), GDP(0), UDP (I), and CDP (4) of A vinelandiiin soil medium containing 0,.5% (w/v)glucose . . . . . . . . . . . .. . .

7. Growth rate .(), concentrations of nucleosidemonophosphates per 10 cells, AMP (®)f, GMP(®), UMP (o), and CMP ('b) of A vinelandiiin soil medium containing 0.5% (w/v)glucose . . . . . . .........

8. Pathways for the utilization of pyrimidinebases and nucleosides and the subsequentinterconversions occurring at thenucleotide level..*.............. . ....

25

27

29

40

V

Page

CHAPTER I

INTRODUCTION

Nucleotides are phosphorylated, heterocyclic compounds

classified as either pyrimidines or purines. Three

pyrimidine bases, uracil, thymine and cytosine, and two

purine bases, adenine and guanine, constitute the major

nitrogeneous bases which make up the nucleotides. The

ribonucleoside triphosphates, uridine triphosphate (UTP),

cytidine triphosphate (CTP), adenosine triphosphate (ATP)

and guanosine triphosphate (GTP) have several crucial

functions within the cell (22) which are well understood.

These include (a) control of individual biosynthetic

pathways; (b) synthesis of RNA and DNA; (c) carriers of

phosphate and pyrophosphate in several important enzymatic

reactions involved in the transfer of chemical energy; and

(d) a coenzyme-like function in which they behave as

energized carriers of specific types of building block

molecules.

The role of ATP is well established. It serves as the

major linking intermediate between energy-yielding and

energy-requiring chemical reactions in cells. Adenosine

triphosphate is the primary and universal carrier of

1

..........

2

chemical energy in cells (24) and is involved in all the

major metabolic sequences in every aspect of metabolism.

The relative concentrations of ATP, ADP, and AMP serve as

the controlling mechanism for many of the metabolic

reactions in all living organisms. These adenosine

nucleotides couple many energy producing and energy

utilizing metabolic reactions stoichiometrically, thereby

making it possible to equate metabolic reactions with the

relative concentrations of phosphorylated adenylates in

terms of the second law of thermodynamics. Atkinson (1)

showed a relationship between the adenosine nucleotide pool

in cells and their physiological condition in terms of an

"adenylate energy charge" (AEC) which was calculated by the

formulaAEC = [ATP1 + 0.5 [ADP1

[ATP] + (ADP] + [AMP]

The AEC reflects the relative number of high energy

phosphate bonds (ATP) to those of lower energy (AMP) in the

adenylate nucleotides in the cell pool (2, 7, 8). Atkinson

(1, 3) also proposed that there was a balance between ATP,

ADP and AMP for maintaining cellular homeostasis. In

reviewing the role of AEC, Atkinson (1, 3) observed that

whereas ATP is involved in providing energy for a wide range

of biological processes, GTP, UTP and CTP provide energy

only for certain specific anabolic reactions. However,

3

other investigators have claimed that a significant portion

of the total energy flux during bacterial growth proceeds

through non-adenosine nucleotide pools (21). Unlike the ATP

pool, the intracellular concentrations of non-adenosine

nucleoside triphosphates fluctuate in direct proportion to

their requirement for biosynthesis (11, 18, 29, 30).

Nucleoside triphosphates have been used as indices of

biomass and metabolic activity. Holm-Hansen (14) determined

total viable microbial biomass by measurement of ATP. Holm-

Hansen and Booth (15) measured ATP in the ocean in efforts

to determine the distribution of living cells in the open

sea. Leung and Schramm (23) suggested that changes among

the different phosphorylated adenosine nucleotides which

form the cell pool occur as a result of perturbations of the

energy yielding metabolism. From a different point of view,

Karl (18) indicated that the intracellular GTP/ATP ratio in

Serratia marinorubra increased in direct proportion to the

rate of cell growth.

Adenylate energy charge is widely accepted as both a

parameter for assessing the physiological condition of cell

populations (6) and as a controlling mechanism for selection

of metabolic pathways (33). Chapman et al. (8) demonstrated

that a wide variety of organisms, both eukaryotes and

prokaryotes, maintain essentially the same adenylate energy

4

charge (AEC) when in the same phase. Actively growing and

dividing cells have an AEC ratio of 0.8 to 0.95; cells in

stationary growth phase maintain a ratio < 0.8, > 0.5; and

scenescent or resting phase cells have a ratio below 0.5.

