Exponentially growing cells of Escherichia coli ML-30 from batch cultures grown at I5C were subjected to cold osmotic shock. This treatment consisted of suspending cells in a hyperosmolar solution of an inert solute containing EDTA at 37°C and then in 0.5 mM MgCl2 at 0°C, This treatment caused a decrease in the initial rate of accumulation of the non-metabolisable solute methyIglucoside. An examination was made of the effects of different growth temperatures on the susceptibility of Escherichia coli to modified shock treatments and on its lipid composition. This allowed an assessment to be made of the importance of lipid unsaturation in predisposing cells to cold osmotic shock. Exponentially growing cells of Saccharomyces cerevisiae NCYG 566 from batch cultures grown at 50°C or from a chemostat culture grown under glucose limitation at 30°C were subjected to a modified form of the cold osmotic shock treatment. This treatment caused a decrease in the initial rate of accumulation of the non-metabolisable solutes glucosamine and 2-aminoisobutyrate. Storage of unshocked batch-grown yeast in buffer at 10°C led to an increase in ability to accumulate glucosamine and further experiments were confined to yeast grown in a chemostat. A study was made of the effect of modifying the cold osmotic procedure applied to yeast, including the osmotic stress, the chelating agents and the cold Mg2+ -containing diluent on viability and solute accumulating ability. Growth of shocked yeast cells in defined medium resembled that of unshocked yeast, but in malt extract-yeast extract-glucose-peptone medium the shocked yeast had a longer lag phase of growth and initially grew at a slower rate. Cold osmotic shock caused the release of low molecular- weight compounds and about 6-8% of the cell protein. The yeast cell envelope enzymes, invertase, acid phosphatase and L-leucine-naphthylamidase were not released when yeast cells were subjected to cold osmotic shock; nor was the cytoplasmic enzyme, alkaline phosphatase.

The lipid compositions of Saccharorrces cerevisiae N.C.Y.C. 366 grown in batch culture at 30°C and 15°C, were compared. The compositions of lipids from cells grown in a chemostat, under glucose limitation and at constant dissolved oxygen tension at two rates at 30°C and one rate at 15°C, were also examined to discover whether the growth temperature-induced changes in the lipid composition of batch-grown cells were caused solely by the change in temperature or whether they were conditioned partially or wholly by the lowering of the growth rate that accompanies a decrease in the growth temperature of batch cultures. Lowering the growth temperature of batch cultures was accompanied by an increase in the contents of total lipids, total fatty acids, triacylglycerols and phospholipid (mainly as phosphatidylcholine), but had little or no effect on the contents of diacylglycerols, free fatty acids, sterols or sterol esters. Analyses of lipids from chonostut-grown cells suggested that the increased synthesis of triacylglycerols was caused mainly by lowering of the growth temperature, but that the increased content of phosphatidylcholine could be attributed mainly to a decrease in growth rate. Lowering the rate at which cells were grown continuously at 30°C caused an increased synthesis of sterol esters whereas lowering the temperature at which cells were grown at a fixed rate was accompanied by a decreased content of both free and esterified sterols. Extracts of all cells examined contained two major sterols, Delta 5,7,22,24(20)-ergostatetraene-3beta-ol and ergosterol. There was little change in either the fatty-acid composition or the degree of unsaturation of the fatty acids in batch-grown or chemostat-grown cells following a change in incubation temperature. Significant proportions of C fatty acids were detected in cells grown in chemostat at 30°C at 0.05h-1. Data on the sterol content obtained by saponification and acid hydrolysis of whole cells are also reported. Preliminary data are reported on the distribution of lipids in fractions obtained by density-gradient centrifugation of lysates of yeast spheroplasts.

No description available.

Several aspects of soft rot of wood and of microfungi associated with the degradation process have been investigated. The physiological properties of five species of microfungi isolated from soft-rotted mine timber have been compared with those of an active soft-rotting species by studying the response of the fungi to temperature, illumination, pH9 carbohydrate source and the presence of copper. Attempts have been made to devise a technique for inducing rapid soft rot of wood in the laboratory with a view to screening potential soft rotting fungi. The techniques investigated basically of two trees, blocks of wood being in contact either with free liquid or agar and inoculated with Chaetomium globosum in both instances. Ecological studies have been carried out on the fungi colonizing wooden fence posts with particular reference to the nature of the fungal flora at various levels in the post and to the possibility of the establishment of a succession of fungi in decaying wood. Microscopic investigations have been carried out on the effect of several soft-rotting fungi on wood and detailed observations have been made of the mechanism of degradation of secondary cell walls of coniferous and non-coniferous woods by hyphae of these fungi under various conditions

No description available.

