Pseudomonas fluorescens biotype F

Metabolism of pyrimidine bases and nucleosides by Pseudomonas fluorescens biotype F Thomas P. West Department of Biological Sciences, lnstitute of Genetics, University of Southern Mississippi, Hattiesburg, Mississippi 39406, U.S.A. Present address: Station Biochemistry, South Dakota State University, Box 217O, Brookings, south Dakota 57007, u.s.A.

Abstract Pyrimidine metabolism in Pseudomonas fluorescens biotype F, and its ability to grow in liquid culture on pyrimidines and related compounds was investigated. lt was found that uracil, uridine, cytosine, cytidine, deoxycytidine, dihydrouracil, dihydrothymine. p-alanine or B-aminoisobutyric acid could be utilized by this pseudomonad as a sole nitrogen source. Only uridine. cytidine, 6-alanine, B-aminoisobutyric acid or ribose were capable of supporting its growth as a sole source of carbon. ln solid medium, the pyrimidine analogue S-fluorouracil or S-fluorouridine could prevent P. fluorescens biotype F growth at a low concentration while a 2O-fold higher concentration of S-fluorocytosine, S-fluorodeoxyuridine or 6-azauracil was necessary to block its growth. The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed. Only cytosine deaminase and nucleoside hydrolase activities could be detected under the assay conditions used. The effect of growth conditions on cytosine deaminase and nucleoside hydrolase levels in the micro-organism was explored. Cytosine deaminase activity was shown to increase if glycerol was substituted for glucose as the sole carbon source or if asparagine replaced (NH4)2SO4 as the sole nitrogen source in each respective medium. ln contrast, nucleoside hydrolase activity remained virtually unchanged whether the carbon source in the medium was glucose or glycerol. A decrease in nucleoside hydrolase activity was witnessed when asparagine was present in the medium instead of (NHo)rSOu as the sole source of nitrogen.

lntroduction Pyrimidine metabolism in pseudomonads has been studied far less than in other bacteria (O'Donovan and Neuhard, 1970). The extensive taxonomic classification study of aerobic pseudomonads by Stanier et al. (1966) did not examine whether pyrimidine bases and nucleosides could support their growth. Later studies have investigated the utilization of pyrimidine bases and nucleosides by aerobic pseudomonads.

In a comprehensive

study of pyrimidine metabolism in

it

was shown that only uracil, cytosine or thymine could support its growth as a sole nitrogen source and that pyrimidines could not sustain $owth ofthis pseudomonad as sole carbon sources (Kelln and $7arren, L974).In Pseudomonas facilis, uracil, dihydrouracil, thymine or dihydrothymine could serve as a sole source of nitrogen (Kramer and Kaltwasser, 1969). A number of studies have investigated the growth of fluorescent pseudomonads on pyrimidines (Fink et a1.,1954; Potter e, a1.,1982; \trflest and Chu, 1985, 1986).

Pseudomonas acidoaorans,

Growth on uridine, cytidine, dihydrouracil or dihydrothymine was observed for Pseudnmonas aerugirnsq Pseudamorws chbroraphis and Psadomoras aureofocieru either a nitrogen or a carbon source. For these pseudgmonads, uracil, thymine or cytosine gupported their growth exclusively as a nitrogen source. Deoxycytidine

as

also supported P. aeruginosa growth only as a nitrogen source. The least versatile

of the fluorescent pseudomonads examined was Pseudomonas putida. It grew

27

Microbios

56 27-36

1988

Published ano O tge8 by The Faculty Press 88 Regent Street, Cambridge, Great Britain

