WO1991019802A1 - Novel bacteriocin from a strain of lactococcus lactis subsp. cremoris - Google Patents
Novel bacteriocin from a strain of lactococcus lactis subsp. cremoris Download PDFInfo
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- WO1991019802A1 WO1991019802A1 PCT/EP1991/001109 EP9101109W WO9119802A1 WO 1991019802 A1 WO1991019802 A1 WO 1991019802A1 EP 9101109 W EP9101109 W EP 9101109W WO 9119802 A1 WO9119802 A1 WO 9119802A1
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- polypeptide
- bacteriocin
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- asn
- leu
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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
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/097—Preservation
- A23C19/10—Addition of preservatives
- A23C19/11—Addition of preservatives of antibiotics or bacteriocins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1322—Inorganic compounds; Minerals, including organic salts thereof, oligo-elements; Amino-acids, peptides, protein-hydrolysates or derivatives; Nucleic acids or derivatives; Yeast extract or autolysate; Vitamins; Antibiotics; Bacteriocins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C11/00—Fermentation processes for beer
- C12C11/003—Fermentation of beerwort
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
- C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
- C12H1/12—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages without precipitation
- C12H1/14—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages without precipitation with non-precipitating compounds, e.g. sulfiting; Sequestration, e.g. with chelate-producing compounds
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- This invention relates to a novel bacteriocin and its isolation, synthesis and use.
- the above microorganism is autolytic when cell concentrations are high and we have discovered that it produces a bacteriocin which is found extracellularly in growth media, for example M17 medium.
- the autolysis of the bacteria can be shown to be due to the lytic properties of the bacteriocin. Lysis is prevented by the addition of proteases that degrade the bacteriocin.
- bacteriocin 11 is used herein to include substances released by bacteria which kill not only the productive organism itself, but also other strains of bacteria, by any mechanism, including lysis.
- the new bacteriocin here concerned has been shown to inhibit the growth of more than 120 strains of lactococci, including strains producing the known bacteriocins diplococcin and nisin.
- the productive organism itself carries a gene coding for an immunity factor providing resistance to the bacteriocin to prevent indiscriminate lysis and the bacteriocin only seems to lyse its productive organism at high cell concentrations.
- the above structure is different from that of nisin and has no meaningful sequence homology with other known polypeptide sequences in the SWISS PROT data bank.
- the lytic activity of the bacteriocin of the invention is substantially greater than that of previously known L.lactis bacteriocins.
- novel bacteriocin is heat stable and retains activity after boiling in water for 30 minutes. This is of value in permitting its use in industrial processes using L.lactis organisms for example cheese and yoghurt manufacture.
- the bacteriocin appears to kill lactococci by lysis. Accelerated lysis of lactococci is beneficial in accelerating cheese ripening and the new bacteriocin is thus of particular application in the production of cheese.
- the lytic activity of the bacteriocin may also be of use in production of cell wall preparations or for liberation of nucleic acid material.
- bacteriocin Since certain bacteria, for example Gram-negative bacteria, are resistant to bacteriocins, negative selection is possible by using the bacteriocin according to the invention to remove certain cells, for example L.lactis. from mixed cell populations e.g. in starter cultures for fermentation.
- addition of the bacteriocin of the invention to the starter culture serves to eliminate foreign organisms and may be effective against, for example, spore forming clostridia or unwanted strains of L.lactis.
- the bacteriocin may advantageously be added to a cheese or yoghurt fermentation at a relatively late stage, after lactic acid, protease and flavour production by the L.Lactis organism has already taken place.
- the bacteriocin may also be used to kill selectively strains of lactic acid producing bacteria in beer and distillery fermentations, since these are attributed in the literature to be the major causes of spoilage in unpasteurised beers and give rise to the greatest proportion of infections during fermentation.
- the invention particularly includes starter cultures of microorganisms containing the bacteriocin as an inhibitor of contaminating lactococcus species.
- Such microorganisms may, for example, be strains of L.lactis resistant to the bacteriocin e.g. the producing organism, so that only unwanted microorganisms are removed from the starter culture, or yeasts of use in beer or distillery fermentations.
- Such starter cultures will normally be in lyophilised form.
