Publicação
Development of an improved method for the detection of Rhodococcus erythropolis based on specific viral proteins
| Resumo: | Rhodococcus erythropolis (R. erythropolis) is a Gram-positive bacterium that can be found widely in nature, especially in polluted areas. It is well-known for its ability to degrade both organic and inorganic pollutants. This makes it an important member of the biological consortium in biological wastewater treatment systems, including activated sludge (AS) systems. However, due to its hydrophobic nature, its overabundance can contribute to AS foaming, which is one of the most significant solid-liquid separation issues in AS systems. AS foaming incidents often lead to foam being discharged with treated effluent, which lowers effluent quality, poses safety risks, and raises concerns about surface water quality and water scarcity. Identifying R. erythropolis is crucial for developing strategies to manage its excessive growth. Current identification methods have limitations, including low sensitivity, modest specificity, and being resource-intensive and time-consuming. A promising solution to this challenge is the utilization of bacteriophage-encoded proteins, particularly endolysins, as innovative bacterial detection systems. The cell binding domain (CBD) of endolysins imparts high affinity and specificity, guiding the endolysin catalytic domain (CD) to its peptidoglycan substrate. Exogenously added, recombinant endolysins can effectively control bacterial growth, and when fused with a reporter as the Green Fluorescent Protein (GFP), they become a reliable detection method. In this study, two endolysin-encoding genes, g114 and g74, from two different phages, were identified and expressed as recombinant proteins (Gp114 and Gp74) in E. coli. However, they were insoluble, showing no lytic activity. To assess the binding efficiency of the CBD and the contribution of the CD, truncations (g114@568, g74@388, and g74@1048) and full-length g114 were expressed in E. coli fused with GFP. All four GFP-fusion proteins exhibited binding to R. erythropolis cells. Enhanced binding was achieved by pre-treating the cells with 1% Triton X-100 for 1 hour at 28°C and 250 rpm before binding. This optimized condition highlighted GFP-gp74@388 as a promising candidate for a novel R. erythropolis detection method in AS foam samples. Nevertheless, additional optimization is imperative to enhance the yield of soluble recombinant endolysins and their binding to bacterial cells, ensuring their suitability for large-scale applications |
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| Autores principais: | Ferreira, Alda de Jesus Gonçalves |
| Assunto: | Rhodococcus erythropolis Activated sludge (AS) Activated sludge foaming Endolysins Green Fluorescent Protein (GFP) Triton X-100 Lamas ativadas Espuma em lamas ativadas Endolisinas Engenharia e Tecnologia |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Rhodococcus erythropolis (R. erythropolis) is a Gram-positive bacterium that can be found widely in nature, especially in polluted areas. It is well-known for its ability to degrade both organic and inorganic pollutants. This makes it an important member of the biological consortium in biological wastewater treatment systems, including activated sludge (AS) systems. However, due to its hydrophobic nature, its overabundance can contribute to AS foaming, which is one of the most significant solid-liquid separation issues in AS systems. AS foaming incidents often lead to foam being discharged with treated effluent, which lowers effluent quality, poses safety risks, and raises concerns about surface water quality and water scarcity. Identifying R. erythropolis is crucial for developing strategies to manage its excessive growth. Current identification methods have limitations, including low sensitivity, modest specificity, and being resource-intensive and time-consuming. A promising solution to this challenge is the utilization of bacteriophage-encoded proteins, particularly endolysins, as innovative bacterial detection systems. The cell binding domain (CBD) of endolysins imparts high affinity and specificity, guiding the endolysin catalytic domain (CD) to its peptidoglycan substrate. Exogenously added, recombinant endolysins can effectively control bacterial growth, and when fused with a reporter as the Green Fluorescent Protein (GFP), they become a reliable detection method. In this study, two endolysin-encoding genes, g114 and g74, from two different phages, were identified and expressed as recombinant proteins (Gp114 and Gp74) in E. coli. However, they were insoluble, showing no lytic activity. To assess the binding efficiency of the CBD and the contribution of the CD, truncations (g114@568, g74@388, and g74@1048) and full-length g114 were expressed in E. coli fused with GFP. All four GFP-fusion proteins exhibited binding to R. erythropolis cells. Enhanced binding was achieved by pre-treating the cells with 1% Triton X-100 for 1 hour at 28°C and 250 rpm before binding. This optimized condition highlighted GFP-gp74@388 as a promising candidate for a novel R. erythropolis detection method in AS foam samples. Nevertheless, additional optimization is imperative to enhance the yield of soluble recombinant endolysins and their binding to bacterial cells, ensuring their suitability for large-scale applications |
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