on uridine, cytidine or dihydrothymine as a carbon source and cytidine, uracil or thymine as a nitrogen source (lU[est and Chu, 1986). Many of the above investigations have explored whether pyrimidine analogues influence pseudomonad growth. The sensitivity to pyrimidine analogues of a microorganism can provide an indication as to which pyrimidine salvage enzymes are present. P. acidovorans, it was found that 5-fluorouracil, 5-fluorocytosine, 5-fluorodeoryuridine, Gazauracil, 6-azarytosine or 5-bromodeoryuridine prevented growth at high concentrations (Kelln and \tr7arren, 1974). In general, the fluorescent pseudomonads P. aeruginosa, P. chlororaphis, P. aureofaciens and P. putida were more resistant to pyrimidine analogues than P. acidooorans (West and Chu, 1985, 1986). It was found that 5-fluoropyrimidine analogues had the maximal inhibitory effect on their growth. In addition, a high concentration of 6-azauracil was found to halt the growth of P. chlororaphis and P. aureoJaciens. Pyrimidine salvage enzyme activities in aerobic pseudomonads have been investigated. Cytosine deaminase, which deaminates cytosine to uracil, has been detected in P. aeruginosa, P. chlororaphis, P. aureofaciens, P. acidooorans and Pseudomonas fluorescens (Kelln and lfarren, 1974; Sakai et al.,19761,Kim et al., 1987). Another salvage enzyme active in pseudomonads is nucleoside hydrolase. This enzyme has been shown to catalyse the hydrolysis of a pyrimidine or a purine ribonucleoside to the respective pyrimidine or purine base and ribose (Terada et al., 1967). Hydrolase activiry has been reported in P. aeruginosa, P. chlororaphis, P. aureofacicns and P. fluorescms (Sakai et al., 1976). Enzyme activities such as uridine phosphorylase, thymidine phosphorylase and cytidine deaminase, which are present in enteric bacteria (O'Donovan and Neuhard 1970), appear to be absent in many pseudomonads (Kelln and \Warren, 1974;Potter et al.,

lt

al., 1985). The purpose of this study was to investigate pyrimidine metabolism in P. fluorescens biotype F. By examining the ability ofthis pseudomonad to utilize 1982; Carlson et

pyrimidines, by testing its sensitivity to pyrimidine analogues and by assaying for its pyrimidine salvage enzyme activities, information regarding pyrimirtine metabolism in this aerobic pseudomonad can be gained. The acquisition of such data could prove relevant to the future taxonomic analysis of aerobic pseudomonads.

Materials and methods Straia and media Pseudomoras fluoresccns biotype F

ATCC 12983 was the strain used in this study

(Hugo and rurner, 1957). The strain was maintained on nutrient agar slants at 4oC throughout the investigation. A minimal medium devised by Stanier (1947) was modified as previously described by West and Chu (1985, 1986). Initially, pyrimidines and related compounds were tested for their ability to support the growth of P. fluorescens as sole nitrogen sources in liquid media. At a concentration of 0.20/0, each compound was substituted for (NH4)2SO4 but the carbon source remained 0.4y0 glucose. The utilization ofthese compounds as sole carbon sources by P. fluorescans

28

Microbios

T. P. West

to support its growth was also investigated in liquid media. The minimal medium was modified in this instance so that it lacked glucose or Na.C6H5O7.2H2O to ensure that they did not compete with the pyrimidine compound (0.270) being tested as a possible carbon source. Pyrimidine analogue sensitivity of P. fluorescezs was determined using the modified minimal medium which contained 0.49o (NHa)2SOo as the nitrogen source and0.4Vo glucose as the carbon source. This medium was supplemented with a pyrimidine analogue at a concentration of l0 p.g/ml or 200 pglml. All analogue sensitivity experiments were performed using solid media where 2Vo agar was added to each medium. Growth determination

The ability of pyrimidines and related compounds to support the growth of Liquid cultures (5 ml) of each nitrogen or carbon source-containing medium were inoculated with approximately 108 cells. Preparation of the inoculum involved growing P. fluoresans was investigated spectrophotometrically at 600 nm.

the pseudomonad in a minimal medium culture to the stationary phase of growth. After collecting the bacterial cells, they were washed with 0.859o Nacl and resuspended in 0.8570 NaCl. The initial ODooo of each inoculated culture was

0.01. The cultures were shaken at200 rpm for 8 days at 30oC. After 8 days of growth at 30oC, the ODuoo of each culture was determined. control cultures for the growth experiments were also performed. one such control culture involved growing the pseudomonad in minimal medium [0.490 glucose as the carbon source and0.4vo (NH4)2so4 as the nitrogen source] for 8 days at 30oC. Another type of control culture involved growing P. fluorescens in medium containing neither a carbon nor a nitrogen source for 8 days at 30oC. Growth was measured by observing the change in OD600 for each control culture. For the pyrimidine analogue sensitivity srudies, a stationary phase minimal medium culture of the pseudomonad strain grown at 30oC was used as the inoculum. An inoculum of approximately 108 cells was spread onto each pyrimidine analogue-containing solid medium plate at 30'C. If visible growth was observed after 48 h, growth was recorded as positive. Preparation of cell extracts

Cell extracts were prepared from cells which had been grown on one of the following three types of media: minimal medium (with (NHa)2SOo as the nitrogen source and glucose as the carbon source), minimal medium containing 0.2% asparagine as the nitrogen source and 0.470 glucose as the carbon source, and minimal medium containing 0.4Vo glycerol as the carbon source and 0.4Vo (NH4)2SO4 as the nitrogen source. Liquid culrures (70 ml) were inoculated with the strain and shaken at 200 rpm at 30oC until cell growth reached the late exponential phase. The cells were harvested by low-speed centrifugation and then washed with 0.8590 NaCl (10 ml). The resulrant cell pellet was resuspended in 2.5 ml of 50 mm Tris-HCl buffer, pH 7.3, and the cells were ultrasonically disrupted for a total of 3.5 min at 0oC. The cell exrract was centrifuged at l2rl00 x g at 4oC for 30 min. The cell-free extract was assayed immediately for each enzyme activity.

29

P. fluorescens biotype F pyrimidine metabolism

Enzyme assays

A

number of pyrimidine salvage enzymes were assayed including cytosine

deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase

and cytidine deaminase. All enzyme assays were performed at 30"C. Cytosine deaminase was assayed using a previous method (West ar al.r 1982) where the assay mix contained 50 mM Tris-HCl buffer, pH 7.3, and 0.5 mM cytosine in a final volume of 0.5 ml. The reaction was halted by the addition of 1 N HCIO. and the enzyme activity was determined by examining the difference in OD2e5

between a control and reaction mix (!(est et al., 1982). A previously devised assay protocol was modiflred to quantitate nucleoside hydrolase activity (Terada et al., 1967). The assay mix (1 ml final volume) contained 50 mM Tris-HCl buffer, pH 7.3,2 mM 2-mercaptoethanol, l0 mM MgCl2, 5 mM uridine and the extract. At selected times, 0.2 ml aliquot of each reaction mix was added to 0.8 ml of 0.5 N HCIO4 to stop the reaction. After the precipitated protein was removed 0.5 ml supernatant was mixed with 0.5 ml I N NaOH. The OD2e6 was measured and an increase of 5.41 was equivalent to the conversion of 1 pmol uridine into uracil (Beck, Ingraham, Neuhard and Thomassen, L972). The reaction mixture for the uridine phosphorylase assay contained 50 mM Tris-HCl buffer, pH 7.3, 2 mM 2-mercaptoethanol, l0 mM MgClr, 10 mM phosphate (pH 7.0), 5 mM uridine and the extract in a final volume of I ml. The phosphorylase assay was performed exactly as described above for the nucleoside hydrolase assay. Thymidine phosphorylase was assayed using the reaction conditions and protocol as given previously by Munch-Petersen (1968). Cytidine deaminase activity was measured as given in a prior procedure (Beck er al.,1972). The assay mix (l ml) of this procedure was modified to contain 50 mM Tris-HCl buffer, pH 7.3,2 mM 2-mercaptoethanol, l0 mM MgClr, 0.5 mM deoxycytidine and the extract. Protein was determined by the procedure of Bradford (1976). Lysozyme served as the standard protein. Specific activity rffas expressed as nmol product/min/mg protein. Chemlcalc

Pnimidine

pyrimidine nucleosides, B-alanine, B-aminoisobutyric acid ribose, deoxyribose, asparagine, 5-fluorouracil, 5-fluorodeoxyuridine, 5-fluorocytosine, 5-bromodeoxyuridine, 5-bromocytidine, 5-bromodeoxycytidine, 5-bromouracil, 6-azauracil, 6-azathymine and 2-mercaptoethanol were purchased from the Sigma Chemical Co., St Louis, Missouri, U.S.A. Both 5-fluorouridine and 6-azacytidine were obtained from the Calbiochem-Behring Corporation, La Jolla, California, bases,

U.S.A. Results

The ability of P.

fluorescezs biotype

F to utilize pyrimidines and related

compounds for growth was studied. First, the growth of this pseudomonad on these compounds as possible nitrogen sources was examined in liquid cultures. As can be seen in Table 1, uracil, uridine, cytosine, cytidine, deoxycytidine,

30

Microbios

T. P. West

1 Ability of Pseudomonas fluorescens biotype F to utilize pyrimidines and related compounds to support its growth (8 days at 3OoC)

Table

Growth

on compound serving as: Carbon source

Nitrogen source

Uracil

1.18 1.12

o.o1

o.03 o.o2 o.77

o.o1 o.o1

1.O7

o.58

o.o7 o.41

o.o1 o.o1

.55 1.20 1.28 1.48

o.o2 o.o5 o.76 o.79

Uridine

Thymine Thymidine Cytosine

I

(ODuoo)

Compound

Cytidine Deoxyuridine Deoxycytidine Dihydrouracil Dihydrothymine p-Alanine P-Aminoisobutyric acid

1

o.72

o.o1

Ribose

1.23

Deoxyribose

o.o1

Growth (ODuoo) of each control culture after 8 days at 3OoC was as follows: minimal medium, 1.36; medium lacking a carbon and a nitrogen source, 0.01. Each value represents the average of two separate determinations.

dihydrouracil, dihy&othymine, B-alanine or B-aminoisobutyric acid could suppofi the growth ofthis pseudomonad as a sole nitrogen source. With respect to possible carbon sources, uridine, cytidine, B-alanine, B-aminoisobutyric acid or ribose were found to support bacterial growth after 8 days at 30'C (Table l). The ability of more pyrimidine compounds to support the growth of this microorganism as a source ofnitrogen rather than as a source ofcarbon is evident. In order to learn additional information about pyrimidine metabolism in P. fluorescens biotype F, the sensitivity of this pseudomonad strain to pyrimidine analogues was examined at two different concentrations. At a concentration of l0 pglml5-fluorouracil or 5-fluorouridine, it was observed that bacterial growth was blocked (Table 2). Growth inhibition of this pseudomonad by the analogues 5-fluorodeoxyuridine, 5-fluorocytosine or 6-azauracil was pronounced at a concentration of 200 pglml (Table 2). The remaining pyrimidine analogues did not appreciably affect the growth of this strain (Table 2).

The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed at 30oc in cell-free extracts of this strain. As can be seen in Table 3, cytosine deaminase and nucleoside hydrolase activities were measurable while uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were not detectable under the assay conditions used at 30oC. It can be seen that

31


Table 2 Effect of pyrimidine analogues on Pseudomona.s fluorescens biotype F growth Growth on analogue at: Pyrimidine analogue

5-Fluorouracil 5-Fluorouridine 5-Fluorodeoxyuridine 5-Fluorocytosine 5-Bromodeoxyuridine 5-Bromodeoxycytidine 5-Bromocytidine 5-Bromouracil 6-Azauracil 6-Azacytidine 6-Azathymine 6-Azauridine

1O

pglml

2OO ltglml

+ +

+

+

+ +

+ + +

+ + +

+

+ +

+

+

Solid minimal medium containing pyrimidine analogue was inoculated with the pseudomonad no growth. and then incubated 48 h at 3OoC, Growth was recorded as follows: +, growth;

-,

nucleoside hydrolase specific activity is significantly higher than cytosine deaminase specific activity. It was thought that the levels of cytosine deaminase and nucleoside hydrolase might vary with the cellular growth conditions. Initially, the activities ofboth enzymes were compared in relation to the nitrogen source present in the medium (Table 4). It can be seen that growth of this strain on the nitrogen source (NH4)2SO4 or asparagine had different effects upon both enzyme activities. With respect to cytosine deaminase activity, it was found to

3

Table

biotype

Pyrimidine salvage enzyme activities of Pseudomonas fluorescens F

Enzyme

Specific activity

1.17

Cytosine deaminase Nucleoside hydrolase Uridine phosphorylase Thymidine phosphorylase

ND

Cytidine deaminase

ND

25.16 ND

Specific activity was expressed as nmol uracil formed/min/mg protein at 30oC. Values shown repr€sent the average of two separate determinations within 1O% error. ND, not detectable under the given assay conditions.

32

Microbios

T. P. West

4 Effect of nitrogen source upon cytosine deaminase and nucleoside hydrolase activities

Table

Nitrogen source

Doubling time

Specific activity of: Cytosine deaminase

Nucleoside hydrolase

(NH4)2SO4

103

1.17

25.1 6

go

2.15

16.18

Asparagine

Doubling time of the strain in liquid culture was expressed in min. Specific activity of both enzymes was expressed as nmol uracil formed/min/mg protein at 30oC. Values shown represent the average of two separate determinations within 10% error.

increase nearly 2-fold when asparagine was substituted for (NHu)rSOu in the medium. In contrast, nucleoside hydrolase activity diminished when asparagine served as the sole nitrogen source instead of(NHo)2Soo. It should be mentioned that growth of this strain on asparagine was slightly faster than on (NHo)rSOo (Table 4). since nitrogen sources were able to influence both pyrimidine salvage activities in this pseudomonad, the possibility existed that a change in carbon source could also alter enzyme levels. As can be seen in Table 5, cells grown on glycerol instead of glucose in the medium maintained higher cytosine deaminase activity. on the other hand this change in carbon source had little effect upon nucleoside hydrolase activity (Table 5). Glycerol decreased the doubling time of the cells relative to that observed on glucose as the carbon source (Table 5). It is nor clear if changes in the pyrimidine salvage enzyme activiries can be attributed to variations in

the growth rate of this strain.

5 Effect of carbon source upon cytosine deaminase and nucleoside hydrolase activities

Table

Carbon source

Doubling time

Specific activity of: Cytosine deaminase

Glucose

103 83

1.17

25.16

1.81

25.31

Glycerol

Nucleoside hydrolase

Doubling time of the strain in liquid culture was expressed in min. Specific activity of both enzymes was expressed as nmol uracil formed/min/mg protein at 30oC. Values shown represent

the average of two separate determinations within

33

1Oo/o

enor.


Discussion The ability of P. fluorescezs biotype F to utilize pyrimidines can be used to differentiate it from the previously investigated fluorescent pseudomonads P. aeruginosa, P. chlororaphis, P. aureofaciens and. P. putida (West and Chu, 1985, 1986). It can be seen that this strain is the only fluorescent pseudomonad which can sustain its growth on deoxycytidine as a nitrogen source but unable to grow on thymine as a source of nitrogen. Moreover, it is the only fluorescent pseudomonad which has been found not to utilize dihydrouracil or dihydrothymine as a carbon source.

Another way to distinguish the pseudomonad in this study from the other fluorescent pseudomonads is to note its sensitivity to pyrimidine analogues. The sensitivity of P. fluoresuns biotype F to pyrimidine analogues was similar to the analogue sensitivities of P. chlororaphis and P. aureofaciens (!7est and Chu, 1985, 1986). These latter micro-organisms were found to be inhibited at low concentrations by 5-fluorouracil or 5-fluorouridine and by high concentrations of 5-fluorocytosine or 6-azauracil. The pseudomonad in this investigation diflered from P. chlororaphis and P. aureofaciens since its growth was inhibited by a high concentration of 5-fluorodeoxyuridine. The reductive pathway of pyrimidine base catabolism is known to occur in P. aeruginosa and P. facilis (Fink et al., 1954; Kramer and Kaltwasser, 1969). In this pathwaR dihydrouracil or dihydrothymine is formed from uracil or thymine, respectively. The final products of the pathway B-alanine and B-aminoisobutyric acid result from the degradation ofdihydrouracil and dihydrothymine, respectively (Vogels and van der Drift, 1976). It would appear that the reductive pathway is operating in P. fluorescezs biotype F since uracil, dihydrouracil, dihydrothymine, B-alanine or B-aminoisobutyric acid sustained its growth as a nitrogen source. Although growth studies are relevant to the taxonomic analysis of fluorescent pseudomonads, they also serve to clarify bacterial pyrimidine metabolism. It was observed that P. fluorescens biotype F grew on cytosine as a nitrogen source and was sensitive to 5-fluorocytosine. These findings indicated cytosine deaminase activity and its presence was confirmed by enzyme assay. This was not surprising since cytosine deaminase activity has been detected in another P. fluoresans strain (Sakai er a1.,1976). The ability of this biotype F strain to catabolize uridine or cytidine was most likely due to a reaction catalysed by nucleoside hydrolase or uridine phosphorylase. After assaying for both enzymes' activities, nucleoside hydrolase activity was detected. Other studies have also reported that nucleoside hydrolase is very active in P. fluorescens extracts (Terada et al., 1967; Sakai er al., L968). Considering the presence of cytosine deaminase and nucleoside hydrolase in P. fluorescens, the absence of cytidine deaminase was not unexpected. Its absence in this species

has also been confirmed previously (Sakai et al., 1976). The inability of P. fluorescens biotype F to use thymidine or deoxyuridine as a carbon source

is directly related to its lack of thymidine phosphorylase activity. Thymidine phosphorylase activity is undetectable in a number of pseudomonad species (Kelln and Warren, 7974; Potter et al., 1982; Carlson et al., 1985).

34

Microbios

T. P. West

Regulation of pyrimidirie biosynthesis at the level of gene expression has been investigated in P. aeruginosa and P. putida but such control did not seem to be significant (Isaac and Holloway, 1968; Condon et a1.,1976). Regulation at the level of enzyme synthesis has not been investigated for the pyrimidine salvage enzymes in the fluorescent pseudomonads. The effect of growth conditions on the pyrimidine salvage enzymes cytosine deaminase and nucleoside hydrolase was investigated. It appeared that cytosine deaminase was influenced by the change in nitrogen or carbon source to a greater degree than was nucleoside hydrolase. From the results, cytosine deaminase synthesis appeared to be repressed by the presence of ammonium ions but catabolite repression of deaminase synthesis also appeared to occur. In contrast, nucleoside hydrolase activity diminished in the absence of ammonium ions which precluded any chance of its repression by these ions. Since the carbon source ribose is a product ofthe hydrolase reaction, it was possible that this enzyme could be subject to catabolite repression. This does not appear to be a factor since hydrolase activity is unaffected by the change in carbon source from glucose to glycerol. Further study on the regulation of gene expression for the fluorescent pseudomonad pyrimidine salvage enzymes would seem to be warranted.

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ISAAC J. H. and Holloway B. W. 1968. Control of pyrimidine biosynthesis in Pseudomonas aeruginosa. J. Bact. 96 1732-41. KELLN R. A. and Warren R. A. J. 1974. Pyrimidine metabolism in Pseudomonas acidovorans.

Can. J. Microbiol. 20 427-33. KIM J. M., Shimizu S. and Yamada H. 1987. Cytosine deaminase that hydrolyzes creatinine to N-methylhydantoin in various cytosine deaminase-forming microorganisms. Arch. Microbiol.

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J. and Kaltwasser H. 1969. Verwertung von pyrimidinderivaten durch Hydrogenomonas facilis. ll. Abbau von thymin und uracil durch wildstamm und mutanten. KRAMER

Arch. Mikrobiol. 69 138-48. MUNCH-PETERSEN A. 1968. On the catabolism of deoxyribonucleosides in cells and cell extracts oI Escherichia coli. Eur. J. Biochem. 6 432-42. O'DONOVAN G. A. and Neuhard J. 1970. Pyrimidine metabolism in microorganisms. Bact. Rev.

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POTTER A. A., Musgrave D. R. and Loutit J. S. 1982. Thymine metabolism in Pseudomonas aeruginosa strain 1 : the presence of a salvage pathway. J. gen. Microbiol. 128 1 391 -40O. SAKAI T.. Watanabe T. and Chibata l. 1968. Metabolism of pyrimidine nucleotides in bacteria.

J. Ferment. Technol. 46 2O2-13.

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SAKAI T., Yu T. and Omata S. 1976. Distribution of enzymes related to cytidine degradation in bacteria. Agric. Biol. Chem. 4O 1893-5. STANIER R. Y. 1 947. Simultaneous adaption: a new technique for the study of metabolic

pathways. J. Bact.54 339-48. STANIER R. Y., Palleroni N. J. and Doudoroff M. 1966. The aerobic pseudomonads: a taxonomic study. J. gen. Microbiol. 43 159-271. TERADA M., Tatibana M. and Hayaishi O. 1967. Purification and properties of nucleoside hydrolase ftom Pseudomonas fluorescens. J. biol. Chem. 242 5578-88. VOGELS G. D. and van der Drift C. 1976. Degradation of purines and pyrimidines by microorganisms. Bact. Rev. 40 403-68. WESTT. P., Shanley M. S. and O'Donovan G. A. 1982. Purification and some properties of cytosine deaminase trom Salmonella typhimurium. Biochim. biophys. Acta 719 251 -e. WESTT. P. and Chu C. 1985. Growth oI Pseudomonas chlororaphis on pyrimidines and pyrimidine analogues. Microbios Letters 3O 73-7. WEST T. P. and Chu C. 1986. Utilization of pyrimidine and pyrimidine analogues by fluorescent pseudomonads. Microbios 47 1 49 -57.

Accepted 4 April 1988

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