- the bacteriocin may be used as a taxonomic tool in the identification of Lactococcus species.
- the new bacteriocin may be isolated from cultures of Lactococcus lactis subsp. cremoris by fractionation of the growth medium whereby fractions enriched in the bacteriocin are collected.
- the organism may be grown in a suitable culture medium, e.g. M17 broth, and the supernatant subjected to fractional precipitation e.g. with ammonium sulphate, followed by chromatography e.g. on carboxymethyl agarose with elution with phosphate buffer and/or on phenylsuperose with gradient elution with phosphate buffer containing increasing concentrations of ethanol.
- the gene coding for the bacteriocin runs from base 375 to base 536.
- the gene coding for the putative immunity factor runs from base 554 to base 847 in a different reading frame.
- Three putative promoter sequences are indicated as PI, P2 and P3 in regions -35 and -10 and ribosome binding sites are indicated as RBS.
- DNA coding for the bacteriocin and for the immunity factor respectively. It will be appreciated that knowledge of the overall amino acid sequence shown in claim 1 and/or the DNA sequence coding for the pro- bacteriocin does not provide an indication of the position of the first codon coding for the mature bacteriocin.
- the invention thus includes not only the DNA sequences shown in Fig. 1 but also sequences which due to the degeneracy of the code, are also capable of coding for the proteins concerned.
- the invention also includes cloning and expression vectors containing the DNA coding for the mature bacteriocin and/or for the immunity factor. Expression vectors appropriate to L. lactis are particularly preferred.
- the invention also includes strains of L. lactis transformed with such vectors.
- the immunity factor according to the invention may be of use in combating the effects of L. lactis bacteriocins, for example, in controlling the effects of the bacteriocin according to the invention.
- the gene coding for the immunity factor may be used as a selective marker in future construction of food grade cloning vector, for example instead of an antibiotic matter.
- the operon shown in Fig. 1 was obtained from the fragmented plasmid DNA of L. lactis cremoris, using a probe comprising all or part of the non-coding DNA strand corresponding to the mature bacteriocin coding portion of the DNA strand shown in Fig. 1.
- the DNA coding for the mature bacteriocin or immunity factor may be incorporated into any convenient cloning vector for amplification and into an expression vector for transformation of host microorganisms such as L. lactis. for example cloning vector pIL253 (A. Chopin, Biochimie 70, 1988, 59-566). Growth under suitable culture conditions will provide the bacteriocin in the growth medium, from which it can be isolated by the techni ues described above.
- strains of L. lactis may be transformed with multiple copies of a plasmid or other vector containing the required DNA sequence to provide an improved strain giving rise to enhanced production of the bacteriocin.
- Such improved strains may provide more rapid lysis and hence accelerated cheese ripening when used in cheese manufacture.
- the strain of L.lactis which produces the bacteriocin and which thus also carries a resistance gene may be provided with such multiple copies of the vector; this will thus be able to proliferate without premature destruction by the bacteriocin.
- the new bacteriocin may also be prepared by chemical synthesis, for example using solid phase synthesis, advantageously using a polypeptide synthesis apparatus, as commercially available.
- active side chain groupings e.g. amino or carboxyl groups
- the final step will be deprotection and/or removal from the inert support to which the polypeptide is attached during synthesis.
- the histidine derivative will have a free ⁇ -amino group while the other reactant will have either a free or activated carboxyl group and a protected amino group.
- the intermediate may be purified for example by chromatography, and then selectively N-deprotected to permit addition of a further N-protected and free or activated amino acid residue. This procedure is continued until the required amino acid sequence is completed.
- Carboxylic acid activating substituents which may, for example, be employed include symmetrical or mixed anhydrides, or activated esters such as for example p-nitrophenyl ester, 2,4,5,trichlorophenyl- ester, N-hydroxybenzotriazole ester (OBt) , N-hydroxy- succinimidylester (OSu) or pentafluorophenylester (OPFP) .
- the coupling of free amino and carboxyl groups may, for example, be effected using dicyclohexylcarbodi- imide (DCC) .
- DCC dicyclohexylcarbodi- imide
- Another coupling agent which may, for example, be employed is N-ethoxycarbonyl-2-ethoxy-l,2-dihydro- quinoline (EEDQ) .
