Thesis--Wisconsin. / Vita. Includes bibliographical references.

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 127-135).

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

No description available.

Thesis (Ph.D.)--University of Wyoming, 2007. / Title from PDF title page (viewed on June 17, 2009). Includes bibliographical references (p. 83-103).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Dysgalacticin is a large, 21.5 kDa bacteriocin that belongs to subgroup B of the class III bacteriocins. It is ribosomally produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 and exerts inhibitory activity mainly against the medically important pathogen Streptococcus pyogenes by a "non-lytic" mechanism. Despite numerous studies of the mechanisms of action of a wide variety of bacteriocins and of the basis of their producer strain self-immunity, relatively little is known about the "non-lytic" class of bacteriocins. The structural gene encoding for dysgalacticin (dysA) was known to be carried on a small, rolling circle plasmid pW2580 (3.04 kb) (Heng et al., 2006). However, the dysgalacticin immunity gene (dysI) had not been identified prior to the present study. The aims of this research were to elucidate the mechanism of action of dysgalacticin against S. pyogenes and to identify the genetic basis and the mechanism of producer strain self-immunity. Recombinantly-produced dysgalacticin was used to determine the mode of action against S. pyogenes. Dysgalacticin was bactericidal for S. pyogenes, increasing the permeability of the cytoplasmic membrane and ultimately leading to leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [�⁴C]serine uptake, a membrane potential-dependent process in S. pyogenes. Interestingly, dysgalacticin inhibited glucose fermentation by non-growing cell suspensions and blocked transport of both glucose and the nonmetabolisable analogue 2-deoxyglucose. This finding indicates that dysgalacticin may target the phosphophenolpyruvate (PEP)-dependent glucose and mannose phosphotransferase system (PTS) of S. pyogenes. Taken together, these data suggest that dysgalacticin targets the glucose-PTS and/or mannose-PTS as a receptor, leading to inhibition of sugar uptake, and a subsequent dissipation of the membrane potential leading to cell death. Complementation studies demonstrated that dysI is located on pW2580. RNA analysis showed that dysI is co-transcribed with genes encoding for the plasmid copy control protein, copG and replication initiation protein, repB. S. pyogenes transformed with a plasmid containing dysI displayed a markedly higher dysgalacticin MIC (1024 nM) than the corresponding dysgalacticin-sensitive, plasmid-negative strain (8 nM). Further studies of this DysI-expressing S. pyogenes showed that membrane integrity, glucose fermentation and [�H]2DG uptake were not affected by dysgalacticin treatment. These findings are consistant with a mechanism whereby the immunity peptide binds to the target-binding site of dysgalacticin, effectively blocking access by the bacteriocin. H₆DysI was found to localise to the cytoplasmic membrane, further indicating that DysI may bind to the proposed target of dysgalacticin, i.e., the membrane-bound glucose-PTS and mannose-PTS. Thus both the mode of action and the producer strain self-immunity of dysgalacticin are likely to be cytoplasmic-membrane based. Homology searching revealed that the bacteriocin SA-M7 produced by M-type 57 S. pyogenes has structural similarities to dysgalacticin, as do two hypothetical proteins, EF1097 and YpkK, of Enterococcus faecalis and Corynebacterium jeikeium, respectively (Heng et al., 2004, 2006). These proteins were all predicted to contain relatively unstructured N-termini and helix-loop-helix structured C-termini. In each case the C-termini contain two conserved cysteine residues that are predicted to form a disulphide bridge. Heterologous expression of SA-M57, EF1097 and YpkK in Escherchia coli demonstrated that all three proteins have antimicrobial activity, but of differeing activity spectra. Reductive-alkylation of SA-M57, EF1097 and YpkK confirmed that their predicted disulphide bonds were essential for biological activity. These proteins were later renamed streptococcin A-M57, enterococcin V583 and corynicin JK respectively. The outcome of preliminary domain-swapping experiments supported the existence of functional domain-type segments in streptococcin A-M57, enterococcin V583, corynicin JK and dysgalacticin. The N-terminal domain of each of these proteins and also the C-terminal domain of corynicin JK were successfully expressed in E. coli. The failure to express the C-termini of the remaining proteins was thought possibly due to toxicity of thses pepetides for the E. coli host. Nevertheless, the C-terminus of corynicin JK displayed an inhibitory spectrum apparently identical to that of the full-length corynicin, indicating that the N-terminus may not always be required for target binding of this class of antimicrobials. Preliminary mode of action studies revealed that streptococcin A-M57, enterococcin V583 and corynicin JK all resemble dysgalacticin in that they exert inhibitory activity by non-lytic means. These results, in combination with the protein structural predictions indicate that dysgalacticin, streptococcin A-M57, enterococcin V583 and corynicin JK are all members of the same basic class of "non-lytic" bactericoicns.

Dysgalacticin is a large, 21.5 kDa bacteriocin that belongs to subgroup B of the class III bacteriocins. It is ribosomally produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 and exerts inhibitory activity mainly against the medically important pathogen Streptococcus pyogenes by a "non-lytic" mechanism. Despite numerous studies of the mechanisms of action of a wide variety of bacteriocins and of the basis of their producer strain self-immunity, relatively little is known about the "non-lytic" class of bacteriocins. The structural gene encoding for dysgalacticin (dysA) was known to be carried on a small, rolling circle plasmid pW2580 (3.04 kb) (Heng et al., 2006). However, the dysgalacticin immunity gene (dysI) had not been identified prior to the present study. The aims of this research were to elucidate the mechanism of action of dysgalacticin against S. pyogenes and to identify the genetic basis and the mechanism of producer strain self-immunity. Recombinantly-produced dysgalacticin was used to determine the mode of action against S. pyogenes. Dysgalacticin was bactericidal for S. pyogenes, increasing the permeability of the cytoplasmic membrane and ultimately leading to leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [�⁴C]serine uptake, a membrane potential-dependent process in S. pyogenes. Interestingly, dysgalacticin inhibited glucose fermentation by non-growing cell suspensions and blocked transport of both glucose and the nonmetabolisable analogue 2-deoxyglucose. This finding indicates that dysgalacticin may target the phosphophenolpyruvate (PEP)-dependent glucose and mannose phosphotransferase system (PTS) of S. pyogenes. Taken together, these data suggest that dysgalacticin targets the glucose-PTS and/or mannose-PTS as a receptor, leading to inhibition of sugar uptake, and a subsequent dissipation of the membrane potential leading to cell death. Complementation studies demonstrated that dysI is located on pW2580. RNA analysis showed that dysI is co-transcribed with genes encoding for the plasmid copy control protein, copG and replication initiation protein, repB. S. pyogenes transformed with a plasmid containing dysI displayed a markedly higher dysgalacticin MIC (1024 nM) than the corresponding dysgalacticin-sensitive, plasmid-negative strain (8 nM). Further studies of this DysI-expressing S. pyogenes showed that membrane integrity, glucose fermentation and [�H]2DG uptake were not affected by dysgalacticin treatment. These findings are consistant with a mechanism whereby the immunity peptide binds to the target-binding site of dysgalacticin, effectively blocking access by the bacteriocin. H₆DysI was found to localise to the cytoplasmic membrane, further indicating that DysI may bind to the proposed target of dysgalacticin, i.e., the membrane-bound glucose-PTS and mannose-PTS. Thus both the mode of action and the producer strain self-immunity of dysgalacticin are likely to be cytoplasmic-membrane based. Homology searching revealed that the bacteriocin SA-M7 produced by M-type 57 S. pyogenes has structural similarities to dysgalacticin, as do two hypothetical proteins, EF1097 and YpkK, of Enterococcus faecalis and Corynebacterium jeikeium, respectively (Heng et al., 2004, 2006). These proteins were all predicted to contain relatively unstructured N-termini and helix-loop-helix structured C-termini. In each case the C-termini contain two conserved cysteine residues that are predicted to form a disulphide bridge. Heterologous expression of SA-M57, EF1097 and YpkK in Escherchia coli demonstrated that all three proteins have antimicrobial activity, but of differeing activity spectra. Reductive-alkylation of SA-M57, EF1097 and YpkK confirmed that their predicted disulphide bonds were essential for biological activity. These proteins were later renamed streptococcin A-M57, enterococcin V583 and corynicin JK respectively. The outcome of preliminary domain-swapping experiments supported the existence of functional domain-type segments in streptococcin A-M57, enterococcin V583, corynicin JK and dysgalacticin. The N-terminal domain of each of these proteins and also the C-terminal domain of corynicin JK were successfully expressed in E. coli. The failure to express the C-termini of the remaining proteins was thought possibly due to toxicity of thses pepetides for the E. coli host. Nevertheless, the C-terminus of corynicin JK displayed an inhibitory spectrum apparently identical to that of the full-length corynicin, indicating that the N-terminus may not always be required for target binding of this class of antimicrobials. Preliminary mode of action studies revealed that streptococcin A-M57, enterococcin V583 and corynicin JK all resemble dysgalacticin in that they exert inhibitory activity by non-lytic means. These results, in combination with the protein structural predictions indicate that dysgalacticin, streptococcin A-M57, enterococcin V583 and corynicin JK are all members of the same basic class of "non-lytic" bactericoicns.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.