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Penicillin-binding protein sensitive to cephalexin in sporulation of Bacillus cereus
Microbiol. Res. (1997) 152,227-232
Microbiological Research C!:) Gustav Fischer Verlag
Penicillin-binding protein sensitive to cephalexin in sporulation of Bacillus cereus Takahisa Miyamoto, Ken-ichiro Yamaguchi, Md. Abu Sayed, Ryo Sasahara, Ken-ichi Honjoh, Shoji Hatano Food Science and Technology, Faculty of Agriculture, Kyushu University, lO-I, Hakozaki 6-chome, Higashi-ku, Fukuoka, 812-81 Japan Accepted: March 10, 1997
Abstract Cephalexin, cefac1or, cefadroxil, and cefotaxime strongly inhibited sporulation of Bacillus cereus ts-4 at 1 Ilg/ml. Cephalexin was most inhibitory on the sporulation of B. cereus when the antibiotic was added at 3 h after induction of sporulation by nutrient downshift technique. Examination of 4' ,6-diamidino2-phenylindole-stained cells by fluorescence-phase contrast microscopy showed that cephalexin inhibited the formation of asymmetric septum. By using [3H]penicillin, eight penicillinbinding proteins (PBPi'i) were detected from the cells of B. cereus ts-4. Among them, four PBPs were also detected in sporulating cells. Affinity of cephalexin to PBPs were measured indirectly by competition for subsequent binding of radioactive penicillin O. Cephalexin strongly bound to PBP 4 with molecular weight of 72,000 in sporulating cells. Key words: Bacillus cereus-sporulation-penicillin binding protein-Cephalexin
Introduction Bacillus cereus is one of the food poisoning bacteria which produces heat resistant enterotoxin and spores (Sinagawa et al. 1991, 1992). The presence of its spores in food is one of the biggest problems in food industry because the spore is resistant to heating, ultraviolet light and drugs. It is very difficult to stelilize food contaminated with spores of B. cereus. We have studied the mechanism of sporulation of B. cereus to develop an effective method to inhibit sporulation. We previously showed the presence of chromosomal proteins that seem to control the initiation of sporulation of B. cereus ts-4 (Miyamoto et al. 1991; Matsuno et al. 1995). Corresponding author: T. Miyamoto
The formation of an asymmetrically situated division septum is an early event of sporulation of Bacillus (Young and Fitz-James 1959). The asymmetric division results in two distinct cells, that have radically different developmental fates (Young and Fitz-James 1959). This sporulation division contrasts with division during vegetative growth of Bacillus, in which the septum is symmetrically situated with respect to the ends of the dividing cell. The penicillin-binding proteins (PBPs) are membrane bound enzymes required for peptidoglycan synthesis and septum formation (Spratt 1983). There are at least seven PBPs produced by vegetative cells of Escherichia coli (Spratt 1975). There are various ~-lactam antibiotics and the target sites of some of them have been studied in detail on E. coli (Spratt 1975). Some of these antibiotics have specific target PBPs, for example, cefaloridine specifically binds to and inhibits PBP 1, mecillinam is specific for PBP 2 and cephalexin for PBP 3 of E. coli (Spratt 1975). The studies on the mechanism of action of penicillin on B. subtilis have shown the presence of PBPs that increase or decrease during sporulation (Buchanan und Sowell 1983 ; Kleppe and Strorninger 1979; Sowell and Buchanan 1983). However, the mechanism of the gene expression for these PBPs at the initial stage of sporulation is not clear. In this paper, we desclibe the effects of various ~-lac tam antibiotics on sporulation of B. cereus. We found that cephalexin, which is specific for PBP 3 of E. coli (Spratt 1975), inhibited the sporulation of B. cereus which had been induced to sporulate by nutrient downshift technique. The presence of PBP that has high affinity to cephalexin and its role in sporulation will be discussed. Microbiol. Res. 152 (1997) 3
227
Materials and methods Bacterial strains. Bacillus cereus ts-4 was a temperature
sensitive mutant for the initiation of chromosome replication delived from B. cereus JCM 2152 which had been obtained from Japan Collection of Microorganisms, Wako, Saitama, Japan (Miyamoto et al. 1991). Culture conditions. B. cereus was cultivated in YEEMM (Matsuno et al. 1995) containing 0.2% (NH4hS04' 1.4% (W/V) K ZHP0 4 , 0.6% KH zP0 4 , 0.1 % sodium citrate· 2H20, 0.02% MgS04 • 7H20, 0.5% glucose, 0.3% glutamic acid, 0.3% L-asparagine, 1% (WN) casamino acids (Difco Laboratories), and 0.02% yeast extract (Wako Pure Chemicals, Osaka, Japan) at 30°C for 24 hr with shaking. To examine the effects of various antibiotics (ampicillin, cloxacillin, cefaloridine, cephalexin, cefaclor, cefadroxil, cefoxitin, and cefotaxi me ) on sporulation, 100 III of the culture was transferred to modified G medium, G medium (Gollakota and Halvorson 1960) supplemented with 0.2% glutamic acid, and cultured at 30°C with shaking. The growth of bacteria was monitored by measuring optical density at 660 nm (OD 660 ). A 11100 G medium (Matsuno et al. 1995), in wich concentrations of ammonium sulfate, yeast extract and glucose were reduced to 111 00 of those of the modified G medium, was prepared and used to induce sporulation of B. cereus. Induction of sporulation of B. cereus. Sporulation was induced by the nutrient downshift technique. B. cereus
was cultured in 200 ml of YE-EMM at 30°C. At midexponential phase of growth, the cells were harvested by centrifugation at 2,000 x g for 10 min at room temperature. The cells were resuspended in 100 ml of 11100 G medium and incubated at 30°C to induce sporulation. Measurement of viable cells and spores. Total viable cells and spores of B. cereus were measured by plating
assay at 20 hr after a start of cultivation or after an induction of sporulation. In measurement of spores, the culture was heated at 85°C for 15 min before platmg. Combination Jluorescence-phase-contrast microscopy.
Bacterial cells, which had been harvested by centrifugation at 1,400 x g for 5 min from 1 ml of a culture, were resuspended in 0.1 ml of water. Then the cell suspension was mixed with 0.1 ml of 4' ,6-diamidino-2-phenylindole (DAPI) solution (50 Ilg/ml). The stained cells and nucleoids were visualized with a Nikon Optiphoto Microscope, x 2,400, by the "fluo-phase" method (Hiraga et al. 1989). Preparation of membranes and assay for PBPs. The membrane of B. cereus was prepared according to the
method of Buchanan (1979) except that the sonication 228
Microbial. Res. 152 (1997) 3
was carried out with a Branson sonifier model 185 at 60 w for ten 30-sec pulses for vegetative cells and twenty 30-sec pulses for sporulating cells. The membranes were frozen in a small volume of 50 mM TrisHCl buffer, pH 7.5, containing 1 mM MgCI 2, 1 mM phenylmethylsulfonylfluoride (PMSF). Proteins were measured by the dye-binding method of Bradford (1976). For the detection ofPBPby radioactive penicillin, the membrane fraction (about 500 Ilg proteins in the same Tris-HCl buffer) was incubated with 44 kEq of a [3H]penicillin G (814 gBq/mmole, Amersham) in a total volume of 30 III for 30 min at 25°C. The binding reaction was stopped by adding 2 III of cold penicillin G (300 mg/ml). The reaction mixture was mixed with 10 III of 0.1 M Tris-HCl buffer, pH 6.8, containing 40% (WN) glycerol, 8% (WN) sodium dodecyl sulfate (SDS), and 0.004% bromophenol blue, and boiled for 3 min. The mixture was then centrifuged at 10,000 x g for 10 min. Five microliters of the sample solution (about 63 Ilg proteins) was loaded onto a lane of a slab gel. Separation of membrane proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemrnli (1970) in a 10% (WN) polyacrylamide gel (l x 90 x 80 mm) containing 0.1 % SDS at room temperature at 25 mA per gel. To measure the bindings of unlabeled ~-lactam antibiotics, the membrane fraction was incubated with various concentrations of cephalexin and ampicillin for 10 min before the addition of the labeled penicillin. The [3H]penicillin-bound proteins in the gel were detected by fluorography (Bonner and Laskey 1974).
Results Effects of various (3-lactam antibiotics on sporulation of B. cereus
To examine the effects of various ~-lactam antibiotics on sporulation, antibiotics were added to the culture of B. cereus ts-4 in G medium at various concentrations at late-exponential phase of growth (OD660 = 0.9). Fig. 1 shows the structures of antibiotics tested. Fig. 2 shows the effects of various ~-lactam antibioties on viability and sporulation of B. cereus ts-4. Ampicillin had no effect on both viability and sporulation of B. cereus ts-4 even at 10 Ilg/ml. Cloxacillin strongy reduced both viability and sporulation. Numbers of viable cells slightly decreased with increase in concentrations of cloxacillin, cefaloridine, cephalexin, cefaclar, cefadroxil, cefoxitin, and cefotaxime. Inhibitory effects on sporulation of ts-4 were remarkable in cephalexin, cefaclor, cefadroxil, and cefotaxime.
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Ant i b jot icc 0 n Ce n t rat ion (~g / m I ) Fig. 2. Dose-response curves of viability and sporulation of B. cereus ts-4 to ~-lactam antibiotics. Antibiotics were added at the late-exponential phase of growth (00660 = 0.9) in G medium. Symbols: 0, viable cells ; . , spores Effect of time of addition of cephalexin on sporulation of B. cereus ts-4 B. cereus ts-4 cells in the exponential phase of growth were harvested and were resuspended and cultured in 11100 G medium to induce sporulation. Cephalexin and ampicillin were added at various times after resuspension and rates of sporulation were measured. Fig. 3 shows the effect of time of addition of these antibiotics on sporulation of B. cereus ts-4. Ampicillin had no effect on the rate of sporulation despite of times of addition at 25 /J-g/ml. When cephalexin was added at 3 hr after re-
suspension, the rate of sporulation fell to the lowest level. The sporulation rate increased at 4 hr. The result indicates that events or proteins exist around 3 hr after resuspension are sensitive to cephalexin. Fig. 4 shows the fluorescence and phase-contrast micrographs of B. cereus ts-4 after the resuspension. Cephalexin and ampicillin were added to the culture at 2hr after resuspension. Examination of DAPI-stained cells showed that the nucleoids had become axial filaments at 2 h after resuspension (Fig. 4 b). An asymmetric septum formation was observed at 7 hr and the completed asymmetric septum enclosed the forespore nucleoids Microbial. Res. 152 (1997) 3
229
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Time after resuspension (hr) Fig. 3. Effects of time of addition of cephalexin and ampicillin on sporulation rate of B. cereus ts-4. The exponentially growing cells of B. cereus ts-4 were harvested and were resuspended and cultured in 11100 G medium to induce sporulation. Cephalexin was added to the culture at various times after resuspension at 0.1 (0), 0.25 (~), and 2 (e) ).lg/ml. Ampicillin was added at 25 ).lg/ml (D). The rates of sporulation were determined 20 hr after the resuspension. Values were expressed as a percentage of the total cell population.
(Fig.4c). The formation of a forespore was apparent in ampicillin-treated and control cells. In contrast, the asymmetric septum formation was completely inhibited by cephalexin at 2 flg/ml (Fig. 4e). The result suggests that cephalexin inhibits the event necessary for the formation of asymmetric septum required to produce a spore.
(a)
(b)
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By using radioactive penicillin G, eight PBPs were detected in vegetative cells of B. cereus ts-4 and their molecular weights were calculated to be 106,000, 83,000, 75,000, 72,000, 63,000, 55,000, 46,000, and 32,000 (Fig. 5). Among these PBPs, bands of PBPs of molecular weights of 106,000, 83,000, 75,000, and 72,000 were decreased or disappeared in the electrophoregram by the prebinding with cephalexin. These bands were not decreased by the prebinding with ampicillin. In sporulating cells, only four bands of PBPs with molecular weights of 75,000, 72,000, 46,000, and 32,000 were detected. The PBPs with molecular weights of 72,000 and 75,000 were disappeared and decreased, respectively, by the prebinding with cephalexin at 1 flg/ml.
Discussion Among antibiotics tested, cephalexin, cefaclor, cefadroxiI, and cefotaxime strongly reduced the number of spore of B. cereus ts-4 than that of viable cells (Fig. 2). Cephalexin, cefadroxil and cefaclor contain a benzyl group on the side chain and amino group on the 9-u-carbon (Fig. 1). Cefotaxime also contains a free amino group in the substituents (Fig. 1). It was possible that the free amino group of these cephalosporins somehow has the potential in giving them binding specificity to
(d)
(e)
1 0 ~m Fig.4. Fluorescence and phase-contrast micrographs of B. cereus ts-4 after induction of sporulation. The exponentially growing cells of B. cereus ts-4 were resuspended and cultured in 11100 G meElium to induce sporulation. Cephalexin and ampicillin were added at 2 hr after the resuspension. (a) Cells at Oh after resuspension. (b) Cells at 2hr. (c) Control cells at 7 hr. (d) Cells in the presence of ampicillin (25 ).lg/ml) at 7hr. (e) Cells in the presence of cephalexin ( 2Ilg/ml) at 7 hr. 230
Microbiol. Res. 152 (1997) 3
Vegetative cells
Sporulating cells
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PBP(s) essential for sporulation of B. cereus ts-4. Although ampicillin contains a free amino group and benzyl group in the side chain (Fig. 1), it was not effective against both growth and sporulation of B. cereus (Fig. 2). B. cereus is a potent ~-lactamase producing microorganism, it produces three ~-lactamases (Nielsen and Lampen 1983). Ampicillin resistance of B. cereus observed in this stq~y should be due to the action of these ~-lactamases. By using radioactive penicillin, eight PBPs were detected in B. cereus ts-4 (Fig. 5). The PBPs with molecular weights of 106,000, 83,000, 75,000, 72,000, 63,000, 55,000,46,000, and 32,000 were numbered as PBP 1, 2, 3, 4, 5, 6, 7, and 8, respectively, according to their decreasing order of mobility in the SDS-PAGE (Fig. 5). In the initial stage of sporulation, at 4 h after resuspension, only four PBPs (PBPs 3, 4, 7, and 8) were detected in the membrane fraction of B. cereus (Fig. 5). The PBP 4 was the sole PBP disappeared by prebinding with cephalexin (Fig. 5). Cephalexin inhibited the asymmetric septum formation in B. cereus (Fig. 4). The drug also inhibited the sporulation of B. megaterium and B. subtilis (data not shown). It has been reported that cephalexin binds to PBP 3 and inhibits septum formation in E. coli (Spratt 1975). Cloxacillin is one of the most effective ~-lactam antibiotics against gram-positive bacteria and is specific to PBP showing the function as PBP 2 of E. coli (Buchanan and Strominger 1976). The drug was effective on both growth and sporulation (Fig. 1). Although it is necessary to test other antibiotics specific to PBP 2, the event related to the PBP 2 seems to be less important than that related to PBP having function as PBP 3 of E. coli in sporulation of B. cereus. Until now, two PBPs from B. subtilis were reported to be homologous to E. coli PBP 3. A homologous protein in B. subtilis (PBP
Fig. 5. SDS-PAGE profiles ofPBPs of B. cereus ts-4 detected by [3H]penicillin. Membrane proteins of the strain were prepared from vegetative cells and sporulating cells by the method described in the text. These proteins were incubated with [3H]penicillin G. After SDS-PAGE of the proteins, PBPs in the gel were detected by fluorography. To measure the bindings of cephalexin (CEX) and ampicillin (AMP) to PBPs, the membrane proteins were incubated with these ~-lac tams at 1 and 10 Ilg/ml before the addition of the radioactive penicillin G.
2B) had shown to be required for vegetative and sporulating septa formation (Daniel et al. 1994). The other homolog, B. subtilis SpoVD, which seems to be evolutionary related to E. coli PBP 3 since their respective genes, spoVD and pbpB, occupy similar chromosomal locations, immediately upstream from a conserved gener called murE (Yanouri et al.1993). Considered from the nature of affinity of PBP to cephalexin, it seems that B. cereus PBP 4 probably has the functional similarity with E. coli PBP 3 and B. subtilis PBP 2 Band/or SpoVD. Suginaka et al. (1972) have reported that five PBP bands were detected in B. cereus, the two more intense than the other three, by electrofocusing. However, they did not refer to the relationship between these PBPs and sporulation. This is the first extensive study of B. cereus PBPs. It seems that the PBP 4 is essential for sporulation-specific septum formation in B. cereus. Cloning and sequencing of a gene coding the PBP 4 will be reported in the near future.
References Bonner, W. M., Laskey, R. A. (1974): A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. 1. Biochem. 46, 83- 88. Bradford, M. M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Buchanan, C. E., Strominger, J. L. (1976): Altered penicillinbinding components in penicillin-resistant mutants of Bacillus subtilis. Proc. Nat. Acad. Sci. USA 73,1816-1820. Buchanan, C. E. (1979): Altered membrane proteins in a mini cell-producing mutant of Bacillus subtilis. J. Bacteriol. 139, 305-307. Microbial. Res. 152 (1997) 3
231
Buchanan, C. E., Sowell, M. O. (1983): Stability and synthesis of the penicillin-binding proteins during sporulation. J. Bacteriol. 156,545-551. Daniel, R. A., Drake, S., Buchanan, C. E., Scholle, R., Errington, J. (1994): The Bacillus subtilis spo VD gene encodes a mother-ceIl-specific penicillin-binding protein required for spore morphogenesis. J. Mol. BioI. 235, 209-220. Gollakota, K G., Halvorson, H. o. (1960): Biochemical changes occurring during sporulation of Bacillus cereus. J. Bacteriol. 79,1-8. Hiraga, S., Niki, H., Ogura, T, Ichinose, c., Mori, H., Ezaki, B., Jaffe', E. (1989): Chromosome partitioning in Escherichia coli: novel mutants producing a nucleate cells. J. Bacteriol. 171, 1496-1505. KJeppe, G., Strominger. J. L., (1979): Studies of the high molecular weight penicillin-binding proteins of Bacillus subtilis. J. BioI. Chern. 254,4856-4862. Laemmli, U. K (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685. Matsuno, K, Miyamoto, T, Yamaguchi, K, Sayed, Md., A., Kajiwara, T, Hatano, S. (1995): Identification of DNA binding proteins changed after induction of sporulation of Bacillus cereus. Biosci. Biotech. Biochem. 59, 231-235. Miyamoto, T, Matsuno, K, Yoshimoto, M., Hatano, S. (1991): Induction of spOlulation during chromosome replication by Bacillus cereus and changes in chromosomal proteins of cells that sporulated. (in Japanese) Nippon Nogeikagaku Kaishi, 65, 1769 -177 6. Nielsen, J. B., Lampen, J. O. (1983): Beta-Iactamase III of Ba-
232
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cillus cereus 569: membrane lipoprotein and secreted protein. Biochemistry 22,4652-4656. Sinagawa, K, Sato, K., Konuma, H., Matsusaka, N., Sugii, S. (1991): Fluid accumulation in mouse ligated intestine inoculated with the vascular permeability factor produced by Bacillus cereus. 1. Vet. Med. Sci. 53, 167-17l. Sinagawa, K, Takechi, T, Matsusaka, N., Sugii, S. (1992): Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody. Can. J. Microbiol. 38, 153-156. Sowell, M. 0., Buchanan, C. E. (1983): Changes in peniciIlinbinding proteins during spOlulation of Bacillus subtilis. J. Bacteriol. 153, 1331-1337. Spratt, B. G. (1975): Distinct penicillin-binding proteins involved in the division, elongation, and shape of Escherichia coli K-12. Proc. Nat. Acad. Sci. USA 72,2999-3003. Spratt, B. G. (1983): Penicillin-binding proteins and the future of p-Iactam antibiotics. J. Gen. Microbiol. 129, 1247-1260. Suginaka, H., Blumberg, P. M., Strominger, J. L. (1972): Multiple penicillin-binding components in Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Escherichia coli. J. BioI. Chern. 247, 5279-5288. Yanouri, A., Daniel, R. A., Errington, J., Buchanan C. E. (1993): Cloning and sequencing of the cell division gene pbpB, which encodes penicillin-binding protein 2B in Bacillus subtilis. J. Bacteriol. 175,7604-7616. Young, I. E., Fritz-James, P. C. (1959): Chemical and morphological studies of bacterial spore formation.: 1. The formation of spores in Bacillus cereus. J. Biophysic. and Biochem. 6,467-489.
Microbiological Research C!:) Gustav Fischer Verlag
Penicillin-binding protein sensitive to cephalexin in sporulation of Bacillus cereus Takahisa Miyamoto, Ken-ichiro Yamaguchi, Md. Abu Sayed, Ryo Sasahara, Ken-ichi Honjoh, Shoji Hatano Food Science and Technology, Faculty of Agriculture, Kyushu University, lO-I, Hakozaki 6-chome, Higashi-ku, Fukuoka, 812-81 Japan Accepted: March 10, 1997
Abstract Cephalexin, cefac1or, cefadroxil, and cefotaxime strongly inhibited sporulation of Bacillus cereus ts-4 at 1 Ilg/ml. Cephalexin was most inhibitory on the sporulation of B. cereus when the antibiotic was added at 3 h after induction of sporulation by nutrient downshift technique. Examination of 4' ,6-diamidino2-phenylindole-stained cells by fluorescence-phase contrast microscopy showed that cephalexin inhibited the formation of asymmetric septum. By using [3H]penicillin, eight penicillinbinding proteins (PBPi'i) were detected from the cells of B. cereus ts-4. Among them, four PBPs were also detected in sporulating cells. Affinity of cephalexin to PBPs were measured indirectly by competition for subsequent binding of radioactive penicillin O. Cephalexin strongly bound to PBP 4 with molecular weight of 72,000 in sporulating cells. Key words: Bacillus cereus-sporulation-penicillin binding protein-Cephalexin
Introduction Bacillus cereus is one of the food poisoning bacteria which produces heat resistant enterotoxin and spores (Sinagawa et al. 1991, 1992). The presence of its spores in food is one of the biggest problems in food industry because the spore is resistant to heating, ultraviolet light and drugs. It is very difficult to stelilize food contaminated with spores of B. cereus. We have studied the mechanism of sporulation of B. cereus to develop an effective method to inhibit sporulation. We previously showed the presence of chromosomal proteins that seem to control the initiation of sporulation of B. cereus ts-4 (Miyamoto et al. 1991; Matsuno et al. 1995). Corresponding author: T. Miyamoto
The formation of an asymmetrically situated division septum is an early event of sporulation of Bacillus (Young and Fitz-James 1959). The asymmetric division results in two distinct cells, that have radically different developmental fates (Young and Fitz-James 1959). This sporulation division contrasts with division during vegetative growth of Bacillus, in which the septum is symmetrically situated with respect to the ends of the dividing cell. The penicillin-binding proteins (PBPs) are membrane bound enzymes required for peptidoglycan synthesis and septum formation (Spratt 1983). There are at least seven PBPs produced by vegetative cells of Escherichia coli (Spratt 1975). There are various ~-lactam antibiotics and the target sites of some of them have been studied in detail on E. coli (Spratt 1975). Some of these antibiotics have specific target PBPs, for example, cefaloridine specifically binds to and inhibits PBP 1, mecillinam is specific for PBP 2 and cephalexin for PBP 3 of E. coli (Spratt 1975). The studies on the mechanism of action of penicillin on B. subtilis have shown the presence of PBPs that increase or decrease during sporulation (Buchanan und Sowell 1983 ; Kleppe and Strorninger 1979; Sowell and Buchanan 1983). However, the mechanism of the gene expression for these PBPs at the initial stage of sporulation is not clear. In this paper, we desclibe the effects of various ~-lac tam antibiotics on sporulation of B. cereus. We found that cephalexin, which is specific for PBP 3 of E. coli (Spratt 1975), inhibited the sporulation of B. cereus which had been induced to sporulate by nutrient downshift technique. The presence of PBP that has high affinity to cephalexin and its role in sporulation will be discussed. Microbiol. Res. 152 (1997) 3
227
Materials and methods Bacterial strains. Bacillus cereus ts-4 was a temperature
sensitive mutant for the initiation of chromosome replication delived from B. cereus JCM 2152 which had been obtained from Japan Collection of Microorganisms, Wako, Saitama, Japan (Miyamoto et al. 1991). Culture conditions. B. cereus was cultivated in YEEMM (Matsuno et al. 1995) containing 0.2% (NH4hS04' 1.4% (W/V) K ZHP0 4 , 0.6% KH zP0 4 , 0.1 % sodium citrate· 2H20, 0.02% MgS04 • 7H20, 0.5% glucose, 0.3% glutamic acid, 0.3% L-asparagine, 1% (WN) casamino acids (Difco Laboratories), and 0.02% yeast extract (Wako Pure Chemicals, Osaka, Japan) at 30°C for 24 hr with shaking. To examine the effects of various antibiotics (ampicillin, cloxacillin, cefaloridine, cephalexin, cefaclor, cefadroxil, cefoxitin, and cefotaxi me ) on sporulation, 100 III of the culture was transferred to modified G medium, G medium (Gollakota and Halvorson 1960) supplemented with 0.2% glutamic acid, and cultured at 30°C with shaking. The growth of bacteria was monitored by measuring optical density at 660 nm (OD 660 ). A 11100 G medium (Matsuno et al. 1995), in wich concentrations of ammonium sulfate, yeast extract and glucose were reduced to 111 00 of those of the modified G medium, was prepared and used to induce sporulation of B. cereus. Induction of sporulation of B. cereus. Sporulation was induced by the nutrient downshift technique. B. cereus
was cultured in 200 ml of YE-EMM at 30°C. At midexponential phase of growth, the cells were harvested by centrifugation at 2,000 x g for 10 min at room temperature. The cells were resuspended in 100 ml of 11100 G medium and incubated at 30°C to induce sporulation. Measurement of viable cells and spores. Total viable cells and spores of B. cereus were measured by plating
assay at 20 hr after a start of cultivation or after an induction of sporulation. In measurement of spores, the culture was heated at 85°C for 15 min before platmg. Combination Jluorescence-phase-contrast microscopy.
