the biological chemistry journal of no. vol 157, of ... · the journal of biological chemistry...

THE JOURNAL O F BIOLOGICAL CHEMISTRY

Printed in U S A . Vol 157, No. 23. Issue of December 10. pp. 14565-14575, 1982

Sugar Transport by the Bacterial Phosphotransferase System PREPARATION AND CHARACTERIZATION OF MEMBRANE VESICLES FROM MUTANT AND WILD TYPE SALMONELLA TYPHIMURIUM*

(Received for publication, September 1, 1981)

Daniel A. BeneskiSg, Thomas P. MiskoS, and Saul Roseman From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

Modifications of published procedures (reviewed by Kaback, H. R. (1974) Science (Wash. D. C.) 186,882-892) were developed for preparing membrane vesicles from Salmonella typhimurium. The preparations consisted largely of closed, unilamellar structures and contained inner membrane with little to no contamination by outer membrane or cell wall. A variety of cytoplasmic proteins was assayed in the membrane preparations, and they were found to be present at low to trace levels, whereas other proteins known to be associated with membranes were found at high levels (with respect to specific activities) in the vesicle preparations. At least 90% of the vesicles appeared to be oriented right-side- out; we do not know whether the remaining 10% represents closed vesicles oriented inside-out or “leaky” right-side-out vesicles.

The vesicle preparations were impermeable to both low and high molecular weight solutes, for example, to both intra- and extravesicular sucrose. In double label experiments, the vesicle volumes were found to be about 6 pl/mg of protein for preparations isolated from the wild type strain, and about 4.5 pl/mg of protein for vesicles isolated from a mutant, SB2950, deleted in ptsH, ptsl, and crr genes (proteins HPr, Enzyme I, and I I P , respectively).

One advantage of S. typhimurium over Escherichia coli for these studies is that the former can be induced to take up phosphoenolpyruvate. This may be the reason that S. typhimurium vesicles transported methyl a-glucoside at 4- to 100-fold the rates reported for vesicles from E. coli, while uptake rates of proline were comparable in the two types of preparations.

Vesicles from strain SB2950 were unable to take up methyl a-glucoside, but the transport (and phosphorylating) system was reconstituted in the vesicles by trap- ping the soluble purified proteins inside the vesicles during preparation of the latter. All three proteins were required for reconstitution.

Studies with intra- and extravesicular soluble proteins of the phosphoeno1pyruvate:glucose phospho-

* This work was supported by Grant CA 21901 from the National Institutes of Health. This is Contribution 1170 from the McCollum- Pratt Institute. This is Paper XXIII in the series, “Sugar Transport by the Bacterial Phosphotransferase System.” The preceding paper in this series is Ref. 44. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Training Grant GM-57 from the National Institutes of Health. The first two authors (D. A. B. and T. P. M.) contributed equally to the work presented in this paper. 0 Present address, Jefferson Medical College of Thomas Jefferson

University, Philadelphia, PA 19107.

transferase system showed that the IIMa” complex, which phosphorylates glucose, 2-deoxyglucose, and other sugars, is symmetrically oriented in the membranes. That is, this complex could phosphorylate 2- deoxyglucose when supplemented with Enzyme I and HPr either inside or outside of the membranes, and the sugar phosphate was found on the same side of the membranes as the soluble phosphotransferase system proteins. The integral membrane protein, II-BGIc, which phosphorylates glucose and methyl a-glucoside, showed contrasting behavior. Methyl a-glucoside phosphate was formed (intravesicularly) only when the soluble proteins (Enzyme I, HPr, and IIIG“) were located inside the vesicles. Thus, II-BG’” appears to be asymmetrically oriented in the membranes.

The phosphotransferase system in bacteria is responsible for the concomitant phosphorylation of sugars and their transport across the cell membrane (1). As described in the accompanying papers (2-4), the complete system in Salmonella typhimurium comprises four components, three of which have been purified and characterized; a considerable amount of kinetic data has been obtained on the interaction between these components and on the transfer of the phosphoryl group from phosphoenolpyruvate to the appropriate sugar. Never- theless, since transport is a vectorial process and purification of the components necessitates their isolation from the cell, it is not possible to study in cellular extracts precisely how the PTS’ mediates translocation of PTS sugars. Furthermore, the exact mechanism by which the PTS regulates the uptake of non-PTS sugars by other membrane transport systems has not been elucidated with purified components. For these reasons we turned to a study of bacterial membrane vesicles, which, since fist described by Kaback and Stadtman ( 5 ) , have proved to be extremely useful for studying a variety of transport systems (6-10). Ideally, the vesicles are closed systems composed of the cytoplasmic membrane, and as such they retain the integral membrane proteins while containing only traces of cytoplasmic proteins. I t is possible to load the vesicles with desired proteins under specified conditions, however, and in this manner to produce a defined system for

’ The abbreviations used are: PTS, phosphoeno1pyruvate:glycose phosphotransferase system; P-enolpyruvate, phosphoenolpyruvate (PEP in Miniprint); PPO, 2,5-diphenyloxazole; dimethyl-POPOP, 1,4- bis-2-(methyl-5-phenyloxazolyl)benzene; KDO, 2-keto-3-deoxy-~-oc- tanoate; HPr, histidine-containing phosphocarrier protein of the phosphotransferase system. The designations of the components of the phosphotransferase system are described in the accompanying manuscript (2). Unless otherwise designated all sugars are of the D configuration and are pyranosides.

14565

14566 Sugar Transport by the Bacterial Phosphotransferase System. XXIII

studying selected aspects of the mechanism of translocation (11, 12). In studying the PTS, for example, the membrane- bound sugar-specific proteins remain associated with the vesicle, while the soluble proteins may be trapped inside. This permits not only the possibility of investigating the interaction of soluble and membrane-bound proteins in a defined setting, but also provides a method for determining the orientation of the respective specific protein in the membrane. Of particular interest also is the possibility of evaluating PTS activities in vesicles obtained from mutants which lack specific PTS proteins.

All of the studies suggested above require the use of well characterized vesicles which are not leaky to high or low molecular weight solutes (at least over the time course of the experiments). Since the preceding papers in this series de- scribe the purification and characterization of PTS components from S. typhimurium (2-4) and also because we have a variety of well characterized mutants of this organism (I), we turned our attention to the preparation of vesicles from S. typhimurium. A major advantage of this organism, as described under "Results," is that, unlike Escherichia coli, it has an inducible P-enolpyruvate transport system (13). To our knowledge, S. typhimurium vesicles have been studied only rarely (6-9, 11, 14-18), and there is no published report in which the characterization of such vesicles with respect to volume, purity, content of soluble cytoplasmic enzymes, etc. is described. In fact, our initial preparations of S. typhimurium vesicles, prepared by standard methods, were quite leaky. We therefore developed modified methods for preparing vesicles from S. typhimurium, and used the vesicles to study transport.

This paper describes the preparation and characterization of vesicles from S. typhimurium. The vesicles transported methyl a-glucoside at a rate much higher than that previously reported with other membrane vesicles. Further, the vesicle preparations appeared to be contaminated with only small amounts of cytoplasmic proteins and outer membrane, they were oriented right-side-out to the extent of at least 90%, and they did not leak low molecular weight solutes. Vesicles prepared from a deletion mutant lacking Enzyme I, HPr, and IIIG1c did not take up methyl a-glucoside, but when these proteins were incorporated into the vesicles, the reconstituted vesicles translocated the glucoside; all of the sugar taken up was phosphorylated. Finally, studies with intra- and extravesicular PTS proteins showed that II-BG1' is asymmetrically located in the membrane, whereas the IIMm complex can react with phospho-HPr on either side of the membrane.

EXPERIMENTAL PROCEDURES'

RESULTS

Uptake Studies-To determine whether transport systems other than the PTS functioned in the vesicles, we measured proline as well as methyl a-glucoside uptake in vesicles of glucose-grown S. typhimurium SB3507. Table V compares initial rates of L-proline and methyl a-glucoside uptake in our vesicles with the rates in vesicles obtained by two other methods. The rates for proline uptake were comparable to those published for several preparations of E. coli vesicles and

* Portions of this paper (including "Experimental Procedures," part of "Results," Tables I-V, Figs. 1-7, and Footnote 3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 81-2152, cite authors, and include a check or money order for $14.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

were higher than values reported for another S. typhimurium preparation (47-50).

More important, the uptake of methyl a-glucoside by S. typhimurium vesicles in our studies was much more rapid than that obtained with vesicles from E. coli (39). The marked increase of methyl a-glucoside uptake in S. typhimurium vesicles was probably due to the presence of the inducible P- enolpyruvate transport system, which provides a high internal concentration of P-enolpyruvate.

Reconstitution of Transport Activity in Vesicles Isolated from PTS Mutants-In an accompanying report (40) and in earlier work (I), we showed not only that the soluble proteins of the PTS are required for sugar uptake by intact cells of S. typhimurium and Staphylococcus aureus, but also that mutant strains lacking the soluble proteins were unable to catalyze facilitated diffusion at a significant rate (51-53), despite the fact that the cells contained the relevant membrane proteins. The accompanying paper (40) further shows that mutants defective in II-BG" will not take up methyl a-glucoside, and those lacking the II'" complex will not take up 2- deoxyglucose. Our model (2) based on the results with intact cells states that the known PTS proteins are required for transport of the PTS sugars, that the sugars are phosphorylated concomitant with uptake, and that no other proteins are required. The intact membrane vesicles above provide an excellent system with which to study this model. For example, strain SB2950 is a mutant with a deletion in Enzyme I, HPr, and 1IIG", but contains the membrane protein II-BGlC. Thus, vesicles isolated from SB2950 should be unable to take up methyl a-glucoside. However, if the soluble PTS proteins could be loaded into the vesicles during their preparation (as described under "Methods"; see Miniprint), then transport of the glucoside should be restored.

Reconstitution experiments were therefore conducted, whereby SB2950 spheroplasts were lysed in the presence of crude extract from wild type cells, or of partially purified Enzyme I, HPr, IIIGIc, or of the homogeneous proteins. In each case, the reconstituted vesicles were able to take up and phosphorylate methyl a-glucoside. The experiment with the homogeneous proteins is shown in Fig. 8. All three proteins were required for uptake, and all of the solute taken up was phosphorylated. When the homogeneous proteins were added to the outside of the vesicles, there was no uptake of methyl a-glucoside and a very low rate of phosphorylation (discussed below). These results therefore support the model (2).

Asymmetry of II-BG" and Symmetry of II" Complex-In preparing bacterial vesicles with the correct orientation, two serious potential problems are (a ) that some inside-out (as well as leaky) vesicles may he produced, and ( b ) that some membrane-bound proteins may be transposed from the inside of the vesicle. As much as 50% for example, of an inner surface enzyme, ATPase, has been reported to undergo this transpo- sition to the outside of the vesicle membranes (54).

The results with the methyl a-glucoside described above strongly suggest that more than 90% of the vesicles are right- side-out and furthermore, that II-BG1' is asymmetrically oriented in the membrane, a conclusion that differs from one previously published (7). Our current conclusions are based on the following.

( a ) The rate of sugar phosphorylation when the proteins are intravesicular is at least 10 times greater than the rates with extravesicular proteins. Furthermore, all of the sugar phosphorylated by intravesicular proteins is simultaneously taken up by the vesicles. There is no uptake when the proteins are extravesicular. (The low rate of sugar phosphorylation in the presence of extravesicular PTS proteins is attributed to a small fraction of leaky or inside-out membrane vesicles.)

Sugar Transport by the Bacterial Phosphotransferase System. XXIII 14567

T 5 I

I

0 0 1 2 3 4 5 6

TIME (min) / c- L _ .

I &I

2 3 4 5 6 TIME (min)

FIG. 8. Reconstitution of the FTS with intravesicular purified PTS proteins. SB2950 spheroplasts from cells grown on D- galactose (see "Bacteria and Media" under "Experimental Proce- dures'') and induced for the P-enolpyruvate transport system were lysed in the presence of 28 p~ homogeneous HPr, 260 units of purified Enzyme I/ml, and 6.8 phf III''c. Vesicles were then isolated as described under "Methods." Reaction mixtures contained 3.1 mM P- enolpyruvate and membrane protein from SB2950 vesicles, as specified below, in final volumes of 800 pl. The mixtures were incubated at 31 "C for 3 min and the reaction was started by adding ['4C]methyl a-glucoside (find concentration. 31 ELM; final specific activity, 2.6 mCi/ mmol). At the indicated times, 100-pl aliquots were either added to ethanol at -78 "C or diluted in 0.1 M LiCl and fdtered. These samples were further processed as described under "Methods." Phosphoryla- tion (a) and transport (0) in the presence of 0.84 mg of membrane protein of vesicles with intravesicular homogeneous HPr, purified Enzyme I, and IIIG'c. Phosphorylation (m), and transport (0) with 0.82 mg of membrane protein of vesicles with intravesicular purified Enzyme I. The inset shows transport (0) and phosphorylation (0) of [14C]methyl a-glucoside in SB2950 vesicles reconstituted with homo- geneously pure Enzyme I, HPr, and IIIGIc. Reaction mixtures contained 6 mM P-enolpyruvate and 1.5 mg of membrane protein in a final volume of 1.5 ml and were incubated at 28 "C for 3 min before the reaction was started by the addition of [I4C]methyl a-glucoside (final concentration, 50 p ~ ; final specific activity, 1.6 mCi/mmol), as described above. The bars give the range of determinations obtained with duplicate vesicle suspensions assayed independently. The hatched area represents the range of transport and phosphorylation of methyl a-glucoside obtained with the following combinations of intravesicular PTS proteins or with SB2950 membranes alone: En- zyme I and HPr; Enzyme I and 1IIG"; or HPr and IIIGIC.

(b) If the conclusion given above is correct, methyl n-glucoside phosphorylation should occur with extravesicular PTS proteins if the membranes are inverted or "inside-out." It has been reported that sonic treatment of membrane vesicles yields vesicles, 50-100% of which are oriented inside-out (47, 54). The SB2950 vesicles described above were therefore sonically treated for increasing periods of time, and as shown in Fig. 9, the sonically treated vesicles actively phosphorylated methyl a-glucoside in the presence of extravesicular PTS proteins. The maximum rate was about 5-fold higher than the control.

French press rupture of membrane vesicles is also reported to invert the membranes (47, 54). In the present case, Fig. 9 shows that this treatment did not increase the rate of phosphorylation above the control value, a result that suggests that'II-BG'' is partially inactivated by the French press treatment. This interpretation is consistent with the fact that no additional phosphorylating activity was observed by sonic treatment of the French press membrane vesicles (Fig. 9).

The same approach was used to study the orientation of

4 -

3 -

n

"0 I 2 3 4 5 6

TIME (min) FIG. 9. Effect of sonic disruption of SB2950 membrane vesi-

cles on methyl a-glucoside phosphorylation. Vesicles were prepared from SB2950 cells grown on 0.2% galactose supplemented with 1% casamino acids and tryptophan at 20 pg/ml and not induced for the P-enolpyruvate transport system. Spheroplasts were lysed by

were sonically treated in 30-s bursts (see "Methods") for the times either the syringe method or French press treatment and vesicles

indicated below. Reaction mixtures contained 5 m~ P-enolpyruvate, 65 M homogeneous HPr, 190 units of purified Enzyme I / d , 10.2 p~ I11 , and membrane protein (0.65 mg, syringe-prepared vesicles; 0.84 mg, French press-prepared vesicles) in a final volume of 500 pl. The mixtures were incubated at 32 "C for 3 min, and the reaction was started by adding ["Clmethyl a-glucoside (final concentration, 30 (LM; final specific activity, 2.1 mCi/mmol). At the indicated times, 100- pl aliquots were added to ethanol at -78 "C and assayed for methyl a-glucoside phosphate as described under "Methods." Vesicles prepared by the syringe method were sonically treated for the following total times: 0 min (A), 0.5 min (O), 1 min (O), 2 min (V), and 8 min (+). Vesicles treated for 1 min were used to measure phosphorylation in the presence of HPr and Enzyme I but without 111''' (X). Vesicles prepared by the French press method were sonically treated for the following total times: 0 min (A), 1 min (W), and 6 min (0).

the IIM"" complex in our vesicles. Since 2-deoxyglucose is an excellent substrate for the IIMa" complex, phosphorylation experiments were conducted with this substrate. Fig. 10 shows that, with intact vesicles, and extravesicular Enzyme I and HPr, there was a very high rate of phosphorylation, consistent with the fact that in intact cells the II'" system is a high capacity system relative to the IIIG1c/II-BG'c system (40). Moreover, mannose completely inhibited 2-deoxyglucose phosphorylation (as expected). Sonic treatment of the vesicles lowered this phosphorylation activity, presumably due to partial inactivation of the IIM"" complex. The French press results were similar to those described with methyl a-glucoside, in that the activity of the vesicles was diminished about 4-fold after passage of the vesicles through the French press, and sonic treatment did not increase the activity.

The vesicles used in these studies were prepared from cells grown on galactose. Since the galactose permease is induced under these growth conditions and it has overlapping substrate specificities with the IIMa" complex (41, 42), it was necessary to rule out any contribution of the galactose per-

tk


mease to our results. For example, in the presence of extravesicular Enzyme I, HPr, and IIIG1c, the galactose permease might be able to catalyze the phosphorylation of glucose or 2- deoxyglucose. Thus, vesicles were prepared from a galactose permease-negative strain and PTS proteins and glucose were added extravesicularly. Under these conditions, the glucose was phosphorylated to the same extent as in the galactose

"O \ 90

70

50

30

IO

I 2

TIME (mid FIG. 10. Effect of sonic disruption of SB2950 membrane ves-

icles on 2-deoxyglucose phosphorylation. The vesicle preparations and reaction mixtures were the same as those in Fig. 9 except that ILI"" was omitted and the substrate was 2-deoxyglucose. The mixtures were incubated at 32 "C for 3 min and the reaction was started by adding ['4C]2-deoxyglucose (final concentration, 0.5 mM; final specific activity, 0.3 rnCi/mmol). Aliquots were removed and assayed as in Fig. 9. Vesicles prepared by the syringe method were sonically treated for the following times: 0 min (A), 1 min (O), and 8 min (0). Vesicles sonically treated for 1 min were used to measure phosphorylation in the presence of 4 mM mannose (X). Vesicles prepared by the French press method were sonically treated for the following times: 0 rnin (A), 1 min (m), and 6 min (0).

permease-positive strain, albeit somewhat more slowly (data not shown).

DISCUSSION

As indicated in the Introduction, our current and future goals are to define precisely how the PTS catalyzes uptake of its substrates and how the PTS regulates the transport of certain non-PTS solutes. To investigate these phenomena, membrane vesicles isolated from wild type and mutant S. typhimurium strains appeared to be ideal systems.

The methods described in this report yield vesicles partic- ularly suitable for such experiments. First, the vesicles are obtained in good yield and appear to be composed almost exclusively of inner membrane, contain only small quantities of cytoplasmic proteins, and are enriched for known inner membrane protein markers. They do not leak high or low molecular weight solutes, and at least 90% are oriented right- side-out (the remaining 10% are either leaky or oriented inside-out or both). Second, an important advantage of S. typhimurium is that it can be induced to generate a transport system (13) which, unlike E. coli, actively takes up phosphoenolpyruvate. Thus, vesicles from E. coli require a very high external concentration of P-enolpyruvate (0.1 M) to drive PTS-mediated sugar transport, whereas low P-enolpyruvate concentrations can be used with S. typhimurium vesicles (Fig. 1). This may be the major reason why the uptake of methyl a-glucoside reported here is so rapid (from 4- to 100-fold greater than the rates observed with other vesicle preparations, Table IV). In any event, for future work involving detailed kinetic analysis of PTS function in membrane vesicles, it is essential that the rate of sugar uptake be the limiting factor, rather than the rate of P-enolpyruvate transport.

After investigating several possible techniques, a satisfac- tory method was found for introducing soluble, homogeneous PTS proteins into the vesicles. The method involves lysis of the spheroplasts to vesicles in the presence of the soluble proteins. Thus, when we lysed spheroplasts of SB2950 (a

Soluble Components of the PTS (PTSso, =PEP, Mg2+, Enzyme I, HPr, III 'Ic

FIG. 11. Schematic representation of the orientation of the glucose-spe- Inside cific proteins of the PTS in S. typhi- muriurn membrane vesicles. The following designations are used: ZZMn" rep- ~-DOOXY- GIG resents the membrane complex (11-A/II- B) which phosphorylates glucose, mannose, 2-deoxyglucose, and several other sugars of the D-glUC0- and D-manno- con- figurations; IZ-BG" is the membrane protein which transfers the phosphoryl group from ZZP" to glucose and methyl a-glucoside; MeGZc is methyl a-glucoside; 2-Deoxy-Glc is 2-deoxyglucose. Sol, soluble; PEP, P-enolpyruvate.

Outside


strain deleted for Enzyme I, HPr, and 1IIG") in the presence of the three required soluble proteins, the resulting vesicles were capable of translocating and phosphorylating methyl a- glucoside. As expected, all three soluble proteins were required to reconstitute this activity. Since fluorescent and EPR spin- labeled active HPr derivatives and a fluorescent labeled IIIGIc are now available (55, 56); reconstitution experiments of the type described here with vesicles and with the labeled proteins should permit detailed analysis of the interactions that occur between PTS proteins inside the vesicles before and during transport of PTS sugars, and possibly interactions with non- PTS solutes as well.

The vesicles permitted a study of the orientation of the two Enzyme I1 complexes and the conclusions are presented in Fig. 11. The IIM"" complex appears to be symmetrically oriented in the membrane, since it can interact with 2-deoxyglucose and phosphorylate this sugar when phospho-HPr is located either inside the vesicles (with concomitant uptake of the sugar) or outside the vesicles (phosphorylation but no uptake of sugar). 1I-BGIc, on the other hand, appears to be oriented asymmetrically in the membrane. In this case, phosphorylation only occurred when the soluble proteins were in contact with the cytoplasmic face of the membrane. Under normal conditions in the cell, II-BG1' faces methyl a-glucoside or glucose on the outside of the membrane and phospho-IIIG" on the inside. Translocation of the sugar with concomitant transfer of the phosphoryl group is then catalyzed by II-BGic.

One physiological consequence of the results just described is that the IIM"" system should be capable of phosphorylating its sugar substrates (with the soluble PTS proteins) when the sugar is located inside the cell. This may have significance in bacterial physiology since free glucose, for example, is released by hydrolases which split disaccharides, such as lactose. Whether the IIIG1c/II-BGic complex can also catalyze a similar intracellular sugar phosphorylation at an effective rate was not tested in the present studies. There is, in fact, controversy in the literature (1) whether intracellular methyl a-glucoside (formed by hydrolysis of methyl a-glucoside phosphate) can be rephosphorylated.

The results in Fig. 9 do not resolve the question. Here, methyl a-glucoside is phosphorylated by sonically treated membrane vesicles supplemented with the soluble PTS proteins and P-enolpyruvate. However, it is not clear from these results whether phosphorylation results from the binding of the sugar substrate and phospho-IIIG" to the same or opposite faces of the membrane.

Thus, vesicles made from S. typhimurium can serve as useful tools in the study of sugar transport in bacteria. Our preparations show the desired physical and biochemical char- acteristics. They have been successfully used in the reconstitution of sugar transport mediated by the PTS as well as in the determination of the asymmetric orientation of II-BG" and the symmetry of the IIMa" complex. These vesicles are likely to prove useful in attempts to answer the many questions remaining concerning the transport and regulatory functions of the PTS.

Acknowledgment-We would like to express our thanks to Cory Singer for his help with some of the assays.

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( T O ~ Y O ) 77, 705-718


51. Simoni, R. D., Levinthal, M., Kundig, F. D., Kundig, W., Ander- 966-976 son, B., Hartman, P. E., and Roseman, S. (1967) Proc. Natl. 54. Futai, M. (1974) J. Membr. Biol. 15, 15-28 Acad. Sei. U. S. A. 58, 1963-1970 55. Grill, H., Weigel, N., Gaffney, B. J., and Roseman, S. (1982) J.

Biol. Chen. 246,5838-5840 56. Hildenbrand, K., Brand, L., and Roseman, S. (1982) J. Biol. 52. Saier, M. H., Jr., Young, W. S., 111, and Roseman, S. (1971) J. Biol. Chem. 257, 14510-14517

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14572 Sugar Transport by the Bacterial Phosphotransferase System. XXIIII

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