Purification and chemical analysis of a bacteriocin from the oral bacterium Streptococcus mutans RM-10

Archs oral Bid. Vol. 21, pp. 721 to 727, 1982 Printed in Great Britain. All rights reserved

0003-9969/82/090721-07$03.00/O Copyright 0 1982 Pergamon Press Ltd

PURIFICATION AND CHEMICAL ANALYSIS OF A BACTERIOCIN FROM THE ORAL BACTERIUM STREPTOCOCCUS MUTANS RM-10 H.

FUKUSHIMA,

SAEKO FUKUSHIMA,

Department

T. UMEMOTO, HIROKO

FUKUHARA

of Bacteriology, Osaka Dental University, Higashi-ku, Osaka 540, Japan

and H.

SAGAWA

l-47 Kyobashi,

Summary-Bacteriocin from supernatant of broth-grown cultures of Streptococcus mutans Rm-10 was purified by ammonium sulphate fractionation, precipitation at pH 3.1, and gel filtration on Sephadex G-200 and Sepharose 2B. The purified preparation was shown to be homogeneous by gel filtration and immunoelectrophoresis. A molecular weight of 973,000 was determined by equilibrium sedimentation with UV scanning. Electron microscopy of this purified preparation revealed a fibrous structure with a homogeneous morphology. The bacteriocin was essentially proteinaceous in nature, containing 90 pg phosphorus and 34 pg hexose per mg of protein, and was found to be rich in aspartic acid, glycine, alanine, valine, leucine and lysine.

INTRODUCTION

Since bacteriocin production from Streptococcus mutans was reported by Kelstrup and Gibbons (1969), much information has accumulated in regard to production, properties, genetics and partial purification of bacteriocins of this species (Delisle, 1975; Hamada and Ooshima, 1975a; Paul and Slade, 1975; Yamamoto et al., 1975; Rogers, 1976a,b; Kelstrup and Funder-Nielsen, 1977; Macrina et al., 1977). In our preliminary report (Fukushima et al., 1978a), it was established that an extracellular bacteriocin can be isolated from the supernatant of Strep. mutans Rm-10 culture fluid with 60 per cent ammonium sulphate precipitation and gel filtration. This crude bacteriocin is sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme and phospholipase C, and bactericidal against all strains of Strep. mutans tested (AHT, HS-6, BHT, FA-1, Ingbritt, GS-5, 10449, Ki-R, SL-1, 6715-15, OMZ 175 and Kl), against Staphylococcus aureus 209P, Actinomyces viscosus ATCC 19246, Actinomyces naeshndii ATCC 12104 and Bacillus subtilis PC1 219, but not Escherichia coli B and Fusobacterium nucleatum ATCC 10953 and 25586. It was resistant to heating at 100°C for 30 min and stable at pH 6.&l 1.0, but labile at pH 2.s5.0. The spectrum of its inhibitory action against a variety of other Gram-positive bacteria is similar to bacteriocins described by other workers (Hamada and Ooshima, 1975b; Rogers, 1976a). The ecological (van der Hoeven and Rogers, 1979) and therapeutic roles of these bacteriocins are of considerable interest. However, bacteriocins of Strep. mutans are not readily obtained in a cell-free state from culture fluids, with a few exceptions (Delisle, 1975; Hamada and Ooshima, 1975a; and Paul and Slade, 1975). MATERIALS

AND

METHODS

Organisms and medium The bacteriocin-producing Strep. mutuns Rm-10 and the highly bacteriocin-sensitive Streptococcus fue-

