WO1994004682A1 - Bacteriocin - Google Patents

Abstract

The present invention provides a polypeptide having or including an amino acid sequence substantially corresponding to all or a portion of the amino acid sequence set out in Figure 1 (SEQ ID NO: 1) and derivatives and fragments thereof having bacteriocin and/or bacteriocin immunity activity.

Description

BACTERIOCIN

The present invention relates to a novel bacteriocin, its isolation, synthesis and use.

Bacteriocins are peptides or proteins released by bacteria which show bactericidal activity towards both the producing strain and/or other bacteria.

The producing organism will generally carry a gene coding for an immunity factor which provides resistance to the bacteriocin, and the producing organisms are usually only affected by their own bacteriocin at high cellular concentrations.

Due to their potential use as antibacterial agents, bacteriocins have been the subject of intensive research. In recent years there has in particular been considerable interest in bacteriocins isolated from lactic acid bacteria (LAB) , in view of their potential utility in the food and brewing industries, in particular the dairy industry.

Thus for example, their selective bactericidal activity renders LAB bacteriocins suitable for use in cheese or yoghurt manufacture or in beer or distillery fermentations.

The LAB bacteriocins appear to be structurally quite different from other bacteriocins eg. the colicins of Eschericia coli. LAB bacteriocins are usually small peptides, seldom containing more than 60 amino acids, while colicins are proteins of 300-800 amino acids. Based on their structure, LAB bacteriocins may be divided into two groups. The first group contains the so-called lantibiotics, which have been known for a long time and include in particular the known bacteriocin nisin, (see for example Gross, et al J. Am. Chem. Soc. 93: 4634-4635, 1971 and Hurst, Adv. Appl. Microbiol. 27: 85-123, 1981). Lantibiotics consist of a polypeptide chain in which certain amino acids have been post translationally modified. The modified amino acids include lanthionine and methyllanthionine, and their precursors dehydroalanine and dehydrobutyrine. Among the antibiotics, nisin is by far the most studied although three new LAB antibiotics have recently been purified and characterised (Mørtvedt et al. , J. Gen. Microbiol. 136: 1601-1607, 1990 and Appl. Environ. Microbiol 3_7: 1829-1834, 1991; Piard et al. , Appl. Environ. Microbiol. 58: 279-284, 1992).

The second group of LAB bacteriocins contains those that consist of one short unmodified polypeptide chain, such as lactococcin A (Holo et al., J. Bacteriol, 173: 3879-3887, 1991; Van Belkum et al. , Appl. Environ. Microbiol. 57: 492-498, 1991), leucoccin A-UAL 187 (Hastings et al., J. Bacteriol. 173: 7491-7500, 1991), lactacin F (Murlane et al., J. Bacteriol. 173: 1779- 1788, 1991 and Appl. Environ. Microbiol 57: 114-121, 1991), pediocin PA-1, sakecin P, and curvacin A. These bacteriocins contain between 35 and 60 amino acid residues, and have a high isoelectric point, often above 10. Many of these bacteriocins share significant amino acid sequence homology over relatively large regions, suggesting that these regions may be of importance for activity. Lactococcin A, which does not share any apparent amino acid sequence homology with other isolated LAB bacteriocins, has been shown to induce cell death by permeabilizing the membrane of susceptible cells (Van Belkum, et al., J. Bacteriol. 173: 7934-7941, 1992) .

The antimicrobial activity of the bacteriocins that have so far been studied is due to the action of a single peptide. We have now found, however, a novel lactococcal bacteriocin, which we have termed lactococcin G, whose activity depends on the complementary action of two distinct peptides.

The novel bacteriocin of the invention has been isolated from Lactococcus lactis and purified and bacteriocin activity was found to be associated with two peptides, designated and β , the peptide of which may appear in two forms, ₁ and α₂, which are identical and may differ only in conformation. A corresponding immunity factor has also been identified.

In one aspect the present invention thus provides a polypeptide having or including the amino acid sequence substantially corresponding to all or a portion of the amino acid sequence set out in Figure 1 (SEQ ID NO: 1) and derivatives and fragments thereof having bacteriocin and/or bacteriocin immunity activity.

Preferably, the said amino acid sequence substantially corresponds to the sub-sequences identified as lag A, lag B and lag C in Figure 1. Peptides and β are believed to be expressed in a "pro" form which is processed to the mature α or β peptide.

According to a further aspect the present invention also provides a polypeptide having or including the amino acid sequence

α,, and α₂ (SEQ ID NO: 2) :

N Gly Thr Trp Asp lie Gly Arg Val

Lys Ala- Met Gly

Asn Gin Ala Ser

- Lys Lys His C

and/or

and derivatives and fragments thereof having bacteriocin activity. 82

- 4 -

In a still further aspect, the invention also provides a polypeptide having or including the amino acid sequence

(SEQ ID NO: 4) and derivatives and fragments thereof having bacteriocin immunity factor activity.

Viewed from another aspect, the invention also provide a bacteriocin comprising peptide chains α and β as designated above or combinations of active fragments or derivatives thereof and having or including the amino acid sequences set out above. In such combinations, peptide α as designated herein may be either peptide α₁ or a₂ or a mixture of the two.

The terms "bacteriocin activity" and "active" are used to denote activity in inhibiting the growth of bacterial species, for example a lactococcus test organism. Bacteriocins may kill or inhibit the growth of bacteria by a number of mechanisms including lysis and it is not intended to limit to any particular type of bactericidal activity. Active polypeptides and derivatives or fragments include those which, whilst on their own do not exhibit bacteriocin activity, contribute to such activity when combined with the complementary (α or β ) peptide or its fragment or derivative.

The term "bacteriocin immunity factor activity" denotes activity in providing immunity to the bacteriocin of the invention.

Derivative sequences included within the scope of the invention include functionally-equivalent sequences modified by single or multiple amino acid substitution, addition and/or deletion and also sequences where the amino acids have been chemically modified, including by glycosylation or deglycosylation. By "functionally equivalent" is meant amino acid sequences having essentially equivalent bacteriocin activity. Such functionally equivalent derivatives may occur as natural biological variations or may be prepared using known techniques, for example functionally equivalent recombinant polypeptides may be prepared using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of amino acids.

As mentioned above, modification of the amino acid sequences to obtain functionally-equivalent derivative sequences may be amino acid substitution, as long as the activity of the polypeptide is not affected. Thus for example, an amino acid may be replaced by another which preserves the physicochemical character of the polypeptide eg. in terms of charge density, hydrophilicity/hydrophobicity, size and configuration. For example A may be replace by G or vice versa, V by A, L or G; K by R; S by T or vice versa; E by D or vice versa; and Q by N or vice versa.

Generally, the substituting amino acid has similar properties eg. hydrophobicity, hydrophilicity, electronegativity, bulky side chains etc. to the amino acid being replaced.

"Addition" derivatives include amino and/or carboxyl terminal fusions, for example by addition of amino acid sequences of up to 300 eg. up to 200 or 100 residues, as well as intrasequence insertions of single or multiple amino acids.

Insertional amino acid sequence derivatives are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Preferably, deletions or insertions are made in adjacent pairs eg. a deletion of two residues or insertion of two molecules. In all cases the proviso is that the modification preserves the activity of the polypeptide.

Derivative sequences falling within the scope of the invention may thus include for example amino acid sequences having at least 60%, eg. at least 70% or 80% sequence homology with the sequences of peptides α_1f α₂ or β set out above. It should be noted however that functionally-equivalent derivative peptides may exhibit overall sequence homology below the given figures, but may still fall within the scope of the present invention where they have conserved regions of homology.

Bacteriocin activity, as mentioned above, requires the complementary action of the and β peptides, a. being more effective when combined with β , than α₂ and the novel bacteriocin as provided according to the invention preferably comprises both a and β peptides.

In tests on Lactococcus Lactis subsp. Lactis 1403 indicator cells, the concentrations of peptides α, and β which inhibited cell growth by 50% were found to be 0.15 and 0.02 nM when the complementing peptide was present in excess. When neither was in excess the concentrations were respectively 0.3 and 0.04 nM. It thus appears that roughly 8 times more of . peptide is needed, and whilst not wishing to be bound by theory, it is possible that and β peptides interact in an approximately 8 to 1 ratio in effecting bacteriocin activity.

A further aspect of the invention thus includes bacteriocin comprising peptides and β or fragments or derivatives thereof in a ratio of 5-10 to 1, preferably 7-9 to 1, especially 8 to 1 respectively.

As judged by its amino acid sequence, peptide . has an isoelectric point of 10.9, extinction coefficient of 1.3 x IO^"4 M^*1 cm^'1, and a molecular weight of 4,346 (39 amino acid residues long) . Similarly, peptide β has an isoelectric point of 10.4, extinction coefficient of 2.4 x 10⁴ M^"1, and a molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for a and β , respectively, were obtained by mass spectrometry. The N-terminal half of both the α and β peptides may form amphiphilic α-helices, suggesting that the peptides are pore-forming toxins that create cell membrane channels through a "barrel-stave" mechanism and the novel bacteriocin of the invention may exert its bactericidal effect in this manner. The C-terminal half of both peptides consists largely of polar amino acids.

Peptides α, and α₂ are believed to represent different forms of the same peptide. Amino acid sequence determination suggests that the amino acid sequences for peptides α, and α₂ are identical and the two peptides were separated by their different behaviour during purification, particularly in reverse phase chromatography. In particular upon rechromatography of purified α, on a reverse phase column, a certain proportion eluted as α₂, suggesting that peptide α₂ derives from α₁. Peptides α₁ and α₂ thus appear to represent the same gene product, but may differ in their configuration in a manner which results in α₂ having a slightly lower affinity for the reverse phase column, and reduced bacteriocin activity when combined with β , than peptide α₁.

A further aspect of the invention provides a composition comprising bacteriocin according to the present invention, together with at least one of a carrier, and/or diluent, or excipient.

Such compositions may have a number of uses, particularly in microbiological processes and the carrier, diluent or excipient may be any such conventional material, chosen according to the proposed end use, for example a sterile liquid medium, buffer etc.

The novel bacteriocin of the invention may be used in industrial processes in which microbial species eg. lactobacteria such as lactococcus, are employed, for example in cheese and yoghurt manufacture.

The bacteriocin of the invention may be employed in fully or partially purified form or directly on the culture supernatant. The latter may be advantageous in certain circumstances, for example when the bacteriocin is to be used to selectively kill clostridia, as described below.

It may in certain cases be desirable to kill or arrest the growth of lactobacterial species for example, lactococcus in cheese ripening, and the new bacteriocin may thus be of particular application in the production of cheese, -or other diary products such as yoghurt.

Alternatively, the bacteriocin may be used to kill undesired or contaminating bacterial cells in various preparations. Such cells may be lactobacteria or they may be other bacterial strains, for example species of Bacillus (eg. B. Cereus) or Clostridium eg. C. tyrobutyricum.

Thus, for example since certain bacteria, for example some Gram-negative bacteria, may be resistant to bacteriocins, negative selection is possible by using the bacteriocin according to the invention to remove certain cells, for example clostridia or strains of L.lactis or other lactobacteria, from mixed cell populations e.g. in starter cultures for fermentation. Where the productive strain of L.lactis is used as the sole or principle organism in an industrial process such as cheese or yoghurt production, addition of the bacteriocin of the invention to the starter culture serves to eliminate foreign organisms and may be effective against, for example, spore forming clostridia or unwanted strains of L.lactis. or other lactobacteria.

The bacteriocin may advantageously be added to a cheese or yoghurt fermentation at a relatively late stage, after lactic acid, protease and flavour production by the L.lactis organism has already taken place.

By keeping the productive strain pure, either in the starter culture or in the milk or other medium, uniformity of production can be improved.

The bacteriocin may also be used to kill selectively strains of lactic acid producing bacteria in beer and distillery fermentations, since these are attributed in the literature to be the major cause of spoilage in unpasteurised beers and give rise to the greatest proportion of infections during fermentation.

Other bacterial species than clostridia or L.lactis may also present problems in the spoiling of foods during processing or manufacture. For example, B. cereus present in foods such as rice may lead to food poisoning.

Strains of clostridiu in particular are known to cause a problem in contaminating food manufacturing processes and may lead for example to the spoiling of cheese. At present, clostridial contamination is dealt with either by treatment with nitrates,- which are presently recognised to have a number of disadvantage, notably from the toxicity point of view or using lysozyme which is not generally effective. The use of bacteriocin according to the invention thus presents a considerable advance over such prior art methods. This is an area of considerable commercial and economic importance and a further aspect of the invention provides use of the novel bacteriocin of the invention in selectively killing undesired or contaminating strains of bacteria, eg. lactic acid bacteria, Clostridia, or Bacillus species in microbiological processes such as fermentation (eg. ethanol fermentation) or food manufacturing processes, eg. in dairy processes such as cheese or yoghurt production.

The invention also includes starter cultures of microorganisms containing the bacteriocin as an inhibitor of contaminating bacterial eg. lactococcus or clostridia species. Such microorganisms may, for example, be strains of L.lactis resistant to the bacteriocin eg. the producing organism, so that only unwanted microorganisms are removed from the starter culture, or yeasts of use in beer or distillery fermentations. Such starter cultures will normally be in lyophilised form.

Further uses of the novel bacteriocin include the production of cell wall preparations or for liberation of nucleic acid material.

The novel bacteriocin of the invention may be isolated from cultures of Lactococcus lactis strain LMG 2081 by fractionation of the growth medium whereby fractions enriched in the bacteriocin are collected. Known fractionation techniques may be applied to obtain the bacteriocin in electrophoretic purity. Thus for example the organism may be grown in a suitable culture medium eg. M17 broth (oxoid) and the supernatent subjected to fractional precipitation eg. with ammonium sulphate followed by chromatography eg. a combination of ion exchange, hydrophobic and reverse phase chromatography.

We have found in particular that the novel bacteriocin may be purified to homogeneity by a simple 4 step purification procedure which includes ammonium sulphate precipitation, followed by cation exchange, octyl sepharose and reverse phase chromatography. Using this procedure up to a 7000-fold increase in specific activity may be obtained.

A further aspect of the invention thus includes a method of isolation of bacteriocin according to the invention, wherein a culture of a microorganism expressing said bacteriocin is subjected to fractionation whereby fractions enriched in said bacteriocin are collected. Preferably, in such a method, the expressing organism is L.lactis strain LMG 2081.

Nucleic acid molecules comprising a nucleotide sequence encoding the novel bacteriocin or its component peptides and/or its corresponding immunity factor (which provides resistance against self-destruction in the producing strain) respectively form further aspects of the invention.

The region of the L. Lactis LMG 2081 genome coding for the novel bacteriocin of the invention and its immunity factor has been identified and sequenced. In particular, we have identified and cloned an operon which includes genes coding for the bacteriocin component peptides in their pro form as well as for a further protein believed to be the immunity factor providing resistance against self-destruction by the bacteriocin. The sequence of the operon is shown in Figure 1 (SEQ ID NO: 5) , which also shows the corresponding predicted amino acid sequence (SEQ ID NO: 1) . The putative promoter region and ribosome binding site are indicated. The gene (designated lag a) coding for the pro-sequence of the α peptide (designated lag A) appears to run from nucleotide 536 and a vertical arrow indicates where the peptide is cleaved between the amino acids corresponding to nucleotides 580-581 to give the mature α peptide.

Translation of the β pro-sequence (designated lag b for the gene and lag B for the pro-peptide) starts at nucleotide 746 and again a vertical arrow indicates where cleavage occurs at the amino acids corresponding to between nucleotides 820-821 to give the mature β peptide. Downstream of the above-mentioned sequence is a sequence comprising a single long open reading frame, starting at nucleotide 1027 and encoding a putative polypeptide of 110 amino acids, starting with LFNN and ending with LFIR, believed to be the immunity factor (designated lag C) .

The genes designated lag d and lag e are believed to code for proteins (designated lag D and lag E) that are involved in the secretion of the mature a and β peptides; the lag d and lag e sequences show homology to genes with such functions in other systems.

Accordingly, a further aspect of the invention provides a nucleic acid molecule comprising a nucleotide sequence which encodes a bacteriocin, its component peptides and/or its corresponding immunity factor, or a fragment thereof, substantially corresponding to all or a portion of the nucleotide sequence as shown in Figure 1 (SEQ ID NO: 5) or a sequence which is degenerate or substantially homologous with or which hybridises with any such sequence.

Such nucleic acid molecules may be single or double stranded DNA, cDNA or RNA, preferably DNA and include degenerate, substantially homologous, and hybridising sequences which are capable of coding for the bacteriocin concerned. "Substantially homologous" as used herein includes sequences displaying at least 60%, preferably at least 70% or 80% sequence homology and also functionally-equivalent allelic variants and related sequences modified by single or multiple base substitution, addition and/or deletion. By "functionally-equivalent" is meant nucleotide sequences encoding polypeptides having essentially equivalent bacteriocin activity.

Nucleic acid sequences which hybridise with the sequence shown in Figure 1 (SEQ ID NO: 5) or any substantially homologous or functionally-equivalent sequences as defined above are also included within the scope of the invention. "Hybridisation" as used herein defines those sequences binding under non-stringent conditions (eg. 6 x SSC 50% formamide at room temperature) and washed under conditions of low stringency (eg. 2 x SSC, room temperature, more preferably 2 x SSC, 42^βC) or conditions of higher stringency (eg. 2 x SSC, 65°C) (where SSC = 0.15M Nacl, 0.015M sodium citrate, PH7.2). Generally speaking, sequences which hybridise under conditions of high stringency are included within the scope of the invention, as are sequences which, but for the degeneracy of the code, would hybridise under high stringency conditions.

Derivative nucleotide sequences capable of encoding bacteriocin or bacteriocin derivatives according to the invention may be obtained by using conventional methods well known in the art. These include site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.

Bacteriocin according to the invention may be prepared in recombinant form by expression in a host cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly defined above, operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule.

Appropriate recombinant DNA techniques are well known in the art and are described for example by Sambrook et al., 1989, (Molecular Cloning, a laboratory manual, 2nd Edition, Cold Spring Harbour Press) .

The bacteriocin so expressed may be a fusion polypeptide comprising all or a portion of the bacteriocin according to the invention and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto. This may for example by β- galactosidase, glutathione-S-transferase, or any of the other polypeptides commonly employed in fusion proteins in the art.

Other aspects of the invention thus include cloning and expression vectors containing nucleic acid molecules according to the invention coding for the bacteriocin and/or for the immunity factor. Expression vectors appropriate to L. lactis are preferred. Such expression vectors include appropriate control sequences such as for example translational (eg. start and stop codes) and transcriptional control elements (eg. promoter-operator regions, ribosomal binding sites, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.

The invention also includes transformed or transfected prokaryotic or eukaryotic host cells, or transgenic organisms containing a nucleic acid molecule according to the invention as defined above. Such host cells may for example be transformed strains of lactic acid bacteria eg. strains of L. lactis.

Also included within the scope of the invention are methods for preparing the polypeptides of the invention comprising culturing a host cell containing a nucleic acid molecule as defined above under conditions whereby said polypeptide is expressed and recovering said polypeptide then produced.

The nucleic acid coding for the new bacteriocin and/or immunity factor may be incorporated into any convenient cloning vector for amplification and into an expression vector for transformation of host microorganisms such as L. lactis. for example cloning vector pIL253 (Simon and Chopin, Biochimie 70, 1988, 59- 566) . Growth under suitable culture conditions will provide the bacteriocin in the growth medium, from which it can be isolated by the techniques described above. Furthermore, host cells such as strains of lactic acid bacteria eg. L. lactis may be transformed with multiple copies of a plasmid or other vector containing the required nucleic acid sequence to provide an improved strain giving rise to enhanced production of the bacteriocin. Such improved strains may provide more rapid killing and hence accelerated cheese ripening when used in cheese manufacture. In particular, the strain of L.lactis which produces the bacteriocin may be provided with such multiple copies of the vector; this will thus be able to proliferate without premature destruction by the bacteriocin.

The new bacteriocin may also be prepared by chemical synthesis, for example using solid phase synthesis, advantageously using a polypeptide synthesis apparatus, as commercially available. In such a synthesis, active side chain groupings (e.g. amino or carboxyl groups) of the respective amino acids will be protected and the final step will be deprotection and/or removal from the inert support to which the polypeptide is attached during synthesis.

In building up the peptide chains, one can in principle start either at the C-terminal or the N-terminal although only the C-terminal starting procedure is in common use.

Thus, one can start at the C-terminal by reaction of a suitable derivative of, for example histidine with a suitable protected derivative of leucine. The histidine derivative will have a free α-amino group while the other reactant will have either a free or activated carboxyl group and a protected amino group. After coupling, the intermediate may be purified for example by chromatography, and then selectively N-deprotected to permit addition of a further N-protected and free or activated amino acid residue. This procedure is continued until the required amino acid sequence is completed.

Carboxylic acid activating substituents which may, for example, be employed include symmetrical or mixed anhydrides, or activated esters such as for example p-nitrophenyl ester, 2,4,5,trichlorophenyl- ester, N-hydroxybenzotriazole ester (OBt) , N-hydroxy- succinimidylester (OSu) or pentafluorophenylester (OPFP) .

The coupling of free amino and carboxyl groups may, for example, be effected using dicyclohexylcarbodi- imide (DCC) . Another coupling agent which may, for example, be employed is N-ethoxycarbonyl-2-ethoxy-l,2- dihydro-quinoline (EEDQ) .

In general it is convenient to effect the coupling reactions at low temperatures, for example, -20°C up to ambient temperature, conveniently in a suitable solvent system, for example, tetrahydro- furan, dioxan, dimethylformamide, methylene chloride or a mixture of these solvents.

It may be more convenient to carry out the synthesis on a solid phase resin support. Chloro- methylated polystyrene (cross-linked with 1% divinyl benzene) is one useful type of support; in this case the synthesis will start the C-terminal, for example by coupling N-protected histidine to the support.

A number of suitable solid phase techniques are described by Eric Atherton, Christopher J. Logan, and Robert C. Sheppard, J. Chem. Soc. Perkin I, 538-46 (1981) ; James P. Tam, Foe S. Tjoeng, and R. B, Merrifield J. Am. Chem. Soc. 102. 6117-27 (1980) ; James P. Tam, Richard D. Dimarchi and R. B. Merrifield Int. J. Peptide Protein Res JL6. 412-25 (1980) ; Manfred Mutter and Dieter Bellof, Helvetica Chimica Acta 67 2009-16 (1984) .

A wide choice of protecting groups for amino acids are known and are exemplified in Schroder, E. , and LUbke, K. , The Peptides, Vols. 1 and 2,. Academic Press, New York and London, 1965 and 1966; Pettit, G.R. , Synthetic Peptides, Vols. 1-4, Van Nostrand, Reinhold, New York 1970, 1971, 1975 and 1976; Houben-Weyl, Methoden der Organischen Che ie, Synthese von Peptiden, Band 15, Georg Thieme Verlag Stuttgart, NY, 1983; The Peptides, Analysis, synthesis, biology 1-7, Ed: Erhard Gross, Johannes Meienhofer, Academic Press, NY, San Fransisco, London; Solid phase peptide synthesis 2nd ed. , John M. Stewart, Janis D. Young, Pierce Chemical Company.

Thus, for example amine protecting groups which may be employed include protecting groups which may be employed include protecting groups such as carbobenzoxy (Z-) , t-butoxycarbonyl (Boc-) , 4-methoxy-2,3,6- trimethyl-benzene sulphonyl (Mtr-) , and 9-fluorenylmethoxycarbonyl (Fmoc-) . It will be appreciated that when the peptide is built up from the C-terminal end, an amine protecting group will be present on the α-amino group of each new residue added and Will need to be removed selectively prior to the next coupling step. One particularly useful group for such temporary amine protection is the Fmoc group which can be removed selectively by treatment with piperidine in an organic solvent.

Carboxyl protecting groups which may, for example be employed include readily cleaved ester groups such as benzyl (-OBZ1) , p-nitrobenzyl (-ONB) , or t-butyl (-tOBu) as well as the coupling on solid supports, for example methyl groups linked to polystyrene.

It will be appreciated that a wide range of other such groups exists as, for example, detailed in the above-mentioned literature references, and the use of all such groups in the hereinbefore described processes fall within the scope of the present invention.

A wide range of procedures exists for removing amine- and carboxyl-protecting groups. These must, however, be consistent with the synthetic strategy employed. The side chain protecting groups must be stable to the conditions used to remove the temporary α-amino protecting groups prior to the next coupling step.

Amine protecting groups such as Boc and carboxyl protecting groups such as tOBu may be removed simultan¬ eously by acid treatment, for example with trifluoro acetic acid.

A further aspect of the invention provides a process for the preparation of bacteriocin polypeptides according to the invention in which a corresponding protected or immobilised polypeptide is subjected to deprotection or removal from a solid support.

The invention will now be described in more detail in the following non-limiting Example with reference to the following Figures in which:-

Figure 2 shows reverse phase chromatography of lactococcin G (fraction IV) . (A) The optical density profile ( ) and propanoi gradient ( —) .

(B) Bacteriocin activity without complementation

( ), with complementation with α, ( • ), and with complementation with β ( O ) . The amount applied on the column represents that obtained from approximately 2 liter culture;

Figure 3 shows reverse phase chromatography of (A) α₂ (B) β , and (C) α_r The optical density profile

( ) and propanoi gradient ( = —) . The bacteriocin activity without complementation (....»....) and with complementation ( O ) with (B) a , or with

(A and C) β . The amount applied on the column represents that obtained from approximately 2 liter culture;

Figure 4 shows the amino acid sequence of ,, α₂ and β (SEQ ID NOS: 2 and 3). N and C indicates the, respectively, N- and C- terminal ends of the peptide;

Figure 5 shows the amount of , and β which in combination inhibited growth of the indicator strain by 50%;

Figure 6 shows an Ed undson α-helical wheel representation -of the amphiphilical region in (A) α₁ and (B) β . For . the amphiphilical region shown starts with residue number 3 and ends with number 27, for β it starts with residue number 8 and ends with number 25. The shaded areas indicate non-polar residues, whereas the light areas indicate polar residues.

Example 1

MATERIALS AND METHODS

Bacterial strains and media

The bacteriocin producer was strain Lactococcus lactis LMG 2081, obtained from J. Narvhus, Agricultural University, As, Norway. The indicator organism used in the bacteriocin assay was lactococcus lactis subsp. lactis IL 1403 (Chopin, et al., Plasmid, 11: 260-263, 1984.) Both strains were grown at 30 " C in M17 broth (Oxoid) without lactose, but supplemented with 0.4% (wt/vol) glucose. The M17 broth was also supplemented with Tween 80 to a final concentration of 0.1% (vol/vol) when culturing strain LMG 2081 for the production of bacteriocin.

Bacteriocin assay

The bacteriocin was quantified in a microtiter plate assay system (Gels, et al., Appl. Environ. Microbiol. 45: 205-211, 1983). Each well of the microtiter plate contained 200 μl of M17 broth (supplemented with 0.4% (wt/vol) glucose and 0.1% (vol/vol) Tween 80) , bacteriocin fractions at two-fold dilutions, and the indicator organism, Lactococcus lactis subsp. lactis IL 1403 (A₆₀₀ = 0.1). The microtiter plate cultures were incubated 3 hours at 30°C, after which growth inhibition of the indicator organism was measured spectrophotometrically at 500 nm b using a Dynatech Microplate Reader. One bacteriocin unit (BU) was defined as the amount of bacteriocin which inhibited growth of the indicator organism by 50% (50% of the turbidity of the control culture without bacteriocin) .

Bacteriocin purification

All the purification steps were performed at room temperature, and all the chromatographic equipment, was obtained from Pharmacia-LKB Biotechnology (Uppsala, Sweden) . The bacteriocin was purified from 2-liter cultures of Lactococcus lactis LMG 2081 grown to the late exponential/early stationary phase. The cells were removed by centrifugation at 4,000 x g for 15 min at 4°C, and 400 g of ammonium sulfate per liter culture supernatant was added. The protein precipitate was pelleted by centrifugation at 7,000 x g for 20 min and solubilised in 20 mM sodium phosphate buffer, pH 5.7 (buffer A, 250 ml per 2 liter culture) (fraction I) . Fraction I was applied to a flow rate of about 10 ml/min to a 7 ml S-Sepharose Fast Flow cation exchange column equilibrated with buffer A. After subsequently washing the column with 20 ml of buffer A, the bacteriocin was eluted from the column with 40 ml 1 M NaCl in buffer A (fraction II) . Ammonium sulfate was added to fraction II to a final concentration of 10% (wt/vol) , after which the fraction was applied at a flow rate of about 4 ml/min to a 2 ml Octyl-Sepharose CL-4B column equilibrated with 10% (wt/vol) ammonium sulfate in buffer A. The column was then washed with 8 ml of buffer A, after which the bacteriocin activity was eluted from the column with 10 ml 70% (vol/vol) ethanol and 30% buffer A (fraction III) . Fraction III was diluted to 50 ml with H-,0 containing 0.1% (vol/vol) trifluoroacetic acid (TFA) and subsequently applied to a C₂/C₁₀ reverse-phase column, PepRPC HR 5/5, equilibrated with 2-propanol/H₂0 (10 : 90), containing 0.1% TFA. The bacteriocin was eluted with a linear gradient ranging from 30 to 50% 2-propanol containing 0.1% TFA (fraction IV) . The Bacteriocin peptides eluting from the reverse phase column (fraction IV) were in some cases diluted 4- 5-fold with H₂0 containing 0.1% TFA and rechromatographed on the reverse phase column. Purified bacteriocin was stored in 50-60% 2-propanol and or ethanol containing 0.1% TFA at -20^βC.

Mass spectroscopy analysis

Mass spectroscoopy analysis of peptides was performed using the Biolon Mass Analyser (Applied Biosystem, Sweden) as described earlier by Sorensen et al., Biomed. Environ. Mass Spectrom. 19: 713-720, 1990. Peptide fractions were dissolved in 50-100 μl TFA containing 20% acetonitrile. From each fraction, 5 μl were loaded to a target and data accumulated for 10 min at 16 kV.

Amino acid sequencing

The amino acid sequence was determined by Edman degradation using an Applied Biosystems (Foster City, Calif.) 477A automatic sequencer with an online 120A phenylthiohydantoin amino acid analyser.

RESULTS

Purification of bacteriocin

Initial screening showed that Lactococcus lactis LMG 2081 produced antagonistic activity towards various LAB and a number of different Clostridia. This strain produced bacteriocin constitutively during growth, although maxium activity was found in the culture medium at the very end of the exponential or early stationary phase of growth. The activity started to decrease after a few hours in the stationary phase. The stability of the bacteriocin in the culture depended on the bacteriocin producing strain. With other strains which produced the same bacteriocin, maximum activity was found in the middle of the exponential phase, whereas little if any activity was found towards the end of this growth phase. This may be due to the cell envelope- associated preteinase produced by these strains, as this proteinase rapidly degrades the bacteriocin when present in the culture media.

The purification scheme developed for isolating the bacteriocin for sequencing is shown in Table 1.

TABLE 1. Purification of Lactococcin G

Vol Total Total activity Sp act^b Increase Yield

Fraction (ml) ^Λ A280^a (BU) sp act (%)

Culture supernatant 2000 67,000 30 x 10 450 100

Ammonium sulfate precipitation (fraction I) 250 1,050 17 X 10^c 16,000 35 57

Binding to cation exchanger (fraction II) 40 5.8 6 X 10' 1 X 10' 2,200 20

Binding to Octyl Sepharose (fraction III) 10 2.5 6 X 10° 2.5 X 10' 5,600 20

Reverse phase chromatography (fraction IV) 1.5 5 x 10^£ 3 x 10" • 6,700 17

^a Total A₂₈₀ is the absorbance at 280 mm multiplied by the volume in ml.

^b Specific activity is bacteriocin units (BU) divided by the absorbance at 280 nm.

Tween 80 (final concentration of 0.1% (vol/vol) was added to the culture medium before ammonium sulfate precipitation in order to obtain binding of the bacteriocin to the cation exchanger for bacteriocin activity, as this increased the sensitivity of the assay 2-10 fold. The bacteriocin was concentrated 8-fold from the culture media by ammonium sulfate precipitation. This resulted in a 30 to 40-fold increase in the specific activity and a recovery of about 60% of the activity (Table 1, fraction 1) . By subsequently binding the bacteriocin in fraction 1 to a cation exchanger and eluting it with 1 M NaCl, an increase in the specific activity of more than 2,000 was obtained (Table 1, fraction II) . From this stage and on the yield remained at about 20% (Table 1) . The specific activity increased 5,000-6,000-fold after binding the bacteriocin in fraction II to Octyl-Sepharose and eluting it with 70% ethanol (Table l, fraction III) . When fraction III was applied to the reverse phase column and eluted with a steep propanoi gradient (6%/ml) , the bacteriocin activity coeluted with an absorbance peak at about 40% propanoi (results not shown) . This absorbance peak, however, did not appear to be entirely homogeneous, as two shoulders could be discerned on both sides of the main peak.

Bacteriocin activity depends on the complementary action of two peptides

Upon rechromatography of fraction IV, this time eluting the bacteriocin activity using a shallow propanoi gradient (0.5%/ml), 4 absorbance peaks were obtained (Fig. 2) . The last 3 of these peaks were termed α₂ β and α₁ in the order of which they eluted together with the α₁ absorbance peak, but the total activity was greatly reduced compared to that which was applied to the column (Fig. 2) . However, upon adding an aliquot of the fraction containing α₁ to each of the column fractions, there was a complete recovery of bacteriocin activity in the fraction containing β (Fig. 1) . Similarly there was a complete recovery of bacteriocin activity in the fraction containing ., and to a lesser extent in the fraction containing α₂, when an aliquot of β was added to each fraction (Fig. 2) .

Each peptide α, α₂ and β was purified to homogeneity by rechromatography on the reverse phase column (Fig. 3). Whereas relatively little activity was seen when each peptide was assayed for bacteriocin activity alone, the activity was recovered when the β peptide was complemented with the α peptide, and to a lesser extent with the α peptide (Fig. 3) . No additional increase in the bacteriocin activity was seen upon adding the α₂ peptide to fractions containing both the β and α, peptides. Thus the complementary action of the two peptides, an α and a β peptide appeared to be necessary to obtain bacteriocin activity.

A small amount of β which presumably had not been entirely separated from α, on the previous reverse phase column, was detected upon rechromatography of α₁ (Fig. 3 C) . A small optical density peak apparently due to α₂ as well as a minor peak eluting ahead of α₂ - similarly to the optical density peak which eluted slightly ahead of ₂ in Fig. 2 - were also detected (Fig. 3 C) . It is unlikely that the presence of these two latter peaks was due to incomplete separation from a. on the previous reverse phase column, since they did not contaminate the β preparation to the same extent (Fig. 3 B) . Upon rechromatography of purified α₁ after storage in 50% propanoi for 3 months at -20°C, 30-40% of the peptides eluted as expected for α,, whereas 30-40% eluted similarly to α₂ in Fig 2. As much as 10% of purified α₁ eluted similarly to α₂ upon rechromatography after storage for 24 h. This suggests that α₂ and the component which eluted ahead of α₂ were derived from α₁. Mass spectroscopy analysis of the α and β peptides

In order to determine the molecular weights of α₁ and β and confirm their purity after the final reverse phase chromatography step (Fig. 3) , these peptides were analyzed by plasma disorption mass spectroscopy. Single peaks were observed with molecular weights of 4376 and 4109 for α and β, respectively.

Amino acid sequence of the α and β peptides

The complete amino acid sequence of the α., and α₂ (39 amino acid residues) and β (35 amino acid residues) peptides (SEQ ID NOS: 2 and 3) are shown in Fig. 4. It appears from the sequences that α₂ is identical to α₁ (Fig. 4) , consistent with the apparent formation of α₂ peptides upon rechromatography of . on the reverse phase column.

Relative amounts of α and β to obtain bacteriocin activity

The concentrations of , and β which in combination inhibited growth of the indicator organism by 50% were determined, and the results were plotted as an isobologram (Fig. 5) . When α, was in excess (greater than 1.3 nM, 0.25 pmoles/well), the presence of β at a concentration of 0.02 nM (0.04 pmoles/well), resulted in 50% growth inhibition (Fig. 5) . Similarly, with an excess of β (greater than 0.45 nM (0.03 pmoles/well), the presence of α, at a concentration of about 0.15 nM (0.03 pmoles/well) resulted in a 50% growth inhibition (Fig. 5) . Thus in order to obtain 50% growth inhibition in the presence of an excess of the complementary peptide, 7-8-fold more α₁ than β was needed. When neither α₁ nor β was in excess, the concentrations which resulted in 50% growth inhibition were 0.3 nM (0.06 pmoles/well) for a. and 0.04 nM (0.008 pmoles/well) for β (Fig. 5) . Again there was 7-8-fold more of α₁ than β. The concentrations which inhibited growth by 50% appeared to be invariant to the number of target cells present (within a 3Ofold range in the cell number) , as the same concentrations of . and β resulted in 50% growth-inhibition irrespective of whether the cell density was such that the A was 0.01 or 0.3.

summary

Three optical density peaks associated with _ bacteriocin activity were obtained upon reverse phase chromatography in the final purification step. The peptides associated with the 3 optical density peaks were termed α_{ιr 2} and β.

The bacteriocin activity was due to the complementary action of an α and the β peptide. In combination with the β peptide, α, gave a mugh higher bacteriocin activity than α₂. Upon rechromatography of purified α, on a reverse phase column, some of it eluted as expected for α₂. This suggests that α, and α₂ may in fact be the same peptide, but that they differ in their configuration in a manner which results in α₂ having a slightly lower affinity to the reverse phase column and reduced activity when combined with β than α₁. This view was supported by the amino acid sequencing data. The α₂ peptide was sequenced and this sequence appeared to be identical to the corresponding sequence of a..

As judged by amino acid sequencing, α, contained 39 amino acid residues and its molecular weight should be 4346. A molecular weight of 4376 was obtained by mass spectrometry indicating that the peptide is not grossly modified. Judging from its sequence, β contains 35 amino acid residues and its molecular weight should be 4110. This is in good agreement with the molecular weight of 4109 obtained by mass spectrometry, indicating that this peptide is not modified. From the amino acid sequence, the isoelectric point and extinction coefficient of α₁ were calculated to be 10.9 and 1.3 x 10⁴ M^"1 cm^"1, respectively. For the β peptide the isoelectric point and extinction coefficient were calculated to be 10.4 and 2.4 x IO⁴ M^"1 cm^"1 respectively.

The amino acid sequence of both α and β is such that these peptides are likely to be pore-forming toxins that create cell membrane channels through a "barrel- stave" mechanism, and thus produce an ionic imbalance in the cell (See Ojcius et al, TIBS, .16 225-229, 1991). A region in α₁ starting with amino acid residue number 3 and ending with residue number 27 may form an amphiphilic α-helix, as is evidence when this sequence is displayed on an Edmundson α-helical wheel (Fig. 6A) . The polar amino acids are found almost completely on one side of the α-helix, whereas the nonpolar residues are found on the opposite side of the helix (Fig. 6A) . The amphiphilic distribution of the amino acids in this region is nearly perfect, the only exception being glycine (residue number 9) which appears on the hydrophobic side (Fig. 6A) . However, glycine may be considered to be relatively neutral with respect to its hydrophilic/hydrophobic character. Moreover, the substitution of one amino acid by one of an opposite hydrophobicity may not represent an intolerable disruption of a peptide's amphiphilic character (see Ojcius, Supra) . The 25 amino acids long amphiphilic region in α₁ may allow peptide-monomers to oligomerize into membrane-spanning pores in such a manner that the non-polar side of the α-helix faces the membrane lipids, whereas the polar side faces towards the center of the pore (see Ojcius (Supra) and Lear et al, Science 240:1177-1181, 1988). The amphiphilic region in a. should be long enough to span a membrane, as a minimum of about 20 residues are needed to form a membrane- spanning α-helix. Ten of the 12 C-terminal amino acid residues, starting with position 28 and ending with 39, in α, are polar, of which the last 5 are basic (Arg-Lys- Lys-Lys-His-COOH) . In this connection it is interesting to note that lactococcin A and lactocin S, bacteriocins produced by Lactococcus lactis subsp cremoris and Lactobacillus sake, respectively, also contain a basic C-terminus, the last two C-terminal amino acids being histidine for both of these bacteriocins, (Holo (Supra) , Mørvedt (Supra) ) . It seems likely that the 11 amino acid residues long polar C-terminal region in α, does not penetrate the membrane. One may speculate that its function might be to recognise bacteriocin-binding sites on the target cells and thereby create a local high concentration of bacteriocin monomers on the outside of the cell membrance. This high concentration near the membrane could then induce oligomerization of the monomers into transmembrane pores. Another possible function of the polar C-terminal region might be to stabilize a correct peptide-configuration in a hydrophilic environment. The first two N-terminal amino acid residues in α₁ are polar, and may possibly be the part of the pore which is located inside the cell.

The situation is similar for the β-peptide. An amphiphilic α-helix may be formed in the region starting with amino acid residue number 8 and ending with residue number 25 (Fig 6 B) . In this region there are two exceptions to a perfect amphiphilic amino acid distribution: glycine (residue 22) which appears on the hydrophobic side and isoleucine (residue 24) which appears on the hydrophilic side. The former should not be a major problem, due to the neutral character of glycine. One would expect that proline at position 11 would cause a break or bend in the α-helix structure of the amphiphilic region. The region spans 18 amino acid residues, which may be somewhat less than required to span the cell membrane. However, in front of the amphiphilic region there are 5 nonpolar amino acids starting with residue number 3 and ending with number 7 which presumably also will be part of the transmembrane region. The hydrophilic amino acid, lysine as found at positions 1 and 2 at the N-terminuε; and only 2 of 10- residues starting with position 26, in the C-terminal part of the molecule are hydrophobic. Similar to α₁, one might expect that these regions will be located outside the membrane, the long polar C-terminal region being on the cell's outside. Although other lactic acid bacteria-produced bacteriocins that have been sequenced do not appear to have such a marked amphiphilic distribution of amino acids as α and β such a distribution is also seen in the C-terminal part of lactococcin A and Lactocin S. There is evidence that lactococcin A, permeabilizes target cell membranes (Van Belkum (Supra) as also appears to be the case for nisin (Sahl et al.. Arch Microbiol 149: 120-124, 1987).

The concentrations of α₁ and β which inhibited growth of the indicator cells by 50% were respectively 0.15 and 0.02 nM when the complementing peptide was present in excess. When neither was in excess the concentrations were, respectively, 0.3 and 0.04 nM. Thus 7-8-fold more of α, than β was needed. If the two peptides associate to the target cells with equal efficiency, this ration may reflect that α₁ and β interact in an approximately 8 to 1 ratio, for instance in pore formation. The presence of approximately 40 β molecules per target cell together with an excess of α, is enough to induce 50% growth inhibition. The number of β molecules that interact with a target cell may possibly be even smaller, since the concentration of bacteriocin which inhibited growth by 50% appeared to be invariant to the number of target cells present within a 30-fold range.

Example 2

Cloning and sequencing of the bacteriocin (lactococcin G (LcnG) ) was carried out as follows. Chromosomal DNA was purified from Lactococcus lactis LMG 2081 according to standard methods, and digested with Spel. Spel fragments were fractionated by agarose gel electrophoresis, and DNA fragments of 3-7 kbp were isolated using Magic Minipreps (Promega) . A sub-genomic library was constructed by ligating the fragments with Spel digested and phosphatase treated lambda ZAP II arms (stratagene) . The clone containing the bacteriocin (LcnG) structural gene and immunity gene was isolated by screening the library with a degenerate oligonucleotide probe deduced from the amino acid sequence of LcnG (Nissen-Meyer et al.). The hybridization was carried out according to standard protocols (Molecular cloning, Eds; Sambrook, et al., supra). Sequences were determined by the chain termination method (Sanger et al.) using sequenase (United States Biochemical Corp.). The sequence obtained is shown in Figure 1 (SEQ ID NO: 5).

Claims

1. A polypeptide having or including an amino acid sequence substantially corresponding to all or a portion of the amino acid sequence set out in Figure 1 (SEQ ID NO: 1) and derivatives and fragments thereof having bacteriocin and/or bacteriocin immunity activity.

2. A polypeptide as claimed in claim 1 having or including the amino acid sequence

. and α₂ (SEQ ID NO: 2):

N Gly Thr Trp Asp

He Gly Arg Val

Lys Ala Met Gly

Asn Gin Ala Ser

Lys Lys His C

and/or

and derivatives and fragments thereof having bacteriocin activity.

3. A polypeptide having or including the amino acid sequence

(SEQ ID NO: 4) and derivatives and fragments thereof having bacteriocin immunity factor activity.

4. A bacteriocin comprising polypeptides α and β or fragments or derivatives thereof as defined in claim 2.

5. A bacteriocin as claimed in claim 4 comprising polypeptides α and β , or fragments or derivatives thereof, in a ratio of 5-10 to 1 respectively.

6. A bacteriocin as claimed in claim 5 comprising polypeptides α and β , or fragments or derivatives thereof, in a ratio of 8 to 1 respectively.

7. A composition comprising a bacteriocin as claimed in any one of claims 4 to 6 and/or a bacteriocin immunity factor as claimed in claim 3 together with at least one of a carrier, and/or diluent, or excipient.

8. Use of a bacteriocin as claimed in any one of claims 4 to 6 in selectively killing undesired or contaminating strains of bacteria in microbiological or food manufacturing processes.

9. A starter culture of microorganisms for use in a microbiological process, comprising a bacteriocin as claimed in any one of claims 4 to 6, said microorganisms being resistant to said bacteriocin.

10. A method of cheese or yoghurt production in which a bacteriocin as claimed in any one of claims 4 to 6 is added to effect lysis of lactic acid bacteria.

11. A method of isolation of a bacteriocin and/or a bacteriocin immunity factor as claimed in any one of claims 3 to 6 wherein a culture of a microorganism expressing said bacteriocin and/or immunity factor is subjected to fractionation whereby fractions enriched in said bacteriocin and/or immunity factor are collected.

12. A method as claimed in claim 11 wherein the microorganism is Lactococcus lactis strain LMG 2081.

13. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide or its derivatives or fragments as claimed in any one of claims 1 to 3.

14. A nucleic acid molecule comprising a nucleotide sequence which encodes a bacteriocin, its component peptides and/or its corresponding immunity factor, or a fragment thereof, substantially corresponding to all or a portion of the nucleotide sequence as shown in Figure 1 (SEQ ID NO: 5) or a sequence which is degenerate or substantially homologous with or which hybridises with any such sequence.

15. An expression or cloning vector comprising a nucleic acid molecule as claimed in claim 13 or claim 14.

16. A host cell or transgenic organism containing a nucleic acid molecule as claimed in claim 13 or claim 14.

17. A host cell as claimed in claim 16 being a lactic acid bacterium.

18. A method for preparing a polypeptide, derivative or fragment as claimed in claim 1, comprising culturing a host cell as defined in claim 16 or claim 17 under conditions whereby said polypeptide is expressed and recovering said polypeptide, derivative or fragment thus produced.

19. A process for the preparation of bacteriocin and/or bacteriocin immunity factor polypeptides as claimed in any one of claims 1 to 3 in which a corresponding protected or immobilised polypeptide is subjected to deprotection or removal from a solid support.