Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The possible use of the bacterially produced antimicrobial peptides, and in particular class IIa bacteriocins as food preservatives is a motivating factor in studies on resistance to them by food-borne pathogens like Listeria monocytogenes. The high frequencies of resistance to class Ha bacteriocins have however sparked concern regarding their adequacy as potential biopreservatives. Activity of these cationic peptides was reported to occur by membrane permeabilisation due to pore formation, which results in the leakage of the intracellular contents followed by cell death. The cell envelope (cell wall and cell membrane) is therefore envisaged as a key site of modification in suscepti bility of bacteria to class Ha bacteriocins. Mutants of the L. monocytogenes 873 isolate, resistant to the class IIa bacteriocin, leucocin A, were generated at the start of the study to complement the existing array of L. monocytagenes wild-type and resistant isolates obtained from other sources. The fifty percent inhibitory concentrations using a highly sensitive and reproducible bioassay were determined. This allowed categorisation of the mutants into intermediate and highly resistant phenotypes. Analysis of the growth patterns of all these strains showed decreased growth rates and higher growth yields for all the resistant strains in general. This provided evidence for possible effects of membrane adaptation and metabolic changes in the resistant strains and prompted further investigation. The major focus of the study on the class Ha resistant mutants were: (1) analysis of membrane compositional changes and factors influencing cell surface charge; (2) assessment of physical changes in the membrane and bacteriocin itself using circular dichroism and fourier transform infrared spectroscopy; (3) and, determination of changes in glucose metabolism. Electrospray mass spectrometry analysis of the major listerial phospholipid, phosphatidylglycerol, revealed that membranes of resistant strains had increased levels of unsaturated and short-acyl-chain phosphatidylglycerol molecular species, indicating more fluid membranes. In addition, treatment with a desaturase inhibitor resulted in increased sensitivity of only the intermediate resistant strains to the class na bacteriocin, leucocin A. This indicated the influence of membrane adaptation in only lower levels of resistance. It is conceivable that more fluid membranes could also impact on decreased stability of pore formation by the bacteriocin. Complementary biophysical studies using fourier transform infrared spectroscopy indicated the possible occurrence of greater membrane fluidity of resistant cells, by the notable shift in the anti symmetric CH2 stretching vibration from 2921 cm-I to 2922 cm-I. Additionally, circular dichroism revealed a decreased a-helical and increased random structure of leucocin A in the presence of listerial liposomes derived from highly resistant cell membrane extracts. It is possible that this may result in reduced activity of the bacteriocin in resistant cell membranes as a-helical stucture is a critical feature for membrane insertion of cationic antimicrobial peptides. Cell surface charge was determined by quantification of alanine and lysine esterification of the anionic cell surface polymer, teichoic acid, and membrane phospholipids respectively. Increased D-alanine, which causes neutralisation of the cell surface, was observed in all resistant cells. A tendency for greater lysine content in membrane phospholipids, which also impacts on neutralisation of the anionic phospholipid of listerial membranes, was observed in highly resistant strains only. This neutralisation of the negative charge of the cell surface may interfere with initial electrostatic interaction of bacteriocin with the cell, and subsequent interactions required for permeabilisation of the cell membrane. These differences in alanine and lysine esterification were not the result of increased expression of certain associated genes (d/tA and /mo1695) and may be the result of post-transcriptional regulation. It was, however, found that all resistant L. monocytogenes strains, including the intermediate resistant strains, exhibited decreased expression of a putative docking molecule, the mannose-specific phosphotransferase system EIIAB subunit (EIlABMan).A clear correlation existed between the levels of resistance and EIIABMandown-regulation. Finally, analysis of the glucose metabolism in highly resistant and wild-type strains, indicated a more efficient metabolism with regards to higher growth yields and ATP yield, in contrast to a lower specific growth rate in a spontaneous and genetically defined (EIlABMan inactivated) highly resistant mutant. The switch in metabolic end-product observed, was attributed to the loss of the glucose transporter, EIlABMan,and may cast doubts on the feasibility of the use of class Ha bacteriocins as food preservatives in light of a stable and efficient resistant phenotype. / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Thesis (M.S.)--University of North Carolina at Wilmington, 2004. / Includes bibliographical references (leaves : [25]-27).

