Browsing by Author "Meyers, Paul"
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- ItemOpen AccessActinomycete biodiversity assessed by culture-based and metagenomic investigations of three distinct samples in Cape Town, South Africa(2011) Davids, Muhammad Saeed; Meyers, PaulAll of the samples used for actinobacterial isolation were subjected to a culture-independent (metagenomic) study. The results provided an explanation for why no actinomycetes were found in the aquatic samples, as all of the sequenced clones were shown to be most closely related to uncultured bacteria. In the terrestrial sample, a total of 120 clones were obtained and all were sent for sequencing.
- ItemOpen AccessAnalysis of Actinobacterial Biodiversity in Marine Sediment from Gericke's Point (South Africa) and Screening of Isolates for Novel Antimycobacterial Compounds(2022) Dreyer, Ashleigh; Meyers, PaulThirty-three (33) presumptive actinobacterial strains were isolated using traditional culturebased techniques from sediment taken from marine habitats (a subtidal zone, a rock pool and a beach area) at Gericke's Point (Garden Route National Park, Sedgefield, South Africa). Twenty-seven (27) of the 33 presumptive actinobacterial isolates were identified to the genus level: 26 Streptomyces strains and one Nocardia strain. The partial 16S-rRNA gene sequences obtained for each confirmed actinobacterial isolate were used to determine their phylogenetic positions within their respective genera. Further investigation of specific isolates was done utilising the gyrB gene to determine whether these isolates are clones. Metagenomic data generated from next-generation sequencing of 16S-rRNA amplicons were used to reveal the actinobacterial biodiversity of the Gericke's Point sediment that was not seen in the culture-dependent part of this study. A total of 1 541 544 actinobacterial partial 16S-rRNA gene sequences were identified using the SILVA 16S-rRNA gene database. Actinobacteria that could not be assigned to a class or order made up ~41% of the total actinobacterial strains found in the Gericke's Point sediment samples. The rest of the identified actinobacterial strains belonged to the orders Candidatus Microtrichales (~45%), Candidatus Actinomarinales (~9%), Propionibacteriales (~3%) and other actinobacterial orders that each made up less than one percent (<1%) of the actinobacterial strains found in Gericke's Point. The other actinobacterial orders include Bifidobacteriales, Euzebyales, Frankiales, Geodermatophilales, Micrococcales, Micromonosporales, Mycobacteriales, Pseudonocardiales, Streptomycetales and Streptosporangiales. This is one of the first detections of Frankiales strains in a marine environment. The majority (99%) of actinobacterial strains identified at Gericke's Point could not be assigned to a known genus. This represents an abundance of novel actinobacterial diversity that has yet to be revealed. Multidrug-resistance in Mycobacterium tuberculosis is a global threat to public health which has increased the need for new antibiotics to treat tuberculosis. In this study, all confirmed actinobacterial isolates and two presumptive actinobacterial isolates (29 strains in total) were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard agar overlay method. To investigate their spectrum of antibiotic activity, all isolates were also screened for activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Twenty-one (21) isolates (20 Streptomyces strains and one unidentified strain) displayed strong to very strong antimycobacterial activity (defined as a zone of growth inhibition of over 2000 mm2 ). In addition, two Streptomyces strains displayed strong to very strong activity against S. aureus ATCC 25923. Compounds that displayed strong antimycobacterial activity were analysed using High Performance Liquid Chromatography-Mass Spectrometry and resulting mass spectra were compared to those of known compounds within the Global Natural Products Social Molecular Networking (GNPS) database. Eighteen (18) strains produced compounds with no matches in the GNPS database indicating these compounds could be novel. One strain produced a potential analogue of abyssomicin L (a rare antibiotic). Overall, the results obtained in this study emphasize the potential of marine environments as a source of novel actinobacteria and novel bioactive compounds.
- ItemOpen AccessAnalysis of actinobacterial biodiversity in reservoir sediment and cave soil and screening of isolates for antimycobacterial activity(2020) Rakiep, Adeebah; Meyers, PaulA total of 56 presumptive actinobacterial strains was isolated from three different samples taken from the Silvermine Nature Reserve (Table Mountain National Park, Cape Town), namely, cave soil, the wall of the cave and sediment from the shallow waters of a reservoir. Twenty nine (29) isolates were successfully identified to the genus level by 16S-rRNA gene analysis: one Micrococcus strain, one Streptacidiphilus strain, one Micromonospora strain and 26 Streptomyces strains. The phylogenetic position of each identified strain within its genus was investigated by generating a phylogenetic tree based on its 16S-rRNA gene sequence. Further analysis of the Streptacidiphilus strain was conducted based on the gyrB gene. Metagenomic analysis was used to further analyse the actinobacterial diversity of the freshwater reservoir sediment from the Silvermine Nature Reserve. A total of 97 16S-rRNA gene clones was obtained from the reservoir sediment sample, RS1, using actinobacteriumspecific 16S-rRNA gene primers S-C-Act-0235-a-S-20-F and S-C-Act-0878-a-A-19-R and each clone was identified using the EzBioCloud database. Analysis based on unique phylotypes in the clone library revealed that 80% of the clone library was composed of actinobacterial strains belonging to the orders Acidimicrobiales, Streptomycetales, Streptosporangiales, Corynebacteriales, Sporichthyales and the family Jatrophihabitandaceae (the remaining 20% was identified as non-actinobacterial strains). The percentage composition of the actinobacterial clonal diversity for each order was as follows: Acidimicrobiales, 56%; Streptomycetales, 29%; Streptosporangiales, 9%; Corynebacteriales, 4%; Sporichthyales, 1% and family Jatrophihabitandaceae, 1%. Rarefaction analysis revealed that the total actinobacterial diversity of the sample was not represented in the clone library. Therefore, further sampling and analysis of the sample site would uncover greater actinobacterial diversity. Thirty seven (37) putative actinobacterial isolates of the 56 that were isolated from the Silvermine Nature Reserve were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard over-lay method. A total of five identified 2 actinobacterial isolates (Streptomyces strains RS6, RS7, RS9, RS13 and RS15) and an unidentified actinobacterium, strain RS4, demonstrated very strong antimycobacterial activity (zone of growth inhibition of over 3000 mm2 ). In addition, 15 of the 37 strains were active against Staphylococcus aureus ATCC 25923 and three were active against Escherichia coli ATCC 25922. Streptomyces strains CS1, CS3, CS12, CS18, CS19, CW5, RS3, RS6, RS9, RS13 and RS15, displaying varying strengths of antimycobacterial antimicrobial activity, were selected for antibiotic extraction from culture broths. The resulting crude extracts were subjected to spot bioautography to test for antibacterial activity. The organic compounds extracted from the cell mass of Streptomyces strain CS3 and the broth fraction of Streptomyces strain RS3 demonstrated strong activity against M. aurum strain A+. Furthermore, the crude extracts of 15 actinobacterial isolates (Micromonospora strain RS10 and Streptomyces strains CS1, CS3, CS12, CS18, CW2, CW5, RS3, RS6, RS7, RS9, RS13, RS15, RS18 and RS19) were additionally tested for antiplasmodial activity against Plasmodium falciparum strain NF54. Seven of these strains showed activity against Plasmodium namely, Streptomyces strains CW2, CW5, RS3, RS7, RS13, RS15 and RS19. Streptomyces strains CW2, CW5 and RS7 displayed the strongest activity against P. falciparum strain NF54 with IC50 values below the guideline threshold of 1000 ng/mL (strain CW2 culture broth crude extract: IC50 40 ng/mL, strain CW5 culture broth crude extract: IC50 128 ng/mL and strain RS7 culture broth crude extract: IC50 70 ng/mL).
- ItemOpen AccessThe characterisation of actinomycetes isolated from diverse South African sources, with emphasis on the genus Kribbella(2007) Kirby, Bronwyn Michelle; Meyers, PaulActinomycetes were isolated from the leaves of indigenous plants, aquatic sediment and soil samples, using alternative isolation methods to select for actinomycetes belonging to the rarer genera. Thirty actinomycete strains belonging to the genera Gordonia, Kineococcus, Kribbella, Micromonospora, Nocardia and Streptomyces were selected for full characterisation. A polyphasic approach combining physiology, chemotaxonomy and phylogenetic analysis was used to characterise these isolates. A number of potentially novel strains belonging to the rarer genera were identified, including two Kineococcus and three Micromonospora strains. Two novel Kribbella species were isolated from soil samples and the species descriptions of Kribbella karoonensis Q41T and Kribbella swartbergensis HMC25T were published in 2006.
- ItemOpen AccessEnhanced phylogenetic analysis and targeted search for the genus Kribbella(2015) Curtis, Sarah Maureen; Meyers, Paul[Not OCR'd]
- ItemOpen AccessExploring the molecular diversity and biomedicinal potential of marine invertebrates and South African actinomycetes for tuberculosis drug discovery(2021) Acquah, Kojo Sekyi; Gammon, David; Beukes, Denzil; Meyers, Paul; Warner, DigbyTuberculosis (TB) which is caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from a single infectious disease and remains a global health threat. Although there is medicine for treating TB, it still kills millions each year. This is due to a lengthy and demanding TB treatment regimen with associated problems of adverse drug-drug interactions and toxicities. Several resistant strains of Mtb, which are difficult and more expensive to treat, have also emerged. Therefore, there is a need to discover new potent and safe TB drugs to effectively treat Mtb with its resistant strains. Less explored and biodiversity-rich ecosystems such as the marine environment and South Africa (SA)'s fauna and flora have been a source of new bioactive natural products (NPs). A range of marine invertebrates and SA actinomycetes were therefore studied for the discovery of new antimycobacterial NPs. Several organic and aqueous marine invertebrate extracts were screened for their in vitro inhibitory activity against the Mtb strain H37Rv. The chemical components of two out of 54 active extracts were dereplicated using 1H NMR, HR-LCMS with GNPS molecular networking. The extracts were subsequently subjected to an activity-guided isolation process to yield heteronemin from Hyrtios reticulatus extract, and bengamides P and Q from Jaspis splendens extract. A new bengamide derivative was putatively identified in the molecular network of Jaspis splendens extract, and its structure predicted based on the similarity of its MS/MS fragmentation pattern to that of other bengamides. The isolated bioactive metabolites and semi-pure fractions exhibited antimycobacterial activity with MIC90 in the range of < 0.24 to 62.50 µg/mL. This is first report of antitubercular activity of bengamides P and Q. In studies on actinomycetes, the organic extract of a liquid culture of the South African Streptomyces strain Muiz4Y exhibited antimycobacterial activity against Mycobacterium aurum strain A+ and Mtb strain H37Rv. HR-LCMS analysis of the crude extract for dereplication suggested the presence of new compounds. A bioactivity, spectroscopy and spectrometry guided isolation procedure led to the isolation of three new natural products, a β-carboline alkaloid (1- (1,2-dihydroxyethyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid), a peptide (N-(2- phenylacetyl)-serine), and a glycosylated lactone (4-O-(β-glucopyranosyl)-5-(hydroxymethyl)-3- (4-methylpentyl)-5,6-dihydro-2H-pyran-2-one), together with known compounds 3,6- bis(phenylmethyl)-2,5-piperazinedione and 2,4,6-triphenylhex-1-ene. The structures of the isolated compounds were elucidated based on spectroscopic methods including 1D and 2D NMR, MS, as well as by comparison with the relevant literature. Only 2,4,6-triphenylhex-1-ene exhibited antimycobacterial activity against Mtb strain H37Rv with an MIC90 of 5.8 µg/mL. The novel rare South African Actinomycete Kribbella speibonae strain SK5 exhibited antimycobacterial activity against Mycobacterium aurum A+. Dereplicating the crude extract of a large-scale culture of strain SK5 using 1H NMR, genome mining and HR-LCMS with GNPS molecular networking showed that it is a prolific producer of hydroxamate siderophores including new congeners. Two new analogues, dehydroxylated desferrioxamines, speibonoxamine and desoxy-desferrioxamine D1, were isolated, together with four known hydroxamates, desferrioxamine D1, desferrioxamine B, desoxy-nocardamine and nocardamine, and a diketopiperazine (DKP). The isolated compounds were characterized by the analysis of HRESIMS and 1D and 2D NMR data, as well as by comparison with the relevant literature. Three new dehydroxy desferrioxamine derivatives were tentatively identified in the molecular network of K. speibonae strain SK5 extracts, and structures were proposed based on their MS/MS fragmentation patterns. A plausible spb biosynthetic pathway was proposed. To the best of our knowledge, this is the first report of the isolation of desferrioxamines from the actinobacterial genus Kribbella. The isolated compounds were inactive against Mtb strain H37Rv and Mycobacterium aurum A+. This study confirmed the marine environment as a source of new antimycobacterial NPs and established South African actinomycete as a source of new bioactive NPs.
- ItemOpen AccessIdentification and preliminary characterization of the 2,5-diphenyloxazole biosynthetic pathway in streptomyces polyantibioticus SPRT(2015) Kemp, Ian Kyle; Meyers, PaulAn antibacterial compound produced by the actinomycete, Streptomyces polyantibioticus SPRT, exhibited antibiosis against Mycobacterium tuberculosis H37RvT (the causative agent of tuberculosis), which prompted interest in its biosynthesis. The antibacterial compound was isolated in a previous study and its structure was determined by X-ray crystallography and nuclear magnetic resonance (NMR) to be 2,5-diphenyloxazole (DPO). Based on the structure of DPO, a biosynthetic scheme for the synthesis of this molecule was proposed, whereby a non-ribosomal peptide synthetase (NRPS) condenses a molecule of benzoic acid with 3-hydroxyphenylalanine. The dipeptide is converted to a diphenyloxazole derivative by heterocyclization and a final decarboxylation step leads to DPO. To determine whether the hypothesis pertaining to the DPO biosynthetic pathway was correct, initial efforts were made to identify the genes coding for benzoic acid synthesis and the DPO NRPS in the S. polyantibioticus SPRT genome using PCR amplification, Southern hybridization and sequencing. This led to the identification of 12 unique adenylation (A) domains (of which one was specific for phenylalanine) and a gene, paaK, encoding a phenylacetate CoA-ligase (PA-CoA), putatively involved in benzoic acid biosynthesis. However, no further sequence information could be obtained for the genes encoding the Phe A domain or PA-CoA and similar attempts to identify other NRPS-associated domains, as well as genes involved in benzoic acid synthesis, proved unsuccessful. In light of these difficulties, the S. polyantibioticus SPRT genome was sequenced and a gene cluster was identified as being responsible for the biosynthesis of DPO using a genome mining approach. However, contrary to the hypothesis that a linear NRPS system for DPO biosynthesis would be identified, the gene cluster exhibited a nonlinear arrangement. The core domains are arranged as A-PCP-C (instead of C-A-PCP) and there is also a stand-alone heterocylization domain, a stand-alone thioesterase domain and an acyl-CoA synthetase putatively involved in activating benzoic acid. Furthermore, there are two NRPS domains in the gene cluster that are believed to be inactive. A possible biosynthetic pathway for benzoyl-CoA production, encoded by a separate gene cluster, was identified based on the genome analysis of S. polyantibioticus SPRT. In order to confirm the involvement of the identified genes in DPO biosynthesis, an intergeneric conjugation protocol was developed for the introduction of plasmid DNA into S. polyantibioticus SPRT and subsequent gene disruption experiments. The putative DPO biosynthetic genes were insertionally activated via homologous recombination and the method for isolating DPO was carried out on each of the mutant strains, after which the extracts were assayed for activity against Mycobacterium aurum A+ using TLC-bioautography analysis. The absence of activity against M. aurum A+ in the extracts from mutant strains S. polyantibioticus ΔA99, S. polyantibioticus ΔCYC and S. polyantibioticus ΔACY suggested the involvement of the A domain encoded by gene SPR_53060, the putative heterocyclization domain encoded by gene SPR_53040 and the acyl-coA synthetase encoded by gene SPR_52860 in the biosynthesis of DPO. However, attempts to identify the genes responsible for benzoic acid biosynthesis proved unsuccessful, as gene disruption did not abolish DPO activity in the S. polyantibioticus ΔLAC, S. polyantibioticus ΔPAAK and S. polyantibioticus ΔCIN mutant strains encoding the putative D-lactate dehydrogenase encoded by gene SPR_60250, the PA-CoA ligase (paaK) encoded by gene SPR_46390 and the cinnamate CoA ligase encoded by gene SPR_60150, respectively. Based on the genome annotation analysis and gene disruption studies, a model for DPO biosynthesis is proposed. At this stage, the model cannot account for the source of benzoic acid, as in vivo gene disruption experiments disproved both of the hypotheses on how benzoic acid is synthesized in S. polyantibioticus SPRT. However, alternative hypotheses regarding the mechanism of benzoic acid biosynthesis in S. polyantibioticus SPRT are proposed and are suggested as the place to start in future studies to elucidate the production of this unusual starter unit in DPO biosynthesis. Furthermore, the identification of the gene cluster responsible for DPO biosynthesis may be used for combinatorial biosynthetic studies to create derivatives of DPO that might be used in the treatment of drug resistant tuberculosis. Lastly, the S. polyantibioticus SPRT genome sequence could be explored for the identification of antibiotic gene clusters for other potential antitubercular antibiotics that this organism produces.
- ItemOpen AccessThe investiation into the synthesis of 2,5-Diphenyloxazole in Streptomyces polyantibioticus SPR(2011) Stegmann, Darren Edward; Meyers, PaulAs part of an antibiotic-screening programme, an actinomycete, Streptomyces polyantibioticus SPRT, was isolated from soil collected from the banks of the Umgeni River, KwaZulu-Natal Province, South Africa. It exhibited antibiosis against M. tuberculosis H37RvT, prompting interest in its antibiotic production. An antibiotic produced by S. polyantibioticus SPRT was isolated and its structure determined by nuclear magnetic resonance (NMR) and X-ray crystallography to be 2,5- diphenyloxazole (DPO). Of great interest is the independent confirmation of the antibiotic activity of DPO and extension of the data to show activity against non-replicating persistent cells of M. tuberculosis. It seems likely that 2,5-DPO is synthesized non-ribosomally by S. polyantibioticus SPRT. It is proposed that DPO is synthesised from the starting units of benzoic acid and -hydroxyphenylalanine or phenylalanine, undergoing peptide bond formation followed by cyclization and decarboxylation to form DPO.
- ItemOpen AccessInvestigating the actinomycete diversity inside the hindgut of an indigenous termite, Microhodotermes viator(2010) Rohland, Jeffrey; Meyers, PaulEver since the discovery of a cellulolytic actinomycete inside the gut of a termite, there has been considerable interest in the metabolic abilities and the diversity of these bacteria within this complex environment. However, until fairly recently, most investigations of termite hindguts involved the higher termites from the Termitidae family. Little attention was paid to the lower termites, and even less to the family Hodotermitidae. The main aim of this project was to explore the actinobacterial diversity of the paunch and colon hindgut regions of one particular member of the family Hodotermitidae, Microhodotermes viator. No previous work of this nature has ever been attempted on this particular termite species.
- ItemOpen AccessInvestigations into actinomycetes isolated from coastal environments, with a special emphasis on the genus Micromonospora(2005) Goodwin, Candice Michelle; Meyers, PaulMarine environments were investigated and actinomycetes were isolated on selective media. Thirty-four (34) actinomycete strains were isolated and identified: 21 Micromonospora strains, 10 Streptomyces strains, and 3 Pseudonocardia strains. A polyphasic approach was employed to determine the novelty of the isolates. Potentially, all 21 Micromonospora strains are novel, as revealed by an original identification scheme developed to assess quickly and easily the novelty of newly isolated environmental Micromonospora strains. Standardized media for testing physiological characters of Micromonospora strains were developed, and additional physiological characteristics of 15 of the validly published members of the genus Micromonospora are described. Furthermore, 14 of the 15 validly published Micromonospora species, and 20 of the 21 environmental Micromonospora isolates grew under anaerobic conditions.
- ItemOpen AccessIsolation and characterisation of antibiotic-producing marine actinomycetes(2003) Porter, Donovan Stuart; Meyers, PaulResistance to antibiotics poses a serious threat to healthcare and new drugs are needed. This is especially true for tuberculosis (TB), which is at epidemic levels in South Africa. Multidrug-resistance in Mycobacterium tuberculosis makes TB more difficult and expensive to treat and increases mortality rates. The surfaces of 12 seaweed species found in South African coastal waters were screened for the presence of antibiotic-producing actinomycetes. Of the 67 strains isolated, 26 exhibited antibacterial activity against Mycobacterium aurum A+ and for Enterococcus faecium VanA. These actinomycete strains were physiologically characterised. Three strains showing very strong antibacterial activity were further characterised by the use of chemical taxonomy, DNA sequencing and scanning electron microscopy and were shown to belong to the genus Streptomyces. A strain not showing activity was shown by the same methods to belong to the genus Micromonospora. Partial purification of the active compounds was carried out on the three strains exhibiting strong antibacterial activity. All were shown to produce moderately to highly polar compounds.
- ItemOpen AccessIsolation and characterisation of indigenous actinobacteria from diverse South African environments(2010) Sfarlea, Iulia; Meyers, PaulOne 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.
- ItemOpen AccessPharmacognostic study of 5 medicinal plant species from Western Cape Province (South Africa) for anti-tubercular activity(2006) Bamuamba, Kapinga Benoit; Meyers, Paul; Gammon, David W; Franca, Marie-Geneviève DijouxIn our search for new anti-tuberculosis lead molecules, five medicinal plant species, Olea capensis (L.l, Tulbaghia alliacea (L.), Inula graveolens (L.), Leyssera gnaphaloides (L.), and Buddleja saligna (L.) were collected in Cape Town and surrounding area and investigated for antimycobacterial activity following report of their therapeutic use in traditional medicine to treat infectious diseases such as tuberculosis. A bioassay guided fractionation of the acetone/water (4:1) crude extracts of O. capensis (leaves) and T. alliacea (rhizomes) showed no activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 252923, and Mycobacterium aurum A+. In contrast, the orgamc fractions (hexane, dichloromethane) of the acetone/water (4: 1) crude extracts of 1. graveolens, L. gnaphaloides, and B. saligna exhibited significant activity against M. tuberculosis H37Rv, M. avium 25291, M. microti ATCC 19422, and M. scrofulaceum ATCC 19987. The isolation and structure determination of the bioactive led to the identification of pentacyclic triterpenoids, ursolic acid (UA) and oleanolic acid as major antitubercular constituents of B. saligna, L. gnaphaloides, and 1. graveolens. The in vitro cytotoxicity assays of the isolated bioactive constituents showed no cytotoxicity against Chinese Hamster Ovarian (CHO) cells line. Subsequently, given the pharmaceutical value of the above finding, a survey on structure activity of pentacyclic triterpenoids was conducted. It was was found, for instance that selective substitutions at C-3 and/or C-28 and the double bond at UA, OA and betulinic and (1) BA) were made in order to improve anti-tumour and anti-HIV activity. However, thought a great number of modified bioactive pentacyclic triterpenoids is reported, none was tested against Mtb. Therefore, this study also explored a new synthetic route (scheme 1) toward a generation of (5), which may allow improving antitubercular, anti-HIV or anti-tumour activity, and/or specificity.
- ItemOpen AccessQuaternary structures of the cyanide dihydratases of Bacillus pumilus C1 and Pseudomonas stutzeri AK61(2003) Berman, Mark Nicholas; Sewell, Bryan Trevor; Meyers, PaulNitrilases catalyse the conversion of a nitrile to its corresponding acid and ammonia by the addition of two water molecules. Cyanide dihydratases, a subgroup of nitrilases, specifically hydrolyse cyanide to formic acid and ammonia. Nitrilases are found in a diverse collection organisms that includes plants, bacteria and fungi. They form one branch a superfamily of structurally related enzymes that are believed to have in common a unique cys-glu-Iys catalytic triad. Many nitrilases exiat as a large molecular weight oligomers of more than 300kDa. In the current study the structures of two cyanide dihydratases, from Pseudomonas stutzeri AK61 and Bacillus pumilus Cl, have solved at a resolution 2.9nm and 32nm respectively by single particle reconstruction from electron micrographs of enzyme particles stained in uranyl acetate. Each enzyme consists of a spiral structure of well-defined length. It is proposed that this arrangement of subunits occurs in many other nitrilases and that a number of unexplained observations in the literature can reconciled by this model.
- ItemOpen AccessScreening environmental actinobacteria for antimycobacterial antibiotics and characterisation of Kribbella stellenboschensis sp. nov(2018) Pelser, James Grant; Meyers, PaulSoil 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.
- ItemOpen AccessScreening of actinobacteria for novel antimalarial compounds(2020) Watson, Daniel John; Wiesner, Lubbe; Meyers, PaulThe success of our first-line antimalarial treatments is threatened by increased drug resistance in Plasmodium parasites. This makes the development of novel drugs critical to combat malaria. Historically, natural products have been an excellent source of novel antimalarial compounds and thus are an ideal place to search for potential drugs. Filamentous members of the bacterial phylum, Actinobacteria, are well-known antibiotic producers, but their antimalarial potential has not been well investigated. This makes these actinobacteria a potentially valuable source of novel antimalarial compounds. To evaluate the antimalarial potential of the filamentous actinobacteria, uncharacterized environmental actinobacterial strains from the Meyers laboratory culture collection, as well as the type strains of new actinobacterial species identified and characterized in the Meyers laboratory, were screened for antiplasmodial activity against drug-sensitive Plasmodium falciparum, NF54. Liquid cultures were extracted using the mid-polar solvent, ethyl acetate, with the aim of discovering drug-like molecules that can be administered orally. Thirty-one strains of actinobacteria belonging to eight genera (Actinomadura, Amycolatopsis, Gordonia, Kribbella, Micromonospora, Nocardia, Nonomuraea, and Streptomyces) were screened revealing fourteen active strains. Eight strains were identified for further study as the displayed antiplasmodial efficacy matching predefined criteria. Of these eight candidates, Streptomyces strain PR3 was selected, as it showed excellent antiplasmodial efficacy, no cytotoxicity against Chinese Hamster Ovary (CHO) or liver HepG2 cell lines, no haemotoxicity, and was easy to culture. Bioassay-guided fractionation of the crude extracts of strain PR3, supported by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis, was conducted to isolate and identify the compounds responsible for the antiplasmodial activity. During purification by solid phase extraction (SPE), a novel class of compounds was isolated. The structure of these compounds was elucidated by HRMS and NMR analysis and determined to be a series of crown ethers with a methylated backbone. These methylated crown ethers (MCE) were not produced by strain PR3, but by the cyclization of polypropylene glycol (PPG) oligomers from Amberlite® XAD-16N 20–60 mesh resin under aqueous conditions. The MCEs displayed weak antiplasmodial activity against P. falciparum NF54, without cytotoxicity against the Chinese Hamster Ovary, HepG2 cell lines, nor human erythrocytes. To the author's knowledge, the MCEs are novel compounds, and this is the first time the cyclization of PPG oligomers into crown ethers has been reported. As the MCEs were not responsible for strain PR3's potent antiplasmodial activity, further study was conducted. Using the Global Natural Product Social molecular networking (GNPS) workflow, genome mining, and NMR analysis, it was revealed that the cyclodepsipeptides, valinomycin, montanastatin, and nine other novel analogues were responsible for the high antiplasmodial activity detected. A review of the literature revealed that the structure of four of these analogues had been predicted, based on MS/MS and the biosynthesis of valinomycin. Using the same described biosynthetic logic and MS/MS analysis, two new cyclodepsipeptides, compounds 1054 and 1068, were elucidated. Unfortunately, chromatographic systems developed were unable to purify the cyclodepsipeptides, and individual evaluation of their antiplasmodial efficacy and host selectivity was not possible. The fraction containing the cyclodepsipeptides exhibited strong antiplasmodial activity against the drug-sensitive, NF54 and multidrug-resistant K1, strains of P. falciparum. No cytotoxicity was displayed against the CHO cell line and no haemotoxicity was seen against human erythrocytes. Moderate toxicity was exhibited against the liver HepG2 cell line; however, the selectivity index of the cyclodepsipeptides suggested that they are selectively targeting the Plasmodium parasites. Overall, these results are positive, and further study of the individual cyclodepsipeptides is warranted. During the investigation, discrepancies were noticed between different fractions in terms of antiplasmodial activity. These fractions contained both the MCEs and, cyclodepsipeptides along with a range of impurities, yet they displayed potent antiplasmodial activity. Further study suggested that combination of the MCEs and cyclodepsipeptides elicits a synergistic response and improves antiplasmodial efficacy. This was determined independently using two models, the fixed-ratio isobologram method and the CompuSyn programme based on the massaction law principle. The workflow developed during this investigation demonstrates how new technologies can be used to dereplicate and elucidate bioactive natural products. This workflow can be utilized to continue this research and identify new natural products that can combat malaria
- ItemOpen AccessSelective Isolation and Characterization of Indigenous Actinobacteria, with Particular Emphasis on the Genus Amycolatopsis(2010) Everest, Gareth John; Meyers, Paul