Lack of Glucose Phosphotransferase Function in Phosphofructokinase Mutants of Escherichia coli

Transcription

1 JOURNAL OF BACTERIOLOGY, My 1976, p Copyright C 1976 Americn Society for Microbiology Vol. 126, No. 2 Printed in U.SA. Lck of Glucose Phosphotrnsferse Function in Phosphofructokinse Mutnts of Escherichi coli R. A. ROEHL AND R. T. VINOPAL* Microbiology Section, Biologicl Sciences Group, University of Connecticut, Storrs, Connecticut 6268 Received for publiction 28 October 1975 Phosphofructokinse (p/ka) mutnts of Escherichi coli re impired in growth on ll crbon sources entering glycolysis t or bove the level of fructose 6-phosphte (nonpermissive crbon sources), but growth is prticulrly slow on sugrs, such s glucose, which re normlly trnsported nd phosphorylted by the phosphoenolpyruvte (PEP)-dependent phosphotrnsferse system (PTS). This is due to mlfunction of the PTS in pfka strins: p/ka mutnts resemble PTS mutnt strins in severl wys. The residul slow growth ofp/ka strins on glucose is dependent on low-ffinity trnsport of free glucose nd phosphoryltion by glucokinse. Estblishment by muttion of n ctive PEP-independent route for glucose entry gretly improves growth ofp/ka strins on glucose. pfka mutnts growing on nonpermissive PTS sugrs probbly lck dequte PEP for PTS function nd my thus be useful in physiologicl studies of the structurlly intct but nonfunctioning PTS. Mutnts of Escherichi coli deficient in the mjor phosphofructokinse (pfa) ctivity (Dfructose-6-phosphte, E.C ; the likely pfka product [47]) re impired in the use of crbon sources entering glycolysis t or bove the level of fructose 6-phosphte (Fig. 1). These crbon sources re referred to s nonpermissive; crbon sources entering glycolysis below the level of fructose 6-phosphte, such s fructose, glycerol, succinte, re permissive for pfka strins. Surprisingly, growth on nonpermissive crbon sources is not uniformly slowed; growth on sugrs nd sugr derivtives trnsported nd phosphorylted by the phosphoenolpyruvte (PEP)-dependent phosphotrnsferse system (PTS; 31, 32), for exmple growth on glucose, mnnose nd mnnitol, is much more severely retrded by muttion t pfka thn is growth on sugrs trnsported in other wys, for exmple glctose (52), lctose, nd glucose 6- phosphte (26, 49). It hs been suggested tht the prticulrly slow growth of pfka mutnts on PTS-trnsported sugrs, such s glucose, is due to lck of PEP for trnsport (18). the rgument is stoichiometric. If hexose phosphte formed in pfka strins is metbolized entirely through the hexose monophosphte (HMP) shunt (Fig. 1), one hexose phosphte would yield one triose phosphte (such s PEP) nd three CO2's (18). The PTS-medited trnsport of glucose is written, glucose + PEP -> glucose 6-phosphte + pyruvte (31, 32). Then, in metbolism of glucose by pfka mutnt, one glucose would yield one pyruvte nd three CO2's. In the bsence of 852 route for converting pyruvte to PEP under these conditions, there would be no triose phosphte for biosynthesis, or, s ws suggested, if triose phosphte (or ny precursor) is used for biosynthesis there would be insufficient PEP for the trnsport of glucose (18). Growth on non-pts crbon source, such s glucose 6-phosphte, would not be subject to this restriction. (By the sme resoning, it should not be possible for n orgnism to metbolize PTS-trnsported sugr by the Entner-Doudoroff pthwy, by which hexose phosphte -* triose phosphte + pyruvte, nd confirmtion of this expecttion is found in the fct tht glucose PTS is not present in wide rnge of strictly erobic bcteri [3], which normlly use glucose by the Entner-Doudoroff route.) In ccord with the stoichiometric PEP limittion explntion of the pfka phenotype is the finding tht muttion to constitutive expression of the glyoxylte shunt, muttion known to suppress the PEP synthetse (pps) mutnt phenotype (Fig. 1), probbly by estblishing n lternte pthwy for the conversion of pyruvte to PEP, lso prtilly suppresses the pfka phenotype on crbon sources trnsported by the PTS (49). However, muttionl loss of the HMP shunt in p/ka strin does not eliminte either growth on glucose 6-phosphte or glyoxylte shunt suppression on glucose, wheres loss of the second, minor, phosphofructokinse ctivity, by muttion t the pfkb locus (49), bolishes both growth on glucose 6-phosphte nd glyoxylte shunt suppression on glucose in pfka strins with functionl HMP shunt (48).

2 VOL. 126, 1976 glucose-6-p gloctose lctose glucose glucose-6-p zw 11HMP IMPAIRED PTS FUNCTION IN E. COLI MUTANTS P-gluconoloctone Wi shunt gluconote-6-p monnose pi /( CO2 pnntsi, fructose-6-p === pentose-p monnitol v, '\ fructose 6.rJpfkA pfkbi fdp fructose-1,6-p glycerol A. dihydroxyocetone - P glyceroldehyde-3-p oxlocetote x phosphoenolpyruvote w lyox ) pyk t pps I shunt GTP I Ip- pyruvte CO2 FIG. 1. Centrl pthwys of sugr metbolism in E. coli. Abbrevitions: fdp, fructose diphosphtse; HMP, hexose monophosphte; ptk, phosphofructokinse; pgi, phosphoglucose isomerse; pps, PEP synthetse; pts, trnsport by PEP-dependent phosphotrnsferse system; pyk, pyruvte kinse; zwf, glucose 6-phosphte dehydrogense. Fructose enters glycolysis mostly s fructose 1,6-diphosphte (11). It then ppers tht in pfka mutnt strin metbolism of hexose phosphte is lrgely by glycolysis, through residul phosphofructokinse ctivity. Wheres stoichiometric inference of PEP shortge during growth on glucose nd other PTS-trnsported sugrs thus cnnot be mde, PTS mlfunction in pfka strins cused by PEP limittion still remins n ttrctive hypothesis, s will be discussed. If filure of PTS function is in fct the cuse of the prticulrly slow growth of pfka strins on glucose nd other PTS sugrs, then pfka mutnts should resemble pts mutnts defective in enzyme I (ptsl) or in the histidine-contining phosphte crrier protein (ptsh) of the PTS (31, 32). Two specific predictions my be mde. First, the estblishment of PEP-independent route for the entry of glucose into the cell, by muttion to constitutive expression of the glctose-specific glctose trnsport system (non-pns, with n ffinity for glucose [52, 53]), should suppress the pfka phenotype on glucose just s it suppresses the pts mutnt phenotype on glucose in Slmonell typhimurium (35, 42). Second, the residul slow growth ofp1 A strins on glucose might be expected to depend on slow, non-pns entry of free glucose into the cell followed by denosine 5'-triphosphte-dependent phosphoryltion, s hs been found for residul growth on glucose of pts mutnts of Slmonell (38) nd of E. coli (7, 14, 45). We confirm these predictions here. MATERIALS AND METHODS Bcteril strins. The strins of E. coli K-12 used re listed in Tble 1, with genotypes nd detils of derivtion. Medi. Miniml medium 7 ws used (4), supplemented with 1,g of thimine hydrochloride per ml, nd the crbon source t.2%, unless otherwise stted. BTYEX 7 medium ws miniml medium with 1 g of tryptone (Difco Lbortories, Detroit, Mich.) nd 5 g of yest extrct (Difco) per liter. Csein hydrolyste medium ws miniml medium with 1 g of Csmino Acids (Difco) per liter. Agr pltes were of these medi with 1.5% gr (Difco). Miniml top gr ws miniml medium with.75% gr. Medi used for phge P1 propgtion nd ssy were those of Lennox (21). Nitrogen-free medium (2) ws supplemented with D-serine t 8 mm s nitrogen source. Genetic procedures. Preprtion of phge P1 lystes, their use in trnsductions, mutgenesis with ethyl methne sulfonte, nd penicillin counterselections were done s described (49). thya mutnts were selected s forming colonies on glycerol pltes with 1 jig of trimethoprim per ml nd 5,ug of thymine per ml (24). A glr' derivtive of K1, strin RR3, ws isolted using ethyl methne sulfonte mutgenesis nd the selection of Buttin (3). The glr' mrker ws moved into other strins by cotrnsduction with thya. Thy+ trnsductnts were scored for the glr llele on tetrzolium overly pltes modified from Sedler et l. (34), by replcing membrne filters with filter pper circles (Shrk skin, Schleicher nd Schuell Co., Keene, N.H.) covered with 3 ml of miniml top gr. glr scoring ws confirmed by glctokinse ssy; glr+ strins were induced more thn 1-fold by ddition of glctose to csein hydrolyste medium (from.2 to.3,mol per min per mg of protein), wheres glrr strins hd high, constitutive ctivities. D-serine deminse mutnts (dsda) were isolted following ethyl methne sulfonte mutgenesis nd penicillin counterselection in nitrogen-free glycerol medium with 8 mm D-serine s nitrogen source (23). Becuse our strins re inhibited by contminting L-serine (6), 25,ug of L-isoleucine per ml ws dded to ll medi contining D-serine. ptsi nd glk muttions were introduced into dsda strins by trnsductionl repir to use of D-serine s nitrogen source. Becuse D-serine is toxic to dsda strins (23) phenotypic expression of dsda + ws permitted by csting the trnsduction mixture in nitrogen-free miniml top gr on nitrogen-free gr plte with lctte s crbon source nd incubting overnight t room temperture before dding lyer of gr with D-serine. Purified ptsl trnsductnts were identified s fructose negtive nd showed the typicl pts phenotype. Purified glk trnsductnts of pfka strins were especilly wek on glucose nd were verified by glucokinse ssy; glk+ strins grown in csein hydrolyste medium hd n ctivity of bout 5 nmol per min per mg of protein nd glk- strins, including DF52, hd undetectble ctivity. Enzyme ssys. Glctokinse nd glucokinse were ssyed by modifiction of the glycerol ki-

3 854 R-OEHL AND VINOPAL J. BACTERIOL. TABLE 1. E. coli K-12 strins used Sex Genotype Derivtion, source, or reference AM1 HfrC pfkal, tona22, lmbd D. G. Frenkel; from K1 (26) DF51 HfrH lci22, ptsi2, rel-1, thi-1, lmbd D. G. Frenkel; formerly MM-6 (14, 45) DF52 HfrH glk, lci22, ptsi2, rel-1, thi-1, lmbd CGSC; formerly GN-2 (14, 45) K1 HfrC tona22, lmbd D. G. Frenkel (2) RR25 HfrC glr', ptkal, tona22, lmbd _b RR3 HfrC pfkal, tona22, lmbd s for RR25, but glr+ trnsductnt, by ssy RR45 HfrC glr', glk, ptkal, tona22, lmbd -C RR47 HfrC glr', pfkal, tona22, lmbd s for RR45, but glk + trnsductnt, by ssy RR53 HfrC glk, pfkal, tona22, lmbd _d RR57 HfrC ptkal, tona22, lmbd s for RR53, but glk+ trnsductnt, by ssy RR65 HfrC ptkal, ptsl2, tona22, lmbd _ e RR67 HfrC p/kal, tona22, lmbd s for RR65, but ptsil trnsductnt Gene designtions re ccording to Tylor nd Trotter (46) except glk, glucokinse (7). All known mrkers re included. Phenotypic bbrevitions use cpitlized gene designtions; Fruc is fructose utiliztion. Other bbrevitions re EMS, ethyl methne sulfonte mutgenesis; pcs, penicillin counterselection; spon., spontneous muttion; trns., trnsduction with phge P1 (followed by donor strin in prentheses). Thus, the second step in construction of RR45 would be red " trnsductnt to dsda + using P1 grown on DF52, identified s glk by ssy nd scored s ble to grow on fructose." CGSC, obtined through the courtesy of B. Bchmnn, Coli Genetic Stock Center, Yle University, New Hven, Conn. From AM1 in two steps; first spon., selection for Thy-'; then Thy+ (GlRC[ssy]) trns. (RR3M). From RR25 in two steps; first EMS, pcs for Dsd-f; then Dsd+ (Glk[ssyl, Fruc+) trns. (DF52). dfrom AM1 in two steps; first EMS, pcs for Dsd-'; then Dsd+ (Glk [ssy], Fruc+) trns. (DF52). '*From AM1 in two steps, the first s in footnote d, the second Dsd+ (Fruc- trns. (DF51). f See genetic procedures. nse ssy of Richey nd Lin (29). Wshed cells were sonicted in.5 M tris(hydroxymethyl)minomethne (Tris) hydrochloride (ph 7.8) buffer with 5 mm MgCl2 nd 1 mm mercptoethnol, nd cellfree extrcts were prepred by centrifugtion for 2 min t 2, x g. The rection mixture (41) contined.25 ml of the soniction buffer with 3.2 mm NF,.2 ml of mm denosine 5'-triphosphte,.5 ml of diluted extrct, nd.2 ml of 6.25 mm D-['4C]glucose or glctose (.5 Ci/mol) (finl volume,.25 ml). Rections were crried out t 25 C. At 5, 1, nd 15 min fter ddition of denosine 5'- triphosphte.5-ml liquot ws withdrwn, pipetted onto diethylminoethyl-ion exchnge filter (Whtmn DE81), nd wshed with 5 ml of 8% ethnol, nd then three 1-ml volumes of H2. Filters, blotted dry, were plced into vil for scintilltion counting. Protein ws ssyed by the Folin method (22). Chemicls. Glucose, glucose 6-phosphte, mnnose, glctose, fucose (ll D isomers), nd D-serine were from Sigm Chemicl Co., St. Louis, Mo. Uniformly '4C-lbeled glucose nd glctose were from New Englnd Nucler Corp., Boston, Mss. Mnnitol nd fructose (D isomers) were from Fisher Scientific Co., Pittsburgh, P. Lctte ws from Fisher or Sigm. Fisher sodium lctte, 7% syrup, contined substnce tht ws somewht inhibitory to pt7a strins (49); the ptsi llele from DF52 eliminted inhibition. Sigm lctic cid, sodium slt, 6% syrup, ws not inhibitory. Trimethoprim ws the gift of R. E. Wolf, Jr. RESULTS Estblishment of PEP-independent route for entry nd phosphoryltion of glucose suppresses the ptfca phenotype on glucose. In their description of pts mutnts of S. typhimurium Simoni et l. reported tht induction of trnsport system for glctose permitted growth of ptsi mutnt on glucose nd referred to similr observtions mde with E. coli mutnts (42). Sier et l. lter found tht muttion t glr, resulting in constitutive production of the glctose-degrdtive enzymes, nd the glctose-specific glctose permese (34, 53), resulted in improved growth ofptsi-ptsh mutnts of Slmonell on glucose (35). Suppression of the pts phenotype in these cses ws presumbly due to trnsport of free glucose by glctose permese, for which glucose is substrte but not n inducer, nd denosine 5'-triphosphte-dependent phosphoryltion by glucokinse. Glucokinse-dependent glucose growth of n E. coli strin lcking both PTS enzymes II for glucose, following induction with glctose, hs been described (1). We tested for similr suppression of the pfa phenotype in E. coli. Initil experiments showed tht n-fucose, nonmetbolizble inducer of the glctose system (42, 53), stimulted growth of p/ka mutnts in glucose medium. For exmple, strin RR3 hd doubling time of 7.4 h in.2% glucose miniml medium, nd the ddition of D-fucose to 3.3 mm decresed this to 5.7 h. We then introduced glr' (constitutive) muttion into pfa, thya strin by Pl-medited trnsductionl repir of thya, using glr'' donor strin. (thya nd gir re c. 5% linked by trnsduction [34].) The glr'' trnsductnts, identified on tetrzolium overly pltes nd verified by glctokinse ssy, were fster growing thn glr + (inducible) trnsductnts on glucose pltes nd in glucose miniml medium (Tbles 2, 3, 4; cf. RR25 LfpkAl, glr'] nd the isogenic glr+ strin RR3). Addition of D-fucose to glucose

4 VOL. 126, 1976 medium shortened the doubling time of glr + but not ofglrc trnsductnts (dt not given; it is not known if our strins hve the,8-methyl glctoside permese [53]). Introduction of glrc lso improved growth somewht on mnnose, which is probbly lso trnsported by the glctose permese (35; but with lower ffinity thn for glucose, 33), but not on mnnitol or on the non-pts sugrs glucose 6-phosphte nd glctose (Tbles 2, 3). Improved growth on lctose (Tble 3) will be discussed. If the glrc muttion suppresses the p1 A phenotype on glucose by estblishing non- PTS route for entry of free glucose, then phosphoryltion of glucose, nd suppression, should be dependent on glucokinse (7, 14, 45). To test this, we introduced glucokinse (glk) muttion into D-serine deminse (dsda) mutnt derivtive of pfka, glrc strin, using trnsductionl repir of dsda. (glk nd dsda re IMPAIRED PTS FUNCTION IN E. COLI MUTANTS 855 linked by P1 trnsduction [7].) The glk trnsductnts (verified by ssy) were in fct very slow growing on glucose (Tble 3; cf. RR45 lpfkal, glrc, glk] nd the isogenic glk + trnsductnt RR47). Growth on mnnose ws not much ffected by introduction of glk (Tble 3), suggesting mnnose trnsport nd ction of mnnokinse (38, 39) rther thn use of contminting glucose in the mnnose. Suppression of ptsi or ptsh mutnt on glucose by glrc muttion would necessrily be by estblishment of n lternte, non-pts route for entry of glucose into metbolism (35). However, glrc suppression of ptka mutnt might be more complex. For exmple, if low PEP levels were limiting growth of ptka strins on glucose, then PEP-independent glucose entry vi the glctose permese would ese PEP restriction nd might support extensive simultneous functioning of the glucose TABLE 2. Colony sizes Genotype Crbon source (colony dim, mm) pfka glr Glucose Mnnose Mnnitol Glycerol Fructose Glctose Lctose K RR RR25 - c Miniml pltes contined the crbon source t.2%. Cells from glycerol cultures were wshed nd diluted in buffer nd spred to give 1 to 1 colonies per plte. After bout 36 h t 37 C verge colony dimeters were obtined using dissecting microscope with micrometer scle. TABLE 3. Doubling times in miniml medi Genotype Crbon source (doubling time, h t 37 C) Glcpt7A gir glk Glucose Mnnose Mnnitol Glycerol Glu-6-P tose Lctose K RR > RR25 - c > RR47 - c + 6.9b 8.2 >5 1.4 NDY ND ND RR45 - c >5 1.2 ND ND ND Miniml medi contined the crbon source t.2%. Inocul were from fresh sttionry cultures in.2% glycerol miniml medium. Absorbncy t 58 nm ws determined periodiclly with Perkin-Elmer spectrophotometer, model 139, for 5-ml cultures in 25-ml flsks on New Brunswick gyrtory shking wter bth nd plotted s semilogrithmic function of time. Abbrevition: Glu-6-P, Glucose 6-phosphte. b RR45 nd RR47 re trnsductnts of dsda derivtive of RR25 obtined by ethyl methne sulfonte mutgenesis. This derivtive ws slow growing in severl medi but retined the glrc mrker. c ND, Not determined. TABLE 4. Effect of sugr concentrtion on colony size Genotype Glucose concn (colony dim, mm) Mnnose concn (colony dim, mm) ptha gir.1%.5%.1%.5% 1.% 2.%.1%.5%.1%.5% 1.% 2.% K NDb ND ND ND ND ND AM ND ND ND ND ND ND RR RR25 - c Miniml pltes contined sugr t the indicted concentrtion. Procedures s in Tble 2. Growth on mnnitol ws not improved by incresed concentrtion (Tble 2). b ND, Not determined.

5 856 ROEHL AND VINOPAL PTS. Tht the PTS plys no essentil role in glucose growth of pfka, glr' strin ws demonstrted by the introduction of ptsi muttion into the pfka, glr', dsda strin by cotrnsduction with dsda; growth in glucose medium ws the sme s tht of n isogenic pts+ trnsductnt (dt not given). Residul growth of pfkca strins on glucose is dependent on glucose concentrtion nd glucokinse. Mutnts of Slmonell nd E. coli defective in enzyme I or the histidine-contining phosphte crrier protein of the PTS grow better on glucose thn on other PTS sugrs, such s mnnitol (9, 38). This is evidently due to entry of free glucose by low ffinity trnsport systems nd internl phosphoryltion by glucokinse. Growth ofptsi mutnts of Slmonell is fster t high glucose concentrtions (42) nd muttionl loss of glucokinse ctivity shrply decreses growth rtes on glucose in pts mutnts of Slmonell (38) nd ofe. coli (7, 14, 45). pfka mutnts ofe. coli lso grow better on glucose thn on mnnitol (26, 49; Tbles 2, 3). Dependence of the growth rte of pfka mutnt, strin AMl, on glucose concentrtion is shown in Tbles 4 nd 5. The doubling time of AMl decreses threefold s glucose concentrtion increses from.2% to 2.5%, wheres the growth rte of wild-type E. coli is independent of glucose concentrtion in this rnge (4, 51; Tble 5). A Linewever-Burk plot of the dt in Tble 5 (Fig. 2) suggests two routes for the entry of glucose into the pfka mutnt, one supporting hlf-mximl growth rte t.3 mm glucose, the other lower ffinity, higher V,,,,,. system with n pprent K,, of bout 4 mm glucose (ffinities estimted grphiclly). The use of ech of these routes is dependent on glucokinse, s shown by the growth response of RR53, glk derivtive of AMl (Tble 5, cf. RR53 nd RR57, n isogenic glk+ trnsductnt; Fig. 2). The glk muttion in n otherwise wild-type strin does not ffect growth rtes on these concentrtions of glucose (7, 14; nd unpublished observtions). The finding tht the residul slow growth of pka mutnt strins on glucose is lrgely dependent on the ction of glucokinse suggests nonphosphorylting routes of glucose entry. However, it would be possible tht PEP spred by glucose metbolism through glucokinse would permit prtil functioning of the PTS system in pfka mutnts, s discussed bove. In this cse, incresing concentrtions of glucose might improve the "pump-priming" of the PTS nd yield Linewever-Burk plot of the generl shpe seen in Fig. 2. Loss of the PTS by muttion should then eliminte t lest the low ffinity trnsport system, s in the cse of glk muttion. A pfka, ptsi double mutnt ws constructed nd compred in growth on vrious concentrtions of glucose to n isogenic ptsi' strin (strins RR65 nd RR67, Tble 5; RR65, Fig. 2. A different pir of isogenic strins gve the sme results, in seprte series of growth o 16 D 2 o do RR65 (pfka, ptsd) RR53(glk,pfkA) AMI (pfka) l - 4 t K 1 (wild type) ~~~A ~~~~~~~~~~~ [glucose]li (mm-1) FIG. 2. Effect of glucose concentrtion on growth rtes ofseverl strins ofe. coli. Plotted from dt in Tble 5. Abbrevitions: glk, glucokinse; ptsi, enzyme I of the PTS. TABLE 5. Effect of glucose concentrtion on doubling time Genotype Glucose concn (doubling time, h. t 37 C) pfka glk ptsi.2%.3%.4%.5%.1%.2%.4%.5% 1.% 2.% 2.5% K AMI RR RR RR RR Miniml medi contined glucose t the indicted concentrtion. Other procedures s in Tble 3. J. BACTERIOL.

6 VOL. 126, 1976 rte mesurements.). Wheres the ptsi lesion does slow growth of pfka strin on glucose mrkedly, the growth response of the double mutnt to chnging glucose concentrtion remins biphsic, with the two trnsport components probbly not chnged; pprent K,,'s for glucose in the double mutnt re estimted s roughly.7 mm nd 22 mm, respectively, from Fig. 2. The effect ofptsi muttion is then principlly on V,,,,,, nd my thus be bsed on ptsincresed sensitivity to glucose inhibition (31, 32, 36) of synthesis or ctivity of two unidentified non-pts trnsport systems crrying glucose, rther thn on direct elimintion of route for glucose entry. Growth of pfka strins on mnnose is less concentrtion-dependent thn growth on glucose (Tble 4). Incresing mnnitol concentrtion does not improve growth ofptha strins on pltes (dt not shown), but this could be due simply to lck of mnnitol kinse. Growth inhibition of pfka strins by nonpermissive crbon sources. Growth ofpts mutnts on permissive crbon sources is typiclly inhibited by PTS sugrs, due to interference with induction of ctbolic enzymes (27, 31, 32, 37). pfka mutnts re lso inhibited by some nonpermissive crbon sources, nd this sensitivity resembles tht ofpts mutnts (27) in tht inhibition ofp/a strins by glucose my under some conditions be overcome by cyclic denosine 3',5'-monophosphte (48). Of course, p1ka mutnts, blocked in mjor metbolic route, might ccumulte phosphorylted intermedites, nd such ccumultion is known often to result in growth stsis (13, for ccumulted glucose 6-phosphte; 1, for number of other references). We hve thus exmined inhibition ofpf A mutnts further. p/ka strins re inhibited on succinte or csein hydrolyste medium by nonpermissive PTS sugrs nd not by glctose or lctose (unpublished observtions), but this ccords simply with the reltive impirment of growth on these sugrs s sole crbon source. Becuse slow growth of p1 A strins on glucose is dependent on glucokinse (Tble 3; Fig. 2), glk muttion should directly or indirectly decrese production of phosphorylted intermedites from glucose. Then, if ccumultion of n orgnic phosphte is the bsis of glucose inhibition of ptka strins, glk muttion should protect ginst it, wheres if glucose inhibition is like tht seen for pts mutnts glk muttion might even increse sensitivity to glucose by excerbting PTS deficiency. A comprison of the sensitivities of the p/ha strin AM1 nd glk derivtive of it to inhibition by PTS sugrs (Tble 6) supports the ltter hypothesis. As IMPAIRED PTS FUNCTION IN E. COLI MUTANTS 857 with pts mutnts (7, 31), the nonmetbolizble glucose nlogues methyl--d-glucoside nd 2-. deoxyglucose lso inhibit p1 A strins (unpublished dt). Surprisingly, glucose-resistnt revertnts of AM1 selected on complex medium include strins evidently mutted t ptsl (H). These strins re lso resistnt to mnnose nd mnnitol nd re completely unble to grow on fructose, mltose, nd lctose, s the result of lesion mpping close to dsda, by trnsduction (dt not shown). ptsi derivtives of AM1 constructed by trnsduction re lso protected ginst inhibition by PTS sugrs (Tble 6). However, the donor ptsi strin, DF51, is lso insensitive to inhibition (Tble 6) nd might be crr mutnt (31, 32, 36, 37). (ptsi [H] mutnts hve been selected s revertnts in cse of fructose toxicity involving ccumulted phosphorylted intermedites [11].) DISCUSSION Wheres our studies support the ide tht PTS mlfunction is n importnt prt of the phosphofructokinse mutnt phenotype, the bsis for the mlfunction is not known. Cses of inhibition of the PTS by ccumulted metbolic intermedites hve been described (16, 17, 25; 8 discusses regultion of glucose PTS ctivity), TABLE 6. Inhibition by PTS-trnsported sugrs Sugr tested Genotype (zone of inhibi- Growth tion, cm ) medium Glu- Mn- Mncose nose nitol K Succ CH7 AMI Succ CH7 2.8 RR Succ CH RR Succ CH7 DF Succ CH7 Cells from fresh sttionry BTYEX7 cultures were wshed in buffer, suspended in 2.5 ml of miniml top gr t c. 5 x 17 cells/ml, nd overlyered onto miniml pltes with.2% sodium succinte or 1.% Csmino Acids s crbon source. Filter pper disks (.635 cm, Schleicher nd Schuell, Inc., no. 74-E) were sturted with 1% solution of the sugr tested nd plced onto n overlyered plte. Incubtion ws t 37 C for 24 h. Abbrevitions: Succ, succinte; CH, csein hydrolyste. b Dimeter of the zone of inhibition round pper disk is given; indictes no visible inhibition t the time of inspection. Inhibition of AM1 by glucose nd mnnose ws seen on CH7 medi t shorter incubtion times; dt re comprtive.

7 858 ROEHL AND VINOPAL but in detil these exmples do not support n explntion of the pfika phenotype s involving dernged "fine control" of the PTS. Lck of dequte PEP for trnsport nd biosynthesis in p1ka strins provided with nonpermissive PTS crbon sources ws suggested by Kornberg nd Smith on the bsis of logiclly ppeling rgument tht is most likely incorrect (Introduction). However, for severl resons PEP limittion remins n ttrctive explntion for the prticulrly slow growth ofpfka strins on PTS sugrs. (i) Kornberg nd Smith showed tht pfka cell suspensions re stimulted in the ccumultion of lbel from rdioctive glucose if preincubted with substnces which cn be converted to PEP or spre its use, such s pyruvte nd mlte (18). The stimultion by pyruvte ws eliminted by introduction of pps muttion (Fig. 1) in ccord with conversion to PEP being required for the effect (18), lthough pyruvte hs been reported to inhibit PTS function in pps strins (25). (ii) Muttion to constitutive expression of the enzymes of the glyoxylte shunt increses growth rtes of p1ka strins on PTS sugrs, glucose, for exmple, supporting s rpid growth rte s tht on glucose 6-phosphte (49). An unregulted glyoxylte shunt would be likely to estblish route for conversion of pyruvte to PEP, nd should t lest decrese use of PEP for nplerotic function (49). (iii) If PEP limittion ffects PTS function in pfka strins, then supplement of end products hving PEP s biosynthetic precursor might spre PEP removl nd stimulte growth on PTS sugrs. Growth ofpfka strins on mnnose is stimulted by ddition of romtic mino cids nd shikimic cid to the medium (49). However, ddition of succinte or mlte, which could hlt removl of PEP for nplerotic function, hs no obvious effect (dt not shown). (iv) PEP insufficiently in pfka strins could not be due simply to metbolism by the HMP shunt (Introduction). Still, if PEP is limiting in growth on PTS sugrs, then ny use of the HMP shunt would worsen PEP shortge. Loss of the shunt, for exmple, by muttion in glucose 6-phosphte dehydrogense (zwf, Fig. 1), might then increse productive metbolism through residul phosphofructokinse nd improve growth on PTS sugrs. This is not ordinrily seen with glucose s crbon source (18, 49), but then growth ofpfka strins on glucose is lrgely dependent on non-pts phosphoryltion; on mnnitol pltes, however, zwf trnsductnts ofptka strins (49), verified by ssy, lwys show slightly but distinctly stronger J. BACTERIOL. growth thn zwf+ trnsductnts (unpublished observtion). Similrly, muttion in phosphoglucose isomerse (pgi, Fig. 1) mrkedly improves mnnitol growth of t lest one pfka mutnt (5). Why should PEP be limiting in pfka mutnts? Altered concentrtions of PEP or its precursors in p1ka mutnts could led to excessive or uncoordinted ctivity of n enzyme cting on PEP (pyruvte kinse, for exmple) or on precursor of PEP, nd this could result in selective effect on utiliztion of PTS sugrs. Wheres lowered PEP concentrtion should not ffect the PTS selectively, the ffinity of the PTS for PEP being comprble to tht of biosynthetic enzymes hving PEP s substrte (5, 19, 44), filure of the PTS would quickly stop production of PEP. If single unknown rection were responsible for most PEP loss some prtil revertnts of p/ka strins might be ltered in tht ctivity. However, one set of 3 independent revertnts selected on mnnose did not include ny of this clss, ll being ssigned to other clsses (48). We re continuing to look for such revertnt nd re conducting direct mesurements of cellulr PEP pools following shift of pfka strins to nonpermissive crbon sources. It is of interest tht growth ofpfka mutnts on lctose, non-pts sugr, is improved by glrc muttion (Tble 3). Lctose is split to glucose nd glctose during metbolism, but improved use of glctose seems n unlikely bsis for the effect becuse glrc does not improve growth on glctose (Tble 3). Phosphoryltion of endogenously generted glucose in E. coli my occur to lrge extent by ction of the PTS; loss of glucokinse results in only 2% decrese in the growth rte on lctose of n E. coli mutnt unble to use glctose (7). p1 A muttion does slow growth on lctose more thn on glctose or glucose 6-phosphte (Tble 3; 49). If PTS phosphoryltion of endogenously generted glucose involves prior excretion (s in the cse of fructose formed from sucrose in fructokinse mutnts of Aerobcter erogenes [15]) then estblishment of non-pts route for glucose entry, s by glr' muttion, would improve lctose growth of pfka strin. Muttion in glctokinse (glk) results in ccumultion of endogenously produced glctose nd induction of glctose permeses (53). The p/ka phenotype on glucose might be expected to be suppressed by glk, butpfka,glk strins used in previous studies (47, 48) were not suppressed. Constitutivity of glctose permese in these strins hs not been checked. It is not cler wht role the PTS plys in the residul slow growth of pfka mutnts on glu-

8 VOL. 126, 1976 cose. The relevnt dt re presented in Fig. 2. Two trnsport systems for glucose (hlf-mximl growth rtes t roughly 4 mm nd.3 mm) re seen to function in pika strins. By contrst, the growth rte of wild-type E. coli on glucose is independent of glucose concentrtion except t very low concentrtions, t which the pprent K,,, for growth in bout 1,uM (4, 51). The PTS shows K,,, for glucose of.4 mm in vitro (19; this vlue my be 1-fold lower when cytoplsmic fctor III prticiptes [32]; low K,,, vlues for glucose PTS trnsport by whole cells re reported in reference 1), hence the higher ffinity route for glucose entry in pfka strins might be the PTS. However, loss of glucokinse slows growth of p/ka strins t ll glucose concentrtions nd results in essentilly concentrtion-independent growth. This indictes tht neither of the two trnsport systems cn be n independently functioning PTS, nd limits possible role for the PTS to (i) crrying out group trnsloction of glucose by use of PEP mde vilble by metbolism through glucokinse, or (ii) fcilitting diffusion of glucose without phosphoryltion (43), or both. Loss of enzyme I of the PTS does not eliminte either the higher or lower ffinity glucose trnsport system, but insted results in decresed ctivity of ech. One explntion of this would be tht the synthesis or ctivity of the two unidentified trnsport systems crrying glucose is more sensitive to glucose inhibition in the ptsi strin (ctbolite repression, inducer exclusion; 27, 31, 32, 36, 37), with the PTS plying no role in trnsport. The higher ffinity system, for exmple, could be the glctose-specific glctose permese (53; cf. RR3 nd RR25, Tble 4; some ptsl mutnts re impired in growth on glctose [12]). However, rguments could lso be mde for residul enzyme I ctivity functioning with PEP spred by ction of glucokinse or for n effect of the ptsi muttion on vilbility of enzymes II (1, 7) for fcilitted diffusion of glucose (43). Glucose concentrtion responses ofp/ka strins lcking one or both of the enzymes II for glucose (7) would help settle this question nd re being crried out. Wheres trnsport of number of sugrs by group trnsloction is the best understood function of the PTS, it is clerly not the only function. Mutnts ltered in components of the P1S re ffected in the regultion of inducible enzyme synthesis, in trnsport of non-pts substrtes, in cyclic denosine 3',5'-monophosphte metbolism, nd in chemotxis (1, 31, 32), thus it is likely tht the PTS plys centrl regultory role (28, 36) not fully understood t present. Wheres mutnt nlysis of the P1S hs IMPAIRED PTS FUNCTION IN E. COLI MUTANTS 859 been vluble in understnding it, the results hve not ll been esy to interpret, in prt becuse muttionl ltertion in one component of very complex system my not leve other components unffected. p/ka mutnts my permit study of structurlly intct but nonfunctioning P1'S once the bsis of P1'S mlfunction in these strins hs been estblished. ACKNOWLEDGMENTS We re grteful to D. G. Frenkel for comments on the mnuscript nd to W. Epstein for communiction of unpublished dt. This investigtion ws supported by the University of Connecticut Reserch Foundtion nd in prt by Ntionl Science Foundtion reserch grnt BMS A2. Addendum in Proof A new pir of isogenic strins ws constructed to eliminte the difficulty noted in footnote b, Tble 3: RR95 (glre, p kal), nd RR93 (glre, glk, p1kal). Doubling times in miniml medi (determined s in Tble 3) were, for RR95, 2.9 h in glucose nd 5.2 h in mnnose; for RR93, 15.2 h in glucose nd 7.4 h in mnnose. Colony dimeters (determined s in Tble 2) were, for RR95,.35 mm on glucose,.35 mm on mnnose; for RR93, less thn.5 mm on glucose, nd.2 on mnnose. LITERATURE CITED 1. Adler, J., nd W. Epstein Phosphotrnsfersesystem enzymes s chemoreceptors for certin sugrs in Escherichi coli chemotxis. Proc. Ntl. Acd. Sci. U.S.A. 71: Bchmnn, B. J Pedigrees of some mutnt strins of Escherichi coli K-12. Bcteriol. Rev. 36: Buttin, G Mechnismes regulteurs dns l biosynthese des enzymes du metbolisme du glctose chez Escherichi coli K-12. II. Le determinisme gen6tique de l r6gultion. J. Mol. Biol. 7: Clrk, D. J., nd. Mloe DNA repliction nd the division cycle in Escherichi coli. J. Mol. Biol. 23: Corwin, L. M., nd G. R. Fnning Studies of prmeters ffecting the llosteric nture of phosphoenolpyruvte crboxylse ofescherichi coli. J. Biol. Chem. 243: Cosloy, S. D., nd E. McFll L-Serine-sensitive mutnts of Escherichi coli K-12. J. Bcteriol. 13: Curtis, S. J., nd W. Epstein Phosphoryltion of D-glucose in Escherichi coli mutnts defective in glucosephosphotrnsferse, mnnose phosphotrnsferse, nd glucokinse. J. Bcteriol. 122: Dietzler, D. N., M. P. Leckie, J. L. Mgnni, M. J. Sughrue, nd P. E. Bergstein Evidence for the coordinte control of glycogen synthesis, glucose utiliztion, nd glycolysis in Escherichi coli. J. Biol. Chem. 25: Epstein, W., S. Jewett, nd C. F. Fox Isoltion nd mpping of phosphotrnsferse mutnts in Escherichi coli. J. Bcteriol. 14: Ferenci, T., nd H. L. Kornberg The utiliztion of fructose by Escherichi coli. Properties of mutnt defective in fructose 1-phosphte kinse ctivity. Biochem. J. 132: Ferenci, T., nd H. L. Kornberg The role of phosphotrnsferse-medited syntheses of fructose 1- phosphte nd fructose 6-phosphte in the growth of Escherichi coli on fructose. Proc. R. Soc. London Ser. B 187:

9 86 ROEHL AND VINOPAL 12. Fox, C. F., nd G. Wilson The role of phosphoenolpyruvte dependent kinse system in,b-glucoside ctbolism in Escherichi coli. Proc. Ntl. Acd. Sci. U.S.A. 59: Frenkel, D. G The ccumultion of glucose 6- phosphte nd its effect in n Escherichi coli mutnt lcking phosphoglucose isomerse nd glucose 6- phosphte dehydrogense. J. Biol. Chem. 243: Frenkel, D. G., F. Flcoz-Kelly, nd B. L. Horecker The utiliztion of glucose 6-phosphte by glucokinseless nd wild-type strins ofescherichi coli. Proc. Ntl. Acd. Sci. U.S.A. 52: Kelker, N. E., nd R. L. Anderson Evidence for vectoril phosphoryltion of D-fructose by intct cells of Aerobcter erogenes. J. Bcteriol. 112: Kornberg, H. L Nture nd regultion of hexose uptke by Escherichi coli. Mimi Winter Symp. 3: Kornberg, H. L Fine control of sugr uptke by Escherichi coli. Symp. Soc. Exp. Biol. 27: Kornberg, H. L., nd J. Smith Role of phosphofructokinse in the utiliztion of glucose by Escherichi coli. Nture (London) 227: Kundig, W., nd S. Rosemn Sugr trnsport. I. Isoltion of phosphotrnsferse system from Escherichi coli. J. Biol. Chem. 246: Kustu, S. G., nd G. F.-L. Ames The hisp protein, known histidine trnsport component in Slmonell typhimurium, is lso n rginine trnsport component. J. Bcteriol. 116: Lennox, E. S Trnsduction of linked genetic chrcters of the host by bcteriophge P1. Virology 1: Loowry,. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: McFll, E Genetic structure of the D-serine deminse system of Escherichi coli. J. Mol. Biol. 9: Miller, J. H Experiments in moleculr genetics. Cold Spring Hrbor Lbortory, Cold Spring Hrbor, N.Y. 25. Morgn, M. J., nd H. L. Kornberg Regultion of sugr ccumultion by Escherichi coli. FEBS Lett. 3: Morrissey, A. T. E., nd D. G. Frenkel Selection of fructose 6-phosphte kinse mutnts in Escherichi coli. Biochem. Biophys. Res. Commun. 32: Pstn, I., nd R. L. Perlmn Repression of /- glctosidse synthesis by glucose in phosphotrnsferse mutnts ofescherichi coli. Repression in the bsence of glucose phosphoryltion. J. Biol. Chem. 244: Peterkofsky, A., nd C. Gzdr Interction of enzyme I of the phosphoenolpyruvte: sugr phosphotrnsferse system with denylte cyclse of Escherichi coli. Proc. Ntl. Acd. Sci. U.S.A. 72: Richey, D. P., nd E. C. C. Lin Importnce of fcilitted diffusion for effective utiliztion of glycerol of Escherichi coli. J. Bcteriol. 112: Romno, A. H., S. J. Eberhrd, S. L. Dingle, nd T. D. McDowell Distribution of the phosphoenolpyruvte: glucose phosphotrnsferse system in bcteri. J. Bcteriol. 14: Rosemn, S A bcteril phosphotrnsferse system nd its role in sugr trnsport. Mimi Winter Symp. 3: Rosemn, S Crbohydrte trnsport in bcteril cells. Metb. Pthwys 6: Rotmn, B., A. K. Gnesn, nd R. Guzmn Trnsport systems for glctose nd glctosides in J. BACTERIOL. Escherichi coli. II. Substrte nd inducer specificities. J. Mol. Biol. 36: Sedler, H., A. Gullon, L. Fiethen, nd P. Strlinger Negtive control of the glctose operon in E. coli. Mol. Gen. Genet. 12: Sier, M. H., Jr., F. G. Bromberg, nd S. Rosemn Chrcteriztion of constitutive glctose permese mutnts in Slmonell typhimurium. J. Bcteriol. 113: Sier, M. H., Jr., nd B. V. Feucht Coordinte regultion of denylte cyclse nd crbohydrte permeses by the phosphoenolpyruvte: sugr phosphotrnsferse system in Slmonell typhimurium. J. Biol. Chem. 25: Sier, M. H., Jr., nd S. Rosemn Inducer exclusion nd repression ofenzyme synthesis in mutnts of Slmonell typhimurium defective in enzyme I of the phosphoenolpyruvte: sugr phosphotrnsferse system. J. Biol. Chem. 247: Sier, M. H., Jr., W. S. Young, III, nd S. Rosemn Utiliztion nd trnsport of hexoses by mutnt strins of Slmonell typhimurium lcking enzyme I of the phosphoenolpyruvte-dependent phosphotrnsferse system. J: Biol. Chem. 246: Sebstin, J., nd C. Asensio Purifiction nd properties of the mnnokinse from Escherichi coli. Arch. Biochem. Biophys. 151: Sheht, T. E., nd A. G. Mrr Effect of nutrient concentrtion on the growth of Escherichi coli. J. Bcteriol. 17: Shermn, J. R., nd J. Adler Glctokinse from Escherichi coli. J. Biol. Chem. 238: Simoni, R. D., M. Levinthl, F. D. Kundig, W. Kundig, B. Anderson, P. E. Hrtmn, nd S. Rosemn Genetic evidence for the role of bcteril phosphotrnsferse system in sugr trnsport. Proc. Ntl. Acd. Sci. U.S.A. 58: Solomon, E., K. Miyi, nd E. C. C. Lin Membrne trnsloction of mnnitol in Escherichi coli without phosphoryltion. J. Bcteriol. 114: Stub, M., nd G. Denes Purifiction nd properties of the 3-deoxy-D-rbinoheptulosonte-7-phosphte synthse (phenyllnine sensitive) of Escherichi coli K-12. I. Purifiction of enzyme nd some of its properties. Biochim. Biophys. Act 178: Tnk, S., D. G. Frenkel, nd E. C. C. Lin The enzymtic lesion of strin MM-6, pleiotropic crbohydrte-negtive mutnt of Escherichi coli. Biochem. Biophys. Res. Commun. 27: Tylor, A. L., nd C. D. Trotter Linkge mp of Escherichi coli K-12. Bcteriol. Rev. 36: Vinopl, R. T., D. Clifton, nd D. G. Frenkel pflea locus ofescherichi coli. J. Bcteriol. 122: Vinopl, R. T., nd D. G. Frenkel pflkb nd ptlec loci of Escherichi coli. J. Bcteriol. 122: Vinopl, R. T., nd D. G. Frenkel Phenotypic suppression of phosphofructokinse muttions in Escherichi coli by constitutive expression of the glyoxylte shunt. J. Bcteriol. 118: Vinopl, R. T., J. D. Hillmn, H. Scliulmn, W. S. Reznikoff, nd D. G. Frenkel New phosphoglucose isomerse mutnts of Escherichi coli. J. Bcteriol. 122: von Meyenberg, K Trnsport-limited growth rtes in mutnt of Escherichi coli. J. Bcteriol. 17: Wilson, D. B Source of energy for the Escherichi coli glctose trnsport systems induced by glctose. J. Bcteriol. 12: Wilson, D. B The regultion nd properties of the glctose trnsport system in Escherichi coli K-12. J. Biol. Chem. 249: