No description available.

No description available.

Mutacin 1140, a class 1 bacteriocin, is produced by Streptococcus mutans and belongs to the type A lantibiotic family. Experiments were done to optimize production of mutacin 1140 in minimal media enabling a more cost efficient downstream purification method. The development of a small volume fermentation method enabled a rapid screen of several variables in a standard shaking incubator. This method provided a fast approach for determining components that promote mutacin 1140 production in minimal media broth. Lactose was determined to be the optimal carbon source for mutacin 1140 production. High concentrations of CaCl2 (0.3% w/v) and MgSO4 (0.77% w/v) promoted an increase in mutacin 1140 production, while ZnCl2 and FeCl3 appeared to impair production. Optimization of mutacin 1140 production in minimal media resulted in more than a 100old increase in production compared to the base medium used to begin our optimizations. The yield has been estimated by RP-HPLC to be 10 mg/L.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Antimicrobial resistance is one of the major problems faced by the medical industry today. The ability of bacteria to rapidly acquire resistance against antibiotics and the over prescription and inappropriate use of antibiotics further exacerbate this crisis. Few new antimicrobials are, however, making it through the drug discovery pipeline. The search and development of novel and effective antimicrobials is therefore of the utmost importance. Lantibiotics are ribosomally synthesized cationic antimicrobial peptides with extensive post-translational modifications. They are active against a wide range of Gram-positive bacteria, including antibiotic-resistant strains. They are characterized by the presence of lanthionine and methyllanthionine rings and have been suggested as alternatives or for use in conjunction with antibiotics against resistant pathogens. Staphylococcus aureus is the most common bacteria isolated from skin and soft tissue infections (SSTIs). Strains of S. aureus have emerged with resistance against antibiotics with the most common being methicillin-resistant S. aureus (MRSA). Several lantibiotics are active against MRSA in vivo and have even shown superior activity to traditional antibiotics. Lantibiotics therefore show much promise for the treatment of SSTIs caused by resistant- and non-resistant S. aureus. In this study the bacterially diverse soil of the Fynbos in the Western Cape was screened for novel antimicrobials. Two antimicrobial producing Bacillus strains were isolated, Bacillus clausii AD1 and Bacillus amyloliquefaciens AD2. Both of these strains produce lantibiotics with B. clausii AD1 producing a known lantibiotic, clausin. B. amyloliquefaciens AD2 produces a novel two-component lantibiotic which was designated amyloliquecidin. The lantibiotic operon of amyloliquecidin was sequenced and annotated. All the genes required for successful production of amyloliquecidin are present in the operon. Amyloliquecidin was characterized in vitro and along with clausin is active against clinical strains of S. aureus (including MRSA), Enterococcus spp., Listeria spp. and beta-haemolytic streptococci. Amyloliquecidin has remarkable stability at physiological pH compared to nisin and clausin. A comparative in vivo murine infection model was used to evaluate the effectiveness of amyloliquecidin, nisin, clausin and Bactroban (commercial S. aureus topical treatment) in treating wound infections caused by S. aureus. All the lantibiotics proved to be just as effective as the Bactroban treatment. Furthermore, the tested lantibiotics did not have a negative influence on the wound closure rates of infected and non-infected wounds. Bactroban had a negative effect on wound healing compared to the lantibiotics. To our knowledge amyloliquecidin is the third two-component lantibiotic isolated from Bacillus. This study represents the first to test the effectiveness of amyloliquecidin in vivo and is one of a handful to test lantibiotics as topical treatments. / AFRIKAANSE OPSOMMING: Antimikrobiese weerstandbiedende bakterieë is op die oomblik een van die grootste probleme in die mediese veld. Die antibiotika krisis word vererg deur die vermoë van bakterieë om vinnig weerstand op te bou teen antibiotika, asook die alledaagse misbruik van antibiotika. Daar is ook ʼn tekort in die hoeveelheid antibiotika wat na die finale fases van ontwikkeling gaan. Om die oorhand teen antibiotika-weerstandige bakterieë te kry is dit van uiterste belang dat meer effektiewe antibiotika ontdek word. Lantibiotika is kationiese antimikrobiese peptiede wat deur die ribosoom gesintetiseer word en bevat ʼn verskeidenheid van modifikasies wat na translasie ingebou word. Hulle word gekarakteriseer deur lanthionien en metiellanthionien ringe. Lantibiotika is aktief teen ʼn verskeidenheid Gram-positiewe bakterieë en kan in kombinasie met antibiotika, of as alternatief gebruik word. Staphylococcus aureus is die mees algemene bakterium wat geassosieer word met vel en sagte weefsel infeksies (VSWIs). Staphylococcus aureus met weerstand teen antibiotika is ook al geïsoleer, die mees algemene weerstandige ras is methisillien-weerstandige S. aureus (MWSA). Lantibiotika is wel aktief teen MWSA in vitro en in vivo, met van hulle wat tot beter aktiwiteit as die voorgeskrewe antibiotika het. Lantibiotika kan dus gebruik word as behandeling vir VSWIs wat veroorsaak word deur weerstandige S. aureus, asook teen nie-weerstandige rasse. In hierdie studie was die bakteriese diverse grond van die Fynbos in die Wes-kaap ondersoek vir bakterieë wat antimikrobiese middels produseer. Twee Bacillus rasse, Bacillus clausii AD1 en Bacillus amyloliquefaciens AD2, wat antimikrobiese middels produseer, is geïsoleer. Bacillus clausii AD1 produseer ʼn bekende lantibiotikum, naamlik clausin. Bacillus amyloliquefaciens AD2 produseer ʼn nuwe twee-komponent lantibiotikum, amyloliquecidin. Die lantibiotikum operon wat verantwoordelik is vir die produksie van amyloliquecidin is geïdentifiseer en geannoteer. Die operon bevat al die gene benodig vir die biosintese van amyloliquecidin. Amyloliquecidin is in vitro gekarakteriseer en het aktiwiteit teen ʼn verskeidenheid Gram-positiewe bakterieë. Amyloliquecidin en clausin is aktief teen S. aureus (insluitend MWSA), Enterococcus spp., Listeria spp. en beta-hemolitiese streptococci wat vanaf infeksies geïsoleer is. Amyloliquecidin is baie stabiel by filologiese pH en aansienlik meer stabiel as nisin en clausin. Die effektiwiteit van nisin, clausin en amyloliquecidin in die behandeling van muis vel infeksies veroorsaak deur S. aureus was vergelyk met die kommersiële behandeling Bactroban. Al drie lantibiotika het die verspreiding van S. aureus met die selfde effektiwiteit as Bactroban belemmer. Geen van die lantibiotika het ʼn negatiewe effek op wond genesing nie. Bactroban, inteendeel, belemmer wond genesing. So ver ons weet is amyloliquecidin die derde twee-komponent lantibiotikum wat uit Bacillus geïsoleer is. Die studie is ook die eerste om die effektiwiteit van amyloliquecidin in vivo te rapporteer, asook ook een van die min studies wat kyk na lantibiotika as behandeling vir topikale infeksies.

La clausine est une bactériocine sécrétée par la souche probiotique Bacillus clausii. Elle est active contre des bactéries multi-résistantes aux antibiotiques, comme le MRSA (Staphylococcus aureus résistant à la méticilline) et le VRE (Enterococcus résistant à la Vancomycine). Nous avons produit, purifié, caractérisé la structure de la clausine et effectué des expériences d'interaction par Résonance Magnétique Nucléaire (RMN) du liquide. La clausine s'est révélée être un nouveau peptide maturé de la famille des lantibiotiques. Il est composé de trois ponts thioéthers ou lanthionines, d'un pont aminovinyle et de trois résidus deshydratés. La clausine possède une structure tri-dimensionnelle bi-modulaire, similaire à la fois à la nisine et à la mersacidine. Comme ces dernières, elle s'est révélée se lier aux lipides bactoprénols, tels que le lipide II (LII) et l'UndécaprénylPyroPhosphate (UPP), empêchant ainsi la formation de la paroi bactérienne. Nous nous sommes ensuite focalisés sur le mode d'action de la clausine sur des modèles de membranes bactériennes. Dans ce but, nous nous sommes basés sur la composition phospholipidique de trois bactéries affectées par la clausine : Staphylococcus aureus, Micrococcus Luteus et Bacillus Megaterium. Nous avons utilisé la RMN du solide du 2H et du 31P pour caractériser l'effet de la clausine sur la dynamique membranaire, ainsi que l'éllipsométrie et l'épifuorescence avec le LII-dansylé pour visualiser son effet sur des monocouches lipidiques. La clausine seule a entraîné une désorganisation membranaire à fort ratio peptide/lipide et formé des domaines. Nous avons souligné l'importance de la tête polaire des bactoprénols sur la dynamique lipidique. Nous avons finalement observé un effet synergique de la clausine avec les bactoprénols, suggèrant la formation de domaines « raft-like » organisés en réseau avec l'UPP ou en fibre avec lelipide II. Nos résultats in vitro permettent d'expliquer les divisions cellulaires aberrantes observées in vivo par un mécanisme de séquestration des bactoprénols par les peptides lantibiotiques. / Clausin is a bacteriocin secreted by the probiotic strain Bacillus clausii. It is active against multi-resistant bacteria to antibiotics, such as MRSA (Methicillin-Resistant Staphylococcus Aureus) and VRE (Vancomycin-Resistant Enterococcus).We have produced, purified, characterized the structure of Clausin and performed binding studies with Liquid-state Nuclear Magnetic Resonance (NMR). Clausin has been shown to be a new mature peptide from the lantibiotic family. It consists of three thioether rings called lanthionines, an aminovinyl ring and three dehydrated residues. Clausin displays a bi-modular three-dimensional structure similar to nisin as well as to mersacidin. It binds as well to bactoprenol lipids, such asLipid II (LII) and UndecaprenylPyroPhosphate (UPP), blocking then bacterial cell wall formation.We have then focused our attention to the action mode of Clausin on bacterial membrane models. For this purpose, we have used the phospholipid composition of three bacteria affected by Clausin: Staphylococcus aureus, Micrococcus Luteus and Bacillus Megaterium. We have used 2H and 31P Solid-state NMR for characterizing the Clausin effect on lipid dynamics, as well as ellipsometry and epifluorescence with dansylated-LII for visualizing its effect on lipid monolayer. Clausin alone has led to membrane desorganization at a high peptide/lipid ratio and formed domains. We have further underlined the importance of the polar head-group of bactoprenols on lipid dynamics. We have finally observed a synergic effect of Clausin with bactoprenols on lipid dynamics suggesting the formation of « raft-like » domains organized in network with UPP or evenfiber with LII. Our in vitro results allow us to explain the aberrant bacterial cell division observed in vivo through a mechanism of bactoprenol sequestration by lantibiotic peptides.

Nisina é um peptídeo antimicrobiano natural produzido por Lactococcus lactis subsp. lactis ATCC 11454 durante a fase exponencial de crescimento. A bacteriocina é usada como conservante natural de alimentos, uma vez que mostra atividade antimicrobiana contra bactérias Gram-positivas e esporos. Tem potencial aplicação em inúmeros campos (farmacêutico, veterinário e cosméticos). O objetivo deste trabalho foi estudar a cinética de crescimento bacteriano e a produção de nisina em biorreator, utilizando leite desnatado diluído, como um meio de cultura a baixo custo. Também foram avaliados os consumos de açúcar e proteína, formação de ácido lático e adsorção de nisina nas células produtoras durante os processos de produção de nisina. Pré-cultivos com 107 UFC.mL-1 de Lactococcus lactis foram cultivados em biorreator de 2 L contendo 25% de leite desnatado diluído em água (1,5 L, pH 6,7). Os ensaios foram esenvolvidos a 30°C por 52 horas, variando a agitação e aeração: (i) 200 rpm (0,0, 0,5, 1,0 e 2,0 L.min-1) e (ii) 100 rpm (0,0 e 0,5 L.min-1). A atividade de nisina foi avaliada pelo método de difusão em ágar, utilizando Lactobacillus sakei ATCC 15521 como microrganismo sensível à ação de nisina. A melhor concentração de nisina (62,68 mg.L-1 ou 2511,89 AU.mL-1), foi obtida em 16 horas, 200 rpm e sem aeração (kLa = 5,29 x 10-3 h-1). A adsorção de nisina nas células produtoras foram baixas (6,8 - 15,1%), quando comparadas com a atividade do sobrenadante. Estes resultados mostraram que o meio de cultivo composto por leite desnatado diluído favoreceu o crescimento celular e produção associada ao crescimento da nisina. Foram realizados estudos preliminares de liofilização (bioconservação) e purificação por cromatografia da nisina produzida em biorreator. A liofilização apresentou perda da atividade de nisina (24,8%), enquanto a purificação por cromatografia de interação hidrofóbica com resina Butyl-Sepharose, recuperou 40% da atividade da biomolécula, mostrando que ambos os processos poderão ser aplicados à bacteriocina. / Nisin is a natural antimicrobial peptide produced by Lactococcus lactis subsp. lactis ATCC 11454 during its exponential growth phase. The bacteriocin is used as natural food preservative due to its antimicrobial activity against Gram-positive bacteria and outgrowth of spores. This property allows its application in numerous fields (pharmaceutical, veterinary and cosmetic). The aim of this work was to study the bacterial growth kinetics of L. lactis and respective nisin production in bioreactor, using diluted skimmed milk as an inexpensive medium. During the production, the consumption of sugar and protein, lactic acid formation and nisin adsorption on the producer strain cells were evaluated. Pre-cultivation with 107 UFC.mL-1 of L. lactis were expanded in a 2 L bioreactor containing 25% diluted skimmed milk in water (1.5 L, pH 6.7). The assays were performed at 30°C for 52 hours, varying agitation and airflow rate: (i) 200 rpm (0.0, 0.5, 1.0 and 2.0 L.min-1) and (ii) 100 rpm (0.0, 0.5 L.min-1). Nisin activity was evaluated through diffusion assays using Lactobacillus sakei ATCC 15521 as sensitive strain. The best nisin concentration (62.68 mg.L-1 or 2511.89 AU.mL-1), was achieved at 16 hours, 200 rpm and with no airflow rate (kLa = 5.29 x 10-3 h-1). The quantity of nisin adsorbed by the producer cells were low (6.8 -15.1%) when compared to the quantity released in the supernatant. These results showed that diluted skimmed milk supported cell growth and growth-associated nisin. Preliminary assays of lyophilization (biopreservation) and purification by chromatography of nisin produced in bioreactor were performed. Lyophilization presented a loss of nisin activity (24.8%) while purification by hydrophobic interaction chromatography with Butyl-Sepharose column recovered 40% of the activity, showing that both processes can be applied to the bacteriocin.

Nisina é um peptídeo antimicrobiano natural produzido por Lactococcus lactis subsp. lactis ATCC 11454 durante a fase exponencial de crescimento. A bacteriocina é usada como conservante natural de alimentos, uma vez que mostra atividade antimicrobiana contra bactérias Gram-positivas e esporos. Tem potencial aplicação em inúmeros campos (farmacêutico, veterinário e cosméticos). O objetivo deste trabalho foi estudar a cinética de crescimento bacteriano e a produção de nisina em biorreator, utilizando leite desnatado diluído, como um meio de cultura a baixo custo. Também foram avaliados os consumos de açúcar e proteína, formação de ácido lático e adsorção de nisina nas células produtoras durante os processos de produção de nisina. Pré-cultivos com 107 UFC.mL-1 de Lactococcus lactis foram cultivados em biorreator de 2 L contendo 25% de leite desnatado diluído em água (1,5 L, pH 6,7). Os ensaios foram esenvolvidos a 30°C por 52 horas, variando a agitação e aeração: (i) 200 rpm (0,0, 0,5, 1,0 e 2,0 L.min-1) e (ii) 100 rpm (0,0 e 0,5 L.min-1). A atividade de nisina foi avaliada pelo método de difusão em ágar, utilizando Lactobacillus sakei ATCC 15521 como microrganismo sensível à ação de nisina. A melhor concentração de nisina (62,68 mg.L-1 ou 2511,89 AU.mL-1), foi obtida em 16 horas, 200 rpm e sem aeração (kLa = 5,29 x 10-3 h-1). A adsorção de nisina nas células produtoras foram baixas (6,8 - 15,1%), quando comparadas com a atividade do sobrenadante. Estes resultados mostraram que o meio de cultivo composto por leite desnatado diluído favoreceu o crescimento celular e produção associada ao crescimento da nisina. Foram realizados estudos preliminares de liofilização (bioconservação) e purificação por cromatografia da nisina produzida em biorreator. A liofilização apresentou perda da atividade de nisina (24,8%), enquanto a purificação por cromatografia de interação hidrofóbica com resina Butyl-Sepharose, recuperou 40% da atividade da biomolécula, mostrando que ambos os processos poderão ser aplicados à bacteriocina. / Nisin is a natural antimicrobial peptide produced by Lactococcus lactis subsp. lactis ATCC 11454 during its exponential growth phase. The bacteriocin is used as natural food preservative due to its antimicrobial activity against Gram-positive bacteria and outgrowth of spores. This property allows its application in numerous fields (pharmaceutical, veterinary and cosmetic). The aim of this work was to study the bacterial growth kinetics of L. lactis and respective nisin production in bioreactor, using diluted skimmed milk as an inexpensive medium. During the production, the consumption of sugar and protein, lactic acid formation and nisin adsorption on the producer strain cells were evaluated. Pre-cultivation with 107 UFC.mL-1 of L. lactis were expanded in a 2 L bioreactor containing 25% diluted skimmed milk in water (1.5 L, pH 6.7). The assays were performed at 30°C for 52 hours, varying agitation and airflow rate: (i) 200 rpm (0.0, 0.5, 1.0 and 2.0 L.min-1) and (ii) 100 rpm (0.0, 0.5 L.min-1). Nisin activity was evaluated through diffusion assays using Lactobacillus sakei ATCC 15521 as sensitive strain. The best nisin concentration (62.68 mg.L-1 or 2511.89 AU.mL-1), was achieved at 16 hours, 200 rpm and with no airflow rate (kLa = 5.29 x 10-3 h-1). The quantity of nisin adsorbed by the producer cells were low (6.8 -15.1%) when compared to the quantity released in the supernatant. These results showed that diluted skimmed milk supported cell growth and growth-associated nisin. Preliminary assays of lyophilization (biopreservation) and purification by chromatography of nisin produced in bioreactor were performed. Lyophilization presented a loss of nisin activity (24.8%) while purification by hydrophobic interaction chromatography with Butyl-Sepharose column recovered 40% of the activity, showing that both processes can be applied to the bacteriocin.

Chez Bacillus subtilis nous avons identifié un complexe protéique membranaire impliquant une protéine inconnue, YteJ, et une autre protéine membranaire, SppA, une signal peptide peptidase également impliquée dans la résistance aux peptides antibactériens de la famille des lantibiotiques. Après délétion des gènes correspondant, nous avons montré que les deux protéines sont impliquées dans cette résistance. Dans la souche ΔsppA, la surexpression ectopique de SppA a non seulement restauré la résistance, mais elle a également induit la formation de cellules allongées, un phénotype supprimé par la surexpression simultanée de YteJ. L'expression de versions tronquées de YteJ a mis en évidence le rôle inhibiteur d'un domaine spécifique de YteJ. Enfin, des études biochimiques in vitro ont confirmé que l'activité de la protéase SppA était fortement réduite par la présence de YteJ, confirmant l'hypothèse d'une inhibition par YteJ. Nos études in vivo et in vitro ont montré que YteJ, via l'un de ses domaines, agit comme régulateur négatif de l'activité protéase de SppA dans ce complexe. En conclusion, nous avons montré que le complexe SppA/YteJ est impliqué dans la résistance aux lantibiotiques à travers l’activité protéase de SppA, elle-même régulée par YteJ. / We have identified a membrane protein complex of Bacillus subtilis involving an unknown protein, YteJ, and SppA, a membrane protein first described as a signal peptide peptidase and later shown to be also involved in the resistance to antibacterial peptides of the lantibiotic family. Using deletion mutant strains, we showed that both proteins are involved in this resistance. In the ΔsppA strain, the ectopic overexpression of SppA not only restored the resistance, it also induced the formation of elongated cells, a phenotype suppressed by the simultaneous overexpression of YteJ. Furthermore, the expression of truncated versions of YteJ pinpointed the inhibitory role of a specific domain of YteJ. Finally, in vitro biochemical studies showed that SppA protease activity was strongly reduced by the presence of YteJ, supporting the hypothesis of an inhibition by YteJ. Our in vivo and in vitro studies showed that YteJ, via one of its domain, acts as a negative regulator of the protease activity of SppA in this complex. In conclusion, we have shown that SppA/YteJ complex is involved in lantibiotic resistance through the protease activity of SppA, which is regulated by YteJ.

Background: Subtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot. Results: In this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed “online” reporter strains to efficiently monitor the dynamics of subtilin biosynthesis. Conclusions: Within this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.

No description available.