Institute for Microbial Biotechnology & Metagenomics

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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
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.
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.
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.
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.
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.
Non-specificity of Staphylococcus generic primers
(Society for General Microbiology, 2003) Ah Tow, Lemese; Cowan, Donald A.
Our results allow us to conclude that there appears to be significant conservation between the tuf genes of Planococcus, Planomicrobium and Staphylococcus spp., and that although the primer set TstaG422/TStag765 has been shown to possess high specificity, its use for the definitive identification of Staphylococcus spp. must be treated with some caution.
PCR-based detection of non-indigenous microorganisms in ‘pristine’ environments
(Elsevier, 2003) Baker, Gillian; Ah Tow, Lemese; Cowan, Donald A.
PCR-based technologies are widely employed for the detection of specific microorganisms, and may be applied to the identification of non-indigenous microorganisms in ‘pristine’ environments. For ‘pristine’ environments such as those found on the Antarctic continent, the application of these methods to the assessment of environmental contamination from human activities must be treated with caution. Issues such as the possibility of non-human dispersal of organisms, stability and survival of non-indigenous organisms in vivo, the sensitivity, reproducibility and specificity of the PCR process (and particularly primer design) and the sampling regime employed must all be considered in detail. We conclude that despite these limitations, PCR and related technologies offer enormous scope for assessment of both natural and non-indigenous microbial distributions.
Purification, crystallization and preliminary X-ray diffraction analysis of thermostable nitrile hydratase: research letter
(Academy of Science of South Africa (ASSAf), 2004) Cowan, Donald A.; Sayed, Muhammed F.; Tsekoa, Tsepo L.; Cameron, Rory A.; Sewell, B. Trevor
Microbial nitrile hydratases are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. Bacillus strain RAPc8 nitrile hydratase has recently been cloned and functionally expressed in E. coli. Here, the purification, crystallization and preliminary X-ray diffraction data of this nitrile hydratase are described. The heterotetrameric enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 30% PEG 400, 0.1 M MES (pH 6.5) and 0.1 M magnesium chloride were selected for X-ray diffraction studies. A data set complete to 2.5 Å was collected under cryoconditions at the in-house X-ray source at the University of the Western Cape. The space group was determined to be primitive tetragonal (P41212) with unit cell dimensions a = 106.61 Å, b = 106.61 Å, c=83.23 Å, = = =90°; with one dimer per asymmetric unit. Solution of the three-dimensional structure via molecular replacement is in progress.
Review and re-analysis of domain-specific 16S primers
(Elsevier, 2003) Baker, Gillian; Smith, Jacques J.; Cowan, Donald A.
The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the “universal” primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective ‘universal’ Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.
The search for the ideal biocatalyst
(Nature Publishing Group, 2002) Burton, Stephanie G.; Cowan, Donald A.; Woodley, John M.
While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.
Selection of Clostridium spp. in biological sand filters neutralizing synthetic acid mine drainage
(Wiley, 2013) Ramond, Jean-Baptiste; Welz, Pamela J.; Le Roes-Hill, Marilize; Tuffin, Marla I.; Burton, Stephanie G.; Cowan, Donald A.
In this study, three biological sand filter (BSF) were contaminated with a synthetic iron- [1500 mg L-1 Fe(II), 500 mg L-1 Fe(III)] and sulphate-rich (6000 mg L-1 SO2/4-) acid mine drainage (AMD) (pH = 2), for 24 days, to assess the remediation capacity and the evolution of autochthonous bacterial communities (monitored by T-RFLP and 16S rRNA gene clone libraries). To stimulate BSF bioremediation involving sulphate-reducing bacteria, a readily degradable carbon source (glucose, 8000 mg L-1) was incorporated into the influent AMD. Complete neutralization and average removal efficiencies of 81.5 (±5.6)%, 95.8 (±1.2)% and 32.8 (±14.0)% for Fe(II), Fe(III) and sulphate were observed, respectively. Our results suggest that microbial iron reduction and sulphate reduction associated with iron precipitation were the main processes contributing to AMD neutralization. The effect of AMD on BSF sediment bacterial communities was highly reproducible. There was a decrease in diversity, and notably a single dominant operational taxonomic unit (OTU), closely related to Clostridium beijerinckii, which represented up to 65% of the total community at the end of the study period.

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