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Don A Cowan

Publications and source records attributed to Don A Cowan.

15 recordsLinked to original sources
PubMed2007

Subtractive hybridization magnetic bead capture: a new technique for the recovery of full-length ORFs from the metagenome.

A new method for the recovery of full-length open reading frames from metagenomic nucleic acid samples is reported. This technique, based on subtractive hybridization magnetic bead capture technology, has the potential to access multiple gene variants from a single amplification reaction. It is now widely accepted that classical microbiological methods provide only limited access to the true microbial biodiversity (less than 1%). The desire to access a higher proportion of the metagenome has led to the development of efficient environmental nucleic acid extraction technologies and to a range of sequence-dependent and sequence-independent gene discovery techniques. These methods avoid many of the limitations of culture-dependent gene targeting.

Chromosome Mapping↗
PubMed2006

Caldalkalibacillus thermarum gen. nov., sp. nov., a novel alkalithermophilic bacterium from a hot spring in China.

A thermophilic, alkaliphilic and catalase-positive bacterium, designated strain HA6(T), was isolated from a hot spring in China. The strain was aerobic and chemo-organotrophic and grew optimally at 60 degrees C, pH 8.5 and 1.5 % (w/v) NaCl. The cells were Gram-positive rods, forming single terminal endospores. The predominant cellular fatty acids were iso-C(15 : 0) and iso-C(17 : 0). The cell-wall peptidoglycan contained meso-diaminopimelic acid. The genomic DNA G+C content was 45.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain HA6(T) formed a distinct lineage within the family Bacillaceae and was most closely related to Bacillus horti K13(T) and Bacillus smithii DSM 4216(T), with sequence similarities of 91.8 and 93.1 %, respectively. On the basis of its physiological and molecular properties, strain HA6(T) should be placed in a novel genus and species, for which the name Caldalkalibacillus thermarum gen. nov., sp. nov. is proposed. The type strain of Caldalkalibacillus thermarum is strain HA6(T) (=CGMCC 1.4242(T)=JCM 13486(T)).

China↗
PubMed2006

Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples.

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at -20 degrees C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

Acanthamoeba↗
PubMed2005

Molecular analysis of the nitrile catabolism operon of the thermophile Bacillus pallidus RAPc8.

The gene cluster containing the nitrile hydratase (NHase) and amidase genes of a moderate thermophile, B. pallidus RAPc8 has been cloned and sequenced. The (5.9 kb) section of cloned DNA contained eight complete open reading frames, encoding (in order), amidase (belonging to the nitrilase related aliphatic amidase family), nitrile hydratase beta and alpha subunits (of the cobalt containing class), a 122-amino acid accessory protein, designated P14K, a homologue of the 2Fe-2S class of ferredoxins and three putative proteins with distinct homology to the cobalt uptake proteins cbiM, cbiN and cbiQ of the S. typhimurium LT2 cobalamin biosynthesis pathway. The amidase and nitrile hydratase genes were subcloned and inducibly expressed in Escherichia coli, to levels of approximately 37 U/mg and 49 U/mg, respectively, without the co-expression of additional flanking genes. However, co-expression of P14K with the NHase structural genes significantly enhanced the specific activity of the recombinant NHase. This is the first description of an accessory protein involved in thermostable NHase expression. Modelling of the P14K protein structure has suggested that this protein functions as a subunit-specific chaperone, aiding in the folding of the NHase alpha subunit prior to alpha-beta subunit association and the formation of alpha(2)beta(2) NHase holoenzyme.

Amidohydrolases↗
PubMed2005

Dissemination and survival of non-indigenous bacterial genomes in pristine Antarctic environments.

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.

Antarctic Regions↗
PubMed2005

Halalkalicoccus tibetensis gen. nov., sp. nov., representing a novel genus of haloalkaliphilic archaea.

A haloalkaliphilic archaeon (strain DS12T) isolated from Lake Zabuye, the Tibetan Plateau, China, was characterized to elucidate its taxonomy. The strain was aerobic, chemo-organotrophic, and grew optimally at 40 degrees C, pH 9.5-10.0 and 3.4 M NaCl. Cells of strain DS12T were non-motile cocci and stained Gram-variable. The major polar lipids of strain DS12T were diphytanyl and phytanyl-sesterterpanyl diether derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. No glycolipids were detected. Phylogenetic analysis revealed that the strain formed a distinct lineage within the family Halobacteriaceae. The low 16S rRNA gene sequence similarity values to its closest relatives (91.5-92.5 %) and its signature bases both suggest that the strain has no close affinity with any members of the family Halobacteriaceae with validly published names. Therefore, it is proposed that strain DS12T (=AS 1.3240T=JCM 11890T) represents the type strain of a novel species in a new genus, Halalkalicoccus tibetensis gen. nov., sp. nov.

Base Sequence↗
PubMed2005

Bacterial diversity in the rhizosphere of Proteaceae species.

The Cape Floral Kingdom is an area of unique plant biodiversity in South Africa with exceptional concentrations of rare and endemic species and experiencing drastic habitat loss. Here we present the first molecular study of the microbial diversity associated with the rhizosphere soil of endemic plants of the Proteaceae family (Leucospermum truncatulum and Leucadendron xanthoconus). Genomic DNA was extracted from L. truncatulum rhizosphere soil, L. xanthoconus rhizosphere and non-rhizosphere soil and used as a template for the polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA gene (rDNA). Construction and sequencing of 16S rDNA libraries revealed a high level of biodiversity and led to the identification of several novel bacterial phylotypes. The bacterial community profiles were compared by 16S rDNA denaturing gradient gel electrophoresis (DGGE). Cluster analysis and biodiversity indices revealed that the rhizosphere soil samples were more similar to each other than to non-rhizosphere soil and the rhizosphere soil contained a bacterial diversity that was richer and more equitable compared with non-rhizosphere soil. A Chloroflexus and an Azospirillum genospecies were restricted to the L. xanthoconus rhizosphere soil and Stenotrophomonas genospecies was identified in all rhizosphere soil samples but was not present in the non-rhizosphere soil. Taxon-specific nested PCR and DGGE-identified differences between the Proteaceae plant rhizosphere soil with a Frankia genospecies restricted the L. truncatulum rhizosphere. Archaea-specific rDNA PCR, DGGE and DNA sequencing revealed that Crenarcheote genospecies were excluded from the plant rhizosphere soil and only present in non-rhizosphere soil.

Bacteria↗
PubMed2004

Endangered antarctic environments.

The Antarctic continent harbors a range of specialized and sometimes highly localized microbial biotopes. These include biotopes associated with desiccated mineral soils, rich ornithogenic soils, glacial and sea ice, ice-covered lakes, translucent rocks, and geothermally heated soils. All are characterized by the imposition of one or more environmental extremes (including low temperature, wide temperature fluctuations, desiccation, hypersalinity, high periodic radiation fluxes, and low nutrient status). As our understanding of the true microbial diversity in these biotopes expands from the application of molecular phylogenetic methods, we come closer to the point where we can make an accurate assessment of the impacts of environmental change, human intervention, and other natural and unnatural impositions. At present, it is possible to make reasonable predictions about the physical effects of local climate change, but only general predictions on possible changes in microbial community structure. The consequences of some direct human impacts, such as physical disruption of microbial soil communities, are obvious if not yet quantitated. Others, such as the dissemination of nonindigenous microorganisms into indigenous microbial communities, are not yet understood.

Antarctic Regions↗
PubMed2003

High 16S rDNA bacterial diversity in glacial meltwater lake sediment, Bratina Island, Antarctica.

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 depth-related composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'G/CGC3'and Msp I. 5'C/CGG3', 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 alpha, gamma, and delta 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.

Actinobacteria↗
PubMed2003

PCR-based detection of non-indigenous microorganisms in 'pristine' environments.

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.

Antarctic Regions↗
PubMed2002

Antarctic Dry Valley mineral soils contain unexpectedly high levels of microbial biomass.

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.

Adenosine Triphosphate↗
PubMed2002

Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile, Bacillus pallidus.

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 degrees C and compared with cultivation within a 1-l stirred tank reactor (STR). Specific growth rate (micro) 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(-1) h(-1), respectively, in shake-flask experiments, whereas corresponding values of 0.09 and 0.018 g l(-1) h(-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 ( micro ) 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.

2-Propanol↗
PubMed2002

The search for the ideal biocatalyst.

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.

Animals↗