Vol. 138, No. 2

JOURNAL OF BACTERIOLOGY, May 1979, p. 345-351

0021-9193/79/05-0345/07$02.00/0

Export of Extracellular Levansucrase by Bacillus subtilis: Inhibition by Cerulenin and Quinacrine M. P. CAULFIELD,t R. C. W. BERKELEY,`* E. A. PEPPER,' AND J. MELLING2 Department of Bacteriology, The Medical School, University ofBristol, Bristol BS8 I TD, England' and Microbiological Research Establishment, Porton Down, Salisbury, Wiltshire SP4 OJG, England2

Received for publication 3 March 1979

Bacillus subtilis B secretes an inducible, extracellular enzyme, levansucrase. Inhibition studies were undertaken to investigate the possible mechanism of release of this enzyme. The antibiotic cerulenin, at a concentration of 10 ,ug/ml, totally inhibited de novo lipid synthesis in B. subtilis B for at least 1 h, while only slightly reducing protein and RNA synthesis. At this concentration cerulenin, added concomitantly with the inducer sucrose, prevented the release of levansucrase for at least 150 min. This was not due to the prevention of inducer uptake by the cells. The release of the enzyme was also independent of cell division. In B. subtilis 1007 the induction of,-galactosidase by 5 mM lactose was not prevented by cerulenin. Preliminary evidence indicated the association of a lipid moiety with the enzyme as it passes through the cytoplasmic membrane. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing protease of Bacillus licheniformis, inhibited levansucrase release from B. subtilis B, but had no effect on lipid synthesis. The mechanism of export of extracellular proteins from most bacteria is obscure. One exoenzyme whose export mechanism has been intensively studied is the penicillinase of Bacillus licheniformis 749/C. This has been shown to have a nascent membrane-bound form (18) which differs structurally from the exoenzyme in that it has a phospholipopeptide attached to the amino-terminal lysine residue of the exoprotein (22, 23), making it hydrophobic in character. The phospholipopeptide is thought to mediate the transfer of the protein from its site of synthesis at the inner surface of the cytoplasmic membrane to the outer surface of this structure, where the additional sequence is cleaved off by a specific protease, thus allowing release of the extracellular enzyme (1, 19). The significance of phospholipopeptides in the process of exoprotein production has recently been reviewed (13). It is concluded that, although there exist small amounts of protein presumed to have phospholipid attachments in membranes of B. subtilis B71, a hydrophobic membrane-bound penicillinase in the membrane of another strain of B. licheniformis, strain 6346/C, and *hydrophobic, membrane-bound amylases in B. amyloliquefaciens and B. subtilis, there is no direct evidence that any of these are phospholipoproteins. The generality of this t Present address: Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854.

export mechanism is therefore in doubt. It is at least possible that the membrane attachment of the B. licheniformis penicillinase has evolved as a response to pressures tending to maximize the protective capacity of the enzyme against wall-active antimicrobial substances. Thus, in choosing an alternative system as a subject for inhibitor studies on export mechanisms of exoproteins, we selected levansucrase as an enzyme for which such an argument could not be advanced. This protein has the additional advantage that it is extremely well characterized in certain strains of B. subtilis (7, 10, 17). Cerulenin is an antibiotic that specifically inhibits the condensation reaction between acyl and malonyl thioesters by acting as a noncompetitive inhibitor of the enzyme of fatty acid chain elongation (6, 20). In Escherichia coli (9), Staphylococcus aureus (4), B. subtilis B42 (21), and other microorganisms (16) its major effect is on lipid synthesis. Thus, it was expected that, if levansucrase is a phospholipoprotein, its synthesis would be prevented by cerulenin, whereas synthesis of enzymes without any attached lipid material would be relatively unaffected. Quinacrine is an inhibitor of proteolytic enzymes and has been reported to interfere with the action of the B. licheniformis 749/C penicillinase-releasing protease (19). If levansucrase is released by a similar protease, it might be expected that quinacrine would also prevent levansucrase release.

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346

CAULFIELD ET AL.

In this paper studies on the inhibition of export of levansucrase by cerulenin and quinacrine

described. (A preliminary report of some of this work has been published [M. Caulfield, I. Chopra, J. Melling, and R. C. W. Berkeley, Proc. Soc. Gen. Microbiol. 3:91-92, 1976]). are

MATERIALS AND METHODS Organisms and growth conditions. B. subtilis B (Department of Bacteriology, University of Bristol, Bristol, England) and B. subtilis 1007 (Meat Research Institute, Langford, Bristol, England) were maintained on nutrient agar. Cultures were grown in a 10% succinate salt medium, containing 7.0 g of K2HPO4, 3.0 g of KH2PO4, 2.0 g of (NH4)2504, 0.05 g of FeCl3.6H20, 0.05 g of MnCl2.6H20, 100 g of sodium succinate, 0.25 g of MgSO4.7H20, and 10 g of peptone (Difco); this medium was made up to 1 liter with distilled water and autoclaved at 121°C for 30 min. Cultures were grown at 37°C on an orbital shaker in 250-ml conical flasks containing 100 ml of medium. Inoculation was with 3 ml of an overnight culture, and growth was measured turbidometrically with a Pye Unicam SP1800 spectrophotometer at 675 mm. Induction of levansucrase. Levansucrase was induced by the addition of sucrose to the culture at an absorbance of 0.2, to give a final concentration of 100 mM.

Cerulenin and nalidixic acid. Cerulenin (Makor Chemicals Ltd., Jerusalem, Israel) was dissolved in absolute alcohol and stored at -20°C in the dark. The solution was equilibrated at 37°C before addition to the cultures. Nalidixic acid (Winthrop Laboratories, Newcastle-Upon-Tyne, England) was dissolved in medium by the dropwise addition of 1.0 N NaOH to give a stock solution of 500 ug/ml. The solution was added to a culture 2 min after the addition of sucrose to give a final concentration in the culture of 25 jig/ml. Quinacrine and o-phenathroline. Quinacrine (Sigma Chemical Co. Ltd.) was dissolved in medium to give a final concentration in the culture of 0.2 or 1.0 mM. o-Phenanthroline (Sigma) was dissolved in absolute alcohol and added to cultures to give a final concentration of either 0.2 or 1.0 mM. Levansucrase assay. Culture samples (5 ml) were centrifuged in a bench top centrifuge, and the supernatant fluids were dialyzed overnight at 4°C against 50 mM potassium phosphate buffer, pH 6.8, before the enzyme activity was assayed by a modification of the method of Dedonder (7), determining the free glucose by an adaptation of the Somogyi-Nelson method (15). Determination of intracellular levansucrase. Culture samples (5 ml) from the cerulenin experiments were divided into two aliquots. One aliquot was used to assay enzyme activity in the culture supernatant, and the other was sonicated, centrifuged, and dialyzed, and the enzyme activity was determined in the cytoplasm-culture supernatant fraction and the particulate cell debris fraction. In the quinacrine experiments the cells were centrifuged and resuspended in 1 M Trishydrochloride buffer before sonication, and the enzyme activities in the cytoplasm and cell debris fractions were determined as described above.

J. BACTERIOL.

,8-Galactosidase assay. The method was essentially that of Herzenberg (12). Labeled sucrose uptake. [U-'4C]sucrose (Radiochemicals Ltd., Amersham, England) at an activity of 5 ,uCi/ml, together with unlabeled sucrose (50 mM), was added to cultures at an absorbance of 0.2, to give a fmal activity of 0.6 IACi/ml. Samples (1 ml) of the culture were filtered through a Whatman GF/C filter presoaked in medium containing 50 mM sucrose and then washed twice with 5 ml of prewarmed (37°C) medium also containing sucrose. (The prewarmed medium was used to prevent cell autolysis brought about by a sudden temperature change.) The filters were then dried, and the radioactivity was determined (4). For the detection of intracellular [6,6'-3H]sucrose the cells, after collection on the filter, were sonicated in 1 M Tris-hydrochloride buffer, pH 8.0, and dialyzed overnight against the same buffer. The diffusible material was then lyophilized and resuspended in a minimum volume of distilled water, and samples (1 ,u) were spotted and run on a paper chromatogram (Whatman no. 1 filter paper; ethyl acetate-acetic acidwater, 9:2:2, containing 2% [wt/vol] phenylboronic acid) together with an internal standard of [U-'4C]sucrose. The dried chromatogram was cut into 2-mm strips, and the radioactivity of each was determined. Protein, nucleic acid, and lipid synthesis. The methods used were essentially those described by Chopra (4). Membrane preparations. The method of Aiyappa et al. (1) was used. Polyacrylamide gel electrophoresis. The system used was that described by Lugtenberg et al. (14). The gels were frozen in dry ice, sliced into 1-mm fractions, thawed, and solubilized by using NCS tissue solubilizer (Amersham/Searle, Bucks, England), and the radioactivity in each fraction was determined. Enzyme units. Units of activity are expressed as katals per milliliter, where 1 katal is equivalent to the transformation of 1 mol of substrate per s. RESULTS

Inhibition of lipid synthesis by cerulenin. The effect of cerulenin on the [3H]acetate incorporation into chloroform-methanol-extractable material was determined (Table 1). Unless otherwise stated, subsequent experiments were TABLE 1. Effect of cerulenin on lipid synthesis' % Inhibition Cerulenin concn (pg/ml) 0 1 5 10 15

0 25 90 100 100

Cerulenin was added to cultures of B. subtilis B growing in the presence of [3H]acetate, and the amount of 3H incorporated over 1 h was determined. The results are presented as the percentage of the 3H counts incorporated into chloroform-methanol-extractable material from the control culture (no cerulenin added).

EXTRACELLULAR LEVANSUCRASE EXPORT

VOL. 138, 1979

camed out by using cerulenin at a concentration which totally inhibited lipid synthe10 of lAg/ml, sis, as measured by [3H]acetate incorporation, for at least 1 h. Growth, protein, and RNA synthesis in the presence of cerulenin. After the addition of cerulenin to a culture of B. subtilis B, the rate of increase of absorbance decreased (Fig. 1). Protein synthesis, measured by [3H]phenylalanine incorporation into hot trichloroacetic acidprecipitable material, was reduced by 28% after 1 h and RNA synthesis, determined by [53H]uracil incorporation into cold trichloroacetic acid-precipitable material, was reduced after 50 min by 43%. Effect of cerulenin on levansucrase re-

TIME (mnns.)

FIG. 1. Growth of B. subtilis B in thepresence and absence of cerulenin. At zero time the culture (I) was divided into two portions, and cerulenin (10 pg/ml) was added to one fraction (0). The growth was followed turbidometrically for 2 h. A675 nm, Absorbance at 675 nm.

347

lease. Cerulenin was added to a culture of B. subtilis B concomitantly with sucrose, and the enzyme concentration in the culture supernatant was measured. In the control culture enzyme was detected in the supernatant fluid after 20 to 40 min (Fig. 2A). In the cerulenin-treated culture there was no apparent release of the enzyme for at least 150 min. An examination of the cytoplasm-culture fluid fraction and the particulate cell debris of cells taken from cultures to which cerulenin and sucrose had been added simultaneously showed no detectable levansucrase activity. In the control culture, to which only sucrose had been added, there was no increase in the levansucrase activity in the cytoplasm-culture fluid fraction compared with the level of enzyme activity in the culture fluid alone, and there was also no detectable activity in the cell debris fraction. Furthermore, when cerulenin was added to cultures already induced for and producing levansucrase, the appearance in the supernatant of this enzyme ceased within 5 min (Fig. 2B). Levansucrase release in the absence of cell division. Nalidixic acid specifically inhibits DNA synthesis (5, 11) and indirectly inhibits cell division. When 25 jig of nalidixic acid per ml was added to a culture of B. subtilis B, the culture absorbance continued to increase at the same rate as the control, but the viable counts no longer increased. Induced levansucrase release occurred normally (Fig. 3). Sucrose uptake in the presence of cerulenin. To determine whether the inducer occurred intracellularly, the uptake of [U-'4C]sucrose was followed (Fig. 4). After 50 min the amount of radioactivity from the sucrose detectable in the cerulenin-treated cells was 67% of 0 -

B

14

X 12

E

>-0

10 0

u 08

2

/O

0.6

0

N

z W

04

0.2 0

20

40 60 80 TIME (mins.)

100

FIG. 2. Effect of cerulenin on the release of levansucrase from B. subtilis B. (A) Cerulenin (10 pg/ml) was added at zero time concomitantly with the inducer sucrose (100 mM), and the appearance of levansucrase in the culture supernatant was followed as described in the text. Culture in the absence (0) and the presence (0) of cerulenin. Error bars represent ±2 standard deviations. (B) Cerulenin (10 pg/ml) was added 65 min (shown by the arrow) after the induction of levansucrase.

348

CAULFIELD ET AL.

120 TIME (mins.)

J. BACTERIOL.

180

FIG. 3. Effect of nalidixic acid on levansucrase release. Nalidixic acid (25 pg/ml) was added to the culture 2 min after the addition of 100 mM sucrose (indicated by the two arrows). Growth (5, 3), viable counts (v.c.) (V, V), and the appearance of levansucrase (@) in the culture supernatant were followed as described in the text. Open symbols represent the control culture, and solid symbols indicate the nalidixic acid-treated culture. A, Absorbance.

Enzyme induction in the presence of cerulenin. In the absence of any known suitable intracellular enzyme inducible by a disaccharide in B. subtilis B, the induction of a,B-galactosidase in B. subtilis 1007 was used as a model system to investigate enzyme induction in the presence of cerulenin. The data shown in Fig. 5 clearly indicate that the antibiotic did not prevent induction of f)-galactosidase by lactose. These results indicated that cerulenin does not stop the appearance of levansucrase in the culture supematant by preventing cell division, by stopping uptake of the inducer, or by interfering with the process of induction itself and that there is no intracellular accumulation of levansucrase in cerulenin-treated cells. Attempts TABLE 2. Detection of intracellular [6,6'-H]sucrose" Uptake conditions

Temp

Treatment

(OC)

Cerulenin

30 -...

20

7

10.

E5

Amt of

Time after addition (min)

[6,6'-3H]sucrose in

cytoplas-

mic fraction (cpm)

37

2 63 10 49 No cerulenin 37 2 140 10 105 No cerulenin 4 2 24 aThe procedure used was that described in the text. All [6,6'-3H]sucrose peaks were coincident with the internal standard [U-'4C]sucrose peaks on the paper chromatograms. Cerulenin (80 iLg/ml) was added at zero time along with [3H]sucrose. C

3

)5

C

2 I-