Bacterial cells, which had been harvested by centrifugation at 1,400 x g for 5 min from 1 ml of a culture, were resuspended in 0.1 ml of water. Then the cell suspension was mixed with 0.1 ml of 4' ,6-diamidino-2-phenylindole (DAPI) solution (50 Ilg/ml). The stained cells and nucleoids were visualized with a Nikon Optiphoto Microscope, x 2,400, by the "fluo-phase" method (Hiraga et al. 1989). Preparation of membranes and assay for PBPs. The membrane of B. cereus was prepared according to the
method of Buchanan (1979) except that the sonication 228
Microbial. Res. 152 (1997) 3
was carried out with a Branson sonifier model 185 at 60 w for ten 30-sec pulses for vegetative cells and twenty 30-sec pulses for sporulating cells. The membranes were frozen in a small volume of 50 mM TrisHCl buffer, pH 7.5, containing 1 mM MgCI 2, 1 mM phenylmethylsulfonylfluoride (PMSF). Proteins were measured by the dye-binding method of Bradford (1976). For the detection ofPBPby radioactive penicillin, the membrane fraction (about 500 Ilg proteins in the same Tris-HCl buffer) was incubated with 44 kEq of a [3H]penicillin G (814 gBq/mmole, Amersham) in a total volume of 30 III for 30 min at 25°C. The binding reaction was stopped by adding 2 III of cold penicillin G (300 mg/ml). The reaction mixture was mixed with 10 III of 0.1 M Tris-HCl buffer, pH 6.8, containing 40% (WN) glycerol, 8% (WN) sodium dodecyl sulfate (SDS), and 0.004% bromophenol blue, and boiled for 3 min. The mixture was then centrifuged at 10,000 x g for 10 min. Five microliters of the sample solution (about 63 Ilg proteins) was loaded onto a lane of a slab gel. Separation of membrane proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemrnli (1970) in a 10% (WN) polyacrylamide gel (l x 90 x 80 mm) containing 0.1 % SDS at room temperature at 25 mA per gel. To measure the bindings of unlabeled ~-lactam antibiotics, the membrane fraction was incubated with various concentrations of cephalexin and ampicillin for 10 min before the addition of the labeled penicillin. The [3H]penicillin-bound proteins in the gel were detected by fluorography (Bonner and Laskey 1974).
Results Effects of various (3-lactam antibiotics on sporulation of B. cereus
To examine the effects of various ~-lactam antibiotics on sporulation, antibiotics were added to the culture of B. cereus ts-4 in G medium at various concentrations at late-exponential phase of growth (OD660 = 0.9). Fig. 1 shows the structures of antibiotics tested. Fig. 2 shows the effects of various ~-lactam antibioties on viability and sporulation of B. cereus ts-4. Ampicillin had no effect on both viability and sporulation of B. cereus ts-4 even at 10 Ilg/ml. Cloxacillin strongy reduced both viability and sporulation. Numbers of viable cells slightly decreased with increase in concentrations of cloxacillin, cefaloridine, cephalexin, cefaclar, cefadroxil, cefoxitin, and cefotaxime. Inhibitory effects on sporulation of ts-4 were remarkable in cephalexin, cefaclor, cefadroxil, and cefotaxime.
O
n
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Fig. 1. Structures of ~-lac tam antibiotics used in this study.
Cefolaxi e Cloxacilli n
10'0 ~
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NII 2
sv'!'rC_CO NH ~ ~ II -.JJ II s
Cefoxi ti n
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Cefalorid i ne Cephalexin
Cefaclor
Cefadroxi l
Cefoxilin
Cefotaxime
109 10 8 10 7 10 6
0
10
0
10
0
10
0
10
0
10
0
10
0
10
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10 100
Ant i b jot icc 0 n Ce n t rat ion (~g / m I ) Fig. 2. Dose-response curves of viability and sporulation of B. cereus ts-4 to ~-lactam antibiotics. Antibiotics were added at the late-exponential phase of growth (00660 = 0.9) in G medium. Symbols: 0, viable cells ; . , spores Effect of time of addition of cephalexin on sporulation of B. cereus ts-4 B. cereus ts-4 cells in the exponential phase of growth were harvested and were resuspended and cultured in 11100 G medium to induce sporulation. Cephalexin and ampicillin were added at various times after resuspension and rates of sporulation were measured. Fig. 3 shows the effect of time of addition of these antibiotics on sporulation of B. cereus ts-4. Ampicillin had no effect on the rate of sporulation despite of times of addition at 25 /J-g/ml. When cephalexin was added at 3 hr after re-
suspension, the rate of sporulation fell to the lowest level. The sporulation rate increased at 4 hr. The result indicates that events or proteins exist around 3 hr after resuspension are sensitive to cephalexin. Fig. 4 shows the fluorescence and phase-contrast micrographs of B. cereus ts-4 after the resuspension. Cephalexin and ampicillin were added to the culture at 2hr after resuspension. Examination of DAPI-stained cells showed that the nucleoids had become axial filaments at 2 h after resuspension (Fig. 4 b). An asymmetric septum formation was observed at 7 hr and the completed asymmetric septum enclosed the forespore nucleoids Microbial. Res. 152 (1997) 3
229
0
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0
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....
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40
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Detection of PBPs of B. cereus by using radioactive penicillin
~
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20 0
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2
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4
5
6
7
Time after resuspension (hr) Fig. 3. Effects of time of addition of cephalexin and ampicillin on sporulation rate of B. cereus ts-4. The exponentially growing cells of B. cereus ts-4 were harvested and were resuspended and cultured in 11100 G medium to induce sporulation. Cephalexin was added to the culture at various times after resuspension at 0.1 (0), 0.25 (~), and 2 (e) ).lg/ml. Ampicillin was added at 25 ).lg/ml (D). The rates of sporulation were determined 20 hr after the resuspension. Values were expressed as a percentage of the total cell population.
(Fig.4c). The formation of a forespore was apparent in ampicillin-treated and control cells. In contrast, the asymmetric septum formation was completely inhibited by cephalexin at 2 flg/ml (Fig. 4e). The result suggests that cephalexin inhibits the event necessary for the formation of asymmetric septum required to produce a spore.
(a)
(b)
(c)
By using radioactive penicillin G, eight PBPs were detected in vegetative cells of B. cereus ts-4 and their molecular weights were calculated to be 106,000, 83,000, 75,000, 72,000, 63,000, 55,000, 46,000, and 32,000 (Fig. 5). Among these PBPs, bands of PBPs of molecular weights of 106,000, 83,000, 75,000, and 72,000 were decreased or disappeared in the electrophoregram by the prebinding with cephalexin. These bands were not decreased by the prebinding with ampicillin. In sporulating cells, only four bands of PBPs with molecular weights of 75,000, 72,000, 46,000, and 32,000 were detected. The PBPs with molecular weights of 72,000 and 75,000 were disappeared and decreased, respectively, by the prebinding with cephalexin at 1 flg/ml.
Discussion Among antibiotics tested, cephalexin, cefaclor, cefadroxiI, and cefotaxime strongly reduced the number of spore of B. cereus ts-4 than that of viable cells (Fig. 2). Cephalexin, cefadroxil and cefaclor contain a benzyl group on the side chain and amino group on the 9-u-carbon (Fig. 1). Cefotaxime also contains a free amino group in the substituents (Fig. 1). It was possible that the free amino group of these cephalosporins somehow has the potential in giving them binding specificity to
(d)
(e)
1 0 ~m Fig.4. Fluorescence and phase-contrast micrographs of B. cereus ts-4 after induction of sporulation. The exponentially growing cells of B. cereus ts-4 were resuspended and cultured in 11100 G meElium to induce sporulation. Cephalexin and ampicillin were added at 2 hr after the resuspension. (a) Cells at Oh after resuspension. (b) Cells at 2hr. (c) Control cells at 7 hr. (d) Cells in the presence of ampicillin (25 ).lg/ml) at 7hr. (e) Cells in the presence of cephalexin ( 2Ilg/ml) at 7 hr. 230
Microbiol. Res. 152 (1997) 3
Vegetative cells
Sporulating cells
CEX AMP I CEX AMPI I 1111 ~II 10 (Ilg/ml) Molecular Cant. 1 10 1 1 0 Cant. 1 10 weight
I
106 , 000 83 , 000"
75.000 ....
72,000"-
63 , 000~
55.000 ..... 46 , 000'32,000'-
PBP(s) essential for sporulation of B. cereus ts-4. Although ampicillin contains a free amino group and benzyl group in the side chain (Fig. 1), it was not effective against both growth and sporulation of B. cereus (Fig. 2). B. cereus is a potent ~-lactamase producing microorganism, it produces three ~-lactamases (Nielsen and Lampen 1983). Ampicillin resistance of B. cereus observed in this stq~y should be due to the action of these ~-lactamases. By using radioactive penicillin, eight PBPs were detected in B. cereus ts-4 (Fig. 5). The PBPs with molecular weights of 106,000, 83,000, 75,000, 72,000, 63,000, 55,000,46,000, and 32,000 were numbered as PBP 1, 2, 3, 4, 5, 6, 7, and 8, respectively, according to their decreasing order of mobility in the SDS-PAGE (Fig. 5). In the initial stage of sporulation, at 4 h after resuspension, only four PBPs (PBPs 3, 4, 7, and 8) were detected in the membrane fraction of B. cereus (Fig. 5). The PBP 4 was the sole PBP disappeared by prebinding with cephalexin (Fig. 5). Cephalexin inhibited the asymmetric septum formation in B. cereus (Fig. 4). The drug also inhibited the sporulation of B. megaterium and B. subtilis (data not shown). It has been reported that cephalexin binds to PBP 3 and inhibits septum formation in E. coli (Spratt 1975). Cloxacillin is one of the most effective ~-lactam antibiotics against gram-positive bacteria and is specific to PBP showing the function as PBP 2 of E. coli (Buchanan and Strominger 1976). The drug was effective on both growth and sporulation (Fig. 1). Although it is necessary to test other antibiotics specific to PBP 2, the event related to the PBP 2 seems to be less important than that related to PBP having function as PBP 3 of E. coli in sporulation of B. cereus. Until now, two PBPs from B. subtilis were reported to be homologous to E. coli PBP 3. A homologous protein in B. subtilis (PBP
Fig. 5. SDS-PAGE profiles ofPBPs of B. cereus ts-4 detected by [3H]penicillin. Membrane proteins of the strain were prepared from vegetative cells and sporulating cells by the method described in the text. These proteins were incubated with [3H]penicillin G. After SDS-PAGE of the proteins, PBPs in the gel were detected by fluorography. To measure the bindings of cephalexin (CEX) and ampicillin (AMP) to PBPs, the membrane proteins were incubated with these ~-lac tams at 1 and 10 Ilg/ml before the addition of the radioactive penicillin G.
2B) had shown to be required for vegetative and sporulating septa formation (Daniel et al. 1994). The other homolog, B. subtilis SpoVD, which seems to be evolutionary related to E. coli PBP 3 since their respective genes, spoVD and pbpB, occupy similar chromosomal locations, immediately upstream from a conserved gener called murE (Yanouri et al.1993). Considered from the nature of affinity of PBP to cephalexin, it seems that B. cereus PBP 4 probably has the functional similarity with E. coli PBP 3 and B. subtilis PBP 2 Band/or SpoVD. Suginaka et al. (1972) have reported that five PBP bands were detected in B. cereus, the two more intense than the other three, by electrofocusing. However, they did not refer to the relationship between these PBPs and sporulation. This is the first extensive study of B. cereus PBPs. It seems that the PBP 4 is essential for sporulation-specific septum formation in B. cereus. Cloning and sequencing of a gene coding the PBP 4 will be reported in the near future.
References Bonner, W. M., Laskey, R. A. (1974): A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. 1. Biochem. 46, 83- 88. Bradford, M. M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Buchanan, C. E., Strominger, J. L. (1976): Altered penicillinbinding components in penicillin-resistant mutants of Bacillus subtilis. Proc. Nat. Acad. Sci. USA 73,1816-1820. Buchanan, C. E. (1979): Altered membrane proteins in a mini cell-producing mutant of Bacillus subtilis. J. Bacteriol. 139, 305-307. Microbial. Res. 152 (1997) 3
231
Buchanan, C. E., Sowell, M. O. (1983): Stability and synthesis of the penicillin-binding proteins during sporulation. J. Bacteriol. 156,545-551. Daniel, R. A., Drake, S., Buchanan, C. E., Scholle, R., Errington, J. (1994): The Bacillus subtilis spo VD gene encodes a mother-ceIl-specific penicillin-binding protein required for spore morphogenesis. J. Mol. BioI. 235, 209-220. Gollakota, K G., Halvorson, H. o. (1960): Biochemical changes occurring during sporulation of Bacillus cereus. J. Bacteriol. 79,1-8. Hiraga, S., Niki, H., Ogura, T, Ichinose, c., Mori, H., Ezaki, B., Jaffe', E. (1989): Chromosome partitioning in Escherichia coli: novel mutants producing a nucleate cells. J. Bacteriol. 171, 1496-1505. KJeppe, G., Strominger. J. L., (1979): Studies of the high molecular weight penicillin-binding proteins of Bacillus subtilis. J. BioI. Chern. 254,4856-4862. Laemmli, U. K (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680-685. Matsuno, K, Miyamoto, T, Yamaguchi, K, Sayed, Md., A., Kajiwara, T, Hatano, S. (1995): Identification of DNA binding proteins changed after induction of sporulation of Bacillus cereus. Biosci. Biotech. Biochem. 59, 231-235. Miyamoto, T, Matsuno, K, Yoshimoto, M., Hatano, S. (1991): Induction of spOlulation during chromosome replication by Bacillus cereus and changes in chromosomal proteins of cells that sporulated. (in Japanese) Nippon Nogeikagaku Kaishi, 65, 1769 -177 6. Nielsen, J. B., Lampen, J. O. (1983): Beta-Iactamase III of Ba-
232
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cillus cereus 569: membrane lipoprotein and secreted protein. Biochemistry 22,4652-4656. Sinagawa, K, Sato, K., Konuma, H., Matsusaka, N., Sugii, S. (1991): Fluid accumulation in mouse ligated intestine inoculated with the vascular permeability factor produced by Bacillus cereus. 1. Vet. Med. Sci. 53, 167-17l. Sinagawa, K, Takechi, T, Matsusaka, N., Sugii, S. (1992): Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody. Can. J. Microbiol. 38, 153-156. Sowell, M. 0., Buchanan, C. E. (1983): Changes in peniciIlinbinding proteins during spOlulation of Bacillus subtilis. J. Bacteriol. 153, 1331-1337. Spratt, B. G. (1975): Distinct penicillin-binding proteins involved in the division, elongation, and shape of Escherichia coli K-12. Proc. Nat. Acad. Sci. USA 72,2999-3003. Spratt, B. G. (1983): Penicillin-binding proteins and the future of p-Iactam antibiotics. J. Gen. Microbiol. 129, 1247-1260. Suginaka, H., Blumberg, P. M., Strominger, J. L. (1972): Multiple penicillin-binding components in Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Escherichia coli. J. BioI. Chern. 247, 5279-5288. Yanouri, A., Daniel, R. A., Errington, J., Buchanan C. E. (1993): Cloning and sequencing of the cell division gene pbpB, which encodes penicillin-binding protein 2B in Bacillus subtilis. J. Bacteriol. 175,7604-7616. Young, I. E., Fritz-James, P. C. (1959): Chemical and morphological studies of bacterial spore formation.: 1. The formation of spores in Bacillus cereus. J. Biophysic. and Biochem. 6,467-489.