Four Fibroporia radiculosa isolates undergoing decay of untreated and 1.2% ammoniacal copper citrate treated wood were evaluated for differential expression of citrate synthase (CS), isocitrate lyase (ICL), glyoxylate dehydrogenase (GLOXDH), succinate/fumarate antiporter (ANTI), and a copper resistance-associated ATPase pump (ATPase). Samples were analyzed at 2, 4, 6, and 8 weeks for oxalate and protein production, enzyme activities, and gene expression. ATPase pump expression was increased in the presence of copper when initial oxalate concentrations were low, suggesting it functions in helping the fungus adapt to the copper-rich environment by pumping toxic copper ions out of the cell. A connection in expression levels between CS, ANTI, ICL, and GLOXDH for the four isolates was found suggesting the production of oxalate originates in the mictochondrial TCA cycle (CS), shunts to the glyoxysomal glyoxylate cycle (ANTI), moves through a portion of the glyoxylate cycle (ICL), and ultimately is made in the cytoplasm (GLOXDH).

Brown rot fungi are notoriously copper-tolerant, which makes them difficult to control with copper-based wood preservatives. Brown rot fungi are also unique because they have evolved a bilateral strategy for decay. Their initial attack involves the production of hydroxyl free radicals to increase wood porosity, followed by an enzymatic onslaught of glycoside hydrolases that free the sugars locked within cellulose and hemicellulose. Our molecular understanding of these biological processes, however, has been hampered by our limited knowledge of the underlying genetic mechanisms. To address this knowledge gap, high-throughput, short-read sequencing was used to conduct a comprehensive analysis of the genomics and transcriptomics of wood decay and copper tolerance in the brown rot fungus Fibroporia radiculosa. The results were impressively informative. In the genomic study, the sequences of 9262 genes were predicted and gene function was assigned to 5407 of the genes. An examination of target motifs showed that 1213 of the genes encoded products with extracellular functions. By mining these genomic annotations, 187 genes were identified with putative roles in lignocellulose degradation and copper tolerance. The transcriptomic study quantified gene expression of the fungus growing on wood treated with a copper-based preservative. At day 31, the fungus was adapting to the preservative, and the wood showed no strength loss. At day 154, the preservative effects were gone, and the fungus was actively degrading the wood, which exhibited 52% strength loss. A total of 917 differentially expressed genes were identified, 108 of which appeared to be regulating wood decay and preservative tolerance. Genes that showed increased expression at day 31 were involved in oxalate metabolism, hydroxyl free radical production by the enzyme laccase, energy production, xenobiotic detoxification, copper resistance, stress response, and pectin degradation. Genes that exhibited higher expression at day 154 were involved in wood polysaccharide degradation, hexose transport, oxalate catabolism, catabolism of laccase substrates, proton reduction, re-modeling the glucan sheath, and shoring up the plasma membrane for acid shock. These newly discovered genes represent a significant step towards accelerating a genome-wide understanding of brown rot decay and tolerance to wood preservatives.

Copper tolerant brown rot fungi are able to depolymerize the structure of wood treated with copper or organic wood preservatives. This research used quantitative polymerase chain reaction (qRT-PCR) combined with RNA-seq to explore what genes of the brown-rot fungus, Fibroporia radiculosa, are expressed when the fungus is overcoming the wood preservatives and decaying the wood. The preliminary study of ACQ-treated wood indicated that the hydrogen peroxide needed for wood decay to proceed may come from AAOX (aryl alcohol oxidase), with oxalate regulation by ODC2 (oxalate decarboxylase), and copper regulation by COP (copper resistance P-type ATPase pump). The principal study measured the expression of ten genes at early, mid, and late stages of decay in wood treated with azole, copper, quat, ACQ, CA, plus untreated. Both AAOX and LCC (laccase) were often expressed at their highest levels early in the decay stages, thus either one or both could be involved in early Fenton chemistry. Expression levels of ICL (isocitrate lyase) and GLOXDH (glyoxylate dehydrogenase) were also highest in early decay stages. Of great interest was the complete lack of expression of the COP gene on copper-treated wood at any decay stage. The most surprising and significant result is the impact the quat-treatment had on the metabolism of the fungus, and lack of impact of the azole-treatment. This research indicates that it is the quat that provides the greatest inhibition of F. radiculosa, more so than the copper. Based on RNA Seq, the total number of genes that were up- or down-regulated on the copper-treatment was 473, with 293 on the quat-treatment, and 185 on the azole-treatment. There were a number of genes with unknown protein functions highly expressed. These data distinctly show that gene expression profiles of F. radiculosa are altered by different wood preservative compositions and the duration of wood decay. These genes and this data needs further analysis and study in order to meet the long term goal of understanding the mechanism of copper-tolerance inFibroporia radiculosa.

Antipredator defenses are ubiquitous in aquatic ecosystems. In the widely studied Chaoborus-Daphnia predator-prey system, Daphnia elicit a variety of phenotypically plastic responses to Chaoborus including: morphological, life history, and behavioral responses. While these inducible defenses benefit the prey, metal contaminants have been shown to interfere with chemosensory functions, thereby inhibiting antipredator defenses and decreasing survivorship. However, in lakes with a history of metal contamination, such as Kelly Lake in Sudbury, Ontario, there is evidence to suggest that Daphnia may have adapted to high, ambient copper concentrations. Using seven distinct Daphnia clones that were hatched from resting eggs from Kelly Lake, we examined morphological and life history traits when clones were exposed to either a nominal concentration of copper, kairomone, or a combination of both. As expected, clones displayed a variety of inducible responses in both kairomone-control and kairomone-copper treatments, which was attributed to genetic variability. Expected trade-offs in life history traits were not always observed, suggesting that inducible traits may be coupled. Furthermore, in contradiction to life history theory, one clone exhibited both increased somatic growth and increased reproductive output, indicating that clones likely adopted adaptive strategies to stressors rather than elicitng trade-offs in traditional traits. Our results indicate that environmentally relevant copper concentrations do not inhibit the induction of antipredator defenses in Daphnia from Kelly Lake, and we conclude that Kelly Lake Daphnia have developed an adaptive tolerance to copper. Adaptation to copper contamination may have implications for resilience in natural Kelly Lake populations. / Thesis (Master, Biology) -- Queen's University, 2012-04-03 19:33:59.137

Sequence analysis of part of a large plasmid carried by Australian environmental isolate of Listeria monocytogenes strain DRDC8 has lead to identification of an islet of genes that encode proteins similar to copper binding and transport genes found in other Gram positive bacteria. Comparative sequence analysis showed that there are at least four genes (pCT0017, pCT0018, pCT0019 and ctpA) on this islet predicted to be involved in copper homeostasis. One of these, ctpA, is predicted to encode a P-type ATPase with a function analogous to CopA, a copper transporting gene in Enterococcus hirae. ORF pCT0017 is likely to be a CopY-like regulatory protein which could control the expression of ctpA. ORF pCT0019 is predicted to be a Cu²⁺ binding protein. In addition, two genes located downstream of the ctpA are predicted to encode a two component regulatory system region. The predicted function of ORF pCT0018 is not clear. A related chromosomal gene (cutR) is predicted to also encode a copper transporting P-type ATPase. To investigate the role of the protein encoded by pCT0018, the growth behavior of L. monocytogenes strain DRDC8, other strains carrying mutations within pCT0018, pCT0019, cutR and ctpA, as well as strains cured of the large plasmid, were grown under conditions of copper stress and starvation. The growth data showed that with the exception of strain DRDC8 and other strains carrying ctpA, most were unable to grow at higher copper concentration (>15 mM CuSO₄) and suggested that the copper homeostasis genes located on the large plasmid are associated with tolerance to high levels of copper. Strain DSE955PL, which carries a cutR mutation and is cured of the large plasmid, was the most sensitive (<5 mM CuSO₄). This indicated that proteins encoded by plasmid genes work synergistically to confer tolerance to copper. Of most interest was the fact that a pCT0018 mutant was more sensitive (<15 mM CuSO₄) to high levels of copper than the wild type parent DRDC8 (<20 mM CuSO₄). This suggested that ORF pCT0018 was necessary for copper tolerance. To investigate the effects of insertion mutations in pCT0017, pCT0018 and ctpA on copper uptake and export, the levels of copper accumulated by these strains was assessed using atomic absorption spectroscopy. A significant difference in copper accumulation among the bacteria strains was observed when either LEB or BHI media were used to culture the bacteria. This data suggested that the growth medium chemicals influence the levels of copper accumulated by cells. However, the effect of these media on bacteria growth rates during copper stress was not significant. Atomic absorption analysis of intracellular copper accumulation suggested that DSE955PL and DSE955 (a chromosome mutant) were able to accumulate copper (80 - 110 mg.gˉ¹ dry weight of cells), whereas DRDC8 and strains carrying mutations in pCT0018, ctpA, and strains cured of the large plasmid, were less able to accumulate copper (30 - 70 mg.gˉ¹ dry weight of cells). This data suggested that cutR may encode a copper export system and that ctpA is involved in copper uptake. To investigate the gene expression profile for pCT0018 under elevated copper, reverse transcriptase PCR was used to detect transcripts encoding pCT0017, pCT0018, pCT0019 and pCT0020 from RNA extracted from L. monocytogenes strain DRDC8 following culture at elevated levels of copper. Although transcripts for each of the target genes were detected, transcription was not responsive to copper, nor was the pattern of transcription consistent with that expected for a single operon. To directly determine whether the protein encoded by the pCT0018 open reading frame was able to bind copper, this gene was cloned in pET15b in frame with an N-terminal Histag and expressed in E. coli. The expressed protein was purified with a Ni-NTA column and shown to contain copper. Attempts to directly show that protein pCT0018 could bind copper by Cu-IMAC were unable to unequivocally show that the protein was immobilized on the column. Purified protein was used to raise a polyclonal antiserum in rabbit and the antiserum was used for Western analysis to test expression of pCT0018 by wild type L. monocytogenes DRDC8 and specific gene mutants. Although the antiserum bound to purified protein, it was not possible to demonstrate binding to native pCT0018 in cell lysates prepared from L. monocytogenes DRDC8. SDS-PAGE of cytoplasmic and cell envelope proteins isolated from L. monocytogenes strains was used to identify proteins expressed in response to copper stress and starvation. No significant differences in protein profiles for cytoplasmic protein were observed. However, copper-immobilized metal affinity chromatography (Cu-IMAC) showed that expression of a number of copper binding proteins were differentially expressed by DRDC8 following growth in copper stress and starvation conditions. Three of these proteins were selected for amino sequence analysis by MALDI-TOFF MS. Two were confirmed to be L. monocytogenes non-heme iron-binding ferritin and a thiol peroxidase, both of which bind copper. The other protein was similar to an unknown protein from L. monocytogenes. Interestingly, no proteins directly implicated with the copper homeostasis islet were identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374407 / Thesis (M.Sc.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

Sequence analysis of part of a large plasmid carried by Australian environmental isolate of Listeria monocytogenes strain DRDC8 has lead to identification of an islet of genes that encode proteins similar to copper binding and transport genes found in other Gram positive bacteria. Comparative sequence analysis showed that there are at least four genes (pCT0017, pCT0018, pCT0019 and ctpA) on this islet predicted to be involved in copper homeostasis. One of these, ctpA, is predicted to encode a P-type ATPase with a function analogous to CopA, a copper transporting gene in Enterococcus hirae. ORF pCT0017 is likely to be a CopY-like regulatory protein which could control the expression of ctpA. ORF pCT0019 is predicted to be a Cu²⁺ binding protein. In addition, two genes located downstream of the ctpA are predicted to encode a two component regulatory system region. The predicted function of ORF pCT0018 is not clear. A related chromosomal gene (cutR) is predicted to also encode a copper transporting P-type ATPase. To investigate the role of the protein encoded by pCT0018, the growth behavior of L. monocytogenes strain DRDC8, other strains carrying mutations within pCT0018, pCT0019, cutR and ctpA, as well as strains cured of the large plasmid, were grown under conditions of copper stress and starvation. The growth data showed that with the exception of strain DRDC8 and other strains carrying ctpA, most were unable to grow at higher copper concentration (>15 mM CuSO₄) and suggested that the copper homeostasis genes located on the large plasmid are associated with tolerance to high levels of copper. Strain DSE955PL, which carries a cutR mutation and is cured of the large plasmid, was the most sensitive (<5 mM CuSO₄). This indicated that proteins encoded by plasmid genes work synergistically to confer tolerance to copper. Of most interest was the fact that a pCT0018 mutant was more sensitive (<15 mM CuSO₄) to high levels of copper than the wild type parent DRDC8 (<20 mM CuSO₄). This suggested that ORF pCT0018 was necessary for copper tolerance. To investigate the effects of insertion mutations in pCT0017, pCT0018 and ctpA on copper uptake and export, the levels of copper accumulated by these strains was assessed using atomic absorption spectroscopy. A significant difference in copper accumulation among the bacteria strains was observed when either LEB or BHI media were used to culture the bacteria. This data suggested that the growth medium chemicals influence the levels of copper accumulated by cells. However, the effect of these media on bacteria growth rates during copper stress was not significant. Atomic absorption analysis of intracellular copper accumulation suggested that DSE955PL and DSE955 (a chromosome mutant) were able to accumulate copper (80 - 110 mg.gˉ¹ dry weight of cells), whereas DRDC8 and strains carrying mutations in pCT0018, ctpA, and strains cured of the large plasmid, were less able to accumulate copper (30 - 70 mg.gˉ¹ dry weight of cells). This data suggested that cutR may encode a copper export system and that ctpA is involved in copper uptake. To investigate the gene expression profile for pCT0018 under elevated copper, reverse transcriptase PCR was used to detect transcripts encoding pCT0017, pCT0018, pCT0019 and pCT0020 from RNA extracted from L. monocytogenes strain DRDC8 following culture at elevated levels of copper. Although transcripts for each of the target genes were detected, transcription was not responsive to copper, nor was the pattern of transcription consistent with that expected for a single operon. To directly determine whether the protein encoded by the pCT0018 open reading frame was able to bind copper, this gene was cloned in pET15b in frame with an N-terminal Histag and expressed in E. coli. The expressed protein was purified with a Ni-NTA column and shown to contain copper. Attempts to directly show that protein pCT0018 could bind copper by Cu-IMAC were unable to unequivocally show that the protein was immobilized on the column. Purified protein was used to raise a polyclonal antiserum in rabbit and the antiserum was used for Western analysis to test expression of pCT0018 by wild type L. monocytogenes DRDC8 and specific gene mutants. Although the antiserum bound to purified protein, it was not possible to demonstrate binding to native pCT0018 in cell lysates prepared from L. monocytogenes DRDC8. SDS-PAGE of cytoplasmic and cell envelope proteins isolated from L. monocytogenes strains was used to identify proteins expressed in response to copper stress and starvation. No significant differences in protein profiles for cytoplasmic protein were observed. However, copper-immobilized metal affinity chromatography (Cu-IMAC) showed that expression of a number of copper binding proteins were differentially expressed by DRDC8 following growth in copper stress and starvation conditions. Three of these proteins were selected for amino sequence analysis by MALDI-TOFF MS. Two were confirmed to be L. monocytogenes non-heme iron-binding ferritin and a thiol peroxidase, both of which bind copper. The other protein was similar to an unknown protein from L. monocytogenes. Interestingly, no proteins directly implicated with the copper homeostasis islet were identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374407 / Thesis (M.Sc.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

<p>The problem is two fold: how does natural selection operate on systems of interacting genes and how does natural selection operate in natural populations. To address the first problem, I have conducted a theoretical investigation into the evolution of control and the distribution of mutations in a simple system of interacting genes, a linear metabolic pathway. I found that control is distributed unevenly between enzymes, with upstream enzymes possessing the greatest control and accumulating the most beneficial mutations during adaptive evolution. To address the second problem, I investigated the evolution of copper tolerance in the common yellow monkeyflower, Mimulus guttatus. I genetically mapped a major locus controlling copper tolerance, Tol1. A Dobzhansky-Muller incompatibility was hypothesized to also be controlled by Tol1, however, we have demonstrated that it maps to another, tightly linked locus, Nec1. Finally, we investigated the parallel evolution of copper tolerance in multiple new discovered mine populations. We found that copper tolerance has evolved in parallel multiple times via at least two distinct physiological mechanisms. In four mine populations, there was a strong signal of selection at markers linked to Tol1, implying that copper tolerance has evolved via the same genetic mechanisms in these populations.</p> / Dissertation