Since this appears to be a general property of all living

things, one should be able to estimate the relative

physiological condition for an entire microbial community by

measuring its AEC.

There are many techniques (5, 9) available for the

separation and quantification of nucleic acids and related

substances: (a) enzymatic analysis where specificity of

enzymes is used to determine the presence of specific

substances (32); for example, the AMP deaminase assay for

determining the concentration of AMP (31); (b) paper

electrophoresis where nucleic acids and their components,

due to a variety of ionizable groups can be separated and

their concentrations measured according to differences in

net charge of the different species at given pH values; (c)

planar chromatography when the stationary phase assumes a

planar arrangement in the form of a sheet of thin film and

the mobile phase migrates by means of capillary action,

separating the various species according to their

differential solubilities in the two interacting phases

(examples of planar chromatography are paper chromatography

5

and thin layer chromatography); (d) column chromatography

where the stationary phase is either a solid packing

material, a solid support that is coated with a sorbent

layer or a gel, any of which can be packed into a tube and a

migrating liquid phase serves to dissolve the various

substances according to their differential solubilities.

Gas chromatography, ion-exchange chromatography and liquid

chromatography are three forms of column chromatography.

Classical liquid chromatography has been improved by the

addition of high pressure on the moving phase (HPLC). In

recent years, the measurement of endogenous ribo- and

deoxyribonucleotide pools has been revolutionized by the

development of HPLC (12). The two most commonly employed

separation techniques involve either reverse-phase

chromatography on octydecylsilica resins (13, 17) or ion-

exchange chromatography on microparticulate anion-exchange

resins (16, 25). Even though reverse-phase chromatography

provides greater sensitivity and more rapid analysis

compared to any ion-exchange chromatographic method

currently available, it lacks the selectivity necessary for

the analysis of the many closely related nucleotides present

in cell extracts (27).

High pressure liquid chromatography has been employed

in this laboratory simply because rapid, efficient, and

6

reliable quantitative nucleotide separations were needed.

In the present study and in previous studies, Partisil 10-

SAX columns were used. These are strong anion-exchange

columns which are packed with particles 10 um in mean size.

Because of the sensitivity of these microparticle packings

to various chemicals, the extraction procedure is crucial

not only in attaining optimal sensitivity and column

efficiency but also in protecting the column for maximal

life (16). It has been found that even trace amounts of

perchlorate may bring about rapid deterioration of this kind

of column. In addition to this, neutralization of

perchloric acid with Tris(hydroxymethyl) aminoethane which

is used in a common, rapid, and simple method of preparing

extracts for use with some columns, cannot be used with

these packings (4). on the other hand, multiple ether

extractions are time consuming and small amounts of

nucleotides may be partitioned into the ether-water

interphase causing loss of some nucleotides and lower total

nucleotide values on analysis. The extraction procedure

described by Khym (20) is best suited for use with these

microparticle, chemically bonded columns. Trichloroacetic

acid (TCA) is used to extract the nucleotides from cellular

material and is subsequently removed from cell extracts with

a Freon-amine solution.

40 . . ......"Now

7

The objective of this investigation was to compare

the profiles of 12 acid-soluble nucleotides (adenosine

5' -triphosphate, adenosine 5' -diphosphate, adenosine

5' -monophosphate, guanosine 5' -triphosphate, guanosine

5' -diphosphate, guanosine 5' -monophosphate, uridine 5'

-triphosphate, uridine 5' -diphosphate, uridine 5' -

monophosphate, cytidine 5' -triphosphate, cytidine 5' -

diphosphate, and cytidine 5' -monophosphate) and energy

charge values during the growth of A. vinelandii on soil

extract medium ammended with 0.5% (w/v) glucose.

-upon

CHAPTER II

MATERIALS AND METHODS

Chemicals and Reagents

Nucleotides, trichloroacetic acid (TCA) and tri-n-

octylamine were purchased from Sigma Chemical Company (St.

Louis, Missouri); monobasic ammonium phosphate from

Mallinckrodt Inc. (Paris, Kentucky): and 1,1,2-trichloro-

1,2,2-trifluoroethane (Freon) from Eastman-Kodak Company

(Rochester, New York). All other chemicals were of

analytical grade and were purchased from Fisher Scientific

Company (Fair Lawn, New Jersey).

