http://hdl.handle.net/2262/190 Microbiology (Scholarly Publications) 2013-06-18T05:18:23Z http://hdl.handle.net/2262/64065 Title: Tup1-Ssn6 and Swi-Snf remodelling activities influence long-range chromatin organization upstream of the yeast SUC2 gene. Author: FLEMING, ALASTAIR Abstract: The traditional model for chromatin remodelling during transcription has focused upon the remodelling of nucleosomes at gene promoters. However, in this study, we have determined that Tup1-Ssn6 and Swi-Snf chromatin remodelling activities extend far upstream of the SUC2 gene promoter into the intergenic region of the Saccharomyces cerevisiae chromosome. We mapped the nucleosomal array over a 7.5 kb region that encompassed the SUC2 gene promoter and upstream region but was devoid of other transcriptionally active genes. Nucleosome positioning over this region was determined under conditions of glucose repression and derepression, and in snf2, ssn6 and snf2 ssn6 mutant strains. A map detailing remodelling events extending as much as 5 kb upstream of the SUC2 gene promoter underlines the roles of the Tup1-Ssn6 and Swi-Snf complexes in respectively organizing and disrupting nucleosome arrays. The gene specificity of these events suggests a role in gene regulation. We propose that long-range chromatin remodelling activities of Swi-Snf and Tup1-Ssn6 may ultimately influence whether the chromosomal state of the SUC2 gene is proficient for transcription. These data raise the possibility that remodelling of extensive chromatin domains may be a general property of the complexes. Description: PUBLISHED 2007-01-01T00:00:00Z http://hdl.handle.net/2262/64051 Title: Extracellular enzyme synthesis in a sporulation-deficient strain of Bacillus licheniformis. Author: FLEMING, ALASTAIR Abstract: A deletion of the spoIIAC gene of Bacillus licheniformis was prepared in vitro by using the splicing-by-overlapextension technique. This gene was introduced into B. licheniformis on a temperature-sensitive plasmid, and following integration and excision from the chromosome, a precisely located deletion on the chromosomal gene was prepared. The mutated bacterium was totally asporogenous and formed abortively disporic cells characterized by asymmetric septa at the poles of the cells. Qualitative plate tests revealed that the bacterium synthesized normal levels of DNase, polygalacturonate lyase, protease, RNase, and xylanase, but the hydrolysis zones due to b-1,3-glucanase and carboxymethyl cellulase activity were smaller in the mutant than in the parent strain. The synthesis of alkaline protease was the same in batch cultures of the mutant and the parent during prolonged incubation for 72 h, but the a-amylase yields were reduced by about 30% by the mutation. Description: PUBLISHED 1995-01-01T00:00:00Z http://hdl.handle.net/2262/62432 Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid Author: GEOGHEGAN, JOAN; FOSTER, TIMOTHY JAMES Abstract: The Sbi protein of Staphylococcus aureus comprises two IgG binding domains similar to those of protein A and a region that triggers the activation of complement C3. Sbi is expressed on the cell surface but its C-terminal domain lacks motifs associated with wall or membrane anchoring of proteins in Gram-positive bacteria. Cell-associated Sbi fractionates with the cytoplasmic membrane and is not solubilised during protoplast formation. S.aureus expressing Sbi truncates of the C-terminal Y domain allowed identification of residues that are required for association of Sbi with the membrane. Recombinant Sbi bound to purified cytoplasmic membrane material in vitro and to purified lipoteichoic acid. This explains how Sbi partitions with the membrane in fractionation experiments yet is partially exposed on the cell surface. An LTA-defective mutant of S. aureus had reduced levels of Sbi in the cytoplasmic membrane. Description: PUBLISHED 2012-01-01T00:00:00Z http://hdl.handle.net/2262/61581 Title: The nucleoid-associated protein HU controls three regulons that coordinate virulence, response to stress and general physiology in Salmonella enterica serovar Typhimurium Author: DORMAN, CHARLES JAMES; HINTON, JAY Abstract: The role of the HU nucleoid-associated proteins in gene regulation was examined in Salmonella enterica serovar Typhimurium. The dimeric HU protein consists of different combinations of its α and β subunits. Transcriptomic analysis was performed with cultures growing at 37°C at 1, 4 and 6 hours post-inoculation with mutants that lack combinations of HU α and HU β. Distinct but overlapping patterns of gene expression were detected at each time point for each of the three mutants, revealing not one but three regulons of genes controlled by the HU proteins. Mutations in the hup genes altered the expression of regulatory and structural genes in both the SPI1 and the SPI2 pathogenicity islands. The hupA hupB double mutant was defective in invasion of epithelial cell lines and in its ability to survive in macrophages. The double mutant also had defective swarming activity and a competitive fitness disadvantage compared to the wild type. In contrast, inactivation of just the hupB gene resulted in increased fitness and correlated with the up-regulation of members of the RpoS regulon in exponential phase cultures. Our data show that HU coordinates the expression of genes involved in central metabolism and virulence genes, and contributes to the success of S. enterica as a pathogen. Description: PUBLISHED; Published online 06 January 2011 2011-01-01T00:00:00Z http://hdl.handle.net/2262/61058 Title: The PolyA tail length of yeast histone mRNAs varies during the cell cycle and is influenced by Sen1p and Rrp6p Author: BOND, URSULA MARY; JAMES, THARAPPEL C Abstract: Yeast histone mRNAs are polyadenylated, yet factors such as Rrp6p and Trf4p, required for the 3′-end processing of non-polyadenylated RNAs, contribute to the cell cycle regulation of these transcripts. Here, we investigated the role of other known 3′-end processing/transcription termination factors of non-polyadenylated RNA in the biogenesis of histone mRNAs, specifically the Nab3p/Nrd1p/Sen1p complex. We also re-evaluated the polyadenylation status of these mRNAs during the cell cycle. Our analysis reveals that yeast histone mRNAs have shorter than average PolyA tails and the length of the PolyA tail varies during the cell cycle; S-phase histone mRNAs possess very short PolyA tails while in G1, the tail length is relatively longer. Inactivation of either Sen1p or Rrp6p leads to a decrease in the PolyA tail length of histone mRNAs. Our data also show that Sen1p contributes to 3′-end processing of histone primary transcripts. Thus, histone mRNAs are distinct from the general pool of yeast mRNAs and 3′-end processing and polyadenylation contribute to the cell cycle regulation of these transcripts. Description: PUBLISHED 2012-01-01T00:00:00Z http://hdl.handle.net/2262/60583 Title: Regulation of transcription by DNA supercoiling in Mycoplasma genitalium: global control in the smallest known self-replicating genome. Author: DORMAN, CHARLES JAMES Abstract: The mollicute Mycoplasma genitalium causes sexually transmitted disease in humans. It has recently come to widespread public attention through its involvement in pioneering synthetic biology experiments. The 580-kilo-base-pair genome of M. genitalium contains just 470 genes, few of which seem to encode conventional transcription regulators. This raises the important question of how does this simple organism control its gene expression? The finding that the transcription of an osmotically inducible gene encoding a lipoprotein is sensitive to antibiotics that inhibit the activity of DNA gyrase, the enzyme that introduces negative supercoiling into DNA, raises the possibility that changes in DNA supercoiling provide a regulatory mechanism for controlling transcription in M. genitalium. Description: PUBLISHED; Published online 02 June 2011 2011-01-01T00:00:00Z http://hdl.handle.net/2262/59847 Title: Synthesis and evaluation of phosphoramidate and phosphorothioamidate analogues of amiprophos methyl as potential antimalarial agents Author: BELL, ANGUS; DEMPSEY, ENDA Abstract: A series of phosphoramidate and phosphorothioamidate compounds based on the lead antitubulin herbicidal agents amiprophos methyl (APM) and butamifos were synthesised and evaluated for antimalarial activity. Of these compounds, phosphorothioamidates were more active than their oxo congeners and the nature of both aryl and amido substituents influenced the desired activity. The most active compound was 46, O-ethyl-O-(2-methyl-4-nitrophenyl)-N-cyclopentyl phosphorothioamidate, which was more effective than the lead compound. Description: PUBLISHED 2011-01-01T00:00:00Z http://hdl.handle.net/2262/59839 Title: Pervasive post-transcriptional control of genes involved in amino acid metabolism by the Hfq-dependent GcvB small RNA. Author: HINTON, JAY Abstract: GcvB is one of the most highly conserved Hfq-associated small RNAs in Gram-negative bacteria and was previously reported to repress several ABC transporters for amino acids. To determine the full extent of GcvB-mediated regulation in Salmonella, we combined a genome-wide experimental approach with biocomputational target prediction. Comparative pulse expression of wild-type versus mutant sRNA variants revealed that GcvB governs a large post-transcriptional regulon, impacting ∼1% of all Salmonella genes via its conserved G/U-rich domain R1. Complementary predictions of C/A-rich binding sites in mRNAs and gfp reporter fusion experiments increased the number of validated GcvB targets to more than twenty, and doubled the number of regulated amino acid transporters. Unlike the previously described targeting via the single R1 domain, GcvB represses the glycine transporter CycA by exceptionally redundant base-pairing. This novel ability of GcvB is focused upon the one target that could feedback-regulate the glycine-responsive synthesis of GcvB. Several newly discovered mRNA targets involved in amino acid metabolism, including global regulator Lrp, question the previous assumption that GcvB simply acts to limit unnecessary amino acid uptake. Rather, GcvB rewires primary transcriptional control circuits and seems to act as a distinct regulatory node in amino acid metabolism. Description: PUBLISHED 2011-01-01T00:00:00Z http://hdl.handle.net/2262/59501 Title: Staphylococcus lugdunensis IsdG Liberates Iron from Host Heme Author: FOSTER, TIMOTHY JAMES; HEILBRONNER, SIMON Abstract: Staphylococcus lugdunensis is often found as part of the normal flora of human skin but has the potential to cause serious infections even in healthy individuals. It remains unclear what factors enable S. lugdunensis to transition from a skin commensal to an invasive pathogen. Analysis of the complete genome reveals a putative iron-regulated surface determinant (Isd) system encoded within S. lugdunensis. In other bacteria, the Isd system permits the utilization of host heme as a source of nutrient iron to facilitate bacterial growth during infection. In this study, we establish that S. lugdunensis expresses an iron-regulated IsdG-family heme oxygenase that binds and degrades heme. Heme degradation by IsdG results in the release of free iron and the production of the chromophore staphylobilin. IsdG-mediated heme catabolism enables the use of heme as a sole source of iron, establishing IsdG as a pathophysiologically relevant heme oxygenase in S. lugdunensis. Together these findings offer insight into how S. lugdunensis fulfills its nutritional requirements while invading host tissues and establish the S. lugdunensis Isd system as being involved in heme-iron utilization. Description: PUBLISHED 2011-01-01T00:00:00Z http://hdl.handle.net/2262/59298 Title: The Sbi Protein Is a Multifunctional Immune Evasion Factor of Staphylococcus aureus Author: SMITH, EMMA JANE; FOSTER, TIMOTHY JAMES Abstract: The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active. Description: PUBLISHED 2011-01-01T00:00:00Z