JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 415-423

Vol. 140, No. 2

0021-9193/79/11-0415/09$02.00/0

Production and Excretion of Cloacin DF13 by Escherichia coli Harboring Plasmid CloDF13 GERDA J. VAN TIEL-MENKVELD,* ANNA REZEE, AND FRITS K. DE GRAAF Department of Microbiology, Biological Laboratory, Vrije Universiteit, 1007 MC Amsterdam, The Netherlands Received for publication 23 August 1979

The production and the mechanism of excretion of cloacin DF13 were investigated in noninduced and mitomycin C-induced cell cultures. A mitomycin C concentration was selected which did not cause lysis of cloacinogenic cells, but at the same time induced a maximal production of cloacin DF13. Native cloacin DF13, possessing killing activity, was first released into the cytoplasm. Shortly thereafter, the bacteriocin was transported through the cytoplasmic membrane and accumulated in the periplasm. Finally, cloacin DF13 was excreted into the culture medium. A small amount of cloacin DF13 remained associated with the cell surface. Producing cells did not become permeable for the cytoplasmic enzyme f8-galactosidase. Apparently the cloacin DF13 leaves the producing cells by an excretion process which is not similar to the mechanism proposed for bacterial secretory proteins. The processes of excretion by producing cells and of uptake by susceptible cells were also not identical because mutant cloacin DF13, which was not transported through the outer membrane into susceptible cells, was excreted like the wild-type cloacin DF13. The composition of the culture medium greatly affected production of cloacin DF13. The presence of sugars known to cause catabolite repression not only inhibited the production but also strongly reduced the excretion of cloacin DF13 into the culture medium. Bacterial cells harboring the non-transmissible bacteriocinogenic plasmid CloDF13 produce a bacteriocin designated as cloacin DF13 (11, 39). Cloacin DF13 kills susceptible cells of Enterobacter and Klebsiella species, because it inhibits protein synthesis and induces a leakage of potassium ions (8, 9, 39). The native cloacin DF13 is produced as an equimolar complex of two polypeptides with molecular weights of 56,000 and 8,500, respectively (6). The high-molecular-weight component inhibits protein synthesis by enzymatic cleavage of 16S rRNA (7, 30), whereas the low-molecularweight component (the immunity protein) functions as an inhibitor of the RNase activity (6, 31). Although much information is available about the mode of action of cloacin DF13 and related bacteriocins (16), little is known about the mechanism and regulation of bacteriocin production and excretion. Treatment of bacteriocinogenic cells with agents known to induce the so-called DNA repair system, like mitomycin C or UV irradiation (45), strongly increases the synthesis of bacteriocins (16, 17). Recently, it has been shown that colicin E3 remains bound to producing cells and that the free colicin E3 present in

the culture medium is released during cell lysis

(24).

To simplify the study of cloacin DF13 production, Escherichia coli K-12 was used as a host for the CloDF13 plasmid, originating from Enterobacter cloacae (39). Since E. coli does not possess specific receptors for cloacin DF13 on its cell surface (32), the excreted cloacin DF13 molecules cannot be adsorbed on and translocated back into these cells. Production and excretion were studied by measuring the amounts of cloacin DF13 present in various subcellular fractions at various time intervals. Furthermore, the effect of catabolite repression on cloacin DF13 production and excretion will be described. MATERIALS AND METHODS Bacterial strain8. The production and excretion

415

of cloacin DF13 was studied in an E. coli K-12 C600 strain harboring the wild-type CloDF13 plasmid or mutant plasmid CloDF13-clpO3 (1). For the ,B-lactamase assay, an E. coli K-12 strain harboring recombinant plasmid CloDF13::Tn9Ol was used (2, 40). These strains were kindly provided by E. Veltkamp. The cloacin DF13-susceptible strain Klebsiella edwardsii subsp. edwardsii was used for the determination of killing activity of cloacin DF13 (9) and for lacunae assays.

416

VAN TIEL-MENKVELD, REZEE, AND DE GRAAF

Media and growth conditions. The media used for cultivation of bacteria were as follows: (i) Lab Lemco broth (LL broth, Oxoid Ltd., London) often supplemented with an extra carbon source at a concentration of 0.5% (wt/vol); (ii) brain heart infusion (BHI, Oxoid), and (iii) minimal medium containing 75 mM phosphate buffer (pH 6.7), 2 g of NH4C1, 5 mg of FeSO4, and 50 mg of MgSO4 per liter supplemented with 0.5% (wt/vol) glucose or Casamino Acids (Difco Laboratories, Detroit, Mich.). For the study of cloacin DF13 production, an overnight culture of E. coli K-12(CloDF13) was harvested and the cell pellet was suspended in prewarmed medium to an optical density (OD) at 660 nm of about 0.12. The culture was then grown at 370C and strongly aerated. The growth of the cultures was measured by following the OD at 660 nm. After about 1 h (OD = approximately 0.27), the culture was divided in two portions. To one portion mitomycin C (Kyowa Hakko, Ltd., Tokyo) was added at a concentration of 0.025 jtg/ ml. Preparation of subcellular fractions. Cells were harvested by centrifugation (5 min, 15,000 x g) and converted to spheroplasts as described by Witholt et al. (43), using 0.25 mM EDTA and 30,tg of egg white lysozyme per ml (EC 3.2.1.17, Boehringer Mannheim, West Germany). The completion of spheroplast formation and the stability of the spheroplasts were checked by phase-contrast microscopy and by their osmotic sensitivity (44). After spheroplast formation was completed, usually within 10 min at 00C, 0.02 M MgCl2 was added to stabilize the spheroplasts, and the suspension was then centrifuged (10 min, 5,000 x g). The supernatant contained the periplasmic fraction (28). The spheroplasts were resuspended in phosphate-buffered saline (PBS: 0.01 M phosphate buffer [pH 7.2] and 0.15 M NaCI), with addition of DNase (EC 3.1.4.5, Boehringer) and RNase (EC 3.1.4.22, Boehringer), both 10 ,ug per ml, and disrupted by sonication (Branson Sonic Power Co., type B12) for 1.5 min at 00C and 85 W. After removal of unlysed cells and spheroplasts (10 min, 5,000 x g), the total cellular membrane fraction was collected by centrifugation for 16 h at 100,000 x g and suspended in PBS. The supernatant contained the cytoplasmic fraction. Total cell lysate was obtained by sonication (three timnes during 1.5 min at 85 W) of intact cells suspended in PBS. Assay for cloacin DF13. (i) Killing activity. The amount of cloacin DF13 active in killing susceptible bacteria was determined by a vial test essentially as described by De Graaf et al. (9). In all experiments the killing activity present in the various subcellular fractions and in the culture medium was expressed in units per milliliter of culture with an OD of 1.0 at 660 nm. One unit represents the amount of cloacin DF13 required to kill 50% of 5 x 108 bacteria under the test conditions. (ii) Immunological activity. The total amount of cloacin DF13 was determined using an enzyme-linked immunosorbent assay essentially as described by Mooi et al. (25). Preparation of antisera against cloacin DF13 was as described previously (6). The amount of cloacin DF13 was estimated with the aid of standard

J. BACTERIOL.

dilution series of cloacin DF13, purified as described

previopsly (6). Assay of lacunae. The number of lacunae-forming cells was determined by a modification of the lacunae technique of Ozeki et al. (33) as described by Kool and Nijkamp (21). Dilutions of cloacin DF13-producing cultures were made in cold PBS. Samples (0.1 ml) of the appropriate dilutions were mixed with 2.5 ml of LL broth soft agar with or without 107 susceptible cells. The mixtures were poured into 10-ml LL broth agar plates. After overnight incubation at 370C, lacunae and viable cells were counted. Assay for 8-galactosidase activity. To determine the capacity of fB-galactosidase (EC 3.2.1.23) synthesis, 1 mM isopropyl-fi-D-thiogalactopyranoside (Serva, Heidelberg, West Germany) was added to the culture together with mitomycin C. ,B-Galactosidase assays were performed as described by Miller (23). The activity was expressed in units per milliliter of culture with an OD of 1.0 at 660 nm. One unit was defined as the amount of enzyme that caused an increase in absorbance at 420 nm of 1.0 per min with 0.4 mg of o-nitrophenyl-,f-D-galactopyranoside (Merck, Darmstadt, West Germany) per ml as substrate. Assay for 8-lactamase activity. fi-Lactamase activity was assayed spectrophotometrically by the procedure described by O'Callaghan et al. (29), using a chromogenic cephalosporin compound 87/312 (a gift from Glaxo Ltd., Hoofddorp, The Netherlands). The ,B-lactamase activity was expressed in units per milliliter of cell culture with an OD of 1.0 at 660 nm. One unit was defined as the amount of enzyme that caused an increase in absorbance at 486 nm of 1.0 per min with 50 ug of cephalosporin per ml.

RESULTS Kinetics of spontaneous and induced production ofcloacin DF13. Production of cloacin DF13 can be induced with mitomycin C (11). The usual concentration of mitomycin C added to induce production (0.2 to 2.0 Ag/ml) strongly increased bacteriocin synthesis (16, 17), but was also very harmful to bacterial growth and viability (16, 17, 24). To minimize the contribution of lysed cells in the measurements of cloacin DF13 production and excretion, a mitomycin C concentration was selected which had a minimal effect on the viability of the cells during the experiments but, at the same time, induced an amount of cloacin DF13 as high as possible. Therefore, the effect of various concentrations of mitomycin C on growth of E. coli(CloDF13) was investigated (Fig. 1A). Based on this experiment, a concentration of 0.025 ,ug of mitomycin C per ml of medium was selected for induction experiments. At this concentration the synthesis of cloacin DF13 was increased 50- to 100-fold compared to noninduced cultures. The number of cloacin DF13-producing cells was determined by the lacunae assay and appeared to be in-

VOL. 140, 1979

PRODUCTION AND EXCRETION OF CLOACIN DF13

increased cloacin DF13 production and the number of lacunae-forming cells in cultures grown in LL broth supplemented with lactate, but had a severe effect on the viability of the cells and caused cell lysis. Induction with lower concentrations of mitomycin C resulted in a strongly diminished production of cloacin DF13 and number of lacunae-forming cells. The intracellular amounts of cloacin DF13 and the appearance of the bacteriocin in the medium were measured (Fig. 2). There was a lag time of about 60 min before the synthesized cloacin DF13 appeared in the culture medium. In noninduced cultures the rate of the synthesis was maximal at the end of the logarithmic growth phase. In induced cultures the maximal rate of synthesis was reached about 60 min after mitomycin C addition. In the stationary phase, synthesis and excretion were very low, and about

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time (miTn) FIG. 1. Effect of various concentrations of mitomycin C on bacterial growth and production of lacunae. E. coli(CloDF13) was grown in LL broth supplemented with 0.5% (wt/vol) lactate. Mitomycin C was added at zero time. (A) Bacterial growth was measured by optical density at 660 nm of the cultures. (B) The numbers of viable cells and lacunae-forming cells were assayed by plating samples in appropriate dilutions without and with susceptible cells, respectively. The mitomycin C concentrations used were: 0, none; 0, 0.010 pg/ml; A, 0.025 pg/ml; *, 0.050 pg/ ml; A, 0.10 pg/ml; and 0.50 pg/ml.

creased from 0.29 x 10" lacunae-forming cells ml in noninduced cultures to 3.1 x 108 in mitomycin C-induced cultures (Fig. 1B). Apparently, the number of cloacin DF13-producing celLs as well as the amount of cloacin DF13 produced per cell is raised about 10 times after induction with 0.025 ug of mitomycin C per ml. Higher concentrations of mitomycin C hardly

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time (min) FIG. 2. Kinetics of cloacin DF13 production. E. coli(CloDF13) was grown in LL broth supplemented with 0.5% (wt/vol) lactate. To one-half of the culture 0.025 pg of mitomycin C per ml was added at 0 min. After various time intervals, samples were taken and centrifuged (5 min, 15,000 x g), and the killing activity present in the supernatant was determined (0). The cell pellets were resuspended in PBS and disrupted by sonication, and the killing activity of the broken cell preparation was determined and expressed as described in the text (U). Solid line, Noninduced production; broken line, mitomycin C-induced production.

418

VAN TIEL-MENKVELD, REZEE, AND DE GRAAF

equal amounts of the killing activity were found in the culture medium and in the cells. The cloacin DF13 detected in cells lysed by sonication was located inside the cell envelope and was not associated with the cell surface, since only little killing activity (less than 7%) could be extracted by washing intact cells with buffered saline (PBS, 0.15 M NaCl) or with high salt (1 M NaCl). Localization of cloacin DF13. To study the excretion process of cloacin DF13, the amount of cloacin DF13 present in different subcellular fractions was determined at various time intervals after the initiation of mitomycin C-induced synthesis of cloacin DF13. The cloacin DF13 synthesized in the cytoplasm accumulated first in the periplasm, followed by excretion into the culture medium (Table 1). Three hours after incubation, 46% of the produced cloacin DF13 was present in the culture medium and 54% remained in the cells. The cellular cloacin DF13 was divided between the periplasm (37%) and the cytoplasm (17%), and a minor part (0.3%) was associated with the total cellular membranes (Table 1). This implied that, in the stationary phase, 83% of the bacteriocin was located outside the cytoplasmic membrane and therefore was transported to this location after synthesis in the cytoplasm. The distribution of cloacin DF13 in noninduced cells (not shown) was about the same as in mitomycin C-induced cells. In the stationary phase, 98% (9.3 kU) of the killing activity present in spheroplasts was actually located within the cytoplasm and only 2% (0.2 kU) was found associated with cellular TABLE 1. Localization of cloacin DF13 in various subcellular fractionsa Killing activity (kU/ml)

Timne Of induction

Cell

(min1)lysate

Cyto- Mem- Pen-

plasm

Cul- Recovery' (%) ture

branes plasm me~~dium I

75 3.8 0.2 12.1 6.6 0.0 60 80 25.7 10.4 0.1 10.2 2.7 100 85 37.0 11.5 0.2 19.8 24.7 140 78 180 36.8 9.3 0.2 19.8 31.2 aE. coli K-12(CloDF13) was grown in LL broth supplemented with 0.5% (wt/vol) of lactate. At indicated time intervals after the addition of 0.025 ,g of mitomycin C per ml, samples were taken and centrifuged to obtain the culture medium fraction. The harvested cells were fractionated into different subcellular fractions. In the different subcellular fractions the killing activity was determined. b Killing activity detected in the cytoplasm, mem-

branes, and periplasm per the total detected in the cell lysate.

J. BACTERIOL.

membranes (Table 1). If, however, spheroplasts were sonicated in buffer without saline, the reverse was observed and the major part of cloacin DF13 was found associated with cellular membranes. Addition of saline before or after sonication of spheroplasts reversed this distribution again. Addition of EDTA had no effect on the distribution of cloacin DF13. These results suggested that the binding of cloacin DF13 to the cellular membranes was weak and nonspecific. Washing of spheroplasts with PBS supplemented with 0.25 M sucrose and 10 mM MgCl2, to stabilize the spheroplasts, did not result in extraction of cloacin DF13. This indicated that the cloacin DF13 detected in spheroplasts was on the inside of the cytoplasmic membrane. As a control, to verify whether the cells or spheroplasts became permeable, the activity of a specific cytoplasmic enzyme, fi-galactosidase, was measured in spheroplasts, the culture medium, and the periplasmic fraction. No significant amount of this enzyme could be detected (Table 2). This implied that cells did not become leaky during cloacin DF13 production and that spheroplasts were not permeable during the isolation procedures. As a control for the presence of periplasmic proteins in the supernatant of spheroplast suspensions, the activity of a TEM-type ,8-lactamase (40) was determined (Table 2). This enzyme was shown to be released from cells after osmotic cold shock (5). More than 90% of this enzyme appeared to be released during the formation of spheroplasts. The results presented thus far suggest that cloacin DF13 is released first into the cytoplasm, thereafter transported into the periplasm, and finally excreted into the culture medium. The possibility must be considered that the distribution of cloacin DF13 over the various, subcellular fractions (Table 1) is not an exact reflection of the total amount of cloacin DF13 since the bacteriocin assay used detects only cloacin DF13 molecules capable of killing susceptible cells. Several secretory enzymes and toxins are known to be synthesized primarily as inactive proenzymes or protoxins, some of which undergo posttranslational modification to the active forn after or during translocation through the cytoplasmic membrane (4, 18, 35, 41). For this reason the same experiment as described in Table 1 was perfonned, but the fractions were also tested immunologically for the presence of cloacin DF13 molecules, using antisera prepared against cloacin DF13. No discrepancies, however, were observed when measuring the cloacin DF13 concentration by either of the two assay procedures. From the comparison ofboth assays, it could be concluded that 1 ng of cloacin DF13

PRODUCTION AND EXCRETION OF CLOACIN DF13

VOL. 140, 1979

TABLE 2. Localization of /3-galactosidase and filactamase in various subcellular fractionsa

fl-galactosidase

fl-lactamase ac-

(U/ml)

(U/ml)

activity

Fraction

_b

+c

tivity

-b

+c

7.08 7.60 Spheroplasts 0.06 0.21 Periplasm 0.11 0.28 6.13 6.27 Culture medium 0.04 0.08 0.26 0.49 The activities were assayed after 180 min of induction in fractions obtained from an E. coli K12(CloDF13::Tn9Ol) culture grown in LL broth with lactate (0.5%, wt/vol). b Without mitomycin C. C With 0.025 ,ug of mitomycin C per ml. a

was equivalent to about 1.2 U, which agrees with previously established results of De Graaf and Klaasen-Boor (6). These observations, together with the presence of high amounts of active cloacin DF13, strongly indicate that native cloacin DF13 is synthesized and released in the cytoplasm of producing cells. Effects of fermentable carbon sources on the production and excretion of cloacin DF13. The production of cloacin DF13 was shown to be highly dependent on the composition of the growth medium (Table 3). In minimal medium containing glucose as the carbon source as well as in complex medium containing glucose (brain heart infusion), the production was rather low compared to the production in media without glucose. This suggested that cloacin DF13 production was subjected to catabolite repression. To investigate this possibility, cloacin DF13-producing cells were grown in LL broth supplemented with different carbon sources (Table 4). The total amount of cloacin DF13 produced in LL broth supplemented with glycerol, galactose, fructose, or glucose was much lower than in LL broth supplemented with lactate, succinate, or the nonfermentable sugar inositol, or in LL broth without any addition, both in induced and in noninduced cultures. Since the same fermentable sugars are known to repress the synthesis of inducible catabolic enzymes like B8-galactosidase (34), this enzyme was used as a control for catabolite repression. Synthesis of Bl-galactosidase was repressed to the same extent as the production of cloacin DF13 (Table 4). Another conclusion that could be drawn from the data presented in Table 4 was that the major part of the cloacin DF13 produced in cells grown in LL broth supplemented with glycerol, galactose, fructose, or glucose is not excreted into the medium. The most striking effect occurred in mitomycin C-induced cells, where only a small percentage of the cloacin DF13 was excreted

419

(see also Table 3). The cellular bacteriocin was located inside the cell envelope, since extraction with high salt did not release a significant amount of cloacin DF13. The localization of the cloacin DF13 produced under conditions of catabolite repression is shown in Table 5. In cells grown in LL broth supplemented with glycerol or glucose, the translocation of cloacin DF13 over the cytoplasmic and the outer membrane seemed to be impaired. In the presence of glycerol or glucose, these cells contained 61 and 70%, respectively, of the total amount of cloacin DF13 in their cytoplasm. About 25% of the cloacin DF13 was present in the periplasm, and a minor part was found excreted into the medium. Addition of cyclic AMP (cAMP) to a culture under catabolite repression is known to increase the level of catabolic enzyme synthesis (34). Consequently, addition of 5 mM cAMP together with mitomycin C stimulated the synthesis of ,8-galactosidase to 80 to 90% of the synthesis in absence of catabolite repression (Table 6). Production and excretion of cloacin DF13, however, were only slightly increased upon addition of cAMP. Addition of higher concentrations of cAMP or repeated additions at various stages of the bacterial growth had no further effect on the synthesis of 8l-galactosidase or cloacin DF13. Excretion of inactive cloacin DF13. Further studies on the mechanism of excretion of cloacin DF13 might be facilitated using mutants affected in this process. Recently the isolation of mutants of the CloDF13 plasmid was described (1). The cloacin DF13 produced by these mutants, designated as clpO3 and clp2l, did not affect susceptible cells, because the translocation of the mutant cloacin from the outer membrane receptor to the cytoplasmic membrane was hampered (15). The possibility that these inactive cloacin DF13 molecules were also hampered in TABLE 3. Production of cloacin DF13 in different growth mediaa Killing activity (kU/ml) Mediumb

Culture Cell medium lysate

Total activity (%)

100C 34.5 35.9 LL broth 1.0 7.4 12 BHI MM + glucose 3.1 8.3 16 62 23.8 20.5 MM + Casaniino Acids a Cloacin DF13 production in E. coli K12(CloDF13) was induced with 0.025 yg of mitomycin C per ml of medium. b BHI, Brain heart infusion; MM, minimal medium. 'Total cloacin DF13 production in LL broth was taken as 100%, and the killing activity was determined after 180 min.

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VAN TIEL-MENKVELD, REZEE, AND DE GRAAF

420

TABLE 4. Effect of different carbon sources on the production of cloacin DF13 and ,B-galactosidasea Induced production

Noninduced production

(killing activity in kU/ml) Culture Cell activity medium lysate

(killing activity in kU/ml) Cell Total acit medium lyat

Added carbon source

C

8-galacto(%)

l00 29.0 loob 28.6 loob 0.92 1.00 99 ND d 29.4 27.9 96 0.90 0.95 Inositol 101 ND 29.5 96 29.0 0.85 1.05 Succinate 100 94 29.8 24.3 104 1.00 1.00 Lactate 1.3 28.2 49 36 42 0.65 0.30 Glycerol' 52 39 2.3 27.6 52 0.70 0.30 Galactose' 52 1.0 37.0 66 52 0.90 0.10 Fructose' 0.35 14.0 25 19 26 0.45 0.05 Glucose' a E. coli K-12(CloDF13) was grown in LL broth supplemented with different carbon sources at a concentration of 0.5% (wt/vol). The production in noninduced cultures was measured at the beginning of the stationary phase, and the induced production was measured at 180 min after addition of 0.025 Lg of mitomycin C per ml. b Total production in LL broth without carbon source was taken as 100%. The ,-galactosidase activity was measured in cell lysates. One hundred percent represented 6.50 U/ml of cell culture. d ND, Not determined. ' The repression of production and excretion of cloacin DF13 by certain carbon sources is not a result of a decreased pH of the growth medium, caused by fermentation. c

TABLE 5. Localization of cloacin DF13 in different subcellular fractions of cells grown with different carbon sourcese Killing activity (kU/ml)b

Added carbon source

Cytoplasm

Mem-

branes

Recovery"

Peri- Culture plasm medium

74 49.50 97 1.70 Glycerol 96 0.34 Glucose 'E. coli K-12(CloDF13) was grown in LL broth supplemented with different carbon sources at a concentration of 0.5% (wt/vol). Mitomycin C (0.025 jig/ ml) was added to induce cloacin DF13 production. b Killing activity was determined after 180 min of

Lactate

13.40 12.80 5.60

0.35 0.04 0.05

21.45 5.88 1.76

induction. 'Killing activity detected in the cytoplasmn, membranes, and periplasm per the total detected in the cell lysate.

their excretion by producing cells was investigated. The presence of cloacin DF13-c1pO3 in the different subcellular fractions and culture medium was determined using the immunological test. No difference, however, could be detected by comparing the results obtained with the wildtype cloacin DF13. This suggested that excretion of cloacin DF13 by producing celLs and uptake of cloacin DF13 by susceptible cells are not identical processes.

DISCUSSION From the results presented in this paper, cloacin DF13 does not appear to be produced and excreted as described for many noncytoplasmic bacterial proteins, which are initially synthe-

TABLE 6. Induced production of cloacin DF13 and B-galactosidase in the presence of cAMpa Activity Killing activity Added carbon source

(kU/Ml)b

Culture Cell medium lysate

Total activity (%)

of,B-galactosidase (%)

31.0 l00C lood 78 50 26.6 89 66 14.6 Fructose 75 26 15.1 Glucose aE. coli K-12(CloDF13) was grown in LL broth supplemented with different carbon sources. cAMP (5 mM) was added at the same time as 0.025 jig of mitomycin C per ml. b Killing activity was determined 180 min after induction. c Total production in LL broth supplemented with lactate was taken as 100%. d One hundred percent represents an enzyme activity of 6.85 U/ml of cell culture.

Lactate

Glycerol

30.00 4.30 1.30 0.08

sized as precursors on membrane-bound poly-

somes and secreted across the membrane concomitant with translation (18, 35-38). Immediately after the onset of cloacin synthesis, the major part of bacteriocin could be detected in an active configuration in the cytoplasm of producing cells. Soon thereafter the cloacin DF13 is translocated to the periplasm, followed by excretion into the medium. Only a minor part of the cloacin DF13 is associated with the bacterial membranes. Whether the cloacin is initially produced with a hydrophobic N-terminal "leader sequence" which is removed during, or shortly after, translocation across the membranes (3, 14, 18, 37, 38) is not known at this moment. How-

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PRODUCTION AND EXCRETION OF CLOACIN DF13

ever, since no significant differences could be detected between measuring the cloacin DF13 concentration either by killing activity assay or by reaction with specific antisera, the possibility of synthesis of inactive precursors is unlikely. Cloacin DF13 possesses a domain type of structure with a hydrophobic N-terminal part and a hydrophylic C-terminal amino acid sequence (10). Possibly, the hydrophobic N-terminal amino acid sequence may serve as a "signal sequence" triggering the cloacin DF13 across the membrane. Obviously this protein-membrane interaction occurs after completion of the protein synthesis. No differences in the time course of cloacin DF13 production or in the localization of the cloacin DF13 were observed between noninduced and mitomycin C-induced producing cultures. After this work was completed, Jakes and Model (20) reported on the mechanism of export of the related bacteriocin colicin E3 as well as colicin El. They concluded that these colicins are not synthesized as precursor proteins with an N-terminal "leader sequence." Colicin E3 appears to leave the producing cells, long after its synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. As with cloacin DF13, initially much of the colicin remains in the cytoplasm and is not associated with the membrane. Comparable conclusions have been reported by Mock and Schwartz (24), working on the mechanism of colicin E3 production. They demonstrated that the major part of the produced colicin appears to remain associated with the producing cells. Half of this cell-bound colicin is extractable from the cells during a high-salt wash. Both groups suggest that cell lysis may be the reason for the presence of free colicin E3 in the medium. In agreement with this conclusion, they show a decrease in optical density in mitomycin C-induced cultures. Both papers mentioned that 0.5 ,ug of mitomycin C has been used to induce colicin production. As reported in this paper, the concentration of mitomycin C is very critical with respect to cell lysis, and 0.5 ,Ag of mitomycin C per ml also results in lysis of cloacin DF13producing cells. To avoid a contribution of mitomycin C-induced lysis of cloacinogenic cells to the release of cloacin DF13 from the cells, we finally used an amount of mitomycin C for cloacin DF13 induction which was 20-fold lower. At this concentration (0.025 ,Lg/ml) no lysis and no release of cytoplasmic enzymes occur, but a high amount of cloacin DF13 is still produced and excreted. Therefore, we believe that under this circumstance cell lysis is not the mechanism by which cloacin DF13 leaves the producing cells. Production and excretion of cloacin DF13 is

421

highly dependent on the composition of the growth medium. Both in noninduced and mitomycin C-induced cultures, cloacin DF13 production is inhibited in the presence of a carbon source known to induce catabolite repression on inducible catabolic enzyme synthesis. It is currently believed that cAMP and its intracellular receptor protein (CRP) are necessary for highlevel expression of catabolite-sensitive operons in vivo and in vitro (34). Addition of cAMP reverses catabolite repression. In agreement with this, the repression of f8-galactosidase by cloacin DF13-producing cells is largely relieved upon the addition of cAMP. However, very little effect is observed on the repression of cloacin DF13 production. Although the literature about the effect of glucose on the production of the colicins is somewhat contradictory (19, 26), the production of colicin El (13, 27) and lb (19), but not of colicin E2 (13), is dependent on a functional cAMP-CRP system. Obviously, the gene encoding for cloacin DF13 is under control of catabolite repression, but, besides the cAMPCRP system, additional effectors probably play an important role in the regulation of the transcription of this gene. Recently, the existence of such additional effectors for genes under catabolite repression, especially permanently, have been described (12, 42). Quite remarkable was the effect of carbon sources on the excretion of cloacin DF13. The presence of fermentable sugars not only repressed the synthesis of cloacin DF13 but also the translocation of the cloacin DF13 over the membranes into the medium, the major part remaining in the cytoplasm. A possible explanation for this phenomenon might be that synthesis of membrane proteins involved in the translocation of the cloacin DF13 across the membrane was also subjected to catabolite repression. The protein composition of the outer membrane, for instance, is strongly dependent on the growth medium (22). Likewise, the presence of glucose in the growth medium might be the reason why the major part of cloacin DF13 remained associated with the producing cells as described by Mock and Schwartz (24). Inactive cloacin DF13 produced by cells harboring a mutated CloDF13 plasmid was exported through the membranes like the wildtype cloacin DF13, although the translocation over the outer membrane of susceptible cells was hampered. Therefore, the processes of excretion by producing cells and of uptake by susceptible cells are not identical. Some mutant colicin E3, inactive in killing susceptible cells, cannot be transported to the cell surface, however, and seems to be blocked at some intermediate stage in the excretion process (24). Since it

422

VAN TIEL-MENKVELD, REZEE, AND DE GRAAF

is unknown why the mentioned mutant colicin is inactive, the question remains whether excretion and uptake share some common properties. The present work provides no support for the involvement of lysis nor for an export mechanism in the excretion process of cloacin DF13 as has been described for some noncytoplasmic proteins. Recently some indications have been obtained for an increase in membrane permeability to certain cations upon the synthesis of a critical concentration of intracellular cloacin (not published). Changes in membrane permeability upon cloacin DF13 production and the effect of carbon sources on this process will be subjects for further studies.

14.

15.

16. 17. 18.

ACKNOWLEDGMENTS

19.

This investigation was supported by the Netherlands Foundation for Biological Research (B.I.O.N.), with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).

20.

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