Isolation of Dicarboxylic Acid- and Glucose-Binding Proteins

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 1976, p Copyright 1976 Americn Society for Microbiology Vol. 128, No. 2 Printed in U.S.A. Isoltion of Dicrboxylic Acid- nd Glucose-Binding Proteins from Pseudomons eruginos M. W. STINSON,* M. A. COHEN, AND J. M. MERRICK Deprtment of Microbiology, School of Medicine, Stte University of New York t Bufflo, Bufflo, New York Received for publiction 3 August 1976 Inducible binding proteins for C4-dicrboxylic cids (DBP) nd glucose (GBP) were isolted from Pseudomons eruginos by extrction of exponentil-phse cells with 0.2 M MgCl2 (ph 8.5) nd by n osmotic shock procedure without ffecting cell vibility. DBP synthesis ws induced by growth on sprtte, - ketoglutrte, succinte, fumrte, mlte, nd mlonte but not by growth on cette, citrte, pyruvte, or glucose. Binding of succinte by DBP ws competitively inhibited by 10-fold concentrtions of fumrte nd mlte but not by vriety of relted substnces. GBP synthesis nd trnsport of methyl -glucoside by whole cells were induced by growth on glucose or pyruvte plus glctose, 2-deoxyglucose, or methyl -glucoside but not by growth on gluconte, succinte, cette, or pyruvte. The binding of rdioctive glucose by GBP ws significntly inhibited by 10-fold concentrtions of glucose, glctose, nd glucose-i-phosphte but not by the other crbohydrtes tested. The binding of glucose by GBP or succinte by DBP did not result in ny chemicl ltertion of the substrtes. In recent yers, considerble ttention hs been focused on the role of periplsmic binding proteins in the trnsport of nutrients by grmnegtive bcteri. Evidence hs ccumulted tht implictes these proteins s substrte recognition components in number of ctive trnsport systems (4, 15, 18, 29). Severl binding proteins hve lso been shown to undergo substrte-induced conformtionl chnges, phenomenon tht my be involved in the function of these proteins in trnsport (4, 6, 34). Binding proteins re esily isolted from enteric bcteri by the osmotic shock procedure of Neu nd Heppel (27); however, ttempts to isolte similr trnsport components from the surfces of unrelted bcteri hve been lrgely unsuccessful. Recently, Cheng et l. (7) demonstrted tht lkline phosphtse could be relesed from the periplsm of Pseudomons eruginos by extrction with 0.2 M MgCl2 without ffecting cell vibility. In this report, we describe the isoltion of inducible C4-dicrboxylic cid- nd glucose-binding proteins (DBP nd GBP) from P. eruginos by using slight modifictions of this extrction procedure. MATERIALS AND METHODS Orgnism nd culturl conditions. P. eruginos MB-720 (derived from ATCC 15692) ws grown in minerl slts medium contining 150 mm K2HPO4- KH2PO4 (ph 6.5), 15 mm (NH4)2SO4, 10,uM FeSO4, 1 mm MgSO4, 100 SlM CCl2, nd n pproprite crbon source (15 to 40 mm). Cultures (400 ml in 2-liter flsks) were incubted on New Brunswick gyrtory shker t 300C until lte logrithmic growth, usully 16 to 18 h. Growth ws followed by mesuring turbidity with Klett-Summerson photoelectric colorimeter (no. 66 filter). Cultures with opticl densities between 250 nd 300 Klett units (2 x 109 cells/ml; 0.7 mg of cell dry weight per ml) were hrvested by centrifugtion t 220C. For studies concerning lkline phosphtse, bcteri were grown in the inorgnic phosphte-deficient medium described by Cheng et l. (8). Binding protein extrction. In erly studies, modifiction of the method described by Cheng et l. (7) for the isoltion of lkline phosphtse from P. eruginos ws used for the extrction of binding proteins. Pcked cells were rpidly suspended (1.4 mg of cell dry weight per ml) in 0.2 M MgCl M tris (hydroxymethyl ) minomethne-hydrochloride buffer, ph 8.5 (extrction buffer). Cell suspensions were incubted t 22 C for 1 h with constnt stirring nd then centrifuged for 20 min t 16,300 x g. The cler superntnt fluids contining periplsmic proteins were filtered on membrne filters (0.45-,m pore size; Millipore Corp.) nd concentrted 20-fold by ultrfiltrtion (Amicon Corp.; Diflo UM-10 filter). The concentrted mteril ws dilyzed t 40C ginst 1 mm MgCl2-10 mm tris(hydroxymethyl)minomethne-hydrochloride buffer, ph 7.5 (TM buffer), for 24 h. In lter studies, the extrction procedure described below ws routinely used since it ws less time consuming nd more reproducible, nd re- 573

2 574 STINSON, COHEN, AND MERRICK sulted in higher yields of binding protein. Pcked log-phse cells were suspended in extrction buffer (14 mg of cell dry weight per ml) nd incubted t 22 C for 30 min with constnt stirring. The cells were collected by centrifugtion nd rpidly suspended t the sme concentrtion in distilled wter. After n dditionl 30 min of mixing t 22 C, the suspension ws centrifuged nd the "shock fluid" ws removed nd combined with the mgnesium extrct. After dilysis ginst TM buffer for 18 to 20 h, the proteins were precipitted with mmonium sulfte (0 to 95% sturtion). The precipittes were dissolved in smll volume of TM buffer nd dilyzed ginst the sme buffer overnight. Extrcted cells were stored frozen t -18 C. Assy of binding ctivity. The binding ctivity of crude mgnesium shock extrcts ws determined by equilibrium dilysis (1, 19, 30). Dilysis bgs (Union Crbide, 8 mm in dimeter) were filled with 0.3 ml of extrct (0.03 to 0.90 mg of protein per ml), which ws dilyzed for 20 to 22 h t 4 C ginst 5 ml of TM buffer contining 0.02% sodium zide nd rdioctively lbeled substrte (2 AM). Binding ctivity ws liner within these protein concentrtions. Specific rdioctivities of the substrtes were: cette, 55.3 mci/mmol; fumrte, 3 mci/mmol; glucose, 100 mci/mmol; 8-hydroxybutyrte, 11.8 mci/mmol; mlte, 35 mci/mmol; nd succinte, 10.5 mci/ mmol. Upon completion of dilysis, 0.1-ml smples were removed from ech solution for liquid scintilltion counting in Nucler-Chicgo Mrk II spectrometer. One unit of binding is equivlent to 1 nmol of substrte bound. Specific ctivities re expressed in units per milligrm of protein. Substrte uptke. The uptke of methyl -glucoside (MeGlc) by P. eruginos cells ws determined using modifiction of the procedures of Guymon nd Egon (13) nd Midgley nd Dwes (25). Bcteri were hrvested from log-phse cultures by centrifugtion nd wshed nd resuspended in minerl medium (2.0 mg of cell dry weight per ml). A 0.5-ml portion ws dded to 10-ml Erlenmeyer flsk contining 0.1 ml of chlormphenicol (2.0 mg/ml) nd 0.3 ml of the minerl slts medium, nd the mixture ws incubted t 30 C on New Brunswick wter-bth shker (200 rpm) for 15 min before ddition of 0.1 ml of 50 mm '4C-lbeled MeGIc (0.7 mci/mmol). At vrious intervls during the incubtion, 0.05-ml smples of the cell suspension were withdrwn nd the cells were collected nd wshed by membrne filtrtion (Millipore Corp.). The filters were then trnsferred to scintilltion vils nd counted in Nucler-Chicgo Mrk II spectrometer. Uptke of substrte is expressed s nnomoles per minute per milligrm of cell dry weight. The rte of uptke ws clculted during the liner period between 0 nd 3 min. Cell extrcts. The cell pellets obtined fter the extrction of binding proteins were suspended in icecold TM buffer (200 mg [wet weight]/ml) nd disrupted ultrsoniclly (Bronwill Corp.). The crude extrcts were centrifuged t 27,000 x g for 15 min t 4 C to remove unbroken cells. The superntnt fluids were removed nd seprted into soluble nd prticulte frctions by centrifugtion t 105,000 x g for 1 h. The resulting pellets were resuspended in n pproprite volume of TM buffer. Enzyme ssys. Glucose dehydrogense (EC ) ws ssyed spectrophotometriclly using the procedure of Huge (14). Glucokinse ctivity ws determined by the method of Hylemon nd Phibbs (16). Succinte dehydrogense (EC ), fumrse (EC ), nd mlte dehydrogense (EC ) ctivities were determined ccording to Dubler et l. (9). Succinte thiokinse (EC ) nd mlic enzyme (EC ) were ssyed ccording to Lo et l. (20). Alkline phosphtse ctivity ws determined t room temperture by monitoring the hydrolysis ofp-nitrophenyl phosphte (12) with Zeiss PMQ II spectrophotometer. Other ssys. Protein ws mesured by the method of Lowry et l. (24), with crystlline bovine serum lbumin s stndrd. Cell dry weight ws determined with distilled wter-wshed cells dried to constnt weight. Chemicls. [1,4-'4C]succinic cid (10.5 mci/ mmol), D-[U-'4C]glucose (200 mci/mmol), nd [1- '4C]fumric cid (3 mci/mmol) were purchsed from ICN Phrmceuticls, Inc. Methyl -D-[U- 14C]glucopyrnoside (3 mci/mmol), [U-'4C]mlic cid (35 mci/mmol), D(-)-3-hydroxy[3-'4C]butyric cid (11.8 mci/mmol), nd [U-'4C]sodium cette (55 mci/mmol) were purchsed from Amershm/ Serle Corp. Chlormphenicol nd 2-deoxy-D-glucose were purchsed from Sigm Chemicls. Unlbeled methyl -D-glucoside ws obtined from Pfnstiehl Lbortories, Inc. All other compounds were from locl distributors nd were nlyticl grde. RESULTS J. BACTERIOL. Extrction of binding proteins nd the effect on cell vibility. The work of Cheng et l. (7, 8) hs clerly shown tht lkline phosphtse is periplsmic protein nd cn be selectively removed from cells by extrction with 0.2 M MgCl2. We hve confirmed these results (Tble 1) nd hve lso demonstrted tht the MgCl2 extrction procedure cn be utilized to extrct GBP nd DBP from glucose- or succinte-grown cells. These extrction procedures did not ffect cell vibility (Tble 1). DBP. The specificity of the DBP from succinte-grown P. eruginos cells ws exmined by equilibrium dilysis (Tble 2). Of the number of rdioctive lignds tested, binding ws detected with succinte, mlte nd fumrte, but not with cette, /3-hydroxybutyrte or glucose. The quntities of C4-dicrboxylic cids bound were very similr, suggesting tht common binding protein for dicrboxylic cids ws present in the extrct. The specificity of DBP ws lso investigted by mesuring the bility of unlbeled compounds to compete with the binding of ["4C]- succinte. Of the substnces tested, only unl-

3 VOL. 128, 1976 P. AERUGINOSA BINDING PROTEINS 575 TABLE 1. Extrction of binding proteins nd lkline phosphtse from P. eruginos nd the effect on cell vibility Tretment Growth on: Peptone Succinte Glucose Vibility Apseb Vibility DBP Vibility GBP (CFU/ml) Units Sp ct (CFU/ml) Units Sp ct (CFU/ml) Units Sp ct 1 mm Mg M 2.0 x 10' x x Tris, ph Mg2T M M 2.1 x x x Tris, ph 8.5 Wter (fter 0.2 M 2.2 x x Mg2+ extrction) Log-phse bcteri were suspended in 1 liter of ech tretment solution (14 mg of cell dry weight/ml of peptone- or glucose-grown cells; 1.4 mg of cell dry weight/ml of succinte-grown cells) for 30 min t 220C nd then centrifuged. The resulting wsh fluids were ssyed for ctivity. Prior to centrifugtion smple of ech cell suspension ws serilly diluted nd plted on nutrient gr. CFU, Colony-forming units. 0 One unit of lkline phosphtse (Apse) is tht ctivity liberting 1,mol of p-nitrophenol per min under defined conditions. TABLE 3. Effect of vrious substnces on succinte TABLE 2. Specificity of DBP binding Substrte Amt bound % Inhibition (nmol/mg of protein) Inhibitors dded Succinte IM 200 AM Fumrte L-Mlte, -fumrte, or Mlte succinte Acette... 0 D-Mlte DL-/3-Hydroxybutyrte Methylsuccinte Glucose Acette 8 18 L-Asprtte <1 50 Binding proteins were extrcted from succintegrown cells. Assys were performed under stndrd Pyruvte <1 32 -Ketoglutrte <1 36 conditions with 2,uM of the indicted '4C-lbeled Mlonte <1 24 compounds s substrte nd 150,ug of cell extrct Isocitrte <1 23 protein. Specific rdioctivities of the substrtes re Oxlocette <1 23 listed in Mterils nd Methods. Citrmlte <1 <1 Citrte <1 <1 DL-Trtrte <1 <1 DL-,8-Hydroxybutyrte <1 <1 DL-Lctte <1 beled succinte, fumrte, nd mlte were strong competitive inhibitors t 10-fold excess (Tble 3). At higher concentrtions (100-fold excess), vriety of other substnces exhibited significnt ffinities for the DBP. To scertin whether binding ctivity could hve been due to dicrboxylte-metbolizing enzymes, the periplsmic extrct ws ssyed for succinte dehydrogense, succinte thiokinse, fumrse, mlte enzyme, nd mlte dehydrogense. As cn be seen in Tble 4, their ctivities were not detected. Furthermore, thin-lyer rdiochromtogrphy of ['4C]succinte fter incubtion with the DBP preprtion showed no chemicl ltertion of the substrte. Induction of DBP synthesis. Induction of DBP ws observed when P. eruginos ws grown on sprtte, fumrte, -ketoglutrte, mlte, mlonte, nd succinte. Much Assys were performed under stndrd conditions with 2,uM ['4C]succinte (10.5 mci/nmol) in the presence of the indicted nonlbeled compounds. Ech rection mixture contined 150 gg of protein. Binding ctivity of DBP in the bsence of inhibitor ws 4,110 cpm/150,ig of protein. smller quntities were detected when the orgnism ws grown on cette, citrte, pyruvte, nd glucose (Tble 5). GBP. The specificity of the GBP ws investigted by mesuring the bility of unlbeled substnces to compete with the binding of [14C]glucose (Tble 6). At 10-fold concentrtions, only unlbeled glucose, glctose, nd glucose-i-phosphte were significnt competitors. At 100-fold excess, vriety of other

4 576 STINSON, COHEN, AND MERRICK TABLE 4. Enzymtic ctivity of DBP preprtions nd whole-cell extrcts Sp ct Enzyme Whole-ctl Whl-el plsmic Periett extrct Succinte dehydrogense 56 NDb Succinte thiokinse 12 ND Fumrse 15 ND Mlte dehydrogense 63 ND Mlic enzyme 16 ND One unit of enzyme is defined s tht quntity tht cused spectrophotometric chnge of 0.01 opticl density unit/min under stndrd ssy conditions. Specific ctivities re expressed s units of enzyme ctivity per milligrm of protein. b ND, No ctivity detected. TABLE 5. Production of DBP by P. eruginos grown on vrious crbon sources Substrte Totl units Sp ct (units/mg of protein) Fumrte Mlte Succinte L-Asprtte Ketoglutr te Mlonte Acette Citrte Pyruvte Glucose Bcteri were grown on 1.6 liters of minerl medium contining 15 mm of the indicted crbon sources nd hrvested in lte exponentil phse (200 Klett units). Binding proteins were isolted s previously described. DBP ssys were performed with 2 AM [14C]succinte nd 150,ug of protein. One unit of binding is equivlent to 1 nmol of substrte bound. sugrs nd glucose derivtives hd significnt ffinity for the binding protein. To eliminte the possibility tht the observed binding ws due to the presence of glucosespecific enzymes, the crude periplsmic extrct ws ssyed for glucokinse nd glucose dehydrogense ctivities. No enzymtic ctivity ws detected (Tble 7). Also, [14Ciglucose exmined by thin-lyer rdiochromtogrphy fter incubtion with GBP ws unchnged. Induction of GBP synthesis nd glucose uptke. The glucose trnsport system of P. eruginos hs been reported to be induced by glctose nd MeGlc during growth on pyruvte or glycerol (13, 16). Glctose is pprently not used by P. eruginos s crbon source for growth. The trnsport system is repressed by cette nd yrious tricrboxylic cid cycle intermedites (13, 25, 26, 28). Mximl production of GBP ws observed when cells were grown on glucose or pyruvte plus glctose, 2-deoxyglucose, or MeGlc (Tble 8). Smll mounts of GBP were detected in cells grown on cette, gluconte, pyruvte, or succinte. The induction of GBP synthesis correlted closely with trnsport ctivity of whole cells. Cells induced for GBP lso demonstrted mximl uptke of '4C-lbeled-MeGlc. Conversely, conditions tht did not induce GBP filed to induce MeGlc uptke. A low level of trnsport ctivity ws, however, detected in TABLE 6. Effect of vrious substnces on glucose binding % Inhibition Inhibitors dded 20,uM 200,M Glucose Glctose Glucose-i-phosphte Glucose-6-phosphte 4 41 Methyl -glucoside 3 40 Gluconte <1 36 Glucosmine < Deoxyglucose <1 19 Fructose <1 15 Mnnose <1 15 Fucose <1 <1 Arbinose <1 <1 N-cetylglucosmine <1 <1 Lctose <1 <1 Assys were performed under stndrd conditions with 2,zM [14C]glucose (100 mci/mmol) in the presence of the indicted nonlbeled compounds. GBP extrct ws used t level of 200,ug of protein per rection mixture. Binding ctivity of GBP in the bsence of inhibitor ws 15,723 cpm/200 Ag of protein. TABLE 7. J. BACTERIOL. Enzymtic ctivity of GBP preprtions nd whole-cell extrcts Sp ct (units/mg of protein) Cell extrct dehydro- Glucokigense Whole cell, soluble Whole cell, prticulte Mg shock fluid One unit of glucose dehydrogense is expressed s tht quntity of enzyme tht oxidizes 1 gmol of glucose per min t room temperture. One unit of glucokinse ctivity is tht mount of enzyme tht reduces 1,umol of nicotinmide denine dinucleotide phosphte per min t room temperture. b ND, No detectble ctivity.

5 VOL. 128, 1976 TABLE 8. Trnsport of MeGlc nd production of GBP by P. eruginos grown on vrious crbon sources GBP Uptke Growth substrte Units/ (nmol/ Units Unts min/mg) Glucose Pyruvte Deoxyglucose + py ruvte MeGic + pyruvte Glctose + pyruvte Gluconte Acette Succinte Bcteri were grown in 1.6 liters of minerl medium contining 20 mm of the indicted crbon sources. Non-metbolizble compounds (2-deoxyglucose, MeGlc, nd glctose) were dded t 10 mm concentrtion in the presence of 20 mm pyruvte. GBP ws isolted nd ssyed s previously described. Trnsport ctivities of untreted cells were determined s in Mterils nd Methods. noninduced cells, pprently result of fcilitted diffusion since MeGlc ws not ccumulted bove the externl concentrtion. This bsl trnsport ctivity hs not been detected by other workers (13, 16, 25), presumbly becuse of less sensitive ssy procedures. DISCUSSION Periplsmic binding proteins for glucose nd for dicrboxylic cids were redily removed from P. eruginos cells by extrction with 0.2 M Mg2+ followed by osmotic shock. The relese of periplsmic proteins occurred without detectble losses in cell vibility. Anlysis of crude extrcts indicted tht cytoplsmic or cytoplsmic membrne enzymes were not relesed by this tretment. The ction of 0.2 M Mg2+ is pprently t the outer cell wll of P. eruginos. Ingrm et l. (17) hve shown tht this tretment dissocites lipopolyscchride from these bcteri nd tht the quntity of lipopolyscchride relesed ws proportionl to the relese of the periplsmic enzyme lkline phosphtse. Extrction with Mg2+ voids problems encountered with the sucrose-ethylenediminetetrcetic cid (EDTA) procedure of Neu nd Heppel (27). Tretment of P. eruginos with EDTA cuses severe dmge to both the cell wll nd cytoplsmic membrne, resulting in lysis (32). Becuse of this effect, periplsmic proteins of P. eruginos hve not received s much ttention s those of enteric orgnisms. The presence of n inducible glycerol-binding protein in EDTA shock fluids P. AERUGINOSA BINDING PROTEINS 577 from P. eruginos hs been reported by Tsy et l. (33). Production of this protein ppered to be requisite for the trnsport of glycerol by intct cells. Periplsmic proteins tht bind dicrboxylic cids nd glucose hve lso been reported in other bcteri. The periplsmic DBP of Escherichi coli ws shown by Lo nd Snwl (22) to bind succinte, mlte, nd fumrte nd monocrboxylic cid 1)-lctte. Two dditionl binding proteins specific for the dicrboxylic cids were solubilized by detergent extrction of the cytoplsmic membrnes of E. coli (21). Genetic nd biochemicl evidence suggested tht these membrne proteins cooperte in some unknown wy in the trnsport of succinte in vivo. Similr membrne proteins hve lso been described in grm-positive bcteri. Fournier nd Prdee (11) hve detected two presumptive trnsport components in the membrnes of L-mlte-induced Bcillus subtilis. Binding specificities were demonstrted for mlte, fumrte, succinte, nd meso-trtrte. 0 A recent study of succinte trnsport in P. putid by Dubler et l. (9) suggests tht common mechnism functions in the uptke of succinte, mlte, nd fumrte. Trnsport ctivity ws induced by ech of these dicrboxylic cids nd by sprtte, lthough sprtte did not compete with succinte in uptke studies. In the present study, growth of P. eruginos on sprtte, -ketoglutrte, nd mlonte in ddition to succinte, mlte, nd fumrte resulted in mximl production of DBP. The former three cids, however, did not compete with succinte binding by extrcts prepred from succinte-, mlte-, fumrte-, sprtte-, -ketoglutrte-, or mlonte-grown cells. A glctose-binding protein, which lso hs high ffinity for glucose, hs been isolted from E. coli (1, 2, 5). This protein ws shown by Boos nd Gordon (5) to be n ctive component of the,8-methylglctoside trnsport system, which medites the trnsloction of f8-methylglctoside, glctose, glucose, n-fucose, nd f-glycerol-glctoside (29). The trnsport of glucose by P. eruginos hs not been intensively investigted. Recent studies indicte tht two distinct mechnisms my be involved. An ctive trnsport system for glucose hs been described by Egon nd ssocites (10, 31), wheres Midgely nd Dwes (25) hve reported on n lternte mechnism, which involves oxidtion of glucose to gluconte by membrne-bound glucose dehydrogense followed by trnsport of gluconte through the cell membrne. Membrne vesicles of P.

6 578 STINSON, COHEN, AND MERRICK eruginos PAO hve been reported to lose their bility to trnsport free glucose (13), suggesting tht some component of the glucose trnsport system, perhps the GBP, ws lost during the preprtion of the vesicles. Wheres there is yet very little direct evidence to implicte the GBP ofp. eruginos in trnsport, it is interesting to note tht induction of GBP nd glucose trnsport re co-regulted. Furthermore, we hve recently isolted trnsportdeficient mutnt tht ppers to lck functionl binding protein. Recent studies by Berger (3) nd Wilson (35) hve reveled mechnisms of energy coupling for the ctive trnsport of vrious mino cids nd sugrs in E. coli. Those systems tht re osmotic shock sensitive (i.e., ssocited with periplsmic binding proteins) utilized phosphte-bound energy to drive trnsport, wheres membrne-bound systems utilized the ctivted membrne stte. In preliminry studies, we hve found tht the ddition of rsente to strved cells in the presence of gluconte or pyruvte completely inhibtted the ctive uptke of MeGlc. This observtion suggests tht glucose trnsport in P. eruginos is driven by the energy of denosine 5'-triphosphte (or derivtive) nd belongs to the binding proteinssocited trnsport system. ACKNOWLEDGMENTS This investigtion ws supported by Public Helth Service generl reserch support nd institutionl fund grnts from the Division of Reserch Resources, Ntionl Institutes of Helth. We thnk Hui-Tng Chng nd Deborh G. Curtiss for their excellent technicl ssistnce. This pper is dedicted to Felix Milgrom of the Stte University of New York t Bufflo on the occsion of the thirtieth nniversry of his reserch ctivities. LITERATURE CITED 1. Anrku, Y Trnsport of sugrs nd mino cids in bcteri. I. Purifiction nd specificity of the glctose- nd leucine-binding proteins. J. Biol. Chem. 243: Anrku, Y Trnsport of sugrs nd mino cids in bcteri. II. Properties of glctose nd leucinebinding proteins. J. Biol. Chem. 243: Berger, E Different mechnisms of energy coupling for the ctive trnsport of proline nd glutmine in Escherichi coli. Proc. Ntl. Acd. Sci. U.S.A. 70: Boos, W Bcteril trnsport. Annu. Rev. Biochem. 43: Boos, W., nd A. S. Gordon Trnsport properties of the glctose-binding protein ofescherichi coli. J. Biol. Chem. 246: Boos, W., A. S. Gordon, R. E. Hll, nd H. D. Price Trnsport properties of the glctose-binding protein of Escherichi coli; substrte-induced conformtionl chnge. J. Biol. Chem. 247: J. BACTERIOL. 7. Cheng, K.-J., J. M. Ingrm, nd J. W. Costerton Relese of lkline phosphtse from cells of Pseudomons eruginos by mnipultion of ction concentrtion nd ph. J. Bcteriol. 104: Cheng, K.-J., J. M. Ingrm, nd J. W. Costerton Alkline phosphtse locliztion nd spheroplst formtion ofpseudomons eruginos. Cn. J. Microbiol. 16: Dubler, R. E., W. A. Toscno, Jr., nd R. A. Hrtline Trnsport of succinte by Pseudomons putid. Arch. Biochem. Biophys. 160: Egon, R. G., nd P. V. Phibbs, Jr Kinetics of trnsport of glucose, fructose nd mnnitol by Pseudomons eruginos. Cn. J. Biochem. 49: Fournier, R. D., nd A. B. Prdee Evidence of inducible L-mlte binding proteins in the membrne of Bcillus subtilis: identifiction of presumptive components of the C4-dicrboxylte trnsport systems. J. Biol. Chem. 249: Gren, A., nd C. Levinthol A fine-structure genetic nd chemicl study of the enzyme lkline phosphtse ofe. coli. I. Purifiction nd chrcteriztion of lkline phosphtse. Biochim. Biophys. Act 38: Guymon, L. F., nd R. G. Egon Trnsport of glucose, gluconte, nd methyl --o-glucoside by Pseudomons eruginos. J. Bcteriol. 117: Huge, J. G Glucose dehydrogense-prticulte. I. Pseudomons species nd Bcterium nitrtum, p In S. P. Colowick nd N. 0. Kpln (ed.), Methods in enzymology, vol. 9. Acdemic Press Inc., New York. 15. Heppel, L. A The concept of periplsmic enzymes, p In L. T. Rothfield (ed.), The structure nd function of biologicl membrnes. 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7 VOL. 128, 1976 P. AERUGINOSA BINDING PROTEINS domons eruginos. Biochem. J. 132: Mukkd, A. F., G. L. Long, nd A. H. Romno The uptke of 2-deoxy-D glucose by Pseudomons eruginos nd its regultion. Biochem. J. 132: Neu, H. C., nd L. A. Heppel The relese of enzymes from Escherichi coli by osmotic shock nd during the formtion of spheroplsts. J. Biol. Chem. 240: Ng, F. M.-W., nd E. A. Dwes Chemostt studies on the regultion of glucose metbolism in Pseudomons eruginos by citrte. Biochem. J. 132: Oxender, D. L Membrne trnsport. Annu. Rev. Biochem. 41: Penrose, W. R., G. E. Nicholds, J. R. Piperno, nd D. L. Oxender Purifiction nd properties of leucine-binding protein from Escherichi coli. J. Biol. 579 Chem. 243: Phibbs, P. V., Jr., nd R. G. Egon Trnsport nd phosphoryltion of glucose, fructose nd mnnitol by Pseudomons eruginos. Arch. Biochem. Biophys. 138: Roberts, N. H., G. W. Gry, nd S. G. Wilkinson The bctericidl ction of ethylenediminetetrcetic cid on Pseudomons eruginos. Microbios 2: Tsy, S. S., K. K. Brown, nd E. T. Gudy Trnsport of glycerol by Pseudomons eruginos. J. Bcteriol. 108: Weiner, J. H., nd L. A. Heppel A binding protein for glutmine nd its reltion to ctive trnsport in Escherichi coli. J. Biol. Chem. 246: Wilson, D. B Source of energy for theescherichi coli glctose trnsport systems induced by glctose. J. Bcteriol. 120: