Uptake of Adenosine 5'-Monophosphate by Escherichia coli

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1 JOURNAL OF BACTERIOLoGY, Feb. 1975, p Copyright 1975 American Society for Microbiology Vol. 11, No. 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 SY3 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 (1) and several hexose phosphates (5, 8, 9, 5). 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 (1). The enzyme 5'-nucleotidase (EC , 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 (). 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 (1) 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-1 strains. Growth conditions. The cells were grown at 37 C in phosphate-buffered minimal M9 medium (). Glucose concentration was.%, the concentration of required amino acids was gg/ml, and of thiamine, Ag/ml. Uptake experiments. Radioactive-labeled compounds were added to exponentially growing cell cultures. In the double-label experiments portions of.5 ml were removed at intervals and filtered through nitrocellulose filters (.45 Am pore size). The filters were immediately washed at room temperature with ml of M9 buffer (M9 medium without glucose) and dried under a lamp. The filtrate was counted by placing 5 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,.1 ml was added to 1. ml of 5% cold trichloroacetic acid, and after at least 3 min the trichloroacetic acid was filtered and the filter was washed with ml of cold water, dried, and counted as described. To measure incorporation by whole cells,.1 ml of the culture was put directly onto a filter, washed with ml of M9 buffer, dried, and TABLE 1. Characteristics of Escherichia coli strains Strain Sex Genotype Refer- AB F- thr ara leu proa lac tsx gal his stra xyl mtl arg thi upp 5A-1 F- thr ara leu proa lac tsx gal his stra xyl mtl arg thi uppa ushe 4K F- serb arga thr leu thi stra 6 4K-6 F- pup udp tpp dra upp thr 6 l leu thi stra a upp, UMP pyrophosphorylase (uracil phosphoribosyl transferase). b ush, Uridine diphosphate sugar hydrolase (5'- nucleotidase). 41

2 4 YAGIL AND BEACHAM J. BACTERIOL. counted. Radiochemicals. [3PJAMP, ammonium salt, [- 3H]AMP, ammonium salt, and [8-14C]adenosine sulfate were purchased from the Radiochemical Centre, Amersham, Bucks, U.K. RESULTS To determine whether AMP is taken up in its intact form or whether it must be dephosphorylated before its uptake, we added to a growing culture of cells a mixture of [3P]AMP and [3H ]AMP labeled at position of the adenine moiety. The disappearance of the label from the medium (Fig. 1A) and its uptake by the cells (Fig. 1B) were followed. Only the tritium label was removed from the medium while being incorpoiated into the cells; the 3P label remained entirely in the medium, and no incorporation was detected. This shows that AMP is dephosphorylated before its uptake. Uptake of inorganic 3P was not observed since the growth medium contained a large excess of unlabeled inorganic phosphate. We have recently described the isolation and properties of 5'-nucleotidase-deficient mutants (). Figure shows the ability to take up and incorporate labeled [3H ]AMP by such a mutant (5A-1) as compared to the parental wild-type strain (AB1157-1). In Fig. A uptake is shown by whole cells, which includes both the cellular pool of the labeled nucleotide and incorporation into nucleic acids. Figure B shows the incorporation into the nucleic acid only (acid-precipitable material). The difference between the values in Fig. A and B is considered as unincorporated soluble label taken up into the cellular 4 [ _ (A) (8) 3 p3 Ẹ'D. _ o a (A) WHOLE CELLS AB (+) 5A-1 (ush) r (B)NUCLEIC ACIDS -. % (C) PCooL I FIG.. Uptake and incorporation of label by a 5'-nucleotidase-deficient mutant (Ush, strain 5A-1) and its Ush+ parental strain (AB1157-1) supplemented with [3H1AMP. Logarithmically growing cultures (-.7 x 18 cells/ml) were labeled with [3H]AMP (.5 MM; 1 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). E - A A FIG. 1. Uptake of label by a culture of strain AB supplemented with a mixture of [3PJAMP (.33 um;.6 qci/umol) and [3H1AMP (.33 um;.63 mci/mgmol). The labeled compounds were added to a growing culture (-4 x 1' cells/ml) and at intervals portions were filtered through a membrane (see Materials and Methods). (A) label remaining in filtrate; (B) label taken up by the cells. pool (Fig. C). 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 can now be taken up in its intact form or must be further cleaved to adenine. Evidence for the latter possibility was already given for cells of Salmonella (15) and membrane vesicles of E. coli (1). To investigate this question we used a mutant strain lacking purine nucleoside phosphorylase activity (Pup-, EC.4..1) which was isolated by the method described by Ahmad

