27? - UNT Digital Library/67531/metadc331468/m2/1/high_re… · Liquid Chromatography as £ Tool for the Measurement of Endogenous Nucleotides in Bacteria. Doctor of Philosophy (Biological

27? A/8 /d

Ato, <25*10

RADIAL COMPRESSION HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY AS A TOOL FOR THE

MEASUREMENT OF ENDOGENOUS

NUCLEOTIDES IN BACTERIA

DISSERTATION

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

Probir Kumar Dutta, M.S

Denton, Texas

August, 1986

r> >r"c> 0

Dutta, Probir K., Radial Compression High Performance

Liquid Chromatography as £ Tool for the Measurement of

Endogenous Nucleotides in Bacteria. Doctor of Philosophy

(Biological S c i e n c e s ) , August, 1986, 117 pp., 6 tables, 20

illustrations, bibliography, 136 titles.

High performance liquid chromatography was used to

measure ribonucleoside triphosphates in microbial samples.

Anion exchange columns in a radial compression module were

used to separate and quantify purine and pyrimidine

ribonucleotides.

Endogenous ribonucleoside triphosphates were

extracted from Escheri chi a coli and Pseudomonas aerugi nosa

using three different solvents, namely trif1uoroacetic

acid (TFA; 0 . 5 M ) , trichloroacetic acid (TCA; 6 per cent

w/v) and formic acid (1.0M). Extracts were assayed for

uridine 5 '-triphosphate (UTP), cytidine 5 1 - t r i p h o s p h a t e

(CTP), adenosine 5'-triphosphate (ATP), and guanosine

5 1 - t r i p h o s p h a t e (GTP) by using anion exchange radial

compression high performance (pressure) liquid

chromatography. The three extraction procedures were

compared for yield of triphosphates. _E. coli , the TFA

extraction procedure was more sensitive and reliable than

TCA and formic acid extraction procedures, but, in P.

aerugi nosa, the best y i e l d s of ATP and GTP were ob ta ined

f o l l o w i n g e x t r a c t i o n w i t h TFA. Y i e l d s of UTP and CTP

increased when e x t r a c t i o n was performed in TCA. These data

i l l u s t r a t e t h a t d i f f e r e n t e x t r a c t i o n procedures produce

d i f f e r e n t measures f o r d i f f e r e n t t r i p h o s p h a t e s , a p o i n t

o f t e n ove r looked .

While assess ing the e f f e c t s of u r a c i 1 - s t a r v a t i o n on a

Pyr" (ur ac i 1 - r e q u i r i ng) mutant of E_. co l i , i t was found

t h a t the endogenous ATP and GTP c o n c e n t r a t i o n s inc reased

1 0 - f o l d a f t e r one hour of i n c u b a t i o n in a u r a c i l - f r e e

medium. This d i scove ry suggests t h a t : (1) the long

s tand ing idea t h a t pur ines c o n t r o l p y r i m i d i n e s (but not

the o ther way around) may have to be m o d i f i e d and (2) the

massive accumula t ion of ATP and GTP under u r a c i l s t r e s s

may p rov ide a new techno logy f o r i n d u s t r i a l p r o d u c t i o n of

adeny la tes and guany la tes .

Because the m a j o r i t y of c e l l u l a r c a t a b o l i c a c t i v i t i e s

i n v o l v e the f o r m a t i o n and h y d r o l y s i s of ATP, w h i l e many

anabo l i c a c t i v i t i e s i n v o l v e the h y d r o l y s i s of GTP, the

GTP/ATP r a t i o and growth r a t e may be q u a n t i t a t i v e l y

l i n k e d . Indeed a p l o t of GTP/ATP r a t i o s aga ins t t i m e ,

produced a s t r a i g h t l i n e on semi log paper . Th is r a t i o may

enable measurement of growth r a t e s of both p ro - and

e u k a r y o t i c c e l l s t h a t are o the rw ise d i f f i c u l t i f not

imposs ib le to assess.

Endogenous nucleotide triphosphate pools were

examined in Yersinia e n t e r o c o l i t i c a grown at three

different temperatures (22°C, 30°C and 37°C). Although 22°C

is thought to be optimal for Y_. enterocol i ti ca,

ribonuc 1 eoside triphosphates were also examined with Y_.

enterocoli ti ca grown at two non-permissive temperatures.

At 22°C, all nucleoside triphosphate pools increased

linearly, but at 30°C and 37°C a profound change in UTP,

ATP and GTP pools followed a brief initial linear

response. The CTP concentration did not increase at 30°C

or 37°C. These data agree with the recent finding that CTP

is not an allosteric inhibitor of aspartate

transcarbamoyl ase for Yersi ni a species. Y_. enterocol i ti ca,

a naturally t e m p e r a t u r e - s e n s i t i v e organism can thus be

used to study the general effects of temperature as a

stress indicator, just as uracil starvation was used

in this study.

Copyright by

Probir Kumar Dutta

1986

TABLE OF CONTENTS

Page

LIST OF TABLES vi

LIST OF ILLUSTRATIONS yit

Chapter

I. INTRODUCTION 1 Cellular Nucleotides and Growth Adenylate Energy Charge and Growth Nonadenine Nucleotides and Growth

(With Special Reference to GTP) Techniques of Separating and Quantifying

Nucleic Acid Related Substances Organization of the Dissertation

II. SEPARATION AND QUANTITATION OF BACTERIAL RIBONUCLEOSIDE TRIPHOSPHATES BY RADIAL COMPRESSION HIGH PRESSURE LIQUID CHROMATOGRAPHY USING TRIFLUOROACETIC ACID AS AN EXTRACTION SOLVENT 23

I ntroducti on Methods Results Discussion

III. ACCUMULATION OF ADENOSINE TRIPHOSPHATE AND GUANOS INE TRIPHOSPHATE IN A URACIL-REQUIRING MUTANT STRAIN OF ESCHERICHIA COLI 41

I ntroducti on Methods Results Discussion

IV. GTP/ATP RATIO, A NEW PARAMETER FOR MEASURING MICROBIAL GROWTH 62

Introducti on Methods Results Discussion

IV

TABLE OF CONTENTS

Page V. TEMPERATURE DEPENDENCY AND

RIBONUCLEOSIDE PRODUCTION IN YERSINIA ENTEROCOLITICA 87

I n t r o d u c t i on Methods Resu l t s D iscuss ion

V I . SUMMARY 1 0 2

BIBLIOGRAPHY 1 0 4

LIST OF TABLES

Table Page

I. Summary of Evidence for Stimulation of Anabolism by GTP 14

II. A Comparision between Thin Layer Chromatography (TLC) and Liquid Chromatography (LC) (Treiber, 1986) . . . 21

III. Yield of Nucleoside Triphosphates in E. coli cells [ymole (g dry wt)" 1] Using Different Extraction Solvents . . . 36

IV. Yield of Nucleoside Triphosphates in P_. aeruqi nosa Cells [ymole (g dry wt)~l] Using Different Extraction Solvents . . . 37

V. Effect of Various Concentrations of TFA on Recovery of Ribonucleoside Triphosphates in £. coli and JP. aeruginosa 39

VI. Accumulation of ATP and GTP Levels Under Various Growth Conditions 55

VI

LIST OF ILLUSTRATIONS

Figure Page

1. Metabolic Pathways of Purine Ribo- and Deoxyribonucleotides in Escherichia coli 5

2. Metabolic Pathways of Pyrimidine Ribo-and Deoxyribonucleotides in Escherichia coli

3. Schematic Diagram Showing the Channeling of Energy by Different Ribo- and Deoxyribonucleoside Triphosphates for Building the Macromolecules Necessary for Growth 9

4. Some Techniques for the Separation and Determination of Nucleic Acid Related Substances 19

5. Chromatogram of the 12 Ribonucleotide Standards 33

6. Elution Profile of the Ribonucleoside Triphosphates 35

7. Chromatogram of the 12 Ribonucleotide Standards 51

8. Elution Profile of the Ribonucleoside Mono-, Di- and Triphosphates from Escherichi a coli Strain TB2 Grown in M9 Medium Containing 0.2% (w/v) Glucose + 0.4% (w/v) Casamino Acids and Uracil ( 50 y g ml "1) 53

9. Elution Profile of the Ribonucleoside Mono-, Di- and Triphosphates from Escheri chi a coli Strain RB2 Cells were Grown in Uracil as for Fig. 8 to 75 Klett Units (KU) 57

10. Interrelationships Between Purine and Pyrimidine Metabolic Pathways for the Production of RNA and DNA in Bacteria . . 59

v 1 i

LIST OF ILLUSTRATIONS

Figure Page

11. Elution Profile of Ribonucleoside Triphosphates Extracted with 6% (w/v) Trichloroacetic Acid from Escherichia col i 71

12. Relationships Between (a) GTP/ATP Ratio versus Time (b) Klett Units versus Time (growth rate), ATP versus Time and GTP versus Time in Escheri chi a coli Growing in M9 + Uracil (50 yg ml -I) 74

13. Regression Plot of Log GTP Concentration (LGTP) versus Time 76

14. Regression Plot of Log ATP Concentration (LATP) versus Time 78

15. Regression Plot of GTP/ATP Ratio (LGA) versus Time Using the Statistical Package for the Social Sciences (SPSS) Chicago, IL 80

16. Karl (1978) Type Plot of GTP/ATP Ratio as a Function of Growth Rate of Escheri chi a coli Cells Growing in Batch Culture Under Four Different Conditions 82

17. Metabolic Reactions Involving ATP and GTP 85

18. Elution Profile of Ribonucleoside Triphosphates for Yersinia enterocoli ti ca 96

19. Effect of Indicator Growth Temperature on the Concentration of (a) UTP and (b) CTP in Yersi ni a enterocoli ti ca . . . . 98

20. Effect of Indicator Growth Temperature on the Concentration of (a) ATP and (b) GTP in Yersinia enterocolitica . . . . 100

v n i

CHAPTER I

INTRODUCTION

Growth in l i v i n g organisms has been def ined as the

"coordinated summation of the biosyntheses" which

u l t i m a t e l y r e s u l t s in the product ion of new c e l l s

(Ingraham et aj_., 1983) . The f o l l o w i n g are some of the

many a v a i l a b l e techniques tha t can be used to measure the

growth of a b a c t e r i a l c u l t u r e : 1) counting v i a b l e c e l l s

based on colony forming a b i l i t y of b a c t e r i a l s t r a i n s ; 2)

counting the t o t a l number of c e l l s ; 3) determining the dry

weight of c e l l s ; 4) measuring biomass by using techniques

of u l t rasound where v a r i a t i o n s in the v e l o c i t y and

a t t e n u a t i o n of pulsed ul t rasound are c o r r e l a t e d to

non-misc ib le substances in l i q u i d c u l t u r e s (Faust & I r i o n ,

1984) ; 5) measuring some c e l l u l a r c o n s t i t u e n t s , f o r

example i n t r a c e l l u l a r DNA, RNA and pro te in using l aser

f low mic ro f luoromet ry ; 6) q u a n t i f y i n g i n t r a c e l l u l a r NADH

using f luoromet ry (Beyeler et al_. , 1981) ; 7) measuring

oxygen and carbon d iox ide concent ra t ion by gas ana lys is

and mass spectrometry (Wang et_ aj_. , 1977; He inz le et al . ,

1983) ; 8) es t ima t ing acid /base product ion by using a pH

e l e c t r o d e (San & Stephanolpoulos, 1984) ; 9) measuring

This d i s s e r t a t i o n fo l lows the format and s t y l e of The Journal of General Mi crobi o logy.

redox potential by rH electrode (Keldgaard, 1977); 10)

measuring heat production using m i c r o c a l o r i m e t r y

(Anantheswaran et_ £l_. , 1984; Bayer & Fuehrer, 1982); 11)

incorporating nucleic acid bases and nucleosides into DNA

(O'Donovan, 1978; Fuhrman & Azam, 1982; Moriarty &

Pollard, 1981) and RNA (Karl, 1979, 1982); 12) o r

incorporating S - S O ^ into protein (Jordan & Likens, 1980;

Cuhel et_ a]_. , 1979); 13) estimating frequency of dividing

cells (Hagstrom et_ _aj_., 1979); 14) measuring ATP to

measure microbial biomass (Holm-Hansen & Booth, 1966); 15)

using adenylate energy charge ratio (Wiebe & Bancroft,

1975); 16) measuring GTP/ATP ratio (Karl, 1978) and 17)

measuring DNA/RNA synthesis ratio (Karl, 1981).

Cellular Nucleotides and Growth

Molecules such as nucleosides and nucleotides are the

major components of all cells. These molecules consist of

either pyrimidines or purines, heterocyclic compounds

having pronounced aromatic c h a r a c t e r i s t i c s . Purines are

regarded as derivatives of pyrimidines as they consist of

two imidazole rings fused together. Three pyrimidine

compounds, uracil, thymine and cytosine and two purine

compounds, adenine and guanine constitute the major

nitrogenous bases found in nucleotides. The nucleoside

5'-triphosphate of the bases adenine, guanine, cytosine

and thymine are required for DNA synthesis. Their

biosyntheses (Fig. 1 & 2) are regulated with great

precision. There are many other nucleotides (Jensen &

Laland, 1960) which occur biologically. Notable among them

are PRPP, IMP, cyclic AMP, cyclic GMP, ppGpp and pppGpp.

The ribonucleoside triphosphate pools, mainly those of

UTP, CTP, ATP and GTP have several crucial functions

within the cell (Lehninger, 1978): (a) nucleotide pools

control individual biosynthetic pathways producing the

substrates for RNA and DNA polymerases; (b) nucleotides

are required for the synthesis of RNA and DNA; (c)

nucleotides are carriers of phosphate and pyrophosphate in

several important enzymatic reactions involved in the

transfer of chemical energy; (d) nucleotides serve as

c o e n z y m e - ! i k e , energized carriers of specific types of

building block molecules.

ATP serves as the major linking intermediate between

energy-yielding and energy-requiring chemical reactions in

cells. ATP is the primary and universal carrier to

chemical energy in cells (Lipmann, 1941). Fig. 3

represents how energy is channelled through various

ri bonucleosi de and d e o x y r i b onucleoside triphosphates in

building m a c r o m o l e c u ! e s , necessary for cell survival of

the living cell.

Adenylate Energy Charge and Growth

The adenine r i b o n u c l e o t i d e s , adenosine triphosphate

Fig. l--Metabo1ic pathways of purine ribo- and deoxyribonucl eotides in Escherichia coli.

tl

J l l f p

M C'u

Fig. 2 - - M e t a b o l i c pathways of p y r i m i d i n e ribo- and d e o x y r i b o n u c l e o t i d e s in E s c h e r i c h i a coli.

t I \ [ | 'Ad.

*0 av|Nn- ^ »Aft » sj. I "4) H

Asp A«r, HCq.-

Si O V t MAh X, 'N „ V »,:>

\

d

J«'N

4NV #S

Fig. 3--Schematic diagram showing the channeling of energy by different ribo- and deoxyribonucleoside triphosphates for building the macromolecules necessary for growth.

Lipids

Polysaccharides Proteins

^ CTP

U T P " | G T P

IHl^ • I > \ IdUTP I 1 UTP 1 / ^ dTTP . • ATP I | dATP | 1 CTP • 1 dCTP § GTP | | dGTP I

RNA's DNA

10

(ATP), adenosine diphosphate (ADP) and adenosine

m o n o p h o s p h a t e (AMP) are associated with every metabolic

sequence in living organisms. These nucleotides

stoichiometrically couple many energy producing and energy

utilizing metabolic reactions. Atkinson (1968) was the

first to show a relationship between the three adenosine

nucleotides in terms of his "adenylate energy charge"

(AEC) concept. AEC can be calculated by the formula,

ATP + 0.5 ADP AEC

ATP + ADP + AMP

In reality, AEC reflects the relative number of high

energy phosphate bonds (anhydride bound phosphate groups)

in the adenylate pool and has been presented in detail by

Atkinson and coworkers (Chapman e_t a_l_. , 1971; Atkinson,

1971; Chapman & A t k i n s o n , 1977). Atkinson (1968, 1977) has

proposed that there is a balance between ATP, ADP and AMP

for maintaining cellular homeostasis. The energy is unity

when the total nucleotide pool is fully phosphorylated to

ATP and zero when the adenine nucleotides are 'empty' and

present only as AMP. Normally, the energy charge of

growing cells is about 0.9 meaning that the adenylate

system is almost completely charged.

The concept of AEC has many limitations. Since AEC is

uni tless, it is of limited use in supplying information

about intracellular nucleotide concentrations or the rate

11

of ATP turnover (Knowles, 1977). Metabolic studies (Leung

& Schramm, 1980) which measure adenine nucleotide levels

of E_. col i suggest that relatively rapid changes in the

pool size occur as a result of perturbation of energy

metabolism. M o r e o v e r , Lowry e_t a_l_. (1971) have shown that

AEC is an insensitive metabolic indicator while Swedes et

al . (1975) have shown for coli that AEC maintains its

normal level even though the adenylate pool level has

fallen below about 30% of its normal value. Thus, it seems

probable that the AEC varies less than the actual

concentrations of most individual m e t a b o l i t e s (Atkinson,

1977). The AEC concept has been critized by Purich & Fromm

(1972) on theoretical grounds, but it should be emphasized

that this concept is important for the understanding of

overall energy metabolism. There are many situations,

however, where the application of AEC requires

modifications or does not even apply (Reich & Sel'kov,

1981). Modifications are generally required when other

nucleotides play an important role. AEC is not applicable

to systems where adenylate kinase is not sufficiently

active, as in the mitochondrial matrix.

Nonadenine Nucleotides and Growth (With Special Reference to GTP)

The nonadenine ribonucleoside triphosphates UTP, CTP

and GTP and the four d e o xyribonucleoside triphosphates are

12

used in m a c r o m o l e c u ! e synthesis. The phosphory1ated

ribonucleosides are analogous in structure to ATP and have

the same A G 0 ' for hydrolysis. Although ATP is the

mainstream carrier of phosphate groups in the cell, the

other types of nucleoside 5'-triphosphates are specialized

to serve in certain biosynthetic pathways. These compounds

acquire their terminal phosphate groups from ATP reactions

catalyzed by a Mg-dependent enzyme called nucleoside

diphosphate kinase. The energy charge response of

nucleoside diphosphate kinase is steeper than those of

typical biosynthetic enzymes. Accordingly, nucleoside

diphosphate kinase is treated as a master enzyme whose

properties undoubtedly control growth rates and contribute

to a hierarchical relationship with intermediary

metabolism. In reviewing the role of AEC, Atkinson (1977)

observed that, whereas ATP is involved in providing energy

for a wide range of biological processes, GTP, UTP and CTP

provide energy for certain anabolic processes. This

segregation of function among nucleoside triphosphates has

been maintained throughout the evolution of life on earth,

suggesting that it has an important biological role and

thus has been selected for such maintenance. The function

suggested by Atkinson (1968) is that the levels of energy

charge of the nonadenine nucleotides may be important in

regulating cellular anabolic activities.

13

A significant portion of total energy flux during

bacterial growth proceeds through nonadenine nucleotide

pools (Lehninger, 1973). Unlike the ATP pool, the

intracellular concentrations of nonadenine triphosphates

fluctuate in direct proportion to their requirement for

biosynthesis (Frazen & Brinkley, 1961; Karl, 1978; Smith &

Maaloe, 1964; Smith, 1979).

It is now well established that GTP, a guanine

nucleotide, present in all cells, activates a wide range

of anabolic processes involved in growth and cell

proliferation by a variety of m e c h a n i s m s . It is therefore

thought to be a mediator of the "pleiotropic response"

(Hershko et_ , 1971). Such activation is of great

importance physiologically in regulating various

activities in intact cells. Table I summarizes some of the

evidence for predicting the role of GTP in the stimulation

of anabolism (Pall, 1985).

GTP levels are controlled intracel1ular1y in a

precisely tuned fashion. There are four mechanisms by

which guanine nucleotide levels vary in response to

physiological changes: (a) cells are able to regulate

anabolic and catabolic pathways for guanine nucleotides

which in turn alter the total concentration of all guanine

nucleotides; (b) cells regulate the energy charge of the

guanine nucleotide pool; (c) cells are able to convert

14

TABLE I

SUMMARY OF EVIDENCE FOR STIMULATION

OF ANABOLISM BY GTP

P r o c e s s s t i m u l a t e d R e f e r e n c e s

P o l y p e p t i d e i n i t i a t i o n

P o l y p e p t i d e e l o n g a t i o n

Con t ro l of p r o t e i n s y n t h e s i s in m i t o c h o n d r i a

I n h i b i t i o n of p r o t e i n s y n t h e s i s

P o l y p e p t i d e t e r m i n a t i o n

P r o t e i n g l y c o s y l a t i o n

S y n t h e s i s of s t a b l e RNAs

E u k a r y o t i c mRNA capp ing

P h o s p h o l i p i d b i o s y n t h e s i s

Polyamine b i o s y n t h e s i s

M i c r o t u b u l e f i l a m e n t assembly

A c t i v a t i o n of c e l l wal l bi o s y n t h e s i s

M a i t r a e t a j . . , 1982; H a s e l k o r n & Rothman-Denes , 1973.

M a i t r a e t a l . , 1982.

F i n z i ej; a_K » 1982.

Pa in & Clemens , 1983.

Ogawa & K a j i , 1975.

G o l d e l a i n e & B e a u f a y , 1983; Pa iement & B e r g e r s o n , 1983.

T r a v e r s ejt a j k , 1980.

S h a t k i n , 1976.

Hashizume £ t a j . . , 1983.

P a u l i n & Poso , 1983.

Lee e_t aj[ . , 1982.

Aloni e t a l . , 1982.

15

6TP/GDP into other guanine-containing compounds and (d)

cells often change the local concentration of GTP or GDP

at one or more of their receptors.

GTP also has been proposed to play the role of second

messenger involved in regulation of polyamines (Bachrach,

1973), intracellular pH (Busa & Nucci tel1i, 1984) and

guanosine polyphosphates of the stringent response

(Gallant, 1979). There are four major lines of evidence to

support the view: (a) there is segregation of function

between adenine nucleotides and nonadenine nucleotides of

phy1ogenetical1y diverse organisms in which the nonadenine

nucleotides are specifically involved in anabolic

processes. This segregation of function is thought to have

been conserved evolutionarily due to regulation of

anabolic activities by a nonadenine energy charge

(Atkinson, 1977); (b) a wide range of anabolic processes

show specific stimulation by GTP in vitro; (c) GTP is

involved in controlling sporulation of microorganisms in

vivo and (d) GTP is an allosteric effector of glutamine

dehydrogenase and other proteins. Thus GTP may be treated

as a regulatory compound.

Techniques of Separating and Quantifying Nucleic Acid Related Substances

There are many techniques that are available for the

separation and quantification of nucleic acid related

16

substances (Chargaff & Davidson, 1972; Colowick & Kaplan,

1957). These methods can be grouped under three general

headi ngs.

Enzymatic Analysis

The specificity of enzymes is used to determine the

presence of substances, for example, luciferase (firefly)

assay to determine ATP (Strehler & McElroy, 1957), AMP

deaminase assay for determining AMP (Smolenski &

S k i adanowski, 1986).

Non-chromatographic Analysis

Paper electrophoresis .--Nuc1eic acids and their

components bear a variety of ionizable groups and it is

therefore possible to separate and determine these

derivatives based on the difference of net charge at a

given pH. This manipulation can be carried out in a short

time and using a very small amount of m a t e r i a l .

Color r e a c t i o n s . - - T h e different reactions has been

reviewed extensively by Schmidt et^ (1957). An example

of this is the reaction by which guanine and xanthine give

a blue color on treatment with folin phenol reagent.

Active c h a r c o a l . - - N u c l e i c acid components are readily

absorbed on active charcoal in acidic solution and can be

eluted with acetone-water or alkaline methanol.

17

Chromatographic Analysis

This method is based on the concept of differential

migration of compounds in a two-phase system. The mobile

phase (solvent)-composed of a liquid or gas, exerts a

driving force to move the sample mixture through a

stationary phase (sorbent)-composed of a liquid or solid.

This method has two general offshoots:

Planar chromatography.--Here the stationary phase

asumes a planar arrangement in the form of a sheet of thin

film. The mobile phase migrates by means of capillary

action. Examples of planar chromatography are paper

chromatography and thin layer chromatography.

