Identification and Characterization of Endophytic Actinobacteria Isolated from Plants and Sponges

Author: Alhnouf Alqurashi

Alqurashi, Alhnouf, 2017 Identification and Characterization of Endophytic Actinobacteria Isolated from Plants and Sponges, Flinders University, School of Medicine

This electronic version is made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact copyright@flinders.edu.au with the details.

Abstract

The majority of biologically active compounds that have been characterised are present in plants, although they are also abundant in microorganisms and other areas where life exists. Therefore, the effect of these metabolites on the existence and functioning of other organisms is important. Most microbial organic molecules that have an interactive function are called secondary metabolites, which consist of diverse chemical structures, their biological activities. Actinobacteria form a large phylum of Eubacteria and are known as prolific producers of these metabolites. The increase in pathogens resistant to antibiotics and the decline in introducing new antibiotics into the market has been the impetus for pharmaceutical companies to search for compounds produced by a range of actinobacterial cultures. This study was carried out to identify and characterize endophytic Actinobacteria isolated from plants and sponges and determine whether they produce any novel compounds, especially antimicrobial activity against test organisms. Thirty- three endophytic Actinobacteria had been already isolated from Australian plants and sponges by previous students in the department of Medical Biotechnology at Flinders University. The isolates were cultured on ISP2 for up to 21 days in order to allow for production of metabolites. The plates containing the culture and agar were extracted with methanol (MeOH) and tested against three bacteria: Staphylococcus aureus, Escherichia coli and Micrococcus luteus. Only one strain ET11 showed a strong activity against the three test organisms compare to the other strains that showed weak activity against one or two of the test microorganisms. ET11 was identified using 16S rRNA gene sequencing (ET11) and found to belong to the Streptomyces genus. BLAST searches based on the almost full length of the 16S rRNA gene sequence, showed that strain ET11 had the highest similarities viii with Streptomyces halophytocola (KLBMP 1284T) (99.78 %) that been isolated from the surface-sterilized stems of a coastal halophyte Tamarix chinensis Lour. Phylogenetic trees reconstructed on the basis of 16S rRNA gene sequences revealed that strain ET11 formed a distinct lineage with S. xiaopingdaonensis DUT 180t and S. halophytocola KLBMP 1284T. Fermentation techniques were used for inducing expression of secondary metabolites from the uncharacterised actinobacteria isolates. By using antimicrobial guided screening, it was determined that few of the isolates produced antimicrobial metabolites. Dominant antagonistic activity was detected from strain ET11 against Gram-positive bacteria. Nine fermentation media were used to cultivate the isolates for the production of antimicrobial metabolites. F33 medium was the most optimal medium for the production of antimicrobial metabolites. Various compounds were detected by using Thin Layer Chromatography (TLC) not identified indicating that endophytic Actinobacteria isolated from plants and sponges are prolific producers of secondary metabolites. Some of the compounds may have antimicrobial activity as some of the isolates cultured in liquid fermentation media and subjected to bioassay showed activity against one of the test microorganisms used in this study.

Keywords: Endophytic Actinobacteria, sponges, secondary metabolites, Australian plants, fermentations, TLC, HPLC, Streptomyces.
Subject: Medical Biotechnology thesis, Medicine thesis

Thesis type: Masters
Completed: 2017
School: School of Medicine
Supervisor: Prof. Chris Franco