Novel bacteriocins, transport and vector system and method of use thereof

Abstract

New bacteriocins capable of inhibiting the growth of bacteria are disclosed, along with methods of obtaining secretion of proteins from lactic acid bacteria, and methods for protecting foodstuffs.

What is claimed:  
     
         1 . An isolated peptide inhibitory to bacterial growth selected from the group consisting of enterocin 900 (SEQ ID NO:28), brochocin-C (SEQ ID NO:23), and muteins thereof.  
     
     
         2 . The peptide of  claim 1 , wherein said peptide is enterocin 900 or a mutein thereof.  
     
     
         3 . The peptide of  claim 1 , wherein said peptide is brochocin-C peptide A, brochocin-C peptide B, or a mutein thereof.  
     
     
         4 . An isolated polynucleotide, said polynucleotide comprising a structural gene sequence which encodes a peptide inhibitory to bacterial growth selected from the group consisting of enterocin 900 (SEQ ID NO:28), brochocin-C (SEQ ID NO:23), and muteins thereof.  
     
     
         5 . The polynucleotide of  claim 4 , further comprising a promoter operable in a host microorganism, operably linked to said structural gene sequence.  
     
     
         6 . The polynucleotide of  claim 5 , wherein said microorganism comprises a lactic acid bacterium.  
     
     
         7 . The polynucleotide of  claim 6 , further comprising a bacteriocin processing peptide operable in said host microorganism, operably linked to said structural gene sequence.  
     
     
         8 . The polynucleotide of  claim 10 , wherein said bacteriocin processing peptide comprises a divergicin A processing peptide.  
     
     
         9 . A secretion vector, for providing secretion of a heterologous protein from a microbial host cell, said secretion vector comprising: 
 a first polynucleotide encoding a heterologous protein;    a second polynucleotide encoding a bacteriocin processing peptide operable in said host cell, operably linked to said polynucleotide encoding said heterologous protein; and    a promoter operable in said host cell, operably linked to said polynucleotide encoding said heterologous protein.    
     
     
         10 . The secretion vector of  claim 9 , wherein said bacteriocin processing peptide comprises a divergicin A processing peptide or an operable mutein thereof.  
     
     
         11 . The secretion vector of  claim 9 , wherein said first polynucleotide encodes a heterologous bacteriocin.  
     
     
         12 . The secretion vector of  claim 11 , further comprising an immunity gene which confers immunity from said heterologous bacteriocin to said host cell.  
     
     
         13 . The secretion vector of  claim 11 , wherein said vector encodes a plurality of different bacteriocins.  
     
     
         14 . The vector pCD3.4 (SEQ ID NO:14).  
     
     
         15 . A host cell transformed with the secretion vector of  claim 12 .  
     
     
         16 . A host cell transformed with a plurality of secretion vectors of  claim 12 .  
     
     
         17 . A host cell transformed with the secretion vector of  claim 13 .  
     
     
         18 . A method for inhibiting the growth of susceptible bacteria in an environment, comprising: 
 providing a microorganism capable of expressing a bacteriocin selected from the group consisting of enterocin 900, brochocin-C, and operable muteins thereof; and    applying said microorganism to said environment in an amount sufficient to inhibit the growth of susceptible bacteria.    
     
     
         19 . The method of  claim 18 , wherein said environment comprises a foodstuff.  
     
     
         20 . The method of  claim 19 , wherein said foodstuff comprises meat.  
     
     
         21 . The method of  claim 18 , wherein said environment comprises a living mammal.  
     
     
         22 . The method of  claim 21 , wherein said environment comprises a food preparation area.  
     
     
         23 . The method of  claim 21 , wherein said microorganism expresses a plurality of bacteriocins.  
     
     
         24 . The method of  claim 21 , wherein said environment comprises a fermentation vessel.  
     
     
         25 . A method for inhibiting the growth of susceptible bacteria in an environment, comprising: 
 providing a bactericin selected from the group consisting of enterocin 900, brochocin-C, and operable muteins thereof; and    applying said bacteriocin to said environment in an amount sufficient to inhibit the growth of susceptible bacteria.    
     
     
         26 . The method of  claim 25 , wherein said bacteriocin inhibits bacteria which cause mastitis.  
     
     
         27 . A method for obtaining secretion of a protein from a microorganism, said method comprising: 
 providing a secretion vector comprising a promoter operable in said microorganism, operably linked to a polynucleotide encoding a bacteriocin processing peptide operable in said microorganism, operably linked to a polynucleotide encoding said protein;    transforming said microorganism with said secretion vector; and    culturing said transformed microorganism under conditions that induce expression and secretion.    
     
     
         28 . The method of  claim 27 , wherein said bacteriocin processing peptide comprises a divergicin A processing peptide.  
     
     
         30 . An expression vector for secreting a heterologous protein in a microorganism host, said vector comprising: 
 a polynucleotide encoding a promoter operable in said host;    a polynucleotide encoding a leucocin A processing peptide;    a polynucleotide encoding said heterologous protein; a polynucleotide encoding lcac (SEQ ID NO:4); and    a polynucleotide encoding lcaD (SEQ ID NO:5).    
     
     
         31 . The vector of  claim 30 , wherein said heterologous protein comprises leucocin A.  
     
     
         32 . An antibody specific for a bacteriocin selected from the group consisting of brochocin-C and enterocin 900.  
     
     
         33 . A method for detecting expression of a bacteriocin in a host cell, said method comprising: 
 providing an antibody specific for a bacteriocin selected from the group consisting of brochocin-C and enterocin 900;    contacting said host cell with said antibody; and    detecting binding or absence of binding between said antibody and said bacteriocin.    
     
     
         34 . An isolated polynucleotide, said polynucleotide comprising a sequence encoding brochocin-C immunity (SEQ ID NO:26) or an operable mutein thereof.  
     
     
         35 . A method for purifying brochocin-C from a microorganism culture, comprising: 
 extracting said microorganism culture with n-butanol;    removing said n-butanol to provide an extract; and    filtering said extract by gel filtration.    
     
     
         36 . A method for purifying enterocin 900 from a solution, comprising: 
 applying said solution to a hydrophobic interaction column;    washing said column with a solution having less than 40% ethanol;    eluting partially purified enterocin 900 from said column with a solution having about 40% or more ethanol;    applying said partially purified enterocin 900 to an ion exchange column;    washing said column with a salt solution having a concentration less than about 150 mM;    eluting enterocin 900 using a salt solution having a concentration of about 200 mM.    
     
     
         37 . The method of  claim 36 , further comprising: 
 applying enterocin 900 to a reverse-phase HPLC column;    washing said column with a solution comprising less than about 50% ethanol; and    eluting enterocin 900 using a solution comprising about 70% ethanol.    
     
     
         38 . A method for inhibiting the growth of susceptible bacteria in an environment, comprising: 
 providing a microorganism comprising a secretion vector, said secretion vector comprising: 
 a first polynucleotide encoding a bacteriocin;  
 a second polynucleotide encoding a bacteriocin processing peptide operable in said host cell, operably linked to said polynucleotide encoding said bacteriocin; and  
 a promoter operable in said host cell, operably linked to said polynucleotide encoding said bacteriocin; and  
   applying said microorganism to said environment in an amount sufficient to inhibit the growth of susceptible bacteria.    
     
     
         39 . The method of  claim 38 , wherein said bacteriocin processing peptide comprises a divergicin A processing peptide or an operable mutein thereof.  
     
     
         40 . The method of  claim 39 , wherein said secretion vector further comprises an immunity gene which confers immunity from said heterologous bacteriocin to said host cell.  
     
     
         41 . The method of  claim 40 , wherein said vector encodes a plurality of different bacteriocins.