Bacillus thuringiensis NEB17 is a plant growth promoting rhizobacterium that produces a compound that directly increases plant growth. The compound is a bacteriocin and we propose the name thuricin 17. Thuricin 17 is a novel peptide inhibiting the growth of Bacillus species/strains, displaying both bactericidal and static effects. Its molecular weight, estimated via SDS-PAGE and verified by MALDI-QTOF mass spectroscopy, is 3162 Da. The partial amino acid sequence was determined and is N-term---WTCWSCLVCAACSVELL, C-term-CAS. Heat and pH stability, production and susceptibility to proteolysis were conducted. Thuricin 17 is active in pH 1.00-9.25, stable above 60°C and produced in the late exponential growth phase. This is the first bacteriocin from a Bacillus PGPR and the first reported to increase plant growth. This work presents an original discovery regarding PGPR mechanisms.

Leucocin A is a class 11a bacteriocin produced by Leuconostoc mesenteroides TA33a that was previously shown to inhibit Listeria monocytogenes. A spontaneous resistant mutant of L. monocytogenes was isolated, and found to be resistant to leucocin A at levels in excess of 2 mg/ml. The resistant mutant had an eight-fold increased binding capacity for leucocin A in comparison to the parental strain. The mutant showed no significant cross resistance to nisaplin or ESFI-7GR. The resistant phenotype had a similar growth rate in monoculture, to the sensitive phenotype. DNA and protein analysis of the resistant and susceptible strains were carried out using silver stained amplified fragment length polymorphism (ssAFLP) and one and two dimensional (2D) SDS polyacrylamide gel electrophoresis (PAGE). Two-dimensional SDS PAGE revealed two differences. The first was a 35 kDa protein which was present in the sensitive but absent from the resistant phenotype and, secondly there was a higher level of expression of a 18 kDa protein in the resistant phenotype compared with the sensitive phenotype. The 35 kDa protein was found to have a 83% homology to the mannose-specific phosphotransferase system IIAB of Streptococcus salivarius. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The winemaking process involves a number of microorganisms, each with its own role. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are Gram-positive bacteria associated with must and wine and perform the malolactic fermentation (MLF), while the acetic acid bacteria (AAB) are Gram-negative bacteria converting ethanol to acetic acid. These microorganisms are present in the cellar and fermentation tanks and can be seen either as beneficial or as wine spoilage microorganisms because, under certain circumstances, they affect the wine quality if they should grow in the wine or must. Strict measures need to be implemented in the cellar during the winemaking process to ensure microbiological stability. This can be achieved through good microbiological practices and, additionally, chemical preservatives. Sulphur dioxide (S02) is widely used as the primary preservative in winemaking. However, consumer resistance has been building up against the use of chemical preservatives, due to the possible health risks and a decrease in nutritional value and sensorial quality of certain foods and beverages. Biopreservation as an alternative to the traditionally-used chemical preservation is a new approach and has been attracting much attention. This implies the use of the natural microflora and/or their antibacterial products, such as bacteriocins and bacteriolytic enzymes (e.g. lysozyme). Bacteriocins from LAB are proteins or protein complexes, produced by Gram-positive bacteria, with antibacterial activity against closely-related Gram-positive species. Lysozyme occurs in substances such as hen egg white and has lytic activity against Gram-positive bacteria. ' The bacteriocins nisin, of the class I lantibiotics, and pediocin PA-1 and leucocin BTA 11a, of the class lIa Listeria-active bacteriocins, have been investigated for the biopreservation of wine. Nisin, however, is the only bacteriocin that has been approved for use as a preservative, while pediocin is likely to follow in approval. Lysozyme has been approved for use in winemaking by the Office International de la Vigne et du Vin (OIV). The main objectives of this study were to determine whether these substances showed any antimicrobial action against wine-associated microorganisms, namely LAB, AAB and yeasts. The stability and suitability of the bacteriocins and lysozyme as antimicrobials in wine was researched, especially when used in combination. Possible synergistic or antagonistic interactions between the bacteriocins were also investigated by means of the microtitre broth dilution method and scanning electron microscopy, as well as at what concentration and combinations the bacteriocins were most effective against increasing LAB concentrations. It was found that nisin, pediocin and leucocin were effective to varying degrees against a test panel of LAB type and reference strains, as well as wine isolates. Nisin repeatedly had the highest level of inhibition against all the LAB tested, followed by pediocin and leucocin. There was no inhibition of the wine-associated AAB and yeasts tested. Pediocin stability was evaluated in simulated wine must and proved to be stable for at least 20 days, without being affected by the sulphur or alcohol content. A low pH, however, led to a more rapid decrease in activity. The same was found for nisin and leucocin in other studies. Combinations of bacteriocins at increasing concentrations were evaluated against increasing concentrations of a LAB wine isolate. When used in pairs (namely, nisinleucocin, nisin-pediocin and pediocin-Ieucocin), the combinations were most effective against lower concentrations of bacteria, namely 102 and 104 cfu/ml. At lower concentrations, the pairs of bacteriocins were much less effective against the higher bacterial concentrations of 106 and 108 cfu/ml. Leucocin-pediocin combinations were the least effective, while nisin-Ieucocin combinations were marginally more effective than the nisin-pediocin combinations. The most pronounced effect was observed when all three the bacteriocins were used together. Combinations of bacteriocins had no inhibitory effect against AAB. Pediocin and lysozyme was used in combination against the same wine isolate, but no conclusive conclusions could be drawn in this experiment. __ Scanning electron microscopy was used to investigate any disturbances in cell morphology when bacteriocins were added to LAB. The above-mentioned LAB was subjected to bacteriocins used singularly and also in combinations of equal amounts of bacteriocins. The action of the bacteriocins led to major disturbances in cell morphology. Once again, the combination of leucocin-pediocin was the least effective, even less so than when the single bacteriocins were used. The nisin-pediocin and nisin-Ieucocin combinations seemed to be more effective in causing cell disturbances and perturbations. The microtitre broth dilution methodwas used to further characterise the nature of the interaction of the pairs of bacteriocins. This test showed clearly that the bacteriocins had definite interactions. By adding one bacteriocin to varying concentrations of another bacteriocin, the inhibitory action of the second bacteriocin was affected, either increasing or decreasing its effectiveness. The most important factor to consider seems to be the ratio at which the bacteriocins should be used together, leading either to synergism or antagonism, and this also implies a very complex interaction. This project indicated that it is indeed possible to use both bacteriocins and lysozyme in "Vine preservation, both being stable in wine environments and effective against LAB without affecting the yeast fermentation. Bacteriocins could also be used in combination, to broaden the inhibition spectrum, as well as possibly increasing the inhibitory potential of the individual antimicrobials. The underlying interactions in such combinations should be carefully researched, however, when considering using combinations of antimicrobials in food and beverage products. Further attention can also be given to finding biopreservatives against the Gram-negative AAB, as well as to research the interaction of the pairs of bacteriocins over time. Another point to consider would be the engineering of yeasts or bacteria to produce these antibacterial substances in situ as part of their metabolism. / AFRIKAANSE OPSOMMING: Daar is 'n verskeidenheid mikroorganismes in die wynrnaakproses betrokke, elkeen met sy eie rol. Giste is vir die alkoholiese fermentasie verantwoordelik, die Gram-positiewe melksuurbakterieë (MSB) wat in mos en wyn voorkom, is vir die appelmelksuurgisting (AMG) verantwoordelik, terwyl die Gram-negatiewe asynsuurbakterieë (ASB) etanol in asynsuur omskakel. Hierdie mikroorganismes is in die wynkelder en fermentasietenke teenwoordig en kan as óf gunstig óf ongunstig beskou word, afhangende van die toestande waaronder hulle groei en hoe die wyn daardeur beïnvloed word. Om mikrobiologiese stabiliteit in wyn te verseker, moet daar streng higiëniese maatreëls in die kelder toegepas word en word daar ook van addisionele chemiese preserveermiddels gebruik gemaak. Swaweidioksied (S02) word tans algemeen as pnmere preserveermiddel in die wynbedryf gebruik. Weens die moontlike gesondheidsrisiko's wat S02 mag inhou en die moontlike verlaging van die voedingswaarde en sensoriese gehalte waarmee dit in sommige voedsel- en drankprodukte geassosieer word, bou daar tans verbruikersweerstand teen die gebruik daarvan as chemiese preserveermiddelop. Biopreservering is 'n alternatief tot hedendaagse chemiese preservering en het reeds baie belangstelling ontlok. Hierdie metode impliseer die gebruik van die natuurlike mikroflora en/of die antimikrobiese produkte van hierdie rnikroërqanisrnes, soos bakteriosiene en bakteriolitiese ensieme (bv. lisosiem). Bakteriosiene van MSB is proteïene of proteïenkomplekse met antimikrobiese aktiwiteit teen naby-verwante Grampositiewe spesies. Lisosiem kom in produkte soos hoendereierwit voor en het litiese aktiwiteit teen Gram-positiewe bakterieë. Die bakteriosiene nisien, wat tot die klas I lantibiotiese bakteriosiene behoort, en pediosien PA-1 en leukosien B-TA11a, wat tot die klas lIa Listeria-aktiewe bakteriosiene behoort, is as moontlike biopreserveringsagense in wyn ondersoek. Nisien is egter tot op hede die enigste bakteriosien wat amptelik vir gebruik as 'n preserveermiddel in voedsel goedgekeur is, terwyl pediosien moontlik sal volg. Lisosiem is vir gebruik in wynmaak deur die Office International de la Vigne et du Vin (OIV) goedgekeur. Die hoofdoelwitte van hierdie studie was om te bepaal of die bogenoemde stowwe antimikrobiese werking teen wyngeassosieerde mikroorganismes het, soos die ongewenste MSB, ASB en giste. Die stabiliteit en geskiktheid van dié bakteriosiene en lisosiem as antimikrobiese middels in wyn is ook ondersoek, veral wanneer hulle in kombinasie vir preservering gebruik is. 'n Mikrotiterverdunningsboeljon-metode en skanderingselektronmikroskopie is gebruik om moontlike sinergistiese en antagonistiese interaksies tussen bogenoemde bakteriosienpare te ondersoek. Terselfdertyd is die effektiefste konsentrasies en kombinasies van bakteriosiene teen stygende MSB-getalle bepaal. Daar is bevind dat nisien, pediosien en leukosien in verskillende mates teen 'n toetspaneel van MSB tipe- en verwysingsrasse, asook MSB-wynisolate, effektief is. Nisien was herhaaldelik die effektiefste teen dié MSB, gevolg deur pediosien en dan leukosien. Die bakteriosiene was nie teen die wyngeassosieerde ASB of giste wat getoets is, effektief nie. Daar is ook bewys dat pediosien vir tot 20 dae stabiel in 'n gesimuleerde wynomgewing was, sonder dat die alkohol- of die swaweldioksiedkonsentrasie 'n invloed op die aktiwiteit gehad het nie. 'n Lae pH het geblyk om die grootste invloed op die afname in aktiwiteit te hê. Hierdie bevindinge ten opsigte van pediosien het die resultate van nisien en leukosien in ander, soortgelyke ondersoeke, bevestig. Die werking van toenemende konsentrasies van bakteriosienkombinasies (as pare van nisien-Ieukosien, nisien-pediosien, leukosien-pediosien, en al drie saam as nisienpediosien- Ieukosien) teen toenemende getalle van In wyngeïsoleerde MSB is geëvalueer. Wanneer die bakteriosiene in pare gebruik is, was die kombinasies die effektiefste teen laer MSB selgetalle (102 en 104 kfe/ml), terwyl dit baie minder effektief teen hoër selgetalle (106 en 108 kfe/ml) was, veral wanneer lae bakteriosienkonsentrasies gebruik is. Die nisien-Ieukosien kombinasiewas tot 'n geringe mate meer effektief as die nisien-pediosien kombinasie. Die leukosien-pediosien kombinasie het die laagste effektiwiteit van.al die pare bakteriosiene wat gebruik is, getoon. Die sterkste werking is waargeneem toe al drie die bakteriosiene saam teen bogenoemde MSB gebruik is. Die bakteriosien kombinasies het geen effek teen ASB gehad nie. Pediosien en lisosiem is ook in kombinasie teen dieselfde wynisolaat gebruik, maar geen oortuigende afleidings kon van hierdie eksperiment gemaak word nie. Skanderingselektronmikroskopie is gebruik om enige morfologiese verandering in die MSB-wynisolaat waar te neem wanneer bakteriosiene daarby gevoeg is. Dieselfde wynisolaat is weer gebruik en bakteriosiene is by die bakterieë gevoeg, enkelvoudig asook in kombinasies (soos voorheen gebruik) teen gelyke hoeveelhede. Die werking van die bakteriosien het gelei na merkbare veranderinge in selmorfologie, en die kombinasie van pediosien-Ieukosien was weereens die minste effektief. Die mikrotiterverdunningsboeljon-metode is gebruik om die aard van die bakteriosieninteraksies verder te karakteriseer. Die toetse het duidelik aangedui dat die bakteriosiene op mekaar reageer. Deur een bakteriosien tot variërende konsentrasies van 'n ander bakteriosien te voeg, is die inhibitoriese werking van die tweede bakteriosien geaffekteer deurdat die effektiwiteit daarvan toegeneem of afgeneem het. Dit het ook geblyk dat die belangrikste faktor wat hier in ag geneem moet word die verhouding is waarteen die bakteriosiene met mekaar gebruik word, aangesien dit tot óf sinergisme óf antagonisme kan lei. Dft dui op 'n baie komplekse interaksie. Die resultate van hierdie projek het dus daarop gedui dat dit inderdaad moontlik is om beide bakteriosien en lisosiem in wynpreservering te gebruik, aangesien beide nie net stabiel in 'n wynomgewing is nie, maar ook effektief is teen MSB sonder dat die gisfermentasies geaffekteer word. Bakteriosiene kan ook in kombinasie gebruik word om die inhibisie spektrum te verbreed, en om ook moontlik die inhibisiepotensiaal van die individuele peptiede te verhoog. Onderliggende interaksies by sulke kombinasies moet egter sorgvuldig ondersoek word wanneer daar oorweeg word om kombinasies van hierdie antimikrobiese middels in voedsel- en drankprodukte te gebruik. Verder moet daar ook aandag geskenk word om biopreserveermiddels te vind wat ook teen die Gram-negatiewe ASB effektief is, asook aan die aard van die verloop van interaksies van pare van bakteriosiene oor tyd. Nog 'n punt om te oorweeg is die manipulasie van giste of bakterieë omdie antimikrobiese peptiede in situ, as deel van hulle metabolisme, te produseer.

No description available.

Batches of fromage blanc, a soft white cheese were prepared from whole pasteurized cow's milk. Processing and formulation methods were used in cheese making to reduce Listeria monocytogenes in artificially contaminated cheese. Treatments implemented included use of additional starter culture in formulation (25% more starter culture than original formulation), use of a higher temperature draining process (at 45oC instead of 22oC), addition of the anti-listerial bacteriocin nisin (Danisco Nisaplin) in formulation at different levels (125 ppm, 250 ppm, 400 ppm), and combinations of these treatments. Characteristics including pH, fat content, protein content, and color were evaluated for each treatment cheese. Statistically significant differences (p<0.0001) were found between the population (log CFU/g) values of L. monocytogenes in the different treatment cheeses and control cheese. Treatments using additional starter culture or higher temperature draining alone were not successful in significantly reducing numbers of L. monocytogenes, but when combined, a 1 log reduction resulted. Of the different concentrations of nisin used in cheese formulation, the level of 250 ppm nisin was used in combination treatments. The treatments using 250 ppm nisin were able to reduce numbers of L. monocytogenes by 2 log 24h after addition. Combination treatments with 250 ppm nisin and additional starter culture in formulation reduced the level of L. monocytogenes by only 1 log, while combination treatments coupling 250 ppm nisin with a higher temperature draining and treatments with 250 ppm nisin, additional starter culture, and a higher temperature draining were able to reduce the pathogen by 2 log. There were statistically significant (p<0.0001) differences found between cheese treatments for values of pH, fat content, and protein content. This soft cheese could be standardized for each of these parameters by the processor before packaging and sale of cheese. There were no statistically significant (p>0.05) differences found between colorimetric values for different cheese treatments. / Master of Science

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2007. / Lactic acid bacteria (LAB) are producers of bacteriocins, ribosomally synthesized antimicrobial peptides. Bacteriocins are secreted into the surrounding environment where they inhibit growth of other bacteria competing for the same nutrients in a particular environment, usually closely related strains. Some of the bacteriocin-sensitive bacteria include food spoilers and - pathogens, which makes bacteriocins potential natural food preservatives. The need for more natural preservation techniques in the food industry is high: Consumers prefer ready-toeat, minimally processed foods containing no chemical preservatives, but at the same time food spoilage and food-related illnesses are areas of big concern. The antibacterial and antiviral properties of some bacteriocins have also made them suitable for controlling bacterial infections, e.g. as part of pharmaceutical ointments. The increasing rate of resistance against antibiotics by micro-organisms has created a market for alternative treatments for infections. Commercial bacteriocin manufacturing proceeds in controlled fermentations or by extraction from plant material. Enterococcus mundtii ST4SA produces a bacteriocin, bacST4SA, with properties giving it potential for use as a food preservative or as part of a pharmaceutical product. In this study, production of bacST4SA by fermentation of low-cost food-grade growth media, sugarcane molasses, corn steep liquor (CSL) and cheese whey, was considered to increase the economic viability of production for food application. Furthermore, individual de Man Rogosa and Sharpe (MRS) medium components, pH and fed-batch fermentation were evaluated to improve bacST4SA activity...

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Please refer to fulltext for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Dental caries is the most common bacterial disease of humans and occurs when oral bacteria produce acids, following their fermentation of dietary carbohydrates. This acid can then cause a localised demineralisation of the tooth surface. A group of seven species of bacteria, collectively known as the mutans streptococci, have been predominantly implicated in the onset of dental caries. In particular, Streptococcus mutans and Streptococcus sobrinus have been shown to be the main aetiological agents of this disease in humans. Most attempts to control the microbial component of caries target these bacteria. The past 50 years has provided considerable information about the pathogenesis of dental caries, the likely route and time of transmission of cariogenic bacteria to susceptible hosts and possible ways of either treating or controlling the onset of this disease. In regards to the latter, many techniques (such as the use of tooth brushes, mouth washes, dental floss and tooth paste) for the control of plaque build-up exist and the examples listed are generally part of a daily routine. However, these techniques need to be applied regularly, and as such only highly-motivated individuals generally experience improved oral health. Therefore, the search for more effective less labour-intensive approaches continues. One area of research is into the potential application of small ribosomally-synthesised antimicrobial peptides, known as bacteriocins. Bacteriocins generally inhibit closely-related species that occupy the same ecological niche. Their relatively-specific targeting, plus the fact that many are remarkably heat and chemically-stable molecules, makes them excellent candidates for possible anti-caries applications. Numerous bacteriocins produced by the lactic acid bacteria have now been identified. Most can be broadly categorised into one of four main classes, of which Class I, the lantibiotics and Class II, the small (<10 kDa) non-modified peptides, contain the most examples. Many screens for anti-mutans streptococcal (MS) bacteriocins have been carried out and it appears that the best source of anti-MS bacteriocins are the mutans streptococci themselves. Research in this laboratory has identified examples of anti-mutans streptococcal bacteriocins produced by both mutans streptococci and non-mutans streptococci. The present study investigated the anti-MS inhibitors produced by two streptococcal strains, S. mutans N and Streptococcus sanguis K11. During the course of this study a third strain, S. mutans UA159, was also studied for its bacteriocinogenic properties. Although S. sanguis K11 produces anti-mutans streptococcal inhibitory activity, this appears only effective against Streptococcus rattus. In addition however, the inhibitory activity of this strain is also directed against all tested strains of Streptococcus agalactiae and ca. 50% of Streptococcus pyogenes. In the present study a 5069 Da novel inhibitory agent (sanguicin K11) was characterised and shown responsible for this unusual inhibitory spectrum. Through reverse genetics the sanK11 locus was identified and shown to encode a Class II type bacteriocin, the first shown to be produced by S. sanguis. Following screens of additional S. sanguis, sanK11 was shown to be present only in strains producing the same type of inhibitory pattern (P-type) as strain K11. The cysteine residues at positions 7 and 38 of the sanguicin K11 propeptide were shown to form a disulphide bridge essential for sanguicin K11 inhibitory activity. S. mutans N and eight other S. mutans strains have been found to have what appears to be the same inhibitory spectrum, which includes members of the mutans streptococci and several other oral streptococcal species. One strain (UA140) of the eight has previously been shown to produce the lantibiotic mutacin I and the non-lantibiotic mutacin IV. S. mutans N was known to produce the non-lantibiotic mutacin N. The current study set out to investigate how two strains, apparently producing completely different bacteriocins could have the same inhibitory spectrum. Reverse genetics identified the mutacin N structural gene (mutN) and mutagenesis studies showed that this bacteriocin was responsible only for the inhibitory activity against mutans streptococci. Further sequencing around the mutN locus identified a second bacteriocin-like locus (mutO) adjacent to mutN. mutO was also identified to have anti-mutans streptococcal inhibitory activity and because of the close proximity of mutO and mutN and given the homology they share with other known two-peptide bacteriocins it seemed probable that mutacins O and N are components of a new member of this special class of bacteriocins (Class IIb, the two peptide bacteriocins) in which the optimal inhibitory activity is dependent on the co-operative activity of the two peptides. Further investigations of strain N examined the expression of mutacins O and N. During a search for a suitable heterologous non-mutacinogenic S. mutans strain to act as an expression host, the genome reference strain, S. mutans UA159 was given consideration. However, contrary to previous reports, this strain was found to exhibit bacteriocin-like inhibitory activity. During a follow-up investigation, strain UA159 was found to inhibit 84 strains representing 11 different species of bacteria, but no inhibition of mutans streptococci was detected. The locus (nlmAB) encoding the two-peptide bacteriocin mutacin IV was identified within the UA159 genome. Using genetic dissection of nlmA and nlmB, the contribution of each peptide was examined and it was found that only the NlmA* propeptide appears to be active, raising doubts as to whether mutacin IV is a bona fide two-peptide bacteriocin. Deletion of the entire nlmAB locus created a mutant strain that exhibited a loss of inhibitory activity against the same 64 strains as was found for the nlmA mutant. A BLASTP search for the consensus leader sequence that precedes the propeptide of Class II bacteriocins, identified ORFs encoding 9 more putative bacteriocin-like peptides. Further genetic dissection identified the SMU.1914c locus as being responsible for the inhibitory activity against a further 15 strains not already sensitive to mutacin IV. SMU.1914c was renamed mutacin V. However, it appears that another as yet unidentified mutacin(s) is also produced by strain UA159 given that three indicator strains still remained sensitive to a double mutant [UA[Delta](1914/NlmAB)] in which both the mutacin IV and putative mutacin V loci were inactivated. Export of Class II bacteriocins has been found to occur by either a SEC-dependent system or via a dedicated peptide ATP Binding Cassette (ABC) transporter. Three potential ABC transporter ORFs were identified in S. mutans UA159. Two (comA and cslA) had the characteristic accessory factor ORF (comB and cslB respectively) located adjacent to the main ABC transporter ORF, while the third ORF763 appeared to lack this. Mutagenesis of each of these five ORFS was carried out and confirmed cslAB to be the ABC transporter involved in the export of the competence stimulating factor, while the function of ORF763 could not be established in this study. Mutagenesis of either comA or comB resulted in a complete cessation of bacteriocin production by the respective mutant strains. Historically, comA and comB is the nomenclature used for loci encoding the exporter of the competence inducing factors in streptococci. In light of this new information, comA and comB were renamed nlmT and nlmE respectively, to account for the newly defined role of this ABC transporter. The present study investigated four bacteriocins two of which (sanguicin K11 and mutacin ON) appear to have some potential for application to anti-caries control, and the others (mutacins IV and V) being shown to be produced by the genome reference strain (UA159). All three mutacins were shown to be exported from their respective producer cells by the NlmTE ABC transporter, while sanguicin K11 is predicted to be exported by a peptide ABC transporter located adjacent to sanK11. Bacteriocins may yet provide a novel alternative for the treatment and control of dental caries. In their favour is that fact that they have relatively narrow defined inhibitory spectra and thus are unlikely to produce widespread changes to plaque ecosystems. Potential uses include as topical agents where bacteriocin preparations could be incorporated into dentrifices such as toothpastes or mouthwashes. Alternatively, streptococci producing anti-mutans streptococcal bacteriocins could be implanted into the oral cavity in strain replacement therapy strategies. There are pros and cons to each technique and the most effective anti-caries control appears more likely to result from "cocktail therapy" where bacteriocins are combined with a number of other anti-mutans streptococcal agents to achieve long-lasting protection against mutans streptococcus proliferation.