Characterization of bacteriocin 423 produced by Lactobacillus pentosus

Van Reenen, Carol A. (Carol Ann) (2000-12)

Thesis (PhD)--University of Stellenbosch, 2000.

Thesis

ENGLISH ABSTRACT: Worldwide, bacteriocins, particularly those produced by food-related lactic acid bacteria, are receiving attention due to the possible use of these peptides as natural preservatives in food, replacing potentially harmful chemical preservatives. Bacteriocins are ribosomally synthesized proteins or peptides that inhibit closely related microorganisms. Most bacteriocins produced by lactic acid bacteria are small, heat resistant peptides that inhibit other Gram-positive bacteria, including food-borne pathogens such as Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Staphylococcus aureus, but do not inhibit Gram-negative bacteria, molds or fungi. Bacteriocins are produced as inactive prepeptides that become active after the N-terminal leader peptide is cleaved off. Small heat resistant bacteriocins are either lantibiotics (Class I), containing unusual posttranslationally modified amino acids, or peptides that are non-Ianthionines (Class II). The Class II bacteriocins are further divided into four different groups: Class lIa, the anti-listerial bacteriocins containing the YGNGV consensus sequence in the N-terminal of the protein, Class lib, bacteriocins consisting of two peptides, Class IIc, bacteriocins that are secreted via the sec pathway, and Class lid, bacteriocins that do not belong in the previous three subgroups. A bacteriocin producing lactic acid bacterium was isolated in our laboratory from traditionally home fermented South African sorghum beer. The producing bacterium was found to be a facultative heterofermentative Lactobacillus sp. and was identified as Lactobacillus plantarum or Lactobacillus pentosus by using the API 50 CHL carbohydrate fermentation system and numerical analysis of total soluble cell protein patterns. RAPD-PCR analysis identified the strain as L. plantarum, but 16S rRNA sequencing confirmed its identification as L. pentosus. The bacteriocin, first designated plantaricin 423 and later bacteriocin 423, was identified as a Class lIa small heat resistant anti-listerial bacteriocin containing the YGNGV consensus motif. Bacteriocin 423 inhibited a variety of Gram-positive bacteria, including Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp., Staphylococcus spp., Bacillus spp., Clostridium spp. and Listeria spp. The bacteriocin was inactivated by proteolytic enzymes and active over a wide pH range (pH 1-10). Bacteriocin 423 lost 50 % of its activity after autoclaving for 15 min at 121°C, but was not affected by lesser heat treatments. Bacteriocin production was increased by optimizing the growth medium, which consisted of glucose, tryptone, yeast extract, potassium phosphate, sodium acetate, ammonium citrate, manganese sulphate, Tween 80 and casamino acids. The bacteriocin was found to be plasmid-encoded. Genetic analysis of the bacteriocin operon indicated a high percentage of homology to the operon of another Class lIa bacteriocin, pediocin PA-1, although the structural genes of the two bacteriocins were markedly different. The structural gene of bacteriocin 423 was amplified by PCR and cloned into a yeastJE. coli vector between the ADH1 promoter and terminator sequences and fused in-frame to the MFa1 secretion signal sequence. Saccharomyces cerevisiae transformed with this plasmid expressed the bacteriocin. The sequence of prebacteriocin 423 (MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVN WGOAFSCSVSHLANFGHGKC) is similar, but not identical to any other reported Class lIa anti-listeria I peptide.

AFRIKAANSE OPSOMMING: Bakteriosiene, veral dié wat deur melksuurbakterieë geproduseer word, wek belangstelling as gevolg van die moontlike gebruik van hierdie natuurlike antimikrobiese proteiëne as preserveermiddels in voedselprodukte, in plaas van potensieël gevaarlike chemiese preserveermiddels. Bakteriosiene is ribosomaal-vervaardigde proteiëne wat naverwante bakterieë inhibeer. Die meeste bakteriosiene wat deur melksuurbakterieë geproduseer word, is klein en hittebestand. Hierdie bakteriosiene inhibeer ander Gram-positiewe bakterieë, insluitend patogene soos Listeria monocytogenes, Bacillus cereus, Clostridium perfringens en Staphylococcus aureus, maar inhibeer nie Gram-negatiewe bakterieë, giste of swamme nie. Bakteriosiene word as onaktiewe prepeptiede geproduseer, wat ge-aktiveer word wanneer die N-terminale leierpeptied afgesplits word. Klein hittebestande bakteriosiene is óf lantibiotika (Klas I), met ongewone aminosure, óf normale peptiede (Klas II). Laasgenoemde klas kan verder in vier groepe verdeel word. Klas lIa is anti-listeriese bakteriosiene met fn YGNGVaminosuurvolgorde in die N-terminale kant van die peptied. Klas lib sluit in bakteriosiene wat uit twee peptiede bestaan. Klas lie is sec-afhanklike bakteriosiene, en Klas lid sluit in al die bakteriosiene wat nie in die eerste drie groepe geklassifiseer kan word nie. 'n Bakteriosien-produserende melksuurbakterie is uit tradisionele tuisgefermenteerde Suid- Afrikaanse sorghumbier geïsoleer. Die bakterie is as 'n fakultatief heterofermentatiewe Lactobacillus sp. geïdentifiseer. Die bakterie is verder as 'n Lactobacillus plantarum of Lactobacillus pentosus geïdentifiseer deur middel van die API 50 CHL-koolhidraat fermentasiesisteem en numeriese analiese van totale oplosbare selproteiënprofiele. Met RAPD-PCR analiese is die organisme as L. plantarum geïdentifiseer, maar 168 rRNA nukleotiedopeenvolging het die identiteit van die organisme as L. pentosus bevestig. Bakteriosien 423, aanvanklik geklassifiseer as plantaricin 423, is fn klein Klas lIa, hittebestande en anti-listeriese bakteriosien met die YGNGV motief, wat verskeie Grampositiewe bakterieë inhibeer. Bakteriosien 423 het verskeie Gram-positiewe organismes geïnhibeer, onder andere Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp., Staphylococcus spp., Bacillus spp., Clostridium spp., en Listeria spp. Proteolitiese ensieme inaktiveer die bakteriosien. Die peptied was oor 'n pH reeks van 1-10 aktief. Outoklavering vir 15 min by 121°C het die aktiwiteit van die peptied halveer, maar die bakteriosien is nie geïnaktiveer met ander hittebehandelings nie. Produksie van die bakteriosien is verhoog deur die groeimedium te optimiseer. Die groeimedium het bestaan uit glukose, triptoon, gisekstrak, kaliumfosfaat, natriumasetaat, ammoniumsitraat, mangaansulfaat, Tween 80 en casaminosure. Die bakteriosien se genetiese determinante is op In plasmied gesetel. Genetiese analiese van die bakteriosien operon het 'n hoë homologie met In ander Klas lIa bakteriosien, pediocin PA-1, getoon, maar die strukturele gene van die twee bakteriosiene verskil merkbaar. Die strukturele geen van bakteriosien 423 is met PKR ge-amplifiseer en in 'n gistE. coli-vektor tussen die ADH1 promotor- en termineerderopeenvolgings, in leesraam met die MFa1 sekresiesein, gekloneer. Saccharomyces cerevisiae wat met hierdie plasmied getransformeer is, het bakteriosien 423 uitgedruk. Die aminosuurvolgorde van prebakteriosien 423 (MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVNWGOAFSCSVSHLANFGHGKC) is verwant aan, maar nie identies aan, ander Klas lIa anti-listeriese peptiede.

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