Carbohydrate Uptake by Escherichia coli HANS L. KORNBERG Department of Bi.ochemistry, University of Cambridge, Cambridge, CB2 1 QW,England
ABSTRACT In contrast to active transport, the uptake of carbohydrates via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) leads to the appearance in the cell of the sugar initially as a 1- or 6- phosphate ester. The components of the PTS that transfer phosphate to the sugar are not absolutely specific for any one sugar. Both their synthesis and their activity are controlled; in the latter, "fine" control, glucose-6-phosphate appears to play an important role. Studies of growth on, and uptake of, galactose by E.coli mutants devoid of components of the PTS and also devoid of active transport systems for galactose, suggest that proteins effecting facilitated diffusion of hexoses may be part of, or be closely associated with, the sugar-specific components of the PTS.
Escherichiu coli exDend metabolic enerev in taking up CarbohGdrates, either by &tive transport or by group translocation. In the former process, the carbohydrate enters the cell chemically unchanged, with concomitant uptake of protons (West, '70; West and Mitchell, '72; Henderson, '74) or expenditure of ATP (Berger, '73; Curtis, '74; Wilson, '74); in the latter, the sugar appears inside the cell initially as a phosphate ester (Rogers and Yu, '62). Since the papers presented in this issue by Doctors Ames, Boos, Heppel and Oxender are all relevant to the mechanisms whereby carbohydrates and amino acids are actively transported by microorganisms, I shall confine myself to a brief survey of the nature and regulation of group translocation, as mediated by the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTS, discovered by Doctor Saul Roseman and his colleagues (Kundig et al., '64) is a multicomponent system, closely associated with the membrane(s) of Gramnegative organisms that dissimilate carbohydrates predominantly via the EmbdenMeyerhof pathway (Romano et al., '70). It comprises (at least) three components that play a role in the uptake of many sugars (Reactions 1 and 2), and other components that exhibit some specificity for a relatively restricted range of sugars (Reactions 3 and 4). J. CELL.PHYSIOL.,89: 545550.
Enzyme I Enzyme I HPrhlP
+
+
PEP
P
+
-+Enzyme I m p
HPr
+HPrNP
+
pyruvate .. (1) Enzyme I
Enzyme I1 - + E n z y m e I I N P
Enzyme I1 N P
+
sugar
-+
sugar
+
+
HPr
(2)
(3)
- P + Enzyme I1
(4)
The main pleiotropic components of Reactions 1 and 2 are (i) an Enzyme I that is phosphorylated at a nitrogen atom of a histidine residue (Stein et al., '74), by phosphoenolpyruvate (PEP), and (ii) a small protein, also containing histidine and capable of being phosphorylated at that residue, designated HPr ("histidine-containing protein") for that reason (Kundig et al., '64). The sugar-specific components that participate in Reactions 3 and 4 are (iii) the sugars themselves and (iv) the appropriate Enzyme(s) I1 that catalyze the transfer of phosphate from the phosphorylated HPr to a terminal hydroxyl group. It has been demonstrated, particularly by genetic analysis, both that one Enzyme I1 can effect the phosphorylation of more than one sugar, and that one sugar can be phosphorylated by more than one Enzyme 11. The phosphorylation of glucose by E . coli, for example, is mediated to a major extent by an Enzyme I1 variously described as the uptake system for methyla-glucoside, specified by a gene thus abbreviated umg (Kornberg and Smith, '72), or as the glucose phosphotransferase component A-abbreviated g p t A (Curtis and Epstein, '70, '75); 545
546
HANS L. KORNBERG
this gene is located at about min 24 on the E.coii genome and is co-transducible with purB. However, wild-type organisms take up about 30% of the glucose they utilize via a different Enzyme 11. This is specified by a gene variously designated p t s X (Kornberg and Jones-Mortimer, ’75), g p t B or m p t (Curtis and Epstein, ’75), located at about min 36 on the E.coli genome and co-transducible with hga and h d g R (JonesMortimer and Kornberg, ’74). In strains of E.coZi that lack alternative routes for glucose uptake, such as the systems concerned in the active transport of galactose, the sequential removal of the functions specified by u m g + and p t s X + also leads sequentially to the loss both of the ability to grow on glucose and the ability to take up labelled glucose (Kornberg and JonesMortimer, ’75). Conversely, the Enzyme I1 specified by p t s X + can phosphorylate to the 6-phosphate not only glucose, glucosamine and mannose (Curtis and Epstein, ’75) but also fructose (Ferenci and Kornberg, ’74; Jones-Mortimer and Kornberg, ’74) which is mainly taken up and phosphorylated to the 1-phosphate ester by a different Enzyme 11, specified by p t s F and located at min 41.5 on the E.coli genome. These Enzymes I1 for the uptake of glucose and fructose are inducible (Kornberg and Reeves, ’72b). However, results obtained with batch cultures may be quantitatively misleading even if they are qualitatively useful. For example, some strains of E.coli (illustrated by strain K2, table 1) appear to be capable of taking up methyl-cuglucoside and glucose at high rates even though they have been grown on other substrates, whereas other strains (illustrated by strain B11, table 1) take up methyl*glucoside and glucose very poorly unless they have been induced by prior exposure to these carbohydrates, and even then the maximal rates of uptake are considerably less than those observed with the K2 strain. Direct measurement of the phosphorylation of methyla-glucoside (or glucose) in these cells, rendered permeable by treatment with toluene, showed that it is the phosphorylation step of the overall uptake process that thus differs in these strains (table 1); moreover, these rates were identical to the rates of methyl-a-glucoside uptake observed with intact cells (Kornberg and Reever, ’72a).
TA.BLE 1
m e c t of carbon source for growth,’ on: (Method A): the initial rate of uptake of metkyla-D- [14C] glucoside by intact cc,lZs, and (Method B ) : the PEP-dependent phosp,horylation of methyla-Dglucoside by toluene-treated cells thus grown ~~
Activity (nmoll min per mg dry wt.)
Strain
Carbon source for growth
Method A Method B
~~~
B11
K2
Glucose Fructose Gluconate Glycerol Glucose Fructose Gluconate Glycerol
15 3 3 3 38 44 24 19
17 3 2 3 56 99 30 23
For details, see Kornb’erg& Reeves, 1972a.
But these differences are not observed with cells grown i n continuous culture with glucose limitation (Herbert and Kornberg, ’76). Under these conditions, samples of either strain, taken from the chemostats, take up glucose at high rates, identical to each other and sufficient to account for the observed growth rates of the organisms over a wide range of doubling times. Glucose limitation thus appears to lead to a marked derepression of the Umg system; this may be a further manifestation of the regulatory component known to be associated with this system (Kornberg and Smith, ’72). Besides these “coarse” controls that govern the rates a t which Enzymes I1 are synthesized by cells growing under different conditions, “fine” controls regulate the rates at which Enzymes 11, already present, function in the cells. This is clearly illustrated by the manner in which one sugar can inhibit the uptake of another, although all the components required for the uptake of that other sugar are abundant, and were previously active, in the cell. For example, when glucose is added to cultures of E. coli growing on fructose (Kornberg, ’72) or on a variety of other sugars (McGinnis and Paigen, ’69; ’73), the organisms virtually cease to take up the previous growth substrate and preferentially utilize glucose. It is not the mere presence of glucose that brings about this dramatic switch: u m g mutants no longer exhibit this preference for glucose, which suggests that either the process of phosphorylation of glucose,
54 7
CARBOHYDRATE UPTAKE BY E . COLI
or the level of glucose-6-phosphate formed, are important to this “fine” control (Kornberg, ’72). Abolition of the ability of glucose to inhibit the uptake of fructose, by removal of Umg function, is not fructose-specific: in umg mutants, glucose is no longer preferred to any other carbon source (Kornberg, ’73). A more specific “fine” control was revealed through study of E.coli mutants that were resistant to hitherto toxic analogues of glucose (such as 2-deoxyglucose and 3-deoxy-3-fluoro-glucose)when growing on fructose, but were still sensitive to these compounds when growing on mannitol, glycerol or lactose (Amaral and Kornberg, ’75). The mutation that gave rise to this phenotype, designated cif (catabolite inhibition of fructose uptake), maps within or very close to the ptsF gene that specifies the high affinity Enzyme I1 for the phosphorylation of fructose to the 1phosphate ester. Unlike their parent organisms, cif-mutants can be induced to synthesize the PtsF-Enzyme I1 for fructose when fructose is added to cultures, even when they are growing on glucose. The cif mutation has thus overcome a “glucose effect” (Magasanik, ’61) that probably operates by inducer exclusion. Normally, glucose impedes the uptake of another sugar and thus impedes also the induction of the enzymes required for the catabolism of that sugar (Lengeler, ’66). It is interesting that cif mutants growing on fructose continue to utilize fructose not only in the presence of glucose but also of glucose6-phosphate: it is possible that the mechanism of inducer exclusion operative in wild-type cells, and that has been altered in cif mutants, involves an interaction of glucose-6-phosphate with a (phosphorylating?) component of the PtsF-system. It is also interesting that Enzyme I mutants of E.coli specifically resistant to inhibition