Bacterial Strain

Azotobacter vinelandii ATCC 12837 used in this study

was obtained from the stock culture collection of the

Department of Biological Sciences, North Texas State

University, Denton, Texas.

Media and Cultures

The organisms were maintained on a Burk's nitrogen-free

agar medium (33) of the following chemical composition in

grams per liter of distilled water: K2HPO4 0.64; KH2PO4 ,

0.16; MgSO4 7H20, 0.2; NaCl, 0.2; CaSOP4 2H2 0, 0.05; Na2MoO ,

7

8

0.001; FeSO , 0.003; agar, 15; and glucose, 5. These were

periodically checked for purity by streaking on Burk's and

nutrient agar plates. All experiments were started by

growing the organism in Burk's liquid medium at 26-28 0C on

the reciprocal shaker (New Brunswick Scientific Co., New

Brunswick, New Jersey). In addition to Burk' s medium, soil

extract medium containing 27mM glucose was prepared by

placing 20 g of finely ground soil in 100 ml of distilled

water in a 100 ml Erlenmeyer flasks. After standing at

room temperature for 1 hour, the mixture was autoclaved for

15 minutes at 121 0C. After standing overnight, the

supernatant was decanted and the soil medium cleared by

centrifugation (10,000 x _, 30 minutes) using the RC-2B

Sorvall refrigerated centrifuge (Du Pont Co., Newtown,

Connecticut) or by filtering through washed 0.45 um filter

membranes (Gelman Sciences, Inc., Ann Arbor, Michigan). The

clear soil extract was sterilized in the autoclave at 1210C

for 15 minutes in sidearm flasks and sterile glucose added

to a final concentration of 5 g per liter of liquid. Cells

of A. vinelandii at mid-logarithmic growth phase from Burk's

medium cultures were inoculated into the soil extract medium

and incubated on the rotary shaker at 26-280C and agitated

at 90 revolutions per minute on the rotary shaker. Growth

9

was monitored by both optical density at 640 nm and by

triplicate plate count on spread plates.

Extraction of Nucleotides

Volumes of 80 ml of bacterial cultures were harvested

and centrifuged at 40 C at 12,000 x g for 4 minutes. After

decanting the supernatant, nucleotides were extracted from

the cell pellet according to the scheme shown in Figure 2.

One ml of ice-cold 12% (w/v) trichloroacetic acid (TCA) was

added to the cell pellet, which was then thoroughly mixed

for 2 minutes in the vortex mixer. This was allowed to

stand at 40 C for 40 minutes before centrifuging at 12,000 x

g for 15 minutes. The clear supernatant was then

neutralized with ice cold Freonamine (20) solution (0.7 M

tri-n-octyl-amine in Freon 113, 1.06 ml amine per 5 ml of

Freon). The sample was then mixed on the vortex mixer for 2

minutes then allowed to separate for 15 miutes at 40C. The

top, aqueous, layer which contained the nucleotide extract,

was removed filtered through a 0.45 um ACRO LCl3 filter

membranes (Gelman Sciences, Ann Arbor, Michigan) and frozen

at -200C until ready for analysis.

Chromatographic Apparatus

'The HPLC equipment (Waters Assoc., Milford,

Massachusetts) consisted of two Model 510 pumps, a Model 680

automated gradient controller, a U6K injector, and a Model

481 LC spectrophotometer. Nucleotides were detected by

monitoring the column effluent at 254 nm with a sensitivity

fixed at 0.05 absorbance units at full scale deflection

(AUFS). Separations were performed on a Waters Radial-Pak

Partisil SAX cartridge (10 cm x 0.08 cm) using a Waters

radial compression Z-Module system.

Chromatographic Conditions

The entire chromatographic system including the column

was stored in 50:50 (v/v) filtered HPLC grade methanol and

filtered, double distilled water (2X) when not in use.

After priming the pumps, the system was flushed with 50 ml

of methanol:water mixture at 3 ml per minute. Next, the

system was thoroughly washed with distilled water with the

initial flow rate at 3 ml per minute. After 10 minutes, the

flow rate was increased to 4 ml per minute. When the back

pressure of the column dropped to 850 pounds per square

inch, the methanol:water mixture was completely washed from

the system. Pump A was then flushed with starting buffer

(filtered ultra pure 7 mM monobasic ammonium phosphate, pH

3.8) followed by pump B which was flushed separately with

final buffer (filtered 250 mM monobasic ammonium phosphate

containing 500 mM potassium chloride, pH 4.5). The LC

spectrophotometer was set at 254 nm and 0.05 AUFS, and the

11

recorder and automated gradient controller were turned on.