Column chromatography.--Here the stationary phase is

either a solid packing material, a solid support that is

coated with a sorbent layer or a gel packed into a tube.

Gas chromatography, ion exchange chromatography and liquid

chromatography are three forms of column chromatography.

Classical liquid chromatography of yesterday has taken the

form of high pressure liquid chromatography (HPLC) today.

There are a number of different techniques available for

HPLC. Notable among them are ion exchange HPLC, size

exclusion HPLC, ion paired HPLC and reversed phase HPLC.

All of these methods of separation of nucleotides are

outlined in Fig. 4.

18

F i g . 4--Some techn iques f o r the s e p a r a t i o n and d e t e r m i n a t i o n of n u c l e i c ac id r e l a t e d substances.

19

Methods of Separation

NONCHROMATOGRAPHIC

D r Paper Electrophoresis ,,

Co

ENZYMATIC CHROMATOGRAPHIC

1

or Reaction

Active Charcoal

Column

, .. (m i r Liquia ion Paper

Exchange

1 Planar

Thin Layer

Classical

[-Ion Exchange (*Cellulose)

-Size Exclusion (*Sephadex)

-Affinity (* Agarose)

•-Reversed Phase (*RPC-5)

Modern ligh Pressure Liquid Chromatography)

-Ion Exchange (*Silica)

-Size Exclusion (* Silica)

-Affinity (* Polyacrylamide)

^Reversed Phase (*Silica/PRPl)

(*) Support material

20

The two most widely adapted techniques used for the

quantitation of nucleic acid related substances are thin

layer chromatography (TLC) and liquid chromatography (LC).

A comparision between TLC and LC is shown in Table I

(Treiber, 1986). HPLC has been used in these studies

because it is reliable, cost effective and less time

consuming.

Organization of the Dissertation

The purpose of this study was to (i) investigate any

possible relation between the growth rate and GTP/ATP

ratio versus time and (ii) to determine the response of

the acid-soluble nucleoside triphosphate pools to stressed

conditions. The study is divided into four chapters as

fol1ows:

1. Separation and quantitation of bacterial

ribonucleoside triphosphates by radial compression high

pressure liquid chromatography using trif1uoroacetic acid

as an extraction solvent (Chapter II).

2. Accumulation of adenosine triphosphate and

guanosine triphosphate in a uraci1-requiring mutant strain

of Escherichia coli (Chapter III).

3. GTP/ATP ratio, a new parameter for measuring

microbial growth (Chapter IV).

4. Temperature dependency and ribonucleotide

production in Yersinia e n t e r o c o 1 i t i c a (Chapter V).

21

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CHAPTER I I

SEPARATION AND QUANTITATION OF BACTERIAL RIBON UCL E OS IDE TRIPHOSPHATES BY RADIAL COMPRESSION HIGH PRESSURE

LIQUID CHROMATOGRAPHY USING TRIFLUOROACETIC ACID AS AN EXTRACTION SOLVENT

INTRODUCTION

For the past twenty years , pur ine and py r im id ine

metabolism in microorganisms has been s tud ied ex tens i ve l y

(Moat & Friedman, 1960; O'Donovan & Neuhard, 1970). Since

i n t r a c e l 1 u l a r pur ine and py r im id ine nuc leo t ide pools

con t ro l the i n d i v i d u a l b i o s y n t h e t i c pathways producing the

subst ra tes f o r RNA (UTP, CTP, ATP and GTP) and DNA (dTTP,

dCTP, dATP and dGTP), accurate measurement of the

endogenous nucleoside t r iphosphates could serve as a

d iagnos t i c too l f o r the es t ima t ion of m i c rob ia l growth

r a t e . Moreover, GTP/ATP r a t i o s ( K a r l , 1978) might also be

a means of assessing growth ra tes in organisms tha t do not

form co lon ies or r e a d i l y lend themselves to convent iona l

growth ra te measurements.

Measurement of endogenous r i b o - and

deoxyr ibonuc leo t ide pools has been r e v o l u t i o n i z e d by the

development (G laser , 1986) of high pressure l i q u i d

chromatography (HPLC). One of the major emphases so fa r

has been r e f i n i n g HPLC procedures (Brown, 1973; Assenza &

Brown, 1983; Hancock & Sparrow, 1984; Boul ieu & Bory,

1985) f o r nuc leo t i de separa t ion . The two most commonly

23

24

employed sepa ra t i on techn iques i n v o l v e e i t h e r

reverse-phase chromatography on o c t y d e c y l s i 1 i c a r e s i n s

(Horvath aj_., 1977; Hoffman & L i a o , 1977) or

ion-exchange chromatography on m i c r o p a r t i c u l a t e

anion-exchange r e s i n s (Har tw ick & Brown, 1975; McKeag &

Brown, 1978) . Even though reverse-phase chromatography

p rov ides g rea te r s e n s i t i v i t y and more r a p i d a n a l y s i s

compared to any ion exchange chromatograph ic method

c u r r e n t l y a v a i l a b l e , i t lacks the s e l e c t i v i t y necessary

f o r the ana l ys i s of the many c l o s e l y r e l a t e d n u c l e o t i d e s

present in c e l l e x t r a c t s (Reiss _t_ _al_. , 1984) .

Fur thermore , very l i t t l e e f f o r t (Chen et^ £l_. , 1977) has

been made in deve lop ing e x t r a c t i o n procedures f o r the

d e t e c t i o n and q u a n t i t a t i o n of low l e v e l s of n u c l e o t i d e

pools in m ic roorgan isms. Dur ing p r imary work in our

l a b o r a t o r y i n v o l v i n g q u a n t i t a t i o n of nuc leos ides and

n u c l e o t i d e s in b a c t e r i a l e x t r a c t s , we devised a novel

e x t r a c t i o n procedure us ing t r i f 1 u o r o a c e t i c ac id (TFA) as

s o l v e n t i ns tead of the more conven t i ona l e x t r a c t i o n

so l ven t s such as t r i c h l o r o a c e t i c ac id (TCA) and f o rm ic

ac id (HCOOH).

TFA i s a c l e a r , c o l o r l e s s , v o l a t i l e l i q u i d w i t h a

very low UV c u t - o f f p o i n t . TFA has been used as a s o l v e n t

system f o r the p u r i f i c a t i o n and a n a l y s i s of pep t ides by

reverse phase HPLC (Mahoney & Hermodson, 1980; Yuan et

25

al., 1982). Various methods of extraction (Brown & Miech,

1972; Lundin & Thore, 1975) for ribonucleosides have been

reported. However, each has some particular drawback with

respect to quantitation at low levels. The present study

was undertaken to find a parallel extraction procedure

using TFA for ribonuc1eoside triphosphates and to compare

it to known extraction procedures using TCA and HCOOH.

This extraction procedure using TFA makes it possible to

detect nanomolar concentrations of ribonucleoside

triphosphates notably ATP and GTP, in microbial samples.

METHODS

Chemi cals

Nucleotides, trichloroacetic acid and tri-n-

octylamine were purchased from Sigma Chemical Company (St.

Louis, MO.); ammonium phosphate- monobasic was purchased

from Mallinckrodt Inc. (Paris, KY). Potassium chloride and

1,1,2-trich1oro-1,2,2- trif1uoroethane (freon) was

purchased from Eastman Kodak Company (Rochester, NY). All

other chemicals were of analytical grade and were

purchased from Fisher Scientific Company (Fair Lawn, NJ).

Bacteri al Strai ns

American Type Culture Collection wild type

Escheri chi a coli Luria strain B and Pseudomonas aeruginosa

were used in these studies.

26

Growth Medium

Bacterial cells were grown in M9 minimal medium

containing in one liter of distilled, deionized water:

N a 2 H P 0 4 , 6; K H 2 P 0 4 , 3; NaCl, 0.5, N H 4 C 1 . Glucose (0.2 per

cent w/v), 2 ml 1M M g S 0 4 . 7 H 2 0 , 0.1 ml of 1M C a C l 2 and

casamino acids (0.4 per cent, w / v ) were added separately.

Growth of Bacteri a

All cultures were grown at 37°C in a Lab Room

Controlled Environmental Room (Labline Instrument, Inc.,

Melrose Park, IL). Liquid cultures were incubated in Klett

Erlenmeyer flasks (Kontes; V i n e l a n d , NJ) in an G10

gyratory shaker (New Brunswick Sci. Co.; Edison, NJ) set

at 120 revolutions per minute. The turbidity was measured

every 20 minutes with photoelectric Klett Summerson

colorimeter (Klett Manufacturing Co., New York, N.Y.)

using a green filter #54 and recorded as Klett Units (KU),

where 1 K U = 1 X 1 0 7 cells m l " 1 . Volumes of 50 ml of bacterial

cultures with a density of 100 KU were harvested and spun

at 4°C at 12000 X g for 2 m inutes. The supernatant was

poured off and cell pellet was used for nucleotide

extracti on.

TFA, TCA, and HC00H Extraction Procedures

One ml of ice cold 0.5M TFA or 6 per cent (w/v) TCA

or 1.0M HC00H was added to the cell pellet which was

27

thoroughly mixed for 2 minutes in a Vortex. It was allowed

to stand at 4°C for 30 minutes before further

centrifugation at 12000 X g for 10 minutes. The clear

supernatant was then neutralized with ice cold freon-amine

(Khym, 1975) solution (0.7 M tri-n-octyl-amine in freon

113, 1.06 ml amine/5 ml freon). The freon-amine mixture

was vortex-mixed for 2 minutes and then allowed to

separate while standing for 15 minutes at 4°C. The top

aqueous layer, which contains the nucleotide pool extract,

was removed, filtered (Gelman, ACR0 LC13, 0.45p) and

frozen at -20°C until ready for use.

Chromatographic Apparatus

The HPLC equipment (Waters Chromatography Division;

Milford, MA) used 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

sensitivity fixed at 0.05 AUFS (absorbance unit full

scale). Separations were performed on a Waters Radial PAK

Partisil SAX Cartridge (0.8 cm. X 10 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

28

methanol arid filtered double-distilled water (2X) when not

in use. After priming the pumps, the system was flushed

with 50 ml of m e t h a n o l : w a t e r mixture at 3 ml m i n " 1 . Next,

the system was thoroughly washed with distilled water with

the initial flow rate at 3 ml min"*. After 10 minutes, the

flow rate was increased to 4 ml m i n " 1 . When the back

pressure of the column dropped to 850 pounds per square

inch, the 50:50 m e t h a n o l : w a t e r 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 S p e c t r o p h o t o m e t e r , set at 254 nm

and 0.05 AUFS, 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 hold

of low concentration buffer.

Nucleotide samples (100 ^1), prepared and obtained

from bacteria as previously described were thawed and

injected into the column. The Partisi1 SAX10 column

(10,000 plates meter 1 ; particle size, 10 pm) was an anion

exchange column in which a quaternary nitrogen is

29

si 1oxane-bonded to Partisil 10 (Silica). The associated

counter-ion was HgPO^". 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 C-5 1 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 7mM N H 4 H 2 P 0 4 (pH=3.8)

buffer (Pogolotti & Santi, 1982). The flow rate was

maintained at 4 ml min""'" and all analyses were done at

ambient temperature. Peaks were integrated either m a n u a l l y

on a Houston Instrument's Omni-Scribe strip chart recorder

or by an Apple lie computer system using an ADALAB dual

slope integrating A/D converter and Chromatochart software

(Interactive Microware Inc., State College, PA). The

sampie peaks were identified by comparing retention times

of appropriate standards. The concentration of the sample

was calculated by comparing its peak height to the

standard for which the concentration was known (1 m M ) and

expressed as micromole (g dry w t ) " 1 . The system was shut

down by first flushing with distilled water and then with

50:50 m e t h a n o l : w a t e r in the same manner used to start the

apparatus.

30

RESULTS

The 8-mm diameter of the radial-compression column

provided greater than six times the surface area of

conventional steel columns. Consequently, the speed of

separation was enhanced more than three times that

originally reported by Hartwich & Brown (1975), without

any appreciable loss in resolution.

