Initial work involved the isolation and characterization of bacteria found on the surfaces in a range of food processing environments. The attachment characteristics of three species of bacteria (S.liquefaciens, S.cohnii and P.fragi) isolated from the same production surface were examined. Mixed culture biofilm development was also modelled using these three organisms. The species were found to have differing attachment abilities and therefore cell surface characteristics such as outer membrane protein and lipopolysaccharide profiles, exopolysaccharide production, and cell surface hydrophobicity and charge were examined to explain these differences. All of these cell surface characteristics were subject to considerable variation in response to the environmental conditions. The outer membrane protein and lipopolysaccharide profiles did not show any relationship to attachment levels, and similarly exopolysaccharide production did not relate to the levels of attachment observed, although exopolysaccharide production was found to be particularly associated with attached cells. The attachment of S. cohnii and P jragi correlated to cell surface hydrophobicity, whilst the attachment of S.liquefaciens was dependent upon cell surface charge.

G protein-coupled receptors (GPCRs) comprise the largest family of cell-surface receptors involved in sensing a multitude of ligands and are consequently attractive pharmacological targets. Their study is complicated by cross-talk between signalling pathways and altered receptor pharmacology due to, for instance, receptor oligomerization. Difficulties in obtaining structural information of the receptors hinder the understanding of oligomerization and therefore it is desirable to develop alternative approaches in which to study this phenomenon. The fungal pheromone GPCRs, STE2 and Mam2, from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively are both known to oligomerize and a GxxxG motif in the first transmembrane (TM) domain of STE2 has previously been shown to mediate receptor oligomerization. Previous work on polytopic proteins suggest that individual TM helices may be treated as individually stable domains, and it may therefore be possible to study oligomerization via single TM peptides as opposed to full-length receptor. This thesis describes the use of STE2 and Mam2 to explore TM helix oligomerization and the effects of mutations on receptor trafficking, localization and cellular signalling. The development of a luminescent reporter assay for Sz. pombe, which proved more sensitive than previously used assays and is capable of generating high-throughput data, is also discussed. It was found that STE2 could couple to the Sz. pombe pheromone-response pathway and mutations in the GxxxG dimerization motif affected both signalling and trafficking. Expression of the first TM GxxxG containing domain of STE2 was insufficient for oligomerization, in line with previous reports suggesting that the presence of the second domain is required for receptor oligomerization. In Mam2, a motif was identified that appeared homologous to the STE2 dimerization motif and mutations of this motif also affected trafficking and signalling. This domain could oligomerize in isolation, and mutations of the motif abolished oligomerization. In contrast the study of more polar TM domains appeared more complicated. These findings suggest that relatively hydrophobic TM domains can be studied as individually stable units, whereas more polar domains may require the presence of other TM domains.

The aim of this study was to understand the ecological interactions of B. pseudomallei, a highly pathogenic potential biological warfare agent, in both soils and bio-aerosols. This study examined the impact of DNA persistence on its detection in environmental samples. The potential for members of the Burkholderiaceae to use persistent extracellular DNA in the process of natural transformation was also determined. Lastly, a comparative analysis of methods using 16S rRNA analysis and metagenomics was also undertaken to evaluate their utility for identification of pathogens in complex samples. Soil microcosms were used to determine the impacts of soil mineralogy on DNA persistence using real-time PCR. This study showed that the presence of clay minerals with contrasting adsorption affinities for DNA resulted in significantly different rates of DNA decay in soil. The capacity for Burkholderia sp. to undergo natural transformation was determined through the use of both chromosomal allelic rescue and nonhomologous plasmid uptake strategies. Transformation was suggested as a result of observations of non-homologous plasmid uptake in B. multivorans 13010. However further work is required in this area. The performance of 16S rRNA sequencing using assays targeting the V1-3 and V4-6 variable regions was assessed using soil samples taken from one location that had undergone contrasting application strategies of veterinary antibiotics. This approach was shown to be able to discern subtle shifts in the relative abundances of certain microbial taxa in response to antibiotic application. Outcomes here support previous observations regarding the increased prevalence of mobile genetic elements (integrons) that harbour antibiotic resistance determinants. For bio-aerosols, samples were acquired from regions of known endemicity of B. pseudomallei (N. Australia) and both 16S rRNA and metagenomic analysis methods were used to determine the fluctuations in microbial diversity and the prevalence of this pathogenic organism. The fluctuations of bio-aerosol microbial diversity and in particular B. pseudomallei, within wet and dry seasons of Northern Australia were identifiable using a combination of 16S rRNA and total community DNA metagenomic analyses. The importance of robust bioinformatic analysis is highlighted.

Chitin is the most abundant nitrogen-containing polymer in nature, with >1x10 10 tonnes produced annually in terrestrial and marine habitats. Chitinolytic bacteria are able to degrade this recalcitrant substrate through a multiplicity of chitinases. A polyphasic approach was taken to studying these organisms within three diverse soil communities. Fluorometric assays employing 4-methylumbelliferyl-labelled chitinooligosaccharides were used to estimate basal soil chitinase activity as well as its chitinolytic potential in response to a- and b-chitin amendment. A molecular approach was adopted to profile the bacterial community and functional chi gene diversity within the soils. Finally, a method of exploring the metaexoproteome, enabling investigation of the dominant chitin degraders at a functional level, was developed and implemented. The metaexoproteome and metaproteome, extracted with an existing method, were compared and used to infer the functional dominance of chitinolytic phyla. The basal chitinase activity in all soils was found to be low, yet chitin amendment rapidly induced chitinases in all soils although intersite differences were seen. b-chitin amendment induced more chitinolytic activity in Cayo Blanco (CB) compared to Sourhope (SH). The Test Soil (TS), a site biannually amended with carapaces, retained higher chitinolytic potential many months after chitin had been consumed. Next-generation pyrosequencing enabled >50% of the potential OTUs present in the soil to be recovered. The 16S rRNA gene analysis of SH revealed dominant phyla to be Proteobacteria, Actinobacteria, and Acidobacteria with little change between amendments. The TS was dominated by the same phyla but saw a proliferation of Actinobacteria with chitin amendment. CB experienced the inverse response to the Test Soil, initially dominated by Actinobacteria only for Proteobacteria to dominate with amendment. Firmicutes were also prevalent with b-chitin amendment. Functional chi gene analysis found Streptomyces-like GH19 chi genes to dominate in both SH and CB. A rare Actinomycete Planobispora dominated chitin-amended TS. This organism is usually found in extremely arid soil. It was not found in the 16S rRNA gene analysis or the metaproteome; further analysis is required to confirm its presence. Streptomyces- like GH18 chi genes only dominated CB with amendment and were absent in SH. A large number of OTUs were identified as uncultured organisms suggesting a large pool of uncharacterized GH18 chi genes. Metaproteomics is the functional analysis of complex communities at a given point in time. The heterogeneity of soil, associated microbial communities, and presence of interfering compounds make the extraction of protein from soil a technical challenge. Chitinases are extracellular and so the metaexoproteome was targeted after development of a novel method that biased extraction towards exoproteins. The protocol successfully extracted the largest soil metaproteome to date. Actinobacterial chitinases were found to be functionally dominant in the Test Soil, especially in response to b-chitin amendment.