Phytophthora capsici is a devastating plant pathogen for which virulence is aided by the secretion of effectors, including cytoplasmic effectors from the CRN (CRinkling and Necrosis) family. CRN effectors were first described in oomycetes by their capacity to induce host cell death. Nevertheless, despite recent efforts aiming to elucidate CRN virulence functions, the virulence relevance of CRN mediated cell death remains unknown. In this thesis, by performing a PCR based random mutagenesis screen on P. capsici CRN effector PcCRN83_152, we showed that PcCRN83_152 cell death is not required for its virulence function. In addition, we demonstrated that PcCRN83_152 interacts with nuclear proteins from the host plants N. benthamiana (NbSIZ1 and NbSLX1) and tomato (SlSIZ1∆867), which we connected to plant immunity processes and PcCRN83_152 mediated phenotypes. Besides increasing our knowledge on P. capsici CRN effectors, in this thesis we also aimed for the identification of new P. capsici effectors. Using a proteomics-based approach, we identified a set of candidate effectors from P. capsici that would not be identified using conventional genomic- and transcriptomic-based studies. In sum, in this thesis we summarise our current understanding of CRN effectors and re-assess some basic assumptions regarding this protein family. Moreover our results pointing to CRN virulence functions independent of cell death phenotypes provide a new conceptual framework for studies aimed at unveiling the virulence functions of cell death inducing CRNs. This knowledge complemented with the identification of PcCRN83_152 plant targets provides great leads to uncover PcCRN83_152 virulence functions. Moreover, the identification of new candidate effectors form P. capsici could be important towards a more global understanding of P. capsici virulence mechanisms.

No description available.

During the studies of interactions on agar media between mycelia of the two tropical agaric fungi, Marasmius pallescens and Marasmiellus troyanus, it was observed that initial deadlock between the mycelial fronts was ultimately followed by overgrowth by M troyanus. This was shown to be the most consistent outcome even when abiotic conditions (pH, aw, temperature) were altered to possibly favor the competitive ability of M pallescens on agar. However, a shift in competitive ability was observed when Birch wood disks precolonized with mycelia of both taxa were confronted in the inoculum ratio of 75:25 M pallescens to M troyanus. In this instance, M pallescens was not only able to retain its resource, but also capture a small portion of resource originally colonized by M troyanus. In studies on the expression of lignocellulose degrading enzymes in liquid cultures ofthese two fungi it was found that when the two taxa were mixed, a rapid increase in laccase and manganese peroxidase activity, but not lignin peroxidase, was detected in the culture supernatant. Even more rapid and elevated induction of laccase was observed when filtersterilized supernatant of M pallescens was added to M troyanus cultures, but the reciprocal experiment (addition ofM troyanus supernatant to M pallescens cultures), did not lead to any increase in laccase activity. Addition of autoclaved supernatant ofM pallescens also induced laccase activity from M troyanus cultures, but over a period of days rather than hours. Although bothM troyamls, and to a lesser extentM pallescens, are able to produce laccases in shaken liquid culture following addition of the inducer 2,5Dimethylalanine, these experiments suggested that the presence of heat-stable and heatlabile laccase inducers, secreted byM pallescens mycelia, led to induction of laccases by M troyanus. ReacSyn™ bioreactors were then successfully used to assay for the induction of laccase and the production of phenolic metabolites in mixed cultures ofM troyamls and M pallescens, utilizing a working volume of 30 ml. The induction of laccase in combined cultures was demonstrated in both shaken ReacSynTM and static ReacSynTM systems, although to a greater extent in the shaken system. A greater change in the quantity and type ofphenolics was detected in combined culture extracts, than in either monoculture control extract. HPLC analysis of extracts produced from culture filtrates from combined cultures and monocultures controls revealed that the phenolic compound that was eluted at eight minutes was produced in highest quantity by M pallescens and appeared to have induced laccase production in M troyanus in combined cultures, and as a result of laccase action could have been partially polymerized. Compounds unique to combined culture profiles were also shown to be present in both the liquid media and the gaseous headspace, when combative interactions between mycelia of both taxa was scaled up to 2 I STR. GC-MS analysis identified a-hydroxyphenylacetic in the culture filtrates of interacting liquid cultures. This compound is a natural mediator of phenolic degradation, and could thus play a role in laccase induction during combative interactions. Moreover, the presence ofan array of volatile compounds unique to combined cultures, suggests that chemicals are involved in offensive/defensive strategies used byM troyamls andM pallescens during mycelial interactions at a distance.

Sgt1, a co-chaperone of Hsp90, plays a fundamental role in plant immunity, protein degradation and kinetochore assembly in yeast. Sgt1 has a multi-domain architecture, including an N-terminal tetratricopeptide repeat (TPR) domain. Through its modular architecture, Sgt1 is believed to bridge the interaction between two key structural components of the yeast inner kinetochore, Skp1 and Ctf13, and Hsp90. This interaction recruits Hsp90 to Ctf13 for subsequent activation into a complex that is competent to bind centromere DNA. This is an essential first step in the assembly of the yeast kinetochore, since activation of Ctf13 is a prerequisite for the subsequent binding of all other components of the kinetochore. The aim of this project was to gain biophysical and structural insight into Sgt1 and the basis of its interaction with Skp1, utilising X-ray crystallography and biophysical methods. Sgt1 exhibits a novel mode of TPR dimerisation in the structure of an Sgt1:Skp1 complex and a second potential mode of dimerisation in the structure of the Sgt1 TPR domain alone. One of these interfaces utilises yeast specific insertions and was confirmed to exist in solution by mutagenesis s tudies targeting the interface. Biophysical studies show that Sgt1 exists predomina ntly as a dimer in solution, but truncating its C-terminal domains exposes regions that promote trimer formation. The structure of the Sgt1:Skp1 complex shows that the interface with Skp1 comprises the concave groove of the TPR domain. Studies of the stoichiometry of Sgt1:Skp1 suggest that the functionally relevant species is 2:1, though the structure shows a fundamental 1:1 interaction. Targeting this interface by mutagenesis disrupts complex formation and show also that a monomer of Sgt1 is sufficient to bind Skp1. Together, this data is used to present an updated model for the role of Hsp90 in kinetochore assembly.

Chapter 1 consists of an introduction to the Myxomycetes (acellular slime-moulds) followed by a description of the culture techniques employed in the study of the organisms. This includes the culture of the haploid amoebae, their crossing to give diploid plasmodia, the culture of plasmodia and the induction of sporulation, and the hatching of spores to give amoebae. Chapter 2 consists of a description of the analysis of fusion behaviour of plasmodia. The behaviour of two plasmodia when they make contact, fusion or non-fusion, was found to be controlled by two genes, four alleles of gene f ( f1 –f4 ) being detected, and two alleles of gene n ( n1 –n2 ). Plasmodia must have the same f genotype for fusion to be possible, and the same n phenotype, ( n2 is dominant to n1 ). One exception to this rule occurs, f3 f3 homozygotes fuse with f4 f4 homozygotes. Based partly on deductions from this exceptional observation, a general model for the mode of action of the f and n genes is proposed. The analysis of fusion type was complicated by the occurence of a recessive allele, qˉ, linked to the f gene. In the homozygous state the qˉ allele results in restricted nutritional behaviour, death occurring on the usual axenic culture medium. This is the first report of linkage in Physarum polycephalum. The genes detected during the analysis of fusion behaviour were made use of in the analysis of senescence. Chapter 3 reports the further analysis of an actidione resistant mutant which, it was hoped, would be of use as a marker in the study of senescence. Chapter 4 reports the application of genetic markers to the study of the ploidy of plasmodia. Chapter 5 begins with an introduction to the phenomenon of senescence. This is followed by the first report of senescence in a Myxomycete. Three main techniques were applied to the analysis of this phenomenon; a) The life-expectancy of heterokaryons formed by fusing equal quantities of plasmodia of known, and disparate, life-expectancy was studied. b) The f gene was used to study the stability of the nuclear ratios of such heterokaryons. c) Various procedures, for example mutagenesis, were applied to plasmodia as a pulse, and the life-expectancy of the treated plasmodia observed to see whether senescence had been affected by the procedure. The data produced by these three techniques suggests the hypothesis that senescence is due to the accumulation of defective mitochondria.