Browsing by Author "Vermeulen, Ross Rayne"
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- ItemHeterologous expression of the class IIa bacteriocins, plantaricin 423 and mundticin ST4SA, in escherichia coli using green fluorescent protein as a fusion partner(Frontiers Media, 2020) Vermeulen, Ross Rayne; Du Preez Van Staden, Anton; Dicks, Leon Milner Theodore, 1961-The antilisterial class IIa bacteriocins, plantaricin 423 and mundticin ST4SA, have previously been purified from the cell-free supernatants of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, respectively. Here, we present the fusions of mature plantaricin 423 and mundticin ST4SA to His-tagged green fluorescent protein (GFP) for respective heterologous expression in Escherichia coli. Fusion of plantaricin 423 and mundticin ST4SA to His-tagged GFP produced the fusion proteins GFP-PlaX and GFP-MunX, respectively. Both fusion proteins were autofluorescent, circumvented inclusion body formation and lowered the toxicity of class IIa bacteriocins during heterologous expression. Not only did GFP-class IIa fusion stabilize heterologous expression and boost yields, the fluorescent intensity of GFP-PlaX and GFP-MunX could be monitored quantitatively and qualitatively throughout expression and purification. This robust fluorometric property allowed rapid optimization of conditions for expression and bacteriocin liberation from GFP via the incorporated WELQut protease cleavage sequence. Incubation temperature and IPTG concentration had a significant effect on bacteriocin yield, and was optimal at 18°C and 0.1–0.2 mM, respectively. GFP-MunX was approximately produced at a yield of 153.30 mg/L culture which resulted in 12.4 mg/L active mundticin ST4SA after liberation and HPLC purification. While GFP-PlaX was produced at a yield of 121.29 mg/L culture, evidence suggests heterologous expression resulted in conformation isomers of WELQut liberated plantaricin 423.
- ItemInvestigating the mode of transcriptional regulation controlling plantaricin 423 expression in Lactobacillus plantarum 423(Stellenbosch : Stellenbosch University, 2019-03) Vermeulen, Ross Rayne; Dicks, Leon Milner Theodore; Rohwer, Johann; Van Staden, Anton du Preez; Deane, Shelly; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The discovery and use of antibiotic therapies was one of the most significant achievements of the twentieth century. However, the current rate at which antibiotic resistance develops heavily outweighs the rate that novel treatments are introduced. Without a doubt, antibiotic resistance is one of the biggest challenges scientists of the twenty-first century are facing. With a global realization of finite resource availability and continuous climate change, the transition toward developing more sustainable systems is becoming a focal point for science, technology, and society as a whole. Such an approach also must be applied in the fight against antibiotic resistance and is already being observed through the emergence of novel fields such as biotherapeutics. Antibiotics have only recently been employed as a therapy, yet antimicrobial compounds and mechanisms for resistance have existed for millennia. Therefore, systems must already exist to ensure the sustainability of an antimicrobial compound’s use by a microorganism within its environment. Such systems are likely to use a diverse array of approaches within a microbiota, with the chemical diversity of antimicrobials being one. While researching the role and effect of antimicrobials within a microbiota may elucidate new approaches and schemes to manage antimicrobial resistance, a diverse group of antimicrobials, known as bacteriocins, has already been discovered. In the past, these antimicrobial peptides have received considerably less attention than antibiotics, however due to the urgent need for alternatives they warrant serious consideration. This study concerns the native transcriptional regulation of a subclass IIa bacteriocin, plantaricin 423, produced by Lactobacillus plantarum 423, one of the strains in the probiotic EntiroTM developed by our research group. The mode of transcriptional regulation for class IIa bacteriocins in the absence of local regulatory genes, as observed for plantaricin 423, is unknown. Through the development of a fluorescent promoter-reporter system, it was observed that the transcriptional regulation of plantaricin 423 responded to manganese-limiting conditions. During this research, significant progress was made for methods concerning bacteriocin classification, heterologous expression, and real-time in vivo transcriptional monitoring. Based on findings obtained using a fluorescent promoter-reporter system and the fact that L. plantarum 423 requires high intracellular Manganese concentrations for aerobic respiration, plantaricin 423 might aid in Manganese acquisition from target cells via cell wall poration. This research represents the first steps towards understanding how L. plantarum 423 and Enterococcus mundtii ST4SA, the other strain in the EntiroTM, interact with each other, the gastrointestinal microbiota and the host. Future research in this direction will be done with the hope of discovering sustainable alternatives to current problems, such as antibiotic resistance.