calis ODU have been described previously (Fukushima et cd., 1980). For production of bacteriocin, strain Rm-10 was grown in trypticase soy broth (TSB, Baltimore Biol. Lab., Cockeysville, Md., U.S.A.). Crude bacteriocin preparation One hundred millilitres of overnight culture of Strep. mutans Rm-IO was inoculated in 101 of fresh TSB and aerobically incubated at 37°C without shaking. When 75 per cent bacterial lysis had occurred (30-36 h after inoculation), cells and cell debris were removed by centrifugation at 12,000g for 50 min. Solid ammonium sulphate (39Og/l of solution) was slowly added to the supernatant solution and, after 16 h of stirring at 4”C, the resulting precipitate was collected by centrifugation at 12,000 g for 50 min. The precipitate was dissolved in 350 ml of 0.05 M trishydrochloride at pH 8.62, and dialysed for 3 days against 20 1 of the same buffer. Bacteriocin assay Samples were assayed for bacteriocin content by a technique essentially the same as that described by Goebel and Barry (1958). A drop of overnight culture of indicator strain was mixed with 4 ml of melted soft agar and poured on the surface of a trypticase soy agar (BBL) plate. Standard drops of serial dilutions of the bacteriocin solution were placed on the seeded plate with a micropipette. After incubation overnight at 37”C, the highest dilution to give a clearly visible area of inhibited growth was determined. The reciprocal of the dilution gave the bacteriocin titre in units/ml. Immunization Purified Rm-10 bacteriocin (2.5 mg, 16.0 arbitrary units (AU)/mg of protein) in Freund’s incomplete adjuvant (Difco, Lab., Detroit, Mich., U.S.A.) was used for primary immunization of rabbits, which then received four intravenous injections of 1.2mg of the bacteriocin for 2 weeks. One week after the last injection, blood was sampled. The titre of the antiserum

721

122

H. Fukushima et ul

obtained by the double-diffusion lony, 1949) was 1: 8.

method

(Ouchter-

Immunoelectrophoresis Immunoelectrophoresis was performed by the method of Cawley (1969) with minor modification. Agarose A-37 (Nakarai Chem., Kyoto, Japan) at a concentration of 1.2 per cent melted in barbital buffer (pH 8.6, u = 0.06) was poured on micro-slides as a layer 2 mm thick. Electrophoresis was carried out at a constant current of 3 mM/plate at room temperature until dye reached the end of the plate. Following the run, a 1 mm-wide longitudinal well was cut and filled with serum. Subsequent immunodiffusion was done in a moist chamber at 4°C for 24 h. Analyrical

centrifuyatiorl

Bacteriocin solution at a concentration of 400&m] was used for analytical centrifugation performed at 320g for 24 h at 10°C in an analytical ultracentrifuge. Model 282 (Hitachi Ltd, Tokyo, Japan), equipped for sedimentation equilibrium with UV scanning. Molecular weight was calculated from an equation with the gradient (dlnC/dr*) obtained on the scanner chart. Electron microscopy Negatively-stained specimens were prepared using a drop of 1 per cent phosphotungstic acid adjusted to pH 7.2. Electron micrographs were taken with an electron microscope, Model HU-12A (Hitachi Ltd, Tokyo, Japan) at an operation voltage of 100 kV. Anal~~tical methods Protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin as the standard. Specific activity of bacteriotin solutions is defined as the number of bacteriocin

1.2 r

Sephodex

G-200

units/mg of protein. Total phosphorus, hexose, pentose and methyl pentose were determined by the methods of Lowry et a/. (1954), Ashwell (1957), Mejbaum (1939) and Dische and Shettles (1948) respectively. For analysis of the amino acid composition, 500 ktl of the diaiysed material containing 12mg/ml of protein was lyophilysed in a hydrolysis tube. The lyophilysed material was then taken up in 6 M HCI, and the tube was sealed and hydrolysed at 100°C for 40 h. The amino acid composition of the hydrolysate was determined on an automatic amino-acid analyser. Mode1 KLA-5 (Hitachi Ltd. Tokyo, Japan). Purijcation

