Contamination from various sources has a huge impact on soil health and microbial community composition. Metal contamination of soil in mining scenarios is of concern and is not adequately addressed, particularly with respect to the microbial community. The mining industry is one of the largest contributors to heavy metal contamination of soil in South Africa, especially since the country is one of the major mining countries in the world. Platinum mining is of special importance, since the largest percentage of the world’s reserves of platinum group metals are found and mined in South Africa. Metals from mining activities become irreversibly immobilized in soil systems because they cannot be degraded and has a huge impact on soil systems. In this study, bacteria was isolated from soil samples collected from a platinum mine tailings dam outside Rustenburg. During the warm sampling season (March 2006) most isolates were found, especially in sites 3 and 4. During the colder and drier season (May 2006) there were less isolates. Most of the isolated cultures also displayed a wide temperature growth range, mostly between 24°C - 37°C. Paenibacillus lautus and Bacillus subtilus DN-10 had a growth range between 5°C - 40°C. Culturable metal tolerant bacteria were isolated, purified and identified using 16S rDNA sequences. Nine different species were found namely Paenibacillus lautus strain DS19, Paenibacillus lautus, Paenibacillus sp. C15, uncultured Paenibacillaceae, Bacillus subtilis strain DN-10, Bacillus sp. KDNB5, Bacillus cereus, Stenotrophomonas maltophilia and Alcaligenes sp. DJWH 146-2. The ability of these strains to tolerate metal concentrations were explored by determining their minimum inhibitory concentrations for a selection of metals e.g. aluminum, barium, cobalt, chromium, cadmium, copper, iron, lead, manganese, nickel and mercury. Most isolates were able to tolerate >5mM of the Al\Ni alloy and cobalt. Transmission electron microscopy was used to determine the location of metals inside bacterial cells and electron dispersive X-ray analysis was used to determine the levels of metals inside microbial cells. Bacillus subtilis DN-10 (LDK0306) showed a high MIC (>5mM) for most metals used, except Hg. This strain also had a high percentage (10.26%) of Pb detected in its cells by EDX. This was the highest percentage detected. Plasmids were extracted from the identified strains and can help gain a better understanding of metal tolerance mechanisms used by these isolates. / Thesis(MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013

Contamination from various sources has a huge impact on soil health and microbial community composition. Metal contamination of soil in mining scenarios is of concern and is not adequately addressed, particularly with respect to the microbial community. The mining industry is one of the largest contributors to heavy metal contamination of soil in South Africa, especially since the country is one of the major mining countries in the world. Platinum mining is of special importance, since the largest percentage of the world’s reserves of platinum group metals are found and mined in South Africa. Metals from mining activities become irreversibly immobilized in soil systems because they cannot be degraded and has a huge impact on soil systems. In this study, bacteria was isolated from soil samples collected from a platinum mine tailings dam outside Rustenburg. During the warm sampling season (March 2006) most isolates were found, especially in sites 3 and 4. During the colder and drier season (May 2006) there were less isolates. Most of the isolated cultures also displayed a wide temperature growth range, mostly between 24°C - 37°C. Paenibacillus lautus and Bacillus subtilus DN-10 had a growth range between 5°C - 40°C. Culturable metal tolerant bacteria were isolated, purified and identified using 16S rDNA sequences. Nine different species were found namely Paenibacillus lautus strain DS19, Paenibacillus lautus, Paenibacillus sp. C15, uncultured Paenibacillaceae, Bacillus subtilis strain DN-10, Bacillus sp. KDNB5, Bacillus cereus, Stenotrophomonas maltophilia and Alcaligenes sp. DJWH 146-2. The ability of these strains to tolerate metal concentrations were explored by determining their minimum inhibitory concentrations for a selection of metals e.g. aluminum, barium, cobalt, chromium, cadmium, copper, iron, lead, manganese, nickel and mercury. Most isolates were able to tolerate >5mM of the Al\Ni alloy and cobalt. Transmission electron microscopy was used to determine the location of metals inside bacterial cells and electron dispersive X-ray analysis was used to determine the levels of metals inside microbial cells. Bacillus subtilis DN-10 (LDK0306) showed a high MIC (>5mM) for most metals used, except Hg. This strain also had a high percentage (10.26%) of Pb detected in its cells by EDX. This was the highest percentage detected. Plasmids were extracted from the identified strains and can help gain a better understanding of metal tolerance mechanisms used by these isolates. / Thesis(MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013

The aim of this study was to determine the effects of the heavy metal pollution on microbial diversity along the gradient from a platinum mine tailings dam using culture-dependent (plating methods) and molecular methods. Tailings and soil samples were collected from seven sites (6 samples per site) at increasing distances from the tailings dam. Samples were collected over a two year period and included two rainy and two dry periods. Concentrations of various heavy metals were determined using an inductively coupled plasma mass spectrometer (ICP-MS). The results demonstrated that seasonal variations in metal concentrations occurred and also that concentrations were significantly different'(P < 0.05) between the experimental sites for each metal. The relative relationship between metals was in the following order: Al > Ni > Cu > Cr. Since soil metal concentration benchmarks for South Africa are lacking, the concentrations were compared to the Canadian microbial benchmarks (MB) and Netherlands maximum permissible concentrations (MPC). Concentrations of most of the heavy metals exceeded the MB and MPC. Levels and diversity of culturable fungi and bacteria at each site were determined using plate count methods. Results indicated that levels of bacteria and fungi were not suppressed by high concentrations of heavy metals. Significantly higher levels (P < 0.05) of fungi were found at the sites on the tailings dam (higher concentrations of heavy metals), compared to sites more than 300 m away. A commonly used soil health index (Shannon-Weaver diversity index) was used to compare microbial community diversity at each site and to evaluate whether or not the heavy metal contamination impacted negatively on these soil bacterial and fungal communities. Shannon-Weaver diversity indices were higher at sites on and close to the tailings dam than sites more than 300 m away. However, ratio of fungal to bacterial levels as determined by plate counts was inconsistent. Representatives of bacterial species that were grouped using colony morphology and whole cell protein profiles were identified by 16S rDNA sequences as Bacillus barbaricus (B. barbaricus) and -Paenibacillus lautus {P. Lautus). Restriction enzyme digest, SDS-PAGE and random amplified polymorphic DNA (RAPD) analyses provided supporting evidence that representatives were correctly grouped. Cluster analysis results demonstrated that the RAPD profiles of the metal tolerant P. lautus representatives were sufficiently dissimilar to discriminate between individuals from the spatially separated sites. The spatially separated sites also represented sites with high and low heavy metal concentrations. Observed genetic variability was thus also associated with varying levels of heavy metals. In conclusion, this study demonstrated the potential of using RAPD analysis as biomarkers for genotoxic effects of heavy metals on bacterial genomes. / Masters / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2009.

Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized. In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network. Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript</p>

The aim of this study was to determine the effects of the heavy metal pollution on microbial diversity along the gradient from a platinum mine tailings dam using culture-dependent (plating methods) and molecular methods. Tailings and soil samples were collected from seven sites (6 samples per site) at increasing distances from the tailings dam. Samples were collected over a two year period and included two rainy and two dry periods. Concentrations of various heavy metals were determined using an inductively coupled plasma mass spectrometer (ICP-MS). The results demonstrated that seasonal variations in metal concentrations occurred and also that concentrations were significantly different'(P < 0.05) between the experimental sites for each metal. The relative relationship between metals was in the following order: Al > Ni > Cu > Cr. Since soil metal concentration benchmarks for South Africa are lacking, the concentrations were compared to the Canadian microbial benchmarks (MB) and Netherlands maximum permissible concentrations (MPC). Concentrations of most of the heavy metals exceeded the MB and MPC. Levels and diversity of culturable fungi and bacteria at each site were determined using plate count methods. Results indicated that levels of bacteria and fungi were not suppressed by high concentrations of heavy metals. Significantly higher levels (P < 0.05) of fungi were found at the sites on the tailings dam (higher concentrations of heavy metals), compared to sites more than 300 m away. A commonly used soil health index (Shannon-Weaver diversity index) was used to compare microbial community diversity at each site and to evaluate whether or not the heavy metal contamination impacted negatively on these soil bacterial and fungal communities. Shannon-Weaver diversity indices were higher at sites on and close to the tailings dam than sites more than 300 m away. However, ratio of fungal to bacterial levels as determined by plate counts was inconsistent. Representatives of bacterial species that were grouped using colony morphology and whole cell protein profiles were identified by 16S rDNA sequences as Bacillus barbaricus (B. barbaricus) and -Paenibacillus lautus {P. Lautus). Restriction enzyme digest, SDS-PAGE and random amplified polymorphic DNA (RAPD) analyses provided supporting evidence that representatives were correctly grouped. Cluster analysis results demonstrated that the RAPD profiles of the metal tolerant P. lautus representatives were sufficiently dissimilar to discriminate between individuals from the spatially separated sites. The spatially separated sites also represented sites with high and low heavy metal concentrations. Observed genetic variability was thus also associated with varying levels of heavy metals. In conclusion, this study demonstrated the potential of using RAPD analysis as biomarkers for genotoxic effects of heavy metals on bacterial genomes. / Masters / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2009.

The genus Ceratomyxa Thélohan, 1892 is one of the largest myxozoan genera, the species of which almost always infect the gall bladder of marine teleosts. Although there are over 180 species known globally, prior to this study only three had been described from Australia. This study explores the systematics, host specificity and species richness of Ceratomyxa species from a diverse range of Queensland marine fishes. The first part of this thesis deals with the issues associated with spore based taxonomy and incorporates the first molecular studies to investigate host specificity and radiation in three common families of Great Barrier Reef (GBR) fishes. Twenty-two new species were described in these chapters, and although they were superficially similar in morphology all were genetically distinct. A focus on Ceratomyxa spp. infecting labrid, pomacentrid and serranid fish revealed tight host specificity of parasite species, confirmed the presence of multiple parasite species in a single host and revealed that no parasite radiation had occurred that could be associated with co-evolution with host families. Exploration of intra- and inter-specific variation by sequencing of multiple replicates from host/parasite combinations allowed interpretation of species boundaries within the system. Ten additional species described in Chapter 5 supported the findings from the labrid, pomacentrid and serranid studies but broadened the host range studied. In total 32 Ceratomyxa species were described from Queensland fishes increasing the number of described species in the genus by 15%. This study also reports on undescribed species collected from a further 70 host species and broadens the known host range to seven families of fishes. A checklist of bivalvulidans from marine teleosts in Australia is compiled of described and undescribed species and highlights the abundance of Ceratomyxa relative to other gall bladder dwelling myxosporeans. A phylogeny of the Ceratomyxa based on SSU rDNA analyses explored the taxonomic integrity of the genus. In general, the morphological diagnostic characters that divide the Myxozoa into genera are not well supported by molecular phylogenetic analyses. The relationship between 42 Ceratomyxa spp. and 36 other marine myxosporeans was examined using Bayesian inference, maximum likelihood and maximum parsimony. Results indicate Ceratomyxa is one of the most cohesive lineages within the Myxozoa and that the freshwater fish parasite, C. shasta, does not represent an independent lineage as suggested in previous studies. The strict host specificity of Ceratomyxa species prompted the investigation of species richness on the GBR. Species accumulation curves were used to explore species richness by using Choa2 and Jackknife1 estimators. The estimates suggested Ceratomyxa is almost as rich as their teleost hosts and that 1,600 species could be present on the GBR and as many as 15,000 species may exist globally. As an unexpected result, Ceratomyxa may be the richest metazoan genus in the sea. In light of what the study revealed, a revision of a second genus within the family Ceratomyxidae, Leptotheca, is presented. The boundaries between Ceratomyxa and Leptotheca were unclear. The diagnostic characters that separate these genera (length to thickness ratios) were found to be plastic and the type species of Leptotheca ultimately fitted the diagnosis for Ceratomyxa. To eliminate confusion between these genera and also between Sphaerospora and Leptotheca, the genus Leptotheca was considered invalid, with all species appropriately assigned to other genera based on morphological and biological characters and supported by genetic evidence.

The genus Ceratomyxa Thélohan, 1892 is one of the largest myxozoan genera, the species of which almost always infect the gall bladder of marine teleosts. Although there are over 180 species known globally, prior to this study only three had been described from Australia. This study explores the systematics, host specificity and species richness of Ceratomyxa species from a diverse range of Queensland marine fishes. The first part of this thesis deals with the issues associated with spore based taxonomy and incorporates the first molecular studies to investigate host specificity and radiation in three common families of Great Barrier Reef (GBR) fishes. Twenty-two new species were described in these chapters, and although they were superficially similar in morphology all were genetically distinct. A focus on Ceratomyxa spp. infecting labrid, pomacentrid and serranid fish revealed tight host specificity of parasite species, confirmed the presence of multiple parasite species in a single host and revealed that no parasite radiation had occurred that could be associated with co-evolution with host families. Exploration of intra- and inter-specific variation by sequencing of multiple replicates from host/parasite combinations allowed interpretation of species boundaries within the system. Ten additional species described in Chapter 5 supported the findings from the labrid, pomacentrid and serranid studies but broadened the host range studied. In total 32 Ceratomyxa species were described from Queensland fishes increasing the number of described species in the genus by 15%. This study also reports on undescribed species collected from a further 70 host species and broadens the known host range to seven families of fishes. A checklist of bivalvulidans from marine teleosts in Australia is compiled of described and undescribed species and highlights the abundance of Ceratomyxa relative to other gall bladder dwelling myxosporeans. A phylogeny of the Ceratomyxa based on SSU rDNA analyses explored the taxonomic integrity of the genus. In general, the morphological diagnostic characters that divide the Myxozoa into genera are not well supported by molecular phylogenetic analyses. The relationship between 42 Ceratomyxa spp. and 36 other marine myxosporeans was examined using Bayesian inference, maximum likelihood and maximum parsimony. Results indicate Ceratomyxa is one of the most cohesive lineages within the Myxozoa and that the freshwater fish parasite, C. shasta, does not represent an independent lineage as suggested in previous studies. The strict host specificity of Ceratomyxa species prompted the investigation of species richness on the GBR. Species accumulation curves were used to explore species richness by using Choa2 and Jackknife1 estimators. The estimates suggested Ceratomyxa is almost as rich as their teleost hosts and that 1,600 species could be present on the GBR and as many as 15,000 species may exist globally. As an unexpected result, Ceratomyxa may be the richest metazoan genus in the sea. In light of what the study revealed, a revision of a second genus within the family Ceratomyxidae, Leptotheca, is presented. The boundaries between Ceratomyxa and Leptotheca were unclear. The diagnostic characters that separate these genera (length to thickness ratios) were found to be plastic and the type species of Leptotheca ultimately fitted the diagnosis for Ceratomyxa. To eliminate confusion between these genera and also between Sphaerospora and Leptotheca, the genus Leptotheca was considered invalid, with all species appropriately assigned to other genera based on morphological and biological characters and supported by genetic evidence.

The genus Ceratomyxa Thélohan, 1892 is one of the largest myxozoan genera, the species of which almost always infect the gall bladder of marine teleosts. Although there are over 180 species known globally, prior to this study only three had been described from Australia. This study explores the systematics, host specificity and species richness of Ceratomyxa species from a diverse range of Queensland marine fishes. The first part of this thesis deals with the issues associated with spore based taxonomy and incorporates the first molecular studies to investigate host specificity and radiation in three common families of Great Barrier Reef (GBR) fishes. Twenty-two new species were described in these chapters, and although they were superficially similar in morphology all were genetically distinct. A focus on Ceratomyxa spp. infecting labrid, pomacentrid and serranid fish revealed tight host specificity of parasite species, confirmed the presence of multiple parasite species in a single host and revealed that no parasite radiation had occurred that could be associated with co-evolution with host families. Exploration of intra- and inter-specific variation by sequencing of multiple replicates from host/parasite combinations allowed interpretation of species boundaries within the system. Ten additional species described in Chapter 5 supported the findings from the labrid, pomacentrid and serranid studies but broadened the host range studied. In total 32 Ceratomyxa species were described from Queensland fishes increasing the number of described species in the genus by 15%. This study also reports on undescribed species collected from a further 70 host species and broadens the known host range to seven families of fishes. A checklist of bivalvulidans from marine teleosts in Australia is compiled of described and undescribed species and highlights the abundance of Ceratomyxa relative to other gall bladder dwelling myxosporeans. A phylogeny of the Ceratomyxa based on SSU rDNA analyses explored the taxonomic integrity of the genus. In general, the morphological diagnostic characters that divide the Myxozoa into genera are not well supported by molecular phylogenetic analyses. The relationship between 42 Ceratomyxa spp. and 36 other marine myxosporeans was examined using Bayesian inference, maximum likelihood and maximum parsimony. Results indicate Ceratomyxa is one of the most cohesive lineages within the Myxozoa and that the freshwater fish parasite, C. shasta, does not represent an independent lineage as suggested in previous studies. The strict host specificity of Ceratomyxa species prompted the investigation of species richness on the GBR. Species accumulation curves were used to explore species richness by using Choa2 and Jackknife1 estimators. The estimates suggested Ceratomyxa is almost as rich as their teleost hosts and that 1,600 species could be present on the GBR and as many as 15,000 species may exist globally. As an unexpected result, Ceratomyxa may be the richest metazoan genus in the sea. In light of what the study revealed, a revision of a second genus within the family Ceratomyxidae, Leptotheca, is presented. The boundaries between Ceratomyxa and Leptotheca were unclear. The diagnostic characters that separate these genera (length to thickness ratios) were found to be plastic and the type species of Leptotheca ultimately fitted the diagnosis for Ceratomyxa. To eliminate confusion between these genera and also between Sphaerospora and Leptotheca, the genus Leptotheca was considered invalid, with all species appropriately assigned to other genera based on morphological and biological characters and supported by genetic evidence.

Wolbachia é uma alfaproteobactéria que apresenta simbiose com uma variedade de artrópodos e nematoides, estando entre os mais abundantes gêneros de bactérias intracelulares já descobertos. Na região Neotropical, os estudos sobre tais bactérias e seus hospedeiros, em especial isópodos terrestres, ainda são incipientes. O presente trabalho teve como objetivos: investigar as espécies de isópodos terrestres neotropicais infectados por Wolbachia; analisar a prevalência de infecção, variação genética e relações filogenéticas das linhagens presentes nessas espécies; investigar a simbiose de Wolbachia em nematoides parasitos de tatuzinhos-de-jardim e inferir sobre as possíveis rotas de transmissão horizontal da bactéria entre os isópodos terrestres e os invertebrados que possuam associações ecológicas com os mesmos. A detecção da bactéria foi realizada através de PCRs diagnósticas, utilizando-se o gene 16S rDNA. A infecção pelo simbionte foi registrada pela primeira vez em Atlantoscia floridana e Burmoniscus meeusei. As linhagens de Wolbachia que infectam as espécies nativas de isópodos terrestres, ao contrário das introduzidas, são muito diversas e não se agrupam dentro do Oniclado. Já as sequências presentes em B. meeusei não são relacionadas a nenhuma outra linhagem presente em crustáceos, e nem mesmo fazem parte de qualquer supergrupo conhecido de Wolbachia. Pela primeira vez foi evidenciada a presença da bactéria em um nematoide da família Mermithidae, Agamermis sp., endoparasito do tatu-bola Armadillidium vulgare. Uma vez que as sequências do parasito e do hospedeiro são idênticas, é possível que um evento de transmissão horizontal tenha ocorrido entre ambos. Por fim, a presença de Wolbachia foi examinada em espécies que possuiam relações ecológicas com os isópodos terrestres (predadores, parasitos, foréticos e animais que vivem sob as mesmas condições ecológicas). Entre as espécies associadas, a infecção foi registrada apenas no nematoide parasito e nos ácaros foréticos. Enquanto as linhagens do isópodo hospedeiro e do nematoide se mostraram muito similares, àquelas dos ácaros foréticos não apresentaram relação filogenética com as de seus forontes Balloniscus glaber. Interessantemente, as sequências presentes nos ácaros são proximamente relacionadas com aquelas de B. meeusei, embora mais estudos sejam necessários para esclarecer tal achado. / Wolbachia is a genus of alfaproteobacteria whose members live in symbiosis with a variety of arthropods and nematodes. It is among the richest genera of intracellular bacteria discovered to date. In the Neotropical region, studies on these bacteria and their hosts, especially terrestrial isopods, are still in the initial stages. The objectives of the present study were: to investigate the species of Neotropical terrestrial isopods infected by Wolbachia; to analyze the prevalence of infection, genetic variation, and phylogenetic relationships of the lineages present in these isopod species; to investigate the symbiosis of Wolbachia in parasitic nematodes of pillbugs; and to provide information to support inferences about the possible routes of horizontal transmission of the bacteria between the terrestrial isopods and the invertebrates that are ecologically associated with them. The bacteria were detected by means of diagnostic PCR’s, using the 16S rDNA gene. Infection by this symbiont was recorded for the first time in Atlantoscia floridana and Burmoniscus meeusei. The lineages of Wolbachia that infect the native species of terrestrial isopods, in contrast to the introduced species, are very diverse and do not group within the Oniclade. The sequences present in B. meeusei are not related to any other lineage present in crustaceans, nor to any other known supergroup of Wolbachia. This study is the first to demonstrate the presence of these bacteria in a nematode of the family Mermithidae, Agamermis sp., an endoparasite of Armadillidium vulgare. Since the sequences from the parasite and the host are identical, it is possible that a horizontal transmission event occurred between the two. Finally, the presence of Wolbachia was examined in species that are ecologically associated with terrestrial isopods (predators, parasites, phoretic species, and animals that live under the same ecological conditions). Among the associated species, the infection was recorded only in the parasitic nematode and in the phoretic mites. Whereas the lineages of the isopod host and of the nematode proved to be very similar, those of the phoretic mites showed no phylogenetic relationship with those of their phoront Balloniscus glaber. Interestingly, the sequences present in the mites are closely related to those of B. meeusei, although further studies are necessary to clarify this finding.

Ageing and senescence remain among the most intriguing questions in biology. Saccharomyces cerevisiae has become well established as a fertile model system for the investigation of ageing. Remarkable conservation has been found to exist between interventions extending lifespan in higher animals and yeast – genetic, chemical, and nutritional – suggesting a network of common regulatory pathways controlling large-scale shifts in gene expression involved in senescence. While it has been proposed that epigenetic regulation controls these shifts, evidence remains incomplete. To address this question, novel longevity mutants were isolated in S. cerevisiae using a purpose-designed high-precision screen based on ageing culture outgrowth. A novel long-lived mutant in uncharacterised gene YDR026C was discovered and found to participate in a pathway distinct from TOR signalling, but share epistasis with the histone deacetylase SIR2&Delta;, a well established regulator of replicative longevity and rDNA maintenance. Through equilibrium density centrifugal separation of culture subpopulations, SIR2&Delta; and Ydr026c&Delta; cultures were found to demonstrate reduced and improved maintenance of post-diauxic quiescence respectively, previously shown to underlie chronological survival in strains including snf1&Delta;. Development of a quantified TUNEL-based assay for genome fragmentation indicated early apoptotic-like behaviour in the SIR2&Delta; strain. Microdissection experiments and sectored-colony assays of strains containing an rDNA-embedded ADE2 reporter determined that Ydr026c&Delta; cells also exhibit extended replicative lifespan, and reduced recombination at the rDNA spacer region hotspot, abrogated in SIR2&Delta; strains. SIR2&Delta; is well established to repress RNA polymerase II-derived transcripts in the rDNA spacer region, including IGS1-R. Northern analysis determined Ydr026c also silences transcription in the spacer, possibly through preventing termination of the main rRNA transcript, interfering with IGS1-R expression. By transformation with a vector overexpressing IGS1-R, partial reconstitution of the SIR2&Delta; phenotype was observed, including rDNA hyperrecombination, shortened replicative longevity, and higher-order chromatin structure restoration. These data suggests a model whereby non-coding rDNA spacer transcripts epigenetically determine rDNA maintenance through recombination, leading to physiological phenotypes of replicative and chronological ageing.