Characterization and mechanism of action of ... - Wiley Online Library

Journal of Applied Microbiology 2000, 89, 361ÿ369

Characterization and mechanism of action of cerein 7, a bacteriocin produced by Bacillus cereus Bc7 J.C. OscaÂriz and A.G. Pisabarro Departamento de ProduccioÂn Agraria, Universidad PuÂblica de Navarra, Pamplona, Spain 151/1/2000: received 28 January 2000, revised 17 April 2000 and accepted 19 April 2000

Cerein 7 is a peptidic antibiotic produced by Bacillus cereus Bc7 (CECT 5148) at the end of exponential growth but before sporulation onset. Cerein 7 has a broad spectrum of antibacterial activity against Gram-positive bacteria, but it is inactive against Gram-negative bacteria. The sequence of its amino-terminal end and its characteristics of hydrophobicity and molecular mass make cerein 7 unique among the bacteriocins produced by the soil bacterium B. cereus. In this paper a further characterization of cerein 7 is presented, it is shown that it can be classi®ed as a Klaenhammer's class II bacteriocin and that its mode of action corresponds to that of a membrane-active compound. J . C . O S C AÂ R I Z A N D A . G . P I S A B A R R O . 2000.

INTRODUCTION

Cerein 7 is a peptidic antibiotic (bacteriocin) produced by Bacillus cereus Bc7 (CECT 5148) that shows a broad spectrum of activity against Gram-positive bacteria but is inactive against Gram-negative ones (OscaÂriz et al. 1999). In this paper we further characterize cerein 7 and study its mode of action. Bacteriocins are ribosomally synthesized peptidic antibiotics produced by a number of different bacteria (Tagg et al. 1976; Klaenhammer 1993; Jack et al. 1995). These peptides belong to a functional group of antibacterial compounds also found in plants (thionins, Broekaert et al. 1995; Broekaert 1997) and animals (defensins, magainins and cecropins, Zasloff 1987; Lee et al. 1989; Lehrer et al. 1993) that share a handful of structural similarities: low molecular weight, thermostability and highly hydrophobic nature. Bacteriocins have been extensively studied because of their potential use as food additives (Lindgren and Dobrogosz 1990) and as antimicrobials in pharmaceuticals used topically (Kolter and Moreno 1992). Bacteriocins produced by Gram-positive bacteria are particularly relevant (Jack et al. 1995), especially those produced by lactic acid bacteria (Klaenhammer 1993) because of their importance in the dairy industry (Delves-Broughton 1990). The vast number of bacteriocins described so far allows different classi®cations of these compounds. The classi®cation of bacteriocins put forward by Klaenhammer (1993) takes into Correspondence to: A.G. Pisabarro, Departamento de ProduccioÂn Agraria, Universidad PuÂblica de Navarra, 31006 Pamplona, Spain (e-mail: [email protected]). = 2000 The Society for Applied Microbiology

account the chemical structure, heat stability, molecular mass, enzymatic sensitivity, presence of modi®ed amino acids and mode of action of these chemicals. Four classes of bacteriocins can be distinguished. (1) lanthibiotics containing the modi®ed amino acid lanthionine. Bacteriocins such as nisin (Hurst 1981), epidermin (Allagaier et al. 1986) and cinnamycin (Kaletta et al. 1991) belong to this group. (2) Low-molecular weight bacteriocins (smaller than 10 kDa) formed exclusively by unmodi®ed amino acids. Within this group speci®c antilisterial compounds (such as enterocin A, Aymerich et al. 1996; pediocin AcH/PA1, Venema et al. 1995), bacteriocins formed by two peptides acting synergistically (such as plantaricins EF and JK, Diep et al. 1996; enterocins L50 A and B, Cintas et al. 1998) and thiol-activated peptides (carnobacteriocin A, Worobo 1994; enterocin B, Casaus 1998) can be found. (3) High-molecular weight bacteriocins: heat labile proteins larger than 30 kDa, such as helveticin J (Joergen and Klaenhammer 1986) and lacticins A and B (Piard 1994) belong to this group. (4) Bacteriocins carrying lipid or carbohydrate moieties such as leuconocin S (Lewus et al. 1992). Bacteriocins show different modes of action. In most cases, the bacterial membrane is their target of action. Other bacteriocins, however, inhibit essential enzymes within the cell such as leuconocin S or pediocin JD (Waite et al. 1998) and colicin E9 (Pommer et al. 1998). The highly hydrophobic nature of bacteriocins allows the interaction of the compound with the lipid bilayer of the cytoplasmic membrane. This interaction can be either nonspeci®c in the case of bacteriocins showing a broad activity spectrum (i.e. pediocin AcH/PA1 or nisin) or receptor

362 J . C . O S C AÂ R I Z A N D A . G . P I S A B A R R O

mediated in the case of species or strain-speci®c bacteriocins (such as lactacin B, Barefoot and Klaenhammer 1983). In some cases, there is an absolute need for the presence of proton motive force to allow the successful interaction of the bacteriocin with the target membrane (Abee et al. 1994). In other cases, the interaction of bacteriocin with the membrane is spontaneous. The outcome of this interaction is the generation of non-speci®c pores that allow an ef¯ux of protons, ions and amino acids but not cytoplasmic proteins. This ef¯ux causes dissipation of the membrane potential and the collapse of the energy generation cellular machinery (Sahl and Brandis 1982). The study of cerein 7's mode of action and its classi®cation in the framework described above will allow us to increase our knowledge about this antibiotic and help us to understand the basis of its broad spectrum of activity. MATERIALS AND METHODS Bacterial strains and culture conditions

Listeria innocua G244 (kindly provided by Dr J.A. VaÂzquez Boland, Fac. Veterinaria, University of Complutense, Madrid, Spain), Micrococcus luteus ATCC 7468 and Staphylococcus aureus ATCC 12600 were cultured in brain heart infusion (BHI) (Biolife, Milan, Italy); Bacillus cereus Bc7 (CECT 5148) which is the cerein 7 producer strain (OscaÂriz et al. 1999) in peptone water (Biolife), and Escherichia coli W7 dapA lysA (Hartmann et al. 1972) in Luria±Bertani (LB) broth (Lennox 1955) (10 g lÿ1 bactotryptone, 10 g lÿ1 yeast extract, 5 g lÿ1 NaCl) supplemented with 10 mg lÿ1 meso-diaminopimelic acid. Solid and soft media were prepared by adding, respectively, 15 or 75 g lÿ1 of agar to the corresponding broth media. All broth cultures were grown at 37 C with vigorous shaking (200 rev minÿ1 in an Innova 3000 culture bath, New Brunswick Scienti®c Co., Edison, NJ, USA). Cerein 7 purification

Cerein 7 was puri®ed as previously described (OscaÂriz et al. 1999). Brie¯y, B. cereus Bc7 was grown in liquid cultures until it reached the early stationary phase, then cells were removed by centrifugation and proteins in the supernatant were precipitated with ammonium sulphate at 65% saturation. The precipitate was dissolved in 20 mmol lÿ1 sodium phosphate-buffered saline pH 68 (PBS) and the volume was reduced to one hundredth of the original supernatant volume by ultra®ltration to minimize the amount of ammonium sulphate present in the sample. Then the bacteriocin was extracted with butanol (2 : 3 ratio of butanol:sample). Afterwards, the butanol was evaporated and the protein dissolved in one hundredth of the original volume of PBS.

Protein concentration was determined according to Bradford (1976) using bovine serum albumin as standard. Cerein 7 activity measure and assay

Cerein 7 activity was measured either on solid or in liquid cultures. For the determination of cerein 7 activity in solid cultures, the spot dilution method was used as previously described (Cintas et al. 1995): twofold dilutions of the cerein sample were spotted onto a Petri dish containing solid culture media overlaid with 106 cells of the indicator strain L. innocua G244. The plates were then pre-incubated at 4 C for 1 h to allow cerein 7 diffusion before they were transferred to 37 C for 16 h to allow the growth of the indicator strain before the diameter of the growth inhibition zones surrounding the antibiotic drops was examined. Antibiotic activity units were measured in solid cultures by spotting 10 ml of twofold dilutions of the cerein puri®ed as described below, onto agar plates. One unit of activity was de®ned as the minimal amount of sample required to produce a detectable inhibitory zone. In order to calculate the minimal inhibitory concentration (MIC) values for cerein 7 against different bacterial species, 2 ml of liquid culture were seeded with 106 cells of the bacteria to be tested. Then, twofold dilutions of cerein 7 were added to the cultures and they were incubated for 16 h before the bacterial mass (determined as optical density at 550 nm, O.D.550) was measured. The antibiotic concentration which produced a 50% reduction in the ®nal O.D.550 of the culture (with reference to the untreated control) was taken as the MIC of the antibiotic for the tested strain. To determine whether cerein 7 had bactericidal or bacteriolytic activity a previously described method was used (Schillinger and Lucke 1989). Brie¯y, a 40-ml culture of the indicator strain (L. innocua G244) grown in BHI broth was incubated at 37 C for 60 min with 200 rev minÿ1 shaking and it was divided in four aliquots. One of them was used as control whereas different amounts cerein 7 were added to the other three. The ¯asks were then incubated again at 37 C for 120 min and samples were removed at different times to measure colony forming units (cfu) on BHI agar plates and optical density. For the assay of murein hydrolytic activity in cerein 7, peptidoglycan agar plates were prepared containing peptidoglycan puri®ed as described elsewhere (Pisabarro et al. 1985) from Escherichia coli W7 and resuspended in PBS containing 75 g lÿ1 of agar. For assaying the activity, cerein 7 dilutions were spotted onto the peptidoglycan plates and the appearance of clear zones was monitored. Commercial lysozyme (Sigma-Aldrich QuõÂmica S.A., Alcobendas, Spain) was used as control. To test the kinetics of action (Zajdel et al. 1985), cultures of different bacterial strains were incubated at appro-

= 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 89, 361ÿ369

CHARACTERIZATION OF CEREIN 7

priate conditions and harvested by centrifugation when they reached an O.D.550 of 06. The cells were immediately washed with saline solution (085% w/v NaCl) and resuspended in PBS 50 mmol lÿ1, pH 68 to an O.D.550 of 25. Cell suspensions of L. innocua G244 and Staph. aureus ATCC 12600, previously incubated at 37 C in BHI, were exposed to various concentrations of cerein 7 and both viability and O.D.550 were recorded after either 5 or 60 min of incubation at 37 C. Chemicals

All the chemicals and enzymes used for Cerein 7 stability analyses were purchased from Sigma-Aldrich QuõÂmica S.A. (Alcobendas, Spain), Merck (Darmstadt, Germany) and Boehringer-Mannheim (Mannheim, Germany). In the case of enzymatic treatments the incubation recommendations indicated by the supplier were followed. RESULTS Characterization of cerein 7

In a previous paper (OscaÂriz et al. 1999) we reported the identi®cation of a new bacteriocin produced by B. cereus Bc7. In order to further characterize this compound a series of experiments aimed to determine some of its physical and chemical properties was carried out. The molecular mass of cerein 7 had been previously determined by mass spectrometry and by polyacrylamide gel electrophoresis (OscaÂriz et al. 1999). The results, however, were somehow contradictory, possibly because of the highly hydrophobic nature of this protein (OscaÂriz et al. 1999). In order to complement the data available regarding the molecular mass of cerein 7, ultra®ltration experiments were performed using Centricon ®lters (Amicon, Inc., Beverly, MA, USA, Amicon 10) which showed that nearly the full cerein 7 activity present in the protein extracts was ®ltered through the 10 kDa but was retained by the 3-kDa

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cut-off membrane ®lters indicating an apparent molecular mass between 3 and 10 kDa. Interestingly, a signi®cant amount of cerein 7 activity was also retained by membrane ®lters with a cut-off limit of 40 kDa (data not shown). This result suggested the association of cerein 7 molecules into large insoluble aggregates. Furthermore, when cerein 7 solutions were subjected to ultracentrifugation (300 000 g, for 3 h at 4 C) a proteinaceous precipitate could be recovered that contained part of the cerein 7 antibiotic activity. To test cerein 7 self aggregation, the effect of nonionic detergents such as Triton X100 on cerein 7 activity was studied. The rationale of this experiment is that cerein 7 aggregation can be the result of hydrophobic interactions. Table 1 shows the results of the recovery of antibacterial activity in the supernatant and precipitate fractions of identical cerein 7 samples dissolved in PBS containing various concentrations of Triton X100 and subjected to the ultracentrifugation conditions described above. The bacterial indicator strain was insensitive to the Triton X100 concentrations used in this assay (data not shown). It was observed that the higher the concentration of Triton X100, the higher the fraction of cerein 7 recovered in the supernatant. Furthermore, the total cerein 7 activity (i.e. supernatant plus precipitate fractions) increased in the samples treated with Triton X100. The proteinaceous nature of cerein 7 was established by the data presented in the report about its discovery (OscaÂriz et al. 1999), yet its biochemical characterization was not complete. There are many reports about peptidic antibiotics containing post-translational modi®cations such as glycosylation and amino-acid modi®cations, as well as about enzymes acting as non-speci®c antimicrobials (i.e. lysozymes against most Gram-positive bacteria). To gain insight into cerein 7 structure, the effect of different enzymatic treatments on the antibiotic activity was studied. Aliquots of cerein 7 were treated with different enzymes according to the manufacturers' speci®cations for 1 h at 37 C (except in the case of proteinase K which was incubated

Table 1 Effect of Triton X100 on the cerein 7 activity against the indicator organism Listeria innocua G244 recovered in the supernatant

and precipitate fractions Triton X100 concentration (v/v)

Supernatant Activity*

Recovery (%)

Activity*

Recovery (%)

Control 0002% 002%

37 50 71

58 72 88

27 19 10

42 28 12

Precipitate

*Activity units determined by spot dilution method as described in Materials and methods. = 2000 The Society for Applied Microbiology, Journal of Applied Microbiology, 89, 361ÿ369


Table 2 Effect of temperature, organic solvents, enzymatic

treatment and environmental conditions on cerein 7 activity

Enzymatic treatment

Organic solvents

Surfactants (02%) Reducing agents Temperature

Autoclaving pH

Treatment

Activity*

Control Trypsin Chymotrypsin Pronase E Proteinase K Aminopeptidase RNase Lysozyme Phosfolipase B a-amylase Acetone Chloroform Acetonitrile Ethanol 2-propanol Butanol Methanol Triton X100 Tween 20 DTT (10 mmol lÿ1) b-mercaptoethanol (10% v/v) 45 60 75 100 121 C, 20 min 20 30 50 70 90 110

19 0 0 0 0 19 19 19 19 19 19 19 19 19 19 19 19 21 19 12 9 19 19 19 19 0 19 19 19 19 19 0

*Activity determined by the diameter of the growth inhibition zone.

at 42 C) and the antibiotic activity remaining after the treatment was estimated by measuring the diameter of the inhibition halos in solid cultures of L. innocua G244. As it was expected, the antibacterial activity of cerein 7 disappeared upon treatment with all the proteases assayed (Table 2). Aminopeptidase was the only exception as no reduction in cerein 7 activity was observed upon treatment with this enzyme. However, the antimicrobial activity was not affected by treatments with RNase, lysozyme, phospholipase B or a-amylase (Table 2), suggesting that a peptidic compound is the only one essential for the antibacterial activity. Gram-positive bacteria produce a wide variety of

extracellular molecules showing antibacterial activity. Among them haemolysins, phospholipases and muramidases are frequently exported to the environment. To test if cerein 7 had haemolysin activity, sheep blood agar plates (Oxoid Ltd) and human and sheep erythrocyte suspensions were treated with the antibiotic and no haemolytic activity was detected in cerein 7 preparations (data not shown). To check if the compound had phospholipase C activity, the antibiotic was spotted onto egg yolk plates (Biolife, Milan, Italy). Again, no phospholipase C activity was detected. Similarly, no murein-hydrolytic activity could be detected in cerein 7 preparations (data not shown). Finally, dilution experiments allowed us to discard the presence of a replicative bacteriophage in the bacteriocin preparation. The stability of cerein 7 activity upon different treatments with organic solvents, surfactants and reducing agents was also studied (Table 2). In each case, equal amounts (35 mg) of cerein 7 were dissolved in a volume of PBS and a volume equal to one tenth of the aqueous phase of the corresponding organic solvent was added and mixed thoroughly. In those cases where the organic solvent could not be mixed with the aqueous PBS solution, a vigorous shaking of the samples was maintained during the experiment. The incubation was carried out for 1 h at 25 C. In many cases (i.e. butanol) the organic solvent extracted the bacteriocin activity from the water phase. All the samples were dried using a Speed-Vac (Savant Instruments Inc., Farmingdale, NY, USA), the protein fraction was resuspended in the original volume of PBS and the antibacterial activity remaining after the treatment was assayed. No reduction in cerein 7 activity was observed to be produced by the treatments with the organic solvents assayed. In a series of similar experiments carried out with nonionic detergents, it was observed that these surfactant agents did not reduce cerein 7 activity. Furthermore, an increase of cerein 7 activity was observed with the treatment with Triton X100. An important reduction of cerein 7 activity was observed, however, when the bacteriocin was treated with reducing agents such as b-mercaptoethanol or DTT indicating that, at least, one disulphide bond is essential for its antibacterial activity. The thermostability of cerein 7 was, in addition, assayed by incubating cerein 7 aliquots at different temperatures for 15 min, and it was found that this bacteriocin could stand treatments up to 100 C for 15 min, although all the antibacterial activity was lost by autoclaving cerein 7 preparations. Finally, the pH range in which cerein 7 was stable was studied, by adjusting the pH of aliquots of cerein 7 dissolved in distilled water to different values within the range between 20 and 110 using HCl and NaOH. The samples were then incubated at 25 C for 2 h and the solvent was evaporated using a SpeedVac. The pellets were resuspended in PBS 20 mmol lÿ1 pH 68 in 1/15 of the original volume. Suitable controls



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Cerein 7 mode of action

Fig. 1 Maximum growth of Staphylococcus aureus ATCC12600 ( & ), Listeria innocua G244 (~), and Micrococcus luteus ATCC

7468 (.) cultures carried out in the presence of different concentrations of cerein 7 after 16 h of incubation at 37 C

were used and the antimicrobial activity was determined on agar plates as described before. It was observed that treatments of the bacteriocin at pH values ranging from 2 to 9 did not affect the ®nal antibiotic activity although a complete loss of activity was observed after the treatment at pH 110.

The MIC of cerein 7 in liquid cultures was determined for some bacterial species. In a previous paper (OscaÂriz et al. 1999) a screening of different bacterial species had revealed that Staph. aureus ATCC 12600 was insensitive to cerein 7 and that M. luteus ATCC 7468 showed the highest sensitivity to this antibiotic. Cultures of L. innocua G244, Staph. aureus ATCC 12600 and M. luteus ATCC 7468 were treated with increasing concentrations of cerein 7 and the O.D.550 of the culture after 16 h of incubation at 37 C was recorded. The MIC value was de®ned as the antibiotic concentration that halved the O.D.550 reached by the culture after the incubation period compared with control cultures. Figure 1 shows that MIC values ranged from 006 mg mlÿ1 in the case of the highly susceptible strain of M. luteus (ATCC 7468) to more than 10 mg mlÿ1 in the resistant strain of Staph. aureus (ATCC 12600). The MIC for the indicator strain used in this work (L. innocua G244) was of 1 mg mlÿ1. In summary, cerein 7 was active at millimolar concentrations (006±1 mg mlÿ1), this activity in the range of ppm indicates its high biological activity. In order to study the mode of action of cerein 7, an exponentially growing culture of the indicator strain L. innocua G244 was treated with different concentrations of cerein 7 and both the cell mass (measured as O.D.550) and the number of viable cells were recorded at different times (Fig. 2). The rate of increase in the optical density of the culture rapidly changed after the addition of cerein 7. At low bacteriocin concentrations (075 mg mlÿ1) only a minor change in the rate of O.D.550 increase was observed, whereas at higher cerein 7 concentrations a sharp stop in cell mass accumulation occurred. The number of viable cells, however, showed a sharp decay after the treatment

Fig. 2 Effect of different cerein 7 concentrations on the mass accumulation (a) and on the number of viable cells (b) of a Listeria innocua G244 culture. The antibiotic is added at time 0 (indicated with an arrow). Cerein 7 concentrations (in mg mlÿ1): 0 ( & ), 075 (~), 100

(!), 175 (.)



Fig. 3 Bactericidal and bacteriolytic effect of cerein 7. (a) Viable number (open symbols) and O.D.550 (®lled symbols) of Listeria innocua

.

(~, ~) and Staphylococcus aureus (*, ) recorded after 5 min incubation of exponentially growing cells in the presence of cerein 7. (b) O.D.550 of L. innocua (~, ~) and Staphylococcus aureus (*, ) recorded after short (5 min, ®lled symbols) and long (60 min, open symbols) incubation in the presence of cerein 7

.

onset which was correlated with the antibiotic concentration present in the culture. These treated cells appeared morphologically normal under light microscopy (data not shown) and represented 052% of the viability of untreated cells (culture use as control) of L. innocua G244 (25 108 cfu mlÿ1). All together, these results indicate that cerein 7 acts as a bactericidal compound even at low antibiotic concentrations. In order to carry out a comparative study on the kinetics of the cerein 7 killing effect on different bacterial species, exponentially growing cells of L. innocua G244 and Staph. aureus ATCC 12600 cells cultured in BHI were treated with different amounts of cerein 7 at 37 C as described in Material and methods, and both viability and O.D.550 values were recorded after 5 min of treatment (short time antibiotic effect). Figure 3(a) shows that after 5 min of incubation no variation in the O.D.550 was observed in any of the two species at any of the antibiotic concentrations used in the experiment. The number of viable cells, however, was reduced in the two samples following similar kinetics, although the ®nal killing effect was higher in the case of Staph. aureus than in the case of L. innocua. This result was unexpected in the case of Staph. aureus as this bacterial species appeared to be resistant to cerein 7 in plate assays (OscaÂriz et al. 1999) as well as in the MIC experiments described above. To study the long-time cerein 7 effect, the O.D.550 was measured in the same cultures after 60 min incubation. Figure 3(b) shows that a drastic decrease in the absorbance was detected in the case of L. innocua which was not observed in the case of Staph. aureus cell suspensions. These results indicate that an extensive cell lysis was triggered in L. innocua after cell death, whereas this cell lysis was not triggered in the case of Staph. aureus, despite

the fact that the cell death produced by cerein 7 in this species is much more pronounced. The drastic killing effect and the hydrophobic characteristics of cerein 7 suggested a mode of action based on pore formation ability for this compound. If this were the case, cerein 7 killing rate would depend on the ionic strength of the environment. To test this dependence, exponentially growing cells were harvested and aliquots of them were resuspended in prewarmed PBS of increasing salt concentration. The suspensions were then treated with 1 mg mlÿ1 of cerein 7 and the number of viable cells after 5 min incubation at 37 C was plotted against the salt concentration. Figure 4 shows that cerein 7 killing rate was inversely dependent on the osmolarity of the resuspension medium. DISCUSSION

Cerein 7 is a peptidic antibiotic produced by Bacillus cereus Bc7 (CECT 5148) at the end of the exponential growth (OscaÂriz et al. 1999). It was de®ned on the basis of its molecular mass (determined by mass spectrometry) and by its aminoterminal sequence, and it corresponded to a new compound not previously found in other species. Here, we present a more comprehensive study of cerein 7 characteristics and mode of action on Gram-positive bacteria. In the initial report on cerein 7 (OscaÂriz et al. 1999), there was a discrepancy between the molecular mass, as determined by mass spectrometry and by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). This discrepancy can be explained on the basis of the abnormal behaviour of some highly hydrophobic proteins in SDS-PAGE experiments (Kaufmann et al. 1984). By means of ultra®ltration experiments, we could con®rm that cerein 7 activity can pass



Fig. 4 Effect of the ionic strength on the activity of cerein 7. The

target cells were resuspended in different phosphate saline buffer concentrations, and were incubated for 5 min in the presence of 1 mg mlÿ1 of cerein 7. (*) Number of viable cells in the untreated control; (.) number of viable cells in the cerein 7-treated samples

through the 10-kDa cut-off membrane although it is completely retained by the 3 kDa, as it was expected. However, it was also observed that a signi®cant portion of the total activity was retained by membrane ®lters with much higher cut-off levels (up to 40 kDa) and that as much as the 40% of the total cerein 7 activity could be recovered from the initial antibiotic preparations by ultracentrifugation (Table 1). These data suggest that high molecular weight cerein 7 aggregates can be formed in aqueous solutions. Cerein 7 aggregates can be partially dissolved adding nonionic detergents to antibiotic preparations. This indicates that the aggregates are maintained mainly by hydrophobic interactions. B. cereus Bc7 was isolated from soil samples and, hence, its natural environment is essentially a polar one. So, formation of cerein 7 aggregates can very likely occur in natural conditions in which a large number of bacteria simultaneously produce the antibiotic as the soil nutrients become limited. These aggregates can prevent diffusion and loss of the antibiotic maintaining its concentration at high levels in the surroundings of the bacterial population. Bacteriocins, by de®nition, are inactivated by at least one protease. Cerein 7 inactivation by proteolytic enzymes

367

allow us to include it in this group of antibacterial compounds. Cerein 7 activity was eliminated by treatments with each of the proteases tested except aminopeptidase, suggesting that the aminoterminal end of the peptide is modi®ed and, insensitive to this exoprotease. However, the N-terminal sequence of cerein 7 could be determined by Edman degradation (OscaÂriz et al. 1999), indicating that the ®rst amino acid was unmodi®ed. Nevertheless, aminoterminal sequencing failed to proceed further than the sixth amino acid, indicating that an amino acid modi®cation can occur at this position that prevents the sequencing cycles to continue. This modi®cation can, also, protect cerein 7 from the exoprotease activity of the aminopeptidase. Taken together, these results suggest that there could be a short amino acid sequence at the N-terminal end of the protein that is not necessary for its activity. In summary, cerein 7 can be classi®ed as a bacteriocin on the basis of its peptidic structure and low molecular mass. Cerein 7 is heat-stable and its antibiotic activity can survive treatments of 100 C for 15 min. It is stable throughout a wide pH range and it is not irreversibly denatured by organic solvents. These data indicate that cerein 7 structure should be very stable or that the protein can fold spontaneously in its correct structure easily. The presence of, at least, one disulphide bond (see Table 2), and the reduced size of this protein can facilitate this stability or spontaneous folding. Bacteriocins have been classi®ed according to different criteria. One of these classi®cations considered size, presence of modi®ed amino acids, heat stability and enzymatic activities to group these antibiotics into four types (Klaenhammer 1993). The size and protein stability data discussed above and the lack of enzymatic activity of cerein 7 allow us to classify this compound as a class II bacteriocin according to Klaenhammer's classi®cation. The mode of action of cerein 7 was studied using L. innocua as the target organism. The rate of both biomass (measured as O.D.550) and the viable cell accumulation was immediately affected by the presence of cerein 7 in exponentially growing cultures (Fig. 2). Sampling times used in this experiment did not allow monitoring of the short-time response of the cells to the antibiotic. Figure 3(a) indicates that cerein 7 killing of L. innocua is already detected after 5 min of treatment and is dependent on the antibiotic concentration present in the culture medium (see also Fig. 2). L. innocua death is followed by a partial cell lysis that can be detected after 60 min of treatment with the antibiotic (Fig. 3b). This lysis, however, is not complete and it appears to be independent of the antibiotic concentration when this is higher than 2 mg mlÿ1. The relationships between cerein 7 killing of L. innocua and the triggering of the autolytic system in this bacteria remains to be elucidated. On this context, some authors (Bierbaum and Sahl 1985; Bierbaum and Sahl 1987; Jack et al. 1995) suggest



that bacteriocin-induced lysis could be due to the liberation of autolytic enzymes, that are usually electrostatically bound to anionic polymers (teichoic and lipoteichoic acids) of the cell wall, that are displaced by cationic bacteriocins from their binding sites. In the case of Staph. aureus, on the other hand, an unexpected response of the bacteria to cerein 7 was observed. Staph. aureus ATCC 12600 was chosen as a model for Gram-positive micro-organisms insensitive to cerein 7 (OscaÂriz et al. 1999). The MIC experiment described in Fig. 1 con®rms this previous observation as well as the lack of cell lysis triggered by cerein 7 (Fig. 3b). Nevertheless, an immediate and quick Staph. aureus cell death was detected upon cerein 7 addition to exponentially growing cells resuspended in PBS (Fig. 3a), and cell death rate was dependent on the antibiotic concentration as it was in the case of L. innocua. We currently cannot explain the apparent contradiction between the data presented in Figs 1 and 3(a), on one hand, and 3B, on the other, concerning Staph. aureus. The study of cerein 7 resistance or tolerance in the Staph. aureus cells recovered after long treatments with cerein 7 revealed that these cells displayed a normal pattern of antibiotic sensitivity (i.e. no growth inhibition zones were detected in spot dilution experiments whereas cell death without lysis was observed in liquid cultures). The molecular basis of this behaviour is not understood and merits further research. The quickness of the bacterial death induced by cerein 7 suggests that it is targeted to the cell wall or to the cytoplasmic membrane. There are many reports on bacteriocins acting on these targets: enterocin B and P (Casaus 1998), plantaricin J (JimeÂnez-DõÂaz et al. 1995) and enterocin A (Aymerich et al. 1996); and, in some cases, their bactericidal activity triggers a concomitant cell lysis such as in the case of nisin A (Bierbaum and Sahl 1985; Bierbaum and Sahl 1987), plantaricin C (GonzaÂlez et al. 1994) and enterocin EFS2 (Maisnier-Patin and Richard 1995) as it is the case with cerein 7. In the case of antibiotics acting at membrane level by pore formation, the killing rate is inversely correlated to the ionic strength of the culture medium (Boman et al. 1994). This is the case with cerein 7 (Fig. 4) which shows maximum lethal activity when the cells are resuspended in distilled water and minimal when they are in a 100-mmol lÿ1 PBS solution. ACKNOWLEDGEMENTS

This work was supported by the research project 779-I of the Departamento de Salud del Gobierno de Navarra and by Funds of the Universidad PuÂblica of Navarra (Pamplona, Spain). JCO holds a research grant from the Universidad PuÂblica de Navarra. The authors thank Dr JoseÂ Antonio VaÂzquez Boland and Dr JoseÂ Leyva for pro-

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