of bacteriocin

The concentrated crude bacteriocin solution from ammonium sulphate fractionation was not retarded on carboxymethyl (CM)-cellulose, but strongly absorbed to diethylaminoethyl (DEAE)- and ECTEOLA-cellulose (SERVA Feinbiochemica, Heidelberg, W. Germany). Moreover, protein was not eluted from the DEAE- and ECTEOLA-cellulose with an increasing salt gradient at constant pH. Therefore. the purification of the bacteriocin was performed by isoelectric precipitation (Holland, 1961) and gel filtration. The crude bacteriocin was dialysed for 24 h against 10 I of 0.05 M citrate buffer, pH 3.1. The precipitate was collected by centrifugation at 46,600 g for 20 mitt, washed several times and dialysed against tris-hydrochloride (pH 8.62) to facilitate solution. The first precipitation at pH 3.1 resulted in a product three times purer without loss of activity. After precipitation at pH 3.1, 105 ml of the material (3.99 mg/ml of protein) was applied in 15 ml portions to a Sephadex G-200 column (2.6 x 1OOcm) and eluted with the same buffer at a flow rate of 15 ml/h. Text Fig. la shows a typical elution profile, i.e. the bacteriocin activity was found in the first fractions containing material which absorbed light at 280nm. The active Sephamse

28 C

0

20

Fig. 1. Filtration profile of Rm-10 described in the text. (O-O),

40

60 Fraction

20

40

60

number

bacteriocin on Sephadex G-200 bacteriocin activity; (t-o),

and Sepharose 2B. Conditions material adsorbing at 280 nm.

are

Purification

Table 1. Yield and specific activity

Stage Supernatant (NH&SOL fraction Precipitation at pH 3.1 Sephadex G-200 Precipitation at pH 3.1 Sepharose 2B * Arbitrary

of Rm-10 bacteriocin procedure

at various

stages in the purification

Total activity (AU)*

Specific activity (AU/mg of protein)

Yield (%)

10,OOo 350 105

4000 3500 3408

0.048 2.42 8.02

100 87.5 85.2

1 51 168

105 105

3360 3200

13.33 15.24

84.0 80.0

280 320

70

2240

16.0

56.0

336

Vol. (ml)

Times purified

units.

fractions were pooled and concentrated by use of polyethylene glycol powder to a final volume of 105 ml. After re-precipitation at low pH, dissolved preparations were applied to a Sepharose 2B column (2.6 x 1OOcm) and eluted with the same buffer, as shown in Fig. lb. Bacteriocin activity was found in the fraction following the first peak. After collection and precipitation, the dissolved material was reapplied to a Sepharose 2B column, resulting in a single active peak, as shown in Fig. lc.

RESULTS

The results of the purification of Rm-10 bacteriocin are summarized in Table 1. The purified bacteriocin had a specific activity 336 times higher than that found in the supernatant of culture fluid. After application to a Sepharose 2B column, 56.0 per cent of the specific activity was recovered. Homogeneity

723

of Strep. mutans bacteriocin

of bacteriocin

Text Fig. 2 is a photograph of an immunoelectrophoresis plate. Rm-10 bacteriocin gave single zones against the homologous antiserum, indicating the immunochemical homogeneity of the antigen. An electron micrograph of negatively-stained bacteriocin is shown in Plate Fig. 3. It appears as aggregates of homologous fibrous structures (Fig. 3a). The arrow in Fig. 3b shows a single fibre of bacteriocin. The elution profile on Sephadex G-75 with 7 M urea or 0.1 per cent sodium dodecyl sulphate (SDS) was the same as that of the control and failed to show subunits.

Molecular weight determination Purified bacteriocin (16.0AU/mg of protein) did not migrate when examined by electrophoresis in 3.75 per cent polyacrylamide gel containing 1 per cent SDS, and so the mol wt of the bacteriocin was first estimated by gel filtration on Sepharose CL-6B(Pharmacia) in 6M guanidine hydrochloride (Ansari and Mage, 1977) using blue dextran (mol. wt 2,OOO,OOO, Pharmacia), thyroglobulin (mol. wt. 669,000, Pharmacia) and ferritin (mol. wt 44O,OOO,Pharmacia) as standards. The active fractions were eluted between blue dextran and thyrogrobulin as shown in Text Fig. 4. The exact mol. wt of the bacteriocin was then determined by analytical centrifugation. The dlnC/dr’ obtained from sedimentation equilibrium with UV scanning was 0.245 (Text Fig. 5), and the mol. wt was calculated to be 973,000. Chemical composition The bacteriocin was essentially protein in nature, containing 9Opg phosphorus and 34pg hexose per of protein. Small amounts of pentose mg (0.163 mg/ml) and methyl pentose (0.107 mg/ml) were blue dextran

