Difference between revisions of "Part:BBa K1954004"
 
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=Mutacin III biosynthetic device=
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__NOTOC__
  
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Mutacin III is a ribosomally synthesized 22 amino acid screw-shaped lanthionine-containing peptide. The biosynthesis of mutacin III involves the expression of the structural gene mutA to make a prepropeptide, comprising a C-terminal propeptide and an N-terminal leader peptide from which the former undergoes processing and the latter is cleaved off before export into the medium (1). The specific post-translational modifications make mutacin III distinct from other bacteriocins and are introduced by enzymes coded for by other genes in the locus (mutBCDP). Basing on sequence homologies of genes in the locus with those of other lantibiotic biosynthetic loci it can be inferred that these enzymes catalyze the dehydration (mutB) and cyclization (mutC) of the propeptide serine and threonine residues which can condense with a neighboring cysteine residue (2) leading to the formation of lanthionine or methyllanthionine (thioether) bridges, respectively. The enzyme coded by mutD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue (3) while the product of mutP is a serine protease which cleaves the leader peptide and is likely the last step in the biosynthesis (4). The mature mutacin III is composed of rings connected by flexible linkers (Fig. 1) which may be important in the mechanism of bacteriocidal activity (5). Following export, the peptide is believed to form transmembrane pores as monomer aggregates leading to membrane disruption and efflux of cellular components (6). The content of anionic phospholipids in the membrane has been suggested to be an important factor influencing initial binding – mutacin III has a net positive charge whereas Gram-positive bacteria have a high relative amount of anionic lipids (7).
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<partinfo>BBa_K1954004 short</partinfo>
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<br>
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<center> https://static.igem.org/mediawiki/2016/a/a7/T--UCL--bacteriocinproducing.png </center>
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<p> <center> Fig. 1. The structure of mature mutacin III thought to be identical to mutacin 1140 (6). </center> </p>
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Mutacin III biosynthetic device
  
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===Sequence and Features===
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<partinfo>BBa_K1954004 SequenceAndFeatures</partinfo>
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===Usage and Biology===
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A decrease in biofilm formation caused by interference with the viability of certain bacterial species presents an approach towards limiting cariogenesis. Our team designed a locus capable of producing and exporting a mature form of an antimicrobial peptide known as mutacin III, first identified in Streptococcus mutans UA787 isolated from a caries-active white female patient in the late 1980s (1). Mutacin III is effective against a wide range of Gram-positive bacteria implicated in dental caries, e.g. other strains of Streptococcus mutans and Actinomyces naeslundii, while Gram-negative bacteria are resistant to inhibition (2).
 
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The biosynthetic locus of mutacin III was designed by our team in a form allowing for high-yield and fine-tuned expression of the peptide (Fig. 2). We placed a strong T7 promoter upstream of mutA to obtain high levels of the propeptide, a repressible pTet promoter upstream of the mutBCDP co-transcription unit and an inducible araBAD promoter for the mutT gene, coding for the ATP-binding-cassette-like transporter of mutacin III. All illegal restriction sites were removed from the endogenous sequences by silent mutagenesis.
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Mutacin III is a ribosomally synthesized 22 amino acid screw-shaped lanthionine-containing peptide. The biosynthesis of mutacin III involves the expression of the structural gene mutA to make a prepropeptide, comprising a C-terminal propeptide and an N-terminal leader peptide from which the former undergoes processing and the latter is cleaved off before export into the medium (1). The specific post-translational modifications make mutacin III distinct from other bacteriocins and are introduced by enzymes coded for by other genes in the locus (mutBCDP). Basing on sequence homologies of genes in the locus with those of other lantibiotic biosynthetic loci it can be inferred that these enzymes catalyze the dehydration (mutB) and cyclization (mutC) of the propeptide serine and threonine residues which can condense with a neighboring cysteine residue (3) leading to the formation of lanthionine or methyllanthionine (thioether) bridges, respectively. The enzyme coded by mutD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue (4) while the product of mutP is a serine protease which cleaves the leader peptide and is likely the last step in the biosynthesis (5). The mature mutacin III is composed of rings connected by flexible linkers (Fig. 1) which may be important in the mechanism of bacteriocidal activity (6). Following export, the peptide is believed to form transmembrane pores as monomer aggregates leading to membrane disruption and efflux of cellular components (7). The content of anionic phospholipids in the membrane has been suggested to be an important factor influencing initial binding – mutacin III has a net positive charge whereas Gram-positive bacteria have a high relative amount of anionic lipids (8).
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<br><br>
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<center><img src =" https://static.igem.org/mediawiki/2016/a/a7/T--UCL--bacteriocinproducing.png"> </center>
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<p> <center> Fig. 1. The structure of mature mutacin III thought to be identical to mutacin 1140 (8). </center> </p>
  
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<center> <img src ="https://static.igem.org/mediawiki/2016/0/07/T--UCL--bacteriocindevice.png" width="50%" height="50%" </center></html>
 
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<p> <center> Fig. 2. Simplified diagram of the mutacin III biosynthetic locus designed by UCL iGEM 2016. </center> </p>
 
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1. Qi F, Chen P, Caufield PW. Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes. Appl Environ Microbiol. 1999 Sep;65(9):3880–7.  
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<br><br>
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2. Cotter PD, Hill C, Ross RP. Food Microbiology: Bacteriocins: developing innate immunity for food. Nat Rev Microbiol. 2005 Oct;3(10):777–88.  
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===References===
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3. Cotter PD, Hill C, Ross RP. Bacterial lantibiotics: strategies to improve therapeutic potential. Curr Protein Pept Sci. 2005 Feb;6(1):61–75.  
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4. Sahl HG, Jack RW, Bierbaum G. Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur J Biochem. 1995 Jun 15;230(3):827–53.  
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<div style="text-align:left">
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5. Moll GN, Roberts GC, Konings WN, Driessen AJ. Mechanism of lantibiotic-induced pore-formation. Antonie Van Leeuwenhoek. 1996 Feb;69(2):185–91.  
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<ol>
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6. Smith L, Zachariah C, Thirumoorthy R, Rocca J, Novák J, Hillman JD, et al. Structure and dynamics of the lantibiotic mutacin 1140. Biochemistry (Mosc). 2003 Sep 9;42(35):10372–84.
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<li>Qi F, Chen P, Caufield PW. Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes. Appl Environ Microbiol. 1999 Sep;65(9):3880–7. </li>
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7. Abee T. Pore-forming bacteriocins of Gram-positive bacteria and self-protection mechanisms of producer organisms. FEMS Microbiol Lett. 1995 Jun 1;129(1):1–9.
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<li>Hillman JD, Johnson KP, Yaphe BI. Isolation of a Streptococcus mutans strain producing a novel bacteriocin. Infect Immun. 1984 Apr;44(1):141–4.</li>
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<li>Cotter PD, Hill C, Ross RP. Food Microbiology: Bacteriocins: developing innate immunity for food. Nat Rev Microbiol. 2005 Oct;3(10):777–88.</li>
 +
<li>Cotter PD, Hill C, Ross RP. Bacterial lantibiotics: strategies to improve therapeutic potential. Curr Protein Pept Sci. 2005 Feb;6(1):61–75.</li> 
 +
<li>Sahl HG, Jack RW, Bierbaum G. Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur J Biochem. 1995 Jun 15;230(3):827–53.</li> 
 +
<li>Moll GN, Roberts GC, Konings WN, Driessen AJ. Mechanism of lantibiotic-induced pore-formation. Antonie Van Leeuwenhoek. 1996 Feb;69(2):185–91.</li> 
 +
<li>Smith L, Zachariah C, Thirumoorthy R, Rocca J, Novák J, Hillman JD, et al. Structure and dynamics of the lantibiotic mutacin 1140. Biochemistry (Mosc). 2003 Sep 9;42(35):10372–84.</li>
 +
<li>Abee T. Pore-forming bacteriocins of Gram-positive bacteria and self-protection mechanisms of producer organisms. FEMS Microbiol Lett. 1995 Jun 1;129(1):1–9.</li>
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</ol>

Latest revision as of 10:26, 21 October 2016


Mutacin III biosynthetic device

Mutacin III biosynthetic device


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1294
    Illegal BglII site found at 2915
    Illegal BglII site found at 3097
    Illegal BglII site found at 3763
    Illegal BglII site found at 3835
    Illegal BglII site found at 4976
    Illegal BglII site found at 5507
    Illegal BamHI site found at 1742
    Illegal BamHI site found at 8141
    Illegal BamHI site found at 8625
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 7976
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 9690
    Illegal SapI site found at 1161
    Illegal SapI site found at 7958
    Illegal SapI.rc site found at 3194


Usage and Biology

A decrease in biofilm formation caused by interference with the viability of certain bacterial species presents an approach towards limiting cariogenesis. Our team designed a locus capable of producing and exporting a mature form of an antimicrobial peptide known as mutacin III, first identified in Streptococcus mutans UA787 isolated from a caries-active white female patient in the late 1980s (1). Mutacin III is effective against a wide range of Gram-positive bacteria implicated in dental caries, e.g. other strains of Streptococcus mutans and Actinomyces naeslundii, while Gram-negative bacteria are resistant to inhibition (2).

Mutacin III is a ribosomally synthesized 22 amino acid screw-shaped lanthionine-containing peptide. The biosynthesis of mutacin III involves the expression of the structural gene mutA to make a prepropeptide, comprising a C-terminal propeptide and an N-terminal leader peptide from which the former undergoes processing and the latter is cleaved off before export into the medium (1). The specific post-translational modifications make mutacin III distinct from other bacteriocins and are introduced by enzymes coded for by other genes in the locus (mutBCDP). Basing on sequence homologies of genes in the locus with those of other lantibiotic biosynthetic loci it can be inferred that these enzymes catalyze the dehydration (mutB) and cyclization (mutC) of the propeptide serine and threonine residues which can condense with a neighboring cysteine residue (3) leading to the formation of lanthionine or methyllanthionine (thioether) bridges, respectively. The enzyme coded by mutD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue (4) while the product of mutP is a serine protease which cleaves the leader peptide and is likely the last step in the biosynthesis (5). The mature mutacin III is composed of rings connected by flexible linkers (Fig. 1) which may be important in the mechanism of bacteriocidal activity (6). Following export, the peptide is believed to form transmembrane pores as monomer aggregates leading to membrane disruption and efflux of cellular components (7). The content of anionic phospholipids in the membrane has been suggested to be an important factor influencing initial binding – mutacin III has a net positive charge whereas Gram-positive bacteria have a high relative amount of anionic lipids (8).

<img src =" T--UCL--bacteriocinproducing.png">

Fig. 1. The structure of mature mutacin III thought to be identical to mutacin 1140 (8).




References

  1. Qi F, Chen P, Caufield PW. Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes. Appl Environ Microbiol. 1999 Sep;65(9):3880–7.
  2. Hillman JD, Johnson KP, Yaphe BI. Isolation of a Streptococcus mutans strain producing a novel bacteriocin. Infect Immun. 1984 Apr;44(1):141–4.
  3. Cotter PD, Hill C, Ross RP. Food Microbiology: Bacteriocins: developing innate immunity for food. Nat Rev Microbiol. 2005 Oct;3(10):777–88.
  4. Cotter PD, Hill C, Ross RP. Bacterial lantibiotics: strategies to improve therapeutic potential. Curr Protein Pept Sci. 2005 Feb;6(1):61–75.
  5. Sahl HG, Jack RW, Bierbaum G. Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur J Biochem. 1995 Jun 15;230(3):827–53.
  6. Moll GN, Roberts GC, Konings WN, Driessen AJ. Mechanism of lantibiotic-induced pore-formation. Antonie Van Leeuwenhoek. 1996 Feb;69(2):185–91.
  7. Smith L, Zachariah C, Thirumoorthy R, Rocca J, Novák J, Hillman JD, et al. Structure and dynamics of the lantibiotic mutacin 1140. Biochemistry (Mosc). 2003 Sep 9;42(35):10372–84.
  8. Abee T. Pore-forming bacteriocins of Gram-positive bacteria and self-protection mechanisms of producer organisms. FEMS Microbiol Lett. 1995 Jun 1;129(1):1–9.