Current project staff
Jacques Smith, PhD student: Metagenomics of Dry Valley soils
Kamini Moodley, MSc student: Phylotypic analysis of Dry Valley soil transects
Jo Rapley, MSc student: Microbiology of Marion Island soils
Rabiu Halimah, Honours student: Novel Antarctic microorganisms
Ashley Uys, Honours student: Phylogenetic analysis of Marion Is. soils

In collaboration with the University of Waikato and Antarctica NZ, we have maintained an active research program on Antarctic microbial diversity [1-5]. We intend to expand our interest in the metagenomics of low temperature environments through participation in the South African National Antarctic Program. We aim to undertake a comprehensive phylogenetic analysis of the bacterial and archaeal diversity in various Antarctic biotopes. We employ ssu rRNA sequence analysis (using bacterial-specific, archaeal-specific and universal primers for PCR) to investigate microbial diversity [6]. Phylotypic diversity is assessed by a combination of direct sequencing, DGGE and ARDRA analyses.
We also use both aerobic isolation and metagenomic cloning strategies to target specific groups of enzymes with biotechnological applications. Our initial focus will be the esterase/lipase group, where cold-active enzymes of novel

 

specificity are required for a variety of applications (chiral separations, ester synthesis). The characterisation and biotransformation activity of these enzymes will be carried out in collaboration with Professor SG Burton, Department of Chemical Engineering, UCT.

 

Current project staff
Sam Muyanga, Post-Doctoral: Gene mining from thermophilic biotopes
Tony Okoh, Post-Doctoral: Analysis of genes identified through metagenomic screening
Patience Basvi, PhD student: Novel enzymes from extremophilic metagenome screening
Mornay du Plessis, PhD student: Generation and annotation of metagenomic sequence data (with Professor W Hide and Ms Z Arieff, UWC)
Kazi Galada, MSc student: Phylotypic analysis of hyperthermophilic biotopes
Hubert Ontong, Honours student: Isolation of novel thermophilic organisms

In this program, we focus on genomes and gene products from bacteria and archaea inhabiting thermophilic and hyperthermophilic environments. Thermostable enzymes from these organisms are a current focus for industrial implementation, largely because of the inherent benefits of

molecular stability with respect to harsh process conditions. This long-term program uses hydro-thermal source material from New Zealand and China (as part of a European Union collaboration with the Universities of Leicester and Seville, Genencor International and the Institute of Microbiology,

Beijing) as a basis for our gene-discovery program. We employ a range of 'metagenomic' methods for the identification of novel genes. These include the preparation of multigenomic libraries [7] and gene-specific PCR methods [8].
We are also developing methods for directly accessing microbial genome ORFs by direct metagenomic sequencing. We intend to use a simplified variant of the 'shotgun' cloning method used for sequencing the Thermoplasma acidophilum genome [9] to obtain Mbp sequence data. The student undertaking this project will be co-supervised by Professor W Hide, SANBI and Dr Z Arieff, a member of academic staff in this Department. The project will include the in silico identification of ORFs, their annotation and subsequent functional verification by PCR cloning and expression. The project focuses specifically on the organic-polymer hydrolase group (protease, lipase, cellulase, mannanase, amylase).

 

Current project staff
William Stafford, Post-Doctoral: Microbial populations associated with endemic fynbos Proteaceae.
Estela Stenico, Post-Doctoral: Biosolubilisation of low rank coals (with Professors Steph Burton and Sue Harrison, UCT)
Pia Wittwer: Research Assistant: Novel oxidative enzymes from soil metagenomes
Joseph Lako, MSc student: Phylotypic analysis of fynbos-associated microbiota
Antoinette van Schalkwyk, PhD student: Engineering microbial strains for phenolics degradation (with Professor Steph Burton, UCT)
Patience Basvi, PhD student: Novel enzymes from extremophilic metagenome screening
Mornay du Plessis, PhD student: Generation and annotation of metagenomic sequence data (with Professor W Hide and Ms Z Arieff, UWC)
Eloise Kuhn, MSc, student: Survival of microorganisms in acid mine drainage-fly ash co-disposal processes.

The ARCAM laboratory has an active research interest in the analysis of national endemic microbial diversity, and the exploitation of microbial genomes as sources of bioproducts and in waste treatment (bioremediation). Several major project proposals in this area are currently under review. For example, we are proposing that the genetic or geographical clusters of the 600+ endemic species of Western Cape Proteaceae will harbour significantly differing rhizosphere microbial populations. Our preliminary analyses will employ ssu rRNA gene amplification together with DGGE analysis.
We will employ similar techniques as part of a collaboration to identify the important prokaryotic and eukaryotic species in wet-land waste treatment systems. This project will investigate changes in microbial distribution between 'pristine' and impacted sites.
Coal mining in South Africa is estimated to produce in excess of 200 ML of acid mine drainage (AMD) per day, while electricity production yields over 30 Mt of ash p.a.

A large number of collieries in South Africa are tied to power stations where the two waste streams, acid mine drainage and fly ash (FA), have the capacity to neutralize each other via co-disposal. As part of a large national collaboration, we will investigate the microbial impacts of this process. This study is highly relevant to the future success of the co-disposal system, as microbial activity may either assist in remediation of the toxic metal ion content or have a detrimental impact through mobilisation of heavy metals.

 

Current project staff
Kenneth Chinkwo, Research Associate: Enzymology of hyperthermophilic Archaea
Tsepo Teskoa, PhD student: Structural analysis and engineering of nitrile hydratases (with Dr Muhammed Sayed, UWC)
Imran Khan, Honours student: Analysis of enzymes from metagenomics screening

We have recently cloned, sequenced and expressed the gene for a thermostable nitrile hydratase [10,11]. Specificity studies show a preference for aliphatic and non-aromatic nitriles, whereas aromatic nitriles act as competitive inhibitors. We have investigated the molecular basis of this specificity by using homology modeling (based on the published Rhodococcus nitrile hydratase crystal structure) and identified a series of hydrophobic binding site residues which we propose are involved in the non-productive binding of aromatic nitrile substrates. These residues are targets for site-specific mutagenesis. The molecular evolution of a thermostable nitrile hydratase with aromatic specificity will not only provide important structure-function relationship information, but the engineered enzyme will have substantial potential in industrial biotransformations (e.g., in the synthesis of acrylamide and nicotinamide). In collaboration with Dr M Sayed (a new member of staff in the Department of Biotechnology), we aim to obtain a crystal structure of the recombinant nitrile hydratase and several site-specific mutants in order to understand (and engineer) the basis of the enzyme's substrate specificity.

We are also interested in identifying additional novel thermostable and cold-active nitrile metabolising enzymes.

Three approaches will be used: (a) Classical screening on selective media, (b) Multigenomic methods (see above) using complementation screening of recombinants on N-minus media, and (c) PCR amplification of homologous genes from community DNA extracts (using conserved primer sequences from nitrile hydratase gene alignments).

References

[1] Sjoling, S. and Cowan, D.A. (2000) Presence of human-specific enteric micro-organisms in current and historic field camp sites. Polar Biology, 23, 644-650.
[2] Cowan, D.A., Mamais, A. Sheppard, D. and Russell, N. (2002) Antarctic Dry Valley mineral soils contain unexpectedly high levels of microbial biomass. Extremophiles, 6, 431-436.
[3] Baker, G, Ah Tow, L, Cowan, DA (2003) Detection of non-indigenous micro-organisms in 'pristine' environments. J. Microbiol. Methods, 53, 157-164
[4] Sjoling, S and Cowan, DA (2003) High 16S rDNA bacterial diversity in maritime lake sediment, Bratina Island, Antarctic, Extremophiles, 7, 275-282.
[5] Ah Tow, L and Cowan, DA (2003) Non-specificity of Staphylococcus generic primers. Microbiology, 149, 1605-1607.
[6] Baker, G, Smith, JJ and Cowan DA Review and re-analysis of Domain-specific 16S primers, J. Microbiol. Methods, 55:451-455.
[7] Wilkinson, D., Jaenicke, T. and Cowan, D.A. (2002) Efficient cloning of DNA from environmental sources. Biotechnol. Lett. 24, 155-161.
[8] Burton, S., Cowan, D.A. and Woodley, J.M. (2002) The search for the ideal biocatalyst. Nature Biotechnology. 30, 35-46
[9] Reupp, A et al. (2000) Genome sequence of Thermoplasma acidophilum. Nature 407: 508-513.
[10] Cameron, RC (2003) Analysis of the nit operon of the thermophile Bacillus pallidus. PhD Thesis, University of London. 248 pp.
[11] Cameron, R, Tsekoa, T and Cowan, DA (2003) Comparative biology of thermophilic and mesophilic nitrile hydratases, Adv. Appl. Microbiol. 52, 123-158.