4 mol. wt 2QO0,OOO !

thyroglobulin mol. wt 669,000 t

i

territin

i

4 mol. wt 440,000

h

+ n :

50

40

Fig. 2. Diagram of immunoelectrophoresis of Rm-10 bacteriocin. Immunoelectrophoresis was carried out at a constant current of 3 mA/plate with barbital buffer (pH 8.6, u = 0.06). Upper and lower well, purified bacteriocin solution (16.0 AU/mg of protein); central longitudinal well, anti-bacteriocin rabbit serum.

Fraction

number

Fig. 4. Elution profile for Rm-IO bacteriocin and standard solution from Sepharose CL-6B in 6 M guanidine HCI. Standard solution contains 1 mg of blue dextran, thyroglobulin and ferritin, respectively. (O---O), Bacteriocin solution; (O--O), standard solution.

H. Fukushima

724 -0.250

x

L

x’

dlnC .0.245 77

d

-

I.250

I1

46

x/ I

I

I

50

I

I 52

f 2h12)

Fig. 5. dlnC/dr’ of Rm-10 bacteriocin obtained from sedimentation equilibrium with UV scanning (280 nm). 100 ~1 of purified bacteriocin (400 fig/ml) was applied to a doublesector cell (Rotor: RAM 60). Centrifugal conditions are described in the text. The dlnC/d? value obtained was 0.245.

also present. In thin layer chromatography, rhamnose, galactose and glucose were detected. The bacteriocin was found to be rich in aspartic acid, glycine, alanine, valine, laucine and lysine, as shown in Table 2. DISCUSSION

Our earlier studies (Fukushima et ul., 1978a; see Introduction) showed that Strep. mutans Rm-IO produced a bacteriocin in a cell-free state when grown in TSB, and that Rm-IO bacteriocin was produced in the cell, not bound on the cell surface, and released from producing cells after cell lysis occurred. This assumption was based on two observations. First, maximum bacteriocin was released when 75 per cent cell lysis occurred. Second, no detectable bacteriocin was obtained from cell extraction (before and after lysis) with 5 per cent NaCl (Jetten, Vogels and de Windt, 1972). Although this bacteriocin was stable physico-chemically, its purification was difficult because the bacteriocin was not eluted from DEAETable

2.

Amino

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

acid composition bacteriocin

of

Rm-10

nmol/mg

Molar ratio

597.8 357.1 296.2 390.3 58.5 450.1 570.8 470.5 115.3 380.1 477.5 167.0 253.1 453.1 149.4 253.5

1.33 0.79 0.66 0.87 0.13 1.00 1.27 1.05 0.26 0.84 1.06 0.37 0.56 1.01 0.33 0.56

et al.

and ECTEOLA-cellulose columns after adsorption, even with increasing salt concentration. We now report that crude bacteriocin is precipitated when dialysed against citrate buffer (pH 3.1) and that the precipitate dissolved without loss of bacteriotin activity when dialysed against tris buffer (pH 8.62). At pH 3.1, bactericidal activity was completely absent from the supernatant of the suspension. This treatment resulted in an increase of specific activity from 2.42 to 8.02 AU/mg. As a result of the whole purification procedure, the specific activity increased 336-fold from that found in the culture supernatant of Strep. mutans Rm-10. This value is three times higher than that of mutacin from Strep. mutans GS-5 (Paul and Slade, 1975) and of sanguicin from Strep. sunguis N-2 (Fujimura and Nakamura, 1979). There are some similarities between GS-5 mutacin and Rm-10 bacteriocin in sensitivity to proteolytic enzymes and resistance to heat. However, the stable pH range and the activity spectra of the two bacteriotins appear to be quite different, indicating that they are separate substances. The purified Rm-10 bacteriocin is considered as a homogeneous substance, judging from the results of chromatography and immunoelectrophoresis studies. Electron microscopic examination supports these findings. Bradley (1967) separated bacteriocins into two main groups-low mol. wt and high mol. wt. Rm-10 bacteriocin appears to belong to the latter. In many cases, the structure of high mol. wt bacteriocins is similar to that of defective phages or phage tails (Kageyama, 1964; Chen and Tai, 1972; Gissman and Lotz, 1975). Strep. mutans Rm-10 was thought to be a lysogenic strain, because mutants derived from strain Rm-10 released defective phage when induced by mitomycin C, although no phage particles were observed after induction of the parent strain (Fukushima, 1976). However, Rm-10 bacteriocin is morphologically different from the phage derived via the mutant (Fig. 3). Amino acid analysis showed that Rm-10 bacteriotin was rich in certain amino acids. Comparison of Rm-10 bacteriocin with that of sanguicin (Fujimura and Nakamura, 1979) and diplococcin from Streptococcus cremoris 346 (Davey and Richardson, 1981) revealed considerable similarities as to major components: aspartic acid, glutamic acid, glycine, alanine, leucine and lysine. This result (Table 2) suggests that Rm-IO bacteriocin is acidic. This is in agreement with the fact that the bacteriocin can be precipitated at pH 3.1 and adsorbed on DEAE- and ECTEOLA-cellulose, but not on CM-cellulose column, and therefore is unlike most other streptococcal bacteriocins. Acknowledgements-We would like to thank Dr J. Kelstrup for his critical reading and correcting of the English version of the manuscript. REFERENCES Ansari A. A. and Mage R. G. 1977. Molecular-weight estlmation of proteins using Sepharose CL-6B in guanidine hydrochloride. J. Chromat. 140, 98-102. Ashwell G. 1957. Calorimetric analysis of sugars. In: Methods in Enzymology III (Edited by Colowick S. P. and Kaplan N. 0.) p. 73. Academic Press, New York. Bradley D. E. 1967. Ultrastructure and . _ _. ___ _.of bacteriophages bacter1oCins. Sact. Kev. 31, 23&314

Purification

of Strep. mutans bacteriocin

Cawley L. P. 1969. Eiectrophoresis and Immunoelectrophoresis. Chap. 1, p.1. Little Brown, Boston, Ma. Chen C. P. and Tai F. H. 1972. Purification and characteristics of Pyocin B39. Antimicrob. Ag. Chemother. 1, 1599163. Davey G. P. and Richardson B. C. 1981. Purification and some properties of Diplococcin from Streptococcus cremoris 346. Appi. Environ. Microbioi. 41, 84-89. Delisle A. L. 1975. Productions of bacteriocins in a liquid medium by Streptococcus mutans. Antimicrob. Ag. Chemother. 8, 707-712. Dische Z. and Shettles L. B. 1948. A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J. biol. Chem. 175, 595-603. Foulds J. 1972. Purification and partial characterization of a bacteriocin from Serratia marcescens. J. Bact. 110, 100-1009. Fujimura S. and Nakamura T. 1979. Sanguicin, a bacteriotin of oral Streptococcus sanauis. Antimicrob. Ag. Chemother. 16, 2621265. Fukushima H. 1976. Thesis. Studies on bacteriouhages of cariogenic streptococci. J. Osaka odont. Sot. 39,. l-29. (In Japanese.) Fukushima H., Fukushima S., Umemoto T., Tsuboi Y. and Sagawa H. 1978a. Studies on bacteriocin from Streptococcus mutans Rm-lO.-Isolation and characterization. J. Osaka odont. Sot. 41,439-446. (In Japanese.) Fukushima H., Fukushima S., Umemoto T., Tsuboi Y., Nishiura 0. and Sagawa H. 1978b. Studies on bacteriotin from Streptococcus mutans Rm-lo.-Influences of cultural conditions to bacteriocin production. J. Osaka odont. Sot. 41, 824830. (In Japanese.) Fukushima H., Tsuboi Y., Fukushima S. and Sagawa H. 1980. The elimination of bacteriocin production in the oral bacterium Streptococcus mutans. Archs oral Biol. 25, 7677771. Gissman L. and Lotz W. 1975. Isolation and characterization of rod-shaped bacteriocin from a strain of Rhizobium. J. gen. Virol. 27, 379-383. Goebel W. F. and Barry G. T. 1958. Colicine K II. The preparation and properties of s substance having colicine K activity. J. exp. Med. 107, 1855209. Hamada S. and Ooshima T. 1975a. Production and properties of bacteriocins (mutacins) from Streptococcus mutans. Archs oral Biol. 20, 641-648. Hamada S. and Ooshima T. 1975b. Inhibitory spectrum of a bacteriocin like substance (mutacin) produced by some strains of Streptococcus mutans. J. dent. Res. 54, 14G-145.

