Isolation and characterisation of indigenous actinobacteria from diverse South African environments

Master Thesis


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University of Cape Town

One soil sample and various indigenous plant and seaweed samples were used to isolate actinobacteria, with a particular focus on isolating rarer actinobacteria (non-Streptomyces species). A total of 169 putative actinobacterial strains was isolated, of which 42 were selected, based on their morphology, for further identification using a rapid molecular identification method and/or 16S rRNA gene sequence analysis. This includes seven strains isolated from various plant and seaweed samples. A total of 28 non-Streptomyces species was identified, with 23 being isolated from the soil sample, four isolated as plant endophytes (strains SM3BL 1, YPC1, YPC2 and YPL 1), and one isolated as a seaweed epiphyte (Y2UE1). Two Streptomyces species were isolated as endophytes (with strain SC1 isolated from a seaweed sample, and YMH1 isolated a plant species). Forty-two strains, including all non-Streptomyces species, were screened for their antibacterial activity against Mycobacterium aurum A+. Six strains showing promising antibacterial activity were selected for antibiotic extraction. The strains were also investigated for their antibiotic biosynthetic potential by PCR screening for the genes for ansamycin, glycopeptide and Type II (aromatic) polyketide antibiotics. Amplification of the AHBA synthase gene (ansamycin biosynthesis) was achieved for strains SE22 and YPC1. Strains SE22 and YMH1 were positive for the presence of the KSα-KSβ gene pair, with strain SE22 potentially producing a Type II (aromatic) polyketide. The gene product of isolate YMH1 was identified as a spore pigment gene. Antibiotic extraction was successful for strains SE22 and YM55, with numerous active compounds isolated from strain SE22, and one active compound isolated from strain YM55. Fifteen strains were selected for full characterisation based on their isolation source, their identification to non-Streptomyces genera and/or their antibacterial activity. These included four Micromonospora strains, three Kribbella strains, three Streptomyces strains, one Actinomadura strain, one Kineococcus strain, one Nocardia strain, one Nonomuraea strain and one Verrucosispora strain. Two previously isolated Microbispora strains were also characterised. The 17 strains were subjecte d to 16S rRNA gene sequence analysis to determine their closest phylogenetic relatives. The use of gyrB gene based phylogeny was also investigated for the two Microbispora isolates, resulting in a more stable phylogenetic tree. However, differences observed between the 16S rRNA and gyrB gene tree topologies suggest that horizontal gene transfer has occurred within the genus. The 17 isolates were distinguished from their closest phylogenetic neighbours through morphological and physiological characteristics. It is likely that the majority of the isolates are novel, although 16 isolates will require DNA-DNA hybridisation studies to determine if they are new species. Actinomadura strain YPC2 may be proposed as a novel species without the need for DNA-DNA hybridisation.