Vol. 137, No. 1
JouRNAL OF BACTZRIOLOGY, Jan. 1979, p. 340-349
0021-9193/79/01-0340/10$02.00/0
Isolation and Characterization of an Endogenous Inhibitor of Protein Synthesis in Escherichia coli K-12 VIRGINIA L. CLARK Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received for publication 27 July 1978
A low-molecular-weight factor was isolated from cell extracts of Escherichia coli K-12. The concentration of the factor in cells was dependent upon nutritional conditions, the concentration being higher in faster growing cells. Treatment of cells with colicin K caused an increase in concentration of the factor. The factor inhibited protein synthesis in E. coli. This inhibition was reversible, apparently because of metabolism of the factor. The inhibition of synthesis of ,B-galactosidase lasted longer than the inhibition of protein synthesis; cyclic AMP eliminated this difference. The factor inhibited the synthesis of /8-galactosidase from preformed lac mRNA, indicating an inhibition of translation. Kinetic studies of the onset of inhibition of ,B-galactosidase synthesis by the factor suggested that the factor may inhibit protein synthesis at the initiation of translation. When Jacob and Monod (10) presented their model of the operon, they postulated that the regulation of gene expression occurred at the level of initiation of transcription. For the lac operon, the repressor binds to the operator sequence of the DNA and prevents transcription; the repressor can be removed by the addition of inducer and transcription proceeds. This simple version of gene regulation was subsequently modified by the discovery (17, 20) of the requirement for cyclic AMP and cyclic AMP-binding protein for optimal levels of transcription. This regulation is also at the level of initiation of transcription. Recently (3, 4, 11, 12), another type of regulation of expression of the tryptophan operon has been described. This regulation, called attenuation, involves the premature termination of transcription. Although there has been little concrete evidence for regulation at the translational level in procaryotes, there may be post-transcriptional control of,B-galactosidase synthesis (8), and current investigations of the bacteriophages T4 (28) and Tl (30) indicate that they shut off host protein synthesis by acting at the level of initiation of translation. I wish to report the isolation from Escherichia coli K-12 of a low-molecular-weight substance that inhibits protein synthesis. Total protein synthesis is shut off; the synthesis of fl-galactosidase is somewhat more sensitive to inhibition than total protein synthesis. The inhibition is reversible, suggesting that the factor is metabolized by the cell. Using a system devised to study effects on translation in vivo (28), I have concluded that the factor probably inhibits protein 340
synthesis at the level of the initiation of translation. This compound appears not to be the same as that described by Ullman et al. (7, 29), but may be similar to microcins, a class of compounds recently discovered in Enterobacteriaceae (1, 2). MATERIALS AND METHODS Materials. Trypsin, soybean trypsin inhibitor, ribonuclease, ribonuclease-free deoxyribonuclease (DNase), and lysozyme were purchased from Worthington Biochemical Corp. Adenosine-3',5'-cyclic monophosphoric acid (cyclic AMP), o-nitrophenyl-f6-D-galactopyranoside (ONPG), isopropyl-,B-D-thiogalactopyranoside (IPTG), DL-7-azatryptophan, 5-methylDL-tryptophan, chloramphenicol, and rifampin were obtained from Sigma Chemical Corp. [U-'4C]-D-glucose, [5-3H]uridine, [U-_4C]-L-proline, and Omnifluor were purchased from New England Nuclear Corp. All other chemicals used were of reagent grade purity. The strong cation exchange resin, AG 50W, and the strong anion exchange resin, AG 1-X8, were obtained from Bio-Rad Laboratories. The activated alumina (type F-20, 80 to 200 mesh) was purchased from Matheson, Coleman and Bell Manufacturing Chemists, and the 3MM chromatography paper was from Whatman, Ltd. Strains and growth media. The strains of Escherichia coli K-12 used in this study were G6 (Hfr thi his) and 3291A (Hfr thi thy reA) from the Luria culture collection. Strain CA8005 UV5 (thi L37 UV5 lac promoter mutant) was a gift from Bonnie Tyler. Cells were grown aerobically at 27°C in the minimal salts medium previously described (19), supplemented with 0.4% glucose, glycerol, L-lactate, or succinate, 1 ,ug of thiamine per ml, and 100 ,g of histidine per ml for strain G6 or 10 ,ug of thymine per ml for strain 3291A. Growth was determined by using a Klett-Sum-
VOL. 137, 1979
ENDOGENOUS INHIBITOR OF PROTEIN SYNTHESIS
merson colorimeter with a no. 54 filter. Protein concentrations were determined by the method of Lowry et al. (13), using bovine serum albumin as a standard. Colicin K studies. The colicin K used in this study was purified as previously described (22). Viability assays, colicin multiplicity, and rates of protein synthesis after colicin treatment were determined as previously described (21, 23). ,B-Galactosidase assay. ,B-Galactosidase activity was assayed in cells treated with toluene ahd sodium dodecyl sulfate (18), measuring hydrolysis of 0.003 M ONPG at 300C. Protein and RNA synthesis determinations. Protein and RNA syntheses were measured by the incorporation of [14C]proline (0.2 mM; 1 ,uCi/ml) or [3H]uridine (0.2 mM; 10 ACi/ml), respectively, into trichloroacetic acid-insoluble material. Samples were removed at various times after the addition of the radioactive label and were added to equal volumes of cold 10% trichloroacetic acid. The trichloroacetic acid precipitates were collected on membrane filters (Millipore Corp., type HA, 0.45 jm, 25 mm) and washed with trichloroacetic acid and water. The filters were dried, and the radioactivity was determined after the addition of a toluene-based scintillation fluid. Biological assay of factor concentration. The determination of the amount of factor in an extract was complicated by the fact that the cells recover from the inhibition by factor (see Results). It qas decided therefore, to include the recovery period in the biological assay procedure. One unit of factor was defined as the amount that gives 50% inhibition of induction of ,8-galactosidase over the 20-min time period chosen arbitrarily for the assay. In the titration assay, each sample is tested at a range of dilutions. The assay was carried out as follows. Various volumes of the factor preparation were added to 6, x 108 cells in a final volume of 3 ml, and the cells were induced for ,Bgalactosidase synthesis for 20 min. The percent inhibition of induction was plotted as a function of the volume of factor added, and the amount of the extract required to, cause 50% inhibition of induction was determined from the graph-and defined as onie unit of factor activity. I estimate that one unit of factor is
approximately 10-7 g/ml. Partial purification of factor. Filtrate containing factor was prepared as follows. Cells at a density of 5 x 108 cells/ml were filtered (Millipore Corp., type HA, 0.45 ,um, 142 mm), washed with complete medium, and suspended in 0.02 to 0.2 times the original volume of complete medium. If the cells were concentrated more than tenfold, the culture was aerated with oxygen. Colicin K was added (multiplicity of about 10) and the cells were incubated at 27°C for 40 min. Tho cells were removed by Millipore filtration, and the filtrate was boiled (100°C, 10 min) to destroy the colicin. Factor was isolated from the cells as follows. The
cells were filtered, washed, suspended, and treated with colicin K as described for the preparations of filtrate. After 40 min of colicin K treatment, the factor was'extracte4 by the addition of cold 100% trichloro-
acetic acid to a final concentration of 10%. The trichloroacetic acid extract was chilled, and the precipitate was removed by centrifugation (10 min, 12,000 x g). The trichloroacetic acid was removed by ether
341
extraction. Further purification of factor from either the filtrate or the cells was as follows. Absolute ethanol was added to the crude preparation to a final concentration of 90%. The solution was chilled, and the precipitate was removed by centrifugation (10 min, 12,000 x g). The supernatant was concentrated to 2 to 4 ml by evaporation and applied to an activated alumina column (1.5 by 30 cm) that had been poured dry. The material was washed into the column with 30 ml of absolute ethanol and eluted with an ethanol-water gradient. The fractions containing factor were pooled, concentrated by evaporation, and spotted on Whatman 3 MM paper. The factor was subjected to descending paper chromatography with an Nbutanol/ethanol/water (40:11:19) (vol/vol/vol) solvent. The paper was dried and cut into 1-cm strips, and each strip was eluted with 1 ml of water. The fractions were assayed for biological activity. The factor migrated at an Rf of 0.3 to 0.4. This purification scheme typically gives approximately 50% recovery of the biological activity and, when ['4C]glucose was used as a carbon source, a 50to 100-fold increase in the specific activity (units of factor per disintegrations per minute) as compared to the crude trichloroacetic acid extract.
RESULTS An inhibitor of ,-galactosidase induction present in colicin K-treated E. coli cultures. In a study of the effects of colicin K on macromolecular synthesis in E. coli K-12, it was observed (C. A. Plate and J. Suit, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, K186, p. 178) that the ability to induce 8-galactosidase synthesis appeared to be more severely inhibited than was protein synthesis (Fig. 1). This rapid loss of enzyme induction could represent an early event in colicin K action, occurring after binding of the colicin but before the death of the cell. Alternatively, it could represent a response of the surviving cells in the culture to some material released from those cells that have been affected by colicin. To test the second alternative, I removed the colicin-treated cells by Millipore filtration and added the filtrate (boiled to destroy the colicin) to untreated cells. This filtrate prevented induction of ,B-galactosidase, the extent of inhibition being dependent upon the volume of filtrate added (Fig. 2). The release of the inhibitory factor into the filtrate is dependent upon the multiplicity of colicin, maximum release being achieved by a multiplicity of about 5 (1% survival). If cultures of colicin-tolerant mutants, which bind colicin but are not killed by it, are treated with colicin, there is no release of inhibitory material, indicating that the factor comes from colicin-killed celLs rather than from the colicin K preparation. The effects of addition of various colicins on the release of factor are summarized in Table 1. Colicins K and El, which kill the cell by dissi-
342
J. BACTERIOL.
CLARK
(see Materials and Methods), I found that measurable inhibitory activity was present in cells that had not been treated with colicin K. The addition of colicin K caused a 20-fold increase in the concentration of the factor as determined by titration, the maximum concentration being
1,0'
_ 1.0 E -j
SURVIVAL + TRYPSIN
4
0
z
IL. 0
CONTROL
z 0.8-
mI ~~~~~~~~~0.5
0
a
0.1
PROTEIN
I.0.6
SYNTHESIS
z
RATE
5w
4t
0.4
-
U-
0
(n~~~~~~~~~~~~~~~n
40.2 0
B -GALACTOSIDASE I NDUCTION
0.011
I
I
6 8 4 .10 12 2 MINUTES AFTER COLICIN K ADDITION FIG. 1. Effect of colicin K on survival, protein synthesis, and ,B-galactosidase activity. Colicin K was added at 0 min to strain G6 at 27°C. Measurements 0
of survival after trypsin treatment (0), incorporation of ['4Cglucose into protein (A), and ability to induce cells for /3-galactosidase activity (A) were performed as described in the text on samples removed at the indicated times.
pating the membrane potential (31) and causing a loss of active transport capability (9, 23), cause the release of factor into the filtrate. Colicin E2 kills cells by degrading DNA (24, 25), whereas colicin E3 kills cells by inhibiting protein synthesis by a specific degradation of ribosomal RNA (5). Neither E2 nor E3 causes the release of factor. Colicins K and El cause an increase in factor concentration. Since colicins K and El cause a loss of active transport, the appearance of the inhibitory factor in the filtrate might be due simply to efflux of the material after dissipation of the membrane potential. Alternatively, the colicin might cause an increase in concentration of the factor. By using extracts made either by boiling or by trichloroacetic acid extraction
30
60
m 90~~~1.
0 90 60 30 TIME (MIN) FIG. 2. Effect offiltrate on /3-galactosidase induction. A culture of strain G6 was treated with colicin K for 30 min at 27°C and Millipore filtered. The filtrate was boiled to destroy the colicin K and added
0
to a fresh culture of G6. The volume offiltrate added to 6 x 10i cells in a final volume of 3 ml was: 0 (0); 0.5 ml (0); 1.0 ml (A); 1.5 ml (A); and 2.0 ml (V). /3Galactosidase activity was determined as described in the text. TABLE 1. Effect of various colicins on inhibitory activity in the filtrate0 Filtrate source
Untreated cells .... Cells treated with:
Rate of,-galactosidase induction (U/min per ml of cells)
4.24
Colicin K .0.10 0.06 Colicin El .... .. Colicin E2 .4.18 4.46 Colicin E3 .... a Cultures of strain G6 were treated with sufficient colicin K, El, E2, or E3 to give 1% survival. The cultures were incubated at 27°C for 40 min, and cells were removed by Millipore filtration. The ifitrate was boiled to destroy the colicin, and equal volumes of filtrate were added to 6 x 10i cells, 2 mM IPTG was added, and induction of f-galactosidase was carried out for 20 min. The /B-galactosidase assay was performed as described in the text.
VOL. 137, 1979
ENDOGENOUS INHIBITOR OF PROTEIN SYNTHESIS
343
achieved by 40 min of colicin treatment at 270C. Extracts from colicin K-treated cells had about 20 10 times the inhibitory capability found in the CONTROL filtrate made from the same culture. Thus, the z-J ±CYCLIC AMP colicin caused both an increase in the intracel- 0 0 lular concentration of factor and the release of 4)E some of this factor into the medium. z E Partial purification of the factor. I have E° U. partially purified the factor either from the filtrate or from the cells of colicin-treated cultures Qo f / + FACTOR 6 by alumina and paper chromatography (see Ma- 0 -/ ± CYCLIC AMP terials and Methods). Radioactively labeled fac4 tor (prepared by treating glucose-grown cells o with colicin K in the presence of ['4C]glucose 0 3 and purified as described above) was bound to U z I I Dowex 50 (H+) and eluted as a single peak of of the All activity. radioactivity and biological I0 following experiments were performed by using wz CONTROL material purified by the alumina-paper chro8 +CYCLIC AMP 0 matography procedure. Effects of the factor on RNA, protein, and IE f8-galactosidase syntheses. I studied the efE 6 0 fects of factor on the syntheses of 8-galactosid4ase, total protein, and RNA as a function of time Z in +FA C TOR (Fig. 3). In strain G6, the factor inhibits protein 0 4 t~CYCLIC AMP and RNA syntheses as well as /8-galactosidase synthesis. The inhibition is spontaneously reversible and, after recovery, synthesis proceeds 3 at the same rate as in the control culture. If factor is added again to the recovered cells, g 12 a second period of inhibition is observed (Fig. 4). This suggests that the recovery from inhibition la is due to the metabolism of the factor and not to 0z either the induction of a mechanism to inacti8 vate the factor (in which case the second period of inhibition would be much shorter) or an al- < E teration in the target of the factor (in which case 6 there would be no second period of inhibition). PThe synthesis of fi-galactosidase recovers from inhibition at a later time than protein synthesis. The addition of cyclic AMP overcomes this de- e4-J 4t layed recovery (Fig. 3). Action of the factor in the reldxed strain 35 3291A. The strain G6 of E. coli K-12 used in the experiment described in Fig. 3 is a stringent strain. Stringency dictates that when protein 60 40 20 0 synthesis is inhibited by amino acid deprivation TIME (MIN) the synthesis of RNA also is inhibited (27). When I tested the effects of factor on the relaxed FIG. 3. Effects of factor on the synthesis of /3-gastrain, 3291A (Fig. 5), both f3-galactosidase and total protein, and RNA in strain G6. lactosidase, was there inhibited but were protein syntheses (0.2 mM, 1 ACi/ml), and no apparent inhibition of RNA synthesis. The Incorporation of ["Cjproline [3H]uridine (0.2 mM, 10 ttCi/ml) and assay of ,8inhibition of RNA synthesis in the stringent galactosidase performed as described in the strain G6, therefore, is probably an indirect ef- text. Cells werewere induced with IPTG at -20 min, and fect of the inhibition of protein synthesis. radioactive labels were added at 0 min. At 10 min Effects of factor concentration on the the following additions were made: no addition (0); syntheses of total protein and of ft-galac- 5 mM cyclic AMP (A); factor (5 U/mi) (0); and factor tosidase. Figure 6 shows the dependency of (5 U/ml) + 5 MM cyclic AMP (A). -
-
a
I
.
344
J. BACTrERIOL.
CLARK
-
o E en cn
a Z
(n D)
Ocy -
J
4~
CONTROL
W Z -J
20j
0
ar
a.
+ FA CTOR M
E E
10E
ov
a:
6
U
z
0
.
4-
80 -
~
ZW "OA-4 oZ EE20
CONTROL C NT 4Ltz
_
JouRNAL OF BACTZRIOLOGY, Jan. 1979, p. 340-349
0021-9193/79/01-0340/10$02.00/0
Isolation and Characterization of an Endogenous Inhibitor of Protein Synthesis in Escherichia coli K-12 VIRGINIA L. CLARK Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received for publication 27 July 1978
A low-molecular-weight factor was isolated from cell extracts of Escherichia coli K-12. The concentration of the factor in cells was dependent upon nutritional conditions, the concentration being higher in faster growing cells. Treatment of cells with colicin K caused an increase in concentration of the factor. The factor inhibited protein synthesis in E. coli. This inhibition was reversible, apparently because of metabolism of the factor. The inhibition of synthesis of ,B-galactosidase lasted longer than the inhibition of protein synthesis; cyclic AMP eliminated this difference. The factor inhibited the synthesis of /8-galactosidase from preformed lac mRNA, indicating an inhibition of translation. Kinetic studies of the onset of inhibition of ,B-galactosidase synthesis by the factor suggested that the factor may inhibit protein synthesis at the initiation of translation. When Jacob and Monod (10) presented their model of the operon, they postulated that the regulation of gene expression occurred at the level of initiation of transcription. For the lac operon, the repressor binds to the operator sequence of the DNA and prevents transcription; the repressor can be removed by the addition of inducer and transcription proceeds. This simple version of gene regulation was subsequently modified by the discovery (17, 20) of the requirement for cyclic AMP and cyclic AMP-binding protein for optimal levels of transcription. This regulation is also at the level of initiation of transcription. Recently (3, 4, 11, 12), another type of regulation of expression of the tryptophan operon has been described. This regulation, called attenuation, involves the premature termination of transcription. Although there has been little concrete evidence for regulation at the translational level in procaryotes, there may be post-transcriptional control of,B-galactosidase synthesis (8), and current investigations of the bacteriophages T4 (28) and Tl (30) indicate that they shut off host protein synthesis by acting at the level of initiation of translation. I wish to report the isolation from Escherichia coli K-12 of a low-molecular-weight substance that inhibits protein synthesis. Total protein synthesis is shut off; the synthesis of fl-galactosidase is somewhat more sensitive to inhibition than total protein synthesis. The inhibition is reversible, suggesting that the factor is metabolized by the cell. Using a system devised to study effects on translation in vivo (28), I have concluded that the factor probably inhibits protein 340
synthesis at the level of the initiation of translation. This compound appears not to be the same as that described by Ullman et al. (7, 29), but may be similar to microcins, a class of compounds recently discovered in Enterobacteriaceae (1, 2). MATERIALS AND METHODS Materials. Trypsin, soybean trypsin inhibitor, ribonuclease, ribonuclease-free deoxyribonuclease (DNase), and lysozyme were purchased from Worthington Biochemical Corp. Adenosine-3',5'-cyclic monophosphoric acid (cyclic AMP), o-nitrophenyl-f6-D-galactopyranoside (ONPG), isopropyl-,B-D-thiogalactopyranoside (IPTG), DL-7-azatryptophan, 5-methylDL-tryptophan, chloramphenicol, and rifampin were obtained from Sigma Chemical Corp. [U-'4C]-D-glucose, [5-3H]uridine, [U-_4C]-L-proline, and Omnifluor were purchased from New England Nuclear Corp. All other chemicals used were of reagent grade purity. The strong cation exchange resin, AG 50W, and the strong anion exchange resin, AG 1-X8, were obtained from Bio-Rad Laboratories. The activated alumina (type F-20, 80 to 200 mesh) was purchased from Matheson, Coleman and Bell Manufacturing Chemists, and the 3MM chromatography paper was from Whatman, Ltd. Strains and growth media. The strains of Escherichia coli K-12 used in this study were G6 (Hfr thi his) and 3291A (Hfr thi thy reA) from the Luria culture collection. Strain CA8005 UV5 (thi L37 UV5 lac promoter mutant) was a gift from Bonnie Tyler. Cells were grown aerobically at 27°C in the minimal salts medium previously described (19), supplemented with 0.4% glucose, glycerol, L-lactate, or succinate, 1 ,ug of thiamine per ml, and 100 ,g of histidine per ml for strain G6 or 10 ,ug of thymine per ml for strain 3291A. Growth was determined by using a Klett-Sum-
VOL. 137, 1979
ENDOGENOUS INHIBITOR OF PROTEIN SYNTHESIS
merson colorimeter with a no. 54 filter. Protein concentrations were determined by the method of Lowry et al. (13), using bovine serum albumin as a standard. Colicin K studies. The colicin K used in this study was purified as previously described (22). Viability assays, colicin multiplicity, and rates of protein synthesis after colicin treatment were determined as previously described (21, 23). ,B-Galactosidase assay. ,B-Galactosidase activity was assayed in cells treated with toluene ahd sodium dodecyl sulfate (18), measuring hydrolysis of 0.003 M ONPG at 300C. Protein and RNA synthesis determinations. Protein and RNA syntheses were measured by the incorporation of [14C]proline (0.2 mM; 1 ,uCi/ml) or [3H]uridine (0.2 mM; 10 ACi/ml), respectively, into trichloroacetic acid-insoluble material. Samples were removed at various times after the addition of the radioactive label and were added to equal volumes of cold 10% trichloroacetic acid. The trichloroacetic acid precipitates were collected on membrane filters (Millipore Corp., type HA, 0.45 jm, 25 mm) and washed with trichloroacetic acid and water. The filters were dried, and the radioactivity was determined after the addition of a toluene-based scintillation fluid. Biological assay of factor concentration. The determination of the amount of factor in an extract was complicated by the fact that the cells recover from the inhibition by factor (see Results). It qas decided therefore, to include the recovery period in the biological assay procedure. One unit of factor was defined as the amount that gives 50% inhibition of induction of ,8-galactosidase over the 20-min time period chosen arbitrarily for the assay. In the titration assay, each sample is tested at a range of dilutions. The assay was carried out as follows. Various volumes of the factor preparation were added to 6, x 108 cells in a final volume of 3 ml, and the cells were induced for ,Bgalactosidase synthesis for 20 min. The percent inhibition of induction was plotted as a function of the volume of factor added, and the amount of the extract required to, cause 50% inhibition of induction was determined from the graph-and defined as onie unit of factor activity. I estimate that one unit of factor is
approximately 10-7 g/ml. Partial purification of factor. Filtrate containing factor was prepared as follows. Cells at a density of 5 x 108 cells/ml were filtered (Millipore Corp., type HA, 0.45 ,um, 142 mm), washed with complete medium, and suspended in 0.02 to 0.2 times the original volume of complete medium. If the cells were concentrated more than tenfold, the culture was aerated with oxygen. Colicin K was added (multiplicity of about 10) and the cells were incubated at 27°C for 40 min. Tho cells were removed by Millipore filtration, and the filtrate was boiled (100°C, 10 min) to destroy the colicin. Factor was isolated from the cells as follows. The
cells were filtered, washed, suspended, and treated with colicin K as described for the preparations of filtrate. After 40 min of colicin K treatment, the factor was'extracte4 by the addition of cold 100% trichloro-
acetic acid to a final concentration of 10%. The trichloroacetic acid extract was chilled, and the precipitate was removed by centrifugation (10 min, 12,000 x g). The trichloroacetic acid was removed by ether
341
extraction. Further purification of factor from either the filtrate or the cells was as follows. Absolute ethanol was added to the crude preparation to a final concentration of 90%. The solution was chilled, and the precipitate was removed by centrifugation (10 min, 12,000 x g). The supernatant was concentrated to 2 to 4 ml by evaporation and applied to an activated alumina column (1.5 by 30 cm) that had been poured dry. The material was washed into the column with 30 ml of absolute ethanol and eluted with an ethanol-water gradient. The fractions containing factor were pooled, concentrated by evaporation, and spotted on Whatman 3 MM paper. The factor was subjected to descending paper chromatography with an Nbutanol/ethanol/water (40:11:19) (vol/vol/vol) solvent. The paper was dried and cut into 1-cm strips, and each strip was eluted with 1 ml of water. The fractions were assayed for biological activity. The factor migrated at an Rf of 0.3 to 0.4. This purification scheme typically gives approximately 50% recovery of the biological activity and, when ['4C]glucose was used as a carbon source, a 50to 100-fold increase in the specific activity (units of factor per disintegrations per minute) as compared to the crude trichloroacetic acid extract.
RESULTS An inhibitor of ,-galactosidase induction present in colicin K-treated E. coli cultures. In a study of the effects of colicin K on macromolecular synthesis in E. coli K-12, it was observed (C. A. Plate and J. Suit, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, K186, p. 178) that the ability to induce 8-galactosidase synthesis appeared to be more severely inhibited than was protein synthesis (Fig. 1). This rapid loss of enzyme induction could represent an early event in colicin K action, occurring after binding of the colicin but before the death of the cell. Alternatively, it could represent a response of the surviving cells in the culture to some material released from those cells that have been affected by colicin. To test the second alternative, I removed the colicin-treated cells by Millipore filtration and added the filtrate (boiled to destroy the colicin) to untreated cells. This filtrate prevented induction of ,B-galactosidase, the extent of inhibition being dependent upon the volume of filtrate added (Fig. 2). The release of the inhibitory factor into the filtrate is dependent upon the multiplicity of colicin, maximum release being achieved by a multiplicity of about 5 (1% survival). If cultures of colicin-tolerant mutants, which bind colicin but are not killed by it, are treated with colicin, there is no release of inhibitory material, indicating that the factor comes from colicin-killed celLs rather than from the colicin K preparation. The effects of addition of various colicins on the release of factor are summarized in Table 1. Colicins K and El, which kill the cell by dissi-
342
J. BACTERIOL.
CLARK
(see Materials and Methods), I found that measurable inhibitory activity was present in cells that had not been treated with colicin K. The addition of colicin K caused a 20-fold increase in the concentration of the factor as determined by titration, the maximum concentration being
1,0'
_ 1.0 E -j
SURVIVAL + TRYPSIN
4
0
z
IL. 0
CONTROL
z 0.8-
mI ~~~~~~~~~0.5
0
a
0.1
PROTEIN
I.0.6
SYNTHESIS
z
RATE
5w
4t
0.4
-
U-
0
(n~~~~~~~~~~~~~~~n
40.2 0
B -GALACTOSIDASE I NDUCTION
0.011
I
I
6 8 4 .10 12 2 MINUTES AFTER COLICIN K ADDITION FIG. 1. Effect of colicin K on survival, protein synthesis, and ,B-galactosidase activity. Colicin K was added at 0 min to strain G6 at 27°C. Measurements 0
of survival after trypsin treatment (0), incorporation of ['4Cglucose into protein (A), and ability to induce cells for /3-galactosidase activity (A) were performed as described in the text on samples removed at the indicated times.
pating the membrane potential (31) and causing a loss of active transport capability (9, 23), cause the release of factor into the filtrate. Colicin E2 kills cells by degrading DNA (24, 25), whereas colicin E3 kills cells by inhibiting protein synthesis by a specific degradation of ribosomal RNA (5). Neither E2 nor E3 causes the release of factor. Colicins K and El cause an increase in factor concentration. Since colicins K and El cause a loss of active transport, the appearance of the inhibitory factor in the filtrate might be due simply to efflux of the material after dissipation of the membrane potential. Alternatively, the colicin might cause an increase in concentration of the factor. By using extracts made either by boiling or by trichloroacetic acid extraction
30
60
m 90~~~1.
0 90 60 30 TIME (MIN) FIG. 2. Effect offiltrate on /3-galactosidase induction. A culture of strain G6 was treated with colicin K for 30 min at 27°C and Millipore filtered. The filtrate was boiled to destroy the colicin K and added
0
to a fresh culture of G6. The volume offiltrate added to 6 x 10i cells in a final volume of 3 ml was: 0 (0); 0.5 ml (0); 1.0 ml (A); 1.5 ml (A); and 2.0 ml (V). /3Galactosidase activity was determined as described in the text. TABLE 1. Effect of various colicins on inhibitory activity in the filtrate0 Filtrate source
Untreated cells .... Cells treated with:
Rate of,-galactosidase induction (U/min per ml of cells)
4.24
Colicin K .0.10 0.06 Colicin El .... .. Colicin E2 .4.18 4.46 Colicin E3 .... a Cultures of strain G6 were treated with sufficient colicin K, El, E2, or E3 to give 1% survival. The cultures were incubated at 27°C for 40 min, and cells were removed by Millipore filtration. The ifitrate was boiled to destroy the colicin, and equal volumes of filtrate were added to 6 x 10i cells, 2 mM IPTG was added, and induction of f-galactosidase was carried out for 20 min. The /B-galactosidase assay was performed as described in the text.
VOL. 137, 1979
ENDOGENOUS INHIBITOR OF PROTEIN SYNTHESIS
343
achieved by 40 min of colicin treatment at 270C. Extracts from colicin K-treated cells had about 20 10 times the inhibitory capability found in the CONTROL filtrate made from the same culture. Thus, the z-J ±CYCLIC AMP colicin caused both an increase in the intracel- 0 0 lular concentration of factor and the release of 4)E some of this factor into the medium. z E Partial purification of the factor. I have E° U. partially purified the factor either from the filtrate or from the cells of colicin-treated cultures Qo f / + FACTOR 6 by alumina and paper chromatography (see Ma- 0 -/ ± CYCLIC AMP terials and Methods). Radioactively labeled fac4 tor (prepared by treating glucose-grown cells o with colicin K in the presence of ['4C]glucose 0 3 and purified as described above) was bound to U z I I Dowex 50 (H+) and eluted as a single peak of of the All activity. radioactivity and biological I0 following experiments were performed by using wz CONTROL material purified by the alumina-paper chro8 +CYCLIC AMP 0 matography procedure. Effects of the factor on RNA, protein, and IE f8-galactosidase syntheses. I studied the efE 6 0 fects of factor on the syntheses of 8-galactosid4ase, total protein, and RNA as a function of time Z in +FA C TOR (Fig. 3). In strain G6, the factor inhibits protein 0 4 t~CYCLIC AMP and RNA syntheses as well as /8-galactosidase synthesis. The inhibition is spontaneously reversible and, after recovery, synthesis proceeds 3 at the same rate as in the control culture. If factor is added again to the recovered cells, g 12 a second period of inhibition is observed (Fig. 4). This suggests that the recovery from inhibition la is due to the metabolism of the factor and not to 0z either the induction of a mechanism to inacti8 vate the factor (in which case the second period of inhibition would be much shorter) or an al- < E teration in the target of the factor (in which case 6 there would be no second period of inhibition). PThe synthesis of fi-galactosidase recovers from inhibition at a later time than protein synthesis. The addition of cyclic AMP overcomes this de- e4-J 4t layed recovery (Fig. 3). Action of the factor in the reldxed strain 35 3291A. The strain G6 of E. coli K-12 used in the experiment described in Fig. 3 is a stringent strain. Stringency dictates that when protein 60 40 20 0 synthesis is inhibited by amino acid deprivation TIME (MIN) the synthesis of RNA also is inhibited (27). When I tested the effects of factor on the relaxed FIG. 3. Effects of factor on the synthesis of /3-gastrain, 3291A (Fig. 5), both f3-galactosidase and total protein, and RNA in strain G6. lactosidase, was there inhibited but were protein syntheses (0.2 mM, 1 ACi/ml), and no apparent inhibition of RNA synthesis. The Incorporation of ["Cjproline [3H]uridine (0.2 mM, 10 ttCi/ml) and assay of ,8inhibition of RNA synthesis in the stringent galactosidase performed as described in the strain G6, therefore, is probably an indirect ef- text. Cells werewere induced with IPTG at -20 min, and fect of the inhibition of protein synthesis. radioactive labels were added at 0 min. At 10 min Effects of factor concentration on the the following additions were made: no addition (0); syntheses of total protein and of ft-galac- 5 mM cyclic AMP (A); factor (5 U/mi) (0); and factor tosidase. Figure 6 shows the dependency of (5 U/ml) + 5 MM cyclic AMP (A). -
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