JOURNAL OF BACTERIOLoGY, Feb. 1975, p. 401-405 Copyright 0 1975 American Society for Microbiology

Vol. 121, No. 2 Printed in U.S.A.

Uptake of Adenosine 5'-Monophosphate by Escherichia coli EZRA YAGIL* AND IFOR R. BEACHAM Department of Biochemistry, George S. Wise Center for Life Sciences, Tel-Aviv University, Tel-Aviv, Israel, and Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, United Kingdom Received for publication 16 September 1974

Adenosine 5'-monophosphate is dephosphorylated before its uptake by cells of Escherichia coli. This is demonstrated by using a radioactive double-labeled culture, and with a 5'-nucleotidase-deficient mutant strain. The adenosine formed is further phosphorolyzed to adenine as a prerequisite for its uptake and incorporation. The cellular localization of the enzymes involved in the catabolism of adenosine 5'-monophosphate is discussed.

In recent years several active transport mechanisms for phosphorylated metabolites have been elucidated, for example L-ai-glycerol phosphate (10) and several hexose phosphates (5, 8, 9, 25). These phosphorylated compounds are taken up by the bacterial cell in their intact form; their specific transport systems were detected in mutant strains impaired in the transport mechanism for the non-phosphorylated forms of the metabolites (for review see reference 18). Adenosine 5'-monephosphate (AMP) can readily serve as a carbon source for cells of Escherichia coli (7) and the adenine moiety is incorporated into the nucleic acids of the cell (21). The enzyme 5'-nucleotidase (EC 3.1.3.5, known also as uridine diphosphate sugar hydrolase) is a surface enzyme located between the two membranes of the cell envelope (11) and it cleaves AMP to adenosine and inorganic phosphate. It is therefore not known whether AMP can enter the cell in its intact form or whether it must be dephosphorylated before its uptake, though the latter possibility is strongly suggested by the finding that mutants deficient in 5'-nucleotidase are unable to use AMP as a carbon source (2). In this communication we provide direct evidence that dephosphorylation by 5'-nucleotidase is obligatory for uptake of the adenosine moiety. Furthermore, we confirm previous reports on Salmonella cells (15) and on membrane vesicles of E. coli (12) that the adenosine formed as a result of the dephosphorylation is further cleaved to adenine before it is incorporated by the cell. MATERIALS AND METHODS Bacterial strains. Table 1 lists the bacterial strains used, which were all E. coli K-12 strains. 401

Growth conditions. The cells were grown at 37 C in phosphate-buffered minimal M9 medium (22). Glucose concentration was 0.2%, the concentration of required amino acids was 20 gg/ml, and of thiamine, 2

Ag/ml. Uptake experiments. Radioactive-labeled compounds were added to exponentially growing cell cultures. In the double-label experiments portions of 0.5 ml were removed at intervals and filtered through nitrocellulose filters (0.45 Am pore size). The filters were immediately washed at room temperature with 20 ml of M9 buffer (M9 medium without glucose) and dried under a lamp. The filtrate was counted by placing 50 uliters onto another filter, which was then dried. The filters were counted in a scintillation spectrometer using toluene scintillation solution. In the experiments labeled with a single radioactive compound, two kinds of samples were taken at intervals: for the measurement of label incorporated into the nucleic acids, 0.1 ml was added to 1.0 ml of 5% cold trichloroacetic acid, and after at least 30 min the trichloroacetic acid was filtered and the filter was washed with 20 ml of cold water, dried, and counted as described. To measure incorporation by whole cells, 0.1 ml of the culture was put directly onto a filter, washed with 20 ml of M9 buffer, dried, and TABLE 1. Characteristics of Escherichia coli strains Strain

AB1157-1 5A-1 4K 4K-26

Sex

Genotype

F- thr ara leu proA lac tsx gal his strA xyl mtl arg thi upp F- thr ara leu proA lac tsx gal his strA xyl mtl arg thi uppa ushe F- serB argA thr leu thi strA F- pup udp tpp dra upp thr l leu thi strA

Refer2 2 26

26

a upp, UMP pyrophosphorylase (uracil phosphoribosyl transferase). b ush, Uridine diphosphate sugar hydrolase (5'nucleotidase).

402

YAGIL AND BEACHAM

counted.

J. BACTERIOL.

60

Radiochemicals. [32PJAMP, ammonium salt, [23H]AMP, ammonium salt, and [8-14C]adenosine sulfate were purchased from the Radiochemical Centre,

(A) WHOLE CELLS

40

Amersham, Bucks, U.K.

