Browsing by Author "Tuffin, Marla"
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Item Actinobacterial diversity of the Ethiopian Rift Valley lakes(University of the Western Cape, 2011) Du Plessis, Gerda; Cowan, D.A.; Tuffin, MarlaThe class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomics studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under "extreme" conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint.Item Assessment of temporal and spatial evolution of bacterial communities in a biological sand filter mesocosm treating winery wastewater(The Society for Applied Microbiology, 2013) Ramond, Jean-Baptiste; Welz, P.J.; Tuffin, Marla; Burton, Stephanie G.AIMS: To assess the impact of winery wastewater (WW) on biological sand filter (BSF) bacterial community structures, and to evaluate whether BSFs can constitute alternative and valuable treatment-processes to remediate WW. METHODS AND RESULTS: During 112 days, WW was used to contaminate a BSF mesocosm (length 173 cm/width 106 cm/depth 30 cm). The effect of WW on bacterial communities of four BSF microenvironments (surface/deep, inlet/ outlet) was investigated using terminal-restriction fragment length polymorphism (T-RFLP). BSF achieved high Na (95·1%), complete Cl and almost complete chemical oxygen demand (COD) (98·0%) and phenolic (99·2%) removals. T-RFLP analysis combined with ANOSIM revealed that WW significantly modified the surface and deep BSF bacterial communities. CONCLUSIONS: BSF provided high COD, phenolic and salt removals throughout the experiment. WW-selected bacterial communities were thus able to tolerate and/or degrade WW, suggesting that community composition does not alter BSF performances. However, biomass increased significantly in the WW- impacted surface sediments, which could later lead to system clogging and should thus be monitored. SIGNIFICANCE AND IMPACT OF THE STUDY: BSFs constitute alternatives to constructed wetlands to treat agri effluents such as WW. To our knowledge, this study is the first unravelling the responses of BSF bacterial communities to contamination and suggests that WW-selected BSF communities maintained high removal performances.Item Bacteriophage diversity in haloalkaline environments(University of the Western Cape, 2013) Nemavhulani, Shonisani; Tuffin, MarlaThere are limited reports on virus population in haloalkaline environments; therefore the aim of this study was to investigate the genetic diversity and biology of bacteriophage communities in these environments. Bacteria were isolated to be used as phage hosts. One bacterium from Lake Magadi and four bacteria from Lake Shala were successfully isolated from sediment samples. A further two Lake Shala bacterial hosts from the IMBM culture collection were also used to isolate bacteriophages. Bacterial isolates were identified to be most closely related to Bacillius halodurans, Halomonas axialensis, Virgibacillus salarius, Bacillus licheniformis, Halomonas venusta, Bacillus pseudofirmus and Paracoccus aminovorans. Bacteriophages were screened using all bacteria against sediment samples from both Lake Shala and Lake Magadi. One phage was identified from Lake Magadi sediments (MGBH1) and two phages from Lake Shala sediments (SHBH1 and SHPA). TEM analysis showed that these phages belong to three different dsDNA phage families; Siphoviridae (MGBH1), Myoviridae (SHBH1) and Podoviridae (SHPA). All phages showed different genome sizes on agarose gel. Due to the small genome size, phage SHPA was chosen for further investigation. Partial, genome sequence analysis showed homology to both bacterial and phage proteins. A further investigation of phage diversity in this environment is essential using metagenomic approaches to understand these unique communities.Item Biodiversity: so much more than legs and leaves(AOSIS OpenJournals, 2013) Cowan, Donald A.; Rybicki, Edward P.; Tuffin, Marla; Valverde, Angel; Wingfield, MichaelMicroorganisms inhabit virtually every possible niche on Earth, including those at the outer envelope of survival. However, the focus of most conservation authorities and ecologists is the ‘legs and leaves’ side of biology – the ‘macrobiology’ that can be seen with the naked eye. There is little apparent concern for the preservation of microbial diversity, or of unique microbial habitats. Here we show examples of the astounding microbial diversity supported by South Africa’s ecosystems and argue that because microbes constitute the vast majority of our planet’s species they should be considered seriously in the