UWCScholar :: Browsing by Author "Cowan, Donald A."

Browsing by Author "Cowan, Donald A."

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16 S rDNA primers and the unbiased assessment of thermophile diversity
(Portland Press, 2004) Baker, Gillian; Cowan, Donald A.
Our understanding of thermophile diversity is based predominantly on PCR studies of community DNA. ‘Universal’ and domain-specific rRNA gene PCR primers have historically been used for the assessment of microbial diversity without adequate regard to the degree of specificity of primer pairs to different prokaryotic groups. In a reassessment of the published primers commonly used for ‘universal’ and archaeal 16 S rDNA sequence amplification we note that substantial variations in specificity exist. An unconsidered choice of primers may therefore lead to significant bias in determination of microbial community composition. In particular, Archaea-specific primer sequences typically lack specificity for the Korarchaeota and Nanoarchaea and are often biased towards certain clades. New primer pairs specifically designed for ‘universal’ archaeal 16 S rDNA sequence amplification, with homology to all four archaeal groups, have been designed. Here we present the application of these new primers for preparation of 16 S libraries from thermophile communities.
Antarctic Dry Valley mineral soils contain unexpectedly high levels of microbial biomass
(Springer Verlag, 2002) Cowan, Donald A.; Mamais, A.; Russell, Nick A.; Sheppard, Devon M.
We have applied bioluminescent ATP detection methods to microbial enumeration in Antarctic Dry Valley mineral soils, and validated our ATP data by two independent methods. We have demonstrated that ATP measurement is a valid means of determining microbial biomass in such sites, and that the desiccated surface mineral soils of the Antarctic Dry Valleys contain cell numbers over four orders of magnitude higher than previously suggested
Balancing redox cofactor generation and ATP synthesis: key microaerobic responses in thermophilic fermentations
(Wiley, 2013) Loftie-Eaton, Wesley; Taylor, Mark; Horne, Kerry; Tuffin, Marla I.; Burton, Stephanie G.; Cowan, Donald A.
Geobacillus thermoglucosidasius is a Grampositive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real-time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up-regulated, the Krebs (tricarboxylic acid or TCA) cycle non-uniformly downregulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down-regulation of the acetate CoA ligase gene (acs) in addition to up-regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down-regulated) by an up-regulated NADH:FAD oxidoreductase and upregulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygenscavenging electron transport chain likely remained active during low redox conditions. Together with the observed up-regulation of a glyoxalase and down-regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic-to-microaerobic switching were consistent with responses to low pO2 stress.
Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile, Bacillus pallidus
(Springer Verlag, 2002) Bustard, Mark T.; Whiting, Samantha; Cowan, Donald A.; Wright, Phillip C.
The aerobic biodegradation of high-concentration, to 24 g l –1 , 2-propanol (IPA) by a thermophilic isolate ST3, identified as Bacillus pallidus , was successfully carried out for the first time. This solvent-tolerant B. pallidus utilized IPA as the sole carbon source within a minimal salts medium. Cultivation was carried out in 100-ml shake flasks at 60°C and compared with cultivation within a 1-l stirred tank reactor (STR). Specific growth rate () was about 0.2 h–1 for both systems, with a maximum cell density of 2.4 x 10 8 cells ml–1 obtained with STR cultivation. During exponential growth and stationary phase, IPA biodegradation rates were found to be 0.14 and 0.02 g l –1h–1, respectively, in shake-flask experiments, whereas corresponding values of 0.09 and 0.018 g l –1h–1 were achievable in the STR. Generation of acetone, the major intermediate in aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Acetone levels reached a maximum of 2.2–2.3 g l–1 after 72 and 58 h for 100-ml and 1-l systems, respectively. Both IPA and acetone were completely removed from the medium following 160 and 175 h, respectively, during STR growth, although this was not demonstrated within shake-flask reactions. Growth of B. pallidus on acetone or IPA alone demonstrated that the maximum growth rate () obtainable was 0.247 h–1 at 4 g l–1 acetone and 0.202 h–1 at 8 g l–1 IPA within shake-flask cultivation. These results indicate the potential of the solvent-tolerant thermophile B. pallidus ST3 in the bioremediation of hot solvent-containing industrial waste streams.
Biodiversity: so much more than legs and leaves
(AOSIS OpenJournals, 2013) Cowan, Donald A.; Rybicki, Edward P.; Tuffin, Marla; Valverde, Angel; Wingfield, Michael
Microorganisms 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.
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.
Comparison of actinobacterial diversity in Marion Island terrestrial habitats
(University of the Western Cape, 2008) Sanyika, Walter Tendai; Cowan, Donald A.; Faculty of Arts
The major aim of this study is to determine and compare the distribution of bacteria and actinobacteria in Marion Island terrestrial habitats.
Dissemination and survival of non-indigenous bacterial genomes in pristine Antarctic environments.
(Springer Verlag, 2005) Ah Tow, Lemese; Cowan, Donald A.
Continental Antarctic is perceived as a largely pristine environment, although certain localized regions (e.g., parts of the Ross Dependency Dry Valleys) are relatively heavy impacted by human activities. The procedures imposed on Antarctic field parties for the handling and disposal of both solid and liquid wastes are designed to minimise eutrofication and contamination (particularly by human enteric bacteria). However, little consideration has been given to the significance, if any, of less obvious forms of microbial contamination resulting from periodic human activities in Antarctica. The predominant commensal microorganism on human skin, Staphylococcus epidermidis, could be detected by PCR, in Dry Valley mineral soils collected from heavily impacted areas, but could not be detected in Dry Valley mineral soils collected from low impact and pristine areas. Cell viability of this non-enteric human commensal is rapidly lost in Dry Valley mineral soil. However, S. epidermidis can persist for long periods in Dry Valley mineral soil as non-viable cells and/or naked DNA.
Efficient molecular cloning of environmental DNA from geothermal sediments
(Kluwer Academic Publishers, 2002) Wilkinson, Dianne E.; Jaenicke, Thomas; Cowan, Donald A.
An efficient and simple method for constructing an environmental library using mechanically sheared DNA obtained directly from geothermal sediments is presented. The method is based on blunt-end modification of DNA fragments followed by 3' -adenylation using Vent DNA polymerase and Taq DNA polymerase, respectively. The prepared DNA fragments are then ligated into a TA cloning vector and used in the transformation of Escherichia coli. This method has been successfully applied to the cloning of ORFs derived from uncultivated prokaryotes present in geothermal sediment.
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.
Exploring diversity and ecology of nonarchaea in hydrothermal biotopes
(University of the Western Cape, 2005) Galada, Ncebakazi; Cowan, Donald A.; Dept. of Biotechnology; Faculty of Science
The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with “universal” archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of “universal” archaeal primers A571F (5’-GCYTAA AGSRIC CGT AGC-3’) and UA1204R (5’-TTM GGG GCA TRCIKA CCT-3’) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermaystems. Upon sequencing of amplicons it was discovered that Chinese and. New Zealandsamples contained novel nanoarchaeal phylotypes. The preliminary nanoarchaeal phylotypes were used to design nanoarchaeal-specific primer N989R (5'-GGT TTC CGG TGT CAG TTC-3'), which was coupled with A571F and used in screening of nanoarchaeotes. The nanoarchaeal phylotypes identified with these primers were further screened by amplified ribosomal DNA restriction analysis (ARDRA), which was used to explore the diversity of these phylotypes. The novel nanoarchaeotes cluster into 9 cosely related clades which may represent separate species. Three of the New Zealand phylotypes form one separate clade which is closely related to the published nanoarchaeotes. The following nanoarchaeal sequences were submitted to the GenBank, TC9F (AY572420), TC11-5 (AY571283), TC11-B6 (AY727890), TC11-B7 (AY727887), TC11-C4 (AY727886), TC11-C6 (AY727889), TC11-C8 (AY727888), AND TC11-D4 (AY727891). Fluorescence in situhybridization was also used to simultaneously visualize, identify and localize nanoarchaeotes.
High 16S rDNA bacterial diversity in glacial meltwater lake sediment, Bratina Island, Antarctica
(Springer Verlag, 2003) Sjoling, Sara; Cowan, Donald A.
