No description available.

Cells that grow by apical extension, such as neurons, pollen tubes, root hairs and fungal hyphae, orient their growth in response to tip contact with physical cues in the environment (thigmotropism). I use Candida albicans, an opportunistic human fungal pathogen, as a model to assess tip re-orientation growth responses after contact. Thigmotropism is associated with virulence (Brand et al., 2008), therefore this thesis aims to characterise the responses that C. albicans displays after contact events in an enclosed chamber featuring various obstacles and shapes. It was found that hyphae grow along surfaces in a nose-down manner, presumably to identify and exploit gaps in the substrate. Further, hyphae preferentially grow nose-down on softer surfaces when given the option of two contrasting surfaces, implying novel substrate sensing mechanisms. Contact-dependent hyphal responses are outlined, where perpendicular contact with an obstacle induced various growth responses after re-orientation. Further, important cytoskeletal regulators of thigmotropism were identified, which subsequently regulate substrate indentations. The applied force generated by hyphal tips was quantified, which was enough to penetrate mammalian membranes without the need of hydrolytic enzymes, and this was modulated by a change in environmental carbon source. This thesis describes several new exploratory behaviours in C. albicans, which may apply to hyphae in general, since behaviours described here have also been observed in other filamentous fungi. Further, the role of septins as regulators of directional growth is discussed. The first chapter describes contact-dependent behaviours that support the ability of hyphae to be opportunistic and exploit their topographical environment to invade surfaces. The second chapter presents a detailed description of how the fungus responds to perpendicular contact events. Finally, the third chapter identifies cytoskeletal regulators important for thigmotropism. Together, this thesis brings together multiple aspects of cell biology and biophysics that apply during polarised tip growth, which adds knowledge to the narrative of why hyphae are such successful space invaders.

Human E. coli O157 isolates were typed by multi locus variable number tandem repeat analysis (MLVA) and household outbreaks were identified. The genetic variation found within the household cases of E. coli O157 was compared to the background variation found in sporadic isolates to determine if the isolates within each household were related to each other or if they were from multiple genotypes. The results showed that 83% of household outbreaks were closely related to each other and were acquired from the same source, whilst the remainder of cases could be a result of multiple strain carriage within the source of infection. Cattle, sheep and human isolates were compared using MLVA and population genetic methods were used to identify the degree of host association between cattle and sheep isolates. This was to determine whether cattle or sheep are the main source of human E. coli O157 infection. The results showed that no host association exists between cattle and sheep isolates. This suggests that cattle and sheep isolates are part of the same population and can circulate between each host. Further, flocks of sheep in Grampian were tested for E. coli O157 whilst they were grazing on pasture in winter, brassicas in spring and on pasture during the summer, to see if a diet of brassicas had an effect on the faecal shedding of E. coli O157 in sheep. Statistical analysis showed significant differences between the shedding of E. coli O157 in sheep grazing on brassicas in spring when compared to sheep grazing on pasture in the summer (P< 0.01) and in winter (P<0.05), suggesting that a diet of brassicas may have an effect on the shedding of E. coli O157 in sheep.

Candida albicans is a fungus that colonizes oral cavity surfaces and is carried by approximately 50% of humans. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans. The objective of this study was to better understand how streptococci communicate with C. albicans in oral biofilms. Mannoproteins comprise a major component of the C. albicans cell wall. Initial aims of the work were to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hypha I functions associated with biofilm community development. A C. albicans mnt1- mnt2L1 mutant, with deleted a1,2-mannosyltransferase genes and thus defective in 0- mannosylation, was abrogated in biofilm formation under various growth conditions, and produced hypha I filaments that were not ·recognized by S. gordonii. Cell wall proteomes of hyphae-forming mutant cells showed reduction, compared to wild type, in a range of protein components including Als1, Als3, Rbt1, Scw1 and Sap9. Hyphal filaments formed by mnt1- mnt2L1 mutant cells, unlike wild type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins, or with S. gordonii Ssp8 partner adhesin. These observations implied that early stage O-mannosylation was critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii, and microbial community development. C. albicans was enhanced in hypha formation when incubated planktonically with S. gordonii. This was supported by transcriptome RNASeq analysis which identified C. albicans genes, including the filamentation and pathogenesis associated genes FRG42, ALS1, CA T1 and TEC1, which were up-regulated in the presence of S. gordonii. Taken collectively these results identify new inter-Kingdom communication mechanisms that provide better understanding of the ways that microbial communities develop, and of potential means to control C. albicans infections.

The seven canonical Sm proteins of <I>Saccharomyces cerevisiae</I> associate with the U1, U2, U4 and U5 spliceosomal snRNAs, and have roles in the biogenesis and localisation of these snRNP particles. The proteins interact extensively with one another in an ordered pathway. In contrast, the Uss1 protein is a non-canonical member of the Sm-like protein family in that it associates with the U6 snRNA. A combination of molecular genetic and biochemical analysis has been used to investigate the molecular associations of the Uss1 protein. Exhaustive two-hybrid screens revealed interactions with other Sm-like proteins, with proteins of the U2 snRNP (and proteins interacting with U2 snRNP proteins) and with proteins not previously reported to be involved in pre-mRNA splicing. A network of potential interactions was produced from these data. Deletion of six open reading frames (ORFs) encoding Sm-like proteins revealed three as essential for cell viability (YBL026w, YER146w and <I>USS2</I>) and three as non-essential (<I>SPB8, </I>YDR378c and YNL147w) suggesting functional redundancies within these non-canonical Sm-like proteins. Since significant levels of U6 snRNA can be precipitated with all 6 proteins, and the Yjr022p (an Sm-like protein identified in two-hybrid screens) the genes encoding these proteins were added to the <I>USS</I> (<I>U</I>-<I>S</I>ix <I>S</I>nRNP) gene class. A subset of these proteins is essential for the stability of U6 snRNA <I>in vivo</I>, with over-production of U6 capable of (partially) rescuing the growth of the conditional strains. All 7 Sm-like proteins which associate with U6 snRNA also co-immunoprecipitate with Uss1p. This combined with the range of interactions revealed by two-hybrid analyses suggests the existence of an Sm-like protein complex (or complexes).

The plant pathogenic basidiomycete, <I>Rhizoctonia solani</I>, contains extrachromosomal double-stranded RNA and DNA elements, but the role of these elements in the biology and pathology of the fungus is uncertain. Aspects of these elements in <I>R.solani</I> and the role of anastomosis (hyphal fusions) in their transmission are examined here. Anastomoses between hyphae, leading to successful cell fusions and death of fused cells (vegetative incompatibility) were observed by video microscopy and by fluorescence microscopy when hyphae were loaded with fluorochromes. However, attempts to monitor organelle transfer were unsuccessful and ultra-violet irradiation of hyphae containing fluorochromes led rapidly to hyphal death. Two strains of anastomosis group (AG) 4 could readily be 'cured' of dsRNA by subculture of hyphal tips, although one strain which contained a 2.5kb DNA element could not be freed in this way, nor by ultra-violet irradiation or heating to 30°C. Several of the resulting hyphal tip subcultures showed an incompatibility reaction when paired with the respective parent strain. These parent-incompatible strains (6 from parent strain PA1 and 6 from parent strain I13) fell into 2 groups - mutually compatible within each group, but incompatible with the other group and the parent. Anastomosis during pairings of strains within any one group never led to a parent-compatible strain when subcultures were taken from the zone of hyphal fusion. There was no evidence that dsRNA influenced compatibility; instead it is suggested that hyphal tip subculturing led to segregation (or expression) of nuclear compatibility genes. Counts of nuclei in tip cells, by DAPI staining and fluorescence microscopy, showed variation in different parts of the fungal colonies, and significant tendency for some juxtaposed branch tips (arising as clusters from a single hypha) to have similar nuclear numbers to one another.

Fumarate reduction in <i>Shewanella </i>is catalysed by a fumarate reductase known as flavocytochrome <i>c₃</i> (Fcc₃). This enzyme consists of three domains: a cytochrome domain containing four <i>c</i>-type heme groups; a flavin domain containing a non-covalently bound FAD; and a mobile clamp domain. Fumarate is saccinate by hydride transfer from the flavin N5 and protonation by the active site acid, Arg402. Access of substrate to the active site in Fcc₃ was believed to be controlled by movement of the clamp domain. To test this assumption, site-directed mutagenesis has been used to create a disulfide bond between the clamp and flavin domains via the double mutation A251C:S430C. The disulfide bond in the mutant enzyme has been confirmed by both crystal structure and Ellman analysis. When the disulfide bond is formed both the steady-state and pre-steady-state rate constants for fumarate reduction fall to 25 and 30% of the wild-type values respectively whilst <i>K_M</i> values for fumarate are unaffected. Deuterium solvent kinetic isotope effects in the mutant enzyme are unchanged from wild-type (8.2 ±0.4 at pL 7.2), indicating that proton and/or hydride transfer is still rate-limiting. These results suggest that clamp domain mobility has little role in controlling fumarate reduction. The reduction of fumarate also requires the delivery of reducing equivalents to the active site. This is facilitated by the four bis-histidine-ligated heme groups within the cytochrome domain. Hemes I, II and III are solvent exposed and therefore able to collect electrons from the electron donor CymA, and deliver them to the FAD via heme IV. Mutations to His61, a ligand to the iron of heme IV, results in a lowering of both the steady-state and pre-steady-state rate constants for fumarate reduction (WT>H61Y>H61M>H61A). Crystal structures of H61A and H61M show that there is an exogenous ligand bound to the heme iron in both cases; acetate and water respectively.

No description available.

The <i>Schizosaccharomyces pombe cdc37</i> gene has been identified and was found to be essential for cell viability. To further study <i>S. pombe</i> Cdc37, a range of mutants were generated using both random and directed mutagenesis. Cdc37 temperature-sensitive mutant cells at the restrictive temperature stop dividing within a single cell cycle. Cdc37 protein levels were not changed at the non-permissive temperature in mutants, indicating that cell inviability arises from defective function of the mutant Cdc37 protein. Morphologically, temperature-sensitive mutant cells arrest with a uniform phenotype, being elongated, characteristic of the <i>cdc </i>phenotype. About 80% of these elongated cells contained a single nucleus with a 2C DNA content, indicating that <i>cdc37</i> temperature-sensitive mutants arrest in G2. The only exception was <i>cdc37-J</i>, where half the cells leaked through to mitosis after 8 hours at the restrictive temperature and displayed divided nuclei and septa, arresting with defects in cytokinesis. Characterisation of the <i>cdc37</i> temperature-sensitive mutants led to the identification of Cdc2 as a client protein and a principal candidate for the cause of the G2 cell cycle arrest. Cdc2 activity was dramatically reduced within one hour at the non-permissive temperature in <i>cdc37</i> temperature-sensitive mutants, but Cdc2 protein levels remained constant. Biochemical and genetic interactions between Cdc37 and Cdc2 were observed, supporting the idea of Cdc2 as a client of Cdc37. Cdc37 is believed to deliver client protein kinases to the co-chaperone Hsp90, forming a heterocomplex. A small fraction of total cellular Cdc37 in <i>s. pombe</i> forms a high molecular weight complex which also contains Hsp90, Cdc37 was found to biochemically and genetically interact with Hsp90, indicating these co-chaperones co-operate in <i>S. pombe.</i>

The SbcCD complex mediates the inviability of long palindromes in <I>Escherichia coli.</I> The eukaryotic homologues, Rad50Mre11, are involved in DSB formation and the human homologues have been implicated in cancer progression. In this work a biochemical characterisation of the hairpin nuclease activity has been undertaken to help elucidate its cellular roles and those of its homologues. It was intended to map the sites of hairpin cleavage and to determine the chemical nature of the cleaved termini. The nuclease requirements for substrate structure were to be examined to test the model for palindrome maintenance. In this regard, it was to be determined whether substrate termini were required. In addition it was intended to discover whether SbcCD possesses a helicase activity. A DNA binding assay was sought to define alternative substrates and thence alternative potential roles of the protein, and to detail the protein substrate interaction. It has been shown that hairpin cleavage occurs immediately 5' of the loop and is followed by stem degradation. Cleavage and degradation have been uncoupled. The cleavage products have 5' phosphate and 3' hydroxyl termini. A dumbell substrate lacking termini was generated and found to be cleaved, supporting the model and suggesting that SbcCD is involved in DSB formation like its homologues. SbcCD has been shown not to posses a helicase activity on a hairpin substrate. DNA binding has been demonstrated by gel filtration and studied by gel retardation. It is proposed that SbcCD may bind its substrate transiently.