No description available.

No description available.

No description available.

No description available.

No description available.

The aim of this study was to understand the microbial communities of deep terrestrial subsurface environments associated with high-organic-matter coal and lignite bearing strata. A sedimentary sequence from the Waikato Basin, New Zealand consisting of interbedded organic-rich layers, sandstones, siltstones and mudstones was drilled. Viable microbial communities were enumerated using most probable number (MPN) series with media selective for a) general heterotrophs b) sulfate-reducing bacteria c) iron(III)- and manganese(IV)-reducing bacteria d) acetogens e) methanogens and f) lignite-reducing bacteria. Subcultures were made from positive MPN enrichment cultures and representative strains were isolated from the sediments. Selected isolates were tested for their metabolic capabilities and physiological characteristics and Molecular genetic techniques were used to investigate the microbial diversity. Viable counts for active metabolic groups ranged from 104 to 105 cells gsed"1 and representatives from 5 bacterial phyla (Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Acidobacterid) were detected. Community size and diversity did not decrease with depth and viable microbes were present in deeply buried and previously heated and possibly sterilised sediments. A large and diverse set of isolates was obtained from the sediments. The collection included several genera previously detected in the deep terrestrial biosphere (Acetobacterium, Sporotalea, Microbacterium, Acidovorax, Sphingomonas) and also genera not typically associated with the deep biosphere (sulfurospirilium). Isolates had a wide range of metabolic capabilities and the collection includes both fermentative and respiring strains. There was also a good overlap between organisms detected using molecular genetic methods and cultivated organisms indicating that these may be important bacteria in situ. There is a substantial and diverse community of Bacteria inhabiting the sediments of the Waikato Basin. Although Archaea were detected in the sediments, none were isolated. The presence of these microbial communities implies that carbon and energy sources must persist within these sediments over millions of years.

My thesis focuses on functions of Rad7/Radl6/AB1: complex (GG-NER complex) that is required for global genome repair (GG-NER), a subpathway of the nucleotide excision repair (NER) pathway, both in vivo and in vitro. Firstly, a putative DNA translocase activity of the GG-NER complex was investigated by using a triple helix strand displacement assay. Previous work in the lab showed that the complex could generate supercoiling in DNA. One way that DNA supercoiling can be induced is via DNA translocase activity. The GGR complex exhibits a similar level of DNA translocase activity to the SV-40 large T-antigen (a well characterised DNA translocase), indicating that the generation of superhelical torsion results from a translocase activity of the GG-NER complex. The activity is required during GGR to facilitate oligonucleotide excision. Secondly, I investigated a putative E3 ubiquitin ligase activity of a complex containing Rad7 and Radl6, and the stability of one of its substrates, Rad4. In response to UV irradiation, the native Rad4 protein which has a half-life of over three hours, is rapidly degraded by the ubiquitin-proteasome pathway. A novel Elongin-Cullin-Socs-box (ECS) type ubiquitin ligase complex, consisting of Rad7, Radl6, Cul3 and Elcl, was identified and was shown to be required for the UV-dependent ubiquitination and degradation of Rad4 in vivo. Furthermore, my data show that the SOCS box domain of Rad7 protein, which is required for the novel E3 ligase activity, was required for the UV-dependent ubiquitination and degradation of Rad4 protein and that Rad4 is a physiological target of this ligase. I showed that this Rad7 containing ubiquitin ligase ubiquitinates Rad4 protein in vitro and that a specific anti-Rad7 antibody inhibited this reaction, suggesting that the SOCS box protein Rad7 is an essential component of this novel ECS type ubiquitin ligase activity. When this ubiquitin ligase is inactive as is the case in the SOCS box mutated rad7 strain (psocs), no significant change in UV survival is observed compared to WT strain. However, when aArad23 mutation is combined with the psocs mutation a significant increase in UV sensitivity is observed in Arad23/psocs strain compared with Arad23 strain. This shows that the effect of the Rad7 containing ECS ligase on UV survival is predominantly observed in the absence of Rad23. I showed that the steady level of Rad4 after UV in psocs/Arad23 strain remains higher than in pRADllArad23 strain, but this does not rescue the UV sensitivity of psocs/Ara/23, indicating that the degradation of Rad4 protein does not correlate with UV survival. My results demonstrate that inducible NER is influenced by the Rad7 ECS ligase complex. Based on my data and other work from our group, it was revealed that ubiquitination of Rad4 in response to UV specifically regulates NER via a pathway that requires de novo protein synthesis, a pathway that is referred to as pathway II. Finally, preliminary experiments were designed and carried out to understand how the ubiquitination of Rad4 by the Rad7 ECS ligase functions in pathway II. In the absence of Rad23, the mRNA level of DDR2 is elevated. Furthermore, Rad23 binds to the promoter region of DDR2 in the absence of DNA damage. This suggests that Rad23 might regulate the transcription of DDR2 by directly binding to the regulatory elements of DDR2. Furthermore, the occupancy of Rad23 at the DDR2 promoter significantly decreased in Aelcl mutant cells, in psocs cells (an K3 ligase mutant), and after cells are exposed to UV, suggesting the possibility that Rad7 HCS ligase regulates a component of the transcriptional response to DNA damage.

Submarine mud volcanoes (MVs) are a type of cold seep environment where sediment, hydrocarbons and other reduced compounds are channelled upwards to the seafloor from significant depth. These sites can be ecological hotspots because of the potential microbial substrates present in the MV ejecta, and are a potential habitat for deep-sourced prokaryotes (Bacteria and Archaea). The microbial communities in sub-surface sediments from four separate MVs in the Gulf of Cadiz (Capt. Arutyunov, Bonjardim, Meknes and Porto) were investigated using a multidisciplinary approach. This involved cultivation and culture-independent molecular genetic-based methods, supplemented by basic pore water geochemistry, activity measurements and direct cell counts. Cultivation-independent 16S rRNA and functional (mcrA and dsrA) gene analyses revealed that the prokaryotes present in the MV sediments were often most closely related to uncultivated organisms. Phylogenetic groups representing major components of the sediment community in these sites included ANME-2a, ANME-la, Miscellaneous Crenarchaeota Group, Deltaproteobacteria and the JS1 candidate division, though community composition varied significantly between MV samples and with depth. Variation in community composition with depth through MV craters paralleled changes in pore water geochemistry indicating this is an important parameter influencing prokaryotic distribution in MV sediments. While containing certain phylogenetic groups 'characteristic' of the deep biosphere, the MV sediments also contained groups commonly associated with near-surface seep environments, suggesting the mud breccia had become colonised by organisms adapted to the present in situ conditions over time. Cultivation analysis showed novel organisms and important functional groups (methanogens and sulphate-reducers) could be cultivated from MV sediment. Pure cultures obtained from Capt. Arutyunov included a putative new species of Arcobacter named "Candidates Arcobacter subtericola" and species belonging to the genera Pseudomonas, Marinobacter, and Halomonas. Enrichments from Meknes contained Bacteria from the groups Bacteroidetes, Fusobacteria, Firmicutes, Spirochaetes, Desulfobulbaceae and Desulfovibrio, and Archaea belonging to the genera Methanogenium and Methanococcoides.

This study demonstrates the effects of different peptones, made from different biological sources and produced by numerous manufacturers, on the growth dynamics and morphology of various bacteria. The effects these differing growth medium constituents have upon the outcome of microbiological procedures, from identification of bacterial species to public health diagnostics, is of great significance. Peptones were assessed as a constituent of the pre-enrichment broth buffered peptone water. Generation times and yields at 24 h were measured using optical density techniques and were significantly affected by the type of peptone employed as nutrient source. Growth characteristics indicated that where peptones performed poorly, this was a result of poor nutrient quantities and not due to the presence of endogenous inhibitors. It is shown that Salmonella exhibit morphological differences, including cultures. which lack flagella and are consequently non-motile, dependent on the peptone constituents of the culture medium. Transfer of a flagellate Salmonella from nutritionally poor media into rich nutrient broth allowed flagella synthesis: indicating that the aflagellate form is still able to produce flagella. Amino acid sequencing of the peptones producing aflagellate organisms showed a relatively low tyrosine concentration: addition of tyrosine and glucose to these media produced flagellate salmonellae. Identification of the Salmonella serotypes is based on flagellar and somatic antigens therefore the absence of flagella may consequently affect complete identification of the serotype. Antibiotic susceptibility of the Enterobacteria was shown to be markedly affected by peptone, causing breakpoints to vary from sensitive to resistant. Thus the inclusion of different, undefined ingredients in growth media has a considerable effect upon the ability of the medium to enumerate bacteria furthermore these medium constituents affect the outcome of scientific research and should be carefully considered before work is commenced.

Repair of UV-induced damage in the cell is crucial to maintain genome integrity and stability. Acetylation of the histone ammo-terminal tails plays key roles in transcription and DNA repair pathways. Acetylation states are determined by a balance between the activities of histone acetyltransferases (HATs) and histone deacerylases (HDACs). This study examined the roles of histone deacetylation in transcription and nucleotide excision repair (NER) in <italic>Saccharomyces cerevisiae</italic> using mutant strains defective in one or more of the genes encoding the deacetylases Rpd3, Hdal, Hosl, Hos2 and Hos3. Single mutations in the genes RPD3 and HDAJ and the various tested combinations of triple mutations did not cause any detectable loss of transcriptional repression of the model MFA2 gene. rpdS and hdal mutants were not found to confer a significant change in sensitivity to UV radiation, however all triple mutations tested resulted in increased UV sensitivity of the cells. Examination of the removal of UV-induced cyclobutane pyrimidine dimers from the genome overall revealed that combined mutations in RPD3, HOSJ and HOS2 or in RPD3, HOSJ and HDAJ resulted in enhanced NER, which intriguingly appears to contradict the UV sensitivity effects. Enhanced NER was also observed at the repressed MFA2 gene for the same triple mutations, indicating roles for the histone deacetylase genes in chromatin alteration during both global and local NER. The NER effects were only observed upon deletion of multiple genes, suggesting a degree of functional redundancy among the deacetylase proteins. Increasing evidence for an epigenetic histone code and interplay between post-translational histone modifications led to speculation that histone phosphorylation may also play a role in NER. Increased UV sensitivity was conferred by specific mutations in the tails of histone H2A and H2B. Genome-wide analysis in a histone mutant with the H2B tail truncated at serine position 125 suggested that any roles for this site in NER are likely to be specific to certain regions of the genome rather than involved in global NER.