The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny. Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out. Results revealed the presence of a highly diverse bacterial population and a significantly depthrelated composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'GVCGC3'and Msp I. 5'CVGG3', BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units). However, only seven archaeal OTUs were detected, indicating low archaeal diversity. Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the a, c, and d subdivisions of Proteobacteria, the Cytophaga–Flavobacterium– Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.
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.
Hypolithic and soil microbial community assembly along an aridity gradient in the Namib Desert
(Springer, 2013) Stomeo, Francesca; Valverde, Angel; Pointing, Stephen B.; McKay, Christopher P.; Warren-Rhodes, Kimberley A.; Tuffin, Marla I.; Seely, Mary; Cowan, Donald A.
The Namib Dessert is considered the oldest desert in the world and hyperarid for the last 5 million years. However, the environmental buffering provided by quartz and other translucent rocks supports extensive hypolithic microbial communities. In this study, open soil and hypolithic microbial communities have been investigated along an East–West transect characterized by an inverse fog-rainfall gradient. Multivariate analysis showed that structurally different microbial communities occur in soil and in hypolithic zones. Using variation partitioning, we found that hypolithic communities exhibited a fog-related distribution as indicated by the significant East– West clustering. Sodium content was also an important environmental factor affecting the composition of both soil and hypolithic microbial communities. Finally, although null models for patterns in microbial communities were not supported by experimental data, the amount of unexplained variation (68–97 %) suggests that stochastic processes also play a role in the assembly of such communities in the Namib Desert.
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 Science
To 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.
The Institute for Microbial Biotechnology and Metagenomics 2009
(UWC, 2010-01) Cowan, Donald A.
The IMBM Brochure (2009) provides a summary of the staffing, activities and outputs of the Institute for the 2009 academic year
Metagenomic methods for the identification of active micro-organisms and genes in biodegradation processes.
(ASM Press, 2007) Cowan, Donald A.; Stafford, William
Metagenomics, gene discovery and the ideal biocatalyst
(Portland Press, 2004) Cowan, Donald A.; Arslanoglu, A.; Burton, Stephanie G.; Cameron, Rory A.; Baker, Gillian; Smith, Jacques J.; Meyer, Quinton
With the rapid development of powerful protein evolution and enzyme-screening technologies, there is a growing belief that optimum conditions for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the ‘ideal biocatalyst’ for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of ‘sequence space’ that is not addressed by traditional screening.
Micro-Eukaryotic diversity in Hypolithons from Miers Valley, Antarctica
(Multidisciplinary Digital Publishing Institute (MDPI), 2013) Gokul, Jarishma K.; Valverde, Angel; Tuffin, Marla; Cary, Stephen Craig; Cowan, Donald A.
The discovery of extensive and complex hypolithic communities in both cold and hot deserts has raised many questions regarding their ecology, biodiversity and relevance in terms of regional productivity. However, most hypolithic research has focused on the bacterial elements of the community. This study represents the first investigation of micro-eukaryotic communities in all three hypolith types. Here we show that Antarctic hypoliths support extensive populations of novel uncharacterized bryophyta, fungi and protists and suggest that well known producer-decomposer-predator interactions may create the necessary conditions for hypolithic productivity in Antarctic deserts.
The microbial composition of a natural methanogenic consortium
(University of the Western Cape, 2005) Mashaphu, Nthabiseng; Cowan, Donald A.; Dept. of Biotechnology; Faculty of Science
Wetlands account for approximately 20% of annual global methane emissions. Many wetlands receive inputs of organic matter, nutrients, metals and various toxic compounds from adjacent agricultural and industrial areas. The present study aimed to investigate the microbial composition of a natural methanogenic consortium. A consortium-based molecular approach to study diversity of methanogenic microbial communities in a natural wetland at the primary inflow was used. Key microorganisms of a nethane producing consortium were identified. Extracted high molecular mss DNA ws analysed by PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rDNA. This study was also aimed to identify syntrophic microorganisms in the wetland system. The data obtained suggest a well established syntrophic relationship within the wetland.

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