by methyla-glucoside when growing on lactose have been described by M. H. Saier and his colleagues. The mutation that confers this specific tolerance to the glucose analogue maps close to, or within, the gene that specifies the uptake system for lactose (Saier et al., ’76). The obvious differences between the active transport of a carbohydrate and its uptake by the phosphotr ansferase system have been taken virtually to exclude any similarity in the mechanisms of these processes. Although Roseman (‘69) originally envisaged the possibility that PTsugars might enter the cells by some (passive diffusion?) carrier and might be phosphorylated in a subsequent “trapping” reaction, this possibility was abandoned by him and his colleagues largely on evidence obtained with S . aureus: mutants devoid of Enzyme I were found to be unable to equilibrate sugars in the medium with those in the cells (Simoni and Roseman, ’73). However, from studies of the uptake of glucose and of its analogues into E.coli strains, Gachelin (’70) concluded that phosphorylation of methyla-glucoside occurred after entry into the cell. More recently, Bourd et al. (‘75) described E.coZi mutants in which the transport of methyla-glucoside by intact cells is low, but extracts of which contain normal phosphotransferase activities. This suggests impairment of a carrier protein involved in the facilitated diffusion (or active transport) of the sugar; this protein is postulated to be specified by a gene tgZ that is closely linked to umg or is an allele of it. We have isolated a similar mutant (KR 161, table 2), suspensions of which do not take up and phosphorylate methyla- [I4C] glucoside but which, when rendered permeable with toluene, readily formed methylaglucoside-6-phosphate in the presence of the analogue and phosphoenolpyruvate. We
TABLE 2
Properties of mutants impaired in the Umg system Organism
K2.lt K2.1.22 KR 161 KR 163
PTS activity of Doubling time (min) on Uptake of toluenized cells with glucose galactose methylff -glucoside glucose methylff -glucoside glucose
60 165 65 155
72 240 78 75
21 1 2 2
38 5 12 4
29 1 34 1
44 6 34 4
Postulated genotype
tgl+ tgltgltgl+
umg+ umgumg+ umg-
548
HANS L. KORNBERG
have also isolated a mutant (KR 163, table 2) which, by these criteria, is U m g - in that it neither takes up methyla-glucoside nor phosphorylates it; however, it appears still to be able to take up at least one analogue of glucose via a protein closely associated with the Umg system, but not requiring phosphorylation of that sugar by the PTS. The glucose analogue thus taken up is galactose (which is a n epimer of glucose at C-4), which is normally actively transported by systems specified by g u l p (Rotman et al., '68), located at about min 55 and cotransducible with fdu (Claudia Riordan and H. L. Kornberg, to be published), and by m g l , located at about min 41 and cotransducible with ptsF (Boos, '69). The strains studied by us [Kornberg and Riordan ('76)l lack these active transport systems, yet grow on galactose at rates strongly dependent on the galactose concentration of the medium. Umg - derivatives of these strains, which, of course, grow more slowly on glucose, also grow very poorly on galactose; reversion to Umg+ or re-introduction of the umg+ allele by recombination restores normal growth on glucose and also restores the pattern of growth on galactose seen with the parent strain. Moreover, the growth on galactose was not affected by loss of Enzyme I function, and while clearly involving a protein closely associated with the Umg-Enzyme I1 for glucose, did not require the phosphotransferase component of that system to be functional. The mutant KR 163 (table 2) differs from other Umg- mutants in retaining the ability to grow on galactose although it has lost virtually all the phosphorylating activity associated with the Umg system: in the interpretation of Bourd et al. ('75), such a mutant might well be Tgl+ Umg-. Indeed, when glucose is added to cultures of this mutant that are growing on galactose, there is a n immediate reduction in growth rate, to that observed with cultures growing on glucose alone (Kornberg and Riordan, '76). This interpretation, that both galactose and glucose can be transported into g u l p m g l strains of E.coli by a carrier closely associated with the Umg system, implies also that the phosphorylation of galactose, by ATP and galactokinase, is much more efficient than the phosphorylation of glucose by ATP and hexokinase; indeed, mutants that, in the
