For the Degree of MASTER OF SCIENCE. Denton, Texas. August, 1988

Transcription

1 379 QUANTITATION OF ENDOGENOUS NUCLEOTIDE POOLS IN Pseudomonas aeruginosa TESIS 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

2 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.

3 TABLE OF CONTENTS List of Tables.....iv Page List of Illustrations v Chapter I. INTRODUCTION & 1 II. MATERIALS AND METODS... Chemicals and Reagents Bacterial Strains Growth Media and Cultures Extraction of Nucleotides Chromatographic Apparatus Chromatographic Conditions Identification and Quantitation of Nucleotides Calculations 14 III. RESULTS IV. DISCUSSION BIBLIOGRAPY... -~~~~~~ iii

4 LIST OF TABLES Table Page I. Genotypes and Phenotypes of Pyrimidine Auxotrophic Mutants in Bacteria II. III. IV. Identification of the Twelve Nucleotide Standards Used in This Work..... Nucleoside Trighosphate in umol (gram dry weight) from P. aeruginosa PAO-1 Wild Type Strain in the Presence and Absence of the Listed Additions. Nucleoside Triphosphates in umol (gram dry weight) -l from P. aeruginosa pyrb Mutant in the Presence of Uracil, Cytosine and Cytidine V. Nucleoside Tri hosphates in umol (gram dry weight)' from P. aeruginosa pyrf Mutant in the Presence and Absence of Uracil, Cytosine and Cytidine VI. VII. Nucleoside Triyhosphates in umol (gram dry weight) from P putida PPN Wild Type Strain in the Presence of the Listed Additions Nucleoside Triyhosphates in umol (gram dry weight)~ from P putida pyrb Mutants in the Presence of Uracil, Cytosine, and Cytidine iv

5 LIST OF ILLUSTRATIONS Figure Page 1. Pyrimidine nucleotide biosynthetic and salvage pathways in Escherichia coli and Salmonella typhimurium Schematic diagram representing the extraction procedure for ribonucleotides from P. aeruginosa Chromatograph of the 12 ribonucleotide standards Elution profile of the ribonucleotides extracted with 6% (w/v) trichloroacetic acid from P. aeruginosa Growth curves for P. aerucinosa PAO-1 wild type strain in succinate minimal (SM) medium Growth curve for P. aeruginosa pyrb strain in succinate minimal (SM) medium at 37C with and without pyrimidines Growth curve for P. aeruginosa yrf strain in succinate minimal (SM) medium at 37C with and without pyrimidines.. 32 v

6 CAPTER I INTRODUCTION The pyrimidine biosynthetic pathway is universal with the same sequence of reactions (Fig. 1) found for every organism (O'Donovan & Neuhard, 197). The first step in the pathway catalyzed by the enzyme carbamoyl phosphate synthetase (E C ; 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 ; ATCase). For arginine biosynthesis, ornithine and carbamoyl phosphate combine to form citrulline and inorganic phosphate. This step is catalyzed by ornithine transcarbamoylase (E C ; OTCase) (Isaac & olloway, 1972). The third step in the pyrimidine pathway, catalyzed by dihydroorotase (E C ; DOase), converts carbamoyl aspartate to the cyclic dihydroorotate by the removal of water (Lieberman & Kornberg, 1953). Dihydroorotate dehydrogenase (E C ; DOdehase) produces orotate in the fourth 1

7 2 Fig. 1. Pyrimidine nucleotide biosynthetic and salvage pathways 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, cytidine 5' -diphosphate; CTP, cytidine 5' -triphosphate.

8 . 3 CTP PyIG CD P SALVAGE PATWAYS UTP CMP SALVAGE PATWAYS- de novo PATWAYS - -- N I CR4 f Cwft I- pyr LUP pyrft AOOOOOO N%%6*1ft UR U 44 - UR - U CMP Orotate pyrd Dihydroorotate t c ~.6 - C CELL. MEMBRANE Arginine Argininosuccinate Carbamoyl Aspartate D Citrulline Q Ornithine pyrb ftcase Aspartate Carbamoyl- (Z) pyra Glutamine ATP CO3 3

9 4 fourth step (Lieberman & Kornberg, 1953). Orotate and phosphoribosyl pyrophosphate (PRPP) combine to form the first pyrimidine ribonucleotide, orotidine-5-monophosphate (OMP) (urlbert and Reichard, 1955) in a reaction catalyzed by orotate phosphoribosyl transferase (EC ; 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 ; 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 ) to form UDP, and then UTP is converted to UTP by the non-specific nucleoside diphosphate kinase (EC ; NDKase). Finally UTP is aminated to produce CTP (Lieberman, 1955) by the enzyme CTP synthetase (EC ; 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.

