Screening environmental actinobacteria for antimycobacterial antibiotics and characterisation of Kribbella stellenboschensis sp. nov
Master Thesis
2018
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University of Cape Town
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Soil was collected from a compost heap in a Mowbray suburban garden and a compost heap in a Plumstead suburban garden. The soil and ‘worm tea’ of a vermiculture farm from the same Mowbray suburban garden were also sampled. Using four different types of media (7H9, CZ, ISP2 and GOT) 135 isolates were putatively identified as actinobacteria based on colony morphology. These isolates were screened for antimycobacterial activity against the test bacterium Mycobacterium aurum A+. A Kribbella strain, isolated and identified by an intern in the lab, and a Micromonospora strain, isolated and identified during the authors Honours project, were also screened for antimycobacterial activity. Sixty-four (64) actinobacterial isolates displayed moderate antibiotic activity or higher (ZOI >1001 mm2 ) based on the standard overlay method. Kribbella strain SK5 displayed very strong antimycobacterial activity (3309 mm2 ). Forty (40) of the actinobacterial strains that exhibited moderate/strong/very strong antimycobacterial activity and/or had interesting morphological features were selected for genus identification via a standard nucleotide-nucleotide blastn analysis of their 16S rRNA gene sequences. Thirty-one (31) strains were identified as Streptomyces species, six strains were identified as Micromonospora species, one strain was identified as a Nocardia species, one strain was identified as a Kitasatospora species, and one strain was identified as a member of the genus Tsukamurella. These isolates were subjected to phylogenetic analysis using the partial 16S rRNA gene sequences. Based on analysis of the 16S rRNA gene sequences, Streptomyces strain PR10 was found to be the most interesting of the Streptomyces isolates and should be pursued as a novel species (99.7% sequence similarity to the top blastn hit and less than 98.8% sequence similarity from the third blastn hit onwards). Further analysis of the gyrase subunit B (gyrB) gene sequence of the Kitasatospora isolate (strain PR3) revealed that the isolate is more closely related to members of the genus Streptomyces. Further evidence to support the assignment of strain PR3 to the genus Streptomyces (rather than Kitasatospora) is that it has two Streptomyces-specific gyrB gene indels signatures. Tsukamurella strain G4 was noted for characterisation as a novel species. The potential for seven isolates to produce ansamycin, glycopeptide, non-ribosomal peptide, and/or TypeII polyketide antibiotics was determined by detection of antibiotic biosynthetic gene clusters using PCR. Strain M27 demonstrated the potential to produce all the aforementioned antibiotics. Strain Y10 demonstrated the potential to produce a non-ribosomal peptide antibiotic. Strains PR10, PR28, PR47 and UK1 demonstrated the potential to produce Type-II polyketide and non-ribosomal peptide antibiotics. The PCR products were sequenced and analysed via blastn to compare them to the known antibiotic biosynthetic gene sequences in the GenBank database. The non-ribosomal peptide synthetase (NRPS) A domain sequences were analysed using the NRPSpredictor2 software to identify the A domain substrate specificity Solvent extraction was done on the broth cultures of Streptomyces strains PR3, UK1 and Y30 and Kribbella strain SK5 to isolate the antimycobacterial compounds. It was found that the cell mass extract of the three Streptomyces isolates had active compounds against M. aurum A+. The culture broth extract of the Kribbella isolate was found to have an active compound against M. aurum A+ and Staphylococcus aureus ATCC 25923. One-dimensional and two-dimensional TLC of the culture broth extract from strain SK5 revealed that a single compound was active against M. aurum A+ and S. aureus ATCC 25923. Nocardamine was purified from the culture broth extract of strain SK5 by Mr Kojo Acquah (PhD student, Department of Chemistry, University of Cape Town). In a side-by-side spot bioautography analysis of the purified nocardamine and the strain SK5 culture broth extract, it was found that the active compound in the culture broth extract was not nocardamine, because nocardamine only had activity against M. aurum A+ while the culture broth extract had activity against M. aurum A+ and S. aureus ATCC 25923. Using the polyphasic taxonomic approach, Kribbella strain SK5 was tentatively characterised as a novel species, for which the name Kribbella stellenboschensis sp. nov. is proposed. The closest phylogenetic relatives were identified as the type strains of Kribbella aluminosa, Kribbella karoonensis, Kribbella pittospori, Kribbella shriazensis, ‘Kribbella sindirgiensis’ and ‘Kribbella soli’. Genetic distances of 0.030 and 0.016 were calculated for ‘K. soli’ and ‘Kribbella sindirgiensis’, respectively, for the concatenated gene sequence of five housekeeping genes (gyrB, rpoB, recA, relA, and atpD). Thus, DNA-DNA hybridisation (DDH) will need to be carried out to confirm that strain SK5 is a separate species. Phenotypic differences were observed between strain SK5 and all the type strains of the most closely related species. Chemotaxonomically, strain SK5 possessed the key characters definitive of the genus Kribbella: i) MK9(H4) as the major menaquinone; ii) LL-diaminopimelic acid as the diagnostic diamino acid; iii) anteiso-C15:0 and iso-C16:0 as the major fatty acids (>10%); and iv) phosphatidylcholine in the polar lipid profile.
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Pelser, J. 2018. Screening environmental actinobacteria for antimycobacterial antibiotics and characterisation of Kribbella stellenboschensis sp. nov. University of Cape Town.