- Chloro- methylated polystyrene cross-linked with 1% divinyl benzene is one useful type of support; in this case the synthesis will start the C-terminal, for example by coupling N-protected histidine to the support.
- amine protecting groups which may be employed include protecting groups which may be employed include protecting groups such as carbobenzoxy (Z-) , t-butoxycarbonyl (Boc-) , 4-methoxy-2,3,6-trimethyl- benzene sulphonyl (Mtr-) , and 9-fluorenylmethoxycarbonyl (Fmoc-) .
- protecting groups such as carbobenzoxy (Z-) , t-butoxycarbonyl (Boc-) , 4-methoxy-2,3,6-trimethyl- benzene sulphonyl (Mtr-) , and 9-fluorenylmethoxycarbonyl (Fmoc-) .
- Z- carbobenzoxy
- Boc- t-butoxycarbonyl
- Mtr- 4-methoxy-2,3,6-trimethyl- benzene sulphonyl
- Fmoc- 9-fluorenylmethoxycarbonyl
- Carboxyl protecting groups which may, for example be employed include readily cleaved ester groups such as benzyl (-0BZ1) , p-nitrobenzyl (-0NB) , or t-butyl (-tOBu) as well as the coupling on solid supports, for example methyl groups linked to polystyrene.
- Amine protecting groups such as Boc and carboxyl protecting groups such as tOBu may be removed simultan ⁇ eously by acid treatment, for example with trifluoro acetic acid.
- acid treatment for example with trifluoro acetic acid.
- the histidine derivative will have a free ⁇ -amino group while the other reactant will have either a free or activated carboxyl group and a protected amino group.
- the intermediate may be purified for example by chromatography, and then selectively N-deprotected to permit addition of a further N-protected and free or activated amino acid residue. This procedure is continued until the required amino acid sequence is completed.
- Carboxylic acid activating substituents which may, for example, be employed ⁇ include symmetrical or mixed anhydrides, or activated esters such as for example p-nitrophenyl ester, 2,4,5,trichlorophenyl-ester, N-hydroxybenzotriazole ester (OBt) , N-hydroxy- succinimidylester (OSu) or pentafluorophenylester (OPFP) .
- OBt N-hydroxybenzotriazole ester
- OSu N-hydroxy- succinimidylester
- OPFP pentafluorophenylester
- the coupling of free amino and carboxyl groups may, for example, be effected using dicyclohexylcarbodi-imide (DCC) .
- DCC dicyclohexylcarbodi-imide
- Another coupling agent which may, for example, be employed is N-ethoxycarbonyl-2-ethoxy-l,2-dihydro- quinoline (EEDQ) .
- Chloro-methylated polystyrene cross-linked with 1% divinyl benzene
- the synthesis will start the C-terminal, for example by coupling N-protected histidine to the support.
- amine protecting groups which may be employed include include protecting groups such as carbobenzoxy (Z-) , t-butoxycarbonyl (Boc-) , 4-methoxy-2,3,6-trimethyl-benzene sulphonyl (Mtr-), and 9-fluorenylmethoxycarbonyl (Fmoc-) .
- Z- carbobenzoxy
- Boc- t-butoxycarbonyl
- Mtr- 4-methoxy-2,3,6-trimethyl-benzene sulphonyl
- Fmoc- 9-fluorenylmethoxycarbonyl
- Carboxyl protecting groups which may, for example be employed include readily cleaved ester groups such as benzyl (-OBZ1) , p-nitrobenzyl (-ONB) , or t-butyl (-tOBu) as well as the coupling on solid supports, for example methyl groups linked to polystyrene.
- Amine protecting groups such as Boc and carboxyl protecting groups such as tOBu may be removed simultan ⁇ eously by acid treatment, for example with trifluoro acetic acid.
- Bacterial strains, media, plasmids, and enzymes The bacterial strains, plasmids and phases used are listed in Table 1. All lactococcal strains were grown in M17 broth (44) and maintained as frozen stocks at -80°C in M17 broth containing 10% glycerol. Escherichia coli DH5 ⁇ was used for propagating p ⁇ C18 and its derivatives. M13 vectors and clones were propagated in 2x YT (2a) with E. coli JM101 as the host.
- T4 DNA ligase T4 polynucleotide kinase
- DNA molecular weight standards were purchased from Bethesda Research Laboratories, Inc. (Gaitherburg, Md.). Calf intestinal alkaline phosphatase, sequence-grade trypsin, and endoprotease glu-C were purchased from Boehringer GmbH (Mannheim, Germany) . Sequenase was obtained from United States Biochemical Corp. (Cleveland, Ohio) .
- L. lactis subsp. cremoris LMG 2130 was grown in M17 broth supplemented with 1% glucose at 38°C in the presence of 0.1 ⁇ xg of novobiocin per ml. Diluted aliquots from this culture were spread on M17 broth-1% glucose plates and incubated at 30°C. Colonies were scored for bacteriocin production.
- Bacteriocin assays Three methods were used to determine bacteriocin activity.
- Colonies of possible bacteriocin-producing bacteria were grown on agar plates overnight. A lawn of 3 ml of M17 soft agar (0.7%) containing 100 ⁇ l of a fresh culture of the indicator organism was poured over a plate. After incubation overnight at 30"C, the colonies were examined for zones of growth inhibition.
- M17 agar plates wells with a diameter of 4 mm were made and filled with bacteriocin solutions. After the liquid had been completely absorbed by the gel. M17 soft agar containing the indicator organism was overlaid on the plates to demonstrate bacteriocin activity as described above.
- Bacteriocin activity was quantified as described by Geis et al. (15) , except that microtiter plates with wells containing 200 ⁇ l of M17 broth were used.
- One unit of bacteriocin activity (BU) was arbitrarily defined as the amount of bacteriocin required to produce 50% growth inhibition (50% of the turbidity of the control without bacteriocin) of L. lactis subsp. cremoris IMN C18 in this assay.
- the bacteriocin was purified from 1-liter cultures of L. lactis subsp. cremoris LMG 2130. The various steps of the purification procedure were carried out at 4°C ⁇ unless otherwise stated. The cells were grown to the early stationary phase, and the bacteria were removed by centrifugation at 10,000 x g for 10 min. The bacteriocin was precipitated from the culture supernatant by the addition of 280 g of ammonium sulfate per liter. Following centrifugation at 10,000 x g for 30 min, the pellet was dissolved in water and adjusted to pH 7.3 by the addition of 0.5 M Na-.HPO ⁇ .
- the eluate from the cation exchanger was applied to a 1- ml Phenyl-Superose column (Pharmacia) equilibrated with 10 mM sodium phosphate (pH 7.3). Following washing with 10 mM sodium phosphate (pH 7.3), elution was carried out with a linear gradient of 0 to 60% ethanol at a flow rate of 0.3 ml/min. Purified LCN-A was stored in 60% ethanol-2.5 mM sodium phosphate (pH 7.3) at -20°C. Protein concentrations were determined spectrophotometrically at 280 nm.
- Plasmid DNA was isolated from L. lactis as described by Klaenhammer (23) .
- Small-scale preparation of E. coli plasmid DNA was performed with GeneClean (BIO 101, La Jolia, Calif.) .
- Large-scaled isolation of plasmids from E. coli was performed by the alkaline lysis method described by Maniatis et al. (25) .
- the M13 plus-strand DNA template for sequencing was prepared from infected 1.5 ml cultures as described previously (2a).
- Enzymes for DNA manipulations were used in accordance with manufacturer's specifications. Plasmid DNA from strain LMG 2130 used for cloning was purified by CsCl isopycnic centrifugation (33) .
- Restriction fragments of the desired size for cloning were isolated and purified from 0.7% agarose gels with Gene-Clean.
- DNA cloned in E. coli was subcloned in lactococci as follows. pUC18 plasmids with inserts were fused to pIL253 by EcoRI digestion and ligation. The resultant constructs were transformed into E. coli. Clones were obtained by selection for erythromycin (300 ⁇ g/ml) and ampicillin (50 ⁇ g/ml) resistance. Plasmid DNA extracted from the clones was used to transform lactococci by electroporation as described by Holo and Nes(20).
- Transformation of E. coli was performed by the method of Hanahan (17) .
- Bio-systems 381A DNA synthesizer 381A DNA synthesizer
- 3 '-ATIGT(T/C)GT(T/C) TG(I/C)TG(T/C)TTICG(I/C)AAICC-5' Colony hybridization was performed as described by Hanahan and Meselson (18) .
- Southern blots were made by vacuum transfer (2016 Vacugene; Pharmacia) of-restriction endonuclease- digested DNA (fractionated on 0.7% agarose gels) to GeneScreen Plus membranes (NEN Research Products, Dupont, Boston, Mass,) (42), Hybridization with the 26- mer oligodeoxynucleotide was performed as described by Church and Gilbert (7) .
- Nucleotide sequencing by the didoxynucleotide method (37) was carried out on restriction fragments cloned into M13mpl8 and M13mpl9 [ ⁇ - 35 S]dATP (600 Ci/mmol; Amersham International, Amersham, United Kingdom) was used for labelling.
- Nucleotide sequence accession number The nucleotide sequence presented in this article has been assigned EMBL accession number M63675.
- the protein is rich in alanine residues (8 of 54) and glycine residues (8 of 54) and contains only three charged amino acid residues.
- the calculated isoelectric point of the bacteriocin was 9.2.
- the extinction co-efficient of LCN-A at 280 nm was estimated to be 1.2 z 10 4 cm “1 M "1 from its content of tryptophan and tyrosine (4) .
- pure LCN-A had a specific activity of about 4.9 x 10 5 BUs/mg. Assuming that the activity of LCN-A was not reduced during purification, strain LMG 2130 produced about 3 mg of LCN-A per liter. By comparison, L.
- lactis subsp. cremoris 346 was found to produce 6 mg of diplococcin per liter (10) .
- the pure bacteriocin was not very soluble in water. Upon storage in aqueous buffers at 4"C, the bacteriocin formed an inactive precipitate. Pure LCN-A could, however, be stored longer than 6 months at -20°C in 60% ethanol containing 2.5 mM sodium phosphate (pH 7.3) without a detectable loss of activity.
- Table 3 shows the sensitivities of various lactococcal strains to LCN-A. Wide variations in sensitivity were found. The most sensitive strains tested appeared to be more sensitive to the bacteriocin when grown in lactic broth (14) than in M17 medium. In lactic broth, 50% growth inhibition of strain NCDO 1198 was observed at a calculated LCN-A concentration of 40 pg/ml, or 7 pM. This amount corresponds to about 400 molecules of LCN-A per CFU in the assay.
- LMG 2131 which did not produce LCN-A was found both to be deprived of the 55-kb plasmid and to give no signal on a Southern blot. Furthermore, Southern analysis of LMG 130 plasmid DNA digests revealed signals from a 4-kb Hindlll fragment a 1.2-kb Hindlll-Rsal fragment and a 0.6-kb Dral fragment. The 4-kb fraction of Hindlll- digested LMG 2130 plasmid DNA was cloned in E. coli with pUCl ⁇ as the vector. Of 1,400 clones, 10 were found to be positive after screening with the oligodeoxy- nucleotide probe.
- the fecombinant plasmid (pONl) from one of these 10 clones was further restricted with Dral and Rsalll.
- the fragments that hybridized to the probe, the 4-kb Hindlll fragment, the 1.2-kb Hindlll-Rsal fragment, and the 0.6-kb Dral fragment were subcloned into M13Mpl8 and M13mpl9 to yield inserts in both orientations.
- Nucleotide sequence of lcnA The Hind III-Rsal fragment was sequenced. The nucleotide sequence of the two consecutive Dral fragments of 625 and 292 nucleotides is shown in Fig. 1. The entire lcnA gene was contained with the 0.6-kb Dral fragment. Computer analysis of the six possible open reading frames (ORFs) revealed long ORFs only on one of the DNA strands. Mature LCN-A of 54 amino acid residues is encoded by the DNA segment from nucleotide positions 316 to 477.
- LCN-A Sensitivity to LCN-A appears to be general among strains of L. lactis. Since this bacteriocin also is highly specific, it may be used for the identification of L. lactis strains.
- LCN-A is a hydrophobic protein. Its hydrophobic character was demonstrated by its high affinity for phenyl-Superose. This matrix is intended for use in hydrophobic interaction chromatography, and most proteins bind to it only at high salt concentrations. LCN-A bound to the column in the absence of salt and could only be eluted as an active bacteriocin by solvents less polar than water.
- nisin has been ascribed to its ability to form pores in cytoplasmic membranes (36) .
- the hydrophobic character of LCN-A suggests that the cytoplasmic membrane may also be the target for this bacteriocin.
- Calculations made as described by Rao and Argos (34) predicted that the stretch from amino acids 30 to 52 in LCN-A can form a membrane-spanning helix (data not shown) .
- the idea that LCN-A acts on the membrane is further supported by the finding that this bacteriocin causes leakage of intracellular components even in hypertonic sucrose-containing media (unpublished data) .
- Secreted proteins are usually synthesized as precursors with a short N-terminal extension called the signal peptide, which promotes secretion and which is removed by specific enzymes (1, 43, 49, 50, 52).
- the signal peptide which promotes secretion and which is removed by specific enzymes (1, 43, 49, 50, 52).
- Comparison of the gene-derived sequence for mature LCN-A with the direct amino acid sequencing data shows that the LCN-A is synthesized as a 75-amino-acid precursor.
- the LCN-A leader peptide of 21 amino acids has a positively charged N-terminus followed by a hydrophobic stretch typical of signal peptides of gram-positive bacteria
- Mature LCN-A has a lysine as its N-terminal amino acid.
- a signal peptidase could cleave the precursor between the two glycines (-2,-1) but not between the glycine and the lysine (-1, +1) .
- This theory may suggest a stepwise processing of the LCN-A precursor in which a 20-amino-acid peptide and then a glycine are removed from the N terminus to yield mature LCN-A of 54 amino acids
- Fig. 1 Three putative promoter elements were found upstream of the lcnA gene (Fig. 1) . Conceivably, transcription initiation could occur 5 to 9 nucleotides downstream of any of the putative Pribnow boxes, yielding leaders of 17 to 33 nucleotides. Overlapping the -10 regions of the putative promoter elements is an inverted repeat sequence that could form a stem-loop structure (Fig. 1) . This structure, with a calculated ⁇ G value of -9.6 kcal/mol (- 40.2 kJ/mol) (45), could represent a Rho- dependent terminator of ORF1.
- Strain LMG 2131 which had lost the lcnA gene, was sensitive to LCN-A. This result suggests that the producing organism harbors a gene(s) encoding immunity to the bacteriocin.
- Strain IL 1403 carrying recombinant plasmid pON7 produced LCN-A and was (by necessity) resistant to the bacteriocin.
- the 1.2-kb (Rsal- Hindlll fragment appears to carry not only the gene encoding LCN-A but also a genetic determinant for resistance.
- the DNA sequence of this fragment shows only one complete ORF in addition to the lcnA gene, this is 0RF2, located downstream of and in the same operon as lcnA.
- ORF2 is the likely candidate to encode an LCN-A immunity function.
- a very similar organization of bacteriocin genes and their corresponding immunity genes has been shown for several E. coli bacteriocins (2, 26).
- 0RF2 with Met at nucleotide position 495, preceded by the possible RBS sequence 5' GGATTAG 3• encodes a hypothetical polypeptide of 98 amino acids.
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JP3510714A JPH06505144A (en) | 1990-06-18 | 1991-06-12 | A novel bacteriocin from Lactococcus lactis subspice cremoris |
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GB909023380A GB9023380D0 (en) | 1990-10-26 | 1990-10-26 | Chemical compounds |
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Cited By (4)
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WO1994004682A1 (en) * | 1992-08-24 | 1994-03-03 | Norwegian Dairies Association | Bacteriocin |
FR2739629A1 (en) * | 1995-10-06 | 1997-04-11 | Systems Bio Ind | USE OF A DRY-DEPENDENT SECRETION SYSTEM FOR SECRETING PROTEIN NORMALLY SECRETED BY A DRY-INDEPENDENT SECRETION SYSTEM, BACTERIA CONTAINING THEM AND THEIR USE |
WO1997018316A1 (en) * | 1995-11-13 | 1997-05-22 | Eijsink Vincent G H | Expression system in microorganism and its use for expressing heterologous and homologous proteins |
US20110129568A1 (en) * | 2008-06-30 | 2011-06-02 | Meiji Dairies Corporation | Process For Producing Fermented Milk And Fermented Milk |
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US5173297A (en) * | 1991-07-01 | 1992-12-22 | Quest International Flavors & Food Ingredients Company Division Of Indopco, Inc. | Bacteriocin from lactococcus lactis subspecies lactis |
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1991
- 1991-06-12 EP EP91910934A patent/EP0535039A1/en not_active Withdrawn
- 1991-06-12 AU AU80816/91A patent/AU8081691A/en not_active Abandoned
- 1991-06-12 CA CA002085580A patent/CA2085580A1/en not_active Abandoned
- 1991-06-12 WO PCT/EP1991/001109 patent/WO1991019802A1/en not_active Application Discontinuation
- 1991-06-12 JP JP3510714A patent/JPH06505144A/en active Pending
- 1991-06-12 NZ NZ238515A patent/NZ238515A/en unknown
- 1991-06-14 IE IE203591A patent/IE912035A1/en unknown
Non-Patent Citations (4)
Title |
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 55, No. 5, May 1989, AMERICAN SOCIETY FOR MICROBIOLOGY, VAN BELKUM M.J. et al., "Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid", pages 1187-1191. * |
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 57, No. 2, February 1991, AMERICAN SOCIETY FOR MICROBIOLOGY, VAN BELKUM M.J. et al., "Organization and Nucleotide Sequences of Two Lactococcal Bacteriocin Operons", pages 492-498. * |
CHEMICAL ABSTRACTS, Vol. 112, No. 11, 12 March 1990, Columbus, Ohio, US, Abstract No. 95145U, MOERTVEDT C. & NES F., "Bacteriocin Produced by a Lactobacillus Strain Isolated from Fermented Meat", page 425. * |
JOURNAL OF BACTERIOLOGY, Vol. 173, No. 12, June 1991, AMERICAN SOCIETY FOR MICROBIOLOGY, HOLO H. et al., "Lactococcin A, a New Bacteriocin from Lactococcus Lactis Subsp. Cremoris: Isolation and Characterization of the Protein and Its Gene", pages 3879-3887. * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994004682A1 (en) * | 1992-08-24 | 1994-03-03 | Norwegian Dairies Association | Bacteriocin |
FR2739629A1 (en) * | 1995-10-06 | 1997-04-11 | Systems Bio Ind | USE OF A DRY-DEPENDENT SECRETION SYSTEM FOR SECRETING PROTEIN NORMALLY SECRETED BY A DRY-INDEPENDENT SECRETION SYSTEM, BACTERIA CONTAINING THEM AND THEIR USE |
WO1997013863A1 (en) * | 1995-10-06 | 1997-04-17 | Systems Bio-Industries | Use of a sec-dependent secretion system for secreting proteins that are usually secreted by a sec-independent secretion system |
US5939317A (en) * | 1995-10-06 | 1999-08-17 | Skw Biosystems | Use of a Sec-dependent secretion system for secreting proteins that are usually secreted by a Sec-independent secretion system, bacteria containing it and their use |
WO1997018316A1 (en) * | 1995-11-13 | 1997-05-22 | Eijsink Vincent G H | Expression system in microorganism and its use for expressing heterologous and homologous proteins |
US6790951B1 (en) | 1995-11-13 | 2004-09-14 | Vincent G. H. Eijsink | Expression system in microorganism and its use for expressing heterologous and homologous proteins |
US20110129568A1 (en) * | 2008-06-30 | 2011-06-02 | Meiji Dairies Corporation | Process For Producing Fermented Milk And Fermented Milk |
Also Published As
Publication number | Publication date |
---|---|
IE912035A1 (en) | 1991-12-18 |
JPH06505144A (en) | 1994-06-16 |
AU8081691A (en) | 1992-01-07 |
CA2085580A1 (en) | 1991-12-19 |
EP0535039A1 (en) | 1993-04-07 |
NZ238515A (en) | 1994-03-25 |
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