WO1997023619A1 - Sequences codant pour de nouvelles bacteriocines - Google Patents

Sequences codant pour de nouvelles bacteriocines Download PDF

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Publication number
WO1997023619A1
WO1997023619A1 PCT/EP1996/005235 EP9605235W WO9723619A1 WO 1997023619 A1 WO1997023619 A1 WO 1997023619A1 EP 9605235 W EP9605235 W EP 9605235W WO 9723619 A1 WO9723619 A1 WO 9723619A1
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Prior art keywords
seq
bacteriocin
polynucleic acid
sequence
protein
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PCT/EP1996/005235
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English (en)
Inventor
Bart Contreras
Luc De Vuyst
Erik Vandamme
Erwin Sablon
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Innogenetics N.V.
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Priority to AU28746/97A priority Critical patent/AU2874697A/en
Publication of WO1997023619A1 publication Critical patent/WO1997023619A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to the field of bacte ⁇ ocins. More particularly the present invention relates to new polynucleic acid sequences encoding new bacte ⁇ ocins and new proteins with bacte ⁇ ocin-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, bacte ⁇ ocins 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, .
  • Lactobacilli are long known for their bacte ⁇ ocin-producing abilities
  • Lb Lactobacillus
  • 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 Bacte ⁇ ocin 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 (Mu ⁇ ana & Klaenhammer, 1987), produced by Lb. Johnsonii; Plantacin B (West & Warner, 1988), Plantancin A (Daeschel et a/.
  • Plantancin S Jimenez-Diaz et a , 1 990
  • Plantancin S Jimenez-Diaz et a , 1 990
  • Reute ⁇ cin 6 Toba et a/. , 1 991 c
  • Lb. reuteri Reute ⁇ cin 6
  • Sakacin A Schott & L ⁇ cke, 1 989
  • Sakacin P Tichaczek et al. , 1 992
  • Lactocin S Mortvedt & Nes, 1 990
  • 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
  • bactenocins have a bactericidal activity towards different food spoilage and/or pathogenic bacteria such as Bacilli, Clost ⁇ dia, Staphilococci and Liste ⁇ ae
  • several bactenocins are small thermostable proteins, facilitating their use as a food additive a food additive in heat-treated food.
  • 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 bacte ⁇ ocin production.
  • the bacte ⁇ ocin coding sequences could also be used in the construction of "food-grade" cloning vectors
  • bacte ⁇ ocin Nism produced by Lactococcus lactis subsp. lactis
  • 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)
  • the bacte ⁇ ocin regulatory signals 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).
  • sequences coding for the transporter proteins could be used to allow secretion and processing of sai ⁇ 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 (Sch ⁇ lein et a , 1 992)
  • HlyD component of the haemoiysin A secretion apparatus of E.coli Schott a , 1 992
  • 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
  • the aim of the present invention is thus to provide polynucleic acid sequences encoding novel bactenocins or bacte ⁇ ocin immunity proteins or transporter proteins
  • Another aim of the present invention is to provide novel purified bactenocins and bacte ⁇ ocin 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:
  • 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.
  • 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 "bacte ⁇ ocin-like" genes followed by a third ORF, presumably an immunity gene. The four bacte ⁇ ocin-like proteins are each characterized by a conserved proteolytic processing site preceded by a conserved prosequence.
  • 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 (Mu ⁇ ana & 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 comp ⁇ ses 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
  • 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.
  • 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 ( u ⁇ ana & Klaenhammer, 1 991 )
  • 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
  • polynucleic acid corresponds to either double-stranded or single-stranded cDNA or genomic DNA, or RNA
  • 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
  • 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,
  • 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
  • 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
  • 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
  • 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
  • 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.
  • 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 hjyb ⁇ dizing 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
  • 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
  • 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).
  • the present invention also relates to a polynucleic acid sequence as defined above comp ⁇ sing 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
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • polynucleic acid sequence of the invention may comprise:
  • vector may comprise a plasmid, a cosmid, a phage or a virus.
  • 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.
  • 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.
  • a coding sequence can include but is not limited to m RNA, DNA (including cDNA), and recombinant polynucleotide sequences.
  • operably linked refers to a juxtaposition wherein the components are figured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of a coding sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • a recombinant vector e.g. a plasmid
  • 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
  • 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.
  • 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 se ⁇ uence 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.
  • a promotor/secretion signal sequence derived from L. amylovorus
  • 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 ⁇ bosome 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.
  • the present invention relates to a host cell transformed with a recombinant vector as defined above
  • the present invention contemplates host cell transformed with a recombinant vector as defined above.
  • 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.
  • 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)
  • 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
  • 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,
  • 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.
  • 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
  • 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 bacte ⁇ ocin and/or bacteriocin immunity activity, and/or transporter activity
  • Said polypeptide according to the present invention is preferably a recombinant polypeptide
  • 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
  • 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
  • mutant 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
  • fragments 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
  • polypeptides and derivatives according to the present invention may be produced by recombinant DNA technology
  • 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
  • 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.
  • polypeptides of the invention can also be prepared in solid phase according to the methods decs ⁇ bed by Atherton and Shepard in their book entitled “Solid phase peptide synthesis” (IRL Press, Oxford, 1 989)
  • 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
  • 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 present invention relates to a polypeptide as defined above comprising in its ammo acid sequence a ⁇ 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
  • 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 bacte ⁇ ocin type 5 as represented in
  • 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 bacte ⁇ ocin 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.
  • 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 bacte ⁇ ocin 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.
  • 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 bacte ⁇ ocin 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.
  • the present invention relates to a composition
  • 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.
  • 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.
  • 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
  • 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 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.
  • 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
  • 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
  • FIG. 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 are examples of 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
  • 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 bacte ⁇ ocin 1
  • 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
  • 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.
  • 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- maxim , 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.
  • a direct, indirect and agar diffusion method was used for the analysis of the anatagonistic activity of the selected bacteria.
  • 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.
  • the MRS-0.2 agarplates were overlayed with 5 ml soft
  • 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 bacte ⁇ ocin-containing MRS medium was spotted (1 0 ⁇ ) 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 ⁇ l 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.
  • LIM KB-1 80 was kept on a suitable temperature.
  • 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).
  • 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.
  • 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
  • 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 ⁇ m-f ⁇ ltre) . This material will be described further as crude bacteriocin (crude lactobin) and stored at -75 °C when not used immediately
  • lactobin preparations were submitted to membrane filtration using an Amicon ultrafiltration device (Amicon Corp , Beverly, MA, USA)
  • lactobin LK-1 80 fraction I 1 00 ⁇ l 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
  • 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 calciumchlo ⁇ de 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/bacte ⁇ ocin reaction mixtures and the controls were incubated for 1 hour at 37 °C and bacteriocin activity was defined using biodosage
  • Lipid- ke substances were analysed via TLC (Thin Layer Chromatography) using the method of Navarre et al. (1 992)
  • T ⁇ cine 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 were : ovalbumin, 43000; carbonic anhydrase, 29000, ⁇ -lactoglobulin, 1 8400; lysozyme, 14300, bovine trypsin inhibitor, 6200; insulin, 3400.
  • 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
  • ammo acid sequence analysis after py ⁇ dylethylation 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
  • 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)
  • DNA fragments were sequenced by the dideoxy chain termination procedure (Sanger et al , 1 977), using the non-radioactive Taq Dye DeoxyTM Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on a ⁇ Applied Biosystems 373A automated DNA sequencing apparatus as described by the manufacturer
  • 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.
  • 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 eryth ⁇ tol; 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.
  • strain 1 80 was 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
  • 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
  • lactobin LK-1 80 in MRS broth 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.
  • Lb. amylovorus LIM KB-1 80 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
  • 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 %)
  • lactobin LK-1 80 fraction I prepared as in section 1 .5
  • a chloroform/methanol mixture (2/1 ) resulted in a bioactive protein precipitate (fraction II)
  • fraction II analysesd via biodosage and T ⁇ cine 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 T ⁇ cine SDS- PAGE) (see also section 1 .6.3)
  • the two smallest bioactive peptides could be separated using Reversed Phase Chromatography with 0.1 % t ⁇ fluoro acetic acid and a linear isopropanol- or acetonit ⁇ lle-gradient (0-1 00 % in 30 minutes).
  • the pure Lactobin preparation (fraction III) was analyzed using laser mass spectrometry resulting in one component with a molecular mass of 4978 0 69.
  • 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
  • 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.
  • 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 were not sensitive to partially pure Lactobin LK-1 80 under the conditions tested (cf. Table 2).
  • phosphate buffer 50 mM, pH 6.5
  • Plasmid analysis and plasmid curing experiments using novobiocine 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.
  • 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.
  • 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) .
  • 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 bacte ⁇ ocin-like proteins were each characterised by a conserved proteolytic processing site preceded by a conserved prosequence.
  • Figure 1 shows the complete sequence as obtained.
  • a 1 kb H ⁇ nc ⁇ -H ⁇ nd ⁇ fragment at the 3' end of the 3.5 kb insert 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).
  • 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.
  • 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.
  • 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.
  • Val (V) Val Met, lie, Tyr, Phe, Leu, Val
  • Gin (Q) Gin, Glu, His, Lys, Asn, Thr, Arg
  • Lys (K) Lys, Arg, Glu, Gin, His
  • Bacillus cereus LMC 6925 Bacillus cereus agar -
  • Daeschel, A.M. ( 1 989). Antimicrobial substances from lactic acid bacteria for use as food preservatives. Food Technology, 43, 91 -94.
  • Nisin properties, biosynthesis and fermentation. In: E.J . Vandamme. (Ed.). Biotechnology of Industrial Antibiotics. Marcel Dekker Inc., New York, 607-628.
  • Lacticin a bacteriocin produced by Lactobacillus delbrueckii subsp lactis. Letters in Applied Microbiology, 12, 43-45
  • Plantacin B a bacteriocin produced by Lactobacillus plantarum NCDO 1193 FEMS Microbiol. Lett., 49, 163-165

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Abstract

L'invention concerne de nouvelles séquences codant pour une nouvelle bactériocine, leur utilisation ainsi que l'utilisation de séquences pour une expression et la sécrétion de protéines homologues et hétérologues. La présente invention concerne, plus particulièrement, un acide polynucléique comprenant: (a) au moins 30 nucléotides contigus d'une séquence choisie dans la séquence de l'acide polynucléique représentée sur la figure 2 (SEQ ID NO: 19), (b) une séquence d'acide polynucléique qui forme un hybride avec l'acide polynucléique comme défini en (a) ou (c) un acide polynucléique avec une séquence dérivée des acides polynucléiques comme défini en (a) ou (b), par dégénérescence, comme cela apparaît dans le code génétique. La séquence de l'acide polynucléique, représentée par SEQ ID NO: 19 sur la figure 2, comprend cinq opérons (voir figure 3). Les deux premiers sont constitués par trois cadres de lecture ouverts: deux gènes du type bactériocine, suivis par un gène de l'immunité. Le troisième opéron est constitué de deux cadres de lecture ouverts, le premier code pour une protéine transporteuse ATP dépendante, le second code pour une protéine accessoire. Le quatrième opéron est constitué de deux cadres de lecture ouverts, un étant un gène du type bactériocine suivi par un gène d'immunité. Le cinquième opéron est constitué par trois cadres de lecture ouverts: deux gènes du type bactériocine, suivis par un gène de l'immunité. Chaque opéron est précédé par un promoteur.
PCT/EP1996/005235 1995-12-22 1996-11-27 Sequences codant pour de nouvelles bacteriocines WO1997023619A1 (fr)

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US7247306B2 (en) 2004-04-30 2007-07-24 Universite Laval Bacteria strain and bacteriocin produced therefrom
CN116676236A (zh) * 2023-07-18 2023-09-01 中国农业大学 一种降解单宁和皂苷的植物乳植杆菌及其应用
WO2024027860A1 (fr) 2022-08-05 2024-02-08 Česká zemědělská univerzita v Praze Composition de bactériocine pour coder une protéine de type linocine-m18 ayant une activité antimicrobienne et son utilisation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7247306B2 (en) 2004-04-30 2007-07-24 Universite Laval Bacteria strain and bacteriocin produced therefrom
WO2024027860A1 (fr) 2022-08-05 2024-02-08 Česká zemědělská univerzita v Praze Composition de bactériocine pour coder une protéine de type linocine-m18 ayant une activité antimicrobienne et son utilisation
CN116676236A (zh) * 2023-07-18 2023-09-01 中国农业大学 一种降解单宁和皂苷的植物乳植杆菌及其应用
CN116676236B (zh) * 2023-07-18 2024-03-08 中国农业大学 一种降解单宁和皂苷的植物乳植杆菌及其应用

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