An initial program with a linear slope (curve profile #6) of

low concentration buffer was run for 10 minutes. A 10-

minute reverse gradient of high concentration buffer was

run, followed by a 10-minute rest with low concentration

buffer.

Identification and Quantitation of Nucleotides

Nucleotide samples of 200 ul each were prepared from

Azotobacter cells as previously described. These were

thawed and injected onto a column of Partisil SAX-10 which

consisted of a silica matrix coated with porous

microparticles of silica of 10 um particle size. The

particles contained fixed-charge quaternary nitrogen groups

and mobile counterions of H2PO4 . Nucleotides bind to the

quaternary nitrogen group with different affinities because

of the functional groups in the bases and the number of

phosphates at the 0-5' of the sugar. A linear gradient

(curve profile #6) of low to high concentration buffer was

applied for 20 minutes, followed by an isocratic period of

10 minutes of high concentration buffer. The column was

regenerated by washing with 30 ml of 7 mM NH4H2PO, pH 3.8

buffer (26). The flow rate was maintained at 4 ml per minute

and all analyses were done at ambient temperature. Peaks

were integrated using a Waters Data Module Model 740

12

attached to a microprocessor 82201. The sample peaks were

identified by comparing their retention times with those of

appropriate standards. The concentrations of nucleotides in

samples were calculated by comparing peak heights to standards

(adenosine 5' -triphosphate, adenosine 5' -diphosphate,

adenosine 5' -monophosphate, guanosine 5' -triphosphate,

guanosine 5' -diphosphate, guanosine 5' -monophosphate,

uridine 5' -triphosphate, uridine 5' -diphosphate, uridine 5'

-monophosphate, cytidine 5' -triphosphate, cytidine 5' -

diphosphate, and cytidine 5' -monophosphate) of known

concentration (0.01 mM) and expressed as nmoles per 108

cells. The system was terminated by first flushing with

distilled water and then with the methanol-water mixture in

the same manner used to start the apparatus.

CHAPTER III

RESULTS

Endogenous ribonucleotides were extracted from

Azotobacter vinelandii cultures according to the scheme shown

in Figure 1. Anion exchange, high performance liquid

chromatography was employed to separate and assay acid-

soluble ribonucleotides in the extracts. Figure 2 shows a

chromatogram of peaks representing the 12 biologically

important ribonucleotide standards. Each ribonucleotide was

identified by its retention time and is listed in Table I.

There is a broad spectrum of compounds in the elution profile

of the acid-soluble extract of A. vinelandii as can be seen

from Figure 3. The cellular ribonucleotides were identified

by comparing the retention times of the extracted compounds

of those of the reference ribonucleotides. Further, the

ribonucleotide content of A. vinelandii was calculated by

comparing the peak heights of the sample ribonucleotides of

those of the standards. The quantities of the individual

base nucleotides are listed as follows: Table II for

adenosine nucleotides, Table III for guanosine nucleotides,

Table IV for uridine nucleotides, and Table V for cytidine

nucleotides. The concentrations of ATP (range: 0.7 to 1.4

13

_________________________________

14

nmoles per 108 cells) were less than those of ADP (range: 0.3

to 3.4 nmoles per 108 cells). Adenosine monophosphate (AMP)

was detected only after 20 to 48 hours of growth and its

concentration ranged from 0.6 to 3.1 nmoles per 108 cells.

During the growth cycle of A. vinelandii, the adenylate

energy charge ratio ranged from 0.27 to 0.84 (Figure 4).

the concentration of GDP (range: 0.4 to 2.0 nmoles per 108

cells) was greater than that for GMP (range: 0.3 to 0.9

nmoles per 10 cells) or GTP (range: 0.1 to 1.0 nmoles per

810 cells). The level of UMP ranged from 0.04 to 23.5 nmoles

per 108 cells and was considerably higher than that of UTP

which ranged from 0.3 to 1.4 nmoles per 10 8 cells or UDP

which ranged from 0.5 to 1.0 nmoles per 108 cells.

It was difficult to obtain reproducible readings for

CTP since the concentration was always extremely low. The

CDP levels ranged from 0.2 to 1.1 nmoles per 108 cells and

the concentration of CMP ranged from 0.5 to 1.5 nmoles per

108 cells (Table V).

The adenylate energy charge was calculated for

different phases of the growth for A. vinelandii. The

azotobacters had an AEC of 0.71 to 0.84 in the logarithmic

phase. When growth ceased, this ratio decreased and was

maintained indefinitely at a value of about 0.6 in the

stationary growth (Figure 4).

15

As can be seen in Figures 5 to 7, the change in all

cellular nucleotides followed a common pattern. The

nucleotide concentrations rose during the logarithmic phase

to reach maximal values and dropped in the stationary phase.

The concentrations of ATP and UTP increased by the same

amount in the logarithmic phase as stationary phase; UTP

pool levels decreased at a faster rate than did the ATP

levels. Figure 5 shows that the GTP pool levels ran

parallel to the growth curve. The rate of change in the

endogenous concentration of ADP, GDP and UDP over the growth

cycle of the organism were very similar. Although there was

a delay in the change of CDP concentration from 12 to 24

hours, the CDP content also increased with growth rate

(Figure 6). Both AMP and UMP levels increased and decreased

in parallel with the concentration of UMP being generally

higher than that of AMP. The GMP and CMP pool levels

increased and decreased less abruptly and their

concentrations were lower (Figure 7).

16

Fig. 1--Schematic diagram representing the extractionprocedure for ribonucleotides from A. vinelandii cultures.

17

Bacterial'Culture

Centrifuge at 12,000 x gfor 5 min at 40C

Supernatant Cell Pellet Capsular Layer

Add TCA (12% w/v)Mix by vortexingAllow to stand for 30 minCentrifuge at 12,000 x gfor 15 min at 40C

Pellet Supernatant

Add Freon amine,vortex, and stand

Bottom Top AqueousLayer Layer contains

nucleotides

Filter

Store at -20 Cuntil analysis

18

Fig. 2--Chromatogram of the 12 ribonucleotidestandards. The numbers refer to the compounds listed inTable I.

19

3Ln

24

0

0 5 10 15 20 25 30

Time, minutes

WNW

20

Fig. 3--Elution profile of ribonucleotides extractedwith 12% (w/v) trichloroacetic acid from A. vinelandii. Thenumbers refer to the compounds listed in Table I.

21

3

N

m0

6

0 5 10 15 20 25 30

T ime, minutes

22

Fig. 4--Growth (o), and energy charge (0) of A.vinelandii in soil medium containing 0.5% (w/v) glucose.

23

0

0

0 0

122

24

- ----

36

- 1.0

-0.8

-0 6

0.4

0.2

0

48

Time, Hrs.

9

r-

u)H0

0

0- 7-

4

6

0

24

Fig. 5--Growth gate (e), concentrations of nucleosidetriphosphates per 10 cells, ATP (C), GTP (0),, and UTP (0x)of A. vinelandii in soil medium containing 0.5% (w/v)glucose.

25

H-

NI

0

0

.- 2.5

- 2.0

005

Time, Hours

.1W00.0

6

02

1.2 24 36 48

4

26

Fig. 6--Growth rate (e), concentrations of nucleosidediphosphates per 10 cells, ADP (@), GDP ($), UDP (0), andCDP (<) of A. vinelandii in soil medium containing 0.5%(w/v) glucose.

27

Vd

Time, Hours

H-

r-4

H

0

0. 7

91

12

6

0 24 36 48

2.0

1.5 co0

0 0

0,0

28

Fig. 7--Growth rgte (o), concentrations of nucleosidemonophosphates per 10 cells, AMP (@), GMP (s), UMP (s), andCMP (<) of A. vinelandii in soil medium containing 0.5 (w/v)glucose.

