Requirement for a Functional Respiration-Coupled D-Fructose

Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp. 1739-1742, May 1974

Requirement for a Functional Respiration-Coupled D-Fructose Transport System for Induction of Phosphoenolpyruvate: D-Fructose Phosphotransferase Activity (Arthrobacter pyridinolis/mutants) ELLEN B. WOLFSON AND TERRY A. KRULWICH Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029 Communicated by Leon A. Heppel, February 8, 1974

ABSTRACT Previous studies have shown that Arthro- A. pyridinolis (3, 5, 6). This system is similar to those de- bacter pyridinolis can transport D-fructose or L-rhamnose scribed by Kaback and coworkers (7) for the transport of using either a phosphoenolpyruvate: hexose phosphotransferase (phosphoenolpyruvate: protein phosphotransferase, various sugars and amino acids in other bacterial species. EC 2.7.3.9) system or a respiration-coupled transport sys- In A. pyridinolis, the respiration-coupled system requires tem which requires the presence of exogenous L-malate. the presence of L-malate (or a precursor thereof) in the me- A mutant, AP4374, which is deficient in the D-fructose- dium (5). This requirement for exogenous oxidizable substrate specific component of the respiration-coupled system can grow on L-rhamnose using the phosphotransferase system, for function of the respiration-coupled system is apparently but cannot grow on D-fructose at all. AP4374 fails to pro- due to failure of A. pyridinolis to accumulate intracellular duce the inducible D-fructose-specific phosphotransferase dicarboxylic acids during metabolism (8). b-Glucose, a-gluco- components (enzyme II and factor III) when grown in the sides (9), and certain amino acids (Blanco, R. and Krulwich, presence of D-fructose. These results indicate a require- in addition to 1-fructose and ment for a functional respiration-coupled transport sys- T., unpublished data), L-rham- tem for induction of the phosphotransferase system. The nose, are transported by the respiration-coupled system. results further suggest that sufficient free D-fructose (or Growth of the wild-type strain on D-glucose or of phospho- D-fructose 6-phosphate derived from it) must be present transferase-negative mutants on D-fructose or L-rhamnose inside the cell in order for induction of the phosphotrans- only occurs if L-malate or a precursor is also present in the ferase system to occur. The entry of sufficient fructose to cause induction of the phosphotransferase system cannot medium. occur by facilitated diffusion in the absence of energy The respiration-coupled system for D-fructose transport coupling. involves a sugar-specific protein in addition to the requisite dehydrogenase and electron transport chain (6). None of the Arthrobacter pyridinolis possesses alternate systems for D- phosphotransferase components is identical to the D-fructose- fructose and L-rhamnose transport. Both of these sugars can specific component of the respiration-coupled system; mu- be transported by a phosphoenolpyruvate: hexose phospho- tants which are deficient in each of the phosphotransferase transferase (phosphoenolpyruvate: protein phosphotransfer- components have been found capable of growing on D-fructose ase, EC 2.7.3.9) system (1-3), such as those extensively in the presence of L-malate (6). However, several mutant studied by Roseman and his collaborators (4), by which strains which, on the basis of in vitro complementation assays, transport of a hexose substrate is catalyzed concomitantly were thought to be deficient in phosphotransferase system with phosphorylation of the hexose. In A. pyridinolis, as in factor III, have subsequently been found to be defective in other species, the reactions of the phosphotransferase system respiration or certain Krebs cycle enzymes. These observa- require four protein components (2): tions suggested that mutations which impair the function phosphoenolpyruvate + phosphocarrier protein enzy I of the respiration-coupled system for D-fructose transport might prevent induction of factor III of the phosphotrans- pyruvate + phosphocarrier protein-phosphate, ferase system. The possible role of D-fructose that is trans- and ported by the respiration-coupled system in induction of the inducible phosphotransferase components has therefore been phosphocarrier protein-phosphate + hexose enym investigated. factor III hexose-phosphate + phosphocarrier protein. MATERIALS AND METHODS Enzyme I and phosphocarrier protein are constitutive, soluble Bacterial Strains. Wild-type A. pyridinolis and mutants proteins which are involved in the transport and phosphoryla- derived from it were used for all the experiments. The mu- tion of both D-fructose and L-rhamnose. Mutants of A. pyri- tant strains employed and previously characterized are as dinolis which are deficient in either enzyme I or phospho- follows. AP100 and AP133 are, respectively, deficient in the carrier protein cannot grow on either D-fructose or L-rham- D-fructose-specific factor III and the D-fructose-specific en- nose as sole carbon source (2, 3). Enzyme II and factor III zyme II of the phosphotransferase system (2). AP243 and are both inducible and sugar-specific proteins; enzyme II AP253 were previously characterized as mutants which are is membrane-bound while factor III is soluble. deficient in two different constitutive soluble phosphotrans- A second transport system for D-fructose and L-rhamnose, ferase components (2). Phosphotransferase activity could not a respiration-coupled transport system, is also present in be restored to either of these strains by addition of purified 1739 Downloaded by guest on October 1, 2021 1740 Biochemistry: Wolfson and Krulwich Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 1. Growth of A. pyridinolis strains AP24S, AP437, brane fractions by centrifugation for 90 min at 150,000 X g. and AP4374 on various sugars in the presence or For assays of complementation between soluble fractions absence of maltte from different strains, each reaction mixture contained 50 ,x1 of the membrane fraction from fructose-grown wild-type Growth (Klett units in 25 hr) cells plus 25,l of each of the two soluble fractions to be tested. of strains: For assays of enzyme II activity, 50 IAl of the membrane Carbon source(s) AP243 AP437 AP4374 fraction from the strain to be tested (fructose + glutamate- grown cells) was added to a reaction mixture containing 50 Fructose (50 mM) 3 3 3 Rhamnose (50 mM) 3 3 360 ,Al of the soluble fraction from fructose-grown wild-type cells. Glucose (50 mM) 3 3 3 The reaction mixture and assay procedure employed were Fructose (50 mM), 465* 25* 21* those described by Tanaka, Lerner, and Lin (11). Protein malate (5 mM) present concentrations were determined by the method of Lowry Rhamnose (50 mM), 355* 355* 350* et al. (12) using lysozyme as a standard. malate (5 mM) present RESULTS Glucose (50 mM), 55* 65* 63* In order to determine whether function of the respiration- malate (5 mM) present coupled transport system for fructose is necessary for induction of phosphotransferase components, it was first necessary * These values represent growth of the hexose; the data were corrected for the turbidity (Klett units) attained by parallel to isolate a mutant which was defective only in the respira- cultures after 25 hr of growth on 5 mM malate alone. tion-coupled transport system. AP437, a double mutant lacking both the fructose-specific component of the respira- enzyme I or histidine-containing phosphocarrier protein tion-coupled system and enzyme I, was previously isolated from Salmonella typhimurium (generously provided by Dr. from phosphotransferase-negative strain AP23 (6). AP437 Saul Roseman). For further characterization of these strains, cannot grow on rhamnose or fructose aloine because of the crude extracts (3 ml) of AP243 and AP253 were incubated enzyme I deficiency. It can grow on rhamnose or glucose in with 150 mg of HCl-washed charcoal (Norite-A) for 15 min the presence of malate, but cannot grow appreciably on fruc- at 0°. The charcoal was then removed by centrifugation tose in the presence of malate because of its deficiency in the for 10 min at 17,000 X g. The resulting supernatant was fructose-specific component of the respiration-coupled system freed of membranes as well as any remaining charcoal by (Table 1). From this double mutant, a strain was isolated centrifugation for 90 min at 150,000 X g. In in vitro comple- which had regained phosphotransferase activity, as shown by mentation assays of phosphotransferase activity (see below), its ability to grow on rhamnose alone, but still lacked the parallel extracts of AP243 and AP253 which were not treated fructose-specific component of the respiration-coupled system. with charcoal had very little activity when assayed sepa- AP437 was treated with ethylmethane sulfonate and then rately but produced an increase of 0.50 nmoles/min per ml plated on minimal medium plus rhamnose. The colonies from of reaction mixture when assayed together. Charcoal-treated these plates were replicated onto plates containing minimal AP243 together with untreated AP253 had only 6% of the medium plus fructose. Growth data for one of the rhamnose- activity of the two untreated extracts together, while char- positive, fructose-negative strains thus isolated (AP4374) coal-treated AP253 and untreated AP243 retained 67% of are shown in Table 1. AP4374 can indeed grow on rhamnose the control activity. From these data and analogous experi- whether or not malate is also present, thus indicating that ments with other phosphotransferase systems (10), it is con- this strain has phosphotransferase activity for rhamnose cluded that AP243 is deficient in enzyme I and AP253 is and hence has regained enzyme I activity. It nonetheless deficient in phosphocarrier protein. Strain AP437 is a double cannot use fructose as sole carbon source and, like its parent mutant, previously described, which is deficient both in en- strain AP437, shows very little utilization of fructose in the zyme I and in the D-fructose-specific component of the res- presence of malate. AP4374 uses glucose in the presence but piration-coupled system (2). not in the absence of malate, as do both its parental strain Mutagenesis of AP437 was carried out using ethylmethane and wild-type cells. sulfonate as previously described (8). It thus appeared that AP4374 possessed the nonsugar- specific phosphotransferase components but, by virtue of Chemicals. The sources of the chemicals used were reported its deficiency in the fructose-specific component of the res- previously (6). The 1-isomers of fructose and glucose and the piration-coupled system, did not exhibit phosphoenolpyru- L-isomers of rhamnose, glutamate, and malate were used in vate: fructose phosphotransferase activity and could not grow all experiments. on fructose alone. Assays of phosphotransferase components Growth Conditions. The media employed have beendescribed were then conducted to determine whether this apparent were at with at lack of phosphotransferase activity was in fact due to a failure elsewhere (1, 8). Cells grown 300 shaking in 200 rpm in a New Brunswick Incubator Shaker. For growth to produce the inducible components the presence of fructose. As shown in from fructose experiments, 300-ml sidearm flasks were employed as pre- Table 2, a soluble cell fraction described + glutamate-grown AP4374 could complement similar frac- viously (8). tions from AP243 (enzyme I-deficient) and AP253 (phospho- Phosphotransferase Assays. For assays of phosphoenol- carrier protein-deficient) for phosphoenolpyruvate: fructose pyruvate: hexose phosphotransferase activity, extracts of phosphotransferase activity. The soluble fraction from AP4374 washed cells were prepared by sonic disruption (8) and were could not complement similar fractions from AP100 (factor freed of whole cells and debris by centrifugation at 18,000 III-deficient) or from glutamate-grown wild-type cells which X g. The extracts were then separated into soluble and mem- have low levels of factor III (2). Downloaded by guest on October 1, 2021 Proc. Nat. Acad. S.i. USA 71 (1974) Induction of Phosphotransferase Activity 1741

TABLE 2. Failure of the soluble fraction from an extract of TABLE 3. Restoration of phosphotransferase activity by A. pyridinolis strain AP4374 to complement those of strain addition of membrane fractions from various mutant strains AP1K00 or glutamate-grown wild-type cells for of A. pyridinolis to the soluble fraction from phosphotransferase activity* fructose-grown wild-type cells* nmole of nmoles of fructose-i- fructose-i- Source of membrane fraction phosphate formed per min phosphate (A. pyridinolie strain) per reaction mixture Source of additional formed per soluble fraction min per reaction AP133 0.05 (A. pyridinolis strain) mixture AP4374 0.11 AP243 0.97 AP243 1.17 AP253 0.65 AP253 0.69 Glutamate-grown 0.23 * Reaction mixtures were the standard phosphotransferase wild-type cells assay mixture (2) except that instead of extract, 50 ,d of the AP100 0.03 soluble fraction from fructose-grown wild-type cells and 50 jsl of the membrane fractions from the mutants indicated (fructose- * Reaction mixtures were the standard phosphotransferase glutamate-grown) were used. The data are corrected for the assay mixture (2) except that instead of extract, 50 ul of the activity of each of the fractions when assayed alone. The protein membrane fraction from fructose-grown wild-type cells, 25 Al content (in mg/ml) of the soluble fraction employed was 7.4, of the soluble fraction from AP4374, and 25 M1 of the soluble and the protein contents of the membrane fractions were AP133, fractions from the mutants indicated (fructose-glutamate-grown) 3.4; AP4374, 7.7; AP243, 5.0; and AP253, 5.6. were used. The data are corrected for the activity of each of the fractions when assayed alone. The protein content (in mg/ml) Presumably when cells of A. pyridinolis are inoculated into of the membrane fraction employed was 6.2, and the protein contents of the soluble fractions were glutamate-grown wild-type, medium containing fructose as sole carbon source, there is 19.3; AP100, 7.1; AP4374, 6.2; AP243, 8.1; and AP253, 5.6. sufficient intracellular malate to allow the requisite amount of fructose transport via the respiration-coupled system to allow induction of the phosphotransferase system. The ob- Assays of enzyme II were also conducted on fructose + servation which prompted this study was that certain Krebs glutamate-grown cells of AP4374 (Table 3). Membrane frac- cycle mutants fail to induce phosphotransferase factor III. tions from such cells were similar to membrane fractions from This observation indicates that not only is the fructose-specific AP133 (enzyme II-deficient) in being unable to restore phos- component of the respiration-coupled system required for phoenolpyruvate: fructose phosphotransferase activity to induction of the phosphotransferase system, but the respira- the soluble fraction from fructose-grown wild-type cells. Thus tion-coupled system must in fact be energy-coupled. In the fructose + glutamate-grown cells of AP4374 appeared to absence of energy-coupling, the fructose-specific component lack both enzyme II and factor III. of the respiration-coupled system, which is constitutive (6), Attempts were made to induce phosphoenolpyruvate: apparently cannot catalyze sufficient facilitated diffusion fructose phosphotransferase activity in AP4374 by growing to provide intracellular inducer for the phosphotransferase the strain on glutamate in the presence of high concentrations system. These observations are similar to those made by of either fructose (100 mM) or fructose-6-phosphate (50 mM). Kusch and Wilson (16) on the induction of fl-galactosidase Phosphotransferase activity for fructose was not induced in a mutant of E. coli which is energy-uncoupled for lactose under such conditions. transport. The absence of both enzyme II and factor III from fructose DISCUSSION + glutamate-grown cells of AP4374 suggests that induction Previous work has shown that in A. pyridinolis, fructose that of these two phosphotransferase components might be co- is transported via the phosphotransferase system is converted ordinate in A. pyridinolis. If so, it should be possible to isolate by that system to fructose-i-phosphate which is then phos- mutants which are constitutive for the fructose-specific en- phorylated to produce fructose-1,6-diphosphate (1). Fructose zyme II and factor III from derivatives of AP4374 which that is transported by the respiration-coupled system enters grow on fructose alone. Such strains would presumably be the cell as free fructose and is converted first to fructose-6- either revertants for the mutation in the respiration-coupled phosphate and then to fructose-1,6-diphosphate (5). We have system or constitutive with respect to the phosphotransferase now shown that the fructose-specific enzyme II and factor components. Studies of the phosphotransferase system of III of the phosphotransferase system fail to be induced by S. typhimurium have shown that the cistrons for enzyme I exogenous fructose (or fructose-6-phosphate) in the absence and histidine-containing phosphocarrier protein are genet- of a functional respiration-coupled transport system for fruc- ically linked (17) and that production of these proteins may tose. This suggests that a sufficient concentration of either be coordinately controlled (18). Perhaps enzyme II and factor free fructose or fructose-6-phosphate must be present inside III are part of another unit of control. the cell in order for induction of the phosphotransferase sys- It will be of interest to determine whether the formation of tem to occur. The requirement for intracellular inducer is in the rhamnose-specific phosphotransferase components are contrast to findings with respect to the hexose-6-phosphate similarly affected by a mutation in the rhamnose-specific transport system in Escherichia coli. The latter system is in- component of the respiration-coupled system for rhamnose duced by exogenous, but not endogenously produced, glucose- transport. While the alternate transport systems for fructose 6-phosphate (13-15). and rhamnose in A. pyridinolis are very similar, there are Downloaded by guest on October 1, 2021 1742 Biochemistry: Wolfson and Krulwich Proc. Nat. Acad. Sci. USA 71 (1974)

some differences, including the apparent constitutive nature 7. Kaback, H. R. (1972) Biochim. Biophys. Ada 265, 367-416. component of the respiration-coupled 8. Wolfson, P. J. & Krulwich, T. A. (1972) J. Bacteriol. 112, of the fructose-specific 356-364. system and inducibility of the analogous rhamnose-specific 9. Sobel, M. E., Wolfson, E. B. & Krulwich, T. A. (1973) J. component (6). Bacteriol. 116, 271-278. 10. Kundig, W. & Roseman, S. (1971) J. Biol. Chem. 246, 1393- This work was supported by Research Grants GB-20481 from 1406. the National Science Foundation and AM-14663 from the Na- 11. Tanaka, S., Lerner, S. A. & Lin, E. C. C. (1967) J. Bacteriol. tional Institutes of Health. 93,642-8. 1. Sobel, M. E. & Krulwich, T. A. (1973) J. Bacteriol. 113, 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, 907-913. R. J. (1951) J. Biol. Chem. 193, 265-275. 2. Wolfson, E. B., Sobel, M. E. & Krulwich, T. A. (1973) 13. Winkler, H. H. (1970) J. Bacteriol. 101, 470-475. Biochim. Biophys. Acta 321, 181-188. 14. Winkler, H. H. (1971) J. Bacteriol. 107, 74-78. 3. Levinson S. L. & Krulwich, T. A. (1974) Arch. Biochem. 15. Dietz, G. W. & Heppel, L. A. (1971) J. Biol. Chem. 246, Biophys. 160,445-450. 2885-2890. 4. Roseman, S. (1969) J. Gen. Physiol. 54, 138S-180S. 16. Kusch, M. & Wilson, T. H. (1973) Biochim. Biophys. Acta 5. Krulwich, T. A., Sobel, M. E. & Wolfson, E. B. (1973) 311,109-122. Biochem. Biophys. Res. Commun. 53, 258-263. 17. Cordaro, J. C. & Roseman, S. (1972) J. Bacteriol. 112, 17-29. 6. Wolfson, E. B., Sobel, M. E., Blanco, R. & Krulwich, T. A. 18. Saier, MI. H., Jr., Simoni, R. D. & Roseman, S. (1970) (1974) Arch. Biochem. Biophys., 160,440-444. J. Biol. Chem. 245, 5870-5873. Downloaded by guest on October 1, 2021