absence of Umg and PtsX activities, transport glucose actively via the GalP and Mgl-systems, grow only slowly and their growth is abolished by mutation of hexokinase (Curtis and Epstein, '75). Moreover, our interpretation accords with the finding (Solomon et al., '73) that the rapid efflux of a labelled sugar from Enzyme I - mutants of E.coli occurs only if the Enzyme I1 for that sugar is present, which bears out their prediction (Tanaka and f i n , '67) that an Enzyme I1 complex may catalyze facilitated diffusion of its substrate across the membrane and without phosphorylation. LITERATURE CITED Amaral, D., H. L. Kornberg 1975 Regulation of fructose uptake by glucose in Escherichia coli. J. Gen. Microbiol., 90: 157-168. Berger, E. A. 1973 Different mechanisms of energy coupling for the active transport of proline and glutamine in Escherichia coli. Proc. Nat. Acad. Sci. USA., 70: 1514-1518. Boos, W. 1969 The galactose binding protein and its relationship to the P-methylgalactoside permease from Escherichia coli. Eur. J. Biochem., 53: 419427. Bourd, G. I., R. S. Erlagaeva, T. N. Bolshakova and V. N. Gershanovitch 1975 Glucose catabolite repression in Escherichia coli K12 mutants defective in methyla-D-glucoside transport. Eur. J. Biochem., 53: 419427. Curtis, S. J . 1974 Mechanism of energy coupling for transport of D-ribose in Escherichia coli. J. Bact., 120: 295-303. Curtis, S. J., and W. Epstein 1970 T w o constitutive P-HPr: glucose phosphotransferases in Escherichia coli K12. Fed. Proc., 30: 1123. 1975 Phosphorylation of D-glucose i n Escherichia coli mutants defective i n glucose phosphotransferase, mannose phosphotransferase, and glucokinase. J. Bact., 122: 1189-1199. Ferenci, T., and H. L. Kornberg 1974 The role of phosphotransferase-mediatedsyntheses of fructose-I-phosphate and fhctose-6-phosphate in the growth of Escherichia coli on fructose. Proc. Roy. S ~ CB., . 187: 105-119. Gachelin, G. 1970 Studies on the a-methylglucoside permease of Escherichia coli. A two-step mechanism for the accumulation of a-methylglucoside-6-phosphate. Eur. J. Biochem., 16: 342357. Herbert, D., and H. L. Kornberg 1976 Glucose transport as rate-limiting step in the growth of Escherichia coli on glucose. Biochem. J., 456: 477-480. Henderson, P. J. F. 1974 Application of the chemiosmotic theory to the transport of lactose, D-galactose, and L-arabinose by Escherichia coli. In: Comparative Biochemistry and Physiology of Transport. L. Bolis, K. Bloch, S. E. Luria and F. Lynen, eds. North-Holland Publ. Co., Amsterdam, pp. 409424. Jones-Mortimer, M. C., and H.L. Kornberg 1974
CARBOHYDRATE UPTAKE BY E. COLI Genetical analysis of fructose utilization by Escherichia coli. Roc. Roy. SOC.B, 187: 121-131. Kornberg, H. L. 1972 Nature and regulation of hexose uptake by Escherichia coli. In: The Molecular Basis of Biological Transport. J. F. Woessner, Jr., and F. Huijing, eds. Academic Press, New York and London, pp. 157-180. 1973 Fine control of sugar uptake by Escherichia coli. Symp. SOC.Exp. Biol., 27: 175193. Kornberg, H. L., and M. C. Jones-Mortimer 1975 P t s X : A gene involved i n the uptake of glucose and of fructose by Escherichia coli. FEBS Letters, 51 : 14. Kornberg, H. L., and R. E. Reeves 1972a Correlation between hexose transport and phosphotransferase activity in Escherichia coli. Biochem. J., 126: 1241-1243. 1972b Inducible phosphoenolpyruvatedependent hexose phosphotransferase activities in Escherichia coli. Biochem. J., 128: 1339-1344. Kornberg, H. L., and C. Riordan 1976 Uptake of glucose into Escherichia coli by facilitated diffusion. J. Gen. Microbiol., 94: 75-89. Kornberg, H. L., and J . Smith 1972 Genetic control of glucose uptake by Escherichia coli. FEBS Letters, 20: 270-272. Kundig, W., S. Ghosh and S. Roseman 1964 Phosphate bound to histidine in a protein as a n intermediate i n a novel phosphotransferase system. Proc. Nat. Acad. Sci., USA, 52: 1067-1074. Lengeler, J. 1966 Untersuchungen zum Glukose Effekt bei der Synthese der Galaktose-Enzyme von Escherichia coli. 2.Vererbungsl., 98:203-229. Magasanik, B. 1961 Catabolite repression. Cold Spring Harbor Symp. Quant. Biol., 26: 249-256. McGinnis, J . F., and K. Paigen 1969 Catabolite inhibition: a general phenomenon in the control of carbohydrate utilization. J . Bact., 100: 902-91 3. 1973 Site of catabolite inhibition of carbohydrate metabolism. J. Bact., 214: 885487. Rogers, D., and S. H. Yu 1962 Substrate spec-
549
ificity of a glucose permease of Escherichia coli. J. Bact., 84:877-881. Romano, A. H., S. J. Eberhard, S. L. Dingle and T. D. McDowell 1970 Distribution of the phosphoenolpyruvate: glucose phosphotransferase system i n bacteria. J. Bact., 104:803-813. Roseman, S. 1969 The transport of carbohydrates by a bacterial phosphotransferase system. J. Gen. Physiol., 54: 138s-180s. Rotman, B., A. K.Ganesan and R. Guzman 1968 Transport systems for galactose and galactosides in Escherichia coli. I1 Substrate and inducer specificities. J. Mol. Biol., 36:247-260. Saier, M. H., B. U. Feucht, M. J. Newman and E. L. Castro 1976 Allosteric regulation of lactose and melibiose transport in Escherichia coli cells and membrane vesicles. Fed. Proc. (in press) Simoni, R. D., and S. Roseman 1973 Sugar transport. VII Lactosetransport in Staphylococcus aureus. J. Biol. Chem., 248: 966-976. Solomon, E., K. Miyai and E. C. C. Lin 1973 Membrane translocation of mannitol in Escherichia coli without phosphorylation. J. Bact., 144: 723-728. Stein, R., 0. Schrecker, H. F. Lauppe and H. Hengstenberg 1974 The staphylococcal PEP-dependent phosphotransferase system: demonstration of a phosphorylated intermediate of the Enzyme I component. FEBS Letters, 42: 98-100. Tanaka, S.,and E. C. C. Lin 1967 T w o classes of pleiotropic mutants of Aerobacter aerogenes lacking components of a phosphoenolpyruvatedependent phosphotransferase system. Proc. Nat. Acad. Sci. USA, 57: 913-919. West, I. C. 1970 Lactose transport coupled to proton movements in Escherichia coli. Biochem. Biophys. Res. Commun., 41: 655-661. West, 1. C., and P. Mitchell 1972 Proton-coupled P-galactoside translocation in non-metabolizing Escherichia coli. J. Bioenerg., 3: 445462. Wilson, D. B. 1974 Source of energy for the Escherichia coli galactose transport systems induced by galactose. J. Bact., 120: 866-871.
ABSTRACT In contrast to active transport, the uptake of carbohydrates via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) leads to the appearance in the cell of the sugar initially as a 1- or 6- phosphate ester. The components of the PTS that transfer phosphate to the sugar are not absolutely specific for any one sugar. Both their synthesis and their activity are controlled; in the latter, "fine" control, glucose-6-phosphate appears to play an important role. Studies of growth on, and uptake of, galactose by E.coli mutants devoid of components of the PTS and also devoid of active transport systems for galactose, suggest that proteins effecting facilitated diffusion of hexoses may be part of, or be closely associated with, the sugar-specific components of the PTS.
Escherichiu coli exDend metabolic enerev in taking up CarbohGdrates, either by &tive transport or by group translocation. In the former process, the carbohydrate enters the cell chemically unchanged, with concomitant uptake of protons (West, '70; West and Mitchell, '72; Henderson, '74) or expenditure of ATP (Berger, '73; Curtis, '74; Wilson, '74); in the latter, the sugar appears inside the cell initially as a phosphate ester (Rogers and Yu, '62). Since the papers presented in this issue by Doctors Ames, Boos, Heppel and Oxender are all relevant to the mechanisms whereby carbohydrates and amino acids are actively transported by microorganisms, I shall confine myself to a brief survey of the nature and regulation of group translocation, as mediated by the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTS, discovered by Doctor Saul Roseman and his colleagues (Kundig et al., '64) is a multicomponent system, closely associated with the membrane(s) of Gramnegative organisms that dissimilate carbohydrates predominantly via the EmbdenMeyerhof pathway (Romano et al., '70). It comprises (at least) three components that play a role in the uptake of many sugars (Reactions 1 and 2), and other components that exhibit some specificity for a relatively restricted range of sugars (Reactions 3 and 4). J. CELL.PHYSIOL.,89: 545550.
Enzyme I Enzyme I HPrhlP
+
+
PEP
P
+
-+Enzyme I m p
HPr
+HPrNP
+
pyruvate .. (1) Enzyme I
Enzyme I1 - + E n z y m e I I N P
Enzyme I1 N P
+
sugar
-+
sugar
+
+
HPr
(2)
(3)
- P + Enzyme I1
(4)
The main pleiotropic components of Reactions 1 and 2 are (i) an Enzyme I that is phosphorylated at a nitrogen atom of a histidine residue (Stein et al., '74), by phosphoenolpyruvate (PEP), and (ii) a small protein, also containing histidine and capable of being phosphorylated at that residue, designated HPr ("histidine-containing protein") for that reason (Kundig et al., '64). The sugar-specific components that participate in Reactions 3 and 4 are (iii) the sugars themselves and (iv) the appropriate Enzyme(s) I1 that catalyze the transfer of phosphate from the phosphorylated HPr to a terminal hydroxyl group. It has been demonstrated, particularly by genetic analysis, both that one Enzyme I1 can effect the phosphorylation of more than one sugar, and that one sugar can be phosphorylated by more than one Enzyme 11. The phosphorylation of glucose by E . coli, for example, is mediated to a major extent by an Enzyme I1 variously described as the uptake system for methyla-glucoside, specified by a gene thus abbreviated umg (Kornberg and Smith, '72), or as the glucose phosphotransferase component A-abbreviated g p t A (Curtis and Epstein, '70, '75); 545
546
HANS L. KORNBERG
this gene is located at about min 24 on the E.coii genome and is co-transducible with purB. However, wild-type organisms take up about 30% of the glucose they utilize via a different Enzyme 11. This is specified by a gene variously designated p t s X (Kornberg and Jones-Mortimer, ’75), g p t B or m p t (Curtis and Epstein, ’75), located at about min 36 on the E.coli genome and co-transducible with hga and h d g R (JonesMortimer and Kornberg, ’74). In strains of E.coZi that lack alternative routes for glucose uptake, such as the systems concerned in the active transport of galactose, the sequential removal of the functions specified by u m g + and p t s X + also leads sequentially to the loss both of the ability to grow on glucose and the ability to take up labelled glucose (Kornberg and JonesMortimer, ’75). Conversely, the Enzyme I1 specified by p t s X + can phosphorylate to the 6-phosphate not only glucose, glucosamine and mannose (Curtis and Epstein, ’75) but also fructose (Ferenci and Kornberg, ’74; Jones-Mortimer and Kornberg, ’74) which is mainly taken up and phosphorylated to the 1-phosphate ester by a different Enzyme 11, specified by p t s F and located at min 41.5 on the E.coli genome. These Enzymes I1 for the uptake of glucose and fructose are inducible (Kornberg and Reeves, ’72b). However, results obtained with batch cultures may be quantitatively misleading even if they are qualitatively useful. For example, some strains of E.coli (illustrated by strain K2, table 1) appear to be capable of taking up methyl-cuglucoside and glucose at high rates even though they have been grown on other substrates, whereas other strains (illustrated by strain B11, table 1) take up methyl*glucoside and glucose very poorly unless they have been induced by prior exposure to these carbohydrates, and even then the maximal rates of uptake are considerably less than those observed with the K2 strain. Direct measurement of the phosphorylation of methyla-glucoside (or glucose) in these cells, rendered permeable by treatment with toluene, showed that it is the phosphorylation step of the overall uptake process that thus differs in these strains (table 1); moreover, these rates were identical to the rates of methyl-a-glucoside uptake observed with intact cells (Kornberg and Reever, ’72a).
TA.BLE 1
m e c t of carbon source for growth,’ on: (Method A): the initial rate of uptake of metkyla-D- [14C] glucoside by intact cc,lZs, and (Method B ) : the PEP-dependent phosp,horylation of methyla-Dglucoside by toluene-treated cells thus grown ~~
Activity (nmoll min per mg dry wt.)
Strain
Carbon source for growth
Method A Method B
~~~
B11
K2
Glucose Fructose Gluconate Glycerol Glucose Fructose Gluconate Glycerol
15 3 3 3 38 44 24 19
17 3 2 3 56 99 30 23
For details, see Kornb’erg& Reeves, 1972a.
But these differences are not observed with cells grown i n continuous culture with glucose limitation (Herbert and Kornberg, ’76). Under these conditions, samples of either strain, taken from the chemostats, take up glucose at high rates, identical to each other and sufficient to account for the observed growth rates of the organisms over a wide range of doubling times. Glucose limitation thus appears to lead to a marked derepression of the Umg system; this may be a further manifestation of the regulatory component known to be associated with this system (Kornberg and Smith, ’72). Besides these “coarse” controls that govern the rates a t which Enzymes I1 are synthesized by cells growing under different conditions, “fine” controls regulate the rates at which Enzymes 11, already present, function in the cells. This is clearly illustrated by the manner in which one sugar can inhibit the uptake of another, although all the components required for the uptake of that other sugar are abundant, and were previously active, in the cell. For example, when glucose is added to cultures of E. coli growing on fructose (Kornberg, ’72) or on a variety of other sugars (McGinnis and Paigen, ’69; ’73), the organisms virtually cease to take up the previous growth substrate and preferentially utilize glucose. It is not the mere presence of glucose that brings about this dramatic switch: u m g mutants no longer exhibit this preference for glucose, which suggests that either the process of phosphorylation of glucose,
54 7
CARBOHYDRATE UPTAKE BY E . COLI
or the level of glucose-6-phosphate formed, are important to this “fine” control (Kornberg, ’72). Abolition of the ability of glucose to inhibit the uptake of fructose, by removal of Umg function, is not fructose-specific: in umg mutants, glucose is no longer preferred to any other carbon source (Kornberg, ’73). A more specific “fine” control was revealed through study of E.coli mutants that were resistant to hitherto toxic analogues of glucose (such as 2-deoxyglucose and 3-deoxy-3-fluoro-glucose)when growing on fructose, but were still sensitive to these compounds when growing on mannitol, glycerol or lactose (Amaral and Kornberg, ’75). The mutation that gave rise to this phenotype, designated cif (catabolite inhibition of fructose uptake), maps within or very close to the ptsF gene that specifies the high affinity Enzyme I1 for the phosphorylation of fructose to the 1phosphate ester. Unlike their parent organisms, cif-mutants can be induced to synthesize the PtsF-Enzyme I1 for fructose when fructose is added to cultures, even when they are growing on glucose. The cif mutation has thus overcome a “glucose effect” (Magasanik, ’61) that probably operates by inducer exclusion. Normally, glucose impedes the uptake of another sugar and thus impedes also the induction of the enzymes required for the catabolism of that sugar (Lengeler, ’66). It is interesting that cif mutants growing on fructose continue to utilize fructose not only in the presence of glucose but also of glucose6-phosphate: it is possible that the mechanism of inducer exclusion operative in wild-type cells, and that has been altered in cif mutants, involves an interaction of glucose-6-phosphate with a (phosphorylating?) component of the PtsF-system. It is also interesting that Enzyme I mutants of E.coli specifically resistant to inhibition
by methyla-glucoside when growing on lactose have been described by M. H. Saier and his colleagues. The mutation that confers this specific tolerance to the glucose analogue maps close to, or within, the gene that specifies the uptake system for lactose (Saier et al., ’76). The obvious differences between the active transport of a carbohydrate and its uptake by the phosphotr ansferase system have been taken virtually to exclude any similarity in the mechanisms of these processes. Although Roseman (‘69) originally envisaged the possibility that PTsugars might enter the cells by some (passive diffusion?) carrier and might be phosphorylated in a subsequent “trapping” reaction, this possibility was abandoned by him and his colleagues largely on evidence obtained with S . aureus: mutants devoid of Enzyme I were found to be unable to equilibrate sugars in the medium with those in the cells (Simoni and Roseman, ’73). However, from studies of the uptake of glucose and of its analogues into E.coli strains, Gachelin (’70) concluded that phosphorylation of methyla-glucoside occurred after entry into the cell. More recently, Bourd et al. (‘75) described E.coZi mutants in which the transport of methyla-glucoside by intact cells is low, but extracts of which contain normal phosphotransferase activities. This suggests impairment of a carrier protein involved in the facilitated diffusion (or active transport) of the sugar; this protein is postulated to be specified by a gene tgZ that is closely linked to umg or is an allele of it. We have isolated a similar mutant (KR 161, table 2), suspensions of which do not take up and phosphorylate methyla- [I4C] glucoside but which, when rendered permeable with toluene, readily formed methylaglucoside-6-phosphate in the presence of the analogue and phosphoenolpyruvate. We
TABLE 2
Properties of mutants impaired in the Umg system Organism
K2.lt K2.1.22 KR 161 KR 163
PTS activity of Doubling time (min) on Uptake of toluenized cells with glucose galactose methylff -glucoside glucose methylff -glucoside glucose
60 165 65 155
72 240 78 75
21 1 2 2
38 5 12 4
29 1 34 1
44 6 34 4
Postulated genotype
tgl+ tgltgltgl+
umg+ umgumg+ umg-
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HANS L. KORNBERG
have also isolated a mutant (KR 163, table 2) which, by these criteria, is U m g - in that it neither takes up methyla-glucoside nor phosphorylates it; however, it appears still to be able to take up at least one analogue of glucose via a protein closely associated with the Umg system, but not requiring phosphorylation of that sugar by the PTS. The glucose analogue thus taken up is galactose (which is a n epimer of glucose at C-4), which is normally actively transported by systems specified by g u l p (Rotman et al., '68), located at about min 55 and cotransducible with fdu (Claudia Riordan and H. L. Kornberg, to be published), and by m g l , located at about min 41 and cotransducible with ptsF (Boos, '69). The strains studied by us [Kornberg and Riordan ('76)l lack these active transport systems, yet grow on galactose at rates strongly dependent on the galactose concentration of the medium. Umg - derivatives of these strains, which, of course, grow more slowly on glucose, also grow very poorly on galactose; reversion to Umg+ or re-introduction of the umg+ allele by recombination restores normal growth on glucose and also restores the pattern of growth on galactose seen with the parent strain. Moreover, the growth on galactose was not affected by loss of Enzyme I function, and while clearly involving a protein closely associated with the Umg-Enzyme I1 for glucose, did not require the phosphotransferase component of that system to be functional. The mutant KR 163 (table 2) differs from other Umg- mutants in retaining the ability to grow on galactose although it has lost virtually all the phosphorylating activity associated with the Umg system: in the interpretation of Bourd et al. ('75), such a mutant might well be Tgl+ Umg-. Indeed, when glucose is added to cultures of this mutant that are growing on galactose, there is a n immediate reduction in growth rate, to that observed with cultures growing on glucose alone (Kornberg and Riordan, '76). This interpretation, that both galactose and glucose can be transported into g u l p m g l strains of E.coli by a carrier closely associated with the Umg system, implies also that the phosphorylation of galactose, by ATP and galactokinase, is much more efficient than the phosphorylation of glucose by ATP and hexokinase; indeed, mutants that, in the
absence of Umg and PtsX activities, transport glucose actively via the GalP and Mgl-systems, grow only slowly and their growth is abolished by mutation of hexokinase (Curtis and Epstein, '75). Moreover, our interpretation accords with the finding (Solomon et al., '73) that the rapid efflux of a labelled sugar from Enzyme I - mutants of E.coli occurs only if the Enzyme I1 for that sugar is present, which bears out their prediction (Tanaka and f i n , '67) that an Enzyme I1 complex may catalyze facilitated diffusion of its substrate across the membrane and without phosphorylation. LITERATURE CITED Amaral, D., H. L. Kornberg 1975 Regulation of fructose uptake by glucose in Escherichia coli. J. Gen. Microbiol., 90: 157-168. Berger, E. A. 1973 Different mechanisms of energy coupling for the active transport of proline and glutamine in Escherichia coli. Proc. Nat. Acad. Sci. USA., 70: 1514-1518. Boos, W. 1969 The galactose binding protein and its relationship to the P-methylgalactoside permease from Escherichia coli. Eur. J. Biochem., 53: 419427. Bourd, G. I., R. S. Erlagaeva, T. N. Bolshakova and V. N. Gershanovitch 1975 Glucose catabolite repression in Escherichia coli K12 mutants defective in methyla-D-glucoside transport. Eur. J. Biochem., 53: 419427. Curtis, S. J . 1974 Mechanism of energy coupling for transport of D-ribose in Escherichia coli. J. Bact., 120: 295-303. Curtis, S. J., and W. Epstein 1970 T w o constitutive P-HPr: glucose phosphotransferases in Escherichia coli K12. Fed. Proc., 30: 1123. 1975 Phosphorylation of D-glucose i n Escherichia coli mutants defective i n glucose phosphotransferase, mannose phosphotransferase, and glucokinase. J. Bact., 122: 1189-1199. Ferenci, T., and H. L. Kornberg 1974 The role of phosphotransferase-mediatedsyntheses of fructose-I-phosphate and fhctose-6-phosphate in the growth of Escherichia coli on fructose. Proc. Roy. S ~ CB., . 187: 105-119. Gachelin, G. 1970 Studies on the a-methylglucoside permease of Escherichia coli. A two-step mechanism for the accumulation of a-methylglucoside-6-phosphate. Eur. J. Biochem., 16: 342357. Herbert, D., and H. L. Kornberg 1976 Glucose transport as rate-limiting step in the growth of Escherichia coli on glucose. Biochem. J., 456: 477-480. Henderson, P. J. F. 1974 Application of the chemiosmotic theory to the transport of lactose, D-galactose, and L-arabinose by Escherichia coli. In: Comparative Biochemistry and Physiology of Transport. L. Bolis, K. Bloch, S. E. Luria and F. Lynen, eds. North-Holland Publ. Co., Amsterdam, pp. 409424. Jones-Mortimer, M. C., and H.L. Kornberg 1974
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