AB 1157-1 (+) RESULTS 20 To determine whether AMP is taken up in its 5A-1 (ush) intact form or whether it must be dephosphorylated before its uptake, we added to a 0 growing culture of cells a mixture of [32P]AMP and [3H ]AMP labeled at position 2 of the .E'D adenine moiety. The disappearance of the label r (B)NUCLEIC ACIDS from the medium (Fig. 1A) and its uptake by 40 0 0 the cells (Fig. 1B) were followed. Only the tritium label was removed from the medium while being incorpoiated into the cells; the 32P 20 o label remained entirely in the medium, and no a incorporation was detected. This shows that AMP is dephosphorylated before its uptake. 0 Uptake of inorganic 32P was not observed since 0. the growth medium contained a large excess of unlabeled inorganic phosphate. We have recently described the isolation and (C) PCooL 2-. properties of 5'-nucleotidase-deficient mutants (2). Figure 2 shows the ability to take up and incorporate labeled [3H ]AMP by such a mutant I0 (5A-1) as compared to the parental wild-type strain (AB1157-1). In Fig. 2A uptake is shown by 0 whole cells, which includes both the cellular 10 5 15 pool of the labeled nucleotide and incorporation MINUTES into nucleic acids. Figure 2B shows the incorporation into the nucleic acid only (acid-precipitaFIG. 2. Uptake and incorporation of label by a ble material). The difference between the val- 5'-nucleotidase-deficient mutant (Ush, strain 5A-1) ues in Fig. 2A and B is considered as unincor- and its Ush+ parental strain (AB1157-1) suppleporated soluble label taken up into the cellular mented with [3H1AMP. Logarithmically growing cul.

_

%

400 [-

-

_

---

(A)

(8)

p300

300

E

2 002

200

tures (-2.7 x 108 cells/ml) were labeled with [3H]AMP (0.5 MM; 21 mCi/gmol) and uptake by whole cells (A) and incorporation into nucleic acids (B) were measured as described in Materials and Methods. Part (C) shows the difference between (A) and (B).

pool (Fig. 2C). It is clear that the uptake of AMP is abolished by the 5'-nucleotidase-deficient mutant strain, which confirms that AMP must be cleaved before its uptake, and that 5'-nucleotidase is specifically involved. The question arises whether the adenosine formed as a result of AMP dephosphorylation MINUTES MINUTES can now be taken up in its intact form or must FIG. 1. Uptake of label by a culture of strain be further cleaved to adenine. Evidence for the AB1157-1 supplemented with a mixture of [32PJAMP (0.33 uM; 22.6 qCi/umol) and [3H1AMP (0.33 uM; latter possibility was already given for cells of 0.63 mCi/Mgmol). The labeled compounds were added Salmonella (15) and membrane vesicles of E. to a growing culture (-4 x 10' cells/ml) and at coli (12). To investigate this question we used a intervals portions were filtered through a membrane mutant strain lacking purine nucleoside phos(see Materials and Methods). (A) label remaining in phorylase activity (Pup-, EC 2.4.2.1) which was filtrate; (B) label taken up by the cells. isolated by the method described by Ahmad A

A

VOL. 121, 1975

403

UPTAKE OF AMP BY E. COLI

and Pritchard (1). This enzyme which phosphorolyzes adenosine to adenine and ribose1-phosphate is inducible by adenosine (19). Figure 3 shows the uptake and incorporation of labeled adenosine and of adenine in an induced culture of Pup- cells as compared to the parental Pup+ strain. As previously, part A shows uptake and incorporation by whole cells, part B shows incorporation only, and part C, which is the difference between A and B, indicates the pool of free label within the cells. Only the uptake of adenosine, and not that of adenine, is

significantly reduced by the Pup- mutation. This clearly demonstrates that purine nucleoside phosphorylase is essential for substantial uptake and incorporation of adenosine, i.e., adenosine must be first cleaved to adenine. The residual incorporation and uptake to labeled adenosine observed in the Pup- strain could either be due to leakiness of the pup mutation or due to uptake of adenosine (20, 21; see Discussion) and its conversion to nucleotides by the appropriate kinases.