29

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31

TABLE II

DISTRIBUTION OF ADENOSINE NUCLEOTIDES, AND THEENERGY CHARGE OF A. VINELANDII GROWN IN SOIL

MEDIUM SUPPLEMENTED WITH 0.5% GLUCOSE

Culture No. Viable AdenylateTime (H) Cells/ml ATPa ADP AMP Energy Charge

0 4.2 x 106

7b8 5.6 x 10 1.2 3.4 - 0.63

812 1.2 x 10 0.7 1.0 -- 0.71

814 1.5 x 10 0.7 0.3 -- 0.85

816 1.7 x 10 0.8 1.0 -- 0.72

20 2.3 x 108 1.6 1.4 0.3 0.71

32 2.9 x 108 1.0 1.4 3.0 0.57

36 3.0 x 108 1.4 1.1 3.1 0.27

48 2.8 x 108 1.0 0.6 0.6 0.59

8 aAdenosine nucleotide10 cells.

bNot detectable.

concentrations in nanomoles per

32

TABLE III

DISTRIBUTION OF GUANOSINE NUCLEOTIDES, AND THE

GUANYLATE ENERGY CHARGE OF A. VINELANDIIGROWN IN SOIL MEDIUM SUPPLEMENTED

WITH 0.5% GLUCOSE

Culture No. Viable GuanylateTime (H) Cells/ml GTPa GDP GMP Energy Charge

60 4.2 x 10

8 5.6 x 10 0.2 2.0 0.8 0.43

12 1.2 x 108 0.1 0.5 0.4 0.38

14 1.5 x 108 0.1 0.4 0.5 0.31

16 1.7 x 108 0.2 0.8 0.6 0.38

20 2.3 x 108 0.3 1.1 0.7 0.41

32 2.9 x 108 1.0 1.2 0.8 0.53

36 3.0 x 108 0.4 1.4 0.9 0.40

48 2.8 x 108 0.7 0.8 0.3 0.65

8 aGuanosine nucleotide10 cells.

concentrations in nanomoles per

33

TABLE IV

DISTRIBUTION OF URIDINE NUCLEOTIDES OF A. VINELANDII

GROWN IN SOIL MEDIUM SUPPLEMENTED WITH 0.5% GLUCOSE

Culture No. ViableTime (H) Cells/ml UTPa UDP UMP

0 4.2 x 106

8 5.6 x 107 1.0 0.9 16.7

812 1.2 x 10 0.4 0.5 6.6

14 1.5 x 108 0.7 0.5 11.6

16 1.7 x 108 0.9 0.5 13.3

20 2.3 x 108 1.4 0.6 0.3

32 2.9 x 108 0.6 1.0 23.5

36 3.0 x 108 1.1 0.8 22.2

48 2.8 x 108 0.3 0.8 0.04

8 aUridine nucleotide concentrations in nanomoles per10 cells.

34

TABLE V

DISTRIBUTION OF CYTIDINE NUCLEOTIDES OF A. VINELANDII

GROWN IN SOIL MEDIUM SUPPLEMENTED WITH 0.5% GLUCOSE

Culture No. ViableTime (H) Cells/ml CTPa CDP CMP

0 4.2 x 106

8 5.6 x10 b 0.6 0.8

12 1.2x108 -- 0.3 0.6

14 1.5x108 -- 1.0 0.5

16 1.7 x 108 0.5

20 2.3x108 -- 0.2 1.1

32 2.9 x 108 __ __ 1.1

36 3.0 x 108 -- 1.1 1.5

48 2.8x108 -- 0.7 0.5

8 aCytidine nucleotide concentrations in nanomoles per

10 cells.

bNot detectable.

CHAPTER IV

DISCUSSION

The endogenous ribonucleoside mono-, di- and

triphosphates were measured by HPLC from exponential and

stationary phase cultures of Azotobacter vinelandii.

Samples were taken at different time-points and the

nucleotide concentrations, in nanomoles per 10 bacteria,

were plotted against time as can be seen in Figures 2, 3,

and 4. The adenylate energy charge was calculated and

plotted against time in Figure 1. Additional details are

given in Tables II, III and V.

Six key points are made regarding these figures and

tables: (1) In all cases the monophosphates concentrations

were greater than the corresponding di- and triphosphates.

(2) The UMP concentration was by far the highest at 23.5

nmoles per 108 cells after 32 hours of growth. (3) The

adenylate concentrations, particularly ATP (at 1 to 2 nmoles

per 108 cells), were far lower than those of Escherichia

coli (19). (4) The CTP concentration could not be con-

sistently quantified under any condition of growth. The CDP

and CMP concentrations also were low. (5) When the adenylate

energy charge was plotted against time, it virtually

35

36

paralleled the growth curve. (6) The guanylate energy

charge did not change significantly with growth rate (Table

III). These six points are now discussed in detail.