Plate

cm. 2719-n

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Herschman H. R. and Helinski D. R. 1967. Purification and characterization of colicin E2 and colicin ES. J. bioi. Chem. 242, 536G5368. Jetten A. M., Vogels G. D. and de Windt F. 1972. Production and purification of a Staphylococcus epidermidis bacteriocin. J. Bact. 112, 235-242. van der Hoeven J. S. and Rogers A. H. 1979. Stability of the resident microflora and the bacteriocinogeny of Streptococcus mutans as factors affecting its establishment in specific pathogen-free rats. Infect. Immun. 23, 206212. Holland I. B. 1961. The purification and properties of Megacin, a bacteriocin from Bacillus megaterium. Biothem. J. 78, 641-648. Kageyama M. 1964. Studies on a pyocin. I. Physical and chemical properties. J. Biochem., Tokyo 55, 49-53. Kelstrup J. and Funder-Nielsen T. D. 1977. Synthesis of bacteriocins in liquid cultures of Streptococcus mutans. J. biol. buccale 5, 99-106. Kelstrup J. and Gibbons R. J. 1969. Bacteriocins from human and rodent streptococci. Archs oral Biol. 14, 251-258. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. 1951. Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Lowry 0. H., Roberts N. R., Leiner K. Y., Wu M. and Farr A. L. 1954. The quantitative histochemistry of brain. I. Chemical methods. J. bioi. Chem. 207, l-17. Macrina F. L., Reider J. L., Virgili S. S. and Kopecko D. J. 1977. Survey of the extrachromosomal gene pool of Streptococcus mutans. Infect. Immun. 17, 215-226. Mejbaum W. 1939. Uber die Bestimmung kleiner Pentosemegen, insbesondere in Deriveten der Adenylfaure. Hoppe Seyier’s Z. physiol. Chem. 258, 117-120. Ouchterlony 0. 1949. Antigenantibody reactions in gels. Acta path. microbial. stand. 26, 5077515. Paul D. and Slade H. D. 1975. Production and properties of an extracellular bacteriocin from Streptococcus mutans bacteriocidal for group A and other streptococci. Infect. Immun. 12, 13751385: Rogers A. H. 1976a. Bacteriocinogenv and the nrouerties of some bacteriocins of Strepto&& mutans: A;chs oral Biol. 21, 99-104. Rogers A. H. 1976b. Bacteriocin patterns of strains belonging to various serotypes of Streptococcus mutans. Archs oral Biol. 21, 243-249. Yamamoto T., Imai S., Nishizawa T. and Araya S. 1975. Production of, and susceptibility to, bacteriocin-like substances in oral streptococci. Archs oral biol. 20,389-391.

1 overleaf.

rt ul

H. Fukushima

726

Plate Fig. 3. Electron (pH 7.2). Fibrous

1

micrographs of Rm-10 bacteriocin stained with 1 per cent phosphotungstic acid structures appear to aggregate with each other (a). The arrow shows a single fibre of bacteriocin (b). x 94,500

Purification

of Strep.

Plate

mutms

1.

bacteriocin

727