DISCUSSION The experiments reported by Lichtenstein, Barner, and Cohen (17) showed that cytidine 5'-monophosphate is dephosphorylated before its uptake by E. coli. We have shown that a purine nucleotide, AMP, is likewise dephosphorylated before uptake, and that 5'-nucleotidase is exclusively involved. For the efficient utilization of the adenosine formed as a result of the dephosphorylation, it must first be cleaved by purine nucleoside phosphorylase to adenine and ribose-1-phosphate. Hochstadt-Ozer (12) likewise showed that membrane vesicles of E. coli cannot take up ° 6 0 adenosine unless it is first cleaved to adenine, 5 0 and the latter is directly converted to AMP by a r4 membrane-associated phosphoribosyl transfer(B) NUClLEIC ACIDS ase. Furthermore, Hoffmeyer and Neuhard (15) E have found that the purine requirement of 2 purine auxotrophs of Salmonella typhimurium, I/ which are also defective in purine nucleoside phosphorylase activity (Pup-), cannot be satis0 fied by adenosine or deoxyadenosine. These investigators have suggested that the metabolic 2 inertness of adenosine in a Pup- mutant strain (C) PC)OL is due to the lack of adenosine kinase. The data of Fig. 3, which show that the Pup- mutation reduces the soluble pool when adenosine is provided (Fig. 3C), suggest that intact adenosine is not taken up by the cell. In contrast, 0 0 6 18 24 30 several investigators have proposed the exist12 6 12 18 24 30 MINUTES ence of an uptake mechanism of adenosine (and FIG. 3. Uptake and incorporation of label by a other nucleosides) (6, 16, 20, 21). According to purine nucleoside phosphorylase-deficient mutant the data discussed above, such an uptake (Pup-, strain 4K-26) and its Pup+ parental strain mechanism is not a major reaction in the utiliza(4K) in cultures labeled with [14C]adenosine or tion of adenosine. Furthermore, its existence ["4C]adenine. The cultures were grown logarithmi- has not yet been directly proven, since in all cally for one generation in the presence of 1 mM reported investigations the nucleosides used adenosine and then centrifuged, washed, and resus- were radioactively labeled in the base moiety pended in fresh M9 minimal medium to a cell density only. The pathway by which AMP is utilized by of 1. 7 x 108 cells/m 1. [14C ]adenosine or [14C ]adenine E. coli is proposed in Fig. 4. (11.3 uM; 5.88 MCi/mmol) were added. Uptake by The cellular localization of 5'-nucleotidase, whole cells (A) and incorporation into nucleic acids (B) were measured as described in Materials and purine nucleoside phosphorylase,. and adenine Methods. Part (C) shows the difference between (A) phosphoribosyl transferase is noteworthy and is pointed out in Fig. 4. All three enzymes are and (B). a

c

/g

0

404

YAGIL AND BEACHAM

J. BACTERIOL.

We thank Nava Silberstein for skillful technical assistance.

5

FIG. 4. Catabolism and uptake of AMP across the cell envelope. Symbols: (OM) outer membrane, (IM) inner membrane, (Ado) adenosine, (A) adenine, (PRPP) phosphoribosyl pyrophosphate, (Ush) UDPsugar hydrolase (5'-nucleotidase), (Pup) purine nucleoside phosphorylase, (Apt) adenine phosphoribosyl transferase.

selectively released into the medium when the cells are osmotically shocked (3, 11, 14, 20), which indicates a "surface" localization. In contrast, when the cells are converted into spherophasts by the action of lysozyme and ethylenediaminetetraacetic acid (a treatment which ruptures the outer membrane; reference 11), 5'-nucleotidase, but not purine nucleoside phosphorylase, is released into the medium (3, 4, 23). This indicates that 5'-nucleotidase is located unbound in the periplasmic space, whereas purine nucleoside phosphorylase, though being surface localized, is not periplasmic. A second line of evidence suggesting that purine nucleoside phosphorylase as well as adenine phosphoribosyl transferase are surface located is provided by the finding that both activities are retained in membrane vesicles formed from osmotically disrupted spheroplasts (12, 13). Taketo and Kuno (23), on the other hand, were unable to detect specific binding of purine phosphorylase to membranes. Thus, although in Fig. 4 these two enzymes are shown in association with the inner membrane, the nature of their surface localization is not yet clear. ACKNOWLEDGMENTS Part of this work was supported by the Science Research Council (United Kingdom). E. Y. was supported by a Fellowship of the European Molecular Biology Organisation.

