Effect of arsenate on inorganic phosphate transport in Escherichia coli.

JOURNAL OF BACTERIOLOGY, Oct. 1980, p. 366-374 0021-9193/80/10-0366/09$02.00/0

Vol. 144, No. 1

Effect of Arsenate on Inorganic Phosphate Transport in Escherichia coli GAIL R. WILLSKYt AND MICHAEL H. MALAMY* Molecular Biology and Microbiology, Tufts University School ofMedicine, Boston, Department of Massachusetts 02111

The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium. The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined. The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio. Growth in arsenate-containing medium was not due to detoxification of the arsenate. Kinetic studies revealed that celLs grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vm,) for Pi, but maintained the same affinity (K.). Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 pM arsenate was determined. The Pstdependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium. The Pi transport-dependent strain ceased growth in arsenate-containing media. This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium. Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide. Previously accumulated arsenate could exchange with arsenate or Pi in the medium.

period in arsenate-containing medium, this strain develops an increased capacity (V..) to transport Pi while maintaining the same affinity (Kin) for Pi. The kinetic analysis of Pi transport characterized Pi transport through these sys- in Pst-dependent cells grown in the presence or tems in cells grown with excess (1 mM) Pi. In absence of arsenate in the medium demonthis paper we report studies of the effect of strated that Pi transport in these cells was alarsenate on Pi transport in strains containing ways sensitive to the arsenate-to-Pi ratio. either the Pit or Pst systems grown in excess MATERIALS AND METHODS phosphate medium. All cells containing a Pit system ceased growth in the presence of 10 mM and media. GR5172 (26) and U7 (1) were arsenate (Fig. 1 in reference 26). We previously theStrains strains dependent upon the Pst system for Pi showed that arsenate is a substrate for the Pit transport, GR5178 (26) was the strain dependent upon system (25). In cells containing only the Pst the Pit system for Pi transport, GR2131 was the strain system, grown in excess phosphate medium, Pi containing both the Pst and Pit transport systems, and transport ceased immediately in the presence of UR1 (1) was the strain lacking both Pi transport a 10-fold or greater excess of arsenate to Pi (26). systems. The miniimal salts medium used was WT as Pst-dependent strains, however, will grow on Pi designated in the accompanying paper (26). Chenicals. The sources of both nonradioactive albeit at a reduced rate in arsenate-containing medium (26). The experiments reported here and radioactive chemicals are given in the accompanywere designed to investigate the development of ing paper (26). Chemical assays. Pi was determined by the proarsenate resistance in the Pst-dependent strain. cedure Berenblum and Chain (2) as previously We will show that the ability of the Pst-de- describedof (26). Arsenate was assayed by the method strain to in the of arsepresence grow pendent of Mitchell (12). A 3-ml sample was mixed with 0.35 nate is not due to detoxification of the arsenate ml of 2.5% (wt/vol) (NH4)sMo70w4H2O in 5N H2S04 in the medium. Rather, after a suitable induction and 0.15 ml of a stock SnCl2 solution (prepared fresh t Present address: Department of Biochemistry and Molec- daily by a 1:200 dilution into water of a stock of 0.4 mg ular Biology, The Biological Laboratories, Harvard Univer- of SnCl2 per ml in concentrated HCl). After 40 min at Wild-type strains of Escherichia coli contain

two major systems for Pi transport: the Pit (Pi transport) and Pst (phosphate specific transport) systems. In the previous paper (26) we

sity, Cambridge, MA 02138.

room temperature, the absorbance was read at 800 366

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nm. Arsenate concentrations were determined by comparison with a standard curve. Transport assays. Phosphate and arsenate uptake assays were done as described in the accompanying paper (26). The working solution of 74As-arsenate was prepared as described for 32Pi to a specific activity of lo' to l04 cpm/nmol. If 74As-arsenate was used, the filters were dried under a heat lamp and immersed in Liquifluor scintillation fluid (New England Nuclear), and the amount of radioactivity was determined in a Beckman LS235 scintillation counter. Exit of radioactive phosphate and arsenate 0 from cells previously exposed to high levels of (0 these isotopes. The procedure for transport assays 0 described above was used. After 5 min of exposure to 400 0.1 high levels of radioactive isotopes, the cells were har400 vested by filtration through membrane filters (MilliE pore Corp., type HA, 0.45-pm pore size) and resuspended rapidly (at time zero) into the original volume ~ 10 (5 to 10 ml) in the presence of the indicated transport C_200, inhibitors. Samples of 0.5 ml were taken before the culture was filtered to determine the cell-associated radioactive isotope before resuspension at time zero, and at various times after resuspension to monitor the efflux of the isotopes. All samples were filtered on membrane filters and washed with 10 ml of WT medium containing 1 mM of the unlabeled isotope. The TIME (hours) filters were then treated, and radioactivity was counted as described above for arsenate uptake studFIG. 1. Effect of the arsenate-to-Pi ratio on the ies. generation time of a Pst-dependent strain. (A) Strain ATP. ATP was assayed by the procedure of Cole et GR5172 (pst' pit) was grown into exponential growth al. (6) as modified and described by Blumberg et al. in glucose minimal medium with 1 mMPi. The culture (3). Calculations were made from the data obtained was divided (at arrow) into four portions, and the during the 20- to 30-s interval after the addition of following additions were made. Symbols: 0, no arextract. A standard ATP curve was prepared with senate addition; El, 10 mM arsenate; A, 50 mM arseeach set of assays. nate; 0, 100 mM arsenate. (B) The generation time calculated from the data ofpart A are plotted as a RESULTS function of the arsenate-to-Pi ratio.

oo

The rate of growth of the Pst-dependent strain (pst+ pit) in arsenate-containing medium was a function of the arsenate-to-Pi ratio. At a given Pi concentration (1 mM), the generation time increased as increasing amounts of arsenate were added to the medium (Fig. 1). To determine if the growth in arsenate medium was a result of detoxification of arsenate, a bioassay for arsenate was used (data not shown). After one to two generations of growth in arsenate-containing medium, pst+ pit cells were harvested by centrifugation and the supernatant was tested for the presence of inhibitory concentrations of arsenate. Growth of the arsenate-sensitive Pit' strain ceased in fresh medium containing arsenate and after the addition of the supematant obtained from the growing culture of Pst-dependent cells. Growth was normal in the original medium without arsenate. Thus, arsenate was still present in a forn toxic to sensitive cells in the medium obtained from pst+pit cells growing in the presence of arsenate. Arsenate accumulation through the two major Pi transport systems. Cells containing

the Pit system (pst+ or pst) accumulated significant amounts of 74As-arsenate during the first 4 min of the standard uptake reaction, whereas cells containing only the Pst system did not accumulate 74As-arsenate (Fig. 2). Arsenate accumulation through the Pit system required a metabolizable carbon source and was inhibited by the sulfhydryl reagent, N-ethylmaleimide (data not shown). In cells containing the Pit system, arsenate or Pi (nonradioactive) could exchange with accumulated 74As-arsenate with a half-time of 1 min (Fig. 3). Kinetics of Pi accumulation in the presence and absence of arsenate. We examined the ability of Pst-dependent and Pit-dependent strains to accumulate Pi when grown and then assayed in the presence or absence of arsenate (Fig. 4). The Pit-dependent strain showed arsenate-sensitive Pi uptake when grown in the absence of arsenate, but accumulated little Pi when assayed after resuspension in arsenate-containing medium. In this arsenate-containing medium, the growth of the Pit-dependent strain

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had ceased. The strain dependent upon the Pst nate. However, when grown in the presence of system also showed arsenate-sensitive Pi uptake arsenate and assayed in the absence of arsenate, when previously grown in the absence of arse- increased Pi accumulation was found (1.6 fold). When the Pst-dependent strain previously grown in the presence of arsenate was assayed Pst Pit in the presence of arsenate, as much as 55% of the Pi accumulation seen in cells grown and assayed in the absence of arsenate was observed. >~60 This amount of Pi accumulation appears to be sufficient to support growth. Inhibition of Pi transport by arsenate in E NE. 50 the Pst-dependent strain. The kinetics of arsenate inhibition of Pi transport were determined in the Pst-dependent Hfr strain, U7. To 40 distinguish between models of competitive and noncompetitive inhibition, the data from a series 30 XPst x pit+ of Pi transport assays (Fig. 5A) were fitted to the x corresponding Michaelis-Menten equation using H-030 a nonlinear least-squares analysis mini'mizing 0n velocity. In all cases the fit was from 3- to 10fold better for the competitive inhibition model. 20 Furthermore, no reasonable theoretical curves for Ki (arsenate) for Pi transport were obtained using the noncompetitive inhibition model. The competitive Ki (arsenate) for Pi transport in strain U7 obtained from this experiment was 23 ,uM arsenate. The kinetic parameters of Pi accumulation were also determined in the pst+ pit strain TIME (MIN) This strain has a Km of 0.6 + 0.03 ,uM FIG. 2. Arsenate accumulation through the Pst GR5172. a and Pi V. of 10.9 ± 0.3 nmol of Pi (mg [dry and Pit P, transport systems. Arsenate transport assays were performed as described in the text using weight]-'min-') grown in medium without 600 uM 74As-arsenate. Symbols: *, GR5172, Pst+; x, arsenate and a Km of 0.4 ± 0.07 uM Pi and a V of 19.7 ± 0.9 nmol of Pi (mg [dry weight]-'min-') GR2131B, the wild-type strain; 4 GR5178, Pit+. -

>0%

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TIME (min) FIG. 3. Arsenate exit through the Pit system. (A) GR2131B (pst' pit+). (B) GR5178 (pst pit*). The standard arsenate transport assay was performed (0) at 660 pM 74As-arsenate. At 2.5 min, 10 mM "1Pi (A) or 73As-arsenate (a) was added.

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TIME (MIN) FIG. 4. Accumulation of 32Pi in cells grown in the presence and absence of arsenate. (A) GR51 72 (pst' pit) assayed using 50 pM 32Pi. (B) GR5178 (pst pit+) assayed using 1(X ,M 32P'. Cells were grown in glucose minimal medium with 1 mM Pi. During exponential growth each culture was divided into two parts and 10 mM arsenate was added to one portion. The procedure for the Pi transport assays is described in the text. If indicated, 1 mM arsenate was added to the uptake assay at zero time. After exposure to arsenate in the growth medium, the cells were washed three times with the original sample volume ofmedium before transport assays. Symbols: cells grown without arsenate in the medium: 0, early exponential cells assayed for Pi transport without arsenate; 0, assayed with arsenate in the uptake medium; E4 late exponential cells assayed without arsenate in the uptake medium; U, assayed with arsenate in the uptake medium. After 3 h in arsenate medium: IA, assayed for Pi transport without arsenate; A, assayed with arsenate in the uptake medium.

in arsenate-containing medium. From these data quired high concentrations of arsenate to load it is clear that the affinity of the Pst transport the Pst-dependent strain with arsenate since the system for Pi is similar in cells grown in both Pst system did not accumulate arsenate if lower media. The increased Pi uptake observed after concentrations were used. Since the phosphate growth of these cells in arsenate-containing me- remained within the cell, while the arsenate dium (Fig. 4) appears to be due to a twofold rapidly left the cell, these data support the idea increase in the capacity (V.) of the Pst trans- that an arsenate exit reaction may be involved port system. in the Pst Pi transport system. It is possible that Pi transport in GR5172 previously grown in the lack of accumulation of arsenate in the pst+ arsenate medium was competitively inhibited by pit strains could result from the activity of a arsenate with a Ki (arsenate) for Pi accumulation rapid exit reaction counteracting the entry reof 39 ,iM arsenate (Fig. 5B). These results indi- action rather than reflect an inherent inability cate that, even after growth in arsenate-contain- of these cells to take up arsenate. Apst pit strain ing medium, Pi transport in this strain was still which could not grow with Pi as the sole phossensitive to the ratio of arsenate to Pi in the phate source also had a functional rapid arsenate uptake assay. exit system (Fig. 7). The strain dependent upon the Pst system Part of the observed arsenate exit mechanism was exposed to normal substrate levels of 32P1, inpst+pit' and pst pit' strains could be due to or high substrate levels of 74As-arsenate, filtered, a loss of 74As-arsenate bound to the cell surface. and suspended in medium in the absence and To determine how much arsenate was cell presence of unlabeled isotope (Fig. 6). No accu- bound, a similar experiment on retention of 74Asmulated 32P, was lost in this assay. The arsenate arsenate was perforned with the pst pit' strain exit reaction in Pst-dependent cells was ex- after exposure to an extremely high level (10 tremely rapid, since essentially all of the arse- mM) of arsenate. Figure 8 shows that, 2 min nate was lost from the cells by the first time after resuspension 50% of the accumulated arpoint after resuspension. These experiments re- senate was still cell associated. Using this assay,

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I/S

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(MM

Pir)'

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( MM Pir I FIG. 5. Determination of the K1 (arsenate) for Pi transport in strains dependent upon the Pst system for P, transport. (A) U7, (pst' pit) grown in minimal medium with glucose and 1 mM Pi but without arsenate. Pi transport was determined using the following concentrations of arsenate: 0, none; x, 15 Ml; EO 30 M; A, 45 pM; 0, 60 W. (B) GR5172 (pst+ pit) grown in glucose medium containing 1 mM P, and 10 mM arsenate. Arsenate added: 0, none; x, 60 W. Pi transport assays were performed as previously described (27).

I/s

the half-time of arsenate efflux was about 0.7 min, which compares favorably with the 1 mi half-time of exit found using 0.6 mM 74As-arsenate with the regular chase protocol (Fig. 3). Cyanide did not prevent the loss of the 'Asarsenate by the pst+pit strain, and the presence of cyanide in the resuspension fluid did not prevent efflux (data not shown). Effect of arsenate on intracellular ATP levels. Representative pst+ pit, pst pit, pst+ pit', and pst pit' strains were employed. In the absence of arsenate in the growth medium, ATP levels rose during exponential growth in all strains and started to drop as stationary phase was approached (Fig. 9). Strains containing the Pit system lost their intracellular ATP within 60 min after the addition of arsenate to the growing cells. Only the ATP levels in the pst pit strain appeared to be unaffected by the addition of

arsenate to the medium. The pst+ pit strain showed a slight loss of internal ATP after the addition of arsenate to the growth medium, and then the ATP levels rose at a slower rate than in celis not exposed to arsenate. These results support the hypothesis that arsenate is able to penetrate the pst+ pit cell and temporarily interfere with the production of intracellular ATP, whereas arsenate cannot enter the pst pit cell. DISCUSSION In this and the accompanying paper (26) we have characterized the Pst and Pit Pi trnsport systems in E. coli cells during balanced growth in excess (1 mM) phosphate media and in the presence and absence of arsenate. (For these studies the two inducible Pi transport systems, glpT and uhp, which would accept Pi as a secondary substrate, were not functioning.)


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The following recapitulates the properties of the Pit and Pst Pi transport systems operating in cells grown in a glucose minimal salts medium with 1 mM phosphate. (i) Pi transport through both systems was sensitive to the energy uncoupler dinitrophenol, and the sulfhydryl reagent N-ethylmaleimide. Only the Pst system was very sensitive to sodium cyanide. (ii) Neither system functioned as an exit system for previously accumulated Pi, and both systems required the presence of a metabolizable energy source for Pi uptake. (iii) Kinetic analysis of Pi transport showed that the Pst and Pit systems were not fully active in a wild-type strain containing both systems. The Pst and Pit systems responded differently to the presence of arsenate. (i) Pi uptake was arsenate sensitive in strains dependent on either system grown in the absence of arsenate. (ii) A Pst-dependent strain grew in arsenate-containing medium but at a reduced growth rate. This was not due to detoxification of arsenate in the medium. Pi uptake increased 1.6-fold during this time, whereas arsenate-resistant Pi uptake developed after exposure to arsenate in the medium. These cells did not accumulate arsenate.

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In the presence of arsenate, the capacity of the Pst system for Pi was increased by a factor of almost two whereas the affinity of the system for Pi remained unchanged. After growth in arsenate-containing medium, the Pst-dependent strain did not become completely arsenate insensitive and Pi transport under these conditions was still competitively inhibited by arsenate. The Pst-dependent strain grown in the presence of arsenate was sensitive to the ratio of arsenate to Pi in both its growth properties and in the kinetics of Pi accumulation (inhibition of Pi transport by arsenate). (iii) Strains containing the Pit system ceased growth in the presence of arsenate. This is consistent with our observation that these cells accumulate arsenate. The increased capacity for Pi transport of the Pst system after exposure to arsenate in the medium can be explained by two basic hypotheses: (i) arsenate competitively inhibits Pi transport through the Pst system and causes derepression of this system due to apparent Pi starvation; or (ii) arsenate is a substrate of the Pst system, which also functions as a rapid arsenate exit system. In both models we would propose that exposure to arsenate leads to the

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TIME AFTER RESUSPENSION (MIN) FIG. 6. Arsenate and Pi exit through the Pst system. The efflux assay using filtered resuspended cells was performed as described in the text using the pst+ pit strain, GR5172. Pi uptake with 100 ,uM 32P used as substrate: *, amount of cell-associated Pi at time zero; *, cells resuspended with 1 mMPi; 0, cells resuspended with no additions. Arsenate uptake: 10 mM 74As-arsenate as substrate: ®, amount of cell-associated arsenate at time zero; A, cells resuspended with 200 mM arsenate; , cell resuspended with no additions.

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arsenate can enter the cell through the Pst system and support model (ii) presented above. However, one cannot rule out the possibility that the addition of arsenate to the medium caused the depression of the Pst system due to phosphate starvation (10, 11, 14-20). The two Pi transport systems differ in their known genetic complexity. The Pit system appears to be coded for by one gene which maps at min 76-77 of the E. coli chromosome (21, 23). Three genetic loci at min 83 of the E. coli chromosome may be involved in the formation of the Pst system. The phoS and phoT genes involved in the Pst transport system (23) also play a role in the regulation of the synthesis of alkaline phosphatase, a periplasmic enzyme whose synthesis is induced by phosphate starvation (7). The phoT gene is necessary for the function of the Pst Pi transport system whereas the phoS gene is required to maintain the specificity of this system (24). The phoS protein (originally isolated by Garen and Otsuji [8]) has

60

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20A~~~~~~ 15 6 8 4 2 TIME (min) FIG. 7. Exit of arsenate in the Pst' Pit and PstI I* Pit- strains. Experimental protocol is described in the legend to Fig. 6. 8 mM 74As-arsenate was used as E 10 substrate. GR5172 (pst' pit): 0, 74As-arsenate cell associated before the assay; 0, resuspended without U) arsenate. URI (pst pit): 0, cell associated arsenate < before assay; 4 resuspended without arsenate. E

-3-2-1 0

formation of more Pst carriers, thereby increasing the capacity (V..) of the Pst system for Pi transport. To differentiate these models we attempted to find evidence that arsenate can enter a cell through the Pst system. Using high levels of radioactive arsenate, there is evidence for the existence of a rapid arsenate exit system in the Pst-dependent strain (Fig. 6 to 8). The rate of ATP formation was examined in strains growing in minimal medium using DL-a-glycerophosphate as the source of phosphorus (Fig. 9). As expected, after the addition of arsenate to the medium, the ATP levels dropped dramatically in strains containing the Pit system. However, also medium also additionof to the arsenate to the medium of arsenate addition thethe decreased the formation of ATP in the pst+ pit strain, whereas the ATP levels in the pst pit strain were unaffected. These results imply that

5

* L -3 -2 -I 0

*

1

2

TIME ( min) FIG. 8. Arsenate exit in a strain dependent on the Pit system. Experimental protocol is described in the legend to Fig. 6. 10 mM 74As-arsenate was used. GR5172 (pst' pit): 0, cell-associated arsenate before resuspension; 0, resuspended in WT medium; 0, 200 mM unlabeled medium withcell-associated resuspended in WT(pst arsearsenate. GR5178 , pit ): resuspended in WT menate before resuspension; 0, dium; U, resuspended in WT medium with 200 mM unlabeled arsenate. (Note change in scale after t 0).


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TIME (hours) FIG. 9. ATP levels in strains grown in the presence and absence of arsenate. ATP assay is described in the text. Cells were grown overnight in minimal medium containing glucose and 1 mM DL -a-glycerophosphate, harvested by low-speed centrifugation, and resuspended in fresh medium. After 2 h of exponential growth, the culture was divided and one portion was adjusted to 10 mM arsenate. Samples were taken at intervals, and ATP levels were determined. Open symbols represent the ATP levels in cells grown in the absence of arsenate. The circled data points represent the zero time point before the addition of arsenate. (A) GR5172 (pst' pit), O and U; GR5178 (pst pit+), V and V. (B) URI (pst pit), A and A; GR2131 (pst pit+), 0 and *.

been unambiguously identified by Gerdes and Rosenberg (9) and Wilisky and Malamy (24, 25) as the periplasmic phosphate-binding protein. Alkaline phosphatase is coregulated with at least three other proteins (one of which is the phoS protein), under the positive regulation of the phoB gene (4, 5). The phoB-controlled proteins are produced constitutively (13,24) inphoS strains or in the absence of the Pst Pi transport system. This effect is not due to lowered internal Pi levels, since significant synthesis of the periplasmic proteins can be observed (i) in the phoT pit' strain which has high intemal levels of phosphate, and (ii) before the intemal Pi level of the cell drops in the phoT pit strain (unpublished observation). In addition, an adenine nu-

cleotide and not Pi has been implicated as the coregulator of alkaline phosphatase synthesis (22), which could indicate that Pi levels in the cell may not directly control alkaline phosphatase synthesis. In phoT strains (mutations inactivating the activity of the Pst Pi transport system isolated as alkaline phosphatase constitutive) and pst strains (mutations inactivating the Pst Pi transport system isolated as arsenate-resistant mutants), the proteins regulated byphoB are produced constitutively. In addition, phoT mutant and pst mutant cells contain different levels of alkaline phosphatase (unpublished observation) which might indicate that these mutations are in different genes. At this time, however, there have been no genetic studies which

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unequivocally demonstrate that pst and phoT are two distinct loci. In strains missing the Pit system the synthesis of proteins regulated by phoB is normal as long as the Pst system is functioning. These observations imply that the Pst system must function to obtain a metabolite required for the normal production of phoBcontrolled periplasmic proteins; only the phosphate-binding protein has been directly implicated in phosphate transport. In addition there are three other periplasmic proteins whose synthesis increases by growing cells in excess phosphate medium (25). These periplasmic proteins may play a role in controlling the activity of the major Pi transport systems in response to the composition of the growth medium. ACKNOWLEDGMENTS This project has been supported by research grants from the American Cancer Society (BC28A) and the Public Health Service, National Institutes of Health (GM14814). UTERATURE CITED 1. Bennett, R. L, and M. H. Malamy. 1970. Arsenate resistant mutants of Escherichia coli and phosphate transport. Biochem. Biophys. Res. Commun. 40:496503. 2. Berenblum, I., and E. Chain. 1938. An improved method for the colorimetric determination of phosphate. Biochem. J. 32:295-298. 3. Blumberg, D. D., C. T. Mabie, and M. H. Malamy. 1976. T7 protein synthesis in F-factor-containing cells: evidence for an episomally induced impairment of translation and its relation to an alteration in membrane permeability. J. Virol. 17:94-105. 4. Bracha, M., and E. Yagil. 1973. A new type of alkaline phosphatase-negative mutant in Escherichia coli K-12. Mol. Gen. Genet. 122:53-60. 5. Brickman, E., and J. Beckwith. 1975. Analysis of the regulation ofEscherichia coli alkaline phosphatase synthesis using deletions and 080 transducing phages. J. Mol. Biol. 96:307-316. 6. Cole, H. A., J. W. T. Wimpenny, and D. E. Hughes. 1967. The ATP pool in Escherichia coli. I. Measurements of the pool using a modified luciferase assay. Biochim. Biophys. Acta 143:445-453. 7. Echols, H., A. Garen, S. Garen, and A. Torriani. 1961. Genetic control of repression of alkaline phosphatase in Escherichia coli. J. Mol. Biol. 3:425-438. 8. Garen, A., and N. Otsuji. 1964. Isolation of a protein specified by a regulatory gene. J. Mol. Biol. 8:841-852. 9. Gerdes, R. G., and H. Rosenberg. 1974. The relationship between the phosphate-binding protein and a regulator gene product from Escherichia coli. Biochim. Biophys. Acta 351:77-89. 10. Gerdes, R. G., K. P. Strickland, and H. Rosenberg. 1977. Restoration of phosphate transport by the phosphate-binding protein in spheroplasts of Escherichia

J. BACTERIOL. coli. J. Bacteriol. 131:512-518. 11. Medveczky, EL, and H. Rosenberg. 1971. Phosphate transport in Escherichia coli. Biochim. Biophysa Acta 241:495-506. 12. Mitchell, P. 1954. Transport of phosphate across the osmotic barrier of Micrococcus pyogenes specificity and kinetics. J. Gen. Microbiol. 11:73-82. 13. Morris, H., M. J. Schlesinger, M. Bracha, and E. YagiL 1974. Pleiotropic effects of mutants involved in the regulation of Escherichia coli alkaline phosphatase. J. Bacteriol. 119:583-592. 14. Rae, A. S., and K. P. Strickland. 1975. Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli. Biochem. Biophys. Res. Commun. 62:568576. 15. Rae, A. S., and K. P. Strickland. 1976. Studies on phosphate transport in Escherichia coli. II. Effects of metabolic inhibitors and divalent cations. Biochim. Biophys. Acta 433:564-582. 16. Rae, A. S., K. P. Strickland, N. Medveczky, and H. Rosenberg. 1976. Studies of phosphate transport in Escherichia coli. I. Reexamination of the effect of osmotic and cold shock on phosphate binding protein. Biochim. Biophys. Acta 433:556-563. 17. Rosenberg, H., G. B. Cox, J. D. Butin, and S. J. Gutowski. 1975. Metabolite transport in mutants of Escherichia coli K-12 defective in electron transport and coupled phosphorylation. Biochem. J. 146:417-423. 18. Rosenberg, H., R. G. Gerdes, and K. Chegwidden. 1977. Two systems for the uptake of phosphate in Escherichia coli. J. Bacteriol. 131:505-611. 19. Rosenberg, H., R. G. Gerdes, and F. ML Harold. 1979. Energy coupling to the transport of inorganic phosphate in E. coli K-12. Biochem. J. 178:133-137. 20. Russell, L. M., and H. Rosenberg. 1979. Linked transport of phosphate, potassium ions and protons in E. coli Biochem. J. 184:13-21. 21. Sprague, G. F., Jr., R. M. Bell, and J. E. Cronan, Jr. 1975. A mutant of Escherichia coli auxotrophic for organic phosphates: evidence for two defects in inorganic phosphate transport. Mol. Gen. Genet. 143:7177. 22. Wilkins, A. S. 1972. Physiological factors in the regulation of alkaline phosphatase synthesis in Escherichia coli. J. Bacteriol. 110:616-623. 23. Wilisky, G. R., R. L Bennett, and KL H. Malamy. 1973. Inorganic phosphate transport in Escherichia coli: involvement of two genes which ply a role in alkaline phosphatase regulation. J. Bacteriol. 113:529539. 24. Willdky, G. R., and M. H. Malamy. 1974. The loss of the phoS periplasmic protein leads to a change in the specificity of a constitutive inorganic phosphate transport system in Escherichia coli. Biochem. Biophys. Res. Commun. 60:226-233. 25. Willsky, G. R., and K. H. Malamy. 1976. Control of the synthesis of alkaline phosphatase and the phosphatebinding protein in Eacherichia coi. J. Bacteriol. 127: 595-609. 26. Willsky, G. R., and K H. Malamy. 1980. Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coi. J. BacterioL 144: 356-365.