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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
No abstract available.
Forest industries are expected to bolster the renewable resource economy, but must contend with ecological challenges in maintaining the long-term fertility of forest plantation soils, and technological challenges in converting forest biomass into industrially relevant sources of carbon and energy. This thesis advances research related to both, first by describing the broad changes in soil microbial communities in the decades following timber harvesting, their implications for soil processes and the influence of biomass retention for mitigation (Chapter 3) and, second, by conducting the first comprehensive culture-independent survey of lignocellulolytic organisms in forest soils to expand knowledge of their diversity and catabolic capabilities (Chapter 4). Analysis of over 1,300 bacterial (16S rRNA gene) and fungal (ITS region) pyrotag libraries demonstrated consistent changes in microbial communities at harvested sites across North America, such as i) the increase of desiccation- and heat-tolerant organisms, ii) the general decline of ectomycorrhizal (EM) fungi with a rise of select EM genera (Suillus and Thelephora), iii) the moderation of population shifts by organic matter retention and iv) changes in the functional character of harvested soils, including reduced methanotrophic populations and cellulolytic activity. Biogeographical differences in community structure revealed the potential for variation in the impacts of harvesting. Overall, a number of taxonomic groups were identified that may be important indicators for assessing the long-term impact of timber harvesting. Stable isotope probing revealed the degradation of model hemicellulose, cellulose and lignin substrates by specialized taxa, active on a sole substrate, and groups capable of degrading all three plant polymers, such as members of Burkholderiales and Caulobacteraceae. Bacterial lignin-degraders were more active than fungi in soil microcosms, represented by taxa with characterized lignolytic capability (Sphingobacteriaceae and Sphingomonadaceae) and novel taxa, such as members of Elusimicrobia and Acidobacteria. Differences in lignocellulolytic populations were observed among ecozones and soil layers. Mineral soils harboured a greater proportion of poorly characterized functional taxa and represent reservoirs of unexplored catabolic diversity. Metagenome assembly was ~3 to 20-fold higher as a result of SIP, providing a trove of sequence data containing carbohydrate- and lignin-active enzymes from lignolytic and cellulolytic taxa for future characterization.
Actinomycetes are an abundant bacterial group in soil, with a critical role in the decomposition of organic matter. Rhodococcus jostii strain RHA1 is of particular interest to the field of bioremediation because it can degrade a broad range of organic compounds, both natural and xenobiotic. Understanding the factors contributing to the desiccation resistance of RHA1 will enrich our basic knowledge of this common soil stress and may help advance bioremediation technologies for contaminated soils subject to droughts.Here I report the first transcriptomic analysis of a Gram-positive bacterium during desiccation. Filtered RHA1 cells incubated at either low relative humidity, as an air-drying treatment, or high relative humidity, as a control, were transcriptionally profiled over a comprehensive time series. Also, the morphology of RHA1 cells was characterized by cryofixation scanning electron microscopy during each treatment. Desiccation resulted in a transcriptional response of 819 differentially regulated genes, 8-times more than in the control. Included among the highly up-regulated desiccation-specific genes was dps1 (induced 33-fold), encoding an oxidative stress protection protein which has not previously been directly associated with desiccation, as well as sigF3 (induced 58-fold), encoding a sigma factor possibly involved in the regulatory response to desiccation.RHA1 mutants with dps1 or both of its dps homologs deleted were challenged with oxidative stressors under a variety of assay conditions. The mutants were also exposed to physiological stresses that generate reactive oxygen species intracellularly, including desiccation. In all cases, the dps− mutants did not have impaired oxidative stress resistance – a novel finding with respect to bacterial dps-null strains. Additionally, the RHA1 dps-null mutant did not have substantially lower survival compared to the wild type when challenged with metal toxicity or DNA-damaging agents or when they were cocultured through multiple cycles of starvation. Nevertheless, expression of RHA1 dps1 in an Escherichia coli dps– mutant restored its hydrogen peroxide resistance. Purified RHA1 Dps1 was shown to have ferroxidase activity and thereby to protect DNA from oxidative damage. The general insensitivity of the RHA1 dps-null mutant may be representative of a large group of Actinobacteria for which robust oxidative stress tolerance is an important adaptation.
Master's Student Supervision (2010 - 2018)
Forests are essential for maintaining global climate and biodiversity, with industrial applications vital to the world economy. Forest soils are inhabited by a highly diverse community of macro- and microorganisms which are responsible for a variety of fundamental ecosystem services such as decomposition, and nutrient cycling. The effects of forest disturbance on soil microorganisms specific to these key processes have yet to be studied thoroughly. Bearing in mind the importance of forest soil organisms, I have identified and investigated the long-term effects of forest disturbance by timber harvesting on bacterial and fungal populations that degrade hemicellulose using molecular techniques coupled to stable-isotope probing (SIP) with ¹³C-hemicellulose. I identified 104 putatively hemicellulolytic bacterial operational taxonomic units (OTUs) and 52 putatively hemicellulolytic fungal OTUs. Based on analysis of ¹³C-enriched phospholipid fatty acids and DNA, harvesting resulted in long-term changes in relative abundances of putatively hemicellulolytic bacterial and fungal populations. Although harvesting resulted in long-term changes in these populations, no statistically significant differences in potential hemicellulolytic activity of the soils was observed, suggesting functional redundancy in this fundamental ecosystem process. Additionally, I identified Methylibium, a genus of facultative methylotrophs as a novel putative hemicellulose degrader. This study is the first to extensively survey both bacterial and fungal soil microorganisms specific to hemicellulose degradation using stable-isotope probing, and to provide evidence for long-term effects of timber harvesting on these populations. These results contribute towards the strategic management of forest ecosystems, and the identification of novel hemicellulolytic organisms in this study will pave new roads for industrial applications of cellulolytic and hemicellulolytic enzymes.
In this study, I investigated two novel transporters associated with cholic acid catabolism in Rhodococcus jostii RHA1. Reverse-transcriptase quantitative-PCR indicated that an ABC transporter was upregulated 16.7-fold and an MFS transporter was upregulated 174-fold during the exponential phase of growth on cholic acid compared to pyruvate. With gene knockout analysis, I discovered that these transporters are required for the reassimilation of distinct cholic acid metabolites. The ABC transporter, encoded by the camABCD genes, was essential for uptake of 12-hydroxy-9-oxo-1,2,3,4,10,19,23,24-octanorcholan-5,22-dioic acid and 12-hydroxy-9-oxo-1,2,3,4,10,19,23,24-octanorchol-6-en-5,22-dioic acid. The MFS transporter, encoded by the camM gene, was essential for uptake of 3,7,12-trihydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,5(10)-trien-22-oic acid. The uptake of these metabolites is necessary for maximal growth on cholic acid: the ΔcamB mutant, lacking the permease component of the ABC transporter, and the ΔcamM mutant, lacking the MFS transporter, only achieved 74% and 77%, respectively, of the final wild type protein yield. These metabolites differ from previously reported cholic acid metabolites from Proteobacteria in that they retain an isopropionyl side chain at the C17 position. This study is the first to demonstrate the function of putative cholic acid genes through targeted mutagenesis, as well as the first to provide evidence for the requirement for transporters involved in cholic acid metabolite uptake. This work highlights the importance and complexity of transport processes associated with bacterial catabolism and may contribute to industrial applications involving bacterial steroid transformation.