Institute for Microbial Biotechnology & Metagenomics

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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.
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.
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.
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.
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.
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.
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.
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.
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.
The upper temperature for life – where do we draw the line?
(Elsevier, 2004) Cowan, Donald A.
The newly isolated hyperthermophilic archaeal strain 121 grows slowly at 121 8C and even survives short periods at 130 8C. This is another organism that grows best at temperatures well in excess of 100 8C! We should not be astonished so much by the numerical increments but by the biochemical implications of this fact, and we should be excited by the scope provided by this and similar organisms to further our understanding of the evolution and adaptation of molecular structures and systems. And what about the upper limit of life? It appears improbable that the end-point of this search is represented by strain 121. The consensus view is that the true upper limit, where the energetic burden imposed by molecular repair and resynthesis becomes unsustainable, will probably lie in the region of 140–150 8C
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.
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.
Metagenomic methods for the identification of active micro-organisms and genes in biodegradation processes.
(ASM Press, 2007) Cowan, Donald A.; Stafford, William
Structure of an aliphatic amidase from Geobacillus pallidus RAPc8
(International Union of Crystallography, 2007) Kimani, Serah W.; Agarkar, Vinod B.; Cowan, Donald A.; Sayed, Muhammed F.; Sewell, B. Trevor
The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase superfamily and catalyzes the conversion of amides to the corresponding carboxylic acids and ammonia. It shows both amide-hydrolysis and acyl-transfer activities and also exhibits stereoselectivity for some enantiomeric substrates, thus making it a potentially important industrial catalyst. The crystal structure of G. pallidus RAPc8 amidase at a resolution of 1.9 A ˚ was solved by molecular replacement from a crystal belonging to the primitive cubic space group P4232. G. pallidus RAPc8 amidase is homohexameric in solution and its monomers have the typical nitrilase-superfamily α-β-β-α fold. Association in the hexamer preserves the eight-layered α-β-β-α:α-β-β-α structure across an interface which is conserved in the known members of the superfamily. The extended carboxy-terminal tail contributes to this conserved interface by interlocking the monomers. Analysis of the small active site of the G. pallidus RAPc8 amidase suggests that access of a water molecule to the catalytic triad (Cys, Glu, Lys) side chains would be impeded by the formation of the acyl intermediate. It is proposed that another active-site residue, Glu142, the position of which is conserved in the homologues, acts as a general base to catalyse the hydrolysis of this intermediate. The small size of the substrate-binding pocket also explains the specificity of this enzyme for short aliphatic amides and its asymmetry explains its enantioselectivity.
Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China
(BioMed Central, 2007) Pagaling, Eulyn; Haigh, Richard D.; Grant, William D.; Cowan, Donald A.; Jones, Brian; Ma, Yanhe; Ventosa, Antonio; Heaphy, Shaun
Background: We are profoundly ignorant about the diversity of viruses that infect the domain Archaea. Less than 100 have been identified and described and very few of these have had their genomic sequences determined. Here we report the genomic sequence of a previously undescribed archaeal virus. Results: Haloarchaeal strains with 16S rRNA gene sequences 98% identical to Halorubrum saccharovorum were isolated from a hypersaline lake in Inner Mongolia. Two lytic viruses infecting these were isolated from the lake water. The BJ1 virus is described in this paper. It has an icosahedral head and tail morphology and most likely a linear double stranded DNA genome exhibiting terminal redundancy. Its genome sequence has 42,271 base pairs with a GC content of ~65 mol%. The genome of BJ1 is predicted to encode 70 ORFs, including one for a tRNA. Fifty of the seventy ORFs had no identity to data base entries; twenty showed sequence identity matches to archaeal viruses and to haloarchaea. ORFs possibly coding for an origin of replication complex, integrase, helicase and structural capsid proteins were identified. Evidence for viral integration was obtained. Conclusion: The virus described here has a very low sequence identity to any previously described virus. Fifty of the seventy ORFs could not be annotated in any way based on amino acid identities with sequences already present in the databases. Determining functions for ORFs such as these is probably easier using a simple virus as a model system.
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.
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
Molecular characterization of the 16S rRNA Gene of helicobacter fennelliae isolated from stools and blood cultures from paediatric patients in South Africa
(Hindawi, 2011) Smuts, H.E.M; Lastovica, Albert J.
Forty strains of H. fennelliae collected from paediatric blood and stool samples over an 18 year period at a children's hospital in Cape Town, South Africa, were amplified by PCR of the 16S rRNA. Two distinct genotypes of H. fennelliae were identified based on the phylogenetic analysis. This was confirmed by sequencing a portion of the beta subunit of the RNA polymerase (rpoB) gene. All isolates from South Africa clustered with a proposed novel Helicobacter strain (accession number AF237612) isolated in Australia, while three H. fennelliae type strains from the northern hemisphere, NCTC 11612, LMG 7546 and CCUG 18820, formed a separate branch. A large (355bp) highly conserved intervening sequence (IVS) in the 16S rRNA was found in all isolates. Predicted secondary structures of the IVS from the 16S rRNA and 23S rRNA were characterised by a primary stem structure formed by base pairing of the 3 and 5 ends and internal loops and stems. This phylogenetic analysis is the largest undertaken of H. fennelliae. The South African H. fennelliae isolates are closely related to an Australian isolate previously reported to be a possible novel species of Helicobacter. This study suggests that the latter is strain of H. fennelliae.
Multilocus sequence typing methods for the emerging Campylobacter species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus
(Frontiers Media, 2012) Miller, William G.; Lastovica, Albert J.
Multilocus sequence typing (MLST) systems have been reported previously for multiple food - and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.

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