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FEMS Microbiology Letters, 1996, vol. 136, pp. 163-168

Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750

Askild Holck, Lars Axelsson and Ulrich Schillinger

ABSTRACT

Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus. Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence was determined by Edman degradation. The peptide consisted of 34 amino acid residues. The calculated M(r) from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry. Divergicin 750 did not show any sequence similarities to other known bacteriocins. The plasmid-located structural gene encoding divergicin 750 (dvn750) was cloned and sequenced. The gene encoded a primary translation product of 63 amino acids with a deduced M(r) = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.

Keywords: Lactic acid bacteria; Carnobacterium diuergens; Bacteriocin; Protein purification; Antimicrobial peptide; Cloning

1. Introduction

Lactic acid bacteria produce a variety of com- pounds with antimicrobial activity [1]. Some of these are proteins or peptides and are termed bacteriocins [2,3]. Bacteriocins from lactic acid bacteria are cur- rently being divided into four classes [4]. Class II bacteriocins are small and have little post-transla- tional modifications. They are usually heat stable, hydrophobic and often act on the cell membrane of susceptible target cells. Bacteriocins are commonly secreted by a dedicated transport and maturation system. The bacteriocins usually inhibit the growth of closely related species. Some bacteriocins also inhibit the growth of pathogens and spoilage organ-isms and may thus be of interest for enhancing food safety and food hygiene [5].

Carnobacteria grow at low temperature, produce relatively little acid and volatile compounds and they generally also have low proteolytic and lipolytic activity. Little is known about bacteriocins from carnobacteria. Some bacteriocin-producing carnobac­teria have been identified [6,7]. We have previously purified and characterised piscicolin 61 from Carnobacterium piscicola LV61 [8]. C. piscicola LV 17 produced three bacteriocins, termed carnobac­teriocin A, BM1 and B2 [9,10] of which carnobacte­riocin A was identical to piscicolin 61. Carnobacteriocin BM 1 and B2 show significant sequence similar­ity and contain the -YGNGVXC- motif typical of bacteriocins active against Listeria. Recently, diver­gicin A, produced by C. dicergens LV 13 was char­acterised [11]. It constitutes an interesting exception in being a small class II-like bacteriocin which uses the cells' sec system for translocation. A lanthion­ine-containing bacteriocin, carnocin UI49 has been purified as well [12].

Here we describe the purification, characterisation and cloning of divergicin 750, a novel bacteriocin from C. diuergens 750.

2. Materials and methods

2.1. Growth of bacteria 

Thirty-seven strains of carnobacteria were screened for production of bacteriocin. Carnobac­terium dicergens 750 from the laboratory stock of Bundesanstalt fur Fleischforschung, Kulmbach. Ger­many, produced divergicin 750. For production of bacteriocin, cells were grown at 25°C overnight in cMRS medium containing Peptone proteose no. 3 (10 g l^-1 ), yeast extract (5 g l^-1), sucrose (20 g l^-1 ), K HPO₄ (2 g l^-1 ), (NH₄)₂ citrate (2 g l ^-1 ), MgSO₄ (0.02 g l^-1), MnSO₄ (0.02 g l^-1 ). pH was adjusted to 8.5 with NaOH and the medium auto­claved for 20 min. In growth kinetic experiments C. diuergens 750 was grown in D-MRS broth [13] adjusted to initial pH 6.6 and pH 8.2. The broths were inoculated at a 0.1% level of overnight cultures and incubated for 48 h at 25°C. At appropriate time intervals the number of colony-forming units and bacteriocin activity in the culture supernatant was determined. Growth of C. dicergens 750 in D-MRS broth of different pH values was monitored using an automated turbidometer, BIOSCREEN C (Labsys­tems, Helsinki, Finland). 10 µl of a ten-fold diluted 24-h culture were added to honeycomb wells con­taining 190 µl D-MRS broth of pH 4.5, 5.0, 6.0. 7.0, 8.0, 9.0, and 10.0. Al! inoculations were done in triplicate and incubation was for 48 h at 30°C. After 14 h and 38 h, samples were withdrawn from a second honeycomb plate and tested for bacteriocin activity. To test the bacteriocin for pH stability, fractions of culture supernatants were adjusted to pH values between 2 and 11 with 5 M NaOH or 2 N HCI. After 1 h of incubation at 20°C, residual activ­ity was examined by use of the agar spot assay.

2, 2. Bacteriocin activity assays

Bacteriocin activity was quantified by using serial dilutions of the bacteriocin in the agar spot test as described previously [14]. One arbitrary unit (AU) was defined as the reciprocal of the highest dilution yielding a definite zone of inhibition on the indicator lawn. Alternatively, bacteriocin activity was quanti­fied in a microliter plate assay system by measuring the growth of the indicator organism in two-fold dilutions of bacteriocin in medium as described pre­viously [15]. When necessary, bacteriocin fractions were adjusted to pH 6.5 and sterilised by filtration through Millipore filters (0.22 µm, Millipore, UK) prior to activity measurements. One bacteriocin unit (BU) was defined as the amount of bacteriocin which inhibited growth of the indicator organism by 50% as compared to a control culture without bacteriocin. Unless otherwise stated C. dicergens L66 was used as an indicator strain.

2.3. Purification and analysis of divergicin 750

Bacteriocin was purified essentially as described previously [161. In short, a 2-1 culture of C. diuer­gens 750 was grown to the stationary phase and cells were removed by centrifugation. The bacteriocin pre-sent in the supernatant fraction was concentrated by ammonium sulfate precipitation and subjected to ion exchange chromatography (S-Sepharose), hydropho­bic interaction chromatography (Octyl-Sepharose) and reversed phase FPLC (Pharmacia). The purified bacteriocin was stable in 40% (v/v) 2-propanol containing 0.1% trifluoroacetic acid at - 20°C.

Amino acid sequencing and mass analysis of puri­fied divergicin 750 were done as described previ­ously [16].

2.4. Recombinant DNA techniques

Basic cloning techniques were used [17]. The probes were end-labelled with ³² P using a terminal transferase end-labelling kit (Amersham, UK). Plas­mid DNA from C. divergens 750 was digested with

various restriction endonucleases and the resulting fragments were separated on agarose gels, blotted onto nylon filters and hybridised with a divergicin 750-specific degenerate oligonucleotide probe. Two partly overlapping fragments, a 2-kb EcoRI frag­ment and a 0.2-kb Mbol fragment, hybridising to the oligonucleotide probe, were purified by excising them from a low-melting-point agarose gel and subse­quently employing the Magic^TM clean-up system (Promega, USA). Cloning was done in Escherichia coli DH5 a using the cloning vector pGEM-7Zf( + ) (Promega). Positive clones were identified by colony hybridisation. The cloned DNA fragments were se­quenced completely on both strands using the Seque­nase version 2.0 DNA sequencing kit (Amersham) and a primer walking strategy. Computer analyses were carried out on an IBM personal computer em­ploying the DNASIS sequence analysis program (Hitachi, Japan) and on a UNIX computer employing the GCG programme package [18].

3. Results

3.1. Production and inhibition spectrum of diver­gicin 750

Growth of C. divergens 750 at different initial pH values of the growth media was measured (Fig. 1). The bacteria grew well at high pH while growth was significantly retarded at pH 5. Bacteriocin was pro­duced over a wide range of pH values and could be detected even down to pH 5. It appeared that diver­gicin 750 was produced in the late exponential phase of growth as the cells entered the early stationary phase and remained fairly stable in the growth super­natant after production (results not shown). When C. divergens 750 was grown at different temperatures, maximum production of bacteriocin occurred at 25°C (results not shown). Bacteriocin production was de­tected down to growth at 4°C. At 37°C no activity was observed. Consequently, cells were grown at initial pH 8.5 and 25°C to enhance the yield of bacteriocin.

Fig. 1. (A) Growth of C. dieergens 750 in D-MRS broth of different pH values as measured by BIOSCREEN automatic tur­bidometric system. (B) Bacteriocin activity in supernatants were measured after 14 h and 38 h of incubation. Activity was mea­sured using the agar spot test.

Some lactic acid bacteria are known to produce more than one bacteriocin. To rule out interference from other inhibitory substances, purified divergicin 750 (see below) was used in determination of the inhibition spectrum (Table 1). Divergicin 750 prefer-

ably inhibited strains of Carnobacterium and Ente­rococcus. Selected strains of Clostridium perfringens and Listeria monocytogenes were also inhibited.

3.2. Purification of dirergicin 750

Divergicin 750 was purified by ammonium sulfate precipitation, sequential cation exchange, hydropho­bic interaction and reversed-phase chromatography (Table 2). The purified bacteriocin appeared homo­geneous when subjected to FPLC-reversed phase chromatography (Fig. 2). Overall, a more that 600-fold increase in specific activity was observed (BU mg^-1 protein). The purified bacteriocin had an activ­ity of approx. 10 000 BU µg ^-1 protein.

The complete amino acid sequence of the purified bacteriocin was determined by Edman degradation. The sequence was identical to that deduced from the structural gene after cloning (see below, Fig. 4).

Divergicin 750 consisted of one polypeptide chain of 34 amino acid residues. No unusual amino acid residues such as lanthionins were found. The calcu­lated M_r of 3447.7 was almost identical to the value of 3448.5 Da obtained by PDMS (Fig. 3). The extra 1 Da in the mass analysis probably originated from protonation of the polypeptide during desorption.

Fig. 2. Purity of divergicin 750 after FPLC reversed phase chro­matography. The bar indicates the bacteriocin collected for N-tenninal sequencing and mass determination

.

Fig. 3. Mass spectrum of the purified divergicin 750.

3.3. Cloning and DNA sequencing of the divergicin 750 structural gene

Hybridisation of a degenerate oligonucleotide probe to plasmid DNA from C. dirergens 750 gave one strong hybridisation signal to a 2-kb EcoRI fragment and a partly overlapping 0.2-kb Mbol frag­ment. The EcoRl and Mbol fragments were cloned and sequenced. The structural gene is presented in Fig. 4. The gene encoded a primary translation prod­uct of 63 amino acids with a calculated M_r = 6789.4. Cleavage of this peptide between amino acid residue 29 and 30 would give the mature divergicin 750. Uncertain amino acid residue determinations in posi­tions 1, 6, 11 and 26 of divergicin 750 were corrobo­rated by deduction from the sequence of the cloned gene. Downstream of the structural gene, a region of dyad symmetry was found.

Fig. 4. Nucleotide sequence of the region encoding the divergicin 750 structural gene (drn750). A proposed ribosome-binding site is underlined. Dyad symmetries are indicated by convergent arrows. Downward pointing arrowhead indicates cleavage site of the leader sequence to give the mature divergicin 750. Boldfaced amino acids were also determined by N-terminal Edman degradation. The EMBL accession number is Z54201.

4. Discussion

Divergicin 750 is a low molecular mass, hy­drophobic and basic peptide and thus shares charac­teristics with other bacteriocins of lactic acid bacte­ria. Divergicin appeared to be produced only in the late exponential phase of growth (results not shown). This is in contrast to many other bacteriocins, for example sakacin A from L. sake Lb706 and sakacin P from L. sake Lb674, which are constitutively synthesized [16,19]. Nothing is known about the mechanism of regulation of divergicin 750.

The bacteriocin was purified by a procedure simi­lar to that used for other bacteriocins [8,19]. During purification a drop in activity was observed after elution from the S-Sepharose column, whilst the activity was regained after the next purification step. The reason for this apparent drop is unknown.

The molecular mass of divergicin 750 as calcu­lated from the deduced amino acid sequence was very close to that determined by PDMS and is consistent with the proposed amino acid sequence. Taken together with the information that no lanthion­ins were detected upon amino acid composition anal­ysis, divergicin 750 appeared not to be subjected to extensive post-translational covalent modifications and thus belonged to the class II bacteriocin group. No sequence similarities of divergicin 750 with other proteins encoded by sequences in the EMBL and GenBank sequence databases were found. Divergicin 750 thus differs from other bacteriocins of the lactic acid bacteria.

A general mechanism of action for the class II bacteriocins has been suggested [20]. In this model the bacteriocin molecules are thought to form am­phiphilic a-helices that combine to make a pore in the cell membrane of susceptible cells. Nothing is known about the mechanism of action of divergicin 750. The region encompassing amino acid residues 7-28 may be able to form an amphiphilic a-helix and may indicate a similar mode of action for diver­gicin 750.

Divergicin 750 is processed from a longer pri­mary translation product. The N-terminal 29 amino acid residues are cleaved off adjacent to a glycine doublet to yield the mature product. This leader sequence, although unusually long, shows typical signatures of bacteriocin leader peptides of the dou­ble-glycine type [21], for example a hydrophilic re­gion at positions - 8 to - 11 flanked by hydropho­bic amino acid residues. Other bacteriocins of lactic acid bacteria with this type of leader invariably have a dedicated secretion system for their export [22]. Indeed, a putative open reading frame starting ap­prox. 1 kb downstream of the structural gene showed a high degree of similarity to bacteriocin ABC ex­porter genes such as sapT [23] (results not shown). This indicates that dvn750 may be part of a larger gene cluster involved in production and secretion of divergicin 750.

Bacteriocins and bacteriocinogenic strains may be used by the food industry as additives to prevent outgrowth of pathogens and spoilage organisms, giv­ing an enhanced control over production processes. The study of the structure of bacteriocins is an important step in establishing knowledge about the parts of the molecule that are responsible for the inhibitory activity. Subsequently, it may be possible to increase the inhibitory spectrum and enhance the activity by changing specific amino acid residues in the bacteriocin molecules.

Acknowledgements

We want to thank Birgitta Baardsen and Merete Bjgrnslett for technical assistance. We also want to thank Dr. K. Sletten at the Department of Biochem­istry, University of Oslo, for carrying out the auto­matic amino acid sequencing, and S. Bayne, Applied Biosystems Division of KEBO lab, Ballerup, for analysing the samples on the Biolon 20 mass anal­yser.

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