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The Antimicrobial Activity of Secondary Metabolites Produced by Bacterial Endophytes Isolated from Pellaea Calomelanos

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The Antimicrobial Activity of Secondary Metabolites Produced by Bacterial Endophytes Isolated from Pellaea Calomelanos COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION o Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. o NonCommercial — You may not use the material for commercial purposes. o ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original. How to cite this thesis Surname, Initial(s). (2012). Title of the thesis or dissertation (Doctoral Thesis / Master’s Dissertation). Johannesburg: University of Johannesburg. Available from: http://hdl.handle.net/102000/0002 (Accessed: 22 August 2017). The antimicrobial activity of secondary metabolites produced by bacterial endophytes isolated from Pellaea calomelanos A Dissertation submitted to the Faculty of Science, University of Johannesburg In fulfilment of the requirement for the award of a Master of Science (MSc) degree: Biotechnology By Siphiwe Godfrey Mahlangu (201125973) Supervisor: Dr Mahloro Hope Serepa-Dlamini APRIL 2019 i Abstract Medicinal plants have been used worldwide as traditional remedies for the treatment of various diseases such as asthma, skin disorders, cardiovascular diseases, gastrointestinal symptoms, respiratory and urinary problems. These plants have shown to synthesize biologically active compounds that can be applied in drug discovery and other industries. The use of medicinal plants has led to deforestation and extinction of plant species; therefore, this has compelled scientists in search for new alternates for drug discovery and development. One of these are endophytes, which are microorganisms associated symbiotically with medicinal plants. Endophytes produce bioactive compounds similar to their plant host; in addition, endophytes produce various bioactive secondary metabolites some of which protect the plant host from pathogens and enhance growth potential of the plant host. This research study aimed at isolating and identifying bacterial endophytes from Pellaea calomelanos, a medicinal plant used for the treatment of headaches, chest and head colds, asthma, nasal and mouth ulcers, and to further evaluate the antibacterial activity of crude extracts produced by the plant and its bacterial endophytes. Six putative bacterial endophytes were isolated from surface sterilized leaves of P. calomelanos and identified using morphological and phenotypic data, the identification was confirmed by the 16S rRNA gene sequence, viz Arthrobacter sp. MHSD1 (MF613647), Pantoea sp. MHSD2 (MF613648), Bacillus infantis strain MHSD3 (MF613649), Pantoea sp. strain MHSD4 (MF613650), Pantoea ananatis strain MHSD5 (MF613651) and Pantoea sp. MHSD6 (MF613652). Crude extracts of the plant and its bacterial endophytes were tested against Gram-negative strains: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 13182); Gram-positive strains: Staphylococcus aureus (NCTC 6571) and Bacillus cereus (ATCC 10876) using the disk diffusion method with Streptomycin used as a positive control and Dimethylsufoxide (DMSO) as a negative control. Methanol extracts of P. calomelanos and ethyl acetate extracts of the six bacterial endophytes showed good antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Staphylococcus aureus and Bacillus cereus. Eighty percent methanol P. calomelanos crude extracts was subjected to liquid chromatography mass spectrometry-ion trap and time-of-flight (LCMS-IT-TOF) and data obtained revealed the presence of two chlorogenic acid compounds, one phenolic acid and one flavonoid compound. The crude extracts of both the plant (methanol crude extracts) and its bacterial endophytes (ethyl acetate extracts) were further analysed using gas chromatography-high resolution time- of-flight mass spectrometer (GC-HRT) to investigate their volatile secondary metabolites and the data generated showed the presence of various volatile secondary metabolites. ii Furthermore, the whole genome DNA of a novel bacterial endophyte, Pantoea ananatis strain MHSD5 was sequenced, assembled and annotated; and the data provided information on genetic components of the bacterium involved in endophytic lifestyle. Keywords: Pellaea calomelanos, bacterial endophytes, bioactive compounds, secondary metabolites, whole genome sequence. iii Declaration I Siphiwe Godfrey Mahlangu student number: 201125973 hereby declare that this dissertation entitled “The antimicrobial activity of secondary metabolites produced by bacterial endophytes isolated from Pellaea calomelanos” is my original work. I declare that the dissertation has not previously been submitted, by me, for a degree at this or any other University; that this is my own work in design and in execution, and that all the material contained therein has been duly acknowledged. SIPHIWE GODFREY MAHLANGU iv Dedication This work is dedicated to my parents Elizabeth and Philemon Mahlangu for their support and sacrifices they have made, and Mr Thabo Peega, the person who encourages me to dream. v Acknowledgements I would like to express my gratitude to the following individuals and organizations for their support throughout this study: This research project was carried out from 2017 to 2018 and most of the laboratory experiments were performed at the University of Johannesburg for which I am extremely thankful to the Department of Biotechnology and Food Technology and my supervisor Dr Mahloro Hope Serepa-Dlamini for giving me the chance to do my MSc studies there. I would like to express my warmest thanks to Dr Mahloro Hope Serepa-Dlamini for introducing me to the fields of Molecular Biology, Genomics and Bioinformatics. Thank you so much for everything. I appreciate all the knowledge, guidance, understanding and the skills you have conferred to me. To Olga Diale and Rebotiloe Morare, thank you guys for the assistance throughout this project. I am thankful to Mr Rudzani Manafe, Mr Alastair Campbell and Mr Mmoloki Majafa for their technical support. I would like to extend my sincere gratitude to Dr Oluwafemi Abedo and Dr Edwin Madala for assisting with LCMS-IT-TOF and GC- HRT analysis. Ms Mokgadi Mphahlele my friend, thanks for all the laughter we shared throughout the research struggle. I appreciate your support, words of encouragement which kept me going up to this far. National Research Foundation (NRF) Masters Scarce Skills for providing me with financial support. My dearest friends Dr Judith Phoku, Mrs Pamela Phungo, Ms Rito Baloyi, Mrs Sibongile Zwane, Dr Thokozile Yende and Dr Keabetswe Masike you are all acknowledged. Ms Mosibudi Malomane thank you so much for assisting with some of the functional assay work. vi Most of all I thank both my parents Mr. and Mrs Mahlangu, my sisters (Thandeka, Victoria and Sibongile) and my cousin Pontsi ‘Emily Olifant’ for their prayers, love and support. I thank Mr Peega for supporting and encouraging me throughout my MSc, thank you for being there from day one. I thank God for guiding me, protecting me and for giving me strength and wisdom. If it had not been for you Lord, I would not have made it this far. vii Research outputs Published manuscripts Mahlangu, S.G. and Serepa-Dlamini, M.H. (2018). The first report of bacterial endophytes from the leaves of Pellaea calomelanos in South Africa. South African Journal of Science, 114, 9-10. Doi.org/10.17159/sajs.2018/4235. Mahlangu, S.G. and Serepa-Dlamini, M.H. (2018). Draft genome sequence of Pantoea ananatis strain MHSD5 isolated from surface sterilized leaves of medicinal plant, Pellaea calomelanos obtained in South Africa. Data in Brief, 19, 1727-1732. Doi.org/10.1016/j.dib.2018.06.039. Manuscripts in preparation for submission Mahlangu, S.G. and Serepa-Dlamini, M.H. The antimicrobial activity of secondary metabolites produced by bacterial endophytes isolated from Pellaea calomelanos. To be submitted to Annals of clinical microbiology and antimicrobials. Conference outputs Poster presentation: Mahlangu, S.G. and Serepa-Dlamini, M.H. The antimicrobial activity of bacterial endophytes isolates from Pellaea calomelanos. University of Johannesburg, Inter-faculty postgraduate symposium, Johannesburg, South Africa, October 2017. Poster presentation: Mahlangu, S.G. and Serepa-Dlamini, M.H. The antimicrobial activity of bacterial endophytes isolates from Pellaea calomelanos. 26th SASBMB- FASBMB (South African Society for Biochemistry and Molecular Biology (SASBMB and the Federation of African Societies of Biochemistry and Molecular Biology (FASBMB)) Conference, North West, July 2018. Poster presentation: Mahlangu, S.G. and Serepa-Dlamini, M.H. Draft genome sequence of Pantoea ananatis strain MHSD5 a bacterial endophyte. 18th Genome Informatics Conference, Wellcome Genome Campus, Cambridge, Hinxton, United Kingdom, September 2018. viii Table of Contents Abstract ........................................................................................................................................... ii Declaration ..................................................................................................................................... iv Dedication ........................................................................................................................................ v Acknowledgements ........................................................................................................................
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