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The arbuscular mycorrhizal (AM) symbiosis is a mutualistic association between plant roots and fungi that belong to the Phylum Glomeromycota. In this symbiosis, the plant provides the fungus with carbohydrate and gets phosphorus in return. Study of these fungi is fettered because of their obligatory symbiotic nature and lack of basic knowledge of their genetics. These fungi produce multinucleate spores and it is not clear whether nuclei within a spore are genetically similar or dissimilar. In this thesis, we discuss techniques that address some fundamental questions about the organization of genetic material in AM fungi. Sequence variants from coding and non-coding genes have been reported from single Glomus isolates but it is not known whether these arise from different nuclei or constitute multiple gene copies within each nucleus. Several alleles (variants) of Binding protein (Bip) gene were observed after cloning this gene from each of the four isolates of G. irregulare studied (Chapter-2). A Sanger sequencing trace of a sample with a mixture of allelic sequences shows two peaks at single nucleotide polymorphism (SNP) positions. A new approach of measuring peak heights was developed that can monitor changes in allelic frequency of selected genes without involving cloning and RFLP-screening, which are both expensive and time consuming. Using this technique addressed two important questions, first that spores are genetically different in a culture Petri plate within and among G. irregulare isolates (Chapter-3) and second, plant hosts have a significant effect in changing the allele frequency of selected fungal genes (Chapter-4). Single nuclei were isolated by Laser Capture Microdissection from spores and hyphae after optimising the experimental conditions to minimise autofluorescence. Sequences of two marker genes BiP and internal transcribed spacer-2 region (ITS2), were amplified from the whole genome amplified DNA of the catapulted single nuclei using multiplex PCR (Chapter-5). This study reports for first time the isolation of single nuclei from spores and hyphae using LCM. Isolation of single nuclei can provide useful information about the genome size, organization of genetic variation and genetic processes that are poorly understood in AM fungi.

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Viable bacteria on hard, open surfaces are usually present alongside some organic (or inorganic) material, typically at the solid-air interface. Even if not multiplying, the presence ofthese viable cells presents a risk to overall hygiene levels. Stainless steel is typically the material ofchoice as a hygienic surface, being hard, inert and easy to clean. Properties ofc1eanability in addition to ease of disinfection are key in the evaluation of hygienic surfaces. The aim of this study was to evaluate the hygienic status and cIeanability ofstainless steel surfaces under test conditions which more closely resemble the environment under investigation, by monitoring survival at the solid-air (rather than solid-liquid) interface, in the presence oforganic material. A range ofmethods were developed: - A synthetic fingerprint emulsion was developed by combining and modifYing published standard sweat and sebum formulations. The formulation allowed survival of large, but decreasing suspended populations ofStaphylococcus aureus and Esche'richia coli, for up to 48h, but on surfaces, no viable cells were recovered - in contrast to observations using real fingerprints. Thus the published formulation, which had been developed for physical/chemical testing, was not appropriate for combination with microorganisms, although was used to evaluate sUrface c1eanability. Results showed the importance of combining p!J,ysical and chemical cleaning strategies. - A 'hard-to-remove' surface soil Cburnt on' milk) was more effectively removed from the smoother oftwo stainless steel surfaces tested (bright annealed), particularly when cleaning parallel to, rather than across, linear surface features. - The requirement for moisture to release active agents from proposed antibacterial surfaces was demonstrated for a silver-containing polymer-coated stainless steel, by monitoring microbial survival at the solid-air interface for up to 3 weeks. Key parameters affecting hygienic status and cleanability were combined to assess the hygienic performance of stainless steel and copper, the latter of which had showed strong antibacterial activity when exposed to cells in nutrient broth. The test included repeated soiling and cleaning cycles, daily for 5 days, with a suspension ofSaureus in bovine serum albumen, inoculated onto three stainless steels of different surface finish, and copper. After the second and subsequent soiling, a layer of material readily stained with the fluorescent dye acridine orange, wasupparent on the copper surface, and any antibacterial effects were absent. Stainless steel, however, remained highly cleanable. A range of methods have been developed which enable the screening of proposed hygienic and/or antibacterial surfaces, combined with a variety ofcleaning and disinfection protocols, under challenging environinental conditions. All commercially available stainless steel finishes tested were comparable in terms of hygienic status (removal ofmicroorganisms), but even a small increase in surface topography (Ra = 20 to 100 nm) resulted in increased retention of organic soil. Thus cleanability remains a key step in ensuring maintenance of hygienic status, and an inert and highly cleanable surface provides the best option in this context.

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This study tested the hypothesis that a relationship exists between taxonomic diversity and antibiotic resistance patterns of filamentous actinomycetes. To this end, 200 filamentous actinomycetes were selectively isolated from a hay meadow soil and assigned to groups based on pigments formed on oatmeal and peptone-yeast extract-iron agars. Forty-four representatives of the colour-groups were assigned to the genera Dactylosporangium, Micromonospora and Streptomyces based on complete 16S rRNA gene sequence analyses. In general, the position of these isolates in the phylogenetic trees correlated with corresponding antibiotic resistance patterns. A significant correlation was found between phylogenetic trees based on 16S rRNA gene and vanHAX gene cluster sequences of nine vancomycin-resistant Streptomyces isolates. These findings provide tangible evidence that antibiotic resistance patterns of filamentous actinomycetes contain information which can be used to design novel media for the selective isolation of rare and uncommon, commercially significant actinomycetes, such as those belonging to the genus Dactylosporangium, a member of the family Micromonosporaceae. A culture-independent, nested PCR procedure based on genus-specific oligonucleotide primers detected the presence of Dactylosporangium strains in 14 out of 21 environmental samples. Clones generated from the 14 positive samples formed novel phyletic lines in the Dactylosporangium 16S rRNA gene tree. Presumptive dactylosporangiae were isolated from 7 of these samples using a medium designed to be selective for members of the genus Dactylosporangium. One hundred and two out of 219 representative presumptive dactylosporangiae were considered to be bona fide members of the genus Dactylosporangium as they gave PCR amplification products with primers specific for this taxon. Representatives of the Dactylsporangium isolates formed distinct phyletic lines in the Dactylosporangium 16S rRNA gene tree were designated as new species, namely Dactylosporangium luridum sp. nov. and Dactylosporangium luteum sp. nov., based on a polyphasic study. Similarly, “Dactylosporangium salmoneum” NRRL B-16294 was validly described as a new species, Dactylosporangium salmoneum sp. nov., nom. rev. In addition, “Dactylosporangium variesporum” NRRL B-16296 was transferred to the genus Saccharothrix as Saccharothrix variisporea corrig. (ex. Tomita et al. 1977) sp. nov., nom. rev. Some of the representative Dactylosporangium isolates inhibited the growth of Bacillus subtilis, Kocuria rhyzophila and Staphylococcus aureus strains, suggesting that novel Dactylosporangium strains might be a rich source of novel antibiotics. Verrucosispora maris AB-18-032, another member of the family Micromonosporaceae, produces atrop-abyssomicin C, the first natural inhibitor of the para-aminobenzoic acid pathway. The self-protective mechanism of this strain was sought by conjugating an atrop-abyssomicin C sensitive Streptomyces griseus strain against a genomic DNA library prepared from V. maris AB-18-032. Seven resultant resistant exconjugants were screened for atrop-abyssomicin C resistance genes using four designed PCR primers. The failure to detect PCR amplification products suggests that the resistance shown by the exconjugants is conferred by mutation within the S. griseus strain or by cloning of unidentified resistance genes from the V. maris strain.

Cellulose is a major component of the plant cell wall and is the most abundant organic molecule in the biosphere. Efficient degradation of this polysaccharide is required if the plant cell wall is to be used as a viable source of renewable biofuels. Bacteria express an arsenal of different cellulases that catalyse the degradation of cellulose. The reason why many different cellulases are expressed rather than one highly active cellulase is unclear, but probably lies in the structural diversity displayed by cellulose, which is much greater than its invariant chemical composition suggests. Part of this work describes a novel cellulase from the plant cell wall degrading bacterium Clostridium thermocellum. This cellulase, CtCel119, is the first of this class of enzyme to display the α8 helical fold, is the founding member of a new GH family, performs catalysis through a possible “Grothuss style mechanism” and shares features typical of lytic transglycoslases. Structural data also seem to suggest that the enzyme may attack a novel structure in crystalline cellulose, which could contribute to understanding why bacteria such as C. thermocellum employ a diverse variety of cellulases. A study on glycoside hydrolase family (GH) 26, which consists mainly of endo-β-1,4 mannanases, was also conducted. The work presented in this thesis focused on two novel members of the family. One component of this section provided a thorough kinetic analysis of mutants of active site residues of a GH26 endo-β-1,4-1,3-glucanase. This identified crucial interactions at the -2 subsite, which contribute to the stabilisation of the 4H3 transition state. The other component of this section was the identification and characterisation of an exo-acting mannanase, CjMan26C, also termed a mannobiohydrolase. CjMan26C is the only mannanase characterised to date to release mannobiose. The mannobiohydrolase displayed an extremely high catalytic efficiency of 3 x 109 min-1 M-1 against mannotetraose. The crystal structure revealed a -1 sugar in a 1S5 pre transition state, providing further support for a B2,5 transition state in GH26. The exo activity was conferred by a four amino acid insertion in loop 3 at the -2 subsite. Mutation of D130, to Gly or Ala, in loop 3 was enough to partially convert the enzyme to an endo-mode of action, while removal of D130 plus two flanking resides caused a full conversion to an endo-mode of action. The gene expansion observed in family GH43 enzymes was also investigated. Eleven genes encoding GH43 enzymes were cloned, expressed and investigated for catalytic activity. Three arabinofuranosidases were characterised, two exo α-1,5-L-arabinofuranosidases and a novel sugar beet arabinan specific α-1,2-L-arabinofuranosidase that could attack both single and double substitutions named CjAbf43B. The crystal structure of CjAbf43B was solved in complex with ligand. The structure revealed a curved binding cleft, around a deep active site pocket, that was specific for the curved nature of sugar beet arabinan backbone. The curved binding cleft also had a groove into which α-1,3-L-arabinofuranosides could be accommodated, explaining how the enzyme has plasticity for single and double substitutions. A β-1,4 xylosidase was also characterised, while four enzymes were identified that displayed „trace‟ activity against xylans. Three of these appeared to display endo-activity, while the fourth enzyme displayed very weak arabinfuranosidase activity against xylans.

In 1967 the International Standards Organisation drew up a method designed to resuscitate a single injured Salmonella cell in 25 g of food. This method takes five days and introduced a pre-enrichment step that allowed injured Salmonella species to recover their resistance to selective agents before they were inoculated in to selective medium. Since 1967 a variety of methods has been developed that shorten the time .taken to detect a positive sample, yet these methods rely on the pre-enrichment step to allow injured Salmonella cells to recover and to allow amplification. This work was carried out to improve recovery ofinjured cells and allow selection during the pre-enrichment phase. Using variants of Buffered Peptone Water as recovery media, it was found that heatinjured Salmonella cells comprised a number of distinct sub-populations, based on their ability to recover in different broths. It was found that anoxic conditions improved recovery of heat-injured cells, while media that allowed rapid growth inhibited recovery. A survey of commercially available BPW found a> 2 10gIO difference in the recovery of heat-injured cells between the best and worst media. To detect Salmonella in food samples it is essential to repress growth of the competitive microflora. An investigation was carried out on autocytotoxic ~galactosides and ~-glucosides based on the biocide, 8-hydroxyquinoline (8HQ). These substrates might be used to selectively inhibit the competitive microflora. Galactosides were chosen to represent substrates transported into the cell by proton symports, and glucosides because they are representative of substrates transported by the phosphotransferase system (PTS). On hydrolysis 8HQ was released into the cytoplasm, but due to its lipophilicity the biocide migrated into the cytoplasmic membrane. The biocide then diffused into the medium until it reached equilibrium on both sides ofthe membrane. This inhibited .the competitive microflora, but would also inhibit the growth of any Salmonellae present. 8HQ glycosides are unsuitable for use in the pre-enrichment phase. In the next stage of this work glycosides of 8-hydroxyquinoline-5-sulphonic acid (8HQ5S) were synthesised. The sulphonate group is ionised at neutral pH and this ensures that on hydrolysis 8HQ5S remains within the cell. It was found that Klebsiella pneumoniae CMCC3077 could take up 8HQ5S-~,D-galactoside and hydrolyse the substrate, but the biocide did not remain within the cell. It is possible that the free 8HQ5S was exported from the cell by efflux pumps. No activity was found for 8HQ5S-~,Dglucoside. However, no free 8HQ5S was found in the medium. It is unknown whether the cell failed to transport and phosphorylate the substrate through the PTS, or failed to hydrolyse the molecule. As these glycosides failed to meet the requirements for autocytotoxic compounds, work was carried out on alaphosphalin. This is a dipeptide analogue that releases Ll- aminoethylphosphonic acid (AEP) on hydrolysis. AEP inhibits alanine racemase and growth. It was shown that alaphosphalin successfully inhibited K. pneumoniae at various inocula, and it was shown that AEP did not inhibit the recovery of heatinjured Salmonella cells. A major limitation to the use of alaphosphalin is that the peptides in BPW competitively inhibit uptake through the di- and oligo-peptide permeases. However, it is possible to synthesise N-linked glycosides that would be taken up by the glycoside permeases. Dr. Bovill of Thermo Fisher Scientific Ltd., has successfully synthesised AEP-~,D-galactoside. This molecule has been shown to be active against coliform bacteria, but inactive against Salmonella species. This molecule successfully meets all the requirements ofautocytotoxic compounds capable of inhibiting the growth of competitive microflora during the pre-enrichment step during detection of Salmonella spp. in foods.