Acetolactate decarboxylase (ALDC) is a bacterial enzyme of the butanediol fermentation pathway that decarboxylates (S)-acetolactate into (R)-acetoin. Remarkably this enzyme also catalyses the decarboxylation of the opposite enantiomer, (R)-acetolactate, to give the same product, (R)-acetoin. It is unusual for an enzyme to convert racemic substrate into an enantiomerically pure product. This unusual stereochemical control has led to extensive study of the ALDC mechanism and the hypothesis that ALDC catalyses the rearrangement of (R)-acetolactate into (S)-acetolactate prior to decarboxylation. The research presented in this thesis sought to gain insight into the molecular mechanism of the ALDC catalysed reaction by using a combination of kinetic and structural techniques. Bacillus subtilis alsD encoding ALDC was cloned into an expression vector and a series of active site mutants were prepared. The activity of mutant AlsD were determined using a circular dichroism based assay, which identified that the two active site glutamates and a basic residue are required for catalysis. A series of chiral transition state analogues were prepared in a two-step synthesis to give enantiomerically enriched 2,3-dihydroxy-2-methylbutanoic acid in reasonable yields. Three of the compounds were identified as competitive inhibitors, co-crystallised with Bacillus brevis ALDC and structures solved to 1.1-1.6 Å. These structures, coupled with inhibition studies and site-directed mutagenesis, provide an improved understanding of the molecular processes involved in the stereoselective decarboxylation of acetolactate. A mechanism for the transformation of each enantiomer of acetolactate is proposed.

Over time and concurrent development of methods to identify and characterise bacteria, the lactic acid bacteria (LAB) have undergone multiple taxonomic revisions. As a result of the revisionary nature of LAB taxonomy, the historical characterisation of Lactobacillus acidophilus has struggled with misidentification and misrepresentation. Now however, due to its global use in food products for both flavour and probiotic effect, L. acidophilus is now one of the most well physiologically characterised Lactobacillus species. Bifidobacterium bifidum and Bifidobacterium animalis subsp. lactis are also LAB that are considered to have probiotic effects. Here modern high-throughput next generation comparative genomic techniques are used alongside conventional biochemical and molecular typing methods to analyse the sub-species level diversity of these three probiotic species. Results Randomly Amplified Polymorphic DNA (RAPD) profile similarity analysis showed limited strain-level diversity of L. acidophilus. A species specific marker test was developed for L. acidophilus and used to search for L. acidophilus in wild rodent and human faeces. No L. acidophilus was detected in wild rodent faeces and its carriage in human faeces was highly variable. High-throughput next generation sequencing was used to resequence the genomes of 28 L. acidophilus isolates. Comparing these sequences indicated a high level of genomic conservation in L. acidophilus, which was reflected by limited phenotypic diversity. Comparative genomics in Bifidobacterium animalis subsp. lactis supported the hypothesis that it is a clonally monophyletic species, whereas B. bifidum strains were genomically diverse. Conclusions Methods for phenotypically characterising and typing LAB have generally been superseded in accuracy by DNA sequence based methods. Probiotic bacteria display a range of subspecies level population structures. Commercial and culture collection L. acidophilus isolates did not significantly differ phenotypically, but were distinct when their genome sequences are compared. B. bifidum was genotypically diverse at the subspecies level, while B. animalis subsp. lactis appeared to be clonally monophyletic. Comparative genomics and genome (re)sequencing of probiotic bacteria will become a “gold standard” method for characterisation and typing of isolates.

Cytokinesis involves abscission of the intracellular bridge between two daughter cells, thus completing mitosis. Membrane traffic is at the heart of mammalian cytokinesis. Rab11-FIP3 (also referred to as FIP3) in complex with Rab11 plays a key role in the delivery and targeting of recycling endosomes to the furrow; this is essential for completion of cytokinesis. FIP3 undergoes spatial and temporal dynamics during mitosis. During metaphase and early anaphase, FIP3 is largely cytosolic, with some localised to endosomal membrane structures. During late anaphase, following furrow initiation, FIP3 localises to the centrosome. At late cytokinesis, FIP3 relocates to the cleavage furrow and midbody. On separation of the daughter cells, FIP3 returns to the centrosome. The regulatory mechanisms governing the dynamics of FIP3 during mitosis are unknown. This work aims to determine if FIP3 can be phosphorylated by the cell-cycle kinases which regulate mitosis, and whether this has an impact on the spatial and temporal dynamics of FIP3. In vitro phosphorylation assays show that FIP3 can be phosphorylated by cyclin B-CDK1, Plk1, Aurora A and weakly by Aurora B. A proteomic approach revealed that, within the limits of our experimental approach, only cyclin B-CDK1 phosphorylated FIP3 significantly, at serine 102. Interestingly, proteomic analysis of FIP3 immunoprecipitated from metaphase cells has identified serine 102, 281, 348 and 451 as sites of potential phosphorylation. Data from this and a collaborating lab offers the hypothesis that FIP3 is phosphorylated during the early stages of the cell-cycle, and that dephosphorylation of FIP3 is the trigger for the association of FIP3 with membranes. A phospho-specific antibody to serine 102 (pS102) detects CDK1 phosphorylated FIP3. Serine 102 is phosphorylated in metaphase and becomes dephosphorylated as the cell progresses through to telophase. Further analysis reveals that cytosolic levels of pS102 peak in metaphase and decrease towards telophase to negligible levels. pS102 is absent in the membrane fraction. This work suggests that FIP3 may be directly phosphorylated by CDK1, at serine 102, in early mitosis. Kinase inhibition studies show that inhibition of CDK1, by the inhibitor BMI-1026, results in a mis-localisation of GFP-FIP3 in HeLa cells. This could also be interpreted as a delay in cytokinesis, since CDK1 inhibition resulted in more cells in telophase displaying GFP-FIP3 in a localisation characteristic of an earlier stage of telophase, compared to the controls. The role of phosphorylation at serines 102, 281, 348 and 451 of FIP3 was investigated by creating phospho-null and phospho-mimetic mutants in the context of GFP-FIP3. It would appear that when mutated singly, the potential phospho-sites of serines 102, 281, 348 and 451 (phospho-mimetic mutant only for serine 451) have no significant effect on the localisation of FIP3 during mitosis, nor do they affect cytokinesis. A phospho-null mutation of serine 451 resulted in poor expression of the protein and an unhealthy population of cells. In summary, the spatial and temporal dynamics of FIP3 may be regulated by phosphorylation. We hypothesise that during prometaphase and metaphase FIP3 is phosphorylated, preventing its association with endosomes. FIP3 is dephosphorylated in late anaphase, allowing it to associate with endosomes and subsequently traffic to the furrow and midbody. We suggest that CDK1 phosphorylates FIP3 in the early stages of mitosis, at serine 102. In conclusion, membrane traffic is central to mammalian cytokinesis and data from this thesis suggests that it may be regulated by the cell-cycle kinases.

Two methionine -lyase gene homologues, mgl1 and mgl2 have been isolated from Trichomonas vaginalis using a degenerate oligonucleotide/PCR approach. Degenerate oligonucleotides designed against cystathionine -lyase from yeast, rat and human were used in the PCR experiments. mgl1 and mgl2 are present at single copy in the T. vaginalis genome and are expressed to give 1.3kb mRNAs. The two genes have extremely short 5' untranslated regions. The predicted molecular mass of MGL1 and MGL2 are 42.9 and 43.1 kDa, respectively. High homology exists at the amino acid level between the two T. vaginalis methionine -lyase gene homologues and methionine -lyase from Pseudomonas putida and cystathionine -lyase from a range of organisms and other related sulphur amino acid-metabolising enzymes. The two methionine -lyase homologues were cloned into expression vectors and recombinant proteins purified and subsequently characterised. Biochemical characterisation of rMGL1 and rMGL2 revealed that both recombinant proteins were able to break down methionine, catabolise homocysteine at high rate and also able to metabolise cysteine and O-acetyl L-serine. Interestingly, the recombinant proteins were not able to break down cystathionine. The two proteins were expressed in T. vaginalis, as judged by ~43 kDa proteins being detected in T. vaginalis lysates and soluble extracts by Western blot analysis. Tritrichomonas foetus and Tritrichomonas augusta do not possess the ability to breakdown homocysteine and are thought not to contain methionine -lyase. Interestingly, however, antibodies raised against rMGL1 and rMGL2 recognised proteins of various molecular weights and upon immunostaining with intact parasites, it is probable that the Golgi apparatus of these parasites were recognised by the anti rMGL1 and rMGL2 sera. For T. vaginalis, immunostaining using the anti rMGL1 and rMGL2 sera revealed a staining of the nucleus and a more general staining of the cytoplasm of these parasites.

Despite the introduction of penicillin, infections caused by Streptococcus pneumoniae are associated with significant morbidity and mortality. As a result, there is an urgent need for successful identification of new drug targets within the organism. This thesis focuses on characterisation of MurM, which initiates the synthesis of branched muropeptides within pneumococcal peptidoglycan. MurM and MurN generate either alanyl-alanine or seryl-alanine appendages on the stem peptide lysine of Lipid II, ultimately resulting in indirect cross-linkage of the cell wall. Inactivation of murMN causes a reversion to penicillin sensitivity in penicillinresistant strains. However, elucidation of the relationship between MurM activity and penicillin-resistance is complicated by some penicillin-sensitive strains, including R6, having an unusually high proportion of indirect cross-linkage in their cell wall. Therefore, MurMR6 has been kinetically characterised with Lipid II, AlatRNAAla and Ser-tRNASer for comparison to MurMPn16 (penicillin-sensitive) and MurM159 (penicillin-resistant). These results confirmed that MurM159 is more catalytically active than MurMPn16. However, in the presence of Ser-tRNASer, the catalytic activity of MurMR6 approaches that of MurM159. Stimulation of MurM by cardiolipin indicates the potential role of pneumococcal membrane phospholipid composition in the regulation of this enzyme. Assessment of MurM substrate specificity was made using misaminoacylated SertRNAAla. Results indicate that Ser-tRNAAla is used more efficiently by MurM providing a link between peptidoglycan biosynthesis and the fidelity of protein synthesis in S. pneumoniae. A 2’-amino minihelix analogue of Ala-tRNAAla inhibits MurM with an IC50 of 0.5 μM demonstrating specific acceptance of the amino acid from the 2’ hydroxyl of the terminal adenine of the tRNA substrate. Crystallisation of MurM in the presence of zinc and subsequent characterisation of its metal-ion binding properties by kinetic analysis, isothermal titration calorimetry and bioinformatics-informed site-directed mutagenesis have identified that this enzyme is zinc-dependent. In combination, these findings have far-reaching implications for future drug design.

The aim of this project was to investigate the possible role of the biofilm matrix as a barrier to drug diffusion in Candida biofilms and in mixed species fungal-bacterial biofilms. The penetration of antifungal agents through single- and mixed-species biofilms containing Candida was investigated using a novel filter disk bioassay. Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine. Drug penetration was more extensive with C. albicans than with the other species and the rates of diffusion of either drug through biofilms of three strains of C. albicans were similar. In all cases, after 3 to 6h the drug concentration at the distal edge of the biofilm was very high (many times the MIC). Nevertheless, drug penetration failed to produce complete killing of biofilm cells. These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms under these conditions. It has been reported that the production of extracellular matrix by Candida biofilms growing under static incubation conditions is relatively minimal, but increases dramatically when developing biofilms are subjected to a liquid flow. In this study, Candida biofilms were grown under flow conditions in a modified Robbins device (MRD). Biofilms of C. albicans grown in the MRD produced more matrix material than those grown statically, and were significantly more resistant (P<0.001) to amphotericin B. Biofilms of C. tropicalis synthesized large amounts of matrix material even when grown statically, and such biofilms were completely resistant to both amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and S. epidermidis RP62A, when grown statically or in the MRD, were also completely resistant to amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and S. epidermidis M7, on the other hand, were completely drug resistant only when grown under flow conditions. Overall, these findings demonstrate that the matrix can make a significant contribution to drug resistance in Candida biofilms, especially under conditions similar to those found in catheter infections in vivo, and that the composition of the matrix material is an important determinant in resistance.