future protection of our genetic resources.Item Characterisation of the arsenic resistance genes in Bacillus sp. UWC isolated from maturing fly ash acid mine drainage neutralised solids(Academy of Science of South Africa, 2010) Musingarimi, Wicleffe; Tuffin, Marla; Cowan, Donald A.An arsenic resistant Bacillus sp. UWC was isolated from fly ash acid mine drainage (FA-AMD) neutralised solids. A genomic library was prepared and screened in an arsenic sensitive mutant Escherichia coli strain for the presence of arsenic resistance (ars) genes. Sequence analysis of a clone conferring resistance to both sodium arsenite and sodium arsenate revealed homologues to the arsR (regulatory repressor), arsB (membrane located arsenite pump), arsC (arsenate reductase), arsD (second regulatory repressor and a metallochaperone) and arsA (ATPase) genes from known arsenic resistance operons. The Bacillus sp. UWC arsRBCDA genes were shown to be arranged in an unusual manner with the arsDA genes immediately downstream of arsC.Item Characterisation of the endophytic bacterial communities associated with South African sorghum plants: looking for potential plant growth-promoting endophytes(University of Western Cape, 2012) Finyom, Cyprien William Bopda; Tuffin, Marla; Ramond, Jean-Baptiste; Cowan, DonThe term endophyte is used to define all microorganisms that, during a part of their life cycle, colonize the internal tissues of a plant host. Many endophytes have been found to promote plant growth by acting either as biocontrol agents, biofertilizers or phytohormone producers. This study aimed to characterise the endophytic microbial community diversity associated with sorghum farmed in South Africa. Members of any common endophytic bacterial species identified during the study might in future studies be developed to improve sorghum production. Sorghum tissues (roots, shoots, stems) were sampled in three South African provinces (Free State, Limpopo and North West), each site being characterised by the use of different agricultural practices. Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analyses were used to characterise the endophytic bacterial communities. The analysis clearly demonstrated that the endophytic bacterial community structure in the three sorghum tissue types differed, suggesting that endophyte colonization is tissue-specific. The endophytic bacterial community structure is quite similar in each tissue when comparing the populations present in the sampling sites. In the sorghum endophytic microbial communities, common bacterial species were identified using molecular tools: The cyanobacterium Synechococcus and Staphylococcus saprophyticus were identified in the root samples. Pantoea sp., Erwinia sp., Enterobacter sp. and Klebsiella sp. were found in all shoot samples. Nocardia fluminea, Bacillus cereus and Microbacterium sp. were isolated as common shoot endophytic bacteria. This study defines, for the first time, the endophytic bacterial species associated with South African sorghum plants. These common endophytic bacterial species can be used to enhance the yield of sorghum crops.Item Cloning and characterization of three compost metagenome-derived α‑L‑Arabinofuranosidases with differing thermal stabilities(University of the Western Cape, 2014) Fortune, Brent Marc; Tuffin, Marla; Huddy, Robert; Smart, MarietteSecond generation biofuels production requires a suite of lignocellulolytic enzymes, acting synergistically to liberate the fermentable monosaccharides contained within agricultural waste materials. The use of thermostable lignocellulosic enzymes in a high-temperature process represents a number of advantages over their respective mesophilic counterparts, including increased solubility of the polymeric lignocellulosic substrates, enhanced enzyme processivity and reduced risk of bacterial and/or phage contamination during fermentation (Turner et al., 2007; Viikari et al., 2007). Alpha-L-arabinofuranosidases (AFases) participate in the deconstruction of lignocellulosic materials by hydrolysing the arabinofuranosyl bonds contained within the hemicellulosic portion of lignocellulose. In this study, three AFases isolated from compost-derived metagenomic DNA were characterised. Three genes derived from the fosmid metagenomic library constructed from 70°C compost were cloned into the pET21a(+) expression vector and expressed in E. coli BL21. The heterologously expressed proteins, AFase_H4, AFase_E3 and AFase_D3, were subsequently purified and their biochemical characteristics determined. All three AFases were