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Regulation of gene expression in Escherichia coli by the local anesthetic procaine
J. Mol.
Hiol.
(1982)
160, 3633367
LETTERS TO THE EDITOR
Regulation
of Gene Expression in Escherichia by the Local Anesthetic Procaine
Procaine trratment of phoA-lac, mall%lnc and ompF-lac results in reduced fl-galactosidase levels. suggesting transcription of these target genes.
colz’
operon fusion strains that procaine inhibits
Local anesthetics have been shown to perturb the translation of exported proteins (Lazdunski & Pages, 1979) and the synthesis and assembly of outer membrane proteins (Halegoua & Inouye, 1979: Pugsley et al., 1980) in Escherichia coli. W:e previously found that growth of E. coli using maltose as sole carbon source was particularly sensitive to inhibition by 20 mM-procaine. In contrast, cells grew on lactose plus procaine at nearly normal rates. The poor growth on maltose was not due to interference with the sugar transport process itself, but rather resulted from inhibition of t)he induction of new transport activity (Granett & Villarejo, 1981). Maltose transport activity is specified by the malB operon at 90 minutes on the E. coli chromosome; the malA operon at 75 minutes encodes the enzymes involved in maltose utilization, amylomaltase and maltodextrin phosphorylase (Debarbouille & Schwartz, 1979). In our experiments, maltose transport and amylomaltase were subject to co-ordinate inhibition by procaine, suggesting that procaine might act on the rnal regulon at the level of gene expression. In agreement with other workers, we found that procaine also blocks the appearance of the outer of alkaline membrane ompF protein (Pugsley et al., 1980) and induction phosphatase in response to phosphate limitation (Tribhuwan & Pradhan, 1977). The specific action of procaine on these gene products could be due to interference with transcription, translation or post-translational processing and translocation. Since operon fusion strains provide a powerful tool for probing transcriptional control (Bassford et al., 1980), we have examined drug effects on gene expression using strains in which normal lac operon structural genes are under the control of the malE, ompF, ompC or phoA promoters. In these strains. figalactosidase activity reflects transcription initiation at the relevant promoter. The operon fusion strains were isolated as described by Casadaban (1976). mall3 is a structural gene in the W&B region (Debarbouille & Schwartz, 1979). In the malE-1a.c operon fusion strain (PB179-23), p-galactosidase activity was induced by addition of 0.20/b (w/v) maltose. In the phoA-lac operon fusion strain (As.2), bgalactosidase was induced by transferring cells to phosphate-limited medium buffered wit’h 0.1 M-Tris. Both strains were provided by P. Bassford in an MC4100 background. ompF and ompC proteins are produced constitutively. The ompF-la,c operon fusion strain (MH513) and ompC-lac (MH225) were also derived from MC4100 and were provided by T. Silhavy. Cultures were grown in a rotary shaker at 37°C in medium 63 (Herzenberg, 1959), supplemented with 1 pg of thiamine per 363 (~22~283e/82/26036:c-O~
$03.00/O
80 1982
Academic
Press
Inc.
(London)
Ltd.
364
S. (:KANETT
ANT) M.
Time
\‘11,1,AREJCf
(hl
PI<:. 1. Inhibition of phoA-/w induction by procainr. (‘ells of the @A-/nc oprrw fusion straiu growing exponentially in M6-glycwol vvt-r harvrstrd 1)~ cwtrifugation and washed free of phosphate. At zwo time. wlls wew transferred to phosphate-frer medium to induce phoA cccpression. At hourly intervals. cell samples wrw withdrawn. toluene-lysed and assayed for /T-galactosidase activity. Phosphate-f&r medium was supplcmrnted with procaine as indicatckd. (0) C”ontrol : ( x ) 10 ITIMprocaine: (0) 15 rnn-prowim; (A) 20 mwprwaine.
ml and @40/, (w/v) glycerol. Media supplemented with procaine.H(‘l was adjusted to pH 7 after drug addition and then autoclaved. Procaine hydrochloride was purchased from Chemical Dynamics Corp., South Plainfield. h’J. /SGalaatosidase activity was measured in toluenized cells (Horiuchi et al., 1962) as the rate of onit’ropheny-p-n-galactopyranoside hydrolysis (Villarejo & Ping, 1978). A unit’ is defined as one micromole of o-nitrophenyl-b-r)-galactopyranoside hydrolyzed per minute. Protein content of whole cells was estimated from cult,ure turbidit,y using a conversion factor of A6a0 = 1 equivalent to 0.23 mg cellular protein per ml of culture. determined by Lowry protein analysis (Lowry et al.. 1951). The effect of procaine on the induction of ,Cgalac:tosidase activity in phoA-lac fusion strains in response to phosphate starvation is presented in Figure 1. Following exposure of the cells to 20 mM-procaine for four hours. /3-galactosidase induction was only 300;;, of the control level. The extent of inhibition of induction was proportional to the concentration of drug used. This action of procaine on phoA gene expression is similar t,o the effect of the drug on the appearance of the authentic gene product, alkaline phosphatase, as described by Tribhuwan K: Pradham (1977). fi-Galactosidase induct’ion w-as essentially complete aft,er one hour in the control culture, but continued at a slow rate in the procaine-treated cells. Procaine also inhibited the induction of fi-galactosidase activity in response to maltose addition in the rnwlE-2nc operon fusion strain (Fig. 2). (‘ells actively growing on glycaerol medium were induced for rrmlE-lac expression by, addition of’ maltose simultaneous wit,h t’he addition of procaine. The extent of p-galactosidase induction is shown in Figure 2 as a function of cell growth for three concentrations of procaine. The induction of mtclE was blocked completely for one t,o two hours in
LETTERS
TO THE
Absorbance
EDITOR
at 600
365
nm
FIG. 2. Inhibition of mall--/nc induction by procaine. Cells of the mrc/IWoc operon fusion strain growing exponentially in M63-glycerol were harvested and transferred to the same medium containing in addition procaine and @2~/, maltose to induce malE: expression. fl-Galactosidase activity was measured on cell samples from control and procaine-supplemented cultures during a 4-h induction period. (0) Control: ( x ) 10 mu-procaine: (0) 1.5 tnM-procaine: (A) 20 mwprocaine.
the presence of 15 or 20 mM-procaine and proceeded slowly thereafter. The block to ma2E expression was not due t’o a general toxicity or inhibition of overall protein synthesis, since cells had accomplished one complete doubling in this period. The specific arrest of malE expression agrees with our previous observation of a block to induction of new maltose transport activity and supports the hypothesis that, procaine inhibits rnac! regulon gene expression. The complete inhibition of ompF-lac expression by procaine is shown in Figure 3(a). fi-Galactosidase activity per ml of culture did not increase following drug addition, illustrating that synthesis of this gene product has ceased despite overall cell growth. In the control cells, continued expression of ompF was demonstrated by increased P-galactosidase levels. The inhibition of ompF-dac expression by procaine supports our earlier observation from pulse-labeling experiments that, procaine blocked appearance of om,pF protein in the membrane (Granett & Villarejo, 1981). In a parallel experiment with an ompC-lac operon fusion strain (Fig. 3(b)), the drug did not inhibit p-galactosidase expression. Again, this observation supports previous pulse-labeling data showing that procaine did not interfere with synthesis of ompC protein (Granett & Villarejo, 1981). Figures 1 to 3 illustrate regulation of expression of phoA, malE and ompF by the local anesthetic procaine. The specificity of the regulation is illustrated by normal ompC-lac expression in the presence of procaine. An additional factor in these experiments was the effect of procaine on the growth rate of the parent strain, MC4100, in M63-glycerol medium. The growth rate of MC4100 was reduced by about 50% in the presence of 20 mw-procaine, whereas Hfr3000, the strain used in our previous experiments, was inhibit)ed by only 14% under the same conditions. 12
366 0.35-(o)
xi ; f 2 ‘; \ UI c c .z zc .’
0.30
-
0.25
-
0.20
-
0.15
-
Absorbance
at 600
nm
Despite the reduction in growth rate, t,he specifics effect’s of the drug on t,he malE-lac and omlpF-lac fusions can be identified by comparing the /Sgalactosidase levels in treated and untreated cells at compamble levels of growth (= equivalent .4600). This approach could not be used with $0~4. since there was negligible growth under conditions of phosphat’e limitation. However. the agreement between the effects on the operon fusions in the MC4100 background and those on the authentic gene products in Hfr3000 supports the validity of t,he operon fusion data. Inhibition of p-galactosidase expression by procaine in three independently isolated operon fusions suggests that procaine blocks transcription of these genes. However. t.he kc control region has been part.ially alt,ered during construction of the fusion strains and the la& ribosome binding sites may also be affected. This distinction is worth considering because the structure of the messenger RNA being translated can regulate t’he efficiency of translation (Tserentant 8 Fiers, 1980). If procaine caused a differential rate of translation of the altered rnRN-4 in the operon fusions, it should also inhibit ompC’-/
LETTERS We fusion
thank Drs strains.
1’. ,J. Hassford
Department of Biochemistry [Tnirersity of California Davis. CA 95616. I’.S.A. Received
8 IIecember
1981,
TO
Jr and
and
and
THE
EDITOR
T. ,J. Silhavy
for
367 generously
providing
the
operon
Biophysics
in revised
form
12 May
1982
REFERENCES Bassford. I’.. Beckwith, J.. Berman, M.. Brickman. E.. Casadaban. M., Guarente, L., SaintGirons. I.. Sarthy, A., Schwartz, M.. Shuman. H. & Silhavy. T. (1980). In TOP Operon (Miller. ,J. & Rrznikoff, W. S.. eds). pp. 245-262, Cold Spring Harbor Laboratory Press. Cold Spring Harbor. Casadaban. M. (1976). J. Mol. Biol. 194, 541-555. Debarbouille. M. & Schwartz. M. (1979). .I. Mol. Rio/. 132, 521-534. Granett. S. 8r Villarejo. M. (1981). J. Bactrriol. 147, 289-296. Halegoua, S. Ri Inouye, M. (1979). J. &lo/. Biol. 130, 39-61. Herzenberg, I,. ,4. (1959). Biochim. Biophys. Acta. 31, 525-539. Horiuchi. T.. Tomizawa, ,J. & Novick, A. (1962). Biochiwt. Biophys. Actn, 55, 152-163. lserentant. D. & Fiers. W. (1980). Gene. 9. I-12. I,azdunski, C. & Pagks, J. (1979). Eur. J. B&hem. 96, 49-57. Lowry. 0. H.. Rosebrough. N. J . . Farr. A. I,. & Randall. R. J. (1951). J. Biol. Chem. 193. 265-275. ? * Pugsley. A. I’., Conrad, II. J., Schnaitman, C. A. & Gregg. T. I. (1980). Bioch,im. Biophys. dcfa. 599. I- 12. Tribhuwan, R. C. & Pradhan. D. S. (1977). J. Bacteriol. 131, 431-437. Villarejo. 1cI. & Ping. C. (1978). Biochem. Rioph,ys. Res. Commun. 82, 935-942.
Edited
by S. Rrenner
Hiol.
(1982)
160, 3633367
LETTERS TO THE EDITOR
Regulation
of Gene Expression in Escherichia by the Local Anesthetic Procaine
Procaine trratment of phoA-lac, mall%lnc and ompF-lac results in reduced fl-galactosidase levels. suggesting transcription of these target genes.
colz’
operon fusion strains that procaine inhibits
Local anesthetics have been shown to perturb the translation of exported proteins (Lazdunski & Pages, 1979) and the synthesis and assembly of outer membrane proteins (Halegoua & Inouye, 1979: Pugsley et al., 1980) in Escherichia coli. W:e previously found that growth of E. coli using maltose as sole carbon source was particularly sensitive to inhibition by 20 mM-procaine. In contrast, cells grew on lactose plus procaine at nearly normal rates. The poor growth on maltose was not due to interference with the sugar transport process itself, but rather resulted from inhibition of t)he induction of new transport activity (Granett & Villarejo, 1981). Maltose transport activity is specified by the malB operon at 90 minutes on the E. coli chromosome; the malA operon at 75 minutes encodes the enzymes involved in maltose utilization, amylomaltase and maltodextrin phosphorylase (Debarbouille & Schwartz, 1979). In our experiments, maltose transport and amylomaltase were subject to co-ordinate inhibition by procaine, suggesting that procaine might act on the rnal regulon at the level of gene expression. In agreement with other workers, we found that procaine also blocks the appearance of the outer of alkaline membrane ompF protein (Pugsley et al., 1980) and induction phosphatase in response to phosphate limitation (Tribhuwan & Pradhan, 1977). The specific action of procaine on these gene products could be due to interference with transcription, translation or post-translational processing and translocation. Since operon fusion strains provide a powerful tool for probing transcriptional control (Bassford et al., 1980), we have examined drug effects on gene expression using strains in which normal lac operon structural genes are under the control of the malE, ompF, ompC or phoA promoters. In these strains. figalactosidase activity reflects transcription initiation at the relevant promoter. The operon fusion strains were isolated as described by Casadaban (1976). mall3 is a structural gene in the W&B region (Debarbouille & Schwartz, 1979). In the malE-1a.c operon fusion strain (PB179-23), p-galactosidase activity was induced by addition of 0.20/b (w/v) maltose. In the phoA-lac operon fusion strain (As.2), bgalactosidase was induced by transferring cells to phosphate-limited medium buffered wit’h 0.1 M-Tris. Both strains were provided by P. Bassford in an MC4100 background. ompF and ompC proteins are produced constitutively. The ompF-la,c operon fusion strain (MH513) and ompC-lac (MH225) were also derived from MC4100 and were provided by T. Silhavy. Cultures were grown in a rotary shaker at 37°C in medium 63 (Herzenberg, 1959), supplemented with 1 pg of thiamine per 363 (~22~283e/82/26036:c-O~
$03.00/O
80 1982
Academic
Press
Inc.
(London)
Ltd.
364
S. (:KANETT
ANT) M.
Time
\‘11,1,AREJCf
(hl
PI<:. 1. Inhibition of phoA-/w induction by procainr. (‘ells of the @A-/nc oprrw fusion straiu growing exponentially in M6-glycwol vvt-r harvrstrd 1)~ cwtrifugation and washed free of phosphate. At zwo time. wlls wew transferred to phosphate-frer medium to induce phoA cccpression. At hourly intervals. cell samples wrw withdrawn. toluene-lysed and assayed for /T-galactosidase activity. Phosphate-f&r medium was supplcmrnted with procaine as indicatckd. (0) C”ontrol : ( x ) 10 ITIMprocaine: (0) 15 rnn-prowim; (A) 20 mwprwaine.
ml and @40/, (w/v) glycerol. Media supplemented with procaine.H(‘l was adjusted to pH 7 after drug addition and then autoclaved. Procaine hydrochloride was purchased from Chemical Dynamics Corp., South Plainfield. h’J. /SGalaatosidase activity was measured in toluenized cells (Horiuchi et al., 1962) as the rate of onit’ropheny-p-n-galactopyranoside hydrolysis (Villarejo & Ping, 1978). A unit’ is defined as one micromole of o-nitrophenyl-b-r)-galactopyranoside hydrolyzed per minute. Protein content of whole cells was estimated from cult,ure turbidit,y using a conversion factor of A6a0 = 1 equivalent to 0.23 mg cellular protein per ml of culture. determined by Lowry protein analysis (Lowry et al.. 1951). The effect of procaine on the induction of ,Cgalac:tosidase activity in phoA-lac fusion strains in response to phosphate starvation is presented in Figure 1. Following exposure of the cells to 20 mM-procaine for four hours. /3-galactosidase induction was only 300;;, of the control level. The extent of inhibition of induction was proportional to the concentration of drug used. This action of procaine on phoA gene expression is similar t,o the effect of the drug on the appearance of the authentic gene product, alkaline phosphatase, as described by Tribhuwan K: Pradham (1977). fi-Galactosidase induct’ion w-as essentially complete aft,er one hour in the control culture, but continued at a slow rate in the procaine-treated cells. Procaine also inhibited the induction of fi-galactosidase activity in response to maltose addition in the rnwlE-2nc operon fusion strain (Fig. 2). (‘ells actively growing on glycaerol medium were induced for rrmlE-lac expression by, addition of’ maltose simultaneous wit,h t’he addition of procaine. The extent of p-galactosidase induction is shown in Figure 2 as a function of cell growth for three concentrations of procaine. The induction of mtclE was blocked completely for one t,o two hours in
LETTERS
TO THE
Absorbance
EDITOR
at 600
365
nm
FIG. 2. Inhibition of mall--/nc induction by procaine. Cells of the mrc/IWoc operon fusion strain growing exponentially in M63-glycerol were harvested and transferred to the same medium containing in addition procaine and @2~/, maltose to induce malE: expression. fl-Galactosidase activity was measured on cell samples from control and procaine-supplemented cultures during a 4-h induction period. (0) Control: ( x ) 10 mu-procaine: (0) 1.5 tnM-procaine: (A) 20 mwprocaine.
the presence of 15 or 20 mM-procaine and proceeded slowly thereafter. The block to ma2E expression was not due t’o a general toxicity or inhibition of overall protein synthesis, since cells had accomplished one complete doubling in this period. The specific arrest of malE expression agrees with our previous observation of a block to induction of new maltose transport activity and supports the hypothesis that, procaine inhibits rnac! regulon gene expression. The complete inhibition of ompF-lac expression by procaine is shown in Figure 3(a). fi-Galactosidase activity per ml of culture did not increase following drug addition, illustrating that synthesis of this gene product has ceased despite overall cell growth. In the control cells, continued expression of ompF was demonstrated by increased P-galactosidase levels. The inhibition of ompF-dac expression by procaine supports our earlier observation from pulse-labeling experiments that, procaine blocked appearance of om,pF protein in the membrane (Granett & Villarejo, 1981). In a parallel experiment with an ompC-lac operon fusion strain (Fig. 3(b)), the drug did not inhibit p-galactosidase expression. Again, this observation supports previous pulse-labeling data showing that procaine did not interfere with synthesis of ompC protein (Granett & Villarejo, 1981). Figures 1 to 3 illustrate regulation of expression of phoA, malE and ompF by the local anesthetic procaine. The specificity of the regulation is illustrated by normal ompC-lac expression in the presence of procaine. An additional factor in these experiments was the effect of procaine on the growth rate of the parent strain, MC4100, in M63-glycerol medium. The growth rate of MC4100 was reduced by about 50% in the presence of 20 mw-procaine, whereas Hfr3000, the strain used in our previous experiments, was inhibit)ed by only 14% under the same conditions. 12
366 0.35-(o)
xi ; f 2 ‘; \ UI c c .z zc .’
0.30
-
0.25
-
0.20
-
0.15
-
Absorbance
at 600
nm
Despite the reduction in growth rate, t,he specifics effect’s of the drug on t,he malE-lac and omlpF-lac fusions can be identified by comparing the /Sgalactosidase levels in treated and untreated cells at compamble levels of growth (= equivalent .4600). This approach could not be used with $0~4. since there was negligible growth under conditions of phosphat’e limitation. However. the agreement between the effects on the operon fusions in the MC4100 background and those on the authentic gene products in Hfr3000 supports the validity of t,he operon fusion data. Inhibition of p-galactosidase expression by procaine in three independently isolated operon fusions suggests that procaine blocks transcription of these genes. However. t.he kc control region has been part.ially alt,ered during construction of the fusion strains and the la& ribosome binding sites may also be affected. This distinction is worth considering because the structure of the messenger RNA being translated can regulate t’he efficiency of translation (Tserentant 8 Fiers, 1980). If procaine caused a differential rate of translation of the altered rnRN-4 in the operon fusions, it should also inhibit ompC’-/
LETTERS We fusion
thank Drs strains.
1’. ,J. Hassford
Department of Biochemistry [Tnirersity of California Davis. CA 95616. I’.S.A. Received
8 IIecember
1981,
TO
Jr and
and
and
THE
EDITOR
T. ,J. Silhavy
for
367 generously
providing
the
operon
Biophysics
in revised
form
12 May
1982
REFERENCES Bassford. I’.. Beckwith, J.. Berman, M.. Brickman. E.. Casadaban. M., Guarente, L., SaintGirons. I.. Sarthy, A., Schwartz, M.. Shuman. H. & Silhavy. T. (1980). In TOP Operon (Miller. ,J. & Rrznikoff, W. S.. eds). pp. 245-262, Cold Spring Harbor Laboratory Press. Cold Spring Harbor. Casadaban. M. (1976). J. Mol. Biol. 194, 541-555. Debarbouille. M. & Schwartz. M. (1979). .I. Mol. Rio/. 132, 521-534. Granett. S. 8r Villarejo. M. (1981). J. Bactrriol. 147, 289-296. Halegoua, S. Ri Inouye, M. (1979). J. &lo/. Biol. 130, 39-61. Herzenberg, I,. ,4. (1959). Biochim. Biophys. Acta. 31, 525-539. Horiuchi. T.. Tomizawa, ,J. & Novick, A. (1962). Biochiwt. Biophys. Actn, 55, 152-163. lserentant. D. & Fiers. W. (1980). Gene. 9. I-12. I,azdunski, C. & Pagks, J. (1979). Eur. J. B&hem. 96, 49-57. Lowry. 0. H.. Rosebrough. N. J . . Farr. A. I,. & Randall. R. J. (1951). J. Biol. Chem. 193. 265-275. ? * Pugsley. A. I’., Conrad, II. J., Schnaitman, C. A. & Gregg. T. I. (1980). Bioch,im. Biophys. dcfa. 599. I- 12. Tribhuwan, R. C. & Pradhan. D. S. (1977). J. Bacteriol. 131, 431-437. Villarejo. 1cI. & Ping. C. (1978). Biochem. Rioph,ys. Res. Commun. 82, 935-942.
Edited
by S. Rrenner