10 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. owever, 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 & olloway, 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 1-fold (i.e., their specific activity increases over 1-fold). At the same time the concentration of nucleoside triphosphates, UTP and CTP, are depleted and drop to near zero levels

11 6 Table I. Genotypes and Phenotypes of Pyrimidine Auxotrophic Mutants in Bacteria.

12 7 C ' C *rr4.4 4 ) p - -r4 r- r- r-4 r- r- r- q.4 r- u Q U u z 4 o o o c e o41 c E-.4 o OP Cd W E.. CnM M4P4 p 4 O En 4) (1) cd Oco U) MCd 4) 44 (1 -r4 4-J X r-4 o 4) P 4'-d P to 4) r- O E "Ag P >-'C PL ' co 4 U ) - QOr-4 TI > ON % O - -'P n oo > Cdo En -* 4 Z o r-4 (1) a 4J d fr- 1-4J C4) U Q z p d Mdm >< 4 4-) cd o4j4-)o J4CU O E- 4 r-4 (1) p p p lc -A >- 4-) C 1 4J ' ', P.N P4 z P644m CO 1< -A - P Z P u aaaa

13 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,

14 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 p 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 (PLC). In recent years, the measurement of

15 1 endogenous ribo- and deoxyribonucleotide concentrations has been revolutionized by the development of PLC (Glaser, 1986). The two most commonly employed separation techniques involve either reverse-phase chromatography on octyldecylsilica resins (offman & Liao, 1977; orvath, Melander & Molnar, 1977) or ion-exchange chromatography on microparticulate anion-exchange resins (artwick & 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, PLC is employed simply because efficient, rapid and reliable quantitative nucleotide separations are needed. In the present study and in previous studies, Partisil 1 SAX columns were used. These are strong anion-exchange columns which are packed with particles 1um 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 (artwick & 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

16 (Table II) adenosine 5'-triphosphate (ATP), adenosine 5' - (GDP), guanosine 5' -monophosphate (GMP), uridine 5' - 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 etherwater 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 diphosphate (ADP), adenosine 5' -monophosphate (AMP) guanosine 5' -triphosphate (GTP), guanosine 5' -diphosphate 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.

17 12 Table II. Identification of the 12 Nucleotide Standards Used in this Work by Their Retention Times (Minutes).

18 13-4-) - Or oin r W r- - NSNS N m* qcs q gc onwwrk- NON S- L E- C-4 L O Fe C. 4-) 4 N NG 4-) rzie l 111 E-4 4-) 4.) - 4J 4- J o 4 - MO -)P 44 d O Cdi4,c Q4 4 4 m )-P Ci4-),4J(,S S Q 4) Wd 4 w 4) (Cl. 4 - U) Q4 o -,, ( cmo 4 En ~ Q 4 U)W4WO M1, 44 M 4 o , 4 O 4., ,, - - Z M 9 ct$-t$,d F -)--Pr: - I 4 I J 4 I I I O I LO LO 1 I I)O UC) o- % %blc (O1 LnOO)LO OO) 4 U (1) ( - M) U)M -U U) E - M)tU Fd - -Fe F Fe- 99Fe d - -) 4d - - () S 5-5 r 1r 4-i-P 4 SQSQ z z - Cc oo-8oc %- CC # -8 M-dy & -Sy&M -'C OS-

19 CAPTER II MATERIALS AND METODS Chemicals and Reagents Nucleotides, trichloroacetic acid (TCA) and tri-noctylamine 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. olloway, 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: Na 2 PO 4, 7.1; K 2 PO 4, 6.8; (N 4 )2So 4, 14