The levels of the monophosphates, AMP, GMP, UMP and

CMP, were greater than the corresponding triphosphates.

This was especially true for AMP. It is likely that this is

attributable to the difficulty in sample preparation for

determining the concentration of nucleotides. No one has

addressed this problem to date. When A. vinelandii cultures

are centrifuged for 5 minutes at 12,000 x g at 40C at the

outset of the preparation (refer to Figure 1), three layers

appear: a cell pellet at the bottom of the tube, a middle

layer of capsular material typical of A. vinelandii, and the

supernatant on top. Despite the short time centrifugation,

the pellet which contains the nucleotides becomes anaerobic

during the centrifugation (anaerobiosis depletes the ATP

concentration rapidly) and the subsequent decanting of the

supernatant and capsular layer. Thus triphosphates,

especially ATP and GTP, are broken down to their mono- and

diphosphates.

The UMP concentration was by far the greatest of the

four monophosphates (at 23.5 nmoles per 10 cells, after 32

hours of growth) while all cytidine nucleotide concentrations

were extremely low. In fact, it was difficult to quantify

37

CTP. Because the CDP and CMP concentrations were also low,

it is likely that all cytosine compounds are converted to

UMP by the very active cytidine deaminase of Azotobacter

(34). Thus CMP from mRNA is broken down to cytidine

(Figure 8). This cytidine is rapidly deaminated to uridine

by cytidine deaminase. The uridine is degraded to uracil by

uridine phosphorylase. All of this uracil is ultimately

converted to UMP by uracil phosphoribosyl transferase. Thus

most of the cytidine compounds ends up as UMP. This would

partially explain the extremely high level of UMP.

The adenylate concentrations, especially ATP, were

somewhat lower than previously reported (19). This can be

explained by the partial anaerobiosis that could have

occurred during the initial centrifugation step. Since the

ADP and AMP were also lowered, the adenylate energy charge

appeared normal and virtually paralleled the growth rate.

The adenylate charge increased from 0.63 at 12 hours to 0.71

at 24 hours; it declined to 0.59 in the stationary phase.

This is in accordance with previously reported results.

Since the adenylate energy charge appeared to be

normal, the guanylate energy charge was scrutinized for any

anomalies. Indeed, the guanylate charge did not change

significantly with growth rate and thus is not considered to

be an important parameter of control.

38

One final point is pertinent. During the measurement

of nucleotides in Azotobacter vinelandii, it was noted that

the procedure for sample preparation was very important in

order to obtain consistent reproducible figures for the

nucleotides. The following precautions should be observed:

(a) ice-cold Freon-amine should be used for neutralization;

(b) ice-cold Freon-amine should be vortex-mixed after

neutralization for 2 minutes and then allowed to settle at

40C for at least 15 minutes; (c) the clear aqueous layer

must be removed gently and carefully without disturbing the

interphase.

Throughout these experiments, a radial-compression

column of 8 mm internal diameter was used. This column

afforded greater than six times the surface area of the

conventional steel columns. Accordingly, the speed of

separation was enhanced more than three-fold that of Hartwick

and Brown in 1975 (16) without any appreciable loss in

resolution.

39

Fig. 8--Pathways for the utilization of pyrimidinebases and nucleosides and the subsequent interconversionsoccurring at the nucleotide level. Genetic symbols for theenzymes are underlined and the gene designations are asfollows: _, uracil phosphoribosyl transferase (EC2.4.2.9); udk, uridine kinase (EC 2.7.1.48); cdd, cytidinedeaminase (EC 3.5.4.5); u, uridine phosphorylase (EC2.4.2.3); cod, cytosine deaminase (EC 3.5.4.1); pyrG, CTPsynthetase (EC 6.3.4.2); ndk, nucleoside diphosphokinase (EC2.7.4.4); pyrH, UMP kinase (EC 2.7.4.4); cmk, CMP kinase (EC2.7.4.18). Abbreviations are CR, cytidine; C, cytosine; UR,uridine; U, uracil.

40

CTP UTP

nda d

CDP UDP

C de nov

CRRPi

Ribose- 1 -P

PRPP

C U

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