379

QUANTITATION OF ENDOGENOUS NUCLEOTIDE

POOLS IN Pseudomonas aeruginosa

THESIS

Presented to the Graduate Council of the

University of North Texas in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Mohammad Entezampour, B. S.

Denton, Texas

August, 1988

Entezampour, Mohammad, Quantitation of Endogenous

Nucleotide Pools in Pseudomonas aeruginosa. Master of

Science (Biology),, August, 1988, 54 pp., 7 illustrations,

7 tables, bibliography, 43 titles.

Nucleotide pools were extracted and quantified from

Pyr+ and Pyr~ strains of P. aerucjinosa. Strains were grown

in succinate minimal medium with and without pyrimidines,

and nucleotides were extracted using trichloracetic acid

(TCA; 6% w/v). The pyrimidine requirement was satisfied by

uracil, uridine, cytosine or cytidine. Pyr~ mutants were

starved for pyrimidines for two hours before nucleotide

levels were measured. This starvation depleted the

nucleotide pools which were restored to wild type levels by

the addition of pyrimidines to the medium. When the

pyrimidine analogue, 6-azauracil, known to inhibit OMP

decarboxylase, was added to cultures of the wild type

strain, the uridine and cytidine nucleotides were depleted

to near zero. Thus, the nucleotide pool levels of

Pseudomonas strains can be manipulated.

TABLE OF CONTENTS

List of Tables.......................

List of Illustrations . . . . . . . .

Chapter

I. INTRODUCTION . .. 0.. .. 0.. .

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

Chemicals and ReagentsBacterial StrainsGrowth Media and CulturesExtraction of NucleotidesChromatographic ApparatusChromatographic ConditionsIdentification and Quantitation

of NucleotidesCalculations

III. RESULTS.............. . . ....

IV. DISCUSSION - - -

BIBLIOGRAPHY................

iii

Page..iv

.. v

.. 0. & 1

14

22

46

51

-~~~~~~ -0 . . . . . .

LIST OF TABLES

Table

I. Genotypes and Phenotypes of PyrimidineAuxotrophic Mutants in Bacteria . ..0.

II. Identification of the Twelve NucleotideStandards Used in This Work . . . . .

III. Nucleoside Trighosphate in umol (gramdry weight) from P. aeruginosaPAO-1 Wild Type Strain in the Presenceand Absence of the Listed Additions .

IV. Nucleoside Triphosphates in umol (gramdry weight) -l from P. aeruginosa pyrBMutant in the Presence of Uracil,Cytosine and Cytidine.............

V. Nucleoside Tri hosphates in umol (gramdry weight)' from P. aeruginosa pyrFMutant in the Presence and Absence ofUracil, Cytosine and Cytidine......

VI. Nucleoside Triyhosphates in umol (gramdry weight) from P putida PPNWild Type Strain in the Presence of theListed Additions

VII. Nucleoside Triyhosphates in umol (gramdry weight)~ from P putida pyrBMutants in the Presence of Uracil,Cytosine, and Cytidine . . . . . . . .

Page

7

13

. . . 36

. . . 38

. . . 41

. . . 43

45

iv

LIST OF ILLUSTRATIONS

Figure Page1. Pyrimidine nucleotide biosynthetic and

salvage pathways in Escherichia coliand Salmonella typhimurium...........3

2. Schematic diagram representing theextraction procedure forribonucleotides from P. aeruginosa . . . 17

3. Chromatograph of the 12 ribonucleotidestandards..................-............ 24

4. Elution profile of the ribonucleotidesextracted with 6% (w/v)trichloroacetic acid from P.aeruginosa ...................... 26

5. Growth curves for P. aerucinosa PAO-1wild type strain in succinate minimal(SM) medium.......................... .28

6. Growth curve for P. aeruginosa pyrBstrain in succinate minimal (SM)medium at 370C with and withoutpyrimidines.-............................. 30

7. Growth curve for P. aeruginosa yrF strainin succinate minimal (SM) mediumat 370C with and without pyrimidines . . 32

v

CHAPTER I

INTRODUCTION

The pyrimidine biosynthetic pathway is universal with

the same sequence of reactions (Fig. 1) found for every

organism (O'Donovan & Neuhard, 1970). The first step in the

pathway catalyzed by the enzyme carbamoyl phosphate

synthetase (E C 2.7.2.5; CPSase) forms carbamoyl phosphate

from bicarbonate, the amide group of glutamine and ATP

(Anderson & Meister, 1966). Carbamoyl phosphate is used to

synthesize both arginine and pyrimidines in approximately

equal amounts (Abdel, .etal., 1969). For pyrimidine

biosynthesis, carbamoyl phosphate condenses with aspartate

to form carbamoyl aspartate and inorganic phosphate (Gerhart

& Pardee, 1962). This step is catalyzed by the enzyme

aspartate transcarbamoylase (E C 2.1.3.2; ATCase). For

arginine biosynthesis, ornithine and carbamoyl phosphate

combine to form citrulline and inorganic phosphate. This

step is catalyzed by ornithine transcarbamoylase (E C

2.1.3.3; OTCase) (Isaac & Holloway, 1972). The third

step in the pyrimidine pathway, catalyzed by dihydroorotase

(E C 3.5.2.3; DHOase), converts carbamoyl aspartate to

the cyclic dihydroorotate by the removal of water

(Lieberman & Kornberg, 1953). Dihydroorotate dehydrogenase

(E C 1.3.3.1; DHOdehase) produces orotate in the fourth

1

2

Fig. 1. Pyrimidine nucleotide biosynthetic and salvagepathways in Escherichia coli and Salmonella typhimurium.Abbreviations are: U, uracil; C, cytosine; UR, uridine; CR,cytidine; OMP, orotidine 5'-monophosphate; UMP, uridine 5'-monophosphate; UDP, uridine 5'-diphosphate; UTP, uridine 5'-triphosphate; CMP, cytidine 5'-monophosphate; CDP, cytidine5' -diphosphate; CTP, cytidine 5' -triphosphate.

SALVAGE PATHWAYS- N

de novo PATHWAYS - --

CTP

PyIG CD P SALVAGE PATHWAYS

UTP CMP

I CR4 f C

I-

pyrH

LUP

pyrFt

CMP

Orotate

pyrD

Dihydroorotate

UR

U 44

tc ~

- UR

- U

- C

CELL.MEMBRANE

Q Ornithine

CarbamoylAspartate

D

pyrB fTCaseAspartate

Carbamoyl- (Z)pyrA Glutamine

ATPHCO3

3

3

Arginine

Arginino-succinate

Citrulline

wft

AOOOOOO

N%%6*1ft .

.6

4

fourth step (Lieberman & Kornberg, 1953). Orotate and

phosphoribosyl pyrophosphate (PRPP) combine to form the

first pyrimidine ribonucleotide, orotidine-5-monophosphate

(OMP) (Hurlbert and Reichard, 1955) in a reaction catalyzed

by orotate phosphoribosyl transferase (EC 2.4.2.10;

OPRTase). Inorganic pyrophosphate is also a product. The

final step on uridine-5'-monophosphate (UMP) biosynthesis is

the decarboxylation of OMP to form UMP (Lieberman et al.,

1955). This sixth step is catalyzed by OMP decarboxylase

(EC 4.1.1.23; OMP decase). The same workers (Lieberman et

al., 1955) showed that the UMP is now converted to UTP in

two kinase steps: first by the highly specific UMP klinase

(EC 2.7.4.4) to form UDP, and then UTP is converted to UTP

by the non-specific nucleoside diphosphate kinase (EC

2.7.4.6; NDKase). Finally UTP is aminated to produce CTP

(Lieberman, 1955) by the enzyme CTP synthetase (EC 6.3.4.2;

CTPSase).

The pathway in Escherichia coli is controlled at

three sites at the level of enzyme activity by feedback

inhibition and by activation. Specifically (i) CPSase is

feedback inhibited by UMP and activated by ornithine

(Pierard, 1966); (ii) ATCase is feedback inhibited by CTP

and activated by ATP (Gerhart & Pardee, 1964); and (iii)

CTP synthetase is inhibited by CTP and activated by UTP

(Long & Pardee, 1967). The E. coli pyrimidine pathway is

also controlled at the level of enzyme synthesis. When E.

5

coli is grown in uracil or other pyrimidines, such as

uridine, cytidine, or cytosine, all enzymes of the pathway

are repressed. Thus, when E. coli is grown in minimal

medium (without uracil) ATCase specific activity is four

times greater than when the cells are grown in minimal medium

plus uracil (Kelln et a., 1975). Table 1 shows the

genotypes of wild type and of mutant strains that lack

pyrimidine enzymes in bacteria. All of these enzymes are

found in E. coli, Salmonella typhimurium and in Pseudomonas

aeruginosa, the organism being studied in this research.

However, there are some major differences in the

pyrimidine pathway of Pseudomonas from the pathway in E.

coli. For instance it has not been possible to repress the

synthesis of the pyrimidine enzymes by growth on exogenous

pyrimidines such as uracil, uridine, cytosine or cytidine

(Condon et al., 1976). The pathway is controlled by

feedback inhibition by UTP rather than by CTP. Indeed UTP

is the primary effector of ATCase in P. aeruginosa (Isaac &

Holloway, 1968; Neumann & Jones, 1964). Mutants exist in

pyrB, pyrD, pyrE and pyrF of P. aeruginosa pathway. These

will be examined in this present study. When Pyr~ mutants of

E. coli are grown in uracil and then starved for uracil, the

enzymes of the pathway become derepressed over 100-fold

(i.e., their specific activity increases over 100-fold). At

the same time the concentration of nucleoside triphosphates,

UTP and CTP, are depleted and drop to near zero levels

6

Table I. Genotypes and Phenotypes of Pyrimidine AuxotrophicMutants in Bacteria.

7

C

*rr4

'H

C

0 0 4

0

.4 0

0) p

0 -

-r4 r- r- r-4 r- r- r- q.4 r-H 0 0 0 u Q U u

4 o o o c e o41 cz

E-.4 o OPCd

0 W EH

..H 0 CnM

HH .. p 0 4 O -4-M4P4

cd 04-1 0

En 0 4) (1)

60 Oco 0U) MCd 4) 44 (1

0X o 0 '-d P-r4 4-J r-4 4) P 4

to 4) 0 r- HO E"Ag P >-'HC

'H U ) QO0r-4 oOPL4 0co TI O 0nH0- > ON % - -'P

4JH d fr- 1 -4J0 C4) U

> 0 Cdo En-* 4 Z o r-4 (1) a

04 4-) 0 cd J04CU E-

Q z p d Mdm ><|o4J4-)O0 0O

0 4 r-4 (1) p p p lC0-A >- 4-) 0 0 C 1

4J 0 0 '0 0 '0 0 0

H, P.N P4 z 0 P644m

0 CO 1< -A - P Z H P

0 0 0 u

00 aaaa

0

8

(Kelln et al., 1975). When uracil is restored to the

culture, the UTP and CTP pools expand to wild type levels

and the enzymes are no longer derepressed (Kelln et al.,

1975). Whey Pyr~ mutants of P aeruginosa are starved for

uracil there is a slight increase (2-fold) in ATCase

specific activity but it has not been determined if UTP and

CTP decreased. In this investigation the levels of UTP and

CTP (as well as other nucleotides) are examined in wild type

nad in Pyr~ mutants of P. aeruginosa and P. putida under

different conditions of growth in order to ascertain the

levels of nucleotides.

Nucleotides are important compounds for the synthesis

of macromolecules in all living cells. Nucleotides are

phosphorylated, heterocyclic compounds classified under two

major groups known as purines and pyrimidines. Two purine

bases, adenine and guanine, and three pyrimidine bases,

uracil, thymine and cytosine are the major nitrogenous bases

for nucleotide make up. The ribonucleoside triphosphates,

uridine triphosphate (UTP), cytidine triphosphate (CTP),

adenosine triphosphate (ATP) and guanosine triphosphate

(GTP) have different important functions within the cell.

These include (a) control of biosynthetic pathways; (b)

synthesis of RNA UTP, CTP, ATP and GTP) and DNA (dTTP, dCTP,

dATP and dGTP); (c) nucleotides are carried of phosphate and

pyrophosphate in several enzymatic reactions involved in the

transfer of chemical energy; (d) and serve as coenzyme-like,

9

energized carriers of specific types of building block

molecules. Several techniques have been developed (Chargeys

& Davidson, 1955) for the separation and quantification of

nucleic acids and related substances (a) enzymatic analysis

where specificity of enzymes is used to determine the

presence of specific substances (Strehler & McElroy, 1957)

(b) paper electrophoresis where nucleic acids and related

compounds, due to a variety of ionizable groups can be

separated and their concentrations measured according to

differences in net charge of the different species at given

pH values; (c) planar chromatrography where the stationary

phase assumes a planar arrangement in the form of a sheet or

thin film and the mobile phase migrates by means of

capillary action, separating the various species according

to their differential solubilities. Planar chromatography

includes thin layer chromatography and paper chromatography;

(d) column chromatography where the stationary phase is

either a solid packing material, a solid support that is

coated with a sorbent latex or gel, any of which can be

packed into a tube, a migrating liquid phase serves to

dissolve the various substances according to their

differential solubilities. Gas chromatography, ion-exchange

chromatography and liquid chromatography are three forms of

column chromatography. Classical liquid chromatography has

been improved by the addition of high pressure on the moving

phase (HPLC). In recent years, the measurement of

10

endogenous ribo- and deoxyribonucleotide concentrations has

been revolutionized by the development of HPLC (Glaser,

1986). The two most commonly employed separation techniques

involve either reverse-phase chromatography on

octyldecylsilica resins (Hoffman & Liao, 1977; Horvath,

Melander & Molnar, 1977) or ion-exchange chromatography on

microparticulate anion-exchange resins (Hartwick & Brown,

1975; McKeag & Brown, 1978). Even though reverse-phase

chromatography provides greater sensitivity and more rapid

analysis compared to any ion-exchange chromatographic method

currently available, it lacks the selectivity necessary for

the analysis of the many closely related nucleotides present

in cell extracts (Reiss, Zuurendonk, & Veech, 1984).

In our laboratory, HPLC is employed simply because

efficient, rapid and reliable quantitative nucleotide

separations are needed. In the present study and in

previous studies, Partisil 10 SAX columns were used. These

are strong anion-exchange columns which are packed with

particles 10um in mean size. Because of the sensitivity of

these microparticle packings to various chemicals, the

extraction procedure is crucial not only in attaining

optimal sensitivity and column efficiency but also in

protecting the column for maximal life (Hartwick & Brown,

1975). It has been found that even trace amounts of

perchlorate may bring about rapid deterioration of this kind

of column. In addition to this, neutralization of

11

perchloric acid with tris (hydroxymethyl) aminoethane which

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

extracts for use with some columns, cannot be used with

these packings (Brown & Miech, 1972). On the other hand,

multiple ether extractions are time-consuming and small

amounts of nucleotides may be partitioned into the ether-

water interphase causing loss of some nucleotides and lower

total nucleotide values on analysis. The extraction

procedure described by Khym (Khym, 1975) is the best studied

for use with these microparticle, chemically bonded columns.

Trichloroacetic acid (TCA) is used to extract the

nucleotides from cellular material and is subsequently

removed from cell extracts with a Freon-amine solution.

The objective of the present investigation was to

compare the profiles of 12 acid-soluble ribonucleotides

(Table II) adenosine 5'-triphosphate (ATP), adenosine 5' -

diphosphate (ADP), adenosine 5' -monophosphate (AMP)

guanosine 5' -triphosphate (GTP), guanosine 5' -diphosphate

(GDP), guanosine 5' -monophosphate (GMP), uridine 5' -

triphosphate (UTP), uridine 5' -diphosphate (UDP), uridine

5' -monophosphate (UMP), cytidine 5' -triphosphate (CMP),

cytidine 5' -diphosphate (CDP), and cytidine 5' -

monophosphate (CMP) during the growth of P. aeruginosa on

succinate minimal medium.

12

Table II. Identification of the 12 NucleotideStandards Used in this Work by Their Retention Times(Minutes).

oInr W

gc oNwwrk- NONr- 0- NSNS N m* qcS q 0 0

E-

C-4H

0

E-4

44H0

0

H

HHd

13

HH

111

0 -H

Or

H4-)

00

0-H

S-

0

4-)

OH

0 0

04 N NG

04-) 4.)00- 0 0 4J 00 04- J0

Cdi4,c m Ci4-),4J(,SQ4 40 4 0)-P S Q 4) 4-) Wd 4-)

0 4) 0 4 - U) Q004 - ,,0 (4 w (Cl . 0 o cmo 40En00 ~ 0 Q4U)W004WO0 M10

, 04040 M 400 0 o 013404 0 0 04, 4 O 04 0.,0 9 .34 040 0.4 04,, 0-H -HZ 0 0 0 0 9-H- -H.- 4 50 M 9 0 cT$-T$ J 40 0 ,d F -)--P-

r: 0- I 4 II I I O I LO LO 1 0 I I)O

UC) o- % %bLC(O1 LnOO)LO OO) 4 U 00(1) (

-H M) U)M -HU U) E0 -H M)tUFd 0 -H 0-HFe 0 0 -H 0 0-H 9 F0 Fe-H 99Fe-H 94- 0-d -H -)0 4d - - 0()0 0 S 5 0 0 - 0 5

-H Cc oO-8OC %- CC# 0-8 M-dy & -Sy&M

OS-0

0

L0

L O

FeC.

rzie

0

4-)

H l

MO0 o4 -H

-)P0

00 r

100r4-i-P

0 0

0 004

0 0

0 0

z zSQSQ

-'C

CHAPTER II

MATERIALS AND METHODS

Chemicals and Reagents

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

octylamine were purchased from Sigma Chemical Company (St.

Louis, Missouri); monobasic ammonium phosphate from

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

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

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

analytical grade and were purchased from Fisher Scientific

Company (Fair Lawn, New Jersey).

Bacterial Strains

Pseudomonas aeruginosa PAO and P. putida PPN wild types

and their Pyr~ mutants pyrB, pyrD, pyrE, and pyrF were

obtained from Professor Bruce W. Holloway, Department of

Genetics, Monash University, Clayton, Victoria, Australia.)

The pyrB mutant strain is the only pyrimidine mutant

available for P. putida.

Growth Media and Cultures

Bacterial cells were grown in Basal Minimal Medium

(Cohen-Bazire, Sistrom, & Stanier, 1957) containing the

following chemical composition in grams per liter of

deionized water: Na2 HPO4 , 7.1; KH2 PO4 , 6.8; (NH4 )2So4 ,

14

15

nitrilotriacetic acid, MgSO4 , 28.9; CaCI2 , 6.67;

(NH4 )6Mo7O 2 4 .4H2 0, 18.5; ZnSO4 . 7H2 0; FeSO4 . 7H20; MnSO4 .

H2 0, CuSO4 . 5H20, Co(NO3 )2 . 6H2 0, Na2 B407.1 0H 2 0, (Hutner's

metals '44'.) was used with succinate as the carbon and

energy source. Pyr~ mutants were supplemented with uracil at

50 ug/ml. These were periodically checked for purity on

basal minimal agar plates. Gram stain tests and other

appropriate morphological observations were made. All

experiments were started by streaking the organism on basal

minimal agar. Cells of P. aeruginosa from a single,

isolated colony were inoculated into basal minimal liquid

medium and incubated on a rotatory shaker at 370C and

agitated at 90 revolutions per minute. P. putida strains

were grown at 300C. Overnight cultures was transferred to a

fresh basal medium and grown to about 100 KU. Growth was

measured by turbidity with a Photoelectric Klett Summerson

Colorimeter (Klett Manufacturing Co., New York, N.Y.) using

a green filter #54 and recorded as Klett Units (IKU=107/ml).

The logarithms of the KUs were plotted against time.

Extraction of Nucleotides

Volumes of 50 ml of bacterial cultures were harvested at

about 100 KU, centrifuged at 40C at 12,000 x g for 5

minutes. After decanting the supernatant, nucleotides were

extracted from the cell pellet according to the scheme shown

in Figure 2. One ml of ice-cold 6% (w/v) trichloroacetic

16

Fig. 2. Schematic diagram representing the extraction

procedure for ribonucleotides from P. aeruginosa cultures.

17

Bacterial Culture

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

Supernatant Cell pellet

Add TCA (6% w/v)Mix by vortexingAllow to stand for 30 minCentrifuge at 12,000 X gfor 15 min at 40C

F- -+

Pellet Supernatant

Add Freon-amine,vortex, and stand

Bottom Top AqueousLayer Layer contains

nucleotides

Filter

Store at -200Cfor analysis

.4&" ilw- 444t w-

18

acid (TCA) was added to the cell pellet, which was then

thoroughly mixed for 2 minutes in the vortex mixer. The

mixture was allowed to stand at 40C for 30 minutes before

centrifuging at 12,000 x g for 15 minutes. The clear

supernatant was then neutralized with ice cold Freon-amine

(Khym, 1975) solution (1.06 ml of 0.7 M tri-n-octylamine in

5 ml of Freon 113). The sample was then mixed on the vortex

mixer for 2 minutes then allowed to separate for 15 minutes

at 40C. The top, aqueous layer, which contained the

nucleotides, was removed, filtered through a 0.45 um ACRO

LCl3 filter (Gelman Sciences, Ann Arbor, Michigan) and

frozen at -200C until ready for analysis.

Chromatographic Apparatus

The HPLC equipment (Waters Assoc., Milford,

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

automated gradient controller, a U6K injector, and a Model

481 LC spectrophotometer. Nucleotides were detected by

monitoring the column effluent at 254nm with a sensitivity

fixed at 0.05 absorbance units at full scale deflection

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

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

radial compressing Z-Module system.

Chromatographic Conditions

The entire chromatographic system including the column

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

19

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

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

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

system was thoroughly washed with distilled water with the

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

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

pressure of the column dropped to 850 pounds per square

inch, the methanol:water mixture was completely washed from

the system. Pump A was flushed with starting buffer

(filtered ultra pure, 7 mM monobasic ammonium phosphate, pH

3.8) followed by pump B which was flushed separately with

final buffer (filtered, 250 mM monobasic ammonium phosphate

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

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

recorder and automated gradient controller were turned on.

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

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

minute reverse gradient of high concentration buffer was

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

buffer.

Identification and Quantitation of Nucleotides

Nucleotide samples of 200 ul each were prepared from

Pseudomonas cells as previously described. These were thawed

and injected onto a column of Partisil SAX-10 which

consisted of a silica matrix coated with porous

20

microparticles of silica of 10 um particle size. The

particles contained fixed-charge quaternary nitrogen groups

and mobile counterions of H2PO4 . Nucleotides bind to the

quaternary nitrogen group with different affinities because

of the functional groups in the bases and the number of

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

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

applied for 20 minutes, followed by an isocratic period of

10 minutes of high concentration buffer. The column was

regenerated by washing with 30 ml of 7mM NH4 H2 PO4 , ph 3.8

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

maintained at 4 ml per minute and all analyses were done at

ambient temperature. Peaks were integrated using a Waters

Data Module 740 attached to a microprocessor. The sample

peaks were identified by comparing their retention times

with those of appropriate standards. The concentration of

nucleotides in samples was calculated by comparing peak

heights to standards (ATP, ADP, AMP, GTP, GDP, GMP, UTP,

UDP, UMP, CTP, CDP, and CMP) of known concentration (0.01

mM) and expressed as nmoles per 108 cells. The system was

terminated by first flushing with distilled water and then

with the methanol-water mixture in the same manner as used

to start the apparatus.

21

Calculations

Moles (gram dry weight)~1 for all nucleotides were

computed as follows:

Sa V 1

X C X XSt Vi Dw

Where Sa = peak height of sample, St = peak height of

standard, C = grams compound in standard/molecular weight of

compound, V = total volume of extract, Vi = volume of

extract injected and Dw = dry weight (Dutta & O'Donovan,

1987).

CHAPTER III

RESULTS

The purpose of this research was to ascertain if the

nucleoside mono-, di-, and triphosphates could be altered in

Pseudomonas strains. To find the answer to this question,

wild type P. aeruginosa, strain PAO-l, and two P. aeruginosa

PAO-1 mutant derivatives namely yrB and pyrF, were

employed. Strains were grown in succinate minimal medium in

the presence and absence of uracil, uridine, cytosine and

cytidine. In addition, the wild type strain was grown in

the presence of 6-azauracil, a pyrimidine analogue known to

inhibit the enzyme OMP decarboxylase in E. coli thereby

starving the cells for pyrimidines. All P. aeruginosa

cultures were grown under identical conditions on a rotary

shaker water bath at 370C. Bases and nucleosides were added

at the rate of 50 ug/ml and pyrimidine auxotrophs were

starved for pyrimidines for two hours at which time samples

were taken. Typically an experiment was carried out as

follows: 100 to 200 ml of succinate minimal medium were

inoculated with the culture to be tested in the presence (at

50 ug/ml) or absence of appropriate additions. The cells

were grown to a density of 100 Klett units (109/ml). Fifty

ml samples of culture were then harvested and treated with

6%(w/v) trichloroacetic acid to extract the nucleotides as

22

23

Fig. 3. Chromatogram of the 12 ribonucleotidestandards. The numbers refer to the compounds listed inTable II.

24

0

.00

cz 2

co

C

D 34t[[4 7

56 8 1

imUe ( nU.s

Time (minutes)

25

Fig. 4. Elution profile of ribonucleotides extractedwith 6% (w/v) trichloroacetic acid from P. aeruginosa. Thenumbers refer to the compounds listed in Table II.

26

(T

Cu0

C

.0CLL

9 110

Time (minutes)

II

27

Fig. 5. Growth curves for P. aeruginosa PAO-1 wildtype strain in succinate minimal (SM) medium at 370 C with

and without the noted additions. 6-Azauracil was added to

the growing culture in SM medium at 90 KU. Symbols andabbreviations are: SM+CAA+U, casamino acid + uracil (0),

SM+U, uracil (a) , SM+C, cytosine (A), SM+CR, Cytidine, (U),

SM+UR, uridine, (a) SM+AzaU, 6-azauracil (A).

28

S.102 A

10

10100 100 200 300 400 500 600

Time (min)

29

Fig. 6. Growth curve for P. aeruginosa pyrB strain inthe succinate minimal (SM) medium at 370 with and withoutpyrimidines. When the cultures reached 100 KU they werestarved for pyrimidines for two hours. Symbols andabbreviations are as in Fig. 5.

0

10

0 400

Time (min)

30

+

200 600 800

a

-I

31

Fig. 7. Growth curve for P. aeruinosa pyrF strain inthe succinate minimal (SM) medium at 37 C with and withoutpyrimidines. When the cultures reached 100 KU they werestarved for pyrimidines for two hours. Symbols andabbreviations are as in Fig. 6.

32

C 10

10

100

0 . 200 400 600 800

Time (min)

33

as described in Materials and Methods section. Nucleotides

were quantitated by HPLC.

Growth curves for the P. aeruginosa wild type PAO-1

strain are shown in Fig. 5 where the log of the Klett Units

(KU) is plotted against time. As can be seen from Fig. 5,

the PAO-l strain grows equally well on succinate minimal

medium and on all supplements. When 6-azauracil was added

(denoted by the arrow in Fig. 5) growth ceased. After one

hour in 6-azauracil a sample was taken for HPLC analysis of

nucleotides.

Growth curves for the P. aeruginosa pyrB strain are

shown in Fig. 6. A previous calculation indicated the

amount of exogenous pyrimidine base or nucleoside that was

needed for the auxutroph to reach 100 KU. This is seen in

Fig. 6 for uracil and uridine, which grow exponentially to

about 100 KU before running out of pyrimidine nutrient.

After two hours of such starvation, samples were taken.

Growth on cytidine was poor as reflected by the slower

growth rate as seen in Fig. 6.

Growth curves for P. aeruginosa pyrF are shown in Fig.

7. In this figure the results are identical to those seen

in Fig. 6. Once again growth on cytidine was poor.

Ribonucleoside mono-, di-, and triphosphates were

measured for each of the growth conditions described in

Figures 5, 6, and 7. Profiles of the 12 ribonucleoside

standards, UMP, CMP, AMP, GMP, UDP, CDP, ADP, GDP, UTP,

34

CTP, ATP AND GTP are shown in Fig. 3. Since growth is

reflected best by the concentration of triphosphate, in this

thesis only the nucleoside triphosphate levels are shown.

Table III shows the nucleoside triphosphates for the

different conditions of growth of the wild type P.

aeruginosa strain PAO-l. As can be seen from Table II the

overall levels of the triphosphates in P. aeruginosa are

similar to those reported earlier for Salmonella typhimurium

by Kelln et al., 1975. When uracil or uridine was added to

the growth cultures, the UTP level increased two-fold from

5.23 umol/(gram dry weight)~ 1 to 10.0 umol(gram dry

weight)-1.

One extraordinary result was obtained as reflected by

the column-headed 6-azauracil in Table III. When this

pyrimidine analogue was added to a growing culture of PAO-1

wild type, growth ceased immediately as reflected by the

growth curve in Fig. 5 (bottom line) but it appears from

TAble III that the nucleoside triphosphates increased

dramatically. The levels seen for UTP and CTP are unusually

high. These findings will be detailed in the Discussion.

Table IV gives the results of the nucleoside

triphosphates found for the P. aeruginosa pyrB mutant. It

can be noted from Table IV that when the pyrB mutant is

starved for pyrimidines, the UTP and CTP levels decreased to

near zero very rapidly. The possible interconversions of

Ma, 'tMd4MM- -ll-, - -,.- -- -.,

35

TABLE III. Nucleoside triphosphates in umole (gram dryweight) from P. aeruginosa PAO-1 wild type strain in thepresence and absence of the listed additions. Supplementswere added to succinate minimal (SM) medium at the rate of50 ug/ml. 6-Azauracil was added to a growing culture at 90Klett Units and sampled after one hour.

0 ZHZQ

4H H32H

ZHQ

04

H

Pa0 wC/

H 04 OP z

W4 F|

H 0 En

U) M r4 '

04 -t-4 I

H . -4t zWQ4

)i z M41 A

0 pq

0

H

N

+

C)

>4u

C.- I

4

+

H'H

0

-H

4

++0

0

rn

9 co 'H

'C'H

os os Co '0H)c% c

co

0

r-H

co C5

'

H

o .HH

LO HH .

LO H

H

LC)

LC'

E-4 -| E-4 pO p| pe

36

37

Tabje IV. Nucleoside triphosphates in umol (gram dryweight) from P. aeruginosa pyrB mutant in the presenceand absence of uracil, cytosine and cytidine. Supplementswere added to succinate minimal (SM) medium at the rate of50 ug/ml and starvation (no supplement added) for pyrimidineswas for two hours.

H-

HH-

N

0H4

ro

N

0 0 r"

0

Fe-r-$

41>4

IwoI

U]

M

0

4-)0H'H

1

a l

+

0

o 'H

'H D

F e

co

N

LO

H.-

r)o 0 ILA

02H w

H r

Fz:

E-4

G HU

2 H

Hm

E-1

00-10

04 014

H 4E9 (1)A

0 04

Hto zw z

CD N

co.0

N

'0

E- E- E- E-

H H H| H

38

C',

.Hr-H

LnH.H

H

H

39

the nucleobases and nucleosides are presented in the

Discussion.

In Table V another P. aeruginosa Pyr~ strain, namely a

pyrF mutant (Fig. 7) was examined. Results seen in Table V

are similar to those of Table IV except that it was

difficult to elevate the UTP level by exogenously added

pyrimidines in the pyrF strains.

In order to compare the results from P. aeruginosa PAO-1

with another Pseudomonas strain, the nucleotides were also

quantitated from wild type P., putida PPN. The results are

shown in Table VI. Table VI shows that the nucleoside

triphosphates of the wild type P. putida are lower in

general than those of P. aeruginosa. In Table VII the

nucleoside triphosphate levels are shown for a P. putida

pyrB mutant. Results for this pyrB mutant of P. putida

shows that uracil is the best (and only) exogenous

pyrimidine that can increase the levels of UTP as reflected

in the UTP level on addition of exogenous uracil (14.1 umol)

(gram dry weight)~1 , cytosine (4.4 umol) and cytidine (3.6

umol).

40

Tabje V. Nucleoside triphosphates in umol (gram dryweight)~ from P. aeruginosa pyrF mutant in the presence andabsence of uracil, cytosine and cytidine. Supplements wereadded to succinate minimal (SM) medium at the rate of 50ug/ml and starvation (no supplement added) for pyrimidineswas two hours.

C

-H-H

>1

C-H

04-)

>4

u

C)

-)

>4

0

0

-)

0

0 q

43

o H

.OC

y ei

41

:z UE-4

H 4H

E-

z zHH

1-11

H

> 4 E-

z

E-4

m,

44 01HM Z

E-4 (01 W

H 0

O- HN ec h

o o k (N

O LO H N4~ CO ~

H Co

o o CNJ

9' r4 N C4N H 0 c4

EP E-q E-4 E-:D u 4 0

42

Table VI. Nucleoside triphosphates in umol (gram dryweight)~ from P. putida PPN wild type strain in thepresence of the listed additions. Supplements were added tosuccinate minimal (SM) medium at the rate of 50 ug/ml.Growth was at 300C for _. putida strains.

E-4

W Er

zH E

0Q>4rzw

E ~H

z~

H rzoZ

HHE ZE-4

E-4 P-

> u:Z H)

0 ok 1:

4J

>1+

4J>4U)

0

41)>4u

u

C-HU)

04)

Co

o Z4 m

+

H

-HC)

C)D

+ -

<C/)

H cc

o o HLu~c c4i

. .J . -H

'0

H

f-

co

N

14N

0

N

c4ci

- E-4 E 4

E U <-4

43

44

Tabje VII. Nucleoside triphosphates in umol (gram dryweight) from P. putida DxrB mutants in the presence ofuracil, cytosine, or cytidine. Growth was at 300 C.

-

H

SH

H FQSE- O

OU

S E-4

H HE- E-4 u8

u: 00'H

HE-4

W AE-1H q E-4 4>

04to M0 PC

e-P\

0

>1

04-)

H

u

0

OH ro

rio

% AD ) LO n

H

LO 00 L OHNC HCOI

H (N

E-4 E-4 E-4 E-P: D u 4 0

45

IM60M!MMMMilidiinNIRMIMEM E|M araniassamment er.asm .....

CHAPTER IV

DISCUSSION

Nucleotides serve two general functions in the cell.

They play key roles in regulating metabolic reactions and

they act as precursors for the synthesis of the nucleic

acids. Metabolic pools of nucleotides and ribonucleic acids

are constantly being synthesized and degraded in bacteria.

The purine and pyrimidine nucleotides are continuously

broken down to free bases, ribose and deoxyribose-l

phosphates. These free bases, ribose and deoxyribose-l-

phosphates are required for salvage pathways to rebuild

nucleotides (Fig. 1). Nucleotides can be synthesized do

novo from glucose and it is known that every organism has a

functional de novo pathway for purine and pyrimidine

nucleotides.

In the past 30 years, purine and pyrimidine metabolism

in microorganisms has been studied extensively (Moat &

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

intracellular purine and pyrimidine nucleotide pools control

the individual biosynthetic pathways producing the

substrates for RNA, accurate measurement of the endogenous

nucleoside triphosphates is very important.

HPLC is both rapid and sensitive and provides the high

degree of resolution necessary to detect and quantitate

46

47

compounds in complex biological samples. As a research

tool, HPLC has been successfully applied to such problems as

detecting nucleotides (Krstulovic e al., 1979) in tissue

extracts and determining alterations in blood nucleotides

and metabolite levels in human disease states (Hartwick et

al., 1979: Payne eta l., 1981). HPLC has been modified

successfully to measure ribo- and deoxyribonucleotides in

bacteria (Dutta & O'Donovan, 1987). But very little effort

(Chen et .a., 1977) has been made in developing extraction

procedures for the detection and quantitation of low levels

of nucleotide pools in microorganisms (Dutta, 1986).

We have used HPLC to answer the question regarding the

concentration and stability of nucleotide pools in the

Pseudomonas. It has been established for several members of

the enteric bacteria, notably for E. coli and S.

typhimurium, that nucleotide pools can be extracted,

separated and accurately quantified. Moreover, it has been

shown by Kelln et al., (1975) that exogenously added bases

and nucleosides (refer to top right of Fig. 1) elevate the

nucleoside triphosphates such that these triphosphates can

act as feedback inhibitors and repressing metabolites in

controlling the -(purine and) pyrimidine biosynthetic

pathways. It has also been established by West (1986;

1987), which nucleic acid bases and nucleosides can satisfy

the requirement of Pseudomonas Pyr~ mutants. However to

date there has been no systematic study on the nucleotide

v MINm- wo

48

pool levels in the Pseudomonas. That is the purpose of the

present research and its results are summarized below.

When wild type P. aeruginosa was grown in succinate

minimal medium its nucleoside triphosphate levels (Tables

III-VII) measured in umoles (gram dry weight)~1 were as

follows: UTP, 5.23 (3.42); CTP, 5.48 (2.21); ATP, 12.1

(10.3) and GTP, 4.53 (3.84). For comparison purposes the

values from Salmonella typhimurium wild type strain grown in

glucose minimal medium are given in parentheses (Kelln et

.al., 1975). The following seven points are made with

respect to the data shown in Tables III-VII.

1) The ribonucleoside mono-, di-, and triphosphates

are present in P. aeruginosa at about the same level as

found in different members of the enteric bacteria as

compared above. (Only the triphosphates, and not the mono-,

and diphosphates, are shown).

2) Addition of exogenous bases and nucleosides altered

the appropriate triphosphates e.g. addition of uracil

increased the UTP pool from 5.23 umole (gram dry weight)~1

to 10.0 umoles (gram dry weight)~ 1 (Table III Line 1.)

Similar results were seen for the UTP pool in both the pyrB

(Table IV, Line 1 plus uracil) and the pyrF (Table V, Line 1

plus uracil).

3) Why Pyr~ mutants of P. aerucinosa were starved for

the pyrimidines, uracil or cytosine, the UTP and CTP pools

49

dropped to zero within two minutes. Refer to Table IV pyrB

UTP, 11.3 umoles (gram dry weight)~i (+uracil); 0.0 umoles

(-uracil), Table V pyrF 14.8 umoles (gram dry weight)~1

(+cytosine); 0.0 umoles (-cytosine).

4) Cytidine satisfies the pyrimidine requirement

poorly as reflected by the minor change in UTP levels on the

addition of exogenous cytidine. Pyr~ mutants of P.

aeruginosa and P. putida do not grown on cytidine (J.

Williams, unpublished results).

5) When a Pyr~ mutant of Pseudomonas was starved for

pyrimidines, the ATP level increased dramatically. A

similar result has been reported for other enteric bacteria

(O'Donovan & Neuhard, 1970).

6) All nucleoside triphosphates of wild type P. putida

strain PPN are lower than the corresponding levels in P.

aeruginosa PAO-1 strain. Compare Table III to Table VI.

7) When 6-azauracil, a pyrimidine analogue, known to

inhibit OMP decarboxylase (Handschumacher, 1960) was added

to growing culture of a P. aeruginosa, growth ceased

immediately even though the UTP, CTP, and ATP pools

increased dramatically. Since 6-azauracil is converted to

6-azaUMP by bacterial extracts (Handschumacher, 1960) and

the 6-azaUMP is a competitive inhibitor of OMP decarboxylase

it follows that the pyrimidine pathway is effectively shut

off. In such cases (e.cg., starvation for pyrimidines) the

ATP pool expands while the UTP and CTP pools decrease

50

simultaneously. One possible explanation for the high

levels of UTP and CTP is that the 6-azaUMP is metabolized

further and converted to 6-azaUTP and 6-azaCTP. These

triphosphate analogues would register as ribotriphosphates

and possibly give the result seen. There is one report

(Brockman & Anderson, 1963) which suggested that Pseudomonas

species can anabolize 6-azaUMP to the triphosphate level.

In summary the question posed at the start of this

thesis is now answered. It is possible to alter nucleoside

triphosphate pools in the pseudomonads. Indeed, the

ribonucleoside mono-, di-, and triphosphates are as

sensitive to manipulation in P. aeruginosa and P. putida as

they are in the enteric bacteria.

ANON

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