20 (N 4 ) 6 Mo 7 O , 18.5; ZnSO ; FeSO ; MnSO nitrilotriacetic acid, MgSO 4, 28.9; CaCI 2, 6.67; 2, CuSO , Co(NO 3 ) , Na 2 B , (utner's metals '44'.) was used with succinate as the carbon and energy source. Pyr~ mutants were supplemented with uracil at 5 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 37C and agitated at 9 revolutions per minute. P. putida strains were grown at 3C. Overnight cultures was transferred to a fresh basal medium and grown to about 1 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=1 7 /ml). The logarithms of the KUs were plotted against time. Extraction of Nucleotides Volumes of 5 ml of bacterial cultures were harvested at about 1 KU, centrifuged at 4 C at 12, 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

21 16 Fig. 2. Schematic diagram representing the extraction procedure for ribonucleotides from P. aeruginosa cultures.

22 17 Bacterial Culture Centrifuge at 12, X g for 5 min at 4 C Supernatant Cell pellet Add TCA (6% w/v) Mix by vortexing Allow to stand for 3 min Centrifuge at 12, X g for 15 min at 4 C Pellet F- -+ Supernatant Add Freon-amine, vortex, and stand Bottom Layer Top Aqueous Layer contains nucleotides Filter Store at -2C for analysis.4&"- 444t ilw-

23 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 4C for 3 minutes before centrifuging at 12, x g for 15 minutes. The clear supernatant was then neutralized with ice cold Freon-amine (Khym, 1975) solution (1.6 ml of.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 4C. The top, aqueous layer, which contained the nucleotides, was removed, filtered through a.45 um ACRO LCl3 filter (Gelman Sciences, Ann Arbor, Michigan) and frozen at -2C until ready for analysis. Chromatographic Apparatus The PLC equipment (Waters Assoc., Milford, Massachusetts) consisted of two Model 51 pumps, a Model 68 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.5 absorbance units at full scale deflection (AUFS). Separations were performed on a Waters Radial-Pak Partisil SAX cartridge (1 cm x.8 cm) using a Waters radial compressing Z-Module system. Chromatographic Conditions The entire chromatographic system including the column was stored in 5:5 (v/v) filtered PLC grade methanol and

24 19 filtered, double distilled water (2x) when not in use. After priming the pumps, the system was flushed with 5 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 1 minutes the flow rate was increased to 4 ml per minute. When the back pressure of the column dropped to 85 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, p 3.8) followed by pump B which was flushed separately with final buffer (filtered, 25 mm monobasic ammonium phosphate containing 5 mm potassium chloride, p 4.5). The LC spectrophotometer was set at 254 nm and.5 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 1 minutes. A 1- minute reverse gradient of high concentration buffer was run, followed by a 1-minute rest with low concentration buffer. Identification and Quantitation of Nucleotides Nucleotide samples of 2 ul each were prepared from Pseudomonas cells as previously described. These were thawed and injected onto a column of Partisil SAX-1 which consisted of a silica matrix coated with porous

25 2 microparticles of silica of 1 um particle size. The particles contained fixed-charge quaternary nitrogen groups and mobile counterions of 2 PO 4. 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 2 minutes, followed by an isocratic period of 1 minutes of high concentration buffer. The column was regenerated by washing with 3 ml of 7mM N 4 2 PO 4, 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 74 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 (.1 mm) and expressed as nmoles per 18 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.

26 21 Calculations Moles (gram dry weight)~ 1 for all nucleotides were computed as follows: Sa V 1 X C X X St 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).

27 CAPTER 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 37 C. Bases and nucleosides were added at the rate of 5 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: 1 to 2 ml of succinate minimal medium were inoculated with the culture to be tested in the presence (at 5 ug/ml) or absence of appropriate additions. The cells were grown to a density of 1 Klett units (1 9 /ml). Fifty ml samples of culture were then harvested and treated with 6%(w/v) trichloroacetic acid to extract the nucleotides as 22

28 23 Fig. 3. Chromatogram of the 12 ribonucleotide standards. The numbers refer to the compounds listed in Table II.

