Riverine environments are stressed and damaged by anthropogenic actions, with chemical contamination a key factor, identified as potentially adversely impacting the aquatic network. Standardised Organisation for Economic Co-Operation and Development (OECD) tests are used to establish biodegradation rates of chemicals in the environment, however these tests are conducted in unrealistic conditions deficient of environmental realism. Increasing the environmental realism of OECD tests has the capacity improve prediction of chemical persistence and identify potential for damage to the environment. Moreover, the interactions between bedform, microbial biofilm communities and chemical biodegradation at the sediment-water interface are not considered in OECD tests. The current project explored the effects of bed-form characteristics on biofilm development and the consequences of sediment on biodegradation at the sediment-water interface. Novel flume systems were designed, constructed and used to develop methodologies to investigate the impact of bed form on hyporheic exchange. River water and sediment were sampled from a stretch of the River Dene (Wellesbourne, UK) and used as microbial inocula in chemical biodegradation studies using a series of specially designed re-circulating flume systems and ex-situ bottle experiments. Flume experimentation quantified the rate of microbial community development, topographical location and effective depth penetration on sediment beds within artificial watercourses, whilst simultaneously identifying the impact that this biofilm development possessed on hyporheic exchange. The effects of light and inoculum source on para-nitrophenol (PNP) biodegradation were also determined following OECD regulatory test protocols in an attempt to evaluate the realism of chemical biodegradation tests. Experimental data showed that microbial development varied on different sediment bed profiles, and that biofilms significantly reduced the rate of hyporheic exchange on small (0.5 mm), but not large (2.0 mm) sediment particle size bed materials. Additionally, this study found that light inhibited PNP degradation in all tests and that sediment sieving utilised in OECD tests decreased rates of biodegradation of PNP. This project revealed the importance of microbial biofilms in determining hyporheic exchange. However, further experimental work is recommended to investigate hyporheic exchange in heterogeneous sediments, whilst developing more inclusive approaches for chemical risk management by Regulatory bodies.

In order to further the study of the molecular biology and biochemistry of the obligate methanotroph Methylococcus capsulatus (Bath) several genetic techniques where investigated. A system for transfer of plasmids from E.coli to M.capsulatus by conjugation using a filter-mating technique was developed, and a variety of broad-host-range plasmids were transferred. Several parameters which could effect the efficiency of plasmid transfer were assessed, including the possible presence of a restriction/modification system, and it was found that transfer was at its' most efficient when M.capsulatus was in early logarithmic growth, transfer took place over 24 hours at 37oC, and the ratio of donor to recipient was at least 1:5. It was also possible to transfer the plasmids RP4 and pULB113 from M.capsulatus to E.coli. The initial development of a vector for analysis of promoter expression in M.capsulatus was attempted. Several plasmids were also investigated for their ability to act as mutagenesis vectors in M.capsulatus. pSUP2021 was found to transfer into M.capsulatus and Tn5, present on the vector, was shown to be inserted into the chromosome. The entire pJFF350 vector was found to be transferred into the chromosome, including Omegon-kanamycin fragment, IS1 ends and RP4-mob fragment. This vector was used to produce novel plasmids containing pJFF350 DNA and M.capsulatus chromosomal DNA. Two vectors, developed for marker-exchange mutagenesis of glutamine synthetase, were tested and found to be unsuccessful. A further vector was developed from pJFF350 and was found to transfer into the chromosome, but as yet its exact position of insertion is unclear. Electroporation was investigated, however, despite the alteration of electrical parameters and the pretreatment of cells no success was achieved. Results appeared to indicate a problem with a restriction/modification system. The mobilization of the chromosome using RP4 prime plasmids and pULBl13 was attempted but was unsuccessful.

The oxidation of methane to methanol by methanotrophs is catalysed by the enzyme methane monooxygenase (MMO). In some methanotrophs two distinct MMOs are found; a membrane bound particulate (pMMO) and a cytoplasmic soluble (sMMO) methane monooxygenase. The intracellular location of MMO is dependent on the copper-to-biomass ratio. pMMO is expressed when the copper-to-biomass ratio is high (>0.25 IlM) and sMMO is expressed when the copper-to-biomass ratio is low «0.25 IlM). Although a great deal of information is known about the expression of MMO with respect to copper, the molecular mechanism regulating this 'copper-switch' is unknown. The aim of this study was to gain further insights into the regulation of MMO expression by copper ions. The complete sMMO operon, including the regulatory genes, mmoR and mmoG were cloned and sequenced from Methylosinus sporium. The genes encoding sMMO are present as single copy in methanotrophs. In this study, duplicate copies of the mmoX gene encoding for the a-subunit of the hydroxylase were characterised at the molecular and biochemical level. Mutational analysis indicated that the second copy was not essential for sMMO expression and also gave insights into the role of the water soluble pigment in siderophore-mediated iron-acquisition. sMMO-minus mutants were complemented by heterologous expression of sMMO genes from Methylosinus trichosporium and Methylococcus capsulatus. These experiments demonstrated the amenability of Ms. sporium to genetic manipulations facilitating its use as an alternative model organism for molecular analysis of MMO regulation. To aid transcriptional analysis of the MMO operons, a series of integrative and broad-host range promoter probe vectors, containing g{p, xylE, kmR or lacZ, were constructed and tested with the copper repressible sMMO 0'54 promoter. The usefulness of these reporter genes for the high-throughput detection of sMMO mutants was also assessed. The expression of LacZ in Mc. capsulatus via the sMMO 0'54 promoter yielded a powerful genetic screen for sMMO mutants. This system was coupled with transposon mutagenesis for surveying the genome on a global scale for sMMO regulatory genes and served as an alternative assay system for detecting sMMO expression. This assay system had the specific advantage in that it was more sensitive and in this context, it was selective for sMMO-minus mutants defective only at the transcriptional level. In collaboration with Robert Csaki (University of Szeged), a transposon mutagenesis protocol was established from which a number of sMMOminus mutants were identified. Genetic tools developed in this study were used to investigate copper mediated transcriptional regulation of the pMMO 0'70 promoter, sMMO 0'54 promoter and its regulatory genes, mmoR and mmoG. Transcription of the pMMO operon was shown to be constitutive. The sMMO 0'54 promoter was reconfirmed to be copper repressible and mmoR and mmoG were shown to be essential for transcription initiation from the sMMO 0'54 promoter, but were not regulated themselves by copper at the transcriptional level. All of these data were confirmed by constructing chromosomal gene fusions with various reporter genes, RT-PCR and RNA dot-blotting. The availability of the Mc. capsulatus genome sequence during this study allowed targeted mutagenesis to be carried out on copper targets responsible for copper transport. 'Knock-out' mutants of a copper transporting gene and a non-ribosomal peptide synthetase gene responsible for the putative biosynthesis of methanobactin, which is believed to be involved in delivering copper to pMMO, were constructed. The phenotypes of these mutants with regards to MMO expression are yet to be analysed.

Obligate micro-parasites must enter and compromise a living host cell in order to reproduce. Distinct from symbiosis and commensalism, parasitism results in host mortality or morbidity; the severity of which may be expressed in terms of virulence. Bacteriophage and viruses are ubiquitous in the environment, and localised co-existence of host with parasite is a priori evidence for mechanisms which act to mitigate virulence. When physical separation of host-parasite is not possible, molecular immunity to infection may be selected. The phage growth limitation (Pgl) system of soil bacterium Streptomyces coelicolor confers resistance to temperate phage ΦC31 and clear plaque (virulent) derivatives. A key component of the Pgl mechanism (pglX) is a putative methyltransferase, which resembles a type I restriction modification protein. Phase variable regulation represents an elegant mechanism for generating bi-directional genetic polymorphism; reversible phenotypic diversity. At ca. 10-3 – 10-4 DNA replications, translational phase variation of pglX results from slipped strand mispairing within a polyG-tract. Potential costs associated with the phage-resistant allele were considered. It was hypothesised that the Pgl phenotypes would be distinct in the absence of ΦC31. Growth differences were observed between phase variant S. coelicolor populations in the absence of ΦC31, indicative of Pgl-derived pleiotropy. Liquid infection studies were executed using virulent phage ΦC31cΔ25, designed to exert various degrees of selective pressure on independent cultures of each host phase variant. Under conditions designed to maximise phage-host encounter, the population and evolutionary dynamics of phage and host were interrogated. It was hypothesised that selection for the Pgl-positive allele would be rapid when host seeds were predominantly Pgl-negative. Shaken liquid microcosms were seeded with host, and exposed to ΦC31cΔ25 at various densities (MOI 10, 0.1, 0.001 and 0). Surviving host were purified of exogenous phage, processed, and progeny spores used to inoculate fresh microcosms. Phage selection was maintained at each respective MOI using ancestral (stock) ΦC31cΔ25. Phage adaptation was predictably constrained, however it was hypothesised that pglX phase variation would prevent ancestral ΦC31cΔ25 extinction at an intermediate MOI. At selected microcosm cycles, host populations were screened for phenotypic resistance to ΦC31cΔ25, and a novel adaptation of dHPLC (Transgenomic Inc) was used to resolve and make semi-quantitative the N+/-1 sized pglX alleles within a mixed PCR product. Using host total community DNA, the contribution of Pgl to supporting free ΦC31cΔ25 in addition to host phage-resistance was ascertained. ΦC31cΔ25 mutants bearing lysogenic characteristics emerged during the first infection cycle; a phenomenon reproduced in soil microcosms. While temperance often gives rise to lytic phage mutants in vitro, the converse was most unexpected, particularly during experimental regimes designed to direct evolution in favour of the host. Mutant ΦC31cΔ25 microcosm isolates produced turbid plaques when assayed using S. lividans TK24, and subsequent S. lividans lysogenic spores were superimmune to infection when screened on ΦC31cΔ25-impregnated agar.

Emerging antibiotic resistance mechanisms threatens the foundation of modern medicine. Growing evidence suggests anthropogenic inputs such as agriculture could form reservoirs of resistant bacteria which could directly or indirectly transfer to humans. Waste Water treatment plants (WWTPs) are an input which contains waste from several sources including that of human, animal and industry, providing a hot-spot for horizontal gene transfer to occur between bacteria from many origins. In this project we evaluated the role of WWTPs in creating environmental reservoirs of antibiotic resistance. An initial study investigated the impacts of WWTP effluent on the antibiotic resistant bacterial load in downstream rivers, particularly focusing on the class 1 integron as a marker for resistance. WWTP effluent was responsible for significantly higher levels of resistant bacteria in downstream river sediments compared to upstream, a result of the introduction and/ or selection for a diverse range of class 1 integrons. A second study investigated the effects of effluent on the clinically important antibiotic resistance gene blaCTX-M-15. Numerous examples of blaCTX-M-15 carried on new genetic contexts in association with new pathogenicity determinants were recovered, as was evidence for transfer of blaCTX-M-15 between diverse bacteria. The prevalence of blaCTX-M-15 as well as the diversity of its carriage were both increased greatly by WWTP effluent. The final study was on the Thames River basin in the UK, where we developed a model with the ability to predict antibiotic resistance load and exposure. This work suggests that WWTP effluent contributes to environmental reservoirs of resistant bacteria which could be of clinical importance. There is a danger that continued expansion of environmental reservoirs of antibiotic resistance will lead to increased therapeutic failure in the clinic and ultimately the end of the antibiotic era.

Ecologically important marine cyanobacteria fuel global geochemical cycles: Synechococcus spp. numerically dominate vast areas of the ocean and specific lineages occupy distinct niches. The ability to respond to the key nutrient iron has been hypothesised to determine how Synechococcus can compete for and occupy various ecological niches. However, the molecular mechanism of uptake, storage and regulation of iron acquisition has not been defined in Synechococcus and even less is known in Synechococcus sp. CC9311, a strain from a highly abundant coastal lineage. The response of Synechococcus sp. CC9311 was investigated using a range of physiological measurements, qPCR and global transcriptional analysis that revealed a marked response to iron deplete conditions. 25% of genes were differentially regulated and were enriched in regions of atypical nucleotide usage, so-called genomic islands, predicted to be hot-spots for horizontal gene transfer. These data add weight to the hypothesis that acquisition of specific genomic islands facilitates niche occupancy for marine Synechococcus. The work identified significant up-regulation of a putative iron regulator protein: a CRP-FNR homologue location in a region with key iron uptake genes. Other unique findings include the up-regulation of putative ferrous iron uptake genes, an interesting finding in light of the presumed reliance of ferric iron uptake in sea water. A system for over-expression and purification of CRP1390 in E. coli was optimised to enable its characterisation, with the aim to identify possible DNA and or iron binding capabilities. A large number of genetic constructs were also implemented to inactive crp1390 and other putative regulators (fur orthologues). This study further elucidated the iron stress response of Synechococcus sp. CC9311, highlighted the role of genomic islands in the adaptation process and reiterated the role of iron on this important group of organisms in light of anthropogenic change of the modern ocean.

This thesis illustrates the characterization and exploitation of inner membrane proteins in Escherichia coli and involves two parallel aspects. Firstly, in order to build the picture of the small inner membrane proteome in Escherichia coli K12, global search for E. coli K12 small inner membrane proteins was performed by means of bioinformatic analyses. The profile that was generated in this study consists of 108 short open reading frames encoding membrane proteins in E. coli str. K12. Additionally, a second list of 138 sequences was produced and is formed by recently identified genomic sequences with predicted transmembrane domains. The generated profile of small (characterized and predicted) membrane proteins facilitated further biochemical analysis of some predicted and hypothetical short open reading frames encoding inner membrane proteins. Two experimental approaches to study small membrane proteins were used. In the first, confirming genomic expression, the expression of short open reading frames encoding membrane proteins was analysed by means of RT-PCR. The second approach, involved cloning the predicted, hypothetical small membrane proteins under arabinose-inducible promoter, pBAD and detecting their membrane localization. These experiments confirmed bioinformatic results and furthermore demonstrated the uselfullness of the approach used. The Bacillus subtilis small inner membrane protein, TatAd was then used as the case study in the exploitation of inner membrane proteins. Recently, it has been shown that E. coli cells can export high levels of heterologous protein in Tat-dependent manner. In this thesis, B. subtilis TatAdCd system in E. coli lacking the native TatABC system was analysed by means of batch fermentation. It was showed, that a heterologus model protein, TorA-GFP, is efficiently exported by the TatAdCd system to the periplasm. Further analysis revealed that while the GFP is initially exported to the periplasm, the mature protein is progressively found in the extracellular medium. It was demonstrated that by the end of a 16 h batch fermentation, ~ 90 % of exported GFP was present in the medium as active mature protein. It was confirmed that total protein profiles of the medium and periplasm were essentially identical, confirming that the outer membrane became leaky during the fermentation process. The downstream processing and sample analysis revealed that GFP export levels were relatively high, with ~ 0.35 g GFP L-1 culture present in the medium. The analysis of bacterial cells under transmission electron microscopy revealed, that the cells remain intact and there is no large-scale release of cytoplasmic contents. Additionally, novel phenotype for E. coli !tat TatAdCd-expressing cells was observed. Cells overexpressing TatAdCd system displayed filamentous phenotype which became more prominent towards the end of the incubation time. The thesis concludes with a discussion of the pharmaceutical and biotechnological relevance of producing recombinant protein in E. coli by the TatAdCd system and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing. Additionally, the advantages and general applicability of global analysis of E. coli small inner membrane proteome are presented.

The translocation of GLUT4 from intracellular stores to the plasma membrane in response to insulin accounts for the large insulin-mediated glucose uptake in muscle and fat tissue. A defect of this translocation mechanism is evident in individuals with insulin resistance and type 2 diabetes. Hence, understanding the molecular basis of GLUT4 localisation and recycling is important in order to assist the design of rational therapies for the treatment of this disease. Here, we have used iodixanol gradient analysis to examine the intracellular distribution of GLUT4. By this method intracellular GLUT4 could be separated into two pools one of which is highly insulin sensitive, and corresponds to "GLUT4 storage vesicles" (GSV), while the other is less insulin sensitive and is of endosomal origin. We further show that during differentiation of 3T3-L1 fibroblasts into adipocytes, the formation of the GSV compartment appears to be driven by the expression of GLUT4.SNARE proteins are involved in the fidelity of GLUT4 translocation, but little is known about how these proteins may be altered in insulin resistance. Using insulin-resistant 3T3-L1 adipocytes, we show that SNARE protein levels are increased. The potential importance of this observation is discussed. Using iodixanol gradient analysis we also found that the downregulation of GLUT4 known to occur in insulin-resistant 3T3-L1 adipocytes is predominantly from the GSV compartment. The implications of these data for the aetiology of insulin resistance are discussed.

Candida species are commonly part of the normal flora in humans; however, they are opportunistic fungal pathogens that are capable of causing a variety of infections in hospitalized and immunocompromised individuals. These infections range from superficial to systemic ones. Many Candida infections involve biofilm formation on the surfaces of implanted devices, such as catheters and prostheses, or host tissues. Candida biofilms are resistant to a range of antifungal agents in current clinical use but the basis of this drug resistance is not clear. The aim of this project was to investigate possible resistance mechanisms using two fungicidal agents, amphotericin B and caspofungin, a new drug reported to have anti-biofilm activity. The activity of amphotericin B and caspofungin at different development phases of Candida biofilms was investigated in vitro. Amphotericin B at two times the MIC (for planktonic culture) had the least effect on Candida biofilms, but at a higher concentration (five times the MIC) it showed relatively high activity against biofilms of C. parapsilosis and C. glabrata, especially at the late development phase. Biofilms of C. albicans were more resistant to amphotericin B throughout development (except for the earliest stage) than the other Candida species. Caspofungin, at two times the MIC, generally exhibited a greater effect on Candida biofilms than amphotericin B although this was not observed with C. parapsilosis biofilms in some development phases. Caspofungin, at five times the MIC, was slightly less effective than at the lower concentration against C. tropicalis in all development phases tested. The species most susceptible to caspofungin throughout biofilm development was C. glabrata. In no case were biofilm cells of any Candida species completely killed by either amphotericin B or caspofungin. The penetration of caspofungin through biofilms of different Candida species was evaluated using an in vitro filter disc bioassay. Caspofungin penetration through biofilms of C. albicans SC5314 was initially faster than C. albicans GDH2346; however, after 6 h drug diffusion was greater with biofilms of strain GDH2346 (70.8% of the control value). Among other Candida species tested, the highest drug penetration was observed with C. glabrata and C. parapsilosis (81.2% and 73.3% of the control value, respectively), while the lowest was seen with biofilms of C. krusei. Biofilms of C. tropicalis also showed poor penetration. Exposure of biofilms of any Candida species to caspofungin (or amphotericin B) in this assay failed to result in complete killing of biofilm cells. However, evaluation of caspofungin activity against biofilms was complicated by the paradoxical phenomenon (reduced activity of the drug at high concentrations, above the minimum inhibitory concentration). Scanning electron microscopy revealed that caspofungin caused more structural damage to biofilm cells and matrix than did amphotericin B; the highest degree of damage due to caspofungin was observed in biofilms of C. glabrata and C. krusei. The presence of a small number of drug-tolerant or persister cells is one possible mechanism of biofilm drug resistance. Biofilms and planktonic cells of five Candida species were surveyed for the presence of persister cell populations after exposure to amphotericin B. None of the planktonic cultures (exponential or stationary phase) contained persister cells. However, persisters were found in biofilms of one of two strains of C. albicans tested and in biofilms of C. krusei and C. parapsilosis, but not in biofilms of C. glabrata or C. tropicalis. Live-dead staining with fluorescein diacetate confirmed these results which do, however, suggest that persister cells cannot solely account for drug resistance in Candida biofilms. If microorganisms exposed to antimicrobial agents undergo a type of programmed cell death or apoptosis, persisters could be variant in which this process has been disabled. Here, specific staining methods were used to investigate the existence of apoptosis in Candida biofilms subjected to different concentrations of amphotericin B. Caspase activity, indicative of apoptosis, was detected with SR-FLICA and D2R fluorochrome-based staining reagents in all of these biofilms. The general inhibitor of mammalian caspases, Z-VAD-FMK, when used at a low concentration (2.5 μM), increased the viability of drug-treated biofilms up to 11.5-fold (P<0.001%). Seven specific caspase inhibitors had different effects on C. albicans biofilm viability, but inhibitors of caspases-1, -9, -5, -3, and -2 all significantly increased cell survival (40-fold, 8-fold, 3.5-fold, 1.9-fