JouRNAL OF BACTRIUoLOGY, July 1977, p. 229-239 Copyright 0 1977 American Society for Microbiology

Vol. 131, No. 1 Printed in U.S.A.

Formation of Sugar Phosphates in Colicin K-Treated Escherichia coli YOHTAROH TAKAGAKI,I* MICHIO MATSUHASHI, JIMPEI YAMASHITA, AND TAKEKAZU HORIO Institute ofApplied Microbiology, The University ofTokyo, Tokyo, Japan 113, and Protein Research Institute, Osaka University, Osaka, Japan 564 Received for publication 27 April 1977

Colicin K greatly decreased the incorporation of 32P-labeled inorganic orthophosphate into nucleotides and nucleic acids, causing a concomitant increase in the formation of 32P-labeled sugar phosphates in sensitive cells of Escherichia coli. These sugar phosphates were formed in aerobically growing cells, as well as in cells under stringent control of ribonucleic acid synthesis. The main 32p_ labeled product was identified as sedoheptulose 7-phosphate in two strains (Bi and K-12 MK-1) and fructose 1,6-diphosphate in one strain (K-12 CP78). The formation of sugar phosphates induced by colicin K was inhibited by carbonyl cyanide m-chlorophenylhydrazone. It was also not observed in N,N'-dicyclohexylcarbodiimide-treated cells or Mg2+-(Ca2+)-adenosine triphosphatase-less mutant (strain K-12 AN120) cells. Thus, the formation of sugar phosphates in colicin K-treated cells is dependent on the formation of adenosine 5'-triphosphate by oxidative phosphorylation. Colicin K is a protein antibiotic with a molecular weight of about 70,000 (25). It becomes attached to the cejl surface (outer membrane) of sensitive cells of Escherichia coli and kills the cells (30, 45). Two important problems about the action of colicin K are (i) how the colicin K molecules interact with the cell surface and (ii) what their biochemical target is. We reported previously that the receptor for colicin K is not essential for its action when the outer membrane of the cell is made permeable to colicin K. This was demonstrated by using ethylenediaminetetraacetic acid-lysozyme-treated "disrupted" spheroplasts of several colicin K-resistant and -tolerant mutants of E. coli K-12 (41), as well as colicin K receptor-deficient, deep rough mutants of E. coli K-12 and B (41; Y. Takagaki and M. Matsuhashi, submitted for publication). Observations supporting this view have also been obtained from studies on colicin K by Smarda and Taubeneck (39) and on other colicins by several investigators (5, 6, 10, 37). Colicin K decreases the intracellular adenosine 5'-triphosphate (ATP) level and inhibits the synthesis of macromolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and proteins (31). Plate et al. (34) proposed that inhibition of the synthesis of macromolecules by colicin K in cells having adenosine triphosphatase (ATPase) activity results from a decrease in the intracellular ATP level. 1 Present address: Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.

Colicin K also causes release of accumulated potassium ions (19, 46), methylthio-,8-n-galactoside (14), and proline (3) from cells and makes the membrane leaky to magnesium ions (27) and chlortetracycline (7). This increase in permeability indicates that the biochemical target of colicin K involves the cytoplasmic membrane. This paper reports the formation of sugar phosphates in colicin K-treated cells and shows that this formation is dependent on oxidative phosphorylation. A preliminary account of this work has appeared (42). MATERIALS AND METHODS Bacterial strains and culture media. The bacterial strains used are listed in Table 1. Bacteria were grown in the following media: Fraser medium (17) containing, in 1 liter of deionized water, 15 g of vitamin-free Casamino Acids (Difco), 30 g of glycerol, 4.5 g of KH2PO4, 10.5 g of Na2HPO4, 1.0 g of NH4Cl, and 0.3 g of MgSO4 7H20; and tris(hydroxymethyl)aminomethane (Tris)-glucose mineral medium (21) containing, in 1 liter of deionized water, 0.1 M Tris-hydrochloride, pH 7.4, 422.5 mg of Na3C6H507. 4H20, 100 mg of MgSO4, 0.405 mg of FeCl3, 1 g of (NH4)2SO4, 2 x 10-3 M potassium phosphate, pH 7.0, and 2 g of glucose. For cultures of CP78 and CP79, Tris-glucose mineral medium was supplemented with 200 mg of threonine, 100 mg of arginine, 200 mg of histidine, 1 mg of vitamin Bl, and 40 mg ofleucine (each per liter). For cultures of AN180 and AN120, half-strength Trisglucose mineral medium was supplemented with 1 g of glucose (final concentration, 0.2%), 50 mg of arginine, and 1 mg of vitamin B,, each per liter, or -

229

230

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TAKAGAKI ET AL.

Type

Strain

TABLE 1. Bacterial strains used Genotypeaa

Refer-

ence

41 leu pro MK-1 ara mtl mal xyl gal lac str W4573 16 thi thr leu arg his lac ara mtl xyl gal mal xr tonA supE relA+ str CP78 16 thi thr leu arg his lac ara mtl xyl gal mal xr tonA supE relA - str CP79 8 arg thi str uncA+ AN180 8 AN120 arg thi str uncA 25 thr pro colK E. coli K-235 AP53 str Bi E. coli B a Abbreviations: leu, leucine; pro, proline; thi, thiamine; thr, threonine; arg, arginine; his, histidine; ara, arabinose; mtl, mannitol; mal, maltose; xyl, xylose; gal, galactose; lac, lactose; Xr, phage lambda resistance; tonA, phage Ti resistance; supE, suppressor of amber mutation; str, streptomycin resistance; colK, colicinogenic factor K-K235. E. coli K-12

nutrient broth described previously (41) was supplemented with 2 g of glucose per liter (final concentration, 0.2%). Preparation of colicins. Highly purified colicin K was obtained from the colicinogenic E. coli AP53 (25). Colicin El was purified from Salmonella typhimurium cysD36 by the same procedure as was used for colicin K. One killing unit (KU) was determined as described by Fields and Luria (14). The preparation of purified colicin K contained 3.3 x 1012 KU per mg of protein. Preparation of cells. Cells were grown in appropriate medium at 37°C with shaking. Washed cells were obtained by harvesting cells in the mid-log phase of growth in Fraser medium, washed twice with cold deionized water, and suspended at a concentration of 10% (wet weight) per volume of cold deionized water for use in experiments. Incorporation of 32p1. 32P-labeled inorganic orthophosphate (32pI) (Institute of Atomic Energy, Tokyo, Japan, carrier free) was purified on a column of Dowex-1 (40). For experiments with washed cells, reaction mixtures contained 50 &mol of Tris-hydrochloride, pH 7.6, 5 ,umol of MgCl2, 6 jAmol of D-glucose, and suspensions of washed cells from Fraser medium (25 mg [wet weight]) in 1 ml. The mixture was preincubated for 6 min, and then 1 jACi of 32Pi in potassium phosphate, pH 7.6 (at the concentration given in the text), was added. Incubation was carried out for 15 min at 30°C with shaking. For experiments with growing cells, 10' cells per ml in Tris-glucose mineral medium were preincubated with additions for 5 min and then mixed with 1 &Ci of 32P1 and incubated for 20 min at 37°C with shaking. The reaction was terminated by adding ice-cold trichloroacetic acid to a final concentration of 5% (wt/vol). The mixture was quickly chilled in an ice bath and left for at least 2 h to complete the extraction. When determining intracellular concentration of 32P-labeled products, the reaction mixture was transferred to a membrane filter (Toyo Roshi TM-2; pore size, 0.45 ,um) and rapidly washed with two 1-ml portions of 50 mM Tris-hydrochloride, pH 7.6, containing 5 mM MgC92. The filter was then transferred to a 10-ml beaker containing 1 ml of icecold 10% (wt/vol) trichloroacetic acid and left in an ice bath overnight for extraction. 32P-labeled organic phosphates were fractioned by

the method of Schneider (38). Nucleotide 32p was estimated after its adsorption on charcoal. Total cold trichloroacetic acid-soluble organic phosphate 32p was estimated by removal of inorganic 32p1 from the trichloroacetic acid-soluble fraction as described by Avron (1). Non-nucleotide organic phosphate 32P was calculated as the difference between total cold trichloroacetic acid-soluble organic phosphate 32p and nucleotide 32p. For determination of 32P-labeled sugar phosphate compounds, cold trichloroacetic acid extracts were first treated with activated charcoal to remove nucleotides. The trichloroacetic acid was then removed by extracting five times with an equal volume of ether; a portion was taken to determine alkali-labile organic phosphates (-"Pi liberated by treatment with 1 M KOH at 30°C for 30 min), and the remainder was used for chromatography and enzyme assays as described below. Chromatography. Descending paper chromatography of32P compounds was performed on Whatman 3MM filter paper with the following solvent systems: I, isobutyric acid-1 M ammonia (5:3, vol/vol) or II, 95% ethanol-1 M ammonium acetate (pH 7.3) (5:2, vol/vol). Two-dimensional polyethyleneiminecellulose thin-layer chromatography was performed as described by Randerath and Randerath (36). Detection of spots and preparation of autoradiograms were carried out by conventional methods. Enzymatic assays. The reaction conditions used for enzymatic assays of phosphate compounds were based on the description of Bergmeyer (2). For the phosphofructokinase reaction, inosine-5'-triphosphate (ITP) was used instead of ATP (4). After completion of reactions, products were separated by paper chromatography on Whatman no. 1 filter paper with solvent system I, and radioactivity was determined. Radioactivity was determined in an end-windowtype Geiger-Muller counter. Protein concentration was determined by the method of Lowry et al. (26), using bovine serum albumin as a standard. Pi concentration was determined by the method of Chen et al. (9). DCCD treatment. Washed cells of strain MK-1 (7 x 108 per ml) were pretreated with 10-4 M N,N'dicyclohexycarbodiimide (DCCD) (dissolved in ethanol; final concentration, 0.1%) at 37°C for 30 min as described by Evans (13). Control cells were also

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SUGAR PHOSPHATES IN COLICIN K-TREATED CELLS

pretreated with 0.1% ethanol at 37°C for 30 min. Chemicals and reagents. Nicotinamide adenine dinucleotide (NAD), reduced NAD (NADH), and nicotinamide adenine dinucleotide phosphate (NADP) were purchased from Oriental Yeast Industry Co., Osaka, Japan, and ATP and adenosine 5'diphosphate (ADP) were from Kyowa Fermentation Industries Co., Tokyo, Japan. ITP was from Boehringer Mannheim Corp., and other nucleotides were commercial products from Sigma Chemical Co. Erythrose 4-phosphate, ribose 5-phosphate, ribulose 5phosphate, xylulose 5-phosphate, and glucose 6phosphate (G6P) were purchased from Sigma. Other sugar phosphates and enzymes were from Boehringer Mannheim. DCCD was purchased from Tokyo Kasei Industry Co., Tokyo, Japan, and carbonyl cyanide m-chlorophenylhydrazone (ClCCP) was from Calbiochem. RESULTS

Formation of trichloroacetic acid-soluble non-nucleotide organic phosphate by colicin K. As previously shown by Nomura and Maeda (31), the incorporation Of 32pI into nucleotides and nucleic acids is significantly inhibited by colicins K and El. We confirmed these effects of colicin K both in cells and in a "disrupted" spheroplast preparation of E. coli K-12 under aerobic conditions (41). The tern "disrupted" spheroplasts is used for a preparation of spheroplasts treated in hypotonic buffer solution containing magnesium ions (41). However, in more detailed experiments on the 32P-labeled fractions in colicin K-treated and untreated cells, 32p, incorporation into total trichloroacetic acid-soluble organic phosphates was always found to be unchanged, whereas a marked decrease in nucleotide formation was TABLE 2. Effect of colicin K and inhibitors on Expt

observed in colicin K-treated cells. Table 2 shows typical results obtained with cells of E. coli strains suspended in high concentration (1010 cells per ml) in Tris-hydrochloride buffer. Previous experiments had shown that 32P1 uptake by intact cells of strain MK-1 was not affected by colicin K under our experimental conditions in 0.2 or 1.25 mM phosphate (data not shown). Table 2 shows that 32P1 incorporation into the trichloroacetic acid-soluble nonnucleotide organic phosphate fraction was increased by colicin K (17 KU/cell). This increase was not observed on addition of ClCCP, pentachlorophenol, or 2,4-dinitrophenol, which are uncouplers of oxidative phosphorylation. Addition of colicin El also resulted in increased 32p, incorporation into the trichloroacetic acid-soluble non-nucleotide organic phosphate fraction (data not shown). The time course of 32P1 incorporation is shown in Fig. 1. Addition of colicin K resulted in an increase in 32P-labeled nonnucleotide organic phosphates, which continued linearly for over 20 min (Fig. 1A). In the absence of colicin K, the increase was less and ceased after 15 min, and on addition of ClCCP, the increase was even less. 32p1 incorporation into the nucleotide fraction was inhibited by both colicin K and ClCCP (Fig. 1B). Identification of trichloroacetic acid-soluble organic phosphates formed in the presence of colicin K. 32P-labeled trichloroacetic acid-

soluble non-nucleotide phosphate compounds were formed by strains MK-1 and Bi in the presence of colicin K. These 32p compounds were identified as follows. Cold trichloroacetic acid extracts of reaction mixtures were sub-

32p, incorporation by cells ofE. coli strainsa

Condition

Strain

231

Cold trichloroacetic acid-soluble organic phosphate Nucleic acidb

Tobal°

NucleoNon-nutideb cleotideb

1

MK-1

Control + Colicin K (50 ,ug/ml) + CICCP (2 x 10-5 M) + Colicin K (50 p,g/ml) and C1CCP (2 x 10-5 M) + Pentachlorophenol (10-3 M) + 2,4-Dinitrophenol (4 x 10-3 M) Glucose omitted

29.7 37.1 16.3 16.2 1.8 4.0 11.3

21.8 7.0 12.2 10.3 0.2 1.9 8.4

7.9 30.1 4.1 5.9 1.6 2.1 2.9

9.1 0.9 2.0 0.8 0.1 0.1 0.4

2

W4573

Control

26.8 18.9 10.7

21.2 4.2 5.9

5.6 14.7 4.8

6.2 0.3 0.8

34.6 Control 29.3 + Colicin K (56 zg/ml) 11.7 + CICCP (5 x 10-5 M) Experimental conditions were as for Fig. 1. Nanomoles of 32p incorporated per milligram of protein per 15 min.

24.7 4.5 8.5

9.9 24.8 3.2

3.8 0.3

+ Colicin K (56 ,ug/ml) + CICCP (5 x 10-5 M) 3 a

b

Bi

1.0

232

TAKAGAKI ET AL.

J. BACTERIOL.

2L5

z~~~~ 52

0

15

0.

T

c

fracton Supnion

fwse

elfM-rw

in Fraser's medium were incubated with

,umnol

32Ji

in 125

of potassium phosphate buffer, pH 7.6. as de-

scribed under Materials and Methods. (A) Non-nucleotide organic phosphate fraction;

fraction. Symbols: 50

/lg

of colicin K per ml;

10-s M

(B) nucleotide

0, control; A, preincubated with

O,

preincubated with 2 x

CICCP.

jected to two-dimensional paper chromatography and thin-layer chromatography on polyeth-

yleneimine-cellulose (36). Figure 2 shows autoradiograms of two-dimensional paper chromatograms of the cold trichloroacetic acid-soluble fractions obtained from cells without (Fig. 2A) and with (Fig. 2B) colicin K treatment. In un-

treated cells,

the

main

products

were

ATP,

other nucleotide triphosphates, and uridine 5'diphosphate

(UDP)-r-glucose.

Small amounts

of sugar phosphates and other glycolytic inter-

mediate phosphates were also formed. In colicin K-treated cells,

most of the incorporated 32p

appeared in one spot in the position of the sugar

monophosphates. (The 32P-labeled spot that has a mobility close to ATP in solvent I, but no

mobility in solvent II, is 32P,

tightly bound to

the paper due to the process that removed sol-

vent I.) These results were confirmed by poly-

ethyleneimine-cellulose thin-layer chromatography. The radioactive spot in the position of the

sugar monophosphates in the experiment with colicin K-treated cells was eluted for chemical

characterization. The phosphate bond was split by alkaline phosphomonoesterase (EC 3.1.3.1).

100°C, the apparent half-decay min for the product of strain MK-1 95 min for that of strain Bi. On in 1 M KOH at 30°C for 30 min, 98%

In 1 M HCl at time was 140

and 90 to incubation

of the phosphate

bond was stable.

However,

when the cold trichloroacetic acid-soluble fraction was treated directly with alkali, about 8 to 20% of the total non-nucleotide organic phos-

phate was unstable (Table 3). This means that some alkali-labile phosphate compounds such as triose phosphate, present in the cold trichloroacetic acid extracts, were destroyed during subsequent paper chromatography in the solvents containing ammonia (47). On treatment of the eluted compounds with phenylhydrazine at room temperature for 2 h (28), a material was formed that gave a spot with higher mobility (Rf = 0.61) on paper chromatography in solvent I. On treatment with phenylhydrazine at 100°C for 2 h, material was formed that gave a new spot with very high mobility (Rf = 0.80). Control experiments using [1-'4C]G6P under the same experimental conditions indicated that these materials were phenylhydrazones (Rf = 0.61) and osazones (Rf = 0.80). The compounds in the non-nucleotide organic phosphate fraction were further identified and measured quantitatively by enzymatic assays. [32P]G6P was assayed by conversion to 6-phosphogluconate with glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and NADP. 32P-labeled fructose 6-phosphate ([32P]F6P) was measured by combined reactions with phosphoglucose isomerase (EC 5.3.1.9), glucose 6-phosphate dehydrogenase, and NADP. Sedoheptulose 7phosphate (S7P) was converted to sedoheptulose 1,7-diphosphate (SDP) with phosphofructokinase (EC 2.7.1.1) and ITP (4). However, F6P was also converted to fructose 1,6-diphosphate (FDP) by this reaction, and the product was, in fact, a mixture of FDP and SDP. These two products could be separately identified by combined reactions with aldolase (EC 4.1.2.13), triose phosphate isomerase (EC 5.3.1.1), a-glycerophosphate dehydrogenase (EC 1.2.1.12), and NADH. The 32P label in [6-32P]FDP should appear in a-glycerophosphate, whereas that in [732P]SDP should appear in erythrose 4-phosphate. Paper chromatography of the reaction mixture showed that all the radioactivity in the region of FDP-SDP disappeared during these enzyme reactions with the appearance of new spots corresponding to a-glycerophosphate and erythrose 4-phosphate. Therefore, [32P]S7P was estimated by substracting [32P]F6P from the radioactivity of SDP plus FDP obtained after the reaction with phosphofructokinase. Other phosphate esters were estimated by counting the radioactivity in spots on the paper chromatogram. Phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate were determined as a mixture on the paper chromatogram. Results of the analysis are shown in Table 3. A correction was made for alkali-labile phosphate compounds measured before chromatography. S7P was the main product in the presence of

-*t .-:,.rs*.,r

VOL. 131, 1977

\..*p.

SUGAR PHOSPHATES IN COLICIN K-TREATED CELLS

colicin K in both strains MK-1 and Bi, with lesser amounts of G6P and other sugar phos-

A'~1

phates.

In all the other strains tested except one, the main product in the presence of colicin K was sugar monophosphate. In the exceptional strain CP78, the main sugar phosphate formed on colicin K treatment was FDP (50%o of total nonnucleotide organic phosphate, see Table 5). This strain was used for the experiments described in the next section on the effect of stringent control of RNA synthesis. Examination of the effect of RNA control. The cells used for the results in Table 2 were grown in enriched medium, washed with deionized water, and then incubated in buffer to measure 32p1 incorporation. Table 2 shows that very little 32P1 was incoxporated into nucleic acids, suggesting that the RNA synthesis in the cells was under stringent control due to the

...

\.^.

_

/

.:

r

e

--

P

..

S

.

3.

/; )

tP

-

: G

-

Qi)P

;/

:>

233

SS

i ... T -;

TABLE 3. Determination of 32P-labeled cold trichloroacetic acid-soluble non-nucleotide organic phosphate compounds formed in the presence of colicin K in E. coli cellsa

2 >i

Amt formed (%) with

Organic phosphate compound

B

E. coli strain: BI

MK-1

Sugar monophosphates G6P 17.0 0.8 F6P 3.9 0 S7P 25.1 63.6 Others 21.5 17.9 PEP + 2PG + 3PGb 6.5 4.4 FDP 0.4 0.5 Alkali unstable 19.2 8.0 Others 6.4 4.8 a 32Pi incorporation was measured as described in the legend of Fig. 2. Samples were prepared and analyzed as described in the text. b PEP, Phosphoenolpyruvate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate.

.Ac :.1.

_.S.: >

..; D

w nt

1:

...

..

FIG. 2. Two-dimensional paper chromatographic separation of 32P-labeled compounds in the cold trichloroacetic acid-soluble fraction. 32P, incorporation was achieved as described in the legend of Fig. 1 except that 10 ,uCi of 32p, with 0.2 p.nol of potassium phosphate buffer, pH 7.6, was added. The cold trichloroacetic acid extract of the reaction mixture was treated five times with an equal volume of ether to remove the trichloroacetic acid, and then the sample

was spotted on paper without removing 32pJ . Descending chromatography was performed for 16 h in the first dimension and 22 h in the second dimension at room temperature. The solvent for the first dimension was removed from the filter paper by washing with acetone. AMP, Adenosine 5'-monophosphate; CMP, cytidine 5'-monophosphate; CDP, cytidine 5'diphosphate; UMP, uridine 5'-monophosphate; GMP, guanosine 5'-monophosphate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; R5P, ribose 5-phosphate; CTP, cytidine 5'-triphosphate; GDP, guanosine 5'-diphosphate; GTP, guanosine 5'triphosphate; UTP, uridine 5'-triphosphate; UDPGlc, UDP-glucose; UDP-GlcNAc, UDP-N-acetylglucosamine. (A) Control cells; (B) Colicin K-treated cells.

234

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TAKAGAKI ET AL.

deprivation of amino acids. This stringent control mechanism is known to also reduce the intracellular level of sugar phosphates (21) and the rate of respiration of the cells (15). Since colicins El and K were reported to restore the reduced rate of respiration (15), it is possible that colicin K restored the reduced sugar phosphate level, and this may have been observed as an apparent increase in the formation of sugar phosphates. It was, therefore, necessary to examine the effect of colicin K on cells in the relaxed state. The effect of washing cells with deionized water could also have caused the increase in colicin K-treated cells, and this was investigated. Table 4 shows results with strain CP78, a stringent strain, and with CP79, which is a relaxed derivative of CP78 (16). Both strains were incubated in culture medium with 32P*. 32P, incorporations of these strains was well studied by Irr and Gallant (21) and Gallant and Harada (18). A correction was made as indicated for growth of the cells during the time of incubation. In CP78 cells, the effect of colicin K on 32I1 incorporation by growing cells was almost the same as that shown in Table 2 for cells under nongrowing conditions. ClCCP inhibited the incorporation into all fractions. When valine was added, the cells shifted to stringent control (18), and the synthesis of nucleic acids was depressed. However, the effect of colicin K on valine-inhibited cells was essentially the same as that on nornally growing cells. In

CP79 cells, the effect of colicin K was identical on CP78 cells, except that addition of valine did not have any effect. Therefore, the increased formation of sugar phosphates caused by colicin K does not result from reversal of the stringent control of the cells. These results also show that the increased formation of sugar phosphates caused by colicin K is not the result of cell damage due to washing of cells and suspending in buffer. The time course of 32p incorporation into the trichloroacetic acid-soluble non-nucleotide fraction by growing CP78 cells was essentially the same as in Fig. 1A (data not shown). In growing cells of CP78, the amount of 32p in the nonnucleotide fraction (corrected for growth) reached a plateau within 10 min after the addition of 32P1 and stayed at this level for at least 40 min after 32 Pi addition. In colicin K-treated cells, 32P1 incorporation into the non-nucleotide fraction increased linearly for at least 40 min after 3pj addition. In growing cells of CP78, complete equilibration of nucleotide triphosphate pools and exogenous 3Pi is reported to be attained within 30 min (18). Distribution of trichloroacetic acid-soluble organic phosphate compounds. The above experiments analyzed the whole reaction mixture including extracellular products. When colicin K-treated cells were separated from the reaction mixture by a membrane filter, about 40 to 50% of 32P label in the trichloroacetic acid-soluble non-nucleotide fraction passed through the to that

TABIS 4. Effect of RNA control on 32p, incorporation by growing celSa Strain

Cold trichloroacetic acid-soluble fraction Addition

Nucleic acidb

Non-nucleoNucleotide", Nucleotideb tideb

CP78 (relA)

Nonec + Colicin K (40 pg/mil) + ClCCP (5 x 10-5 M) + Valine (500 Ag/ml) + Valine (500 pg/ml) and colicin K (40 pg/ml) + Valine (500 pg/ml) and ClCCP (5 x 10-5 M)

19.2 7.5 2.6 15.3 7.6 2.1

10.7 19.1 1.5 5.1 15.6 1.1

16.1 0.7 0.3 3.1 1.3 0.5

CP79 (relA-) Nonec 20.8 9.2 15.2 + Colicin K (40 pg/ml) 7.4 19.3 0.8 + ClCCP (5 x 10-5 M) 4.8 1.0 3.6 + Valine (500 pg/ml) 21.4 12.7 17.9 + Valine (500 pg/ml) and colicin K (40 pg/ml) 7.8 18.1 2.0 + Valine (500 Ag/mi) and CICCP (5 x 10-5 M) 5.2 0.5 3.3 a Reactions were performed with growing cells in Tris-mineral medium as described under Materials and Methods. b Nanomoles of phosphate incorporated per 109 cells per 20 min. C Correction was made for cell growth.

VOL. 131, 1977

SUGAR PHOSPHATES IN COLICIN K-TREATED CELLS

filter, indicating the excretion of these phosphate compounds (Table 5). Nucleotides were not excreted by colicin K-treated cells, and the excretion of trichloroacetic acid-soluble non-nucleotide organic phosphate compounds in control cells was slight. Fields and Luria (15) reported the excretion of some intermediate of glycolysis from colicin El- or colicin K-treated cell. Table 5 also shows 32P incorporation into various intracellular trichloroacetic acid-soluble organic phosphate compounds in control and colicin K-treated cells. Nucleotides were estimated by determining the radioactivity in the spots on a polyethyleneimine-cellulose layer chromatogram, and non-nucleotides were estimated as described above. Colicin K treatment resulted in the decrease of ATP, other nucleotide triphosphates and UDP-glucose, but an in-

235

crease of radioactivity

in ADP was observed. In colicin K-treated cells, sugar monophosphates are excreted more efficiently than other nonnucleotide compounds. The main 32P-labeled product of colicin K-treated CP78 was FDP both in growing cells (Table 5) and in valine-inhibited cells (data not shown). Studies on the origin of the phosphate moiety of sugar phosphates formed in colicin Ktreated cells. Washing cells with deionized water is known to remove intracellular potassium ions (12). This results in the inhibition of the potassium-requiring enzyme pyruvate kinase (44) (EC 2.7.1.40) and thus results in partial inhibition of glycolytic ATP synthesis. Therefore, the increased formation of sugar phosphates in colicin K-treated washed cells suggests that the phosphate moiety of sugar phosphates may be derived from ATP generated by

TABLz 5. Incorporation of 32p into trichloroacetic acid-soluble organic phosphate compoundsa Expt (1) Washed cells of MK-1c

Location Intracellular

Fraction Nucleotide

Compound"

Total

ATP GTP UTP UDP-glucose ADP

(2) Growing cells of CP78d

Control 24.1

Colicin K 7.5

6.9 3.3 2.3 3.1 0.9

1.0 0.5 0.3 0.1 1.2

Non-nucleotide

Total Sugar monophosphate FDP PEP+2PG+3PG

6.3 1.3 0.5 1.7

13.1 7.1 1.0 3.5

Extracellular

Non-nucleotide

Total

1.6

12.1

Intracellular

Nucleotide

Total

15.0

6.0

5.3 2.7 2;0 3.0 0.4

1.1 0.8 0.2 0.5 0.7

Total Sugar monophosphate FDP PEP+2PG+3PG

8.4 1.5 2.4 0.5

11.8

Total

0.6 0.3 0.3 0

ATP GTP UTP

UDP-glucose ADP Non-nucleotide

Extracellular

Non-nucleotide

0.7 8.4 1.7

8.7 4.0 FDP 1.8 PEP+2PG+3PG 2.9 a Reactions were performed as in Fig. 1 for experiment 1 or as in Table 4 for experiment 2. The reaction was stopped by transferring the mixture onto a membrane filter as described in Materials and Methods. " GTP, Guanosine 5'-triphosphate; UTP, uridine 5'-triphosphate; PEP, phosphoenolpyruvate; 2PG, 2-

Sugar monophosphate

phosphoglycerate, 3PG, 3-phosphoglycerate. c Nanomoles of 32p incorporated per milligram of protein per 15 min. d Nanomoles of 32p incorporated per 109 cells per 20 min. In control preparations, correction was made for cell growth.

236

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TAKAGAKI ET AL.

oxidative phosphorylation. Alternatively, colicin K may inhibit oxidative ATP formation while stimulating residual glycolytic formation of ATP, which is then converted to sugar phosphates. Mg2+-(Ca2+)-activated ATPase (EC 3.6.1.3) is known to convert respiratory energy to ATP (8). Therefore, on inhibition of this ATPase by its specific inhibitor DCCD (13) or by the use of an uncA mutant defective in Mg2+-(Ca2+)-ATPase (8), phosphate metabolism of cells can be examined in the absence of oxidative phosphorylation. If the sugar phosphates are formed in the presence of colicin K from ATP generated by oxidative phosphorylation, their formation should be prevented by inhibiting Mg2+-(Ca2+)ATPase or using uncA cells. Conversely, if colicin K stimulates anaerobic phosphorylation while blocking oxidative phosphorylation, inhibition or a defect of Mg2+-(Ca2+)-ATPase should have no effect on the formation of sugar phosphates by colicin K. Table 6 shows that the addition of colicin K to DCCD-treated cells did not result in accumulation of sugar phosphates. The amount of DCCD used to treat cells was assumed to specifically inhibit Mg2+-(Ca2+)-ATPase (13). DCCD alone severely inhibited 32P, incorporation, indicating that 32p, is incorporated mostly via oxidative phosphorylation in washed cells. [14C]proline uptake was inhibited by colicin K in DCCDtreated cells (data not shown), showing that colicin K exerted its effect on DCCD-treated cells. Table 7 shows that colicin K treatment did not result in formation of sugar phosphates in the Mg2+-(Ca2+)-ATPase-less mutant AN120, originally isolated by Butlin and others (8). The decrease in 32P incorporation into the cold trichloroacetic acid-insoluble fraction shows that colicin K affects AN120. 32P, incorporation into nucleotide was decreased to a third in the cells grown in a half-strength Tris-glucose mineral medium supplemented with glucose and TABLE 6. Effect of colicin K on Cells

Control cells DCCD-treated cells

slightly increased in the cells grown in nutrient broth supplemented with glucose. Kopecky et al. (24) showed that the amount of ATP in colicin K-treated AN120 cells decreased to a third in low KCl medium and increased slightly in high KCI medium, and these results seem to be comparable with our results. Since colicin K is known to destroy the permeability barrier of potassium ion (19, 46), it is likely that in low potassium medium the drainage of potassium ions from cells treated with colicin K inhibited glycolytic ATP synthesis, whereas in high potassium medium the potassium concentration is high enough to sustain glycolytic ATP synthesis. Increased formation of 32P-labeled sugar phosphates and decreased 32P incorporation into nucleotide was observed when AN180, an isogenic uncA strain (8), was treated with colicin K both in a half-strength Tris-glucose mineral medium supplemented with glucose and in nutrient broth supplemented with glucose. The above results provide strong evidence for the conclusion that the ATP used for the formation of sugar phosphates in the presence of colicin K was supplied by oxidative phosphorylation and not solely by the stimulation of glycolytic ATP formation. Further support for this conclusion was obtained in the following experiment. For the results shown in Fig. 3, cells were preincubated with colicin K (17 KU/cell) at 30°C for 6 min and then with 32p, for 5 min, and then CICCP was added. On the addition of CICCP, the formation of sugar phosphates stopped immediately, but the nucleotide level was not significantly affected (data not shown). It is known that colicin K requires energy for exerting its primary effect, and CICCP inhibits this initial reaction (23, 32). However, the incubation of cells with colicin K (17 KU/cell) for 11 min at 30°C is considered to be sufficient time for colicin K to exert its irreversible lethal effect on the cells (33), and therefore, subsequent addition of CICCP should not prevent colicin K

32P, incorporation by DCCD-treated cellsa

Addition

None + Colicin K (56 ,ug/ml)

Cold trichloroacetic acid-soluble fractionb Cold trichloroacetic acid-insoluble fracNon-nucleotionb Nucleotide tide

7.50 1.93

0.44 5.06

5.11 0.95

None 2.20 0.96 0.93 + Colicin K (56 ,tg/ml) 1.62 1.38 0.71 a Washed cells of strain MK-1 were treated with DCCD as described under Materials and Methods and incubated with 1 ,uCi of 32p, in 0.5 Mimol of potassium phosphate, pH 7.6, for 20 min. b Nanomoles of phosphate incorporated per 109 cells per 20 min.

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SUGAR PHOSPHATES IN COLICIN K-TREATED CELLS

TABLE 7. Effect ofcolicin K on 32P, incorporation by Mg21-(Ca2+)-ATPase-less mutant AN120 cellsa Expt 1

2

Cold trichloroace- Cold tritic fractaddiolu etic acidAddition Nucleo- Non-nu- insoluble tide cleotide fraction" 21.4 13.3 7.0 Nonec + Colicin K (56 4.2 3.1 5.4 Jg/ml)

acid-solublechoa-

9.3 10.8

Nonec + Colicin K (56

15.4 15.8

6.6 3.2

AgIm1) In experiment 1, a 1-ml culture of AN120 growing in half-strength Tris-glucose mineral medium (see Materials and Methods) was incubated with 1 iLCi of "P, for 20 min. In experiment 2, a 1-ml culture of AN120 growing in nutrient broth (see Materials and Methods) was incubated with 2 jaCi of2P, for 20 min. ° Nanomoles of 3P incorporated per 109 cells per 20 min. c Correction was made for cell growth. I

15 IL~~~~~~~~~

~

@z b~ ~/ IL

::13 / US0

I

6

5

-s--16

15

TIME (( MIN)

FIG. 3. Effect of ClCCP on 3TP, incorporation into the trichloroacetic acid-soluble non-nucleotide organic phosphate fraction in colicin K-treated cells. Cells of strain MK-1 were prepared and tested for 32Mp incorporation as described in Fig. 1. Symbols: A, colicin K (50 pg/ml) added 6 min before 32pJ (control); V, colicin K added 6 min before, and CICCP (2 x 10-5 M) added 5 min after (arrow), 32pb O, CJCCP (2 x 10-5 M) added 6 min before 32Pi

from exerting its effect. Thus, the inhibition of sugar phosphate formation by CICCP in Fig. 3 should be the result of inhibition of oxidative

phosphorylation by CICCP. DISCUSSION The results obtained in this work indicate that, in colicin K-treated cells, sugar phosphates are formed both in cells washed with deionized water and suspended in buffer and in cells that are growing. This sugar phosphate formation was independent of the regulatory mechanism exerted by stringent control of RNA synthesis and was observed only when

237

oxidative phosphorylation was active. Accumulation of trichloroacetic acid-soluble intermediates in the presence of colicins K and El was first observed by Fields and Luria (15) in cells cultured with [14C]glucose. They reported that a considerable amount of the accumulated compounds was susceptible to alkaline phosphatase, and some internediates of glycolysis were excreted. We found, in the present work, that the main 32P-labeled product accumulated in colicin K-treated cells of strain B1 and MK-1 was S7P, an intermediate of the pentose phosphate shunt (20), and the 32P-labeled product formed in strain CP78 was FDP. About 40 to 50% of the trichloroacetic acidsoluble non-nucleotide organic phosphate compounds was excreted from colicin K-treated cells. It is difficult to provide a definite explanation from the present study for the mechanism of the increased sugar phosphate formation in colicin K-treated cells. However, one can speculate that the increase in 32P incorporation into sugar monophosphates is due to the result of the increased phosphorylation of glucose by 32p_ labeled phosphoenolpyruvate via the phosphoenolpyruvate:hexose phosphotransferase system. This interpretation is supported by the observation of Jetten (22) that colicin K or El treatment increased phosphorylation of amethylglucoside in wild-type cells but did not increase in enzyme I-less mutant. Labeling of the phosphate moiety of phosphoenolpyruvate requires [-y-nPIATP-linked phosphorylation catalyzed by phosphofructokinase; this activity is insensitive to allosteric inhibition by ATP in aerobically grown E. coli K-12 cells (43). The amount of phosphofructokinase is low in aerobically grown cells (43), and the amount of accumulated F6P and G6P seems to have exceeded the catalytic activity of phosphofructokinase in colicin K-treated cells. These compounds, therefore, tend to flow to the pentose phosphate shunt and thus contribute to the accumulation of S7P. The accumulation of sugar monophosphate, S7P in particular, may also reflect the effect of colicin K on synthetic processes. The effect of colicin K on cytoplasmic membranes may result in the inhibition of lipopolysaccharide synthesis, which would lead to an accumulation of S7P, its precursor (11). The inhibition of protein synthesis should lead to inhibition of de novo synthesis of aromatic amino acids from erythrose 4-phosphate. Strain CP78 was the only exception among the strains tested: it produced FDP as the main product in colicin Ktreated cells. This may be due to a reduced ability of CP78 to produce S7P; this interpretation would be consistent with the suggestion

238

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TAKAGAKI ET AL.

that there is a correlation between the amount of lipopolysaccharide and the lambda receptor (35), which is lacking in this strain. The experiments with DCCD-treated washed cells and the addition of ClCCP to washed cells damaged irreversibly by colicin K showed that oxidative phosphorylation is necessary for the increased formation of sugar phosphates in colicin K-treated cells. This absolute requirement for oxidative phosphorylation was confirmed by the fact that the Mg2+-(Ca2+)-ATPase-less mutant (uncA) cells, which are adapted to supply all the required ATP by glycolysis, did not produce the increased level of sugar phosphates in response to colicin K. The formation of sugar phosphates in colicin K-treated cells is therefore clearly dependent on oxidative phosphorylation. Although it is clear that oxidative phosphorylation is operative in colicin K-treated cells, the possibility that it is partly inhibited had to be considered. However, on the basis of the following results, we would like to postulate that oxidative phosphorylation is not significantly inhibited by colicin K. Hirata et al. (19) reported data which showed that oxidative phosphorylation in vitro in a cell-free system was not affected by colicin K. We confirmed this result in our preliminary experiments by using a cytoplasmic membrane system prepared essentially according to Mizushima and Yamada (29). Oxidative phosphorylation in this system was sensitive to CICCP but was not affected by colicin K (data not shown). Although P/O ratios in these experiments were low (0.15), this evidence supports our speculation that oxidative phosphorylation is not significantly inhibited by colicin K treatment. The increased formation of sugar phosphates in colicin K-treated cells is dependent on oxidative phosphorylation at the cytoplasmic membrane, so that we favor the idea that this fornation reflects the action of colicin K on the membrane of the cells. One possible explanation is that ATP formed at the cytoplasmic membrane is somehow concentrated at or near the membrane in aerobically growing cells; this ATP is released into the cytoplasm by the change of membrane conformation due to the action of colicin K and becomes accessible to phosphofructokinase. ACKNOWLEDGMENTS We are grateful to B. Maruo for valuable discussions. We also thank G. E. Gerber for the critical reading of the manuscript, M. Futai for providing strains AN180 and AN120, Y. Nishikawa for providing strain B1, and K. Kunugita for preparing the purified colicin K. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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