29 24 co. C cz 2 D 3 4 t[[ imue ( nu.s Time (minutes)

30 25 Fig. 4. Elution profile of ribonucleotides extracted with 6% (w/v) trichloroacetic acid from P. aeruginosa. The numbers refer to the compounds listed in Table II.

31 26 II (T Cu C C LL Time (minutes)

32 27 Fig. 5. Growth curves for P. aeruginosa PAO-1 wild type strain in succinate minimal (SM) medium at 37 C with and without the noted additions. 6-Azauracil was added to the growing culture in SM medium at 9 KU. Symbols and abbreviations are: SM+CAA+U, casamino acid + uracil (), SM+U, uracil (a), SM+C, cytosine (A), SM+CR, Cytidine, (U), SM+UR, uridine, (a) SM+AzaU, 6-azauracil (A).

33 28 S.12 A Time (min)

34 29 Fig. 6. Growth curve for P. aeruginosa pyrb strain in the succinate minimal (SM) medium at 37 with and without pyrimidines. When the cultures reached 1 KU they were starved for pyrimidines for two hours. Symbols and abbreviations are as in Fig. 5.

35 + 3 a I Time (min)

36 31 Fig. 7. Growth curve for P. aeruinosa pyrf strain in the succinate minimal (SM) medium at 37 C with and without pyrimidines. When the cultures reached 1 KU they were starved for pyrimidines for two hours. Symbols and abbreviations are as in Fig. 6.

37 32 C Time (min)

38 33 as described in Materials and Methods section. Nucleotides were quantitated by PLC. 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 PLC 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 1 KU. This is seen in Fig. 6 for uracil and uridine, which grow exponentially to about 1 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,

39 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., 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 1. 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-, - -, ,

40 35 TABLE III. Nucleoside triphosphates in umole (gram dry weight) from P. aeruginosa PAO-1 wild type strain in the presence and absence of the listed additions. Supplements were added to succinate minimal (SM) medium at the rate of 5 ug/ml. 6-Azauracil was added to a growing culture at 9 Klett Units and sampled after one hour.

41 N Z ZQ 4 32 ZQ C) >4 u os os Co ' c)c% 4 C.- I 4 co co C 5 ' Pa wc/ 4 OP z W4 F ' o. En U) 4 ' M r 4 -t-4 I 4 - r- LO. LC). -4 WQ4 t z )i z M 41 A pq ++ o ' 9 c 'C ' LO LC' rn 4 E-4 po p - E- pe

42 37 Tabje IV. Nucleoside triphosphates in umol (gram dry weight) from P. aeruginosa pyrb mutant in the presence and absence of uracil, cytosine and cytidine. Supplements were added to succinate minimal (SM) medium at the rate of 5 ug/ml and starvation (no supplement added) for pyrimidines was for two hours.

43 >4 - - N 4 ro 2 w Fz: Fe -r-$ N r" N r U] IwoI G U E-4 Ln. co LO.- E m ) M ' 1 r) o ILA co. N 4 E9 (1)A 4 to z w z a l o ' ' D F e C',. r- CD N ' E- E- E- E-

44 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).

45 4 Tabje V. Nucleoside triphosphates in umol (gram dry weight)~ from P. aeruginosa pyrf mutant in the presence and absence of uracil, cytosine and cytidine. Supplements were added to succinate minimal (SM) medium at the rate of 5 ug/ml and starvation (no supplement added) for pyrimidines was two hours.

46 C ec h E-4 :z U4 >1 O- N E- 4-) Cu - o o k (N z z >4 > 4 E- -) C) O LO N 4~ CO ~ 1-11 E-4 m, > M Z z E-4 (1 W o o CNJ Co -) 9' r4 N C4 N c4 o q.oc y ei 43 EP E-q E-4 E- :D u 4

47 42 Table VI. Nucleoside triphosphates in umol (gram dry weight)~ from P. putida PPN wild type strain in the presence of the listed additions. Supplements were added to succinate minimal (SM) medium at the rate of 5 ug/ml. Growth was at 3C for _. putida strains.

48 J E-4 W Er >1 cc z E 4J >4 U) ~ E rzoz Q>4rzw E-4 P- z~ 41) >4 uc - U) u Co o o L u~c c4i..j. - > E ZE-4u :Z ) ok 1: 4) - C) ' f- 14 N c4 ci co o Z 4 m C)D N N + U <-4 <C/) - E-4 E 4 E

49 - 44 Tabje VII. Nucleoside triphosphates in umol (gram dry weight) from P. putida DxrB mutants in the presence of uracil, cytosine, or cytidine. Growth was at 3 C.

50 IM6M!MMMMilidiinNIRMIMEM E M araniassamment er.asm S SE- FQO >1 OU S E-4 E- E-4 u8 ' u: 4-) u % AD ) LO n LO L O NC COI W AE-1 q E-4 4> 4 to E-4 M PC e-p\ (N O ro rio E- 4 E-4 E-4 E-P : D u 4

51 CAPTER 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-lphosphates 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 3 years, purine and pyrimidine metabolism in microorganisms has been studied extensively (Moat & Friedman, 196; O'Donovan & Neuhard, 197). 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. PLC is both rapid and sensitive and provides the high degree of resolution necessary to detect and quantitate 46

52 47 compounds in complex biological samples. As a research tool, PLC 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 (artwick et al., 1979: Payne eta l., 1981). PLC 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 PLC 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. owever to date there has been no systematic study on the nucleotide v m- MINwo

53 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 (1.3) 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 1. 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

54 49 dropped to zero within two minutes. Refer to Table IV pyrb UTP, 11.3 umoles (gram dry weight)~i (+uracil);. umoles (-uracil), Table V pyrf 14.8 umoles (gram dry weight)~ 1 (+cytosine);. 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, 197). 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 (andschumacher, 196) 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 (andschumacher, 196) 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

55 5 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

56 BIBLIOGRAPY Abd-El-Al, A. D., Kessler, D.P., & Ingraham, J. L. (1969). Arginine-auxotrophic phenotype resulting from a mutation in pyra gene of Escherichia coli. Journal of Bacteriology 97, Anderson, P. M. & Meister, A. (1969). Control of Escherichia coli carbamyl phosphate synthetase by purine and pyrimidine nucleotides. Biochemistry 5, Assenza, S. P. & Brown, P. R. (1983). Reversed-phase retention of nucleic acid components. Separation and Purification Methods 12, Boulieu, R. & Bory, C. (1985). igh performance liquid chromatographic method for the analysis of purine and pyrimidine bases, ribonucleosides in biological fluids. Journal of Chromatography 399, Brown, P. K., & Miech, R. P. (1972). Comparison of cell extraction procedures for use with high pressure liquid chromatography, Analytical Chemistry 44, 172. Brown, P. R. (1973). igh Pressure Liquid Chromatography: Biomedical and Biochemical Applications. New York: Academic Press. Brockman, R. W. & Anderson, E. P. (1963). Pyrimidine analogues. p In Metabolic Inhibitors, pp Edited by R. M. ochster and J.. Quastels. New York: Academic Press. Chargeys, E. & Davidson, J. N. (1955). The Nucleic Acids, New York: Academic Press, Inc., Volume 1. Chen, S. C., Brown, P. R., & Rosie, D.. (1977). Extraction procedures for use prior to PLC nucleotide analysis using microparticle chemically bonded packings. Journal of Chromatographic Sciences 15, Cohen-Bazire, C., Sistrom, W. R. & Stanier, R. Y. (1957). Kinetic studies of pigment synthesis by nonsulfur purple bacteria. Journal of Cellular and Comparative Physiology 49,

57 52 Colowick, S. P. & Kaplan, N.. (eds), (1957). Methods in Enzymology, k, Academic Press, Inc., New York. Dutta, P. K. (1986). "Radial Compression igh Performance Liquid Chromatography as a Tool for the Measurement of Endogenous Nucleotides in Bacteria," Doctoral Dissertation, North Texas State University, Denton, Texas, Dutta, P. K. & O'Donovan, G. A. (1987). Separation and quantitation of bacterial ribonucleoside triphosphate extracted with trifluroacetic and by anion exchange PLC. Journal of Chromatography, 385, Gerhart, J. C. & Pardee, A. B. (1962). The enzymology of control by feedback inhibition. Journal of Biological Chemistry 237, Gerhart, J. C. & Pardee, A. B. (1964). Aspartate transcarbamylase, an enzyme designed for feedback inhibition. Federation Proceedings 23, Glaser, V. (1986). Chromatography: Primer and Current Practice, Bio-Technology 4, artwick, R. A., Assenza, S. P., & Brown, P. R. (1979). Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography. Journal of Chromatography 186, artwick, R. A. & Brown, P. R. (1975). The performance of microparticle chemically bonded anion exchange resins in analysis of nucleotides. Journal of Cromatography 112, 651, 662. offman, N. E. & Liao, J. C. (1977). Reverse phase high performance liquid chromatography separations of nucleotides in the presence of solvophobic ions, Analytical Chemistry 49, orvath, C., Melander, W., & Molnar, 1. (1977). Liquid chromatography of ionogenic substances with nonpolar stationary phase. Analytical Chemistry 49, urlbert, R. B. & P. Reichard. (1955). The conversion of orotic acid to uridine nucleotides in vitro. Acta Chemica Scandinavia 9,

58 53 Isaac, J.. & olloway, B. W. (1968). Control of pyrimidine biosynthesis in Pseudomonas aeruginosa. Journal of Bacteriology 96, Isaac, J.. & olloway, B. W. (1972). Control of arginine biosynthesis in Pseudomonas aeruginosa. Journal of General Microbiology 73, Karl, D. M. (1978). Occurrence and ecological significance of GTP in the ocean and in microbial cells. Applied and Environmental Microbiology. 36, Kelln, R. A., Kinahan, J. J., Foltermann, K. F., & O'Donovan, G. A. (1975). Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine. Journal of Bacteriology 124, Khym, J. X. (1975). An analytical system for the rapid separation of tissue nucleotide at low pressures on conventional anion exchangers. Clinical Chemistry 21, Krstulovic, A. M., artwick, R. A., & Brown, P. R. (1979). Reversed-phase liquid chromatographic separation of 3',5'-cyclic ribonucleotides. Clinical Chemistry 25, Leung,. B. & Schramm, V. L. (198). Adenylate degradation in E. coli. Journal of Biological Chemistry 255, Lieberman, I. (1955). Enzymatic amination of uridine triphosphate to cytidine triphosphate. Journal of the American Chemical Society 77, Lieberman, I., Kornberg, A. & Simms, E. S. (1955). Enzymatic synthesis of pyrimidine nucleotides. Orotidine-5' -phosphate and uridine-5' -phosphate. Journal of Biological Chemistry 215, Long, C. W. & Pardee, A. B. (1976). Cytidine triphosphate synthetase of Escherchia coli, B. I. Purification and kinetics. Journal of Biological Chemistry 242, McKeag, M. & Brown, P. R. (1978). Modification of high pressure liquid chromatographic nucleotide analysis. Journal of Chromatography 152,

59 54 Moat, A. G. & Friedman,. (196). The biosynthesis and interconversion of purines and their derivatives. Bacteriological Reviews 24, Neumann, J. & Jones, M. E. (1964). End product inhibition of asparate transcarbamylase in various species. Archives of Biochemistry and Biophysics 14, O'Donovan, G. A. & Neuhard, J. (197). Pyrimidine metabolism in microorganisms. Bacteriological Reviews 34, Payne, S. M. & Ames, B. N. (1982). A procedure for rapid extraction and high-presure liquid chromatographic separation of nucleotides and other small molecules from bacterial cells. Analytical Biochemistry 123, Pierard, A. (1966). Control of the activity of Escherichia coli carbamyl phosphate synthetase by antagonistic allesteric effectors. Science 154, Pogolotti, A. L. Jr. & Santi, D. L. (1982). igh pressure liquid chromatography-ultraviolet analysis of intracellular nucleotides. Analytical Biochemistry 126, Reiss, P. D., Zuurendonk, P. F., & Veech, R. L. (1984). Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high presure liquid chromatography. Analytical Biochemistry 14, Strehler, B. L. & McElroy, W. D. (1975). Assay of adenosine triphosphate. Methods of Enzymology 3,