shown to be active between pH 4.0 and 6.0. AFase_H4 and AFase_E3 displayed the highest activity at 60oC, while AFase_D3 had an optimum temperature at 25oC. Furthermore, the three AFases had differing thermostability profiles. In particular, AFase_E3 maintains 100% residual activity following 60 min incubation at 80oC and 24 hour incubation at 60oC. All three AFases have activity upon p-Nitrophenyl-arabinofuranoside and none against a range of alternative p-Nitrophenylglycosidic substrates. Phylogenetic analysis of the catalytic domain, identified within the amino acid sequences of the AFases, suggests that these AFases belong to glycoside hydrolase (GH) family 51. The difference in the AFase primary amino acid sequence motifs were used to infer differences in thermostability. All three AFases indicated similar biochemical and biophysical characteristics. AFase_E3 was determined to be the most thermostable amongst all three AFases. In the assessment of the three AFases respective suitability for inclusion in thermogenic bioethanol production processes, AFase_E3 was concluded as a suitable candidate for hydrolysis and synergistic testing on natural substrates. AFase_D3 possess the potential to be included in novel mesophilic industrialized process.Item Cloning, expression and characterization of thermostable YdaP from Bacillus licheniformis 9A(Acta Biochimica Polonica, 2017) Lako, Joseph D. Wani; Yengkopiong, Jada P.; Stafford, William H. L.; Tuffin, Marla; Cowan, Don A.The Bacillus licheniformis ydaP gene encodes for a pyruvate oxidase that catalyses the oxidative decarboxylation of pyruvate to acetate and CO2. The YdaP form of this enzyme was purified about 48.6-folds to homogeneity in three steps. The enzyme was recovered in a soluble form and demonstrated significant activity on pyruvate using 2, 6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor. HPLC analysis of the YdaP-enzyme catalysed conversion of pyruvate showed acetate as the sole product, confirming the putative identity of pyruvate oxidase. Analysis of the substrate specificity showed that the YdaP enzyme demonstrated preference for short chain oxo acids; however, it was activated by 1% Triton X-100. The YdaP substrate-binding pocket from the YdaP protein differed substantially from the equivalent site in all of the so far characterized pyruvate oxidases, suggesting that the B. licheniformis YdaP might accept different substrates. This could allow more accessibility of large substrates into the active site of this enzyme. The thermostability and pH activity of the YdaP enzyme were determined, with optimums at 50ºC and pH 5.8, respectively. The amino acid residues forming the catalytic cavity were identified as Gln460 to Ala480.Item Development of an actinobacteria based in vitro transcription and translation systems(University of the Western Cape, 2015) Maake, Takalani Whitney; Tuffin, Marla; van ZylHeterologous metagenomic screening strategies have relied largely on the construction of DNA libraries and screening in Escherichia coli to access novel enzymes. There is an increased demand for the identification of novel lignocellulose degrading enzymes with enhanced biochemical properties which are suitable for applications in industrial processes; biofuels being one of them. The use of heterologous gene expression in function based metagenomic studies has resulted in the discovery of enormous novel bioactive compounds. However, there are limitations associated with using E. coli as a heterologous host which does not allow transcription and translation of all genes in the metagenome. E. coli can only express 40% of the environmental DNA because of promoter recognition, codon usage, and host toxicity of gene products. Therefore alternative strategies for expressing or producing novel enzymes are needed, which can also be employed in metagenomic gene discovery. In vitro protein synthesis is an important tool in molecular biology and used to obtain proteins from genes for functional and expression studies. These systems may hold the key to unlock more of the potential in metagenomic DNA. The broader aim of the study is to develop non- E. coli based cell-free protein synthesis systems to further the metagenomics screening. In this study, Rhodococcus erythropolis H8 was evaluated for its suitability in cell-free expression. Crude extracts containing the macromolecular components (70S or 80S ribosomes, tRNAs, initiation, elongation and termination factors) fromR. erythropolis were prepared using existing crude extract based cell-free protein synthesis (CFPS) protocols. Three genes were selected and used as templates for synthesis: cell11, xp12 and acetyl xylan esterase (axe10), all previously isolated from metagenomic libraries screened inE. coli. As judged by zymograms and enzyme assays, all enzymes were successfully expressedfrom their native promoters and in recombinants clones using the PtipA promoter, and wereactive. Furthermore, the amounts of XP12 protein produced using pFos-XP_12 was 1.2mg/mlfrom E. coli and 1.67mg/ml from R. erythropolis CFPS, showing that the R. erythropolismachinery was more efficient in the expression of XP12 than the E. coli machinery. To the best of our knowledge this is the first demonstration of a cell-free expression using an actinomycete.Item The diversity of key anabolic genes in antarctic hypolithons(2009) Makhalanyane, Thulani Peter; Cowan, D.A.; Tuffin, MarlaAntarctica is known for its pristine environments. A variety of unsuitable environmental conditions were once thought to render the continent unsuitable for sustaining life. However, metagenomic data have revealed a wealth of species diversity in a range of biotopes.Hypolithons, photosynthetic communities which live under translucent rocks in climatically extreme environments, are an important input source for both carbon (C) and nitrogen (N) in this hyperarid desert environment. Microbial contribution to biogeochemical cycling resulting in fixation of both C and N remains poorly understood. Moreover, there is a reported close interplay between both cycles, with nitrogen being reported to be a limiting factor in carbon assimilation.In this study the diversity of C and N fixing organisms was investigated by using the cbbL and nifH genes as phylogenetic and functional markers. High Molecular weight metagenomic DNA and RNA was extracted from hypolithons. PCR amplification was carried out using cbbL (800 bp for red-like, 1,100 bp for green-like) and nifH (360 bp) gene specific primers.Resultant PCR products were used to construct libraries which were screened for correct sized inserts. Restriction Fragment Length Polymorphism (RFLP) was used to de-replicate clones prior to sequencing. Phylogenetic positions from both clone libraries were established by aligning nucleotide sequences and constructing similarity trees using NJ clustering methods.BLASTn results indicated the presence of previously uncultured organisms which contain cbbL and nifH genes. BLASTn results were characterized by low percentages of maximum identity (typically <95%), a potential indicator of novel taxa. Sequences from respective libraries clustered with cyanobacteria such as Nostoc, Scytonema, and Tolypothrix and α-, β-, and γ-Proteobacteria such as Azotobacter, Agrobacterium and Mesorhizobium. Generally sequence results indicate a largely homogenous, being dominated by specific taxa. Each group may contain potential keystone species, essential for both biogeochemical cycling in oligotrophic environment.Item Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius(Springer Verlag, 2013) Van Zyl, L.J.; Taylor, M.P.; Eley, K.; Tuffin, Marla; Cowan, Donald A.This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans . Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75×105 M−1 s−1 at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45–55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host’s transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35±0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.Item Eukaryotic diversity of miers valley hypoliths(University of the Western Cape, 2012) Keriuscia Gokul, Jarishma; Cowan, D. A.; Tuffin, Marla; Stomeo, F.The extreme conditions of Antarctic desert soils render this environment selective towards a diverse range of psychrotrophic microbial communities. Cracks and fissures in translucent quartz rocks permit an adequate amount of penetrating light, sufficient water and nutrients to support cryptic microbial development. Hypolithons colonizing the ventral surface of these quartz rocks have been classified into three types: cyanobacterial dominated (Type I),moss dominated (Type II) and lichenized (Type III) communities. Eukaryotic microbial communities were reported to represent only a minor fraction of Antarctic communities. In this study, culture independent techniques (DGGE, T-RFLP and clone library construction) were employed to determine the profile of the dominant eukaryotes, fungi and microalgae present in the three different hypolithic communities. The 18S rRNA gene (Euk for eukaryotes), internal transcribed spacer (ITS for fungi) and microalgal specific regions of the 18S rRNA gene, were the phylogenetic markers targeted for PCR amplification from hypolith metagenomic DNA. Results suggest that the three hypolith types are characterized by different eukaryotic, fungal and microalgal communities, as implied by nMDS analysis of the DGGE and T-RFLP profiles. Sequence analysis indicates close affiliation to members of Amoebozoa, Alveolata, Rhizaria (general eukaryote), Ascomycota (fungal) and Streptophyta (microalgal). Many of these clones may represent novel species. This study demonstrates that Dry Valley hypolithons harbour higher eukaryote diversity than previously recognised.Each hypolithon is colonized by specialized microbial communities with possible keystone species. The ecological role of the detected microorganisms in the hypolith environment is also theorized, and a trophic hierarchy postulated.Item Extremophiles in biofuel synthesis(Taylor and Francis Group, 2010) Barnard, Desire; Casanueva, Ana; Tuffin, MarlaThe current global energy situation has demonstrated an urgent need for the development of alternative fuel sourcesto the continually diminishing fossil fuel reserves. Much research to address this issue focuses on the developmentof financially viable technologies for the production of biofuels. The current market for biofuels, defined as fuelproducts obtained from organic substrates, is dominated by bioethanol, biodiesel, biobutanol and biogas, relying onthe use of substrates such as sugars, starch and oil crops, agricultural and animal wastes, and lignocellulosic biomass.This conversion from biomass to biofuel through microbial catalysis has gained much momentum as biotechnologyhas evolved to its current status. Extremophiles are a robust group of organisms producing stable enzymes, which areoften capable of tolerating changes in environmental conditions such as pH and temperature. The potentialapplication of such organisms and their enzymes in biotechnology is enormous, and a particular application is inbiofuel production. In this review an overview of the different biofuels is given, covering those already producedcommercially as well as those under development. The past and present trends in biofuel production are discussed,and future prospects for the industry are highlighted. The focus is on the current and future application ofextremophilic organisms and enzymes in technologies to develop and improve the biotechnological production ofbiofuels.Item Functional characterisation of a novel ferulic acid esterase from malawian hot spring metagenome(2011) Ngobeni, Rhulani; Tuffin, Marla; Bauer, R.; Cowan, D.A.There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to theα/β hydrolase fold-3 domain protein of Burkholderia sp. The RHgene34 protein contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into Escherichia coli Rosetta. p-Nitrophenol (p-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. Kinetic analysis revealed that RHgene34 had a broad range activity on the p-Npesters, from C2 - C14. RHgene34 operates optimally at 45 ºC, pH 9.0 and has a half life of 30 mins at 45 ºC. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.Item Functional characterisation of a thermophilic cellulase from a Malawian metagenomic library(2013) January, Timna; Tuffin, MarlaBiofuels are currently recognised as the most viable source of energy to replace depleting fossil fuel reserves, with bioethanol the most popular alternative alcohol fuel. Producing bioethanol from agricultural waste residues is a feasible socio-economic industrial process. Lignocellulose, from which plant material is composed, is highly recalcitrant to enzymatic degradation and therefore requires a suite of enzymes for complete hydrolysis of the biomass. Metagenomes, particularly from extreme environments, represent an unlimited resource for the discovery of novel biocatalysts for inclusion in industrial processes. Here we report on the cloning and functional characterisation of a novel thermophilic cellulase identified by the functional screening of a Malawian, hotspring sediment metagenomic library. The gene encoding the cellulase, celMHS, composed of 2,705 nucleotides and encoded a polypeptide of 905 amino acids with a predicted molecular mass of about 98 kDa. The in silico translated protein, CelMHS, contained a putative transmembrane domain, a family 4 carbohydrate binding motive (CBM 4), a truncated glycoside hydrolase family 42 (GH42) domain and a N-terminal region that does not have sequence similarity to any previously described domains. Functional characterisation of the recombinant CelMHS demonstrated that the protein displayed an optimal pH of 6.0 and temperature of 100°C. CelMHS had high specific activity toward substrates comprising of β-1,4 linked glucose subunits such as carboxymethyl cellulose, β-D-glucan from barley and lichenan, however, some activity was also observed against avicel, a crystalline cellulose substrate. HPLC analysis of the hydrolysis products produced by CelMHS indicates that this particular enzyme prefers longer chain oligosaccharides. This is, to the best of our knowledge, the first investigation describing the cloning and characterization of a carbohydrate hydrolysing enzyme comprised of the unique sequence architecture: a partial GH42 catalytic domain, a CBM 4 and a unique N-domain sequence. Key words: cellulose, cellulases, lignocellulosic biomass, bioethanol, saccharification, hydrolysis, metagenomic library, thermophilicItem High-level diversity of tailed phages, eukaryote-associated viruses, and virophage-like elements in the metaviromes of Antarctic soils(American Society for Microbiology, 2014) Zablocki, Olivier; van Zyl, Lonnie; Adriaenssens, Evelien M.; Rubagotti, Enrico; Tuffin, Marla; Cary, Stephen Craig; Cowan, Donald A.The metaviromes of two distinct Antarctic hyperarid desert soil communities have been characterized. Hypolithic communities, cyanobacterium-dominated assemblages situated on the ventral surfaces of quartz pebbles embedded in the desert pavement, showed higher virus diversity than surface soils, which correlated with previous bacterial community studies. Prokaryotic viruses (i.e., phages) represented the largest viral component (particularly Mycobacterium phages) in both habitats, with an identical hierarchical sequence abundance of families of tailed phages (Siphoviridae>Myoviridae>Podoviridae). No archaeal viruses were found. Unexpectedly, cyanophages were poorly represented in both metaviromes and were phylogenetically distant from currently characterized cyanophages. Putative phage genomes were assembled and showed a high level of unaffiliated genes, mostly from hypolithic viruses. Moreover, unusual gene arrangements in which eukaryotic and prokaryotic virus-derived genes were found within identical genome segments were observed. Phycodnaviridae and Mimiviridae viruses were the second-mostabundant taxa and more numerous within open soil. Novel virophage-like sequences (within the Sputnik clade) were identified. These findings highlight high-level virus diversity and novel species discovery potential within Antarctic hyperarid soils and may serve as a starting point for future studies targeting specific viral groups.Item Identification and characterisation of hemicellulases from thermophilic Actinomycetes(University of the Western Cape, 2010) Matthews, Lesley-Ann A.; Cowan, Donald A.; Bauer, Rolene; Easton, Samantha; Tuffin, Marla; Faculty of ScienceTo ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. α-L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones.Item Identification of endophytic bacterial communities associated with South African crops: sorghum bicolor (L. Moench), pennisetum glaucum and arachis villosulicarpa(University of the Western Cape, 2014) Maropola, Mapula Kgomotso Annah; Tuffin, MarlaIn this study, the diversity of endophytic bacteria associated with food crops, sorghum (Sorghum bicolor L. Moench), pearl millet (Pennisetum glaucum L.) and groundnut (Arachis villosulicarpa) is investigated using culture-independent techniques: terminal retriction fragment length polymorphism (t- RFLP) and next generation sequencing (NGS). The first objective of this study was to investigate the effect of different DNA extraction protocols on mDNA yield and quality, as well as the diversity of endophytic bacteria retrieved from root and stem tissues (0.1g or 0.3g) of sorghum, pearl millet and groundnut. Protocols used include two classical methods (CTAB- and SDS-based) and five commercial kits: MoBio PowerPlant Pro® DNA Isolation Kit, Qiagen DNeasyR Plant Mini Kit, Fermentas GeneJET Plant Genomic DNA Purification Kit, MoBio PowerSoilTM DNA Purification Kit and MoBio UltraClean® Soil DNA Isolation Kit. Eletrophoresis and the Nanodrop were used to determine DNA yield and purityItem Identification of rhizospheric microorganisms associated with sorghum(University of the Western Cape, 2012) Tshabuse, Freedom; Tuffin, Marla; Raymond, J.B.; Cowan, D.A.Approximately 50% of sorghum (Sorghum bicolour (L.) Moench) produced globally is used as human food, with 95% of its total consumption occurring in Africa. Unfortunately, sorghum crops are prone to pathogenic attack, notably leading to a reduction in production yields. Generally, chemical agents are used as fertilizers and/or biocides to increase crop production. However, these chemicals can have a detrimental environmental impact including the eutrophication of fresh water and marine ecosystems. Thus, there is increased interest in plant growth promoting rhizobacteria (PGPR), as an alternative to chemicals, to facilitate eco-friendly biological control of soil-borne pathogens. PGPRs colonize the plant root system (i.e rhizosphere and rhizoplane) and promote growth and production yields essentially via the biological control of plant pathogens and their role in the nutrient cycles (e.g N fixation). The aim of this study is to characterize the microbial communities associated with sorghum in South Africa, and to identify common bacteria which could further be developed and applied to improve sorghum growth and yield. Sorghum rhizospheric environments (rhizoplane and rhizosphere) were collected from three sites characterized by different agricultural practices (Free State, Limpopo and North West). Denaturing gradient gel electrophoresis (DGGE) and Terminal-restriction fragment length polymorphism (T-RFLP) were used to identify microbial community molecular fingerprints. Sorghum-associated microbial communities were found to be different in all rhizospheric soil samples which could be explained by differences in soil chemistry, agricultural practices and geographical location. The analyses also clearly demonstrated that the sorghum bacterial community structures were similar in the rhizoplane, indicating the strong influence that the sorghum plant has in determining the rhizoplane colonizers. The archaeal community structure from rhizoplane and rhizosphere in each sampling site were dissimilar, which could be explained by differences in soil type and/or agricultural practices. Both the T-RFLP and DGGE analyses revealed that Bacillus sp. were consistently associated with South African Sorghum, Arthrobacter sp. were detected in the rhizoplane, while Uncultured archaea were detected in the rhizoplane of sorghum. These microorganisms represent valuable targets for engineering to promote growth and yield in sorghum.Item Isolation and characterisation of a xylanase producing isolate from straw-based compost(University of the Western Cape, 2012) Mutengwe, Rudzani Ruth; Cowan, D.A.; Bauer, R.; Tuffin, Marla; Smart, M.Lignocellulosic biomass, a waste component of the agricultural industry, is a promising source for use in bioethanol production. Due to a complex structure, the synergistic action of lignocellulosic enzymes is required to achieve complete digestion to fermentable sugars. This study aimed to isolate, identify and characterise novel lignocellulase producing bacteria from thermophilic straw-based compost (71°C). Colonies with different morphological characteristics were isolated and screened for lignocellulosic activity. A facultative aerobic isolate RZ1 showed xylanase, cellulase and lipase/esterase activity. In addition to these activities, it was also able to produce proteases, catalases, amylases and gelatinases. RZ1 cells were motile, rod-shaped, Gram positive and endospore forming. The growth temperature of isolate RZ1 ranged from 25-55°C with optimal growth at 37°C. The 16S rRNA gene sequence was 99% identical to that of Bacillus subtilis strain MSB10. Based on the biochemical and physiological characteristics and 16S rRNA gene sequence, isolate RZ1 is considered a member of the species B. subtilis. A small insert genomic library with an average insert size of 5 kb was constructed and screened for lignocellulosic activity. An E.coli plasmid clone harbouring a 4.9 kb gDNA fragment tested positive for xylanase activity. The xyl R gene was identified with the aid of transposon mutagenesis and the deduced amino acid sequence showed 99% similarity to an endo-1-4-β-xylanase from B. pumilus. High levels of xylanases were produced when isolate RZ1 was cultured (37°C) with beechwood xylan as a carbon source. On the other hand, the production of xylanases was inhibited in the presence of xylose. Marked xylanase activity was measured in the presence of sugarcane bagasse, a natural lignocellulosic substrate. While active at 50°C, higher xylanase activity was detected at 37°C. Isolate RZ1 also produced accessory enzymes such as β-xylosidases and α-L-arabinofuranosidases, able to hydrolyse hemicellulose.