Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis

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dc.contributor.advisorCoyne, Vernonen_ZA
dc.contributor.authorGildenhuys, Carinen_ZA
dc.date.accessioned2014-07-30T17:37:43Z
dc.date.available2014-07-30T17:37:43Z
dc.date.issued2014-07-30
dc.descriptionIncludes abstract
dc.descriptionIncludes bibliographical references
dc.description.abstractGracilaria gracilis that grows naturally at Saldanha Bay, South Africa is economically important as a source of agar. The Gracilaria yields from natural beds at Saldanha Bay are however unreliable, and consequently the South African Gracilaria industry has experienced a number of setbacks over the years. The only way a consistent supply can be assured is by mariculture to supplement the natural harvests. In 1993 the Seaweed Research Institute (SRU) found that mariculture of G. gracilis in Saldanha Bay is feasible but that there is potential to improve yields by technical research and development (Anderson et al.1996a). Jaffray and Coyne (1996) developed a pathogenicity assay that demonstrated that agarolytic bacteria isolated from Saldanha Bay Gracilaria induced disease symptoms such as thallus bleaching, while non-agarolytic isolates did not. It is thought that unfavorable environmental conditions such as elevated water temperature and nutrient depletion, which occur during the summer months in the surface layers of the water column in Saldanha Bay, induce the onset of agarase production or result in changes in the bacterial community structure in which agarase-producers become more dominant. By using the pathogenicity assay, Jaffray and Coyne (1996) identified the highly agarolytic Gracilaria gracilis pathogen, Pseudoalteromonas sp. LS2i. The aim of this study was to characterize the bacterial pathogen, Pseudoalteromonas sp. LS2i to further our understanding of virulence regulation and specifically, the role of the agarase enzymes in the process of seaweed-pathogen interaction. Two agarolytic clones, pEB1 and pJB1, were obtained after constructing and screening a Pseudoalteromonas sp. LS2i genomic library in Esherichia coli. Restriction enzyme mapping suggested that both clones contain the same agarase gene. Southern hybridization studies confirmed the origin of the cloned DNA and sequencing studies revealed the 1062 bp ORF, putative promoter region, putative ribosome binding site and putative transcriptional start point of the cloned agarase gene. The ORF showed sequence identity to several other β-agarases and was identified as a member of the GH-16 family of glycoside hydrolases. The agarase was purified from the E. coli JM109 (pEB3) transformant. The molecular weight was estimated to be 39 kDa by SDS-PAGE. Zymogram analysis confirmed that the purified protein is agarolytic and TLC analysis revealed that the predominant end-products of agar hydrolysis are neoagarohexaose and neoagarobiose, which indicates the same mode of action as that observed for the agarase produced extracellularly by Pseudoalteromonas sp. LS2i.en_ZA
dc.identifier.apacitationGildenhuys, C. (2014). <i>Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/4266en_ZA
dc.identifier.chicagocitationGildenhuys, Carin. <i>"Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2014. http://hdl.handle.net/11427/4266en_ZA
dc.identifier.citationGildenhuys, C. 2014-07-30. Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Gildenhuys, Carin AB - Gracilaria gracilis that grows naturally at Saldanha Bay, South Africa is economically important as a source of agar. The Gracilaria yields from natural beds at Saldanha Bay are however unreliable, and consequently the South African Gracilaria industry has experienced a number of setbacks over the years. The only way a consistent supply can be assured is by mariculture to supplement the natural harvests. In 1993 the Seaweed Research Institute (SRU) found that mariculture of G. gracilis in Saldanha Bay is feasible but that there is potential to improve yields by technical research and development (Anderson et al.1996a). Jaffray and Coyne (1996) developed a pathogenicity assay that demonstrated that agarolytic bacteria isolated from Saldanha Bay Gracilaria induced disease symptoms such as thallus bleaching, while non-agarolytic isolates did not. It is thought that unfavorable environmental conditions such as elevated water temperature and nutrient depletion, which occur during the summer months in the surface layers of the water column in Saldanha Bay, induce the onset of agarase production or result in changes in the bacterial community structure in which agarase-producers become more dominant. By using the pathogenicity assay, Jaffray and Coyne (1996) identified the highly agarolytic Gracilaria gracilis pathogen, Pseudoalteromonas sp. LS2i. The aim of this study was to characterize the bacterial pathogen, Pseudoalteromonas sp. LS2i to further our understanding of virulence regulation and specifically, the role of the agarase enzymes in the process of seaweed-pathogen interaction. Two agarolytic clones, pEB1 and pJB1, were obtained after constructing and screening a Pseudoalteromonas sp. LS2i genomic library in Esherichia coli. Restriction enzyme mapping suggested that both clones contain the same agarase gene. Southern hybridization studies confirmed the origin of the cloned DNA and sequencing studies revealed the 1062 bp ORF, putative promoter region, putative ribosome binding site and putative transcriptional start point of the cloned agarase gene. The ORF showed sequence identity to several other β-agarases and was identified as a member of the GH-16 family of glycoside hydrolases. The agarase was purified from the E. coli JM109 (pEB3) transformant. The molecular weight was estimated to be 39 kDa by SDS-PAGE. Zymogram analysis confirmed that the purified protein is agarolytic and TLC analysis revealed that the predominant end-products of agar hydrolysis are neoagarohexaose and neoagarobiose, which indicates the same mode of action as that observed for the agarase produced extracellularly by Pseudoalteromonas sp. LS2i. DA - 2014-07-30 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2014 T1 - Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis TI - Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis UR - http://hdl.handle.net/11427/4266 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/4266
dc.identifier.vancouvercitationGildenhuys C. Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2014 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/4266en_ZA
dc.language.isoeng
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherCell Biologyen_ZA
dc.titleInvestigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilisen_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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