3 VOL. 11, 1975 UPTAKE OF AMP BY E. COLI 43 and Pritchard (1). This enzyme which phosphorolyzes adenosine to adenine and ribose- 1-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 6 5 a r4 E c (B) NUClLEIC ACIDS (C) PC)OL /g FIG. 3. Uptake and incorporation of label by a purine nucleoside phosphorylase-deficient mutant (Pup-, strain 4K-6) and its Pup+ parental strain (4K) in cultures labeled with [14C]adenosine or ["4C]adenine. The cultures were grown logarithmically for one generation in the presence of 1 mm adenosine and then centrifuged, washed, and resuspended in fresh M9 minimal medium to a cell density of 1. 7 x 18 cells/m 1. [14C ]adenosine or [14C ]adenine (11.3 um; 5.88 MCi/mmol) were added. 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). I/ 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 (, 1; 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 (1) likewise showed that membrane vesicles of E. coli cannot take up adenosine unless it is first cleaved to adenine, and the latter is directly converted to AMP by a membrane-associated phosphoribosyl transferase. Furthermore, Hoffmeyer and Neuhard (15) have found that the purine requirement of purine auxotrophs of Salmonella typhimurium, which are also defective in purine nucleoside phosphorylase activity (Pup-), cannot be satisfied by adenosine or deoxyadenosine. These investigators have suggested that the metabolic inertness of adenosine in a Pup- mutant strain 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, several investigators have proposed the existence of an uptake mechanism of adenosine (and other nucleosides) (6, 16,, 1). According to the data discussed above, such an uptake mechanism is not a major reaction in the utilization of adenosine. Furthermore, its existence has not yet been directly proven, since in all reported investigations the nucleosides used were radioactively labeled in the base moiety only. The pathway by which AMP is utilized by E. coli is proposed in Fig. 4. The cellular localization of 5'-nucleotidase, purine nucleoside phosphorylase,. and adenine phosphoribosyl transferase is noteworthy and is pointed out in Fig. 4. All three enzymes are

4 44 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, ), 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, 3). 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 (1, 13). Taketo and Kuno (3), 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 A map of four genes specifying enzymes involved in catabolism of nucleosides and deoxynucleosides in Escherichia coli. Mol. Gen. Genet. 14: Beacham, I. R., R. Kahana, L. Levy, and E. Yagil Mutants of Escherichia coli K-1 "cryptic" or deficient in 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3-nucleotidase (cyclic phosphodiesterase) activity. J. Bacteriol. 116: Beacham, I. R., E. Yagil, K. Beacham, and R. H. Pritchard On the localization of enzymes of deoxynucleoside catabolism in Escherichia coli. FEBS Lett. 16: Cerny, G., and M. Teuber Comparative polyacrylamide electrophoresis of periplasmic proteins released from gram-negative bacteria by polymyxin B. Arch. Mikrobiol. 8: Dietz, G. W., and L. A. Heppel Studies on the uptake of hexose phosphates. III. Mechanism of uptake of glucose-i-phosphate in Escherichia coli. J. Biol. Chem. 46: Doskocil, J Inducible nucleoside permease in Escherichia coli. Biochem. Biophys. Res. Commun. 56: Eggleston, L. V., and H. A. Krebs Permeability of Escherichia coli to ribose and ribose nucleotides. Biochem. J. 73: Fraenkel, D. G., F. Falcoz-Kelley, and B. L. Horecker The utilization of glucose-6-phosphate by glucokinaseless and wild type strains of Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 5: Hagihira, H., T. H. Wilson, and E. C. C. Lin Studies on the glucose-transport system in Escherichia coli with a-methylglucoside as substrate. Biochim. Biophys. Acta 78: Hayashi, S. I., J. P. Koch, and E. C. C. Lin Active transport of L-a-glycerophosphate in Escherichia coli. J. Biol. Chem. 39: Heppel, L. A The concept of periplasmic enzymes, p In L. I. Rothfield (ed.), Structure and function of biological membranes. Academic Press Inc., New York. 1. Hochstadt-Ozer, J 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. 47: Hochstadt-Ozer, J., and E. R. Stadman 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. 46: Hochstadt-Ozer, J., and E. R. Stadman 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. 46: Hoffmeyer, J., and J. Neuhard Metabolism of exogenous purine bases and nucleosides by Salmonella typhimurium. J. Bacteriol. 16: Komatsu, Y., and K. Tanaka A showdomycinresistant mutant of Escherichia coli K-1 with altered nucleoside transport character. Biochim. Biophys. Acta 88: Lichtenstein, J., H. D. Barner, and S. S. Cohen The metabolism of exogenously supplied nucleotides

5 VOL. 11, 1975 UPTAKE OF AMP BY E. COLI 45 by Escherichia coli. J. Biol. Chem. 35: Lin, E. C. C The genetics of bacterial transport systems. Annu. Rev. Genet. 4: Munch-Peterson, A On the catabolism of deoxyribonucleosides in cells and cell extracts of Escherichia coli. Eur. J. Biochem. 6: Petersen, R. N., J. Boniface, and A. L. Koch Energy requirements, interactions and distinctions in the mechanisms for transport of various nucleosides in Escherichia coli. Biochim. Biophys. Acta 135: Petersen, R. N., and A. L. Koch The relationship of adenosine and inosine transport in Escherichia coli. Biochim. Biophys. Acta 16: Pritchard, R. H., and K. G. Lark Induction of replication by thymine starvation at the chromosome origin in Escherichia coli. J. Mol. Biol. 9: Taketo, A., and S. Kuno Internal localization of nucleoside catabolic enzymes in Escherichia coli. J. Biochem. 7: Taylor, A. L., and C. D. Trotter Linkage map of Escherichia coli strain K-1. Bacteriol. Rev. 36: Winkler, H. H A hexose-phosphate transport system in Escherichia coli. Biochim. Biophys. Acta 117: Yagil, E., and A. Rosner Phosphorolysis of 5- fluoro-'-deoxyuridine in Escherichia coli and its inhibition by nucleosides. J. Bacteriol. 18: