The present invention relates to the field of bacteπocins. More particularly the present invention relates to new polynucleic acid sequences encoding new bacteπocins and new proteins with bacteπocin-transporter and processing activity, as well as the use of said sequences for heterologous protein expression, and protein secretion.

Lactic acid bacteria are known to produce a whole range of antagonistic substances such as lactic acid, acetic acid, hydrogen peroxide, diacetyl, acetaldehyde, bacteπocins and bacteriocin-like substances (Klaenhammer, 1 988, Daeschel, 1989, Schillinger, 1990, Piard and Desmazeaud, 1 992a, 1992b, De Vuyst & Vandamme, 1 994a, 1 994b) Bactenocins are proteins or protein complexes (protein aggregates, lipo- and/or glycoproteins, . ) with a bactericidal activity, especially against closely related sensitive strains (Tagg et al , 1 976) Among the lactic acid bacteria, Lactobacilli are long known for their bacteπocin-producing abilities Several publications describe the production of bactenocins or bacteriocin-like substances by Lactobacillus (Lb) strains, such as Lactacin B (Barefoot & Klaenhammer,

1983) and Acidofilucine A (Toba et al. , 1 991 a), produced by Lb. acidophilus; Brevicin 37 (Rammelsberg & Radler, 1990), produced by Lb. brevis, Caseicin 80 (Rammelsberg & Radler, 1990) and Caseicin LHS (Dicks et al. , 1 992), produced by Lb. casei; Curvacin A (Tichaczek et a/. , 1992), produced by Lb. curvatus; Lacticin A and B (Toba et al , 1 991 b), produced by Lb. Delbrueckii subsp. lactis, Bacteπocin 466 (De Klerk & Smith, 1 967) produced by Lb fermentum, Lactocin 27 (Upreti & Hinsdill, 1 973), Helveticin J (Joerger & Klaenhammer, 1986) and Helveticin V- 1 829 (Vaughan et a/. , 1 992), produced by Lb. helveticus; Lactacin F (Muπana & Klaenhammer, 1987), produced by Lb. Johnsonii; Plantacin B (West & Warner, 1988), Plantancin A (Daeschel et a/. , 1 990) and Plantancin S (Jimenez-Diaz et a , 1 990), produced by Lb. plantarum; Reuteπcin 6 (Toba et a/. , 1 991 c), produced by Lb. reuteri; and

Sakacin A (Schillinger & Lύcke, 1 989), Sakacin P (Tichaczek et al. , 1 992) and Lactocin S (Mortvedt & Nes, 1 990), produced by Lb. Sake No bactenocins produced by Lb amy/ovorus could be discovered sofar

The antimicrobial properties of the bactenocins produced by lactic acid bacteria could be of economical importance since these bactenocins could be used as food preservatives

Several bactenocins have a bactericidal activity towards different food spoilage and/or pathogenic bacteria such as Bacilli, Clostπdia, Staphilococci and Listeπae Furthermore, several bactenocins are small thermostable proteins, facilitating their use as a food additive

a food additive in heat-treated food. Alternatively, the lactic acid bacteria producing these bactenocins could be used for the development of new starter cultures for use in food preservation via in situ bacteπocin production. The bacteπocin coding sequences could also be used in the construction of "food-grade" cloning vectors For the moment only the bacteπocin Nism, produced by Lactococcus lactis subsp. lactis, is commercially produced and applied worldwide as a biological food preservative (Hurst, 1 981 , Rayman & Hurst, 1 984, Delves-Broughton, 1 990, De Vuyst & Vandamme, 1 994c)

On the other side, the bacteπocin regulatory signals (promotors, terminators and signal sequences) could be used for the production of homologous and/or heterologous proteins in lactic acid bacteria ( for an overview of the biotechnological potential of lactic acid bacteria see Gasson, 1 993). Also, the sequences coding for the transporter proteins could be used to allow secretion and processing of saiα proteins The secretion of most bactenocins depends on the presence of ATP-dependent ABC transporter proteins Those proteins are characterized by six transmembrane domains, a carboxy-termtnal ATP-bmding cassette and a N-terminal proteolytic domain (Havarstein et al , 1 995) They belong to a superfamily of cytoplasmic membrane translocators shown to be involved in the signal sequence-independent secretion and processing of bactenocins of the double-glycme peptide type. The present invention also deals with a protein that shows homology with HlyD component of the haemoiysin A secretion apparatus of E.coli (Schυlein et a , 1 992) These so-called accessory proteins are characterized by a unique, N-terminal transmembrane domain and a hydrophilic carboxy-terminus They are predicted as integral proteins of the cytoplasmic membrane thought to facilitate signal sequence-independent secretion.

Lactic acid bacteria are G.R.A.S. organisms ((generally .Regarded s Safe) and could be preferred to other bacteria such as E.coli as a production organism for proteins which will be used as human therapeutics. The use of lactic acid bacteria for heterologous protein expression could also lead to the development of a new generation of "live" oral vaccins

(Iwaki, 1 990; Boersma, 1 992, Marteau, 1 993)

The aim of the present invention is thus to provide polynucleic acid sequences encoding novel bactenocins or bacteπocin immunity proteins or transporter proteins

Another aim of the present invention is to provide novel purified bactenocins and bacteπocin immunity proteins, and ATP-dependent transporter proteins and their accessory proteins.

Another aim of the present invention is to provide ammo acid sequences for the

same.

Another aim of the present invention is to provide recombinant polypeptides for the same, as well as methods for preparing the same.

Another aim of the present invention is to provide recombinant vectors comprising a polynucleic acid sequence as defined above from the coding and/or non-coding regions thereof for cloning and/or homologous or heterologous protein expression and/or secretion purposes.

Another aim of the present invention is to provide probes or primers derived from said polynucleic acid sequences. Another aim of the present invention is to provide host cells transformed with said recombinant vectors.

Another aim of the present invention is to provide methods for producing heterologous or homologous recombinant proteins using said transformed host cells.

Another aim of the present invention is to provide for the use of said purified and/or recombinant polypeptides in microbiological or food manufacturing purposes.

Another aim of the present invention is to provide starter cultures for such uses.

The present invention relates more particularly to a polynucleic acid comprising:

(a) at least 30 contiguous nucleotides of the polynucleic acid as shown in Figure 1

(SEQ ID NO 1 ), or Figure 2 (SEQ ID NO 1 9), or, (b) a polynucleic acid which hybridizes to the polynucleic acid as shown in Figure

1 (SEQ ID NO 1 ) , provided that nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included, or Figure 2 (SEQ ID NO 1 9), or as defined in (a), or,

(c) a polynucleic acid with a degenerate sequence as a result of the genetic code to the polynucleic acid sequences as defined in (a) or (b) and which code for a polypeptide as defined.

The polynucleic acid sequence represented as SEQ ID NO 1 in Figure 1 shows a polynucleic acid of 3654 nucleotides length. The 5' part of this sequence contains part of the insertion element IS 1 201 , which is not related to the present invention. The nucleotides 1 to 445 are therefore excluded (see figure 3). The 3" part of this sequence contains part of a polylinker, which is not related to the present invention. The nucleotides

3587 to 3654 are therefore excluded (see figure 3). This sequence was also found to comprise two operons each consisting of three open reading frames. Each operon is preceded by a promoter. Each operon consists of two "bacteπocin-like" genes followed by a third ORF, presumably an immunity gene. The four bacteπocin-like proteins are each

characterized by a conserved proteolytic processing site preceded by a conserved prosequence. The nucleotide and deduced am o acid sequence of both operons are also shown in Figure 1 : SEQ ID NO 2 which codes for bacteriocin 1 (LbnA I ; SEQ ID NO 3) of operon 1 , SEQ ID NO 4 which codes for bacteriocin 2 (LbnA2; SEQ ID NO 5) of operon 1 , SEQ ID NO 6 which represents bacteriocin 1 (LbnB I ; SEQ ID NO 7) of operon 2, SEQ ID

NO 8 which codes for bacteriocin 2 (LbnB2; SEQ ID NO 9) of operon 2, SEQ ID NO 1 0 which codes for immunity protein LbiA (SEQ ID NO 1 1 ) of operon 1 , SEQ ID NO 12 which codes for immunity protein LbiB (SEQ ID NO 1 3) of operon 2. The regulatory sequences are also shown in Figure 1 : SEQ ID NO 14 shows the operon 1 promoter sequence, SEQ ID NO 1 5 shows the operon 2 promoter sequence, SEQ ID NO 1 6 shows the operon 1 promoter and signal sequence, SEQ ID NO 17 shows the operon 2 promoter and signal sequence. The closest related sequence was found to be the Lactacin F operon (Muπana & Klaenhammer, 1 991 ).

The polynucleic acid sequence represented as SEQ ID NO 1 9 in figure 2 shows a polynucleic acid of 8738 nucleotides length, and comprises the nucleotides 446 to 3587 of SEQ ID NO 1 as represented in Figure 3 and comprising the two operons as defined above, followed by a sequence that compπses three additional operons, two operons consisting of two open reading frames, and one operon consisting of three open reading frames. Each operon is preceded by a promoter The first additional operon (operon 3) consists of an ORF that codes for an ATP-dependent signal sequence-independent transporter protein followed by an ORF that codes for an accessory protein. A second additional operon (operon 4) consists of one "bacteriocin-like " ORF followed by an ORF, presumably an immunity gene. A third additional operon (operon 5) consists of two "bacteriocin-like" ORFs followed by an ORF, presumably an immunity gene. The nucleotide and deduced ammo acid sequence of the three operons are also shown in Figure 2: SEQ

ID NO 20 which codes for a transporter protein (LbnT; SEQ ID NO 21 ) of the operon 3, SEQ ID NO 22 which codes for an accessory protein (LbnE, SEQ ID NO 23) of operon 3, SEQ ID NO 24 which codes for a bacteriocin (LbnC, SEQ ID NO 25) of operon 4, SEQ ID NO 26 which codes for an immunity protein (LbiC; SEQ ID NO 27) of operon 4, SEQ ID NO 28 which codes for bacteriocin 1 (LbnD 1 ; SEQ ID NO 29) of operon 5, SEQ ID NO 30 which codes for bacteriocin 2 (LbnD2; SEQ ID NO 31 ) of operon 5, SEQ ID NO 32 which codes for an immunity protein (LbiD; SEQ ID NO 33) of operon 5. The regulatory sequences are also shown in Figure 2: SEQ ID NO 34 shows promoter sequence of the operon 3 encoding the transporter protein LbnT and the accessory protein LbnE; SEQ ID NO 35 shows the

promoter sequence of operon 4 encoding bactenocines LbnC, SEQ ID NO 36 the promoter sequence of operon 5 encoding bactenocines LbnD 1 en 2, SEQ ID NO 37 shows the promoter and signal sequence of bacteriocin C; SEQ ID NO 38 shows the promoter and signal sequence of bacteriocin D. The closest related sequence was found to be the Lactacin F operon ( uπana & Klaenhammer, 1 991 )

According to a preferred embodiment, the present invention also relates to part of a polynucleic acid as defined above, particularly parts which can regulate the transcription and/or translation of heterologous sequences

The term "polynucleic acid" corresponds to either double-stranded or single-stranded cDNA or genomic DNA, or RNA

The polynucleic acids of the invention are to be understood as also comprising the degenerate nucleic acids of the nucleic acid coding for any of the polypeptides of the invention as disclosed below

The term "hybridizes" refers to conventional hybridization conditions known to the man skilled in the art, preferably to stringent hybridization conditions (see f i Maniatis et al ., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory,

1 989) in which the sequence coding for Lactacin F (Muπana and Klaenhammer, 1 991 ) will not hybridize.

The polynucleic acid according to option (a) as set out above can be prepared according to the procedures as set out in the Examples section of the present invention

Alternatively, said polynucleic acids may also be derived according to any other procedure obvious from the teaching of the present invention or any other procedure known to the man skilled in the art for preparing polynucleic acid sequences

The polynucleic acids according to options (b) and (c) can be prepared by applying techniques of hybridization, cloning and/or recombinant expression known to the man skilled in the art.

The polynucleic acids according to option (c) may be derived by cloning equivalents of the polynucleic acids as depicted in SEQ ID NO 1 , provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included , and/or of SEQ ID NO 1 9 This may be achieved by screening libraries containing polynucleic acids of other related bacteria with probes derived from the polynucleic acid as given in SEQ ID NO 1 , provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included, and/or SEQ ID NO 1 9 Alternatively, said equivalents may be cloned by performing an amplification reaction, such as PCR of mRNA to obtain amplified products, with primers essentially comprising a

nucleotide sequence which is part of the polynucieic acid sequence given in SEQ ID NO 1 and/or SEQ ID NO 1 9. The polynucleic acids according to (c) may also be provided by classical chemical synthesis methods of oligo- or polynucleotides generally known by the person skilled in the art. Also included in the present invention are polynucleic acid sequences comprising a sequence of preferably 1 0, 1 1 , 1 2, 1 3, 14, 1 5, 1 6, 1 7, 1 8, 1 9, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 500, 1000 or more contiguous nucleotides selected from the sequence as depicted in Figure 1 (SEQ ID NO 1 ), provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included, and/or selected from the sequence as depicted in Figure 2 (SEQ ID NO 1 9), or their complement, or sequences hjybπdizing thereto under stringent hybridization conditions, or sequences which are degenerate as a result of the genetic code to any of the foregoing sequences

Especially preferred parts of the polynucleic acid sequences of the present invention include regulatory sequences such as promoter sequences, transcriptional initiation and termination sequences, and translational initiation and termination sequences, signal sequences, etc. These parts can be used according to the present invention for expression of heterologous sequences in different bacterial hosts, such as lactic acid bacteria The present invention particularly relates to parts of any of SEQ ID NO 1 to 38, provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 as presented in SEQ ID NO 1 are not included

Alternatively, the present invention also relates to a part of the polynucleic acids of the present invention which codes for at least part of a polypeptide according to the present invention The present invention particularly relates to part of any of SEQ ID NO 1 to 1 3 and SEQ ID NO 1 9 to 33, provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 as presented in SEQ ID NO 1 are not included

According to another embodiment, the present invention also relates to any combination of polynucleic acid parts as described above (coding with non-coding or exclusively combinations of coding or exclusively combinations of non-coding parts).

According to another embodiment, the present invention also relates to a polynucleic acid sequence as defined above compπsing at least 1 0 or more contiguous nucleotides, for use as a specific or unique hybridization probe for detecting the presence of any target sequence comprising a polynucleic acid according to the present invention, as defined above

The term "probe" refers to single-stranded sequence-specific oligonucleotides which

have a sequence which is sufficiently complementary to a target sequence to be detected or cloned. Probes may be labelled according to any of the techniques known in the art. Preferably these probes are about 10 to 50 nucleotides long According to the hybnzation solution used, these probes should be hybridized at appropriate temperatures and be of appropriate length to attain sufficient specificity

According to another embodiment, the present invention relates to polynucleic acid sequences as defined above, for use as a specific or unique primer for amplification of any polynucleic acid sequence according to the present invention, as defined above.

The term "primer" refers to a single-stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension product Preferably, the primer is about 10 to about 50 nucleotides long Specific length and sequences will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength The amplification method used can be any method known in the art.

The fact that amplification-primers do not have to match exactly with the corresponding template sequence to warrant proper amplification, providing that an exact match at the last three nucleotides at the 3' end of the primer is maintained, is amply documented in the literature (Kwok et al., Nucleic Acids Research 1 8.999-1005, 1990;

Sommer and Tautz, Nucleic Acids Research 1 7, 6749, 1 989)

Probes and primers according to these aspects of the present invention may be used to isolate equivalents of the polynucleic acid sequence as defined above in bacteria other than Lactobacillus amylovorus as specified above. According to another embodiment, the present invention relates to a recombinant vector, particularly for cloning and/or expression, with said recombinant vector comprising a vector sequence, itself comprising a polynucleic acid sequence according to the present invention, as defined above, or fragments thereof.

It is to be understood that said polynucleic acid sequence of the invention may comprise:

(a) only regulatory elements/sequences capable of providing for the expression and/or secretion of certain (heterologous) coding sequences by specific host cells, or,

(b) only coding sequences which are operably linked regulatory sequences present

in the vector sequence, and/or sequences coding for proteins that are capable of providing for the secretion of expressed proteins,

(c) both regulatory and coding sequences capable of providing for the expression and/or secretion of certain products encoded by said polynucleic acids (homologous expression).

The term "vector" may comprise a plasmid, a cosmid, a phage or a virus. Preferably said vector will be a plasmid. Particularly preferred plasmids (or vectors) for the expression of coding sequences derived from the polynucleic acid sequences according to the present invention include for instance pGKV21 0 or any other suitable vector known in the art. The term "coding sequence" refers to a polynucleic acid sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5 '-termιnus and a translation stop codon at the 3'- termmus. A coding sequence can include but is not limited to m RNA, DNA (including cDNA), and recombinant polynucleotide sequences.

The term "recombinant" refers to the fact that a fusion is being made between polynucleic acids from different origins.

The term "operably linked" refers to a juxtaposition wherein the components are figured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of a coding sequence.

The term "regulatory sequence" or control sequence refers to those sequences which control the transcription and/or translation of the coding sequences; these may include but are not limited to promoter sequences, transcriptional initiation and termination sequences, and translational initiation and termination sequences. In addition, control sequences refer to sequences which control the processing of the polypeptide encoded within the coding sequence; these may include, but are not limited to sequences controlling secretion, protease cleavage, and glycosylation of the polypeptide.

It should be understood that the signal sequence (and the signal peptide encoded by it) of the proteins encoded within the sequences of the invention in itself form an aspect of the invention, and it is contemplated that they (it) may be inserted upstream of DNA sequences coding for other proteins or peptides so as to obtain secretion of the resulting products from the cell. More particularly, these signal sequences may be used for the following host systems: Bacillus, Lactobacillus, Lactococcus and other Gram positive bacteria.

According to a particularly preferred embodiment, the present invention relates to a recombinant vector as defined above, more particularly a plasmid, comprising a nucleotide sequence encoding a heterologous or homologous protein or peptide which is desired to be expressed, and/or secreted. Examples of such proteins or peptides may include, but are not limited to, any of the polypeptides or peptides characterized by an ammo acid sequence encoded by any of the polynucleic acids as defined above, more particularly a part of the polynucleic acid sequence as depicted in SEQ ID NO 1 , provided that the nucleotides 1 to 445 and 3587 to 3654 are not included, and/or a part of the polynucleic acid sequence of SEQ ID NO 1 9 (homologous protein or peptide), or polypeptides or peptides encoding for instance mouse

TNF and other cytokines from mammalian origin, or any other heterologous proteins or peptides.

Accordmg to another embodiment, the present invention relates to a recombinant vector, more particularly a plasmid, comprising a promotor sequence included in any of the polynucleic acid sequences according to the present invention, as defined above.

More particularly, the present invention relates to a recombinant vector (e.g. a plasmid) comprising a promoter sequence derived from L amy/ovorus, selected from the sequences present on the nucleotide sequence as depicted in SEQ ID NO 1 , provided that the nucleotides 1 to 445 and nucleotrdes 3587 to 3654 are not included, and/or from the sequences present on SEQ ID NO 1 9, or a derivative thereof as defined above

According to another embodiment, the present invention relates to a recombinant vector, more particularly a plasmid, comprising a promotor/secretion signal sequence included in any of the polynucleic acid sequences according to the present invention, as defined above, and/or a sequence that codes for a transporter protein according to the present invention, as defined above.

More particularly, the present invention relates to a recombinant vector, more particularly a plasmid, comprising a promotor/secretion signal sequence derived from L. amylovorus, selected from the sequences present on the nucleotide seαuence as depicted in SEQ ID NO 1 , provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included, and/or from SEQ ID NO 1 9, or a derivative thereof as defined above.

In order to carry out the expression of the polypeptides of the invention in bacteria such as E. coli or B.subtilis or in eukaryotic cells such as in S cerevisiae, or in cultured vertebrate or invertebrate hosts such as insect cells, Chinese Hamster Ovary (CHO), COS, BHK, and MDCK cells, the following steps are carried out

transformation of an appropriate cellular host with a recombinant vector, in which a nucleotide sequence coding for one of the polypeptides of the invention has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a prokaryotic host, an appropriate πbosome binding site (RBS), enabling the expression in said cellular host of said nucleotide sequence, culture of said transformed cellular host under conditions enabling the expression of said insert. According to yet another aspect, the present invention relates to a host cell transformed with a recombinant vector as defined above

More particularly, the present invention contemplates host cell transformed with a recombinant vector as defined above. Such host cells include Gram positive hosts such as

Lactococcus spp., Bacillus spp.. Streptococcus spp. and Lactobacillus spp. and more preferably Lactococcus lactis, Streptococcus gordonu, Lactobacillus acidophilus, L. acidophilus, L. casei, but also for instance S. cerevisiae.

Most particularly, the host cell is L.amylovorus LIM KB-1 80 as deposited under No. LMG P- 1 31 39 on January 1 1 , 1 993 in the BCCM-LMG culture collection (Laboratory for Microbiology, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium) According to yet another embodiment, the present invention relates to a method for producing a desired heterologous or homologous protein or peptide in a host cell as defined above, comprising at least the following steps

- transforming said host cell with any recombinant vector, more particularly a plasmid, as defined above, - culturing the transformed host cell in a suitable medium under conditions allowing expression of said protein or peptide, and,

- recovering the expressed protein or peptide from said host cell or said medium According to yet another embodiment, lactic acid bacteria transformed as set out above, and secreting the appropriate heterologous proteins can be used as an 'oral vaccine', in which said proteins are the antigens used for immunization, or as a medicament in which said proteins directly interfere with a toxic substance or protein as for instance the cholera toxin.

According to a preferred embodiment, the present invention relates also to a polypeptide in substantially pure form comprising an ammo acid sequence substantially

corresponding to any of the ammo acid sequences encoded by a polynucleic acid sequence according to the present invention, as defined above, or derivatives or fragments thereof having bacteriocin and/or bacteriocin immunity activity and/or transporter activity

More particularly the present invention relates also to a polypeptide in substantially pure form comprising an ammo acid sequence substantially corresponding to any of the ammo acid sequences encoded by a polynucleic acid sequence as set out in SEQ ID 1 , provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included and/or of SEQ ID NO 1 9, or derivatives and fragments thereof having bacteπocin and/or bacteriocin immunity activity, and/or transporter activity Said polypeptide according to the present invention is preferably a recombinant polypeptide

The expression "an ammo acid sequence substantially corresponding to " refers to an ammo acid sequence which is at least 90%, preferably 95%, more preferably 98% or more homologous to any of the ammo acid sequences as set out in SEQ ID NO 1 when nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included and/or to any of the am o acid sequences encoded by SEQ ID NO 1 9

The term "derivatives" corresponds to mutems or homologoues of the ammo acid sequences as depicted in SEQ ID NO 1 , provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included, and/or of the sequences as depicted in SEQ ID NO 1 9, which (i) are derived from other bacterial strains (purified naturally occuring derivatives), or (n) which have in their ammo acid sequence insertions, substitutions or deletions in comparison to the ammo acid sequences encoded by SEQ ID NO 1 provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included and/or in comparison to the ammo acid sequences encoded by SEQ ID NO 1 9, which do not influence the bacteriocin or bacteriocin immunity or transporter activity of said polypeptides or fragments, or (in) which are encoded by polynucleic acid sequences according to the present invention which are degenerate as a result of the genetic code to any of the polynucleic acid sequences as depicted in SEQ ID NO 1 , provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included, and/or to SEQ ID NO 1 9 It is to be understood that the derivatives within the scope of the present invention have retained the bacteπocin and/or bacteriocin immunity and/or transporter protein activity

It should be understood that also the polynucleic acid sequences encoding such

"derivatives" are within the scope of the present invention

The term "mutein" referred to above corresponds to polypeptides which are derived

from the polypeptides of the invention and which include ammo acid substitutions, deletions or insertions compared to the polypeptides of the invention encoded by SEQ ID NO 1 , provided that the nucleotides 1 to 445 and nucleotides 3587 to 3654 are not included, and/or to the ammo acid sequences encoded by SEQ ID NO 1 9. Table 1 gives an overview of the am o acid substitutions which could be the basis of some of the mute s as defined above

The term "fragments" or peptides refers to smaller portions of about 5 to about 50 am o acids in length of any of the above-mentioned polypeptides or their derivatives

The latter polypeptides and derivatives according to the present invention may be produced by recombinant DNA technology The derivatives and particularly the fragments according to the present invention may also be prepared by classical chemical synthesis methods The synthesis can be carried out in homogenous solution or in solid phase For instance, the synthesis technique in homogenous solution which can be used is the one described by Houbenweyl in the book entitled "Methode der organischen Chemie" (Method of organic chemistry) edited by Wunsh, vol. 1 5-1 et II THIEME, Stuttgart, 1 974 The polypeptides of the invention can also be prepared in solid phase according to the methods decsπbed by Atherton and Shepard in their book entitled "Solid phase peptide synthesis" (IRL Press, Oxford, 1 989)

The expression "bacteriocin activity" is referred to in the introduction and may be measured by any method known in the art, and as depicted in the examples

The expression "bacteriocin immunity activity" is the activity that confers resistance towards the action of bactenocines and may be assessed by any method known in the art

The expression "transporter activity" is referred to in the introduction and may be assessed by means of measuring the secretion of bacteriocin activity According to a more specific embodiment the present invention relates to a polypeptide as defined above comprising in its ammo acid sequence aπ ammo acid sequence of a bacteriocin type 1 as represented in SEQ ID NO 2 to 3 or derivatives or fragments thereof having bacteriocin activity

The present invention also relates to a polypeptide as defined above comprising in its am o acid sequence an ammo acid sequence of a bacteriocin type 2 as represented in

SEQ ID NO 4 or 5 or derivatives or fragments thereof having bacteriocin activity

The present invention also relates to a polypeptide as defined above comprising in its ammo acid sequence an ammo acid sequence of a bacteriocin type 3 as represented in SEQ ID NO 6 or 7 or derivatives or fragments thereof havmg bacteriocin activity

The present invention also relates to a polypeptide as defined above comprising in its ammo acid sequence an amino acid sequence of a bacteriocin type 4 as represented in SEQ ID NO 8 or 9 or derivatives or fragments thereof having bacteriocin activity.

The present invention also relates to a polypeptide as defined above comprising in its ammo acid sequence an ammo acid sequence of a bacteπocin type 5 as represented in

SEQ ID NO 24 or 25 or derivatives or fragments thereof having bacteriocin activity

The present invention also relates to a polypeptide as defined above comprising in its am o acid sequence an ammo acid sequence of a bacteriocin type 6 as represented in SEQ ID NO 28 or 29 or derivatives or fragments thereof having bacteπocin activity The present invention also relates to a polypeptide as defined above comprising in its ammo acid sequence an ammo acid sequence of a bacteriocin type 7 as represented in SEQ ID NO 30 or 31 or derivatives or fragments thereof having bacteriocin activity

The present invention also relates to a polypeptide as defined above comprising in its ammo acid sequence a bacteriocin type 1 and/or bacteriocin type 2 and/or a bacteriocin type 3 and/or a bacteriocin type 4 and/or a bacteriocin type 5 and/or bacteriocin type 6 and/or a bacteriocin type 7 and/or derivatives or fragments thereof having bacteriocin activity.

According to yet another embodiment, the present invention relates to a polypeptide as defined above comprising in its am o acid sequence an ammo acid sequence of a bacteriocin immunity protein type 1 as represented in SEQ ID NO 1 0 or 1 1 or derivatives or fragments thereof having bacteriocin immunity activity

The present invention relates also to a polypeptide as defined above comprising in its ammo acid sequence an ammo acid sequence of a bacteriocin immunity protein type 2 as represented in SEQ ID NO 1 2 or 1 3 or derivatives or fragments thereof having bacteriocin immunity activity

The present invention relates also to a polypeptide as defined above comprising in its ammo acid sequence an ammo acid sequence of a bacteπocin immunity protein type 3 as represented in SEQ ID NO 26 or 27 or derivatives or fragments thereof having bacteriocin immunity activity. The present invention relates also to a polypeptide as defined above comprising in its ammo acid sequence an am o acid sequence of a bacteriocin immunity protein type 4 as represented in SEQ ID NO 32 or 33 or derivatives or fragments thereof having bacteriocin immunity activity.

The present invention relates also to a polypeptide as defined above comprising in

its ammo acid sequence an am o acid sequence of a bacteriocin immunity protein type 1 and/or a bacteriocin immunity type 2 protein and/or a bacteriocin immunity protein type 3 and/or a bacteriocin immunity type 4 and/or derivatives or fragments thereof having bacteriocin immunity activity. According to yet another embodiment, the present invention relates to a polypeptide as defined above comprising in its ammo acid sequence an ammo acid sequence with a putative transporter activity as represented in SEQ ID NO 20 or 21 and/or with an accessory function for this transporter activity as presented in SEQ ID NO 22 or 23.

The present invention relates also to a polypeptide as defined above comprising in its ammo acid sequence an ammo acid sequence of a bacteπocin type 1 and/or bacteriocin type 2 and/or a bacteriocin type 3 and/or a bacteriocin type 4 and/or bacteriocin type 5 and/or a bacteriocin type 6 and/or a bacteriocin type 7 and/or a bacteriocin immunity protein type 1 and or a bacteriocin immunity type 2 protein and/or a bacteriocin immunity protein type 3 and or a bacteriocin immunity type 4 protein and/or an ATP-dependent signal sequence-independent transporter protein and/or its accessory protein and/or derivatives or fragments thereof having a bacteriocin and/or a bacteriocin immunity activity, and/or transporter activity.

According to yet another embodiment, the present invention relates to a composition comprising at least one bacteriocin as defined above and/or at least one bacteriocin immunity factor as defined above together with at least one of a suitable carrier, and/or diluent, or excipient, with said carriers, diluents and excipients being well known in the art.

According to another aspect, the present invention relates to the use of a bacteriocin or composition as defined above for selectively killing undesired or contaminating strains of bacteria in microbiological or food manufacturing processes or any other use known for bactenocins in the art as specified in the introduction.

According to yet another embodiment, the present invention relates to a starter culture of microorganisms for use in a microbiological process, comprising at least one bacteriocin or bacteriocin immunity protein as defined above, said microorganisms of said starter culture being resistant to said bacteriocin

Said bacteriocin is thus used to prevent the growth of bacteπocm-sensitive bacteria The present invention also relates to a method of isolation of a bacteriocin and/or a bacteriocin immunity factor as defined above 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

The present invention relates more particularly to a method as defined above wherein the microorganism is Lactobacillus amylovorus LIM KB- 1 80

FIGURE LEGENDS

Figure 1 : The polynucleic acid sequence represented as SEQ ID NO 1 in Figure 1 shows a polynucleic acid of 3654 nucleotides length This sequence was found to comprise two operons each consisting of three open reading frames Each operon is preceded by a promoter. Each operon consists of two "bacteriocin-like" genes followed by a third ORF, presumably an immunity gene. The four bacteriocin-like proteins are each characterized by a conserved proteolytic processing site preceded by a conserved prosequence. The nucleotide and deduced am o acid sequence of both operons are also shown in Figure 1 : SEQ ID NO 2 which codes for bacteriocin 1 (LbnAI , SEQ ID NO 3) of operon 1 , SEQ ID NO 4 which codes for bacteriocin 2 (LbnA2; SEQ ID NO 5) of operon 1 , SEQ ID NO 6 which represents bacteriocin 1 (LbnBI ; SEQ ID NO 7) of operon 2, SEQ ID NO 8 which codes for bacteriocin 2 (LbnB2, SEQ ID NO 9) of operon 2, SEQ ID NO 1 0 which codes for immunity protein LbiA (SEQ ID NO 1 1 ) of operon 1 , SEQ ID NO 1 2 which codes for immunity protein LbiB (SEQ ID NO 1 3) of operon 2. The regulatory sequences are also shown in Figure 1 . SEQ ID NO 14 show the operon 1 promoter sequence, SEQ ID NO 1 5 shows the operon

2 promoter sequence, SEQ ID NO 1 6 shows the operon 1 promoter and signal sequence, SEQ ID NO 1 7 shows the operon 2 promoter and signal sequence

Figure 2- The polynucleic acid sequence represented as SEQ ID NO 1 9 in Figure 2 shows a polynucleic acid of 8738 nucleotides length, and comprises the 3654 nucleotides as represented in Figure 2 and comprising the two operons as defined above, followed by a sequence that comprises three additional operons, two operons consisting of two open reading frames and one operon consisting of three open reading frames. Each operon is preceded by a promoter. The first additional operon (operon 3) consists of one ATP- dependent transporter protein and one accessory protein. Operon 4 consists of one " bacteriocin-like" gene followed by a second ORF, presumably an immunity gene. The four

"bacteriocin-like" proteins are each characterized by a conserved proteolytic processing site preceded by a conserved prosequence. The nucleotide and deduced ammo acid sequence of both operons are also shown in Figure 2. SEQ ID NO 20 which codes for a transporter protein (LbnT; SEQ ID NO 21 ) of operon 3, SEQ ID NO 22 which codes for an accessory protein (LbnA; SEQ ID NO 23) of operon 3, SEQ ID NO 24 which represents bacteπocin 1

(LbnC; SEQ ID NO 25) of operon 4, SEQ ID NO 26 which codes for an immunity protein

(LbiC; SEQ ID NO 27) of operon 4, SEQ ID NO 28 which codes for bacteriocin 1 (LbnD 1 ; SEQ ID NO 29) of operon 5, SEQ ID NO 30 which codes for bacteriocin 2 (LbnD2; SEQ ID NO 31 ) of operon 5, SEQ ID NO 32 which codes for immunity protein (LbiD; SEQ ID NO 33) of operon 5. The regulatory sequences are also shown in Figure 2. SEQ ID NO 34 show promoter sequence of the third operon encoding the transporter protein LbnT and the accessory protein LbnE, SEQ ID NO 35 shows the promoter sequence of the fourth operon encoding LbnC and LbiC, SEQ ID NO 36 the promoter sequence of the fifth operon encoding bactenocines LbnD 1 en 2, SEQ ID NO 37 shows the promoter and signal sequence of bacteriocin C, SEQ ID NO 38 shows the promoter and signal sequence of bacteriocin D

Figure 3: Schematic represention of the organisation of the operons coding for the bactenocins, the respective immunity genes, the transporter and the accessory protein and how they are defined by SEQ ID NO 1 and SEQ ID NO 1 9 The IS element and the polylmker as defined by the borders of SEQ ID NO 1 are disclaimed

EXAMPLES

1 . MATERIALS AND METHODS

1 .1 . Bacterial strains and media

The bacterial strains, the coresponding media and the growth conditions are depicted in tables 1 &2. All bacterial strains were kept at -75 °C as frozen cultures in the appropriate medium containing 25 % glycereol. Before experimental use, cultures were re- inoculated twice. The transfer inoculum used was 1 % (v/v). Agar containing media were prepared by adding 1 ,5 % granular agar (Oxoid) to liquid medium.

1 .2. Isolation of lactic acid bacteria

Samples were taken from freshly diluted Corn Steep Liquor (CSL), pH 3.8, 50 ° C, originating from the starch processing company Cerestar N.V. They were subsequently incubated for 6 hours at 45 °C and streaked on MRS-agar plates (De Man, Rogosa, Sharpe- mediu , Oxoid) supplemented with 0.01 % cycloheximide (to prevent yeast growth). The plates were incubated anaerobically overnight at 45 °C (anaerobic jar). Appearing colonies were picked and remcubated anaerobically overnight at 45 °C. The colonies were subsequently analysed on GRAM-staining, sporeformation, katalase- and oxidase activity. Non-sporeforming, GRAM-positive, katalase-negative and oxidase-negative bacteria were further analysed with the API 50 CH-testkit and on their anatagonistic characteristics.

1 .3. Bacteriocin detection and measurement

For the analysis of the anatagonistic activity of the selected bacteria, a direct, indirect and agar diffusion method was used. 1 2 selected lactic acid bacteria were used as indicator organisms (see strains marked with asterisk in table 1 ). An E. coli strain was used as a GRAM-negative control. The indirect method used was a modification of the method described by Mayr-Harting et al. ( 1 972). Overnight cultures of the bacteria to be

tested were diluted in a sterile fysiological solution, plated out on MRS-plates ( 1 /5 % w/v) supplemented with 0.2 % glucose (further marked as MRS-0.2), and incubated for 24 hours at 45 °C. The plates were subsequently overlayed with a soft MRS-agar layer (0.7 % agar) inoculated with an exponential growing culture of the indicator organism, and further incubated for 1 2 hours. The same overlay-technique was used when the tested strains were streaked out on solid agarplates. Large clear inhibition zones could be observed when Lb.helveticus ATCC 1 5009 was used as an indicator organism . This strain was subsequently used whenever routine bacteriocin activity determinations had to be carried out. In the agar diffusion method, the MRS-0.2 agarplates were overlayed with 5 ml soft

MRS-0.2 agar inoculated with 1 50 μ\ of an exponential growing indicator strain culture. Subsequently, 7 mm holes were drilled in the agarlayer and 45 μl of culture supernatant of the potential inhibiting strain were added. This culture supernatant was brought to pH 6.5 to rule out any inhibition by lactic acid. The plates were incubated for 24 hours at 45 °C and analysed on the formation of inhibition zones.

Bacteriocin activity was tested semi-quantitatively via application of the critical dilution method used for testing bactenocins (Mayr-Harting et a/., 1 972) . A twofold dilution series of filtre-sterilised bacteπocin-containing MRS medium was spotted (1 0 μ) on exponential growing cells of Lb.helveticus ATCC 1 5009 used as the indicator organism. The indicator strain was prepared by growing the strain to an OD (600 nm) of 0.3 and the subsequent addition of 1 50 μl cell suspension to 3.5 ml top agar (MRS). The plates were incubated for 24 hours at 45 °C. The activity was defined as the inverse value of the largest dilution still showing inhibiting activity against the indicator organism. The activity was expressed in activity units (AU) per milliliter.

1 .4. Bacteriocin production

In order to study the kinetics of bacteriocin production, a 7.5 I fermentor (New Brunswick Bioflo II), containing 5 I MRS broth, was used. The high volume of broth in the fermentor ensured micro-aerophilic conditions. No aeration of the fermentor took place. A slight agitation (50 rpm) was used to homogenize the culture fluid. No pH control took place. The fermentor was inoculated with 50 ml of an overnight culture of Lb. amylovorus

LIM KB-1 80 and was kept on a suitable temperature. For this overnight culture, a stock culture was used (kept at -80°C) to inoculate 1 0 ml MRS broth and grown overnight at

37 °C. The culture was subsequently re-inoculated ( 1 vol%) in 1 0 ml sterile MRS broth ( 1 2 hours, 37 °C). Finally, 1 vol % of this culture was used to inoculate 50 ml sterile MRS broth. This 50 ml was used as the final inoculum of the bacteriocin fermentation. During this fermentation samples were taken at regular time intervals in order to measure the OD (600 nm) and the bacteriocin titer (AU/ml) Samples to be tested for bacteriocin activity were first centrifuged to discard the cells the supernatant was subsequently filter sterilised (0.2 μm filters).

In order to optimalise the bacteriocin production in M RS broth, the lactobin producing strain Lb. amylovorus LIM KB-1 80 was grown at different temperatures (1 5, 20, 30, 37, 45 and 50°C), different starting pH (2.0, 3.0, 4.0, 5 0, 6 0, 7 0, 8 0, 9.0 and

10.0) and in the presence of different carbon sources (cf. table 7, final concentration = 1 or 2 %). Several fermentations were also carried out at constant pH (4.5, 5.0, 5.5, 6.0 and 6.5) and with different initial glucose concentrations

1 .5. Isolation of crude lactobin LK-180

Crude culture filtrates, originating from fermentor cultures (MRS, 5 I, 37 °C), were harvested after 1 2-14 hours of fermentation. Cells were discarded after centrifugation (10 mm. , 5000 g) and the supernatant was brought to pH 6.5 (with 30% NaOH) and filter sterilised (0.2 μm-fιltre) . This material will be described further as crude bacteriocin (crude lactobin) and stored at -75 °C when not used immediately

1 .6. purification of crude lactobin

1 .6.1 . ultrafiltration

In order to augment the bacteriocin concentration and to remove low molecular weight contaminants, the lactobin preparations were submitted to membrane filtration using an Amicon ultrafiltration device (Amicon Corp , Beverly, MA, USA)

1 .6.2. ammoniumsulphate precipitation

1 00 ml crude bacteriocin (pH 6.5, 4°C) preparations were saturated to 1 0, 20, 30, 40, 50 and 60% ammoniumsulphate respectively These solutions were incubated

overnight at 4° C with gentle stirring. The solutions were subsequently centrifuged ( 1 0 mm , 4°C) whereby 2 phases where formed, an oil-like upper layer and an aquaous lower phase The oil-like substance at the surface of the solution was isolated and resuspended in a minimal volume of MilliQ or 50 mM sodiumphosphate buffer (pH 6 5) Both the oil-like layer and the supernatant were analysed for possible bacteriocin activity using the semi- quantitative biodosage-method (see 1 3 ) The oil-like substance obtained after ammoniumsulphate precipitation will be designated further as lactobin LK-1 80 (fraction I )

1 6 3. extraction using organic solvents

Two different methods were applied for the partial purification of the bacteriocin

1 00 μl bacteriocin containing solution (lactobin LK-1 80 fraction I) was resuspended in 25 volumes of a Chloroform/methanol mixture (2/1 ) and incubated at 4° C for 1 hour A precipitate was collected after centrifugation at 1 3,000 rpm for 1 0 m This treatment was repeated and the resulting pellet was dissolved in sterile MilliQ This precipitate will be designated further as partially pure lactobin LK-1 80 (fraction II) To this supernatant 0.2 volumes of water were further added and centrifuged for 30 mm after mixing The lower organic phase was dried under vacuum and the pellet was resuspended in a minimal volume of chloroform After addition of 4 volumes of diethylether, the mixture was incubated for

1 hour at -20°C.

1 00 l desalted bacteriocin solution (desalted lactobin LK-1 80) fraction I was resuspended in 2 volumes of a water saturated ethanol/diethylether mixture (1 /3) The liquid phases were separated via centrifugation ( 10 mm , 1 3,000 rpm) Subsequently, the organic phase was shaked out 3 times with 100 μl water and finally dried by vacuum The bioactivity of all fractions was analysed at all times All fractions were also analysed on their protein content (via Tπcine SDS-PAGE) and fatty acid content (via TLC)

1 .7. effect of hydrolytic enzymes on bacteriocin activity

Crude, partially purified and purified lactobin LK- 1 80 was treated with different hydrolytic enzymes Trypsin, chymotrypsin (Sigma C-7762), proteinase K (Sigma P-6556), protease type IX (Sigma P-61 41 ) and protease type XII were resuspended in 50 mM sodiumphophate buffer (pH 7.0) Lipase (Boehringer 644072) was resuspended in 50 mM

sodium phosphate buffer (pH 7.0) containing 5 mM calciumchloπde The enzymes were used in a final concentration of 1 mg/ml. Control samples consisted of buffers, heatinactivated ( 1 5 mm., 1 00 °C) enzymes in buffer and crude bacteriocin in buffer respectively. De buffer/enzyme/bacteπocin reaction mixtures and the controls were incubated for 1 hour at 37 °C and bacteriocin activity was defined using biodosage

1 .8. chromatographic purification

Chromatographic purification was carried out on an FPLC device (Pharmacia-LKB,

Uppsala, Sweden) with different chromatographic columns ^• gelpermeation chromatography

(Superdex™ 75 HR 1 0/30 and Superdex™ 200 HR 5/5), anion- (Mono Q HR 5/5) and cation exchange (MonoS HR 5/5) chromatography and reversed phase chromatography

(Pep RPTM HR 5/5) .

1 .9. protein determination

Protein concentrations were defined using the method of Bradford (Biorad kit, CA, USA). Bovine serum albumin (BSA) was used as a standard

1 .10. analysis of the lipid-like material

Lipid- ke substances were analysed via TLC (Thin Layer Chromatography) using the method of Navarre et al. (1 992)

1 .1 1 . Tricine SDS-PAGE

Tπcine SDS-PAGE was carried out according to Schagger & von Jagow ( 1 987). Polyacrylamide concentrations in the stacking- and separating gel were 9.6 and 1 6% respectively. PAGE was carried out at a constant voltage of 30 V during 1 hour followed by 90 V during 1 8 hours. Gels were stained using Coomassie Blue R (Sigma, St. Louis, Missouri, USA), silver (Silver stain kit, Biorad, CA, USA), Sudanblack B (Sigma) or Oilred 0 (Sigma). Protein standards and their respective molecular weights (Dalton) were : ovalbumin, 43000; carbonic anhydrase, 29000, β-lactoglobulin, 1 8400; lysozyme, 14300,

bovine trypsin inhibitor, 6200; insulin, 3400. In order to demonstrate bioactivity, gels were washed overnight with sterile MilliQ (refreshed regularly), transfered to a MRS agarlayer and overlayed with a thin layer of soft MRS agar inoculated with the sensitive Lb.helveticus ATCC 1 1009 strain

1 .12. amino acid sequence analysis

The ammo acid sequence analysis after pyπdylethylation (Amons et al , 1 984) was carried out using Edman degradation (Edman et al , 1 967) on an Applied Biosystems 477A protein sequencer (Applied Biosystems, Foster City, CA, USA) using protocols supplied by the manufacturer

1 .13. electrospray mass spectrometry

Approximately 1 00 pmole sample was dissolved in 1 0 l 50% acetonitnlle/ 1 % formic acid in water en subsequently injected in the electrospray source of a VG BIO-Q triple quadrupole mass spectrometer (VB Biotech, Altπchman, UK) The sample was was pumped with a sale of 5μl/mιn ( 140 A delivery system, Applied Biosystems, Foster City, CA) The capptlary tip was adjusted at a voltage of 4.1 kV, the sample cone voltage was

51 V. The mass spectrometer, wherefrom only the first quadrupole was used, was adjusted to scan masses ranging from 650 to 1 350 Da in 9 seconds Data were collected during 2 minutes. The mass spectrometer was calibrated with horse heart myoglobin (Sigma, St Louis, MO)

1 .14. nucleotide sequence analysis

DNA fragments were sequenced by the dideoxy chain termination procedure (Sanger et al , 1 977), using the non-radioactive Taq Dye Deoxy™ Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on aπ Applied Biosystems 373A automated DNA sequencing apparatus as described by the manufacturer

1 .15. analysis of the antimicrobial activity spectrum

Cells from 1 ml of an overnight culture of the sensitive strain Lb.helveticus LMG 641 3 or other tested strains were harvested via centrifugation. cell pellets were washed twice with 50 mM sodium phosphate buffer (pH 6.5), resuspended in 0 1 ml crude lactobin LK-1 80 (200 AU/ml against Lb.helveticus ATCC 1 1 009 or any other sensitive micro- organism) and incubated at 37 °C. Samples were taken after 1 5, 2 5, 3.5, 4 5 and 5.5 hours incubation and the percentage of living cells was defined

2. SCREENING FOR BACTERIOCIN PRODUCING LACTIC ACID BACTERIA ISOLATED FROM CORN STEEP LIQUOR

Both the direct and indirect (platingmethod, 1 0 μl spot of culture supernatant, isolation of separated colonies) methods were used to screen 240 lactic acid bacteria isolated from fresh corn steep liquor (50 °C, pH3 8) for their antagonistic activities. Isolation of these strains was already described in section 1 .2. of material and methods The indicator strains used are depicted in table 2. In order to restrict acid production an MRS medium with low glucose content (MRS-0.2) was used In order to rule out inhibition through production of hydrogen peroxide, the first incubation was carried out under anaerobic conditions. In total 3 Lactobacillus strains (strains 1 72, 1 74 and 1 80) producing inhibition zones against other Lactobacilh (Lb.delbruecku subsp. bulgancus LMG 6901 , Lb.de/brueckn subsp. lactis LMG 7942, Lb.helveticus LMG 641 3) were found A sleight inhibition against Lb. acidophilus LMG 7943 could also be noted. Culture filtrates (brought to pH 6.5) of these 3 strains, found positive in the agarspot test, were subsequently analysed using the agardiffusionmethod. Only 2 strains ( 1 74 and 1 80) were able to produce inhibitionzones on agarplates in this test. The same Lactobacillus as described above were sensitive. The other tested Gram-positive and Gram-negative micro-organisms (see table 2) were not inhibited.

3. CHARACTERISATION OF THE LB.AMYLOVORUS STRAIN 180 ON SPECIES LEVEL

Strain 1 80 is a facultative anaerobic. Gram-positive rod It is catalase-negatif, produces only D(-) lactic acid (end pH = 3 60) and produces no gass from glucose The temperature range for active frowth is situated between 20°C and 45 °C No growth could be seen at 1 5 °C or 50°C. Active growth could also be detected in MRS medium

supplemented with 10% NaCl or 5% ethanol. The fermentation patteren for carbon source utilisation (as tested via API) was negative for rhamnose, trehalose, arabmose, xylose, raffmose, mannitol, sorbitol, glycerol and erythπtol; and positive for glucose, lactose, cellobiose, maltose, mannose, sucrose, amylose and salic e Growth on N- acetylglucosamine and amygdaline was delayed These data suggest that strain 1 80 behaves as a homofermentative Lactobacillus strain closely related with Lb.delbruecku subsp. bulgancus and Lb. acidophilus. Comparative protein electrophoresis data obtained from the Laboratory for Microbiology (University Ghent, K L Ledeganckstraat, 35, Ghent, Belgium) suggested a similar relationship However, only a 60% similarity could be found with Lb. acidophilus LMG 7943 T No homology could be established with these

Lactobacillus strains using DNA/DNA hybridisations Finally, 16S rRNA sequence analysis showed strain 1 80 to be a Lactobacillus amylovorus (99.8% homology). From now on, this strain will be designated Lb amylovorus LIM KB 1 80 Strain 1 80 could indeed acidify (although slowly) amylose as the sole carbon source ( 0.5 % in M RS broth), a pH of 3.9 was reached after 120 hrs. of fermentation The strain has strong aggregating capabilities

(imediate sedimentation) Lb. amylovorus LIM KB- 1 80 was deposited in the BCCM-LMG culture collection of the Laboratory for Microbiology (University Ghent, K.L Ledeganckstraat, 35, Ghent, Belgium) as number LMG P-1 31 39

4 IDENTIFICATION OF THE INHIBITING SUBSTANCE OF LB.AMYLOVORUS LIM KB- 180: Effect of hydrolytic enzymes on lactobin LK-180 activity

The effect of different enzymes on the inhibiting substance produced by Lb. amylovorus LIM KB- 1 80 was analyzed using the crude bacteriocin preparations described in paragraph 1 .5., partially pure lactobin preparations as described in paragraph 1 6.3. (cf. Table 4) or pure lactobin preparations as detailed in paragraph 1 .7. Neither the proteases (active or inactive), nor the used buffers had any inhibitory effect on the growth of the indicator strain. Treatment of the culture supernatant with trypsin, chymotrypsin, protease type IX, protease type XII and protease K destroyed the antagonistic activity, suggesting a proteinaceous nature for the substance Treatment with protease free lipase had no effect, suggesting that a possible lipid component is not necessary for activity Treatment with -amylase and lysosym had no effect, suggesting that neither a carbohydrate nor peptidoglycan component is not necessary for activity Treatment with catalase had also no effect, so the inhibiting substance is not likely to be hydrogen

peroxide. Bacteriophage induction as a possible cause of inhibition could be ruled out since the inhibiting substance could diffuse through an agar layer and was resistant to heat treatment. So it can be concluded that the inhibiting substance produced by Lb. amylovorus LIM KB-1 80 can be identified as a bacteriocin, further designated lactobin LK-1 80.

5. KINETICS OF LACTOBIN FERMENTATION

The activity of lactobin LK-1 80 in MRS broth as measured with the critical dilution method (1 .3.) exhibits primary metabolite kinetics. The growth of Lb. amylovorus LIM KB- 1 80 in MRS broth at 37 and 45 °C resulted in a detectable amount of antagonistic substance in the supernatant after 3 hrs. of incubation (early exponential growth phase). This activity reached its maximum (1 600 AU/ml) after 1 0- 1 2 hrs incubation (late exponential growth phase). At that moment the culture had a pH of 3.55. The inhibiting substance decreased during stationary phase; after 24 and 30 hrs incubation the activity was respectively 75 and 50% of the original activity. The data obtained from f ermentations carried out at 1 5, 20, 30, 37, 45 and 50 °C are combined in Table 5. Growth and bacteriocin production takes place in a temperature window of 37-45 °C.

Growth is maximal at 45 °C; bacteriocin production (and inactivation at prolonged fermentation runs) occurs more rapidly at 37 °C.

Analogous results were obtained with fermentation runs carried out in "semi¬ synthetic" medium (MRS broth without pepton and 'Lab Lemco') . Optimal production of lactobin LK-1 80 in semi-synthetic medium was obtained when using 2 % glucose as carbonsource with a constant pH of 5.5 and at a temperature of 37 ° C. Under these conditions a maximal antimicrobial titer of 800 AU was obtained at an 0D _60o of 0.8. Using the semi-synthetic medium minimises the interaction with other peptides during purification. The influence of the initial pH is represented in Table 6. Maximal bacteriocin production was obtained when the fermentation was carried out with an initial pH of 5.0- 6.0.

The influence of different carbonsources on growth and lactobin production by Lb. amylovorus LIM KB-1 80 is represented in Table 7. Growth and lactobin production take place using glucose ( 1 and 2 %), maltose (2 %), sucrose (2 %), cellobiose ( 1 %), mannose

( 1 %) and amylose (1 %) . The use of fructose ( 1 %), maltose (1 %) and sucrose (1 %) leads to growth but not to lactobin production. Growth nor bacteriocin production could

be detected when using lactose (1 and 2 %), fructose (2 %), rhamnose ( 1 %), arabinose ( 1 %), xylose (1 %), raffinose ( 1 %), sorbose ( 1 %) and the sugar alcohols sorbitol ( 1 %), mannitol (1 %), adonitol ( 1 %), xylitol ( 1 %) and inositol ( 1 %)

6. PURIFICATION OF LACTOBIN LK-180

6.1 Ammoniumsulphate precipitation

The treatment of culture supematant with ammonium sulphate resulted in the clear separation of two phases following centrifugation ^■ floating material (fraction 1 ) and supernatans. All of the bacteriocin activity ( 1 00 %) could be removed from the broth at a saturation level of 35 % ammoniumsulphate. However, only 1 7-37 % of the bacteriocin activity in the starting material could be recovered

6.2 Extraction with organic solvents

Treatment of lactobin LK-1 80 fraction I (prepared as in section 1 .5) with a chloroform/methanol mixture (2/1 ) resulted in a bioactive protein precipitate (fraction II) (analysed via biodosage and Tπcine SDS-PAGE) Addition of a water saturated ethanol/diethylether mixture (1 /3) resulted in a separation in two phases whereby the bioactive protein is present in the aqaeous phase (analysed via biodosage and Tπcine SDS- PAGE) (see also section 1 .6.3)

6.3 Purification of Lactobin

Purification of crude and chloroform/methanol extracted Lactobin LK- 1 80 was attempted using different chromatographic techniques. The use of gel permeation, reversed phase or ion exchange chromotography did not lead to the purification of crude Lactobin due to both the characteristics of the antagonistic component and the presence of contaminating fatty acids. These fatty acids could be removed using a methanol/chloroform extraction. However, following this extraction, again no purification could be achieved using gelpermeation or ion exchange chromatography; probably due to the strong aggregating properties of lactobin. On the other hand, Reversed Phase Chromatography resulted this time in an efficacious separation of the different components Unfortunately,

its usage for large-scale puπfiaction was limited due to the low lactobin concentrations in the chloroform-extracted preparation. For this reason, the chloroform-extracted lactobinpreparations were first enriched using ultrafiltration columns with a cut-off of 1 00,000 Da. (Amicon Corp., Berverly, MA, USA). This techique alowed us eventually to remove all components interfering with mass-spectrometry.

All of the bioactivity was retained in the retentate (fraction III), the molecular weight of the bioactive component as estimated on PAGE not withstanding Using this method the Lactobin preparation could be concentrated 20-fold After concentration the lactobin appeared as a yellowish-brown substance. As a control the filtrate was dried under vacuum, resuspended in a minimal volume of sterile MilliQ and tested for bioactivity using biodosage. Analysis of the Lactobin preparation on PAGE and visualisation using Coomassie Brilliant Blue R-250 or silver stain showed that no contaminating proteins were present any more The presence of bioactive Lactobin in the resulting polyacrylamide gel could be confirmed by overlay with the indicator organism In most cases, using the overlay technique, 2 and sometimes 3 bioactive bands running very closely together could be visualised on the polyacrylamidegel. It is for the moment not clear what the relation is between the different bands and which of these bioactive bands corresponds with the protein that was purified. The two smallest bioactive peptides could be separated using Reversed Phase Chromatography with 0.1 % tπfluoro acetic acid and a linear isopropanol- or acetonitπlle-gradient (0-1 00 % in 30 minutes).

7 FYSICOCHEMICAL PROPERTIES OF LACTOBIN LK-180

^* pH-stabihty

Exposing the bacteriocin to different pH values (range 2 0-9 0, room temperature) did not reduce the activity. At a pH of 1 0 0-1 2 0 the activity was reduced with 50 % Lactobin LK-1 80 did not lose its activity after long-term storage at -75 °C and lyophi sation.

* Heat stability

Heat treatment during 5, 1 0, 20, 30, 45 and 60 mm at 60 and 1 00°C had no influence on the bioactivity of culture supernatans and partially pure Lactobin LK-1 80. No loss of activity could be observed after autoclaving the material for 20 m at 1 21 ° C These observations clearly show that Lactobin LK-1 80 is a heatstable protein

* UV-spectrum

The UV adsorption spectrum of partially pure Lactobin LK- 1 80 showed maximal adsoprtion at 21 0 nm, a characteristic of peptide bonds. There was almost no adsoption at 280 nm, indication that almost no aromatic ammo acids are present.

* Mass analysis

The pure Lactobin preparation (fraction III) was analyzed using laser mass spectrometry resulting in one component with a molecular mass of 4978 0 69.

* Ammo acid analysis

The N-terminal ammo acid sequence of the pure lactobin preparation (fraction III) was determined by pyridyl ethylation (Amons et al , 1 984) followed by Edman degradation

(Edman et al., 1 967). The 48 first N-terminal am o acids could be determined in this way These ammo acids are shown in next figure using single letter code.

NRWTNAYSAALGCAVPGVKYGKKLGGVHGAVIGGVGGAAVCGLAGYVR (SEQ ID

NO 1 8)

This am o acid sequence could be confirmed by the corresponding nucleic acid sequence isolated from chromosomal DNA of Lb. amylovorus LIM KB-1 80. Starting from the nucleic acid sequence, 2 additional C-terminal ammo acids and a 1 5 ammo acid prosequence could also be determined.

8. BIOLOGICAL PROPERTIES OF LACTOBIN LK-180

8.1 Inhibitionspectrum

The antimicrobial effect of Lactobin LK-1 80, partially purified via ammonium sulphate precipitation, on different Gram-positive and Gram-negative (cf Tables 1 and 2) indicator strains was analysed using the critical dilution method. The inhibiting activity of Lactobin LK-1 80 was restricted to specific lactic acid bacteria, more precisely to Lb. delbrueckii subsp. lactis LMG 7942, Lb. delbrueckn subsp bulgaricus LMG 6901 , Lb. helveticus ATCC1 5009, Lb. helveticus LMG 641 3, Lb. plantarum LMG 6907 and Lb plantarum LMG 1284. The producer was not inhibited by its own bacteriocin, suggesting the presence of an immunity factor. Inhibition could also be seen against Enterococcus

faecium LMG 8149 and Enterococcus faecalis LMG 8146. Other tested Gram-positive and Gram-negative bacteria (including several food spoiling and/or pathogenic species) were not sensitive to partially pure Lactobin LK-1 80 under the conditions tested (cf. Table 2).

8.2 Mechanism of action

Addition of the bacteriocin (200 AU/ml) to a cell suspension of Lb. helveticus

ATCC 1 5009 at a concentration of 1 .1 x 1 0 ⁷ CFU/ml in phosphate buffer (50 mM, pH 6.5) reduced the amount of living cells with 1 log-cyclus within a time-interval of 90 mm. in the presence of 200 AU/ml. During the experiments there was no change in optical density and no cell lysis was taking place, suggesting a bactericidal but no bacteπolytic mode of action

9. GENETIC DETERMINANTS

9.1 Chromosomal location of the genetic determinants

Plasmid analysis and plasmid curing experiments using novobiocine (200 μg/ml) showed the presence of a single plasmid of 23 MDa, named pBC 1 80, in the Lactobin producing Lb. amylovorus LIM KB-1 80 strain The genetic determinants for Lactobin production and -immunity are not present on this plasmid and are therefore located on the chromosome.

9.2 Isolation of the structural gene for Lactobin LK-180

Starting from the N- and C-terminal am o acid sequence of the purified Lactobin LK-1 80, two degenerate 32-mer oligonucleotides were designed These oligonucleotides were used in a PCR reaction on chromosomal DNA isolated from Lb amylovorus LIM KB- 1 80. Two different PCR fragments could be isolated, resulting from this reaction They had respective lengths of 1 36 bp and 90 bp These fragments were subsequently subcloned in a pBluescript SK( + ) vector (Stratagene Cloning Systems, La Jolla, CA, USA) and sequenced (Sanger et al. , 1 977). It could be confirmed that the 1 36 bp fragment consisted of the coding sequence of the Lactobin structural gene whereas the 90 bp fragment consisted of only part of the Lactobin stuctural gene, most probably originated

from aspecific hybridisation of the synthetic degenerate N-termmal 35-mer probe.

9.3 isolation of the full-size structural preLactobin gene and surrounding sequences

Using the 1 36 bp fragment as a probe, a 3.5 Kb Hindlll fragment could be isolated from Hindlll digested chromosomal DNA from Lb. amylovorus LIM KB-1 80 could be subcloned in a pBluescript SK( + ) vector (Stratagene Cloning Systems, La Jolla, CA, USA) .

Starting from vector bearing the cloned 3.5 Kb Hindlll fragment, nested deletions were generated using Exolll exonuclease. The resulting deletion clones were subsequently sequenced according to Sanger et al. ( 1 977) . Using this method, up to 80% of the DNA sequence of the 3.5 Kb fragment could be elucidated . The remaining 20 % of the sequence could be determined using subcloned 700 bp Hindlll-Nael and 400 bp Xbal-Kpnl fragments and additional internal sequencing primers. Analysis of the generated DNA sequence information revealed the presence of two bacteriocin operons each consisting of three reading frames. Each operon was preceded by a promotor and these promotors showed a 80 % homology. Each operon consists of two "bacteriocin-like" genes followed by a third ORF, presumably an immunity gene. The four bacteπocin-like proteins were each characterised by a conserved proteolytic processing site preceded by a conserved prosequence. Figure 1 shows the the complete sequence as obtained.

9.4 Chromosome walking

In a first round of chromosome walking, a 1 kb Hιnc\\-Hιnd\\\ fragment at the 3' end of the 3.5 kb insert (see 9.3) was ³² P-labelled and used as a homologous probe for screening a HincW chromosomal library of L. amylovorus LMG P- 1 31 39 (Stratagene Cloning Systems, La Jolla, CA, USA). This led to the isolation of a HincW fragment of approximately 2.3 kb, including the 1 kb overlap with the previously isolated 3.6 kb Hind\\\ fragment harboring the two lactobin operons. Furthermore, a 3.4 kb HindUl fragment, having a 560 bp overlap with the newly obtained 2.3 HincW fragment was isolated in another round of chromosome walking.

Analysis of the resulting nucleotide sequence revealed three additional operons which were named 3, 4 and 5 respectively. Operon 3 contains two open reading frames of 720 and 1 98 ammo acids, respectively. The 720 am o acid protein was designated as LbnT and corresponds to a family of related ATP-dependent bacterial ABC transporters

involved in signal sequence-independent transport. The protein of 1 98 ammo acids, LbnE displayed homology with members of the HlyD family, although limited to its N- and C- terminal domains. These data suggest a signal sequence independent secretion mechanism for the export of the class lib bactenocins encoded by the lactobin operons. Operon 3 is followed by bacteriocin operons 4 and 5. Operon 4 contains two open reading frames. The first one encodes a one-component class II bacteriocin, followed by an ORF that is presumably encoding the corresponding immunity gene. Operon 5 contains three open reading frames. The first two ORFs encode third class Mb bactenocins, followed by an ORF that is presumably encoding the corresponding immunity gene The putative promoter upstream of operon 5 (a bacteriocin operon) displays no homology with the other potential promoters found in the bacteriocin cluster.

TABLE 1 : Examples of Amino acid substitutions in the muteins

Am o acids Synonymous groups

Ser (S) Ser, Thr, Gly, Asn

Arg (R) Arg, His, Lys, Glu, Gin

Leu (L) Leu; lie. Met, Phe, Val, Tyr

Pro (P) Pro, Ala, Thr, Gly

Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gin

Ala (A) Ala, Pro, Gly, Thr

Val (V) Val, Met, lie, Tyr, Phe, Leu, Val

Gly (G) Gly, Ala, Thr, Pro, Ser

He (I) lie, Met, Leu, Phe, Val, lie, Tyr

Phe (F) Phe, Met, Tyr, lie, Leu, Trp, Val

Tyr (Y) Tyr, Phe, Trp, Met, He, Val, Leu

Cys (C) Cys, Ser, Thr, Met

His (H) His, Gin, Arg, Lys, Glu, Thr

Gin (Q) Gin, Glu, His, Lys, Asn, Thr, Arg

Asn (N) Asn, Asp, Ser, Gin

Lys (K) Lys, Arg, Glu, Gin, His

Asp (D) Asp, Asn, Glu, Gin

Glu (E) Glu, Gin, Asp, Lys, Asn, His, Arg

Met (M) Met, lie. Leu, Phe, Val

TABLE 2 : EFFECT OF LACTOBIN LK-180 ON SELECTED LACTIC ACID BACTERIA

indicatororganism Cultivation Sensitivity medium + for incubation lactobin LK- temperature 180

Lb. acidophilus LMC 7943 ^' MRS, 37°C

Lb. brevis LMC 6906 MRS, 37°C

Lb. brevis LMC 7761 MRS, 37°C

Lb. casei subsp. casei ATCC 7469 ^' MRS, 37°C

Lb. casei subsp. casei LMC 6904 MRS, 37°C

Lb. casei subsp. rhamnosus LMC 6400 MRS, 37°C

Lb. delbrueckii subsp. bulgaricus LMC MRS, 37°C +

6901

Lb. delbrueckii susp. delbrueckii ^' LMC MRS, 37°C

6412

Lb. delbrueckii subsp. lactis LMG 7942 MRS, 37°C +

Lb. fermentum LMG 6902 MRS, 37°C

Lb. helveticus ATCC 15009 ^' MRS, 37°C +

Lb. helveticus LMC 6413 MRS, 37°C +

Lb. jensenii LMC 6414 ^' MRS, 37°C

Lb. plantarum LMC 6907 MRS, 37°C +

Lb. plantarum LMC 1284 MRS, 37°C +

Lb. plantarum LMC 8155 MRS, 37°C

L. lactis subsp. lactis ML8 ^' MRS, 30°C indicator organism cultivation Sensitivity medium + for incubation lactobin LK- temperature 180

Leuc. mesenteroides subsp. MRS, 30°C - mesenteroides ATCC 12291 ^"

Leuc. mesenteroides subsp. MRS, 30°C - mesenteroides LMC 7939

P. acidilactici ATCC 8042 ^' MRS, 37°C -

TABL E 3 : EFFECT OF LACTOBIN LK-180 ON SELECTED INDICATOR STRAINS indicator organism Cultivation medium + Sensiti¬ incubation vity for temperature lactobin LK-180

- c + bacteria

Bacillus cereus LMC 6923 Bacillus cereus agar ±

Bacillus cereus LMC 6924 Bacillus cereus agar -

Bacillus cereus LMC 6925 Bacillus cereus agar -

Bacillus subtilis LMG 8197 Bacillus cereus agar -

Clostridium perfringens LMC - 11284

Enterococcus faecium LMC 8149 Slanetz & Bartley agar +

Enterococcus faecalis LMC 8146 Slanetz & Bartley agar +

Listeria innocua LMC 11387 Oxford agar -

Listeria monocytogenes LMC Oxford agar - 10470

Liste Oxford agar - ria eceiigeri LMC 11386

Listeria welsh imeri LMC 11389 oxford agar - staphylococcus aureus LMC 8224 Baird-Parker agar ±

- c - bacteria

Enterobacter aerogenes LMG Nutrient agar - 2094

Escherichia coli Nutrient agar -

Pseudomonas fluorescens LMC Nutrient agar -

1794

Pseudomonas aeruginosa LMG Nutrient agar - 68029 salmonella enteritidis LMG 10395 Brilliant green agar -

Salmonella typhimurium LMG Brilliant green agar - 10396

Yersinia enterocolitica IP 383 Yersinia selective agar -

Yersinia enterocolitica WS24/92 Yersinia selective agar -

Yersinia enterocolitica IP 1103 Yersinia selective agar -

Yersinia enterocolitica WA 289 Yersinia selective agar -

TABLE 4 : Sensitivity of crude LACTOBINE LK-180 for hydrolytic enzymes

TABLE 5 : OVERVIEW OF LACTOBACILLUS SP. 180 FERMENTATIONS AT DIFFERENT TEMPERATURES

TABLE 6 : INFLUENCE OF THE INITIAL pH ON GROWTH AND LACTOBINPRODUCTION WITH Lb. AYMYLOVORUS LIM KB-180

(MRS-broth; 45 C; 12 h fermentation)

initial pH OD-600 AU/ml

5.00 1.99 3200 6.00 1.90 3200 7.00 0.87 400

TABLE 7 : INFLUENCE OF THE CARBON SOURCE ON GROWTH AND

LACTOBIN PRODUCTION WITH Lb. AMYLOVORUS LIM KB -180 (MRS-broth; 45 °C; 12 h fermentation)

Carbon source PH OD-600 AU/ml glucose 4.55 1.735 800 maltose 5.70 0.587 0 sucrose 6.80 0.233 0 lactose 7.10 0.040 0 fructose 5.65 0.564 0 cellobiose 6.95 0.16 100 rhamnose 7.00 0.09 0 arabinose 7.00 0.08 0 xylose 5.97 0.100 0 mannose 5.97 0.510 200 raffinose 7.00 0.112 0 amylose 5.85 0.645 200 sorbose 7.00 0.07 0 aesculin 6.50 0.14 0 sorbitol 7.00 0.07 0 mannitol 7.00 0.07 0 adenitol 7.00 0.06 0 xylitol 7.00 0.06 0 inositol 7.00 0.07 0

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