The range of buffer pH used in this study was 3.8 to

4.5. It was observed that the resolution of the nucleoside

mono-, di- and triphosphates was very sensitive to the pH

of the high-phosphate buffer B. F u r t h e r m o r e , the use of a

continuous phosphate gradient for elution presented

certain difficulties in the analysis of intracellular

nucleotide pools. A continuous gradient was necessary for

resolution of nucleoside triphosphates which eluted late

but a gradient delay is required for the optimal

resolution of those which eluted early. This was the

reason for my not being able to resolve mono- and

diphosphates in bacterial extracts. In addition, it is

well known that uv-absorbing impurities exist in phosphate

buffers which, when operating in a gradient mode up to

high buffer c o n c e n t r a t i o n s , cause rising baselines and

limit the sensitivity of such systems during the high

ionic strength phase of the gradient. To avoid such

problems, I mixed KC1 (Kodak) with analytical grade

31

NH^^PO^ ( M a l l i n c k r o d t ) . The r e s u l t a n t b u f f e r m i x t u r e had

less uv -absorb ing i m p u r i t i e s and d id not r e q u i r e the

t ed ious p u r i f i c a t i o n procedure of Shmukler (1970) . F i g . 5

shows the HPLC p r o f i l e of the s tandard n u c l e o t i d e m i x t u r e

on P a r t i s i l SAX us ing the system desc r ibed above.

T y p i c a l l y TCA i s used in our l a b o r a t o r y f o r

e x t r a c t i n g b a c t e r i a l n u c l e o t i d e p o o l s . Since the use of

t h i s e x t r a c t i o n procedure proved inadequate f o r measuring

r i b o n u c l e o s i d e t r i p h o s p h a t e s , e s p e c i a l l y ATP and GTP,

d i f f e r e n t e x t r a c t i o n so l ven t s were t h e r e f o r e t e s t e d .

I n i t i a l r e s u l t s of repeated runs f o l l o w i n g TFA e x t r a c t i o n

of r i b o n u c l e o t i d e s from E_. c o l i produced a much g r e a t e r

inc rease in y i e l d s than TCA or HCOOH e x t r a c t i o n procedures

(Table I I I ) . TFA e x t r a c t i o n procedures r e s u l t e d in b e t t e r

peak he igh ts ( F i g . 6 ) , a major c r i t e r i o n f o r comput ing

c o n c e n t r a t i o n s a c c u r a t e l y . The same e x t r a c t i o n procedures

were used w i t h TFA as the e x t r a c t i n g s o l v e n t in _P.

aerug i nosa. Resu l ts (Table IV) showed t h a t y i e l d s of ATP

and GTP s i m i l a r l y inc reased f o l l o w i n g TFA e x t r a c t i o n

a l though UTP and CTP were best e x t r a c t e d f o l l o w i n g i n i t i a l

t r ea tmen t w i t h TCA.

D i f f e r e n t c o n c e n t r a t i o n s of TFA were next t e s t e d to

a s c e r t a i n the op t ima l e x t r a c t i o n c o n c e n t r a t i o n . In

p r i n c i p l e , the lowest c o n c e n t r a t i o n of TFA should be used

to avoid q u a n t i t a t i v e loss of n u c l e o t i d e s . As can be seen

32

Fig. 5--Chromatogram of the 12 ribonucleotide standards. Column, radial compression Partisil 10-SAX cartridge; temperature, ambient; detector sensitivity, 0.05 AUFS. Absorbance recorded at 254 mM N H . H ? P 0 . containing 500 mM KC1 pH 4.5. Flow rate maintained at 4 ml mi n"

33

0).

03 o (/)

c D >

o c CO

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10 15 20 Time ( m i n u t e s )

34

F ig . 6 - - E l u t i o n p r o f i l e of the r i bonuc leos ide t r i phospha tes . (A) Standard r ibonuc leos ide t r i phospha te mix ture (100 m) c o n s i s t i n g of 10 M of each n u c l e o t i d e . (B) to (D) Sample (100 fi l ) from Escher ich ia c o l i c e l l s a f t e r e x t r a c t i o n using TCA (6% w / v ) , HC00H (T7M) and TFA (0.5M) r e s p e c t i v e l y . Condi t ions as in F ig . 5 legend.

35

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36

TABLE I I I

YIELD OF NUCLEOSIDE TRIPHOSPHATES IN E. COLI

CELLS [yMOLE (G DRY WT)_ 1J USING

DIFFERENT EXTRACTION SOLVENTS

TCA HCOOH TFA (6% w / v ) ( l .OM) (0 .5M)

UTP 2. ,04 1. .76 4. .6

CTP 1. .01 1. .21 1. .84

ATP 3. ,04 3. ,32 5. ,28

GTP 1. ,48 i k 2. , 96

( * ) no t d e t e c t a b l e

37 TABLE IV

YIELD OF NUCLEOSIDE TRIPHOSPHATES IN P. AERUGINOSA

CELLS (JJMOLE (G DRY WT)'^| USING

DIFFERENT EXTRACTION SOLVENTS

TCA HCOOH TFA (6% w/v) (1.0M) (0.5M)

UTP 2.6 2.1 1.64

CTP 1.04 0.96 0.52

ATP 4.2 4.1 4.84

GTP 2. 68 1.9 2.96

Note: The Pseudoroonas aeruginosa were grown at 37°C in glucose minimal medium and did not reach the density of 109 cells ml~l. This is due to the fact that P. aerugi nosa does not use glucose as a source of carbon very well. The pool levels are accordingly, slightly low.

38

from Table V, in E_. coli 0.5M TFA gave highest readings

for UTP, CTP and ATP while the highest value for GTP was

obtained using 1.OM TFA. In £. aerugi nosa however, the

highest values of ATP and GTP were obtained following

extraction with 0.25M and 1.0M TFA respectively. These

data therefore clearly illustrate the importance of

extraction procedures for individual ribonucleoside

triphosphates, a point that has often been overlooked.

DISCUSSION

The procedure for initial sample preparation is a

critical step in the quantitation of bacterial

ribonucleoside triphosphates for avoiding potential

sources of error. The resolution of peak heights or peak

areas is not consistent and thus reproducible values for

the ribonucleoside triphosphates are not found. To avoid

such discrepancies the following guidelines are

recommended: (a) that ice cold freon-amine be used for

neutralization; (b) that cold freon-amine neutralized

extract be vortex-mixed sufficiently (2 minutes) for

complete mixing and thereafter allowed to settle at 4°C

for at least 15 minutes. The clear aqueous layer should be

gently and carefully removed so that the interphase is not

disturbed. Often, the aqueous layer looks clear even

though the interphase is mixed with it. This interphase

layer, which was composed of micellar aggregrates produced

39

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40

anomalous peaks.

The mariner in which TFA interacts with bacterial

nucleosides, allowing them to retain their solubility, is

not known. TFA is a weak acid and thus degrades

triphosphates to a much lesser extent than TCA or HCOOH.

It is also possible that freon-amine neutralization of TFA

is complete. This is probably not the case for TCA or

HCOOH. The basic criteria characteristic of an effective

extraction solvent are: (a) sensitivity (b) stability (c)

ease of sample recovery because of the volatility of

solvent (d) clear and colorless and (e) easily removable

by freon-amine solutions. The solvent TFA has all of these

characteristics. I have developed this procedure to

measure ATP and GTP pools of bacterial strains. However,

this method can be used to investigate many other aspects

of purine and pyrimidine metabolism in bacteria and other

organ i sms.

The experiments described in Chapters III, IV and V

were actually carried out before the experiments described

in this chapter (Chapter II). For this reason TFA was not

used throughout.

CHAPTER III

ACCUMULATION OF ADENOSINE TRIPHOSPHATE AND GUANOSINE TRIPHOSPHATE IN A URACIL-REQUIRING MUTANT

STRAIN OF ESCHERICHIA COLI

INTRODUCTION

Purine and pyrimidine nucleotides are important

compounds for the synthesis of all macromo1 ecu 1es in

living cells. Dynamic changes and adjustments of the

intracel1ular chemical composition of nucleotides in

living cells evoke metabolic reorientation of essential

m e t a b o l i t e s , alter the genetic expression of the genome

and induce changes in macromo1 ecu 1ar syntheses.

A supply of ribonuc1eoside and d e o x y r i b o n u c l e o s i d e

5'-triphosphates is required for the biosynthesis of

nucleic acids. The synthesis of these compounds occurs in

two main stages: (a) the formation of the purine and

pyrimidine ring systems and their conversion into parent

ribonucleoside monophosphates and (b) a series of

interconversions involving the reduction of

ribonucleotides to form a balanced set of

d e o x y r i b o n u c l e o s i d e triphosphates. This balance is

maintained by feedback control of biosynthesis based on

end-product inhibition, whereby normal bacterial cells

match purine and pyrimidine synthesis to the rate of

purine and pyrimidine removal into newly synthesized

41

42

nucleic acids of the cell (Gerhart, 1970). The balance is

maintained in a more permanent manner by repression of the

synthesis of the purine and pyrimidine biosynthetic

enzymes in bacteria.

The pyrimidine biosynthetic pathway is universal and

is obligately required for the synthesis of nucleotide

precursors of RNA and DNA (O'Donovan & Neuhard, 1970). The

occurrence of bacterial mutants with an absolute

requirement for pyrimi dines shows that an alternative to

the de novo pathway for the generation of pyrimidine

nucleotides must exist. The fact that uracil, cytosine,

uridine, cytidine, deoxyuridine and deoxycytidine satisfy

the pyrimidine requirement of such mutants (Cohen, 1953;

Moore & Boylen, 1955) further indicates that considerable

interconversions may occur among the compounds.

Accordingly, isotope studies show that any of the above

mentioned pyrimidine compounds is equally effective as a

precursor for all the pyrimidine moieties of RNA and DNA

(Bolton, 1954; Bolton & Reynard, 1954; Siminovitch &

Graham, 1955).

ATP was first isolated from muscle extracts by

Lohmann in 1929. ATP is involved in many of metabolic

reactions as well as being a key intermediate in the

transfer of energy in all living organisms (Atkinson,

1977) while GTP has been shown to be a central regulator

43

of c e l l u l a r anabol ism ( P a l l , 1985).

I n d u s t r i a l l y , nuc leos ides and n u c l e o t i d e s produced by

microorganisms are w ide l y used as food f l a v o r i n g

substances (Ogata, 1975; Nakao, 1979(1 ) ; Kempler, 1983) .

Fur thermore , n u c l e o t i d e s and r e l a t e d compounds are used as

a n t i b a c t e r i a l , a n t i v i r a l , a n t i f u n g a l , a n t i p r o t o z o a l ,

a n t i l e u k e m i c and an t i t umor agents . A d d i t i o n a l l y ,

n u c l e o t i d e s are i nvo l ved in blood p l a t e l e t a g g r e g a t i o n ,

immunosuppression and neurost imu1 a t i o n . Phospho ry la t i on of

AMP to ATP by yeast e x t r a c t s was f i r s t s t ud ied by

Lutwak-Mann & Mann (1935) . L a t e r , Ostern et a l . (1938)

showed t h a t adenosine or AMP was phosphory la ted to ADP and

ATP coupled w i t h g l y c o l y s i s in y e a s t . In 1960, C a n e l l a k i s

et aj_. d i scovered t h a t o ther nuc leos ide d i - and

t r i p h o s p h a t e s were formed from t h e i r co r respond ing

nuc leos ides and nuc leos ide monophosphates. Based on these

f i n d i n g s , Toch ikura et a 1 . (1967) developed an improved

process to produce ATP in a molar y i e l d of 72 per cent by

p h o s p h o r y l a t i o n of adenosine of 5'-AMP w i t h bake rs ' yeas t

d r i e d w i t h acetone. By 1970, p r o d u c t i o n of CTP, UTP and

GTP from t h e i r co r respond ing mononuc leot ides was r epo r t ed

by a number of researchers (Ki t a j i m a et_ a_l_. , 1970; S h i r o t a

et aj_., 1971) .

D i r e c t p r o d u c t i o n of ATP from adenine was c a r r i e d out

by Tanaka £t_ a]_. (1968) and Nara £ t (1967, 1968). When

44

adenine was added to the c u l t u r e s of Brev i b a c t e r i um

ammoniagenes, ATP, ADP and AMP were produced. S i m i l a r l y ,

a d d i t i o n of guanine led to the p roduc t i on of GTP, GDP and

GMP. Kawaguchi £ t &]_. (1970) a lso showed t h a t GTP, GDP and

GDP-mannose were produced in a i r d r i e d c e l l s of b a k e r ' s

yeast from GMP and g lucose .

The present work examines the e f f e c t of u r a c i l

s t a r v a t i o n on pu r ine and p y r i m i d i n e metabo l i sm. I t a lso

p rov ides an e n t i r e l y new scheme f o r the e f f i c i e n t

p roduc t i on of ATP and GTP us ing s p e c i a l l y c o n s t r u c t e d

mutant s t r a i n s of b a c t e r i a .

METHODS

Chemi ca l s

N u c l e o t i d e s , t r i c h l o r o a c e t i c ac id and t r i - n -

oc ty lam ine were purchased from Sigma Chemical Company ( S t .

L o u i s , MO.). Ammonium phosphate-monobasic was purchased

from M a l l i n c k r o d t I n c . ( P a r i s , KY) and 1 , 1 , 2 - t r i c h 1 o r o -

1 , 2 , 2 - t r i f 1 u o r o e t h a n e ( f r e o n ) was purchased from Eastman

Kodak Company (Roches te r , NY). A l l o the r chemica ls were of

a n a l y t i c a l grade and were purchased from F ishe r S c i e n t i f i c

Company ( F a i r Lawn, NJ) .

B a c t e r i a l S t r a i n

A mutant s t r a i n of E_. co l i TB2 was p rov ided by Dr.

O'Donovan (Department of B i o l o g i c a l Sc iences , Nor th Texas

45

State U n i v e r s i t y ) for use in this work. This strain has a

genotype of JE. col i K12 pyrBI argF argl.

Growth Medium


containing in one liter of distilled, deionized water:

N a 2 H P 0 4 , 6; K H 2 P 0 4 , 3; NaCl, 0.5, N H 4 C 1 , and uracil (50 \xg

m l " 1 ) . Glucose (0.2 per cent w/v), 2 ml 1M M g S 0 4 . 7 H 2 0 , 0.1

ml of 1M CaC12 and casamino acids (0.4 per cent w/v) were

added separately.

Growth of Bacteria




Erlenmeyer flasks (Kontes; Vineland, NJ) in an G10

gyratory shaker (New Brunswick Sci. Co.; Edison, NJ) set

at 120 revolutions per minute. The turbidity was measured

every 20 minutes with a photoelectric Klett Summerson

colorimeter (Klett Manufacturing Co.; New York, N.Y.)

using a green filter #54 and recorded as Klett units (KU),

where 1KU=1X10^ cells/ml. Volumes of 50 ml of bacterial

cultures at different stages of the exponential phase were

harvested, actual Klett units recorded, and spun at 4°C at

12000 X g for 2 minutes. The supernatant was decanted and

cell pellet was used for nucleotide extraction.

46

The culture was not allowed to grow once the cells

reached a density of 85 Kletts. A 200 ml sample of this

culture was then centrifuged, washed, and the pellet was

resuspended in 200 ml of the same medium without uracil.

The cells at this stage showed a density of 25 Kletts. The

cells were shaken for one hour before harvesting in the

same manner as before.

Trichloroacetic Acid (TCA) Extraction Procedure

One ml of ice cold 6 per cent (w/v) TCA was added to

the cell pellet which was thoroughly mixed for 2 minutes

in a Vortex. The mixture was allowed to stand at 4°C for

30 minutes before further centrifugation at 12000 X g for

10 minutes. The clear supernatant was then neutralized

with ice cold freon-amine (Khym, 1975) solution (0.7 M

tri-n-octyl-amine in freon 113, 1.06 ml amine/5 ml freon).

The freon-amine mixture was vortex-mixed for 2 minutes and

then allowed to separate while standing for 15 minutes at

4°C. The top aqueous layer, which contains the nucleotide

pool extract, was removed, filtered (Gelman, ACR0 LC13,

0.45|i) and frozen at -20°C until needed.


The HPLC equipment (Waters Chromatography Division;


Model 680 automated Gradient Controller, a U6K injector

47

and a Model 481 LC S p e c t r o p h o t o m e t e r . Nucleotides were




Partisil SAX Cartridge (0.8 cm X 10 cm) using a Waters

Radial Compression Z - M o d u l e System.




methanol and filtered double distilled water (2X) when not


with 50 ml of methanol:water mixture at 3 ml m i n - 1 . Next,


the initial flow rate at 3 ml min After 10 m i n u t e s , the

flow rate was increased to 4 ml m i n " 1 . When the back











48

(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 hold

with low concentration buffer.

Nucleotide samples (100 pi), prepared and obtained

from bacteria as previously described, were thawed and

injected onto the column. The Partisi1 SAX10 column

(10,000 plates meter"^; particle size, 10 pm) was an an ion


si1oxane-bonded to Partisil 10 (Silica). The associated

counter-ion was H^PO^". Nucleotides bind to the quaternary



phosphates at the C-5 1 of the sugar. A linear gradient




regenerated by washing with 30 ml of 7mM N H 4 H 2 P 0 4 (pH=3.8)

buffer. The flow rate was maintained at 4 ml min"''" and all

analyses were done at ambient temperature. Peaks were

integrated either manually on a Houston Instrument's

OmniScribe strip chart recorder or by an Apple lie

computer system using an ADALAB dual slope integrating A/D

converter and Chromatochart software (Interactive

Microware Inc., State College, PA). The sample peaks were

49

identified by comparing retention times of appropriate

standards (Fig. 7). The concentration of the sample was

calculated by comparing its peak height to the standard

for which the concentration was known (1 m M ) and expressed

as mi cromo1e (g dry wt) 1 . The system was shut down by

first flushing with distilled water and then with 50:50

m e t h a n o l : w a t e r in the same manner used to start the

apparatus.

RESULTS

The ATP and GTP levels of the culture were monitored

during initial growth in M9+ uracil+ casamino acid medium

using high pressure liquid chromatography. Fig. 8 shows a

typical profile of the nucleotides from E. coli strain

TB2. The levels of ATP and GTP continued to accumulate

linearly reaching a maximum when the cells reached a

density of 75 Kletts at the end of 265 m inutes. Cells were

grown to various densities from 60-100 Kletts units. The

precipitous increase in ATP concentration occurred to the

greatest extent when uracil was removed from strain TB2 at

75 KU. At this stage, the cells were harvested,

centrifuged, washed and resuspended in a medium without

uraci1.

The bacterial suspension at this stage contained 2.5 8 1

X 10 cells ml" . The cells were allowed to grow for one

hour, at which time a density of 3.0 X 1 0 8 cells m l " 1 was

50

F i g . 7--Chromatogram of the 12 r i b o n u c l e o t i d e s tanda rds . Cond i t i ons as in F i g . 5 legend except t h a t the f l ow r a t e was ma in ta ined at 4 .5 ml min .

51

JL 10 1'5 20

T ime ( m i n u t e s )

52

j • F i J : 8 - - E l u t i o n p r o f i l e of the r ibonuc leos ide mono-and t r iphosphates from Escher ich ia c o l i S t r a i n T R ? '

grown in M9 medium conta in ing 0 . 2 % (w /vT^Tucose + 0 4%

as in n g ? m 7 n i e g e n d S U r a C n < 5 ° M ™' K C o n d ' t i o " s

53

03 o CO

c D >

O c 03 JO

o CO -Q

03

CO LL D <

in o

t 10 15 Time ( m i n u t e s )

20 25

54

attained. When the ATP and GTP pools were measured for

this uracil-less culture, a 6-fold increase was noted when

compared to cells having the same density during initial

growth in presence of uracil (Table VI). However, the ATP

and GTP increased only 2-fold over the peak accumulation

during initial growth in presence of uracil (Fig. 9 and

Table VI). The ATP and GTP levels started decreasing after

reaching their peak as growth continued in the absence of

uracil in the medium.

DISCUSSION

Elevation and accumulation of ATP and GTP at molar

concentrations under uracil starvation is noteworthy. This

study suggests that nucleoside triphosphate levels can be

increased specifically whenever cells are subjected to

stress. It is important to discuss probable reasons for

such a high accumulation of ATP and GTP. Older literature

(Henderson et a]_., 1977) leaves no doubt that purines

regulate pyrimidine synthesis but that pyrimidines do not

regulate the synthesis of purines. The results reported

here however may cause this view to be m o d i f i e d . A logical

interpretation can be offered in terms of the old concept

wherein the ATP concentration is taken as the key mediator

in metabolic control (Atkinson, 1977). As can be seen in

Pyr mutant (Fig. 10), uracil is an absolute requirement

for the ultimate production of UTP and CTP to be used for

TABLE VI

ACCUMULATION OF ATP AND GTP LEVELS 9

UNDER VARIOUS GROWTH CONDITIONS

M9 + Casamino acids'3 30 11.61

55

Actual K1 ett

Growth condition units ATP GTP

M9 + Uracil + Casamino acids 30 1.92 0.74

4.49

M9 + Uracil + Casamino acids 75 5.79 2.03

(a) Expressed as mole (g dry wt)~*

(b) Cells starved for uracil at this stage

56

Fig. 9 — Eluti on profile of the ribonucleoside m o n o - , di- and triphosphates from Escherichia coli Strain TB2 cells were grown in uracil as tor Fig. F T o 75 Klett units (KU|. They were harvested, washed and resuspended at 25KU ml in the same medium without uracil. They were starved for 1 hr. Conditions as in Fig. 7 legend.

57

— CO o CO

c 3 > O c CO JD L. o CO

JD CO

CO LL D <

lO o

S/l/

Q. I -<

JUrU,

Q. H 0

10 15 20 Time ( m i n u t e s )

25

58

Fig. 10 Interrelationships between purine and pyrimidine metabolic pathways for the production of R N A and D N A in bacteria.

59

J ) N A •* t K / /

" V /

J. \

/

^ / y / / /

/ / y

/ /

\

dGTP

dCTP—> dUTP—• dUMP—»dTMP

>CTP

Ajj)P-*dADP dGDP*G|P

AMR.

Ribose 5 P

Orotic ATCase

.

Pentose Phosphate Pathway

60

t he s y n t h e s i s o f RNA. When u r a c i l i s removed, RNA

s y n t h e s i s s t o p s , RNA i s degraded and n u c l e o t i d e s

accumu la te . Thus the c e l l s t a r t s p r o d u c i n g and

a c c u m u l a t i n g mass ive q u a n t i t i e s of ATP and GTP. I f t h i s

were a w i l d t ype c e l l ( i . e . Pyr ) , a l l f o u r n u c l e o s i d e

t r i p h o s p h a t e s would accumu la te . However, s i n c e s t r a i n TB2

i s a u r a c i 1 - r e q u i r i n g m u t a n t , then UTP and CTP do no t

accumu la te . F u r t h e r m o r e , s i n c e RNA i s not p roduced , v i t a l

ATP and GTP r e q u i r i n g s teps i n p r o t e i n b i o s y n t h e s i s no

l o n g e r occur such t h a t they do not r e q u i r e ATP and GTP.

Thus, a l l c e l l u l a r ATP and GTP a c c u m u l a t e s . Taken t o g e t h e r

t h i s mass ive a c c u m u l a t i o n of ATP and GTP i s a l o g i c a l

e x t e n s i o n of the u r a c i l s t a r v a t i o n .

To my know ledge , t h i s s t u d y i s the f i r s t of i t s k i n d

t o p r o v i d e a new d i r e c t i o n f o r t he m a n u f a c t u r e of

a d e n y l a t e s and g u a n y l a t e s i n d u s t r i a l l y . Kawamoto e t a_l_.

(1970) was ab le t o show t h a t a u r a c i 1 - r e q u i r i n g mutan t of

A r t h r o b a c t e r p a r a f f i neus accumula ted o r o t i c ac i d and

o r o t i d i n e when grown on n - p a r a f f i n as a s o l e carbon

s o u r c e . Furuya e_t jal_. (1971 & 1973) used a

decoy i n i n e - r e s i s t a n t mutant of jJ. ammoni agenes t o show

d i r e c t p r o d u c t i o n of 5 ' - g u a n i n e n u c l e o t i d e s f rom a

c a r b o h y d r a t e by mixed c u l t i v a t i o n of 5 ' -XMP a c c u m u l a t i n g

s t r a i n and 5 *-XMP c o n v e r t i n g m u t a n t . But no s tudy so f a r

has shown such an e x t e n s i v e a c c u m u l a t i o n of ATP and GTP

61

can occur in a u r a c i 1 - r e q u i r i n g s t r a i n . A l t hough t h i s

t e c h n i q u e of p r o d u c i n g ATP and GTP c e r t a i n l y shows g r e a t

p romise f o r the f u t u r e , s i m i l a r work shou ld be conduc ted

on i n d u s t r i a l s t r a i n s such as those of B. ammoniagenes and

Corynebac te r iu rn q lu tamicum u s i n g l a r g e s c a l e f e r m e n t a t i o n

f o r i n d u s t r i a l a d a p t a t i o n . F i n a l l y , my t e c h n i q u e pu ts

f o r w a r d the concep t of u s i n g mutan t s t r a i n s of b a c t e r i a i n

t he f i e l d of b i o t e c h n o l o g y . Th i s t e c h n i q u e m igh t

r e v o l u t i o n i z e the p r e s e n t a v a i l a b l e t e c h n i q u e s of

p r o d u c t i o n of n u c l e i c ac i d r e l a t e d s u b s t a n c e s .

CHAPTER IV

GTP/ATP RATIO, A NEW PARAMETER FOR MEASURING MICROBIAL GROWTH

INTRODUCTION

During the past few years, several techniques have

been developed to determine the growth rates of microbial

populations with special attention being given to

macromolecular (DNA, RNA and protein) synthesis. This is

because of the striking similarities between the synthesis

of proteins and nucleic acid and the rate of cell growth

(Kjeldgaard, 1967; Maaloe & Kjeldgaard, 1966; Nierlich,

1978). The rate of synthesis of these macromolecules is

regulated by varying the number of sites of polymerization

and not by varying the concentration of substrates. The

number of replicating forks determines the rate of

synthesis of DNA; the number of ribosomes determines the

rate of synthesis of proteins; and the number of active

RNA polymerase molecules determines the rate of synthesis

of RNA. It is also known that ability to synthesize purine

and pyrimidine ring systems de novo is universal in

nature. To synthesize their major chemical constituents,

microorganisms require a source of energy which is

normally achieved through particular energy-rich

compounds, notable among them being ATP and GTP.

Atkinson (1968) was among the first to provide a

62

63

physiological rationale to measure growth rates in terms

of "adenylate energy charge" (AEC) which is defined as

[(ATP) + 0 . 5 ( A D P ) ] / [ ( A T P ) + (ADP) + (AMP)]. AEC is a linear

measure of the amount of metabolic energy stored in the

adenine nucleotide pool. Metabolic studies Leung &

Schramm, 1980) which measured the adenine nucleotide

levels of Escherichi a coli have suggested that relatively

rapid changes can occur in the pool size as a result of

perturbations of energy metabolism. However, since AEC is

unitless, it is of limited use in supplying information

about intracellular nucleotide concentrations or the rate

of ATP turnover (Knowles, 1977). Furthermore, Lowry £t aj_.

(1971) have argued that the AEC is an insensitive

metabolic indicator since changes in AEC actually

overshadow much larger changes in absolute ATP/ADP or

ATP/AMP ratio to which the enzymes are actually

responding. Despite these objections, an overwhelming

proportion of published data suggest that the relative

molar concentration of the adenine nucleotides in actively

metabolizing cells is maintained within the stringent

limits predicted by Atkinson's hypothesis (Karl, 1980).

In reviewing the role of AEC, Atkinson (1977)

observed that whereas ATP was involved in providing energy

charge for a wide range of biological processes, GTP, UTP

and CTP provided energy for anabolic processes. This

64

segregation of function among nucleoside triphosphates has

been maintained throughout the evolution of life on earth,

suggesting that it has an important biological role and

thus has been selected for such maintenance. The function

suggested by Atkinson (1968) was that the levels of energy

charges of the non-adenine nucleotides may be important in

regulating cellular anabolic activities.

A survey of the literature in recent years suggested

that GTP activated many different anabolic processes by a

variety of mechanism. This led Pall (1985) to hypothesize

that GTP and high guanine nucleotide energy charge had a

general role in promoting anabolic activities and that

such activation was of physiological significance in

regulating these activities in intact cells. Thus it

seemed to be important to examine the GTP/ATP ratios and

to correlate these ratios with microbial growth rates in

nature.

METHODS

Chemi cals

Nucleotides, trichloroacetic acid and tri-n-

octylami ne were purchased from Sigma Chemical Company (St.

Louis, MO); ammonium phosphate-monobasic was purchased

from Mai 1inckrodt Inc. (Paris, KY) and 1,1,2-trich1 oro-

1,2, 2 - t r i f 1 u o r o e t h a n e (freon) was purchased from Eastman

65

Kodak Company (Rochester, NY). All other chemicals were of

analytical grade and were purchased from Fisher Scientific

Company (Fair Lawn, NJ).

Bacterial Strain

Wild type E. coli Luria strain B used in these

studies was obtained from the American Type Culture

Col 1ecti on.

Growth Medium


containing in g liter" 1 of d i s t i l l e d , deionized water:

N a 2 H P 0 4 , 6; K H 2 P 0 4 , 3; NaCl , 0.5, N H 4 C 1 , and uracil (50 ng

m l " 1 ) . Glucose (0.2 per cent w/v), 2 ml 1M M g S 0 4 . 7 H 0, 0.1

ml of 1M C a C l 2 and casamino acids (0.4 per cent w/v) were

added separately.

Growth of Bacteria




flasks in an G10 gyratory shaker (New Brunswick Sci. Co.;

Edison, NJ) set at 120 revolutions m i n " 1 . The turbidity

was measured every 20 minutes with photoelectric Klett

Summerson colorimeter (Klett Manufacturing Co., New York,

N.Y.) using a green filter #54 and recorded as Klett units

(KU), where 1 KU = 1 X 1 0 7 cells/ml. Volumes of 50 ml of

66

b a c t e r i a l c u l t u r e s at d i f f e r e n t stages of the exponen t i a l

phase were ha rves ted , ac tua l K l e t t u n i t s r eco rded , and

spun at 4°C at 12000 X g f o r 2 m inu tes . The superna tan t

was poured o f f and c e l l p e l l e t was used f o r n u c l e o t i d e

e x t r a c t i on.

T r i c h l o r o a c e t i c Acid (TCA)

E x t r a c t i o n Procedure

One ml of i ce co ld 6 per cent (w/v ) TCA was added to

the c e l l p e l l e t which was t h r o u g h l y mixed f o r 2 minutes in

a V o r t e x . I t was a l lowed to stand at 4°C f o r 30 minutes

be fo re f u r t h e r c e n t r i f u g a t i o n at 12000 X g f o r 10 m inu tes .

The c l e a r superna tan t was then n e u t r a l i z e d w i t h ice co ld

f reon-amine (Khym, 1975) s o l u t i o n (0.7M t r i - n - o c t y l - a m i n e

in f reon 113, 1.06 ml amine/5 ml f r e o n ) . The f reon-amine

m i x t u r e was vo r tex -m ixed f o r 2 minutes and then a l lowed to separa te on s tand ing f o r 15 minutes at 4°C. The top

aqueous l a y e r , which con ta ins the n u c l e o t i d e pool e x t r a c t ,

was removed, f i l t e r e d (Gelman, ACR0 LC13, 0 .45p) and

f r ozen at -20°C u n t i l ready f o r use.


The HPLC equipment (Waters Chromatography D i v i s i o n ;

M i l f o r d , MA) used cons i s t ed of two Model 510 pumps, a

Model 680 automated Grad ien t C o n t r o l l e r , a U6K i n j e c t o r

and a Model 481 LC Spec t ropho tometer . Nuc leo t i des were

de tec ted by m o n i t o r i n g the column e f f l u e n t at 254 nm w i t h

67



Parti si 1 SAX Cartridge (0.8 cm X 10 cm) using a Waters

Radial Compression Z-Module System.




methanol and filtered double-distilled water (2X) when not


with 50 ml of m e t h a n o l : w a t e r mixture at 3 ml m i n " 1 . Next,


the initial flow rate of 3 ml m i n " 1 . After 10 m i n u t e s , the

flow rate was increased to 4 ml min 1 . When the back











(curve profile #6) of low concentration buffer for 10

minutes was run. This was followed by a 10 minute reverse

68

of high concentration buffer and a 10 minute hold at

initial conditions was always run.

Nucleotide samples (100 jil), prepared and obtained

from bacteria as previously d e s c r i b e d , were thawed and

injected onto the column. The Partisil SAX10 column

(10,000 plates m e t e r - 1 ; particle size, 10 n m ) was an anion


si 1oxane-bonded to Partisil 10 (Silica). The associated

counter-ion was H 2 P 0 4 . Nucleotides bind to the quaternary



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




regenerated by washing with 30 ml of 7mM NH^h^PO^ (pH=3.8)

buffer. The flow rate was maintained at 4 ml m i n " 1 and all

analyses were done at ambient temperature. Peaks were

integrated either manually on a Houston Instrument's

Omni-Scribe strip chart recorder or by an Apple lie

computer system using an ADALAB dual slope integrating A/D

converter and Chromatochart software (Interactive

Microware Inc., State College, PA). The sample peaks were

identified by comparing retention times of appropriate

standards. The concentration of the sample was calculated

69

by comparing its peak height to the standard for which the

concentration was known ( l m M ) and expressed as micromole

(g dry wt)" 1. The system was shut down by first flushing

with distilled water and then with 50:50 methanol:water in

the same manner used to start the appartus.

RESULTS

E. coli was grown aerobically at 37°C in four

different conditions: (a) Glucose minimal (M9) medium

alone, (b) M9 + uracil, (c) M9 + casamino acids and (d) M9

+ fructose. Acid soluble endogenous nucleotide pools were

extracted and measured from cells harvested from each of

these growth conditions (Fig. 11). I was interested in

determining any relation that existed between

ribonucleoside triphosphates and growth rate. Many earlier

studies (Wiebe & Bancroft, 1975; Swedes et al_., 1975) used

the AEC to measure the growth rate of natural microbial

populations. Another study identifying ATP and GTP as

parameters of growth was that of Karl (1978). In his study

on the marine bacterium, Serrati a mari norubra, Karl (1978)

discovered that intracellular GTP/ATP ratio increased in

direct proportion to the rate of cell growth; in other

words, when Karl plotted GTP/ATP ratio against generations

hour""'', a straight line was obtained. I wished to expand

the results obtained by Karl (1978) to microorganisms in

general especially those which did not lend themselves to

70

F i g . 11 — E l u t i on p r o f i l e of r i b o n u c l e o s i d e t r i p h o s p h a t e s e x t r a c t e d w i t h 6 % ( w / v ) t r i c h l o r o a c e t i c acid f r o m E s c h e r i c h i a c o l i . C o n d i t i o n s as in F i g . 5 l e g e n d .

71

<1) co o CO

c D > o c co JD v. O c/) X) CO

CO Li. D <

in o

T ime ( m i n u t e s )

72

e a s y g r o w t h r a t e m e a s u r e m e n t (such as t h e i n a b i l i t y to

f o r m c o l o n i e s ) . W h e r e a s K a r l ' s ( 1 9 7 8 ) r e s u l t s s h o w e d t h e

i m p o r t a n c e of t h e G T P / A T P r a t i o , t h e y did not m a k e g r o w t h

r a t e m e a s u r e m e n t s any e a s i e r for o r g a n i s m s s u c h as f u n g i ,

p l a n t s , e u k a r y o t i c c e l l s and b a c t e r i a t h a t did not f o r m

c o l o n i e s ( e . g . S t r e p t o m y c e s ) . I w a n t e d to f i n d out if t h e

G T P / A T P r a t i o , w h e n p l o t t e d a g a i n s t t i m e , c o u l d be u s e d as

an i n d i c a t o r of g r o w t h . T h e p u r p o s e of t h i s c h a p t e r w a s to

e x p l o r e t h i s q u e s t i o n . F i g s . 1 2 - 1 5 s h o w t h e c o n c e n t r a t i o n

in [ n m o l e (g d r y w t ) " 1 ] of A T P , G T P and G T P / A T P p l o t t e d

a g a i n s t t i m e w h e n JE. col i c e l l s w e r e g r o w n in M 9 m e d i u m

p l u s u r a c i 1 .

T h e f o l l o w i n g p o i n t s w e r e m a d e a b o u t F i g s . 1 2 - 1 5 : (a)

G T P and A T P c o n c e n t r a t i o n s i n c r e a s e d e x p o n e n t i a l l y w i t h

t i m e for all c o n d i t i o n s . T h i s c o n f i r m e d r e c e n t r e s u l t s of

M u n c h - P e t e r s e n ( 1 9 8 3 ) . ( b ) W h e n G T P and A T P w e r e p l o t t e d

as the G T P / A T P r a t i o a g a i n s t t i m e , an e x p o n e n t i a l l i n e w a s

f o u n d . (c) T h e s l o p e of t h e a b o v e l i n e , p l o t t i n g G T P / A T P

v e r s u s t i m e , p a r a l l e l e d the g r o w t h c u r v e . F i g . 16 is a

K a r l - t y p e p l o t of t h e G T P / A T P r a t i o p l o t t e d a g a i n s t

g e n e r a t i o n t i m e h o u r " ^ as a c h e c k of m y d a t a . It s h o w s

t h a t the l i n e a r i t y a p p l i e d a l s o for E_. col i g r o w n u n d e r

f o u r d i f f e r e n t g r o w t h c o n d i t i o n s .

D I S C U S S I O N

T h e c o m p o s i t i o n of t h e acid s o l u b l e n u c l e o t i d e pool

73

F i g . 12 — Re 1 a t i o n s h i p s between (a) GTP/ATP r a t i o versus t ime (b) K l e t t u n i t s versus t ime (growth r a t e ) . ATP versus t ime and GTP versus t ime ii? E s c h e r i c h i a c o l i growing in M9 + uracTI (TO" pg ml )~

74

2 0 . 4

^ifowth

3*1.0

5 0 i o o m Time (min)

75

Fig. 13--Regression plot of log GTP concentration (LGTP) versus time. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) Chicago, IL.

76

CL c/> CO LU

3-

CL b-CD CO **

O CO oo > \ IE CO < •3Z

CO O >

3E»-CD»—• •h (/) CC

QCLU o>

CNIH-< IjlJ I— CO CO < LUGO -J< UJX CKLU

I X CO I— wa: olO coz

O

CL

CM CO

co in

in CO

CO CM

OO o

o> o>

4 .. .. .. .. + .. .. •• •• + .... 4 in in in CM H CO oo CO CO

+ . . . . . . . . + . . . + . . • • • + in O) in CO in CM if) •** in

if)

o O)

o o o o

0

CO

G O ) LU —« —J o M O < o I- • 1 CSI

COO coo

«H O o o

Z CO Olulu

Q-U.C0 l-O — CD lu -J Q. LU O

LL. ' —J

Oooco

CO o o i -

o h O O ) co a> m »-h cn Ovi H- O < H-coo LU

Za: — 0 < O H D O O CO Q n CO CO CM LU O) CC Q£ O « LU OO or co in • CD

D o -

O Z C 0 -JO — C L H h

h- CL CO < LU LU-J O cOLUtt: < 0£ LU U K t -o z

( D U H

J O h Q .

77

Fig. 14--Regression plot of log ATP concentration (LATP) versus time. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) Chicago, IL.

78

O. oo

(/> UJ

3

o. h-

co <

o moo > \ 2oo <

•32

00 O

> -2 K OQM M GO

OC OC UJ o >

</) CO <

UJ00 - J<

LUX OCUJ

h-X • X

Wl-00 QC OLO 00 2

o

Q-

.. .. + .. .. • • • • + • • • • • • + in IT) in CM in CD H CO H O

H

+ • • • • .. . + • • • • + o> in CO o h- o

o

1 00 K CSI O

CO O H o

• in

o in HI H 00

CO in oaf) H UJM

-JO MO

- in < o 1- •

00 1 CM

H * • * ^ O H o>

O —• MO

m OO

O) ro UJ •H r

in M CO »-H

zoo OLUUJ

in CLU.00

o » - o -ro < UJ —« UJ -J a.

CO r ujO Csl M u. • —1 H 1- Ooooo

CO in O co -—•

H CO O H h ^ O CM oo in in H MO) CM

r^ h- O H < H b-

00 Q UJ

in Z QC —' 0 < CO

CM MZ)0) M 00OCD «—< OOOOO

oo UJ M o or a: H O i

UJ CM oc a>

in CO

CO QO)<—» o UJ • • H H- UJ

o> h-CD OZoo

-JO'—* CL •H h-

h- Q. in 00 < LU

UJ —1 o «** oo lu a: C7> < Qd LU

Oceh-O OZ o> CO O H

—J < H- CL

79

Fig. 15--Regressi on plot of GTP/ATP ratio (LGA) versus time using the Statistical Package for the Social Sciences (SPSS) Chicago, IL.

80

Q. CO \

CO >

CO

o CO oo > \

ZOO <

• a z

CO o

> X H

CD HH 1-iCO

Od ft: LU

o>

UJ CSIJ-

<

LU h-CO CO <

LU CO - » <

LU X QC LU •—

X t X

COK CO QC Q-O c o z

UJ r

< o

o -J CL

CM (O

in

CO in

in CO

<o Z (SI »-«

00 o

+ • • • • .. + .. .. • • • • + • • • • • • • + • • • • + • • • • • • • +

in in uo in in in in CM <j> in <SJ 00 l x h- ** H (D in o

i <** in i in iO

o> o

o O)

o o o

o t-4 CO

liJ *H -JO MO < o

i n s o r ) eo (VI o o o o

co • Z UJ LU o

U.C0 <o — o UJ -J o.

LU O U. -J Ococo

CO (J O) — •H -H H- <£> </) OO OO HO)H I- o <

h-COQ

UJ Z a: ^ O < LT) »—« id in co Oc\i coco en LU oo ft: cc O i lu r-ft: co

co •<j>

OO) —

O Z 0 0 -JO —' a_ H H-

h- Q-C0<LU LiJ -J O co lu a: <Q£ LU U K I -

o z CD O H

J O <

81

Fig. 16--Kar1 (1978) type plot of GTP/ATP ratio as a function of growth rate of Escherichia coli cells qrowinq in batch culture under four different conditions.

82

Q_ h-<C

QL. I— CD

Generations hr

83

of E. coli was studied with various media which support

different rates of growth. The general increase in the

nucleotide content observed during log phase is compatible

with the increase in the total soluble nitrogenous

compounds. The ATP content per cell also exhibited

approximately the same increment of change.

There is no doubt that many cellular catabolic

reactions involve the formation or hydrolysis of ATP while

many endergonic anabolic sequences are coupled to the

hydrolysis of nonadenine nucleoside triphosphates

(Fig. 17). GTP is required for activation and

interconversion of carbohydrate precursors for cell wall

biosynthesis, for RNA transcription and protein

biosynthesis. At least two molecules of GTP are hydrolysed

for each peptide bond that is formed during the

polypeptide elongation cycle of protein synthesis

(Lucas-Lenard & Lipman, 1971). It is apparent from the

data presented here that intracellular GTP concentrations

increase in direct proportion to the cellular growth rate

(doubling time) when normalized to cellular growth rate.

I have shown that when the log of the ATP or GTP

concentration is plotted against time, a straight line is

found. Neither line parallels precisely the growth rate

curve. However, when the GTP/ATP ratio is plotted versus

time, a line with a much closer fit to the growth curve

84

F i g . 1 7 - - M e t a b o l i c r e a c t i o n s i n v o l v i n g ATP and GTP,

85

ENERGY METABOLISM

-•Adenosine triphosphate

-•Adenosine Diphosphate'

Ribonucleic acid

Group-trarisfer coenzymes

Purine deoxyribonucleotiaes

Histidine

Purine deoxyribonucleotiaes

ENERGY METABOLISM

""Guanosine triphosphate

1—^Guanosine diphosphate"

Ribonucleic acid

Group-transfer coenzymes

Purine aeoxyribonucleotides

Folate

Purine deoxyr ibonucleot ides

86

(Figs. 12) is observed. Since, GTP concentration

approximately reflects protein synthesis in exponeni ally

growing cells and ATP concentration is constant at 2mM for

all cells at all growth rates (Franzen & Binkley, 1961),

it was reasonable to assume that the GTP concentration,

normalized by dividing it by ATP concentration, should

closely reflect the growth rate. This is precisely what I

found. Thus, by plotting GTP/ATP ratios versus time one

can estimate the growth rate.

Ecologically, my study should also prove useful for

measurement of biomass and biomass conversion of

heterotropica 11y growing cells. The analysis of GTP/ATP

ratios for microbial communities offers promise as a

diagnostic tool for the estimation of community growth

rate by microbial ecologists. Additionally, the GTP/ATP

ratio should provide insight into growth activity of

microbial systems in response to effects of pollutants

such as mercury, herbicides, etc. Finally, since GTP/ATP

ratio is a function of cellular growth rate, it can be

used to the measure growth rate of any cells that are

otherwise difficult if not impossible to assess.

87

CHAPTER V

TEMPERATURE DEPENDENCY AND RIBONUCLEOSIDE PRODUCTION IN YERSINIA ENTEROCOLITICA

INTRODUCTION

Y e r s i n i a e n t e r o c o l i t i ca causes a wide v a r i e t y of

d iseases in humans. Common c l i n i c a l syndromes i nc l ude

g a s t r o e n t e r i t i s , a r t h r i t i s , and mesen t r i c l ymphaden i t i s

(Bo t t one , 1977; Swaminathan et^ a_l_., 1982) . In a d d i t i o n ,

b i o c h e m i c a l l y a t y p i c a l s t r a i n s have been assoc ia ted w i t h

eye, t h r o a t , wound and u r i n a r y t r a c t i n f e c t i o n s (Bot tone

e_t £ l_. , 1974) . Y e r s i n i a spec ies share many tempera ture

dependent b i o l o g i c a l a t t r i b u t e s (Bot tone et_ aj_. , 1979) .

Among the in v i t r o b i o l o g i c a l t r a i t s inc reased by lowered

i n c u b a t i o n tempera ture (25°C) are f l a g e l l a s y n t h e s i s and

a c e t y l - m e t h y l c a r b i n o ! p roduc t i on (Sonnenw i r th , 1974) ,

e n t e r o t o x i n syn thes i s (Pai & Mors, 1978; Pai £ t aj_. , 1978;

F ree ly e_t aj_. , 1979 & Franc is £ t , 1980) , a b i l i t y to

adhere to and pene t ra te e p i t h e l i a l c e l l s (Lee et a l . ,

1977) , r e s i s t a n c e to serum b a c t e r i c i d a l a c t i v i t y ( N i l e h n ,

1973) and p roduc t i on of b a c t e r i o c i n l i k e substances

(Bot tone _et _al_. , 1979) . Y e r s i n i a spec ies a lso have a broad

range of tempera ture r e l a t e d phenotyp ic t r a i t s . Among the

l a t t e r , expressed at 25°C but not at 37°C, are growth on

va r ious e n t e r i c media (Chester and S t o t z k y , 1976) ,

88

expanded f e r m e n t a t i o n c a p a c i t y (Bo t tone et_ aj_. , 1974;

Chester & S t o t z k y , 1976) and inc reased r e s i s t a n c e to

a m p i c i l l i n , c a r b e n i c i 1 1 i n , ch lo ramphen ico l and the

aminog lycos ides (Chester & S t o t z k y , 1976) . Zink et a l .

(1980) s t u d i e s the presence of p lasmids in Y e r s i n i a grown

at 28°C.

N i lehn (1969) r epo r t ed t h a t most s t r a i n s of _Y.

e n t e r o c o l i t i c a cou ld grow in the tempera ture range of

25°C-39°C but not at 43°C. The v i a b l e count of ten of the

330 c l i n i c a l i s o l a t e s of Y_. e n t e r o c o l i t i c a , i n o c u l a t e d

i n t o raw or cooked beef or po rk , inc reased s i g n i f i c a n t l y

when s to red at 7°C f o r 10 days (Hanna et^ £l_. » 1977).

Su ther land & Varnen (1977) r e p o r t e d t h a t the growth range

of _Y. e n t e r o c o l i t i ca in n u t r i e n t b ro th was 1°C-44°C.

I t i s a l ready known t h a t in a batch c u l t u r e ,

tempera ture changes do not p rov ide changes in the con ten t

of RNA and ribosomes but do a l t e r the r a t e of p r o t e i n

s y n t h e s i s (Mandelstam e_t a_l_. , 1982) . Y_. e n t e r o c o l i t i ca

t h e r e f o r e seemed to be the organism of cho ice to s tudy the

e f f e c t of tempera tu re on the c o n c e n t r a t i o n of the

r i b o n u c l e o s i d e t r i p h o s p h a t e s . Such an i n v e s t i g a t i o n would

enable us to p rov ide an i n s i g h t i n t o the mechanisms of

tempera ture dependent e n t e r o t o x i g e n i c i t y w h i l e u n r a v e l l i n g

some of i t s e p i d e m i o l o g i c a l puzz les as w e l l .

89

METHODS

Chemi cals

Nucleotides, trichloroacetic acid and

tri-n-octylamine were purchased from Sigma Chemical (St.

Louis, MO). Ammonium phosphate-monobasic was purchased

from Mallinckrodt Inc. (Paris, KY) and 1,1,2-trichloro-

1,2,2-trifluoroethane (freon) was purchased from Eastman

Kodak Company (Rochester, NY). All other chemicals were of

analytical grade and were purchased from Fisher Scientific

Company (Fair Lawn, NJ).

Bacterial Strain

ATCC strain of Y_. enterocol i ti ca was used in these

studi es.

Growth Medium

Bacterial cells were grown in M9 minimal medium which

contained in g liter"^" of distilled, deionized water:

N a 2 H P 0 4 , 6; K H 2 P 0 4 , 3; NaCl, 0.5, and N H 4 C 1 , 1. Glucose

(0.2 per cent w/v), 2 ml 1M M g S 0 4 , 0.1 ml of 1M CaC1 2 and

casamino acids (0.4 per cent w/v) were added separately.

Growth of Bacteria

All cultures were grown at either 22°C, 30°C and 37°C

in a Lab Room Controlled Environmental Room (Labline

Instrument, Inc., Melrose Park, IL). Liquid cultures were

incubated in Klett flasks in an G10 gyratory shaker (New

90

Brunswick Sci. Co.; Edison, NJ) set at 120 revolutions

minute"*. The turbidity was measured every 20 minutes with

a photoelectric Klett Summerson colorimeter (Klett

Manufacturing Co., New York, N.Y.) using a green filter

#54 and recorded as Klett units (KU), where 1KU = 1 X 10^

cells ml~*. Volumes of 50 ml of bacterial cultures at

various stages of the exponential phase were harvested,

actual Klett units recorded and the culture was spun at

4°C at 12000 X g for 2 minutes. The supernatant was poured

off and cell pellet was used for nucleotide extraction.

Trichloroacetic Acid Extraction Procedure

One ml of ice cold 6 per cent (w/v) TCA was added to

the above cell pellet which was then vortexed for 2

minutes and allowed to stand at 4°C for 30 minutes before

further centrifugation at 12000 X g for 10 minutes. The

clear supernatant was then neutralized with ice cold

freon-amine (Khym, 1975) solution (0.7 M tri-n-octyl-amine

in freon 113, 1.06 ml amine/5 ml freon). The freon-amine

sample mixture was vortex-mixed for 2 minutes and then

allowed to separate for 15 minutes at 4°C. The top aqueous

layer, which contains the nucleotide pool extract was

removed, filtered (Gelman, ACR0 LC13, 0.45^) and frozen at

-20°C until ready for use.

91


The HPLC equipment (Waters C h r o m a t o g r a p h y Division;


Model 680 automated Gradient C o n t r o l l e r , a U6K injector

and a Model 481 LC Spectophotometer. Nucleotides were


sensitivity fixed at 0.05 AUFS. Separations were performed

on a Waters Radial PAK Partisil SAX Cartridge (0.8 cm X 10

cm) using a Waters Radial Compression Z - M o d u l e System.


The whole chromatographic system including the column

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

filtered double distilled water when not in use. The

system was flushed with 50 ml of m e t h a n o l : w a t e r mixture at

3 ml min"* after priming the pumps. Next, the system was

thoroughly washed with water keeping the initial flow rate

at 3 ml min~*. After 10 m i n u t e s , the flow rate was

increased to 4 ml min""''. When the back pressure of the

column dropped to 850 pounds per square inch, the 50:50

m e t h a n o l : w a t e r 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 c h l o r i d e , pH 4.5). The LC

92

S p e c t r o p h o t o m e t e r was s e t a t 254 nm and 0.05 AUFS,

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 m inutes. A 10

minute reverse gradient of high concentration buffer was

run followed by a 10 minute hold of low concentration

buffer.

Nucleotide samples (100 pi) obtained from bacteria

were injected onto the column. The Partisil SAX10 column

(10,000 plates m e t e r - 1 ; particle size, 10 pm) is an anion


si 1oxane-bonded to Partisil 10 (Silica). The counter-ion

associated with it is H 2 P 0 4 . The 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 C-5 1 of the sugar. A linear

gradient (curve profile #6) of low to high concentration

buffer was applied for 20 m i n u t e s , followed by an

isocratic period of 10 minutes of high concentration

buffer. The column was regenerated by washing with 30 ml

of 7mM N H 4 H 2 P 0 4 (pH=3.8) buffer. The flow rate was

maintained at 4 ml m i n " 1 and all analyses were done at

ambient temperature. Peaks were integrated either m a n u a l l y

on a Houston Instrument's OmniScribe strip chart recorder

or by an Apple lie computer system using an ADALAB dual

93

slope integrating A/D converter and Chromatochart software

(Interactive Microware Inc., State College, PA). The

sample peaks were identified by comparing retention times

of appropriate standards. The concentration of the sample

was calculated by comparing its peak height to the

standard for which the concentration was known (1 mM) and

expressed as micromole (g dry wt)~^. The system was shut

down by first flushing with water and then with 50:50

methanol:water in the same manner used to start the

apparatus.

RESULTS

In Chapter III, the effect of stress caused by uracil

starvation on a Pyr" (uraci1-requiring) mutant strain of

E. coli was investigated. In this chapter, the naturally

temperature-sensitive organism, Y_. enterocol i ti ca, was

used to study similar effects of stress caused by growth

at a non-permissive temperature and by growth in a medium

that was deficient in nutrients for Y_. enterocol i ti ca

(since, Y_. enterocol i ti ca requires a highly enriched

growth medium such as Brain Heart Infusion Agar for

optimal growth). I observed a huge increase in ATP and GTP

when I starved the Pyr" E_. coli strain for uracil. I

wanted to observe the effect of temperature stress on a

Pyr" strain of Y_. enterocol i ti ca.

A typical chromatogram of the endogenous

94

ribonucleotides isolated from Y_. enterocol i ti ca is seen in

Fig. 18. The effect of growth of Y_. enterocol i ti ca was

examined at different temperatures (Figs. 19 & 20). When

the concentration of nucleoside triphosphates was plotted

against time there was a linear increase in all nucleoside

triphosphates at 22°C, the optimum temperature for Y_.

enteroco1itica. However, when the concentration of

nucleotide was plotted against time for growth at 30°C and

37°C, there was a profound change in the nucleoside

triphosphate pools after a brief initial linear response.

ATP, GTP and UTP increased sharply in the late exponential

phase (Figs. 19 & 20). The CTP concentration did not

increase at 30°C or at 37°C. It should be pointed out that

the medium chosen for growth of Y_. enterocol i ti ca was

limiting and was not optimal as I wished to study the

organism under nutrient stress as well as under

temperature stress to parallel the uracil starvation seen

in Chapter III.

DISCUSSION

When a pyrimidine auxotroph of E_. coli was starved

for uracil there was a 10 fold increase in the

concentration of ATP and GTP. Because starvation was for

uracil, there was no increase in UTP and CTP. This was the

expected result. In this study, Y_. enterocol i ti ca, a

naturally temperature sensitive organism, was starved for

95

Fig. 18--Elution profile of ribonuc1eoside triphosphates for Yersinia enterocolitica. Conditions as in Fig. 5 legend.

96

d) 03 o (/>

c 3 >

o c co n V-o CO -Q co

CO u. D <

to o

"2V "2I5" "3o~ 10 Time ( m i n u t e s )

97

Fig. 19--Effect of indicator growth temperature on the concentration of (a) UTP and (b) CTP in Yersinia enterocoli ti ca.

98

(a) UTP

Time (min)

(b CTP

100 200 300 Time (min)

99

Fig. 20--Effect of indicator growth temperature on the concentration of (a) ATP and (b) GTP in Yersinia enterocolitica.

100

(a) ATP

(b) GTP

L31

Time (min)

>

a 2| U)

<D

O 1-

30°C 37°C

/

"20D 3 S 0 -

Time (min)

101

multiple nutrients by (a) growth at optimal temperature

(22-25°C) and (b) growth in a medium that was limiting for

required nutrient. The results seen in Figs. 19 & 20,

suggested that there was balanced growth at 22°C even in

the limiting medium. At 30°C and 37°C, the concentration of

triphosphates increased rapidly after a brief period of

linearity. This suggested that the Y_- enterocol i ti ca was

under stress when grown at 30°C and 37°C. Indeed ATP, GTP

and UTP concentration did increase dramatically.

Surprisingly, the CTP concentration did not increase

at 30°C or at 37°C. This helped to explain a previously

published anomalous result obtained by Wild et al_. (1980)

where the effect of CTP on aspartate transcarbamoylase

(ATCase) from Y_« enterocol i ti ca was studied. In all

enteric bacteria studied to date CTP was a potent feedback

inhibitor of ATCase. In _Y. enterocol i ti ca, there appeared

to be no effect. If as I have observed here that CTP

concentration is very low in Y_. enterocol i ti ca when grown

at 30°C, the temperature of assay by Wild et_ jil_. (1980),

the lack of feedback inhibition seems quite reasonable.

Indeed UTP, present in significant concentration at 30°C,

and not CTP, was found to be the inhibitor.

CHAPTER VI

SUMMARY

The pu r i ne and p y r i m i d i n e d_e novo pathways are

u n i v e r s a l in n a t u r e . Products of these pathways are the

immediate s u b s t r a t e s f o r RNA s y n t h e s i s and u l t i m a t e l y f o r

DNA syn thes i s ( F i g s . 1 & 2 ) . E x p o n e n t i a l l y growing c e l l s

must con ta i n a balanced supply of the f ou r r i b o n u c l e o s i d e

t r i p h o s p h a t e s , UTP, CTP, ATP and GTP, whose p r e c i s e

measurement i s needed to assess the p h y s i o l o g i c a l s t a t e of

the c e l l . HPLC i s the c u r r e n t method of cho ice f o r

s e p a r a t i n g the q u a n t i f y i n g n u c l e o t i d e s and i t has been

used f o r t h a t purpose th roughout t h i s i n v e s t i g a t i o n .

The c u r r e n t s t a t u s of the problem i s desc r ibed in the

I n t r o d u c t i o n (Chapter I ) . In measuring n u c l e o t i d e s w i t h

HPLC a new s o l v e n t system, namely t r i f l u o r o a c e t i c a c i d ,

was d i s cove red . I t i s s u p e r i o r to e x i s t i n g s o l v e n t systems

such as t r i c h l o r o a c e t i c ac id and f o rm ic ac id (Chapter I I ) .

When Pyr" E s c h e r i c h i a c o l i c e l l s were s ta rved f o r u r a c i l a

1 0 - f o l d inc rease in ATP and GTP occur red (Chapter I I I ) .

When GTP/ATP r a t i o s were p l o t t e d aga ins t t i m e , a s t r a i g h t

l i n e was formed. Thus GTP/ATP r a t i o s can be used as a

measure of growth r a t e . Yers i n i a e n t e r o c o l i t i ca was used

as a tempera ture s e n s i t i v e organism to s tudy the e f f e c t of

growth at a non-perm iss i ve tempera ture on n u c l e o t i d e pools

(Chapter V ) .

102

103

From these HPLC studies, the following conclusions

can be made: (a) Trif1uoroacetic acid is a superior

nucleotide solvent for nucleotide extraction; (b)

Starvation of a Pyr" mutant for uracil causes increased

production of ATP and GTP and thus offers a new technology

for the production of adenylates and guanylates

industrially; (c) GTP/ATP ratios taken at different times

can be used to measure growth in organisms that do not

readily lend themselves to easy growth measurement and

(d) Growth of Y_. enterocol i ti ca at non-permissive

temperatures can be used to evaluate nucleotide

perturbations in the control of the pyrimidine pathway.

1 0 4

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