Browsing by Author "Smith, Jacques J."

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    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.
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    Microbial diversity and gene mining in Antarctic Dry Valley mineral soils
    (University of the Western Cape, 2006) Smith, Jacques J.; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
    Soil communities are regarded as among the most complex and diverse assemblages of microorganisms with estimated bacterial numbers in the order of 109-1 cells.g. Studies on extreme soils however, have reported lower cell densities, supporting the perception that the so-called extreme environments exhibit low species diversity. To assess the extent of microbial diversity within an extreme environment, the mineral soils of the Dry Valleys, Ross Dependency, Eastern Antarctica were investigated using 16S rDNA analysis. Three mineral soils designated MVG, PENP and BIS were analysed, each differing with respect to altitude, protein, lipid, water and DNA content. The mid-altitude sample, MVG, yielded the highest levels of DNA and the low altitude BIS soil contained the highest levels of protein, lipid and water. 16S clone libraries were constructed and 60 unique clones were identified and sequenced. BLASTn analysis revealed eight phylogenetic groups with Cyanobacteria, Actinobacteria and Acidobacteria representing the majority. The Cyanobacterial phylotypes were unique to the desiccated high-altitude soils of the PENP sample, suggesting a soil-borne Cyanobacterial population. 21% of the phylotypes identified were assigned as ‘uncultured’. DNA isolated from the Antarctic mineral soils was also used to construct a metagenomic clone library consisting of 90700 clones with an average insert size of 3.5 kb, representing an estimated 3.4% of the available metagenome. Activity-based screening of the library for genes conferring lipolytic activity yielded no positive clones. It is suggested that the failure to produce positive clones might be a result of insufficient nucleotide coverage of the metagenomic DNA. The metagenomic DNA extracted from the Dry Valley mineral soils was further analyzed using PCR. Two sets of degenerate primers based on conserved regions within lipolytic genes were used to target lipase and esterase genes. One set of primers was selected from a previous study. A second primer set was designed manually from amino acid alignments of true lipase genes from family I, sub-families I-VI. PCR analysis resulted in nine partial gene fragments varying between 240 bp and 300 bp. Bioinformatic analysis revealed that all nine partial gene fragments harboured α/β-hydrolase motifs, putatively identifying two esterases and three lipases from both bacterial and fungal origin.
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    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.