LITERATURE CITED 1. Ahmad, S. I., and R. H. Pritchard. 1969. A map of four genes specifying enzymes involved in catabolism of nucleosides and deoxynucleosides in Escherichia coli. Mol. Gen. Genet. 104:351-359. 2. Beacham, I. R., R. Kahana, L. Levy, and E. Yagil. 1973. Mutants of Escherichia coli K-12 "cryptic" or deficient in 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3-nucleotidase (cyclic phosphodiesterase) activity. J. Bacteriol. 116:957-964. 3. Beacham, I. R., E. Yagil, K. Beacham, and R. H. Pritchard. 1971. On the localization of enzymes of deoxynucleoside catabolism in Escherichia coli. FEBS Lett. 16:77-80. 4. Cerny, G., and M. Teuber. 1972. Comparative polyacrylamide electrophoresis of periplasmic proteins released from gram-negative bacteria by polymyxin B. Arch. Mikrobiol. 82:361-370. 5. Dietz, G. W., and L. A. Heppel. 1971. Studies on the uptake of hexose phosphates. III. Mechanism of uptake of glucose-i-phosphate in Escherichia coli. J. Biol. Chem. 246:2891-2897. 6. Doskocil, J. 1974. Inducible nucleoside permease in Escherichia coli. Biochem. Biophys. Res. Commun. 56:997-1003. 7. Eggleston, L. V., and H. A. Krebs. 1959. Permeability of Escherichia coli to ribose and ribose nucleotides. Biochem. J. 73:264-270. 8. Fraenkel, D. G., F. Falcoz-Kelley, and B. L. Horecker. 1964. The utilization of glucose-6-phosphate by glucokinaseless and wild type strains of Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 52:1207-1213. 9. Hagihira, H., T. H. Wilson, and E. C. C. Lin. 1963. Studies on the glucose-transport system in Escherichia coli with a-methylglucoside as substrate. Biochim. Biophys. Acta 78:505-515. 10. Hayashi, S. I., J. P. Koch, and E. C. C. Lin. 1964. Active transport of L-a-glycerophosphate in Escherichia coli. J. Biol. Chem. 239:3098-3105. 11. Heppel, L. A. 1971. The concept of periplasmic enzymes, p. 223-247. In L. I. Rothfield (ed.), Structure and function of biological membranes. Academic Press Inc., New York. 12. Hochstadt-Ozer, J. 1972. The regulation of purine utilization in bacteria. IV. Role of membrane-localized and pericytoplasmic enzymes in the mechanism of purine nucleoside transport across isolated Escherichia coli membranes. J, Biol. Chem. 247:2419-2426. 13. Hochstadt-Ozer, J., and E. R. Stadman. 1971. The regulation of purine utilization in bacteria. II. Adenine phosphoriboyltransferase in isolated membrane preparations and its role in transport of adenine across the membrane. J. Biol. Chem. 246:5304-5311. 14. Hochstadt-Ozer, J., and E. R. Stadman. 1971. The regulation of purine utilization in bacteria. III. The involvement of purine phosphoribosyl transferases in the uptake of adenine and other nucleic acid precursors by intact resting cells. J. Biol. Chem. 246:5312-5320. 15. Hoffmeyer, J., and J. Neuhard. 1971. Metabolism of exogenous purine bases and nucleosides by Salmonella typhimurium. J. Bacteriol. 106:14-24. 16. Komatsu, Y., and K. Tanaka. 1972. A showdomycinresistant mutant of Escherichia coli K-12 with altered nucleoside transport character. Biochim. Biophys. Acta 288:390-403. 17. Lichtenstein, J., H. D. Barner, and S. S. Cohen. 1960. The metabolism of exogenously supplied nucleotides

VOL. 121, 1975

UPTAKE OF AMP BY E. COLI

by Escherichia coli. J. Biol. Chem. 235:457-465. 18. Lin, E. C. C. 1970. The genetics of bacterial transport systems. Annu. Rev. Genet. 4:255-262. 19. Munch-Peterson, A. 1968. On the catabolism of deoxyribonucleosides in cells and cell extracts of Escherichia coli. Eur. J. Biochem. 6:432-442. 20. Petersen, R. N., J. Boniface, and A. L. Koch. 1967. Energy requirements, interactions and distinctions in the mechanisms for transport of various nucleosides in Escherichia coli. Biochim. Biophys. Acta 135:771-783. 21. Petersen, R. N., and A. L. Koch. 1966. The relationship of adenosine and inosine transport in Escherichia coli. Biochim. Biophys. Acta 126:129-145. 22. Pritchard, R. H., and K. G. Lark. 1964. Induction of

23. 24.

25. 26.

405

replication by thymine starvation at the chromosome origin in Escherichia coli. J. Mol. Biol. 9:288-307. Taketo, A., and S. Kuno. 1972. Internal localization of nucleoside catabolic enzymes in Escherichia coli. J. Biochem. 72:1557-1563. Taylor, A. L., and C. D. Trotter. 1972. Linkage map of Escherichia coli strain K-12. Bacteriol. Rev. 36:504-524. Winkler, H. H. 1966. A hexose-phosphate transport system in Escherichia coli. Biochim. Biophys. Acta 117:231-240. Yagil, E., and A. Rosner. 1971. Phosphorolysis of 5fluoro-2'-deoxyuridine in Escherichia coli and its inhibition by nucleosides. J. Bacteriol. 108:760-764.

Uptake of adenosine 5'-monophosphate by Escherichia coli.

Adenosine 5'-monophosphate is dephosphorylated before its uptake by cells of Escherichia coli. This is demonstrated by using a radioactive double-labe...
611KB Sizes 0 Downloads 0 Views