Vol. 174, No. 10
JOURNAL OF BACrERIOLOGY, May 1992, p. 3407-3410
0021-9193/92/103407-04$02.00/0 Copyright © 1992, American Society for Microbiology
Abnormal Fractionation of 3-Lactamase in Escherichia coli: Evidence for an Interaction with the Inner Membrane in the Absence of a Leader Peptide GREGORY A. BOWDEN, FRAN(OIS BANEYX, AND GEORGE GEORGIOU* Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712 Received 19 November 1991/Accepted 13 March 1992
Essentially all exported proteins in Escherichia coli are synthesized as precursors containing a leader peptide (22, 25, 28). The most important function of the leader peptide is probably to maintain the precursor in an export-competent conformation, a process which is facilitated by interactions with chaperonins. According to the kinetic competition model suggested by Pugsley (20) and elaborated by Hardy and Randall (10), protein precursors destined for secretion are able to bind to chaperonins, such as GroEL and SecB, partly because they exhibit slower folding kinetics. Cytoplasmic proteins, on the other hand, reach their native state much more rapidly and do not have an opportunity to enter the export pathway. Certain mature proteins expressed without a leader peptide and a few bona fide cytoplasmic polypeptides are found together with periplasmic components following cell fractionation by osmotic shock. Proteins which appear in the osmotic shock fraction in the absence of a leader peptide include Vitreoscilla hemoglobin, human proapolipoprotein, and mouse interleukin-13 expressed in E. coli and the native cytoplasmic proteins thioredoxin and elongation factor Tu (2, 11, 13, 23). Even though cell fractionation techniques, particularly osmotic shock, are prone to artifacts (18), it is conceivable that the unexpected fractionation behavior of the above proteins stems from their atypical localization within the cell. For example, there is evidence that thioredoxin is found predominantly in adhesion sites between the inner and outer membranes (2). In this report, we show that P-lactamase expressed without a functional leader peptide is found associated with the cytoplasmic membrane and the periplasmic fraction, provided that the cells are grown under conditions which prevent "trapping" of the protein within cytoplasmic inclusion bodies. These results suggest that certain mature secreted proteins are able to interact with the secretory apparatus at a low efficiency even in the absence of a leader peptide. Release of A(-20,-1) 13-lactamase by osmotic shock. Plas*
Corresponding author.
mid pGB1 is a pBR322 derivative containing a Neor gene, a deletion of the -20 to -1 region of the f-lactamase leader peptide [A(-20,-1) 13-lactamasel, and a tac promoter upstream of the P-lactamase gene (5). Cultures of E. coli RB791 (lacI9) transformed with pGB1 were grown in minimal medium, and the synthesis of ,-lactamase was induced by the addition of different concentrations of isopropyl-p-thiogalactoside (IPTG), as described previously (5). The cells were harvested following overnight growth, fractionated by cold osmotic shock using the procedure of Nossal and Heppel (17), and then lysed by passage through a French press. 3-Lactamase in the osmotic shock fraction and in the lysed cells was measured as described previously (24). The sum of activities obtained after cell fractionation agreed, within 10%, with the values determined from assays of total-cell lysates. In all experiments, the release of ,B-galactosidase activity (15) into the osmotic shock fluid was very low, indicating negligible cell lysis (Table 1). The efficiency of cell fractionation was estimated from the release of the periplasmic enzyme marker cyclic phosphodiesterase (9). Growth at 37°C results in extensive aggregation and the formation of inclusion bodies in the cytoplasm of E. coli (5). Surprisingly, in cultures induced with IPTG, part of the ,-lactamase activity appeared in the osmotic shock fluid. Growth at 23°C and induction with a lower concentration of IPTG (0.05 mM) favor the formation of the soluble, enzymatically active protein (Table 1 and Fig. 1) (4, 7). Under these conditions, osmotic shock resulted in quantitative release of A(-20,-1) P-lactamase from the cells. The same percentage (88 + 5) of the total A(-20,-1) ,B-lactamase activity was released by osmotic shock from cells harvested 2, 4, 6, and 8 h after induction with IPTG. Finally, similar results were observed with other E. coli strains such as MC4100 (3) and SF103 (la) (data not shown). A(-20,-1) ,-lactamase was isolated from the osmotic shock fluid of cells grown at 23°C by immobilized zinc affinity chromatography on a chelating Sepharose 6B column (1). The N-terminal sequence was Met-Arg-Ile-, followed by the amino acids of the mature 3-lactamase, as expected from 3407
Downloaded from http://jb.asm.org/ on January 18, 2019 by guest
13-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [A(-20,-1) 13-lactamasel was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of A(-20,-1) ,-lactamase conferred a 25-fold increase in the 50%o lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature 13-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.
3408
NOTES
J. BACT1ERIOL. TABLE 1. Production and distribuion of soluble A(-20,-1) ,B-Lactamase (mean ±SEM)
IPTG concn (mM)
Total activity (U/ml)
23°C
0 0.05 0.1
10 0.8 125 + 1.6 43 0.7
% Total activity in osmotic shock fraction (mean ±
% Release into osmotic shock fluid 37°C
0.1 0.02 10 ± 0.4 6 0.3
P-lactamase in RB791(pGB1)a SEM)
3-Galactosidase
Cyclic phosphodiesterase
23°C
37°C
23°C
37°C
23°C
37°C
13.1 2 85.7 ± 0.6 91.9 2.1
0 35 ± 2.6 71.5 1.6
73.5 1.5 74.5 + 3.5 74 4
NDb 47.6 ± 2.1 57.2 ± 3.2
0 3
0.2 ± 0.08
3.6 ± 1.1
3.3 ± 0.6
0
a Cultures induced with the IPTG concentrations shown and grown overnight at 23 or 37°C were fractionated by osmotic shock. ND, not determined.
b
Purified B-lac.
[IPTGJ:
-0.05 mM- -0.05 mMFIG. 1. Distribution of P-lactamase in RB791(pGB1) cells fractionated by osmotic shock. The cells were grown overnight at 23 and 37°C and induced with the indicated IPTG concentrations. Samples corresponding to the same volume of culture were loaded on a sodium dodecyl sulfate-15% polyacrylamide gel and detected by Western blotting. P, osmotic shock supernatant; C, cytoplasmic fraction; I, insoluble fraction. f-lac., j-lactamase. -0-
into the spheroplast supernatant could have been tightly bound to the external surface of the cytoplasmic membrane. When whole spheroplasts were incubated in isotonic buffer (100 mM Tris-HCl [pH 7.0], 10 mM MgCl2, 0.3 M sucrose) containing 50 ,ig of penicillin G per ml, we detected 5.1 U of I-lactamase activity per ml of culture, which corresponds to almost 30% of the total activity in cell lysates (18.1 ± 2.0 U/ml of culture). Since penicillin G is excluded from the cytoplasm, the ,-lactamase activity in intact spheroplasts must arise from protein which is bound to the external side of the cytoplasmic membrane and therefore is exposed to the external fluid. The subcellular localization of A(-20,-1) 1-lactamase was probed further by determining its accessibility to proteolytic digestion following incubation with 100 ,ug of proteinase K or trypsin per ml. The reactions were stopped at different times by the addition of 10 mM phenylmethylsulfonyl fluoride or 200 p.g of soybean trypsin inhibitor per ml, respectively. P-Lactamase activity was measured in intact and lysed spheroplasts, and subsequently the samples were loaded on 15% polyacrylamide gels (14). In control experiments, the protease and inhibitor were added simultaneously at time zero. Treatment of intact spheroplasts with proteinase K resulted in a very rapid decrease of 3-lactamase activity (Fig. 2). This was surprising because the P-lactamase activity that could be detected only in lysed spheroplasts must be located within the cytoplasm where it should be protected from protease digestion by the presence of the cytoplasmic membrane. Microscopic examination of the proteinase K-treated cells did not reveal any cell lysis. The amount of the A(-20,-1) P-lactamase in the soluble fraction of spheroplasts that had been treated with proteinase K was monitored by Western blotting (immunoblotting) (27). Even after 15 min of incubation with the protease, at which point no enzymatic activity remained, the intensity of the ,-lactamase band was reduced by no more than 30% (Fig. 2A). No proteolytic fragments or change in the mobility of the remaining A(-20,-1) 1-lactamase band could be detected. Digestion of intact spheroplasts with trypsin gave results very similar to the proteinase K experiments (Fig. 2B). The above observations further underline the aberrant cellular localization of A(-20,-1) 13-lactamase in E. coli. The fact that the protein is completely inactivated in spheroplasts incubated with proteases indicates that at least some amino acid segment must be exposed to the external side of the cytoplasmic membrane and thus becomes susceptible to degradation. Previous studies have shown that small deletions at the amino-terminal end of the mature ,B-lactamase reduce the enzymatic activity by almost 1,000-fold (19). Consequently, cleavage of a few amino acids could cause
Downloaded from http://jb.asm.org/ on January 18, 2019 by guest
the DNA sequence, indicating that the protein in the osmotic shock supernatant had not been subjected to N-terminal processing. Cell fractionation by spheroplasting and proteinase K accessibility. The osmotic shock procedure can induce membrane alterations which cause the artifactual release of certain cytoplasmic proteins from the cell (18). Therefore, to determine the true location of A(-20,-1) ,-lactamase more precisely, we examined the distribution of I-lactamase activity in spheroplasts and its accessibility to externally added proteases. Cultures grown at 23°C with 0.05 mM IPTG were harvested in late exponential phase (2 h after the addition of inducer) and converted to spheroplasts as described earlier (4, 16). Microscopic observation of the cells and the distribution of the periplasmic protein acid phosphatase (pH 2.5) (8) indicated that more than 85% of the cells had been converted to spheroplasts. In addition, less than 2% of the total activity of the cytoplasmic marker enzymes lactate dehydrogenase and 3-galactosidase (15) was found in the spheroplast supernatant. The total 3-lactamase activity in cell lysates was 18.1 ± 2.0 U/ml of culture. Of this, 2.5 U/ml of culture, i.e., 13.5% of the total activity, was found in the spheroplast supernatant, whereas 15 U/ml of culture could be detected only after the spheroplasts had been lysed in a French press. Thus, the total activity recovered from the supernatant and spheroplast fractions (17.5 U/ml) is equal, within experimental error, to the value obtained in whole-cell lysates. Part of the A(-20, -1) 1-lactamase which was not released
VOL. 174, 1992
NOTES
IN
I
s
Iaw ttSpb_mla
LydSphd
us
0 27* 0.5
p
de
IN
Strain
0 5 10 15 (4* (i)_-0 5min 2.4t 1.0
76
4
(% ofat
TABLE 2. Ampicillin LD50 for different strains grown at 23°C with and without IPTG
+ PK
*PK
A.
a
0.5
mused
3409
10.mi 0 * 0.5 au.b) 1.5
15mi 0
RB791(pGB1) RB791(pGAPz) MC4100(pGB1)
KB4(pGB1)
Ampicillin LD50 (,ug/ml) With 0.05 mM Without IPTG IPTG
JOURNAL OF BACrERIOLOGY, May 1992, p. 3407-3410
0021-9193/92/103407-04$02.00/0 Copyright © 1992, American Society for Microbiology
Abnormal Fractionation of 3-Lactamase in Escherichia coli: Evidence for an Interaction with the Inner Membrane in the Absence of a Leader Peptide GREGORY A. BOWDEN, FRAN(OIS BANEYX, AND GEORGE GEORGIOU* Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712 Received 19 November 1991/Accepted 13 March 1992
Essentially all exported proteins in Escherichia coli are synthesized as precursors containing a leader peptide (22, 25, 28). The most important function of the leader peptide is probably to maintain the precursor in an export-competent conformation, a process which is facilitated by interactions with chaperonins. According to the kinetic competition model suggested by Pugsley (20) and elaborated by Hardy and Randall (10), protein precursors destined for secretion are able to bind to chaperonins, such as GroEL and SecB, partly because they exhibit slower folding kinetics. Cytoplasmic proteins, on the other hand, reach their native state much more rapidly and do not have an opportunity to enter the export pathway. Certain mature proteins expressed without a leader peptide and a few bona fide cytoplasmic polypeptides are found together with periplasmic components following cell fractionation by osmotic shock. Proteins which appear in the osmotic shock fraction in the absence of a leader peptide include Vitreoscilla hemoglobin, human proapolipoprotein, and mouse interleukin-13 expressed in E. coli and the native cytoplasmic proteins thioredoxin and elongation factor Tu (2, 11, 13, 23). Even though cell fractionation techniques, particularly osmotic shock, are prone to artifacts (18), it is conceivable that the unexpected fractionation behavior of the above proteins stems from their atypical localization within the cell. For example, there is evidence that thioredoxin is found predominantly in adhesion sites between the inner and outer membranes (2). In this report, we show that P-lactamase expressed without a functional leader peptide is found associated with the cytoplasmic membrane and the periplasmic fraction, provided that the cells are grown under conditions which prevent "trapping" of the protein within cytoplasmic inclusion bodies. These results suggest that certain mature secreted proteins are able to interact with the secretory apparatus at a low efficiency even in the absence of a leader peptide. Release of A(-20,-1) 13-lactamase by osmotic shock. Plas*
Corresponding author.
mid pGB1 is a pBR322 derivative containing a Neor gene, a deletion of the -20 to -1 region of the f-lactamase leader peptide [A(-20,-1) 13-lactamasel, and a tac promoter upstream of the P-lactamase gene (5). Cultures of E. coli RB791 (lacI9) transformed with pGB1 were grown in minimal medium, and the synthesis of ,-lactamase was induced by the addition of different concentrations of isopropyl-p-thiogalactoside (IPTG), as described previously (5). The cells were harvested following overnight growth, fractionated by cold osmotic shock using the procedure of Nossal and Heppel (17), and then lysed by passage through a French press. 3-Lactamase in the osmotic shock fraction and in the lysed cells was measured as described previously (24). The sum of activities obtained after cell fractionation agreed, within 10%, with the values determined from assays of total-cell lysates. In all experiments, the release of ,B-galactosidase activity (15) into the osmotic shock fluid was very low, indicating negligible cell lysis (Table 1). The efficiency of cell fractionation was estimated from the release of the periplasmic enzyme marker cyclic phosphodiesterase (9). Growth at 37°C results in extensive aggregation and the formation of inclusion bodies in the cytoplasm of E. coli (5). Surprisingly, in cultures induced with IPTG, part of the ,-lactamase activity appeared in the osmotic shock fluid. Growth at 23°C and induction with a lower concentration of IPTG (0.05 mM) favor the formation of the soluble, enzymatically active protein (Table 1 and Fig. 1) (4, 7). Under these conditions, osmotic shock resulted in quantitative release of A(-20,-1) P-lactamase from the cells. The same percentage (88 + 5) of the total A(-20,-1) ,B-lactamase activity was released by osmotic shock from cells harvested 2, 4, 6, and 8 h after induction with IPTG. Finally, similar results were observed with other E. coli strains such as MC4100 (3) and SF103 (la) (data not shown). A(-20,-1) ,-lactamase was isolated from the osmotic shock fluid of cells grown at 23°C by immobilized zinc affinity chromatography on a chelating Sepharose 6B column (1). The N-terminal sequence was Met-Arg-Ile-, followed by the amino acids of the mature 3-lactamase, as expected from 3407
Downloaded from http://jb.asm.org/ on January 18, 2019 by guest
13-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [A(-20,-1) 13-lactamasel was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of A(-20,-1) ,-lactamase conferred a 25-fold increase in the 50%o lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature 13-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.
3408
NOTES
J. BACT1ERIOL. TABLE 1. Production and distribuion of soluble A(-20,-1) ,B-Lactamase (mean ±SEM)
IPTG concn (mM)
Total activity (U/ml)
23°C
0 0.05 0.1
10 0.8 125 + 1.6 43 0.7
% Total activity in osmotic shock fraction (mean ±
% Release into osmotic shock fluid 37°C
0.1 0.02 10 ± 0.4 6 0.3
P-lactamase in RB791(pGB1)a SEM)
3-Galactosidase
Cyclic phosphodiesterase
23°C
37°C
23°C
37°C
23°C
37°C
13.1 2 85.7 ± 0.6 91.9 2.1
0 35 ± 2.6 71.5 1.6
73.5 1.5 74.5 + 3.5 74 4
NDb 47.6 ± 2.1 57.2 ± 3.2
0 3
0.2 ± 0.08
3.6 ± 1.1
3.3 ± 0.6
0
a Cultures induced with the IPTG concentrations shown and grown overnight at 23 or 37°C were fractionated by osmotic shock. ND, not determined.
b
Purified B-lac.
[IPTGJ:
-0.05 mM- -0.05 mMFIG. 1. Distribution of P-lactamase in RB791(pGB1) cells fractionated by osmotic shock. The cells were grown overnight at 23 and 37°C and induced with the indicated IPTG concentrations. Samples corresponding to the same volume of culture were loaded on a sodium dodecyl sulfate-15% polyacrylamide gel and detected by Western blotting. P, osmotic shock supernatant; C, cytoplasmic fraction; I, insoluble fraction. f-lac., j-lactamase. -0-
into the spheroplast supernatant could have been tightly bound to the external surface of the cytoplasmic membrane. When whole spheroplasts were incubated in isotonic buffer (100 mM Tris-HCl [pH 7.0], 10 mM MgCl2, 0.3 M sucrose) containing 50 ,ig of penicillin G per ml, we detected 5.1 U of I-lactamase activity per ml of culture, which corresponds to almost 30% of the total activity in cell lysates (18.1 ± 2.0 U/ml of culture). Since penicillin G is excluded from the cytoplasm, the ,-lactamase activity in intact spheroplasts must arise from protein which is bound to the external side of the cytoplasmic membrane and therefore is exposed to the external fluid. The subcellular localization of A(-20,-1) 1-lactamase was probed further by determining its accessibility to proteolytic digestion following incubation with 100 ,ug of proteinase K or trypsin per ml. The reactions were stopped at different times by the addition of 10 mM phenylmethylsulfonyl fluoride or 200 p.g of soybean trypsin inhibitor per ml, respectively. P-Lactamase activity was measured in intact and lysed spheroplasts, and subsequently the samples were loaded on 15% polyacrylamide gels (14). In control experiments, the protease and inhibitor were added simultaneously at time zero. Treatment of intact spheroplasts with proteinase K resulted in a very rapid decrease of 3-lactamase activity (Fig. 2). This was surprising because the P-lactamase activity that could be detected only in lysed spheroplasts must be located within the cytoplasm where it should be protected from protease digestion by the presence of the cytoplasmic membrane. Microscopic examination of the proteinase K-treated cells did not reveal any cell lysis. The amount of the A(-20,-1) P-lactamase in the soluble fraction of spheroplasts that had been treated with proteinase K was monitored by Western blotting (immunoblotting) (27). Even after 15 min of incubation with the protease, at which point no enzymatic activity remained, the intensity of the ,-lactamase band was reduced by no more than 30% (Fig. 2A). No proteolytic fragments or change in the mobility of the remaining A(-20,-1) 1-lactamase band could be detected. Digestion of intact spheroplasts with trypsin gave results very similar to the proteinase K experiments (Fig. 2B). The above observations further underline the aberrant cellular localization of A(-20,-1) 13-lactamase in E. coli. The fact that the protein is completely inactivated in spheroplasts incubated with proteases indicates that at least some amino acid segment must be exposed to the external side of the cytoplasmic membrane and thus becomes susceptible to degradation. Previous studies have shown that small deletions at the amino-terminal end of the mature ,B-lactamase reduce the enzymatic activity by almost 1,000-fold (19). Consequently, cleavage of a few amino acids could cause
Downloaded from http://jb.asm.org/ on January 18, 2019 by guest
the DNA sequence, indicating that the protein in the osmotic shock supernatant had not been subjected to N-terminal processing. Cell fractionation by spheroplasting and proteinase K accessibility. The osmotic shock procedure can induce membrane alterations which cause the artifactual release of certain cytoplasmic proteins from the cell (18). Therefore, to determine the true location of A(-20,-1) ,-lactamase more precisely, we examined the distribution of I-lactamase activity in spheroplasts and its accessibility to externally added proteases. Cultures grown at 23°C with 0.05 mM IPTG were harvested in late exponential phase (2 h after the addition of inducer) and converted to spheroplasts as described earlier (4, 16). Microscopic observation of the cells and the distribution of the periplasmic protein acid phosphatase (pH 2.5) (8) indicated that more than 85% of the cells had been converted to spheroplasts. In addition, less than 2% of the total activity of the cytoplasmic marker enzymes lactate dehydrogenase and 3-galactosidase (15) was found in the spheroplast supernatant. The total 3-lactamase activity in cell lysates was 18.1 ± 2.0 U/ml of culture. Of this, 2.5 U/ml of culture, i.e., 13.5% of the total activity, was found in the spheroplast supernatant, whereas 15 U/ml of culture could be detected only after the spheroplasts had been lysed in a French press. Thus, the total activity recovered from the supernatant and spheroplast fractions (17.5 U/ml) is equal, within experimental error, to the value obtained in whole-cell lysates. Part of the A(-20, -1) 1-lactamase which was not released
VOL. 174, 1992
NOTES
IN
I
s
Iaw ttSpb_mla
LydSphd
us
0 27* 0.5
p
de
IN
Strain
0 5 10 15 (4* (i)_-0 5min 2.4t 1.0
76
4
(% ofat
TABLE 2. Ampicillin LD50 for different strains grown at 23°C with and without IPTG
+ PK
*PK
A.
a
0.5
mused
3409
10.mi 0 * 0.5 au.b) 1.5
15mi 0
RB791(pGB1) RB791(pGAPz) MC4100(pGB1)
KB4(pGB1)
Ampicillin LD50 (,ug/ml) With 0.05 mM Without IPTG IPTG
