Gene. and fl-methyl-l-arabinoside competitively inhibit the uptake of L-arabinose. arabinose metabolism, including arae. Although

Transcription

1 JOURNAL OF BACERIOLOGY, Aug. 1972, p Copyright American Society for Microbiology Vol. 111, No. 2 Printed in U.S.A. A Second Transport System for L-Arabinose in Escherichia coli B/r Controlled by the arac Gene CANDICE E. BROWN AND ROBERT W. HOGG Department of Microbiology, Case Westem Reserve University, Cleveland, Ohio Received for publication 9 May 1972 Escherichia coli B/r possesses two active transport systems for L-arabinose, both of which are regulated by arac, the regulatory gene for the L-arabinose operon. The system with the higher affinity for L-arabinose has a Km for initial uptake of L-arabinose of 8.3 x 10-6 M; the system of lower affinity has a Km of 1.0 x 10-1 M. The two systems can also be distinguished by differences in their response to analogues that act as competitive inhibitors of initial uptake. D- Galactose strongly inhibits L-arabinose uptake by the high affinity system but only weakly inhibits such uptake by the low affinity system. D-Fucose, D-xylose, and fl-methyl-l-arabinoside competitively inhibit the uptake of L-arabinose by both systems to approximately the same extent. D-Glucose and L- lyxose do not inhibit either system. On the basis of kinetic evidence and the properties of mutants lacking one or the other of the two systems, it has been concluded that the high affinity uptake system involves the L-arabinose binding protein. Kinetic studies have shown that the Km for L-arabinose uptake by the high affinity system resembles the Km for binding of L-arabinose by the binding protein, and both have similar K, values for the inhibitory substrates tested. A consideration of L-arabinose transport-deficient mutants has demonstrated that the previously described arae mutants lack the low affinity system but retain the high affinity system and have normal levels of the L-arabinose binding protein. Mutants described in this communication which lack only the high affinity system either contain no detectable L-arabinose binding protein or possess an immunologically cross-reacting material that is reduced in its ability to bind L-arabinose. These observations support a role for the binding protein in L-arabinose uptake. An uptake system for L-arabinose in Escherichia coli B/r was reported in 1964 by Novotny and Englesberg (12). These workers documented an inducible, energy-dependent uptake system for L-arabinose with a Km for initial uptake of 1.25 x 10-i M. Uptake of L- arabinose via this system could be abolished by mutations at a locus designated arae, which was located on the E. coli chromosome at a point other than the site of the L-arabinose operon (Isaacson and Englesberg, Bacteriol. Proc., p. 113, 1964). Later studies by Hogg and Englesberg demonstrated the E. coli B/r possesses an L-arabinose binding protein that binds L-arabinose with a Km of 5 x 10-6 M (8). Their studies with mutant strains indicated that the binding protein is not the product of any of the known genes concemed with L- 606 arabinose metabolism, including arae. Although the binding protein was not the product of the arae gene, it was postulated that it participated in the transport of L-arabinose. Schleif (16) reported that E. coli K-12 possesses an L-arabinose operon similar to that of E. coli B/r, but that two uptake systems controlled by the regulatory gene arac appear to be present with Km values for L-arabinose uptake of 5 x 10-5 and 3 x 10-6 M. He also isolated an L-arabinose binding protein similar to the one described by Hogg and Englesberg for E. coli B/r. It was therefore of interest to determine whether E. coli B/r possesses two L- arabinose uptake systems, controlled by the regulatory gene arac, and whether or not the L-arabinose binding protein plays a role in

2 VOL. 111, 1972 transport. To determine whether two separate transport systems were present, kinetic studies of initial rates of L-arabinose uptake were performed, and mutants lacking each of the two systems were obtained and studied. The mutants were characterized with respect to kinetics of uptake and also assayed for the presence or absence of the L-arabinose binding protein. A preliminary account of this work has been reported previously (C. E. Brown and R. W. Hogg, Bacteriol. Proc., p. 126, 1971). MATERIALS AND METHODS Chemicals and reagents. L-Arabinose-1-"4C was obtained from Calbiochem. Paper chromatography in water-saturated butanol, followed by autoradiography, indicated that all of the radioactivity was associated with L-arabinose. D-Fucose and L-arabinose were from the Sigma Chemical Co., D-xylose and D-galactose from Difco Laboratories, and L- lyxose and fl-methyl-d-galactoside from Mann Research Laboratories. fl-methyl-l-arabinoside was synthesized from L-arabinose by the method of Cadotte et al. (2). Bacterial strains. All strains used were derivatives of E. coli B/r and are described in Table 1. Mutants and their properties, specifically derived and relevant to this report, are described in Tables 3 and 4. Media, growth conditions, and uptake assays. Cells were grown in minimal salts containing 1% Casamino Acids. Induction was initiated by addition of 0.4% L-arabinose. After three generations of growth the cells were centrifuged, washed three SECOND L-ARABINOSE TRANSPORT SYSTEM TABLE 1. times in three culture volumes of minimal salts, and suspended to a density of 109 cells/ml in minimal salts containing Casamino Acids (1%) and chloramphenicol (50 Ag/ml). The suspended cells were maintained at 25 C in a shaking water bath. The assay was initiated by addition of L-arabinose-1-"4C (1 gci/mmole) to the desired external concentration, and uptake was terminated by pipetting a 1-ml sample onto a 0.4-jum pore size membrane filter (Millipore Corp.). The filtered cells were washed with 1 ml of room temperature minimal salts, dried, placed in scintillation vials containing 0.4% 2,5-diphenyloxazole and 0.01% 1, 4-bis-2-(5-phenyloxazolyl)-benzene in toluene, and counted in a Packard Tri-Carb liquid scintillation spectrometer. Appropriate corrections for L-arabinose adhering nonspecifically to cells treated with 10% Formalin at 0 C have been included in the calculation. Initial rates of uptake (30-sec samples) were used in calculating all kinetic parameters. Genetic methods. Cells were mutagenized by the ethyl methane sulfonate method of Osborn (13). Transducing lysates of phage Plbt were prepared as described by Gross and Englesberg (6). Transductions were performed by infecting recipient bacteria, suspended in absorption medium (9.015 M CaCl2, 0.03 M MgSO4) at 6 x 108 cells/ml, with donor phage at a multiplicity of The resulting suspension was incubated for 20 min at 37 C and plated on appropriate selective media. Transductants were purified on the selective media and tested for lysogeny by spotting on sensitive bacterial lawns. Thymine auxotrophs were selected by the method of Lomax and Greenberg (11). Strains in which thymine auxotrophs were desired were grown in minimal medium, and 0.1 ml was spread on minimal glucose agar plates containing 20 gg of thymine per Bacterial strains Strain Genotype Arabinose pheniotype Origin or reference UPlOOl F- (wild type) Ara+ Gross and Englesberg (6) UP1041 F-, araa39 Ara-, isomeraseless Sheppard and Englesberg (17) UP1 181 F-, araa89, thr, leu Ara-, isomeraseless Englesberg collection UP1276 F-, aracc67 Ara+, constitutive for Englesberg et al. (4) isomerase, kinase, epimerase, and permease UP1654 F-, arae201 Ara-, permeaseless Hogg and Englesberg (8) UP1664 F-, araa2, arae201 Ara-, isomeraseless, Isaacson and Englesberg (Bacteriol. permeaseless Proc., p , 1964) SB1094 F-, araa719 (deletion Ara-, pleiotrophic nega- Sheppard and Englesberg (17) of arac and arao) tive SB5313 F-, araa39, arae201 Ara-, isomeraseless, Hogg and Englesberg (8) permeaseless CW1000 F-, araa89, aracc67 Ara-, isomeraseless, con- Hogg (unpublished data) by transstitutive for kinase, duction UP1276 x UP1181 epimerase, and permease CW2000 F-, leu, thi Ara+ R. B. Helling, Univ. of Michigan a The mutation previously designated arael has been renumbered (ara E201) to conform to the nomenclature of Demerec. 607

3 608 BROWN AND HOGG J. BACTERIOL. ml and 5 ug of trimethoprim per ml. After 48 hr of growth colonies resistant to trimethoprim were restreaked on the same medium. Mutants were transferred to minimal medium with or without thymine to identify auxotrophs. Equilibrium dialysis assay for the L-arabinose binding protein. The assay and purification of the L-arabinose binding protein have been described by Hogg and Englesberg (8). Inhibiting sugars were added to the extemal buffer at 10-2 M, and K, values calculated as described below. Detection of cross-reacting material (CRM). Stationary-phase cells were washed in minimal salts, suspended in tris(hydroxymethyl)aminomethane buffer (0.05 M) containing 10-1 M 2-mercaptoethanol and 10-3 M MgCl2 at ph 7.6 and disrupted by sonic oscillation. The resulting suspension was centrifuged at 15,000 x g for 30 min, and the supernatant fluid was collected. CRM was detected by the Ouchterlony procedure (14) using antibody to the L-arabinose binding protein prepared as described by Hogg (7). Calculation of Km and K, values. Km values were calculated by the method of Epstein et al. (5). The initial uptake rate of the high affinity system was determined at low external L-arabinose concentrations at which only this system functions. The Km and Vmax for the high affinity uptake system were then calculated from a Lineweaver-Burk type plot. These constants were then used to correct the uptake rates at higher external L-arabinose concentrations, at which both the high affinity and low affinity uptake systems function. The corrected values for uptake at higher external substrate concentrations were plotted, and the Km and Vmax for the low affinity system were determined. Inhibition constants (K,) were determined by adding the inhibiting sugar at 10-2 M 15 sec before the addition of L-arabinose-1-"C to the assay tube. The method of Cirillo (3) was used to calculate K, values, after first determining graphically that the inhibition was competitive. patterns of competitive inhibition by analogues of L-arabinose, and (iv) the isolation of mutants from the strain SB5313 (araa39 ara- E201) background which completely lack the ability to accumulate L-arabinose. Uptake of L-arabinose by the singly mutant strain UP1041 (araa39) was considered in detail. Utilization of L-arabinose during uptake studies was prevented by the nonsense mutation araa39 in the isomerase gene. The uptake of L-arabinose by this strain shows biphasic kinetics (Fig. 2). Km and Vmax values for each of the two processes can be determined as described in Materials and Methods and are tabulated in Table 2. We propose to designate the genetic locus involved in production of the 4 3 Min (x 0-3) TIME (SECONDS) FIG. 1. Uptake of L-arabinose by strains normal for uptake (0) UP 1041 (araa39) induced, and by the transport defective strain (0) SB5313 (araa39 ara- E201). Min = molar internal concentration of L- arabinose. -L- RESULTS Kinetic studies of L-arabinose uptake. Isaacson and Englesberg (Bacteriol. Proc., p. 113, 1964) demonstrated that mutations at the arae locus abolished the ability of their strain UP1664 (E. coli B/r araa2) to accumulate L- arabinose. However, when the arae mutations isolated by these workers were introduced by transduction into another background, strain UP1041 (E. coli B/r araa39), the arae transductants retained some capacity to accumulate L-arabinose (Fig. 1). A possible explanation for this observation is that the araa39 background contains a second L-arabinose transport system also induced by L-arabinose. To test this possibility we have considered (i) the kinetics of uptake in strains UP1041 (araa39) and SB5313 (araa39 arae201), (ii) the regulation of induction of the transport system, (iii) the differing -I I /Mex FIG. 2. Double reciprocal plot of L-arabinose uptake in strain UP1041 (araa39). V expressed as moles of L-arabinose taken up per liter per 30-sec interval. M1,, and Mex = molar internal and external L-arabinose concentration.

4 VOL. 111, 1972 SECOND L-ARABINOSE TRANSPORT SYSTEM TABLE 2. Km values for uptake of L-arabinose Low affinity uptake High affinity uptake Strain Arabinose genotype system system Km X 10-4 M Vmaxa Km X 1O-6 M Vmax UP1041 araa UP1041 araa39 (uninduced) Ob SB1094 araa39 araca CW1000 araa89 aracc (uninduced) SB5313 araa39 arae CW2004 arae CW2012 arae201, araf CW2022 araf404c a Expressed as millimoles per liter per 30 sec. b No uptake detectable above that of Formalin-treated cells. c araf = high affinity uptake. 609 high affinity system (Km, 8.3 x 10-6 M) araf. The low affinity system (Km, 1.0 x 10-' M) is considered equivalent to that previously designated by the genetic locus arae (Fig. 2 and Table 2). Initial rate studies with the strain SB5313 (araa39 arae201) show that a mutation in the arae locus leads to loss of the low affinity system but does not affect uptake by the high affinity uptake system (Table 2). The following observations suggest that both systems are controlled by the regulatory gene arac. (i) No active transport of L-arabinose occurs in uninduced cultures (UP1041, Table 2). (ii) Deletion of the arac gene results in a complete loss of both systems (SB1094, Table 2), and (iii) both uptake systems function in the absence of the inducer, L-arabinose, in strains constitutive for the enzymes of the L- arabinose operon (CW1000,Table2). Genetic studies of L-arabinose uptake. In order to select mutants completely lacking all L-arabinose transport capacity, it was advantageous to begin with an arae201 mutant in a background permitting L-arabinose metabolism. On tetrazolium-arabinose agar medium growth of a strain capable of accumulating and metabolizing L-arabinose results in the formation of white colonies, whereas strains defective in their ability to metabolize arabinose or completely lacking uptake capacity produce red colonies. Mutants partially defective in their L-arabinose uptake ability would be expected to be intermediate in color. To obtain a mutant capable of metabolizing L-arabinose though partially defective for uptake, arae201 was cotransduced with thymine into a thy auxotroph derived from strain CW2000, (leu thc) as described in Materials and Methods (Table 3). Growth of the parental strain (leu thi thy) on tetrazolium-arabinose agar medium resulted in white colonies, whereas the arae201 thy+ transductants were magenta. This arae201 thy+ transductant was mutagenized with ethyl methane sulfonate, and red colonies were selected on tetrazolium-arabinose agar medium (Table 3). The red colonies were assayed for their ability to accumulate L-arabinose, and six independent mutants were isolated which had reduced uptake of arabinose compared with its parental strain arae201 (CW2004). These mutants are designated as high affinity uptake deficient and by genotype as araf. This class is not composed of arac mutants, since all six of the red colonies were shown to have wild-type levels of L-arabinose isomerase. Kinetic assay of arae, araf double mutants indicated that no active transport of L-arabinose was detectable, though such strains grow slowly on L-arabinose as sole carbon source (Table 4). Uptake of arabinose was linear with time, suggesting that passive diffusion can occur at a rate sufficient to support slow growth. When the six mutants were tested for the presence of the L-arabinose binding protein with specific antibody, four contained CRM. Cell-free extracts from three of the CRM-producing mutants CW2009, CW2010 and CW2013 were found to bind L- arabinose at levels significantly below extracts of the parental strain CW2004 (Table 4). An extract of the fourth CRM-producing mutant (CW2012) did not bind L-arabinose. The remainder lack CRM and are incapable of binding L-arabinose under conditions of equilibrium dialysis. To observe the properties of a strain deficient only in high affinity uptake, a functional arae gene was transduced into the uptake de-

5 610 BROWN AND HOGG J. BACTERIOL. TABLE 3. Derivation of strains lacking uptake systems for L-arabinosea Color response Strains Genotype on tetrazolium- Method of construction arabinose CW2000 Wild-type White R. B. Helling, Univ. of Michigan CW2001 thy White From CW2000 by trimethoprim CW2004 arae201 Magenta From CW2001 by cotransduction with thy using Plbt grown on UP1654 CW2009 through arae201, araf404,"thy Red From CW2004 by EMSC muta- CW2014 genesis CW2017 arae201, araf404 Red From CW2010 by trimethoprim CW2022 araf404 White From CW2017 by cotransduction with thy using Plbt grown on CW2000 a All strains are F-, leu, thi derivatives of E. coli B/r. araf = high affinity uptake. c Ethyl methane sulfonate. TABLE 4. Properties of mutants deficient in low affinity and high affinity L-arabinose uptake L-Arabinose-l-'1C bound Produces Generation time Strain Genotype in on L-arabinose (counts per min per mg Ouchterlony test (min) of protein) CW2000 ara Not tested CW2004 arae CW2009 arae201 araf401" CW2010 arae201 araf CW2013 arae201 araf4o CW2014 arae201 araf CW2011 arae201 araf403 _ CW2012 arae201 araf a Cross-reacting material. baraf = high affinity uptake. ficient double mutant arae, araf (CW2012). Thymine auxotrophs of the arae, araf parent were derived as described in Materials and Methods and transduced to thy+ with a lysate of phage prepared on a wild-type strain. The selected thy+ clones were screened for cotransductants containing a functional arae gene by selecting the white colonies from among the parental red colonies on tetrazolium-arabinose agar medium (Table 3). The presence of the arae gene and the absence of the araf gene were confirmed by analysis of the kinetics of L- arabinose uptake. This mutant strain, CW2022 (araf404), demonstrates that the low affinity uptake system (arae) can function independently with unaltered kinetic properties in the absence of the high affinity uptake system (Table 2). Inhibition studies were conducted in strains containing only one of the two systems to distinguish between them further. Figure 3 shows a typical double reciprocal plot for the high affinity system in araa39 arae201 with competitive inhibition by D-galactose, D-xylose, D- fucose, and fl-methyl-l-arabinoside. Not shown, but falling on the control line, are the results for L-lyxose and D-glucose, which did not inhibit uptake at 102 M. A summary of K1 values (Table 5) shows that D-galactose inhibits the high affinity system more efficiently than the low affinity system. D-Fucose, D-Xylose, and fl-methyl-l-arabinoside inhibit both uptake systems to the same extent.,b-methyl- D-galactoside is a very weak inhibitor of the high affinity uptake system. The specificity of inhibition of binding of L-arabinose by the L- arabinose binding protein (Table 5) in equilibrium dialysis assays is similar to that of the high affinity uptake system, including the strong inhibition by D-galactose, which supports the genetic evidence implicating the binding protein in high affinity L-arabinose

6 VOL. 111, 1972 SECOND L-ARABINOSE TRANSPORT SYSTEM The "low" affinity uptake system appears to be identical with the arae permease originally 10- described by Isaacson and Englesberg (Bacteriol. Proc., p. 113, 1964). The high affinity 9 system is absent in mutants which have been 8- designated araf, which have the added property of having altered or no L-arabinose 7-6- binding protein. Although the map location of the L-arabinose binding protein has not been so 5. determined, it is known that it is not located 4. A at the site of the L-arabinose operon or the 3. arae gene locus. 2' Both uptake systems appear to be controlled I by the regulatory gene arac of the L-arabinose operon. This conclusion is based on their in- -I duction by L-arabinose, their absence in uninduced cells, and their constitutive synthesis in 10-5/ M ex L-arabinose constitutive regulatory mutants. FIG. 3. Double reciprocal plot c)f the uptake of L- The results are in agreement with those of arabinose by the high affinity uptake system in Schleif (16) who has reported that E. coli K-12 strain SB5313 (araa39 arae201) i? n the presence and possesses two uptake systems for L-arabinose, absence of various competitive inzhibitors. No addi- both apparently under arac control. Lin (10) tions (0); in the presence of D-fU cose (0); D-Xylose (0); D-galactose (U); suggested that the second transport system for,-methyl- L- Glucose and L-lyxose give result, arabinoslde (A). D- L-arabinose (the high affinity system of this s equivalent to no addition. M1n and Mex = molar internal and external paper) might be the jl-methyl-galactoside permease. Singer and Englesberg (18) find L-arab- L-arabinose concentration. inose uptake to be mediated by this uptake TABLE 5. K, values for competitive inhibitors of L- system in arac- strains after extended induction with L-arabinose. However, since,b-methylarabinose D-galactoside and D-glucose (both substrates K, (moles/liter) for upttake for the fl-methyl-d-galactoside permease) are poor Lw High K (moles/liter) inhibitors of the high affinity uptake sysforbindingtby tem considered in this paper (Table 3) and Inhibitor affinity syasffiitty L-arabinose since uptake is regulated by the arac gene, it system in srstem in binding is unlikely that it is the fl-methyl-d-galactoside araf404 arael protein (CW 2022) (SBE53 01 permease. 13) Inhibition studies indicate that both sys- D-Fucose 2.6 x 10-'I 1.1 xi10-' tems, controlled by arac, are relatively insen- D-Galactose 55.5 x x I 0-'4 5.5 x 10-4 sitive to differences at the C-1 position (,Bf-Methyl-L X X I x 10-' methyl-l-arabinoside), C-4 (D-xylose), and C-6 arabinoside (D-fucose). However, the high affinity system is D-Xylose 14.6 x x: 10-' 11.0 X 10-4 less sensitive to C-6 differences than the low B-Methyl-D- > x: 10-4 >10-2 affinity system, since the presence of a hydroxgalactoside L-LyxOSe >10-2 > 10- > ymethyl group at C-6 in D-galactose prevents - 2 it from competing effectively in the low af- D-Glucose > 10-2 > >>10-2 >10-2 finity system. Both systems are sensitive to the change of the C-3 hydroxyl group to its epimeric form, since L-lyxose is not an inhibitor. The results for the low affinity system transport. agree with those of Novotny and Englesberg DISCUSSIOI (12) who found that D-xylose and D-fucose were Evidence is presented for the presence of competitive inhibitors of uptake for the pertwo transport systems for L,-arabinose in E. mease designated by arae. They concluded coli B/r. Both transport systems are induced that it was uncertain whether D-glucose and D- coordinately with the arabiniose operon. Be- galactose were competitive inhibitors, and L- cause of the differences in JKm values for L- lyxose and f-methyl-l-arabinoside were not arabinose uptake, these have been designated tested. the "high" and "low" affinity uptake systems. Three observations suggest that the binding. 611

7 612 BROWN AND HOGG J. BACTERIOL. protein may function in the high affinity L- arabinose uptake system. First, the Km of the high affinity uptake system is the same as the Km for the binding of L-arabinose by the binding protein. Secondly, the specificities of inhibition of the binding protein parallel those of high affinity system, but not those of the low affinity system. Finally, when independent mutants that lack the high affinity uptake system are selected, half of them have concomitantly lost CRM for the L-arabinose binding protein and the other half possess CRM which is altered in its ability to bind L-arabinose. The results presented in this communication support a model for L-arabinose transport in E. coli B/r that involves two independent uptake systems controlled by the arac gene, either of which can function independently in a cell. The uptake system described by Novotny and Englesberg (12) and designated by the gene arae represents a low affinity uptake process. The high affinity uptake system described in this paper and designated araf minimally contains the L-arabinose binding protein as a functional unit. The fact that all characterized mutations of the high affinity uptake system appear to have altered arabinose binding protein and that all mutations of the low affinity uptake system appear to be located in the arae genetic locus does not exclude the possible involvement of other transport components in either system. However, if other specific proteins assist in the transport process one would except to have detected classes of mutants other than araf in the selection procedure used. Multiple uptake systems for metabolites such as amino acids and carbohydrates have been frequently observed in bacteria. Galactose uptake in E. coli is facilitated by four uptake systems, one of which (the f0-methyl-dgalactoside permease) appears to involve the galactose binding protein (9). The four systems engaged in galactose uptake are unlinked on the chromosome and may also be differentiated on the basis of substrate and inducer specificity. The fl-methyl-d-galactoside uptake system is linked to the galactose binding protein, and both are controlled by a regulatory gene mglr (9). As well as this genetic evidence for the involvement of the galactose binding protein in the uptake of methyl galactosides, kinetic studies indicate identical substrate specificities for binding and uptake. Similar evidence suggests that certain amino acid binding proteins are involved in transport. In Salmonella typhimurium Ames and Lever (1) report the presence of two histidine binding proteins that mediate separate paths of histidine uptake. The loss of either binding protein by mutation results in the simultaneous loss of its respective uptake system. One of these histidine binding proteins has been purified by Rosen and Vasington (15), who demonstrated that osmotic shock reduced histidine uptake and caused the loss of the binding protein considered in their studies. The glutamine binding protein of E. coli (19) also appears to have a role in transport, based on similar evidence of loss of transport concomitant with loss of binding protein following osmotic shock. In addition, mutants having 10% of the glutamine binding protein levels found in wild-type cells have only 10% the rate of initial uptake of glutamine. The results of this paper indicate that there exists a similar involvement of the L- arabinose binding protein in transport. The chromosomal location of the gene for the L- arabinose binding protein and of the high affinity uptake system are currently being determined. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant 1-RO1-AM from the National Institute of Arthritis and Metabolic Diseases, and by a Public Health Service research career program award 1-K4-GM from the National Institute of General Medical Sciences. C.E.B. was a predoctoral fellow of the National Science Foundation. LITERATURE CITED 1. Ames, G. F. L., and J. Lever Components of histidine transport: histidine binding protein and hisp protein. Proc. Nat. Acad. Sci. U.S.A. 66: Cadotte, J. E., F. Smith, and D. Spriestersbach A new synthesis of glycosides. J. Anier. Chem. Soc. 74: Cirillo, V. P Relationship between sugar structure and competition for the sugar transport system in bakers' yeast. J. Bacteriol. 95: Englesberg, E., J. Irr, J. Power, and N. Lee Positive control of enzyme synthesis by gene C in the L- arabinose system. J. Bacteriol. 90: Epstein, E., D. W. Rains, and 0. E. Elzam Resolution of dual mechanisms of potassium absorption by barley roots. Proc. Nat. Acad. Sci. U.S.A. 49: Gross, J., and E. Englesberg Determination of the order of mutational sites governing L-arabinose utilization in Escherichia coli B/r by transduction with phage Plbt. Virology 9: Hogg, R. W "In vivo" detection of L-arabinosebinding protein, CRM-negative mutants. J. Bacteriol. 105: Hogg, R. W., and E. Englesberg L-Arabinose binding protein from Escherichia coli B/r. J. Bacteriol. 100: Lengeler, J., K. 0. Hermann, H. J. Unsold, and W. Boos The regulation of the,b-methyl-d-galactoside transport system and of the galactose binding protein of Escherichia coli K12. Eur. J. Biochem. 19: Lin, E. C. C The genetics of bacterial transport systems. Annu. Rev. Genet. 4:

8 VOL. 111, Lomax, M. S., and G. R. Greenberg Characteristics of the deo operon: role in thymine utilization and sensitivity to deoxyribonucleosides. J. Bacteriol. 96: Novotny, C. P., and E. Englesberg The L-arabinose permease system in Escherichia coli B/r. Biochim. Biophys. Acta 117: Osbom, M. J Isolation of phage resistant mutants of Salmonella typhimurium, p In E. F. Neufeld and V. Ginsburg (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. 14. Ouchterlony, Antigen-antibody reactions in gels. Arkiv Kemi 26: Rosen, B. P., and F. D. Vasington Purification and characterization of a histidine-binding protein SECOND L-ARABINOSE TRANSPORT SYSTEM 613 from Salmonella typhimurium LT-2 and its relationship to the histidine permease system. J. Biol. Chem. 246: Schleif, R An L-arabinose binding protein and arabinose permeation in Escherichia coli. J. Mol. Biol. 46: Sheppard, D., and E. Englesberg Further evidence of positive control of the L-arabinose system by gene ara C. J. Mol. Biol. 25: Singer, J., and E. Englesberg Arabinose transport in ara C- strains of Escherichia coli B/r. Biochim. Biophys. Acta 249: Weiner, J. H., and L. A. Heppel A binding protein for glutamine and its relation to active transport in Escherichia coli. J. Biol. Chem. 246: