Release of Periplasmic Enzymes and Other Physiological Effects of P-Lactamase Overproduction in Escherichia coli

George Georgiou” and Michael L. Shuler Department of Chemical Engineering, Cornell University, lthaca, New York 14853

David B. Wilson Section of Biochemistry, Molecular and Cell BiologK Cornell University, lthaca, New York 14853

Accepted for publication September 9, 1987

When Escherichia coli containing the plasmid ptac l l is induced with 10-4M isopropyl-p-thiogalactopyranoside (IPTG), 90% of the p-lactamase activity of an overnight culture is present in the medium. The high extracellular activity of p-lactamase does not result from cell lysis but from an increase in the permeability of the outer mem- brane. The excreting cells release several other periplas- mic enzymes into the extracellular f luid and are more sensitive to lysis by detergents. It was also shown that in these cells the level of two membrane proteins, OmpA and OmpC, is decreased. None of these phenomena were observed wi th the plasmid pDW17, which has a mutation in the tac promoter that reduces its activity to one fourth of the tac promoter.

INTRODUCTION

Escherichiu coli has been used extensively as the host or- ganism for the production of heterologous proteins, both in the laboratory and by industry. The widespread use of E. coEi results from the greater knowledge of its physiology and genetics compared to other organisms. This knowledge has been exploited to facilitate the cloning and high-level expres- sion of heterologous proteins. However, E. coli, as many other gram-negative organisms, is not normally capable of protein excretion i.e., the release of proteins into the extra- cellular medium. ’,’ Excretion of the target polypeptide is highly desirable from a biotechnological point of view for several reasons. First, it simplifies the recovery and purifica- tion of the product polypeptide. Second, the excreted product is much more resistant to proteolytic degradation since E . coli does not excrete proteases into the extracellular medium. Finally, excretion of the product is a prerequisite for the development of more efficient immobilized cell bioreactors .

* Present address: Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712.

Biotechnology and Bioengineering, Vol. 32, Pp. 741-748 (1988) 0 1988 John Wiley 81 Sons, Inc.

The inability of E . coli to excrete proteins results from the presence of an outer membrane that is not permeable to proteins. While proteins cannot normally transverse a lipid bilayer, there exists an elaborate mechanism for the transport of certain proteins across the inner membrane. The translo- cation of proteins across the inner membrane is called se- cretion. Secreted proteins are localized either in the outer membrane or the periplasmic space and perform a variety of important functions such as nutrient transport and os- m~regulation.~’~ Proteins that are destined for secretion are synthesized in precursor form; i.e., they contain an amino acid extension consisting of approximately 20-30 amino acids (the signal sequence) at their amino terminal.’ The precursor form of a protein is able to transverse the inner membrane of the cell envelope. Once on the other side, the signal sequence is cleaved, and the mature form of the protein folds to its native conformation. However, the presence of the outer membrane prevents the release of protein into the extracellular fluid. One way for a protein to be excreted is to increase the permeability of the outer membrane to allow the polypeptide to diffuse into the cul- ture medium.

There are a number of chemical and physical methods that can be used to increase the permeability of the outer mem- brane, including osmotic shock6 and heat treatment.’ These methods are difficult to reproduce on a large scale and often result in the release of undesired contaminants such as endo- toxins. Alternatively, the use of E. coli mutants with a de- fective membrane (“leaky has been suggested, but such mutants have not yet been exploited on a large scale. In this work we demonstrate that the permeability of the outer membrane can be increased so that periplasmic enzymes are released into the medium. The induction of synthesis of the periplasmic enzyme p-lactamase from the strong tuc promoter results in excretion of 90% of the active enzyme

CCC 0006-3592/88/060741-08$04.00

and partial excretion of other periplasmic proteins. Further we show that high-level synthesis of p-lactamase is a nec- essary condition for excretion. We have also characterized some aspects of the physiology of the cells and the effect of p-lactamase overproduction on the synthesis and export of two outer membrane proteins.

MATERIALS AND METHODS

Strains and Plasmids

The Escherichiu coli strains that were used in this work are listed in Table I. The plasmid ptac 11 was produced from pBR322 by inserting a 350 bp tac promoter between the EcoRI and the Hind111 sites and was obtained from J. H. Richards, Caltech." The plasmid pDW17 is described in the text. The plasmid pKN was constructed by: inserting a BamHI fragment containing the near gene from pNEO (Pharmacia) into the BamHI site of ptacll. '

- Growth and Fractionation of Cultures

Unless otherwise specified, cells were grown in M9 media (initial pH 7.0) supplemented with 0.2% fructose, 0.2% casein, acid hydrolysate, and 50 p g / m L ampicillin. P-Lactamase synthesis was induced by adding isopropyl- P-thiogalactopyranoside (IPTG) (final concentration of 10-4M) to growing cultures at 37°C when they reached an OD, of 0.45. After 24 h of growth, 5 mL cells were cen- tifruged at 10,OOOg for 10 min. The supernatant was saved, and the pellet was washed with 0.33M Tris-HC1 buffer (pH 6.5) and subjected to osmotic shock essentially as de- scribed by Neu and Heppel., After the final centrifugation, the cell pellet was resuspended in 3 mL of 50mM phos- phate buffer (pH 6.5) and broken by two passages through a French press at 20,000 psi. The lysates were centrifuged at 13,OOOg for 10 min to remove insoluble material.

Detergent Sensitivity

Approximately 3 h after induction 5-mL samples were centrifuged, washed once in 20mM Tris-HC1 buffer (pH 7.5), and resuspended in the same buffer to give an OD,,, near 1.0. SDS (0.1% final concentration) or Na-

Table I. Escherichia coli strains

Strain Description Source

RB791 lucl 4L 8 Ref. 10 MG1693 thyA715, h- GCSC 641 1 D403 MG1693 l a c - , F'(luclq, lacc) This study JM I09 recA, A(lac, p ro ) , endAl, Ref. 28

gyrA96, thi-1, hsdRI7, supE44, relAl , F'(traA36, proAB +, l a d q ,

zAM15

Iky207c 207c melBl, thi, l a d , phoS, phoT, J-C. Lazzaroni

D207c 207c, F'(lacIq, lac+) This study

deoxycholate (0.25%) was added, the cells were incubated at 37"C, and the decrease in optical density was monitored.

Enzyme Assays

The assay solution for p-lactamase contained 0.5 mg/mL penicillin G (potassium salt) in 50mM phosphate buffer, pH 6.5. The reaction was followed by measuring the de- crease in OD,,, at 25°C. A unit of activity was defined as 1 pmol penicillin G hydrolyzed/min. Under these condi- tions the specific activity of pure p-lactamase is 3550 U/mg protein. The activity of cyclic phosphodiesterase and glucose- 6-phosphate dehydrogenase were measured as described in references 11 and 12, respectively. Ribonuclease I was as- sayed essentially as described by Weigand and Rothfield' using [8-H3]-polyA (1.63 mCi/mg, Amersham Corp.) as the substrate. A unit of ribonuclease I corresponds to 1 p g polyA hydrolyzed/min. The activity of the acid phosphatase with a pH optimum at 4.5 was determined from the rate of hydrolysis of paranitrophenyl phosphate (pNPP) in 0.1M acetate buffer, pH 4.5, by monitoring the change in opti- cal density at 410 nm." The acid phosphatase with a pH optimum at 2.5 was assayed as described by Dassa and Boquet.13 A unit of either enzyme was defined as the amount causing an increase in OD,,, of 1.0 per 20 minutes. P-Galactosidase was assayed by measuring the rate of hy- drolysis of ONPG in z-buffer.', A unit of activity was de- fined as the amount of enzyme causing a AOD,,,/min of 0.001. Protein was determined by the method of Bradford. l5 Specific activities of enzymes in cultures were reported as total units of enzyme per milligram of total sol- uble protein, i.e., the sum of the protein in the supernatant and intracellular fractions.

Labeling of Cultures and lmmunoprecipitations

Cells were grown in minimal medium containing 0.032M NaH,PO,, 0.064M K,PO,, 0.02M (NH,),SO,, 3 x lO-,M MgSO, * 7H,O, 10-6M ZnCl,, 10-6M FeSO,, 0.2% fruc- tose, and 50 pg/mL ampicillin supplemented with 19 amino acids (all except methionine) at a final concentration of 50 pg/mL each. Synthesis of p-lactamase was induced with IPTG as before. Half an hour after induction, aliquots were diluted fivefold in fresh media and grown to mid- exponential phase. Cells were labeled with 20 pCi/mL

S-methionine (>600 Ci/mmol) for 20 s. The chase was initiated by adding 1 .O mg/mL final concentration of unla- beled methionine. Samples ( I .O mL) were withdrawn at the indicated times and injected into 110 p L 100% TCA at 0°C in Eppendorf centrifuge tubes to stop the processing of the precursors. The proteins were pelleted by centrifugation, washed with ethanol and then with ether, lyophilized, and fi- nally solubilized by boiling for 3 min in 150 pL 50mM of Tris-HC1 buffer, pH 8.0, containing 1 .O% SDS and 50mM dithiothreitol. Samples (20 pL) were reacted with antisera specific for OmpA, OmpC, or p-lactamase." The immune complexes that formed were precipitated with 30 pL of a 10% solution of heat-inactivated Staphylococcus uureus

35

742 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 32, SEPTEMBER 1988

cells in buffer containing 0.5% Triton, 0.5% SDS, 20mM Tris-HC1, pH 7.5, and 50mM NaCl. After 30 min incuba- tion at 37”C, the precipitates were centrifuged and washed twice with 0.01M Tris-HC1 buffer, pH 7.5, containing 0.02% SDS, 0.1OM NaCl, and 0.1 mM EDTA. The cells were finally resuspended in electrophoresis loading buffer and heated for 7 min at 95°C to dissociate the immune complexes.

Table 11. and P-galactosidase.a

Effects of induction on extracellular activity of p-lactamase

p-lactamase p-galactosidase

Total Total specific specific

Optical activity activity density at Excretion (U/mg total Excretion (U/mg total induction (%) protein) (%) protein)

Other Methods

Recombinant DNA techniques were carried out essentially as described by Maniatis et al.I8

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 12% gels as described.” For autoradiog- raphy, gels were treated with EnH3ance (New England Nu- clear) according to the manufacturer’s instructions, dried, and exposed to preflashed X-ray-sensitive film at - 70°C for 16-30 h.

RESULTS

When cells containing ptacll are induced with 10-4M IPTG during exponential growth, a high level of p-lactamase is synthesized. We have reported earlier that approximately 50% of the total p-lactamase precipitates within the periplas- mic space as inclusion bodies,” and the rest is present in soluble form. The insoluble 6-lactamase in inclusion bodies is completely inactive. In contrast, the soluble p-lactamase retains its full enzymatic activity (when the enzyme in the soluble fraction was purified to homogeneity as judged by SDS-PAGE its specific activity was approximately 3500 U/mg). The enzymatic activity was used to monitor the amount of soluble P-lactamase released in the growth medium. It was observed that after overnight growth of fully induced cultures containing the plasmid ptac 11, up to 97% of the total activity of p-lactamase was present in the culture supernatant. The specific activity and the percent- age of excretion of the soluble p-lactamase vary with the op- tical density of the culture at the time of induction (Table 11). Cultures induced in early exponential phase released 26% of the activity of the cytoplasmic enzyme P-galactosidase into the extracellular medium, suggesting that some cell lysis occurred. Induction in the mid-exponential phase gave the highest specific activity of p-lactamase, and only 7% of the 0-galactosidase activity was found in the medium. This indicates that the extracellular p-lactamase activity in cultures induced in the mid-exponential phase did not result from cell lysis. The presence of IPTG did not induce higher levels of P-galactosidase (Table 11). This phenomenon was shown to be strain specific. Both a higher basal level of /?-galactosidase and induction by IPTG were observed in other strains transformed with ptac 11.

Effects of Level of p-Lactamase Synthesis on Excretion

It was observed that plating of induced cultures on mini- mal plates containing IPTG results in cell killing. In order

uninduced 4 41 I .5 228 0.100 97 265 26 245 0.250 96 342 12 295 0.450 96 492 7 280 0.670 84 42 1 7 257 0.960 33 250 3.5 233 1.300 14 259 1 .O not determined

a RB791 (ptacll) was grown in M9 medium and induced at indicated OD,. Samples (5 mL) were collected 24 h after induction, centrifuged, washed once in 0.05M phosphate buffer, and treated as described in Ma- terials and Methods.

to understand the causes of p-lactamase excretion, IPTG- resistant mutants were selected by plating induced cells on minimal media containing 0.2% fructose, 50 pg/mL ampi- cillin, and 5 x 10-4M IPTG. Spontaneous mutants were isolated and grown in liquid media with and without IPTG. Several mutants that showed high levels of p-lactamase without significant excretion were isolated. One of these strains, D17, was used to prepare plasmid DNA (pDW17). Transformants containing plasmid pDW17 exhibited the mu- tant phenotype. Overnight cultures of pDW 17 containing cells induced with 10-4M IPTG produced 50% less p-lac- tamase activity than RB791 (ptacll), and SDS-PAGE of the insoluble fractions of RB791 (pDW17) cells indicated that they do not contain inclusion bodies. The lower level of p-lactamase synthesis suggested that the plasmid pDW17 might contain a mutation in the tuc promoter up- stream from the p-lactamase gene. To localize the muta- tion more precisely, different restriction fragments from pDW17 were ligated to the corresponding large fragments of pKN. The plasmids thus generated were transformed into RB79 1, and ampicillin-resistant transformants were selected. By measuring the level of excretion of p-lacta- mase in induced cells carrying these plasmids, it was shown that the mutation lies in the tuc promoter of pDW17. The EcoRI-Hind111 fragment of pDW17 was subcloned into an M13 single-stranded vector and sequenced. It was found that pDW17 contains a deletion of an adenine residue in the -35 region of the tuc promoter (i.e., the nucleotide in the -62 position of ptacll). These results demonstrate that the observed absence of excretion in cells transformed with pDW17 was caused by a down-promoter mutation. Fur- thermore, when cells containing ptac l l were partially in- duced by using a lower concentration of IPTG ((1.0 X

10-4M), both the total activity and the excretion of /?-lac- tamase decreased.20 Thus, a high level of p-lactamase syn- thesis appears to be a necessary condition for excretion and for inclusion body formation.

GEORGIOU, SHULER, AND WILSON: p-LACTAMASE OVERPRODUCTION 743

Excretion in Other E. coli Strains

In order to determine whether excretion is effected by the properties of the E . coli host strain, several other strains were transformed with ptac 11. Ampicillin-resistant trans- formants could only be obtained when the cells contained a l ad4 allele. Furthermore, some of the strains tested (e.g., a ladq derivative of the E. coli leaky mutant 207c) did not give any transformants. In contrast, pDW17 gave normal frequencies of transformation in all strains tested, except HB101. Cells transformed with ptacll or pDW17 were grown in liquid media and induced with IPTG. All strains that contained ptac 1 1 were found to excrete a large percent- age of their p-lactamase activity into the culture supernatant (Table 111). Excretion also occurred in induced cultures of D403 containing the plasmid pDW17. Apparently, even moderate levels of p-lactamase are sufficient to induce ex- cretion in this strain.

Table 111. plasmid combinations."

Total p-lactamase activity and excretion in different strain-

Total p-lactamase Strain Plasmid activity (U/ml) Excretion, %

RB791 pBR322 13 4

PKN 175 88

pDW17 84 11

ptac 11 168 92

pDW17 89 6 JM109 ptac 1 1 134 91

D403 ptac 11 172 83 pDW17 137 96

a Cells grown in M9 medium and induced at mid-exponential phase. Activity of P-lactamase determined as described in Materials and Methods.

Table IV. Viable counts.a ~~ ~~

Optical densty at Total colonies/ Amp' colonies/ induction mL culture mL culture

Physiology of Excreting Cells

Since overproduction of p-lactamase induces excretion in several different E . coli K-12 strains, the physiology of the excreting cells was investigated further. The effect of dif- ferent carbon sources was examined in liquid cultures of RB791 (ptacll). Growth in M9 media supplemented with 0.2% glucose, maltose, galactose, lactose, or glycerol gave similar levels of total p-lactamase activity and excretion (G. Georgiou, unpublished results). However, in M9 media with succinate as the carbon source and in LB broth sup- plemented with either glucose, fructose, or glycerol, a sig- nificant decrease in the level of p-lactamase was observed. In both cases the pH of the culture increased significantly during growth.

The effect of p-lactamase overproduction on the viabil- ity of the cells was tested by plating aliquots from overnight cultures that were induced at different optical densities (Table IV). It was observed that the number of viable counts decreased in induced cultures. The reduction in viable counts seems to correlate with an increase in the level of p-lacta-

uninduced 3.4 x lo9 3.3 x 109 0.220 2.3 x 109 7.0 X lo8 0.300 1.5 x lo9 not determined 0.440 1.0 x loq 3.6 X 10' 0.620 6.5 X 10' 2.9 X 10' 1 .om 1.3 x 109 1.1 x lo9

a Cells grown in M9 medium and induced at indicated ODm. Viable count determinations performed on LB plates with or without 50 pg/rnL ampicillin. Numbers reported represent average of at least three plates.

mase specific activity. However, the total amount of soluble protein in induced and uninduced cultures did not differ significantly (Table V), indicating that induction did not cause a general inhibition of protein synthesis. It is possible that the decrease in viable counts may be a consequence of the elongated morphology of the cells since it was found that the induced cells are approximately 3-5 times longer than uninduced cells.'' In addition, it was found that the number of colonies on selective plates was lower than on nonselec- tive plates, indicating that the plasmid is unstable in induced cells. This is consistent with earlier observations that in-

Table V. Activities and excretion of different enzymes.' ~~ ~~

Uninduced ptac 11 Induced ptacl 1 Induced pDW17

Total Excretion Total Excretion Total Excretion activity (8) activity (%) activity (%)

P-Lactamase Cyclic phosphodiesterase Acid phosphatase, pH 4.5 Acid phosphatase, pH 2.5 RNAase I P-Galactosidase G-6-P-Dehydrogenase Protein

40.5 4 14 5 11 8 7 0 4.5 9

114 1.5 83.5 0.5 0.37 1.5

516 9 5 . 5 3.5 3.0

48.3 139

0.38

92 22 60 47 45

7 8.5

32

243 14 10.3 6.8 5.7

160 74 0.35

6 8.5

20 6.7

12 0 0 7

a Distribution of enzyme activities in RB791 cells carrying ptacll of pDW17. Cells were grown on minimal medium with 0.2% fructose and casein acid hydrolysate and induced at an OD6o - 0.450. G-6-P-Dehydrogenase: glucose-6-phosphate dehydrogenase. Total activity ex- pressed in U/mg protein. Protein refers to soluble cellular protein (mg/mL culture).

744 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 32, SEPTEMBER 1988

0 0 duced RB791 (ptacll) cells cannot be maintained in continu- -

ous cultures.” In contrast, induced cultures of pDW17 did not exhibit plasmid instability.22

The synthesis of p-lactamase in RB791 cells containing either ptac I1 or pDW17 was initiated immediately after the

onset of the stationary phase in pDW17 cells (3.5 h after induction) but continued for at least 4 more hours in the excreting cultures although at a reduced rate. Appreciable extracellular activity could not be observed until 1 h after the

m b addition of IPTG (Fig. 1). It was essentially complete at the

c 0 .- -c-l

0 2 m::

8

2 addition of IPTG. More than 50% of the activity that was found in the medium was released early in the stationary phase. In order to determine whether the specificity of excre-

.z 8

a

tion changed with time, supernatant fractions were concen- trated and subjected to SDS-PAGE. The gels were stained with coomasie brilliant blue, destained, and scanned with a densitometer. I t was found that at 4, 6, 8, and 24 h after in- duction, the percentage of the total extracellular protein that was p-lactamase was 90, 76, 60, and 46% respectively. Thus, it appears that excretion of p-lactamase occurs first, but it is soon followed by the release of other proteins. The distribution of proteins in the periplasmic and extracellular fractions of overnight cultures was examined by SDS-PAGE and is shown in Figure 2. A number of proteins are released into the extracellular fluid from induced ~ ~ 7 9 1 (ptacl 1) cells but not from uninduced cultures or in induced pDW17 cells. Twenty-four hours after induction p-lactamase makes up approximately 46% of the protein in the supernatant of induced RB791 (ptacll) cells and around 40% of the osmotic shock fraction of pDW17 containing cells. As mentioned

m (u g

0

0 2 4 6 8 10

time (hr) Figure 1. Time course of p-lactamase activity and excretion. Open sym- bols: p-lactamase specific activity, U/mg total protein. Closed symbols: percentage of excretion. (0, 0) RB79lptacll; (A, A) RB791pDW17.

Figure 2. SDS-PAGE of osmotic shock fluid and supernatant fractions. RB791 cells containing either ptacll or pDW17 grown in M9 or LB media as described and induced at midexponential phase. Lanes: 1-4, osmotic shock fluid; 5-8 supernatants; 1 , 5 , ptacll uninduced; 2, 6 , ptacll induced; 3, 7, pDW17 induced; 4, 8, as before but grown in LB media. Molecular weight mark- ers (in kd) are given on the left: p-lac: mature P-lactomose.

GEORGIOU, SHULER, AND WILSON: P-LACTAMASE OVERPRODUCTION 745

in the preceding, the level of p-lactamase in cells grown in LB media is significantly reduced (Fig. 2, lanes 4 and 8).

To determine whether other periplasmic enzymes are re- leased into the medium along with p-lactamase, cells were subjected to osmotic shock, and the activities of several en- zymes were determined (Table V). With induced RB791 (ptacll) cells the release of p-lactamase was accompanied by significant leakage of the periplasmic enzymes: RNAase I, two acid phosphatases with pH optima of 2.5 and 4.5, and to a smaller extent, cyclic phosphodiesterase. The total specific activity of all these enzymes in induced RB791 (ptacll) cultures was about 40% less than that of uninduced cultures or induced RB791 (pDW17) cells, although the amount of cellular protein was approximately the same in each culture. About one-third of the total protein of in- duced RB791 (ptacll) cells was found in the medium. This high percentage results from the release of p-lacta- mase and other periplasmic enzymes into the medium as well as lysis of about 7% of the cells.

The nonspecific release of a number of periplasmic en- zymes is an indication that the permeability of the outer membrane of the E . coli cell envelope was altered. Cells possessing an altered outer membrane frequently exhibit an increased sensitivity to various toxic agents that are other- wise excluded from the cell interi~r.’~ The sensitivity of the excreting cells to low concentrations of detergents was tested (Fig. 3). It was found that excreting cells readily lysed when exposed to as little as 0.1% SDS or 0.25% deoxycholate while virtually no cell lysis was observed in nonexcreting cells under identical conditions. However, neither lysozyme (1 mg/mL) nor up to 20mM EDTA caused cell lysis. Nei- ther the sensitivity to SDS nor the release of p-lactamase

was affected by the presence of lOmM Mg2+ in the medium. Addition of magnesium has been reported to decrease the permeability of the outer membrane in several E . coli mu- tants having a defective cell envelope structure.23

Processing of Secreted Proteins in Excreting Cells

In some cases the overproduction of a secreted protein jams the secretion machinery of the cell and inhibits the cor- rect localization of other exported proteins The high level of p-lactamase synthesis might affect the processing of outer membrane proteins causing the nonspecific leakage of periplasmic enzymes. The kinetics of p-lactamase pro- cessing were determined by pulse labeling and immunopre- cipitation. Processing of the precursor form of p-lactamase in pDW17 cells is approximately 80% complete after 2 min. This is comparable to the processing of p-lactamase in pBR322-containing cells,25 although the level of synthesis in induced pDW17 cells is higher. On the other hand, the p-lactamase precursor in induced RB79 1 (ptac 11) cells was processed very slowly to the mature form (Fig. 4). In these cells the precursor could be detected for more than an hour after the addition of the chase (unpublished data). The slow processing of p-lactamase did not interfere with the processing of two membrane proteins, OmpA and OmpC (Fig. 4). Processing of either protein was essen- tially complete 20 s after the end of the labeling period both in excreting and nonexcreting cells. However, the in- tensity of the OmpA and OmpC bands from excreting cells was reproducibly lower than in the nonexcreting control. These results suggest that the level but not the export of outer membrane proteins is affected by the overproduction

I I I

0 20 40 60 time min

Figure 3. Decrease in OD, of cells exposed to detergents 3 h after in- duction, (open symbols, RB791pDW17; closed symbols, RB79lptacll): (0, 0) 0.1% sodium dodecyl sulfate (SDS); (A, A) 0.25% deoxycholate.

746 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 32, SEPTEMBER 1988

Figure 4. Autoradiograms of pulse chase experiments. Cells labeled with "S methionine for 20 s and samples were with- drawn at 0, 20, 40, 60, and 120 s (lanes 1-5, respectively) after start of the chase. p-lactamase, OmpA, and OmpC immuno- precipitated with respective antisera as described. Precipitated proteins subsequently resolved in 12% SDS-polyacrylamide gels and proteins visualized by fluorography. Upper panel, p-lactamase; middle panel, OmpA (lowest band probably corresponds to p-lactamase precursor, which cross-reacts with OmpA antiserum): lower panel, OmpC; p, precursor form; m, mature form.

of p-lactamase. [To further examine the protein composi- tion of outer membranes from induced and uninduced cells, outer membrane fractions were prepared by both the sucrose gradient and differential detergent solubilization methods.'' Analysis of outer membrane proteins by SDS-PAGE from samples corresponding to the same cell mass indicates that the amount of most major outer membrane proteins in in- duced cells is reduced by approximately 40-60% (data not shown). However, differences in the surface-to-volume ratio of induced and uninduced cells make these data hard to interpret.]

DISCUSSION

Although E . coli does not normally excrete proteins into the extracellular fluid, release of periplasmic proteins into the medium has been reported for several mutants with a de- fective outer membrane.23 More recently, activation of the kil gene has been shown to mediate the nonspecific release of a penicillinase in the culture medium.26 Lazzaroni et al.27 have reported that cells transformed with multicopy plas- mids carrying the phoA gene and grown in alkaline media released a large percentage of the alkaline phosphatase into the media. They reported that amplification of alkaline phosphatase synthesis was necessary for excretion, in agreement with our observations with j3-lactamase. How- ever, the specificity of excretion and pH dependence of this process suggest that alkaline phosphatase release pro- ceeds through a different mechanism than the one investi- gated in this work.

In this study we have shown that induction of p-lactamase synthesis from the strong tuc promoter results in the release of more than 90% of the total j3-lactamase soluble activity into the medium. No significant excretion was observed when the tuc promoter was not fully induced, nor in cells where the synthesis of p-lactamase is directed by the weaker promoter in pDW17. Thus, it can be concluded that high- level synthesis of p-lactamase is a necessary condition for excretion. The excretion of p-lactamase is also accompa- nied by an increased sensitivity of the cells to detergents and the nonspecific release of other periplasmic enzymes into the medium. These results suggest that the penrreabil- ity of the outer membrane of the excreting cells has been affected. In addition, it was shown that the rate of synthe- sis of two outer membrane proteins, OmpA and OmpC, is decreased in excreting cells as is the synthesis of all the periplasmic enzymes that were tested (Table V). Based on these observations, we propose the following preliminary model to explain the events that lead to excretion. Induc- tion of high-level p-lactamase synthesis interferes with the synthesis of cell envelope proteins. This results in changes in the outer membrane that ultimately affect its permeabil- ity. According to this model, cell growth is necessary for excretion. This is consistent with the observed lag between p-lactamase induction and the onset of excretion as well as the decrease in the level of excretion in cells induced at the onset of the stationary phase.

It has been shown previously that the synthesis of export- defective proteins causes a generalized block of protein ~ecretion. ' . '~.*~ Based on these observations, Ito et a]. l6 con-

GEORGIOU, SHULER, AND WILSON: P-LACTAMASE OVERPRODUCTION 747

cluded that there must exist a common step in the secretion of all exported proteins. Consequently, an event that causes the accumulation of the precursor of one protein should af- fect the processing of other secreted proteins. Anba et al? have shown that overproduction of the periplasmic PhoS protein affects not only its own processing but also the pro- cessing of p-lactamase and PhoA, two other periplasmic enzymes. However, in this study we have found that high- level synthesis of p-lactamase inhibits the maturation of its own precursor to a much greater extent than it inhibits the processing of the outer membrane proteins OmpA and OmpC. One possible explanation of these results is that the inhibition of processing of periplasmic enzymes does not interfere with the secretion of outer membrane proteins. While other explanations cannot be ruled out at this point, it is interesting that our results seem to contradict the existence of a common step in the secretion of all exported E . coli proteins.

In summary, we have found a new way to induce the re- lease of periplasmic proteins into the extracellular medium. Inducible excretion can be particularly advantageous for the large-scale production of secreted proteins. The physiology of the excreting cells has been characterized, and a prelimi- nary model was proposed to explain the sequence of events that lead to secretion. The understanding of the physiol- ogy of the excreting cells has already been exploited to achieve the continuous production of genetically engineered p-lactamase in an immobilized cell bioreactor.”

We are grateful to A. Said and R. Yocum of Biotechnica Interna- tional for sequencing the plasmid pDW17 DNA, H. Anraku and S . Mizushima for donating the OmpA and OmpC antisera, respec- tively, and J-C. Lazzaroni and J. Richards for providing E . coli strains. The technical assistance of Diana Irwin is gratefully ac- knowledged. This work was supported in part by National Science Foundation grant ECE 8513612 and a grant from the Cornell Bio- technology Center. One of us (G. G.) was supported by grant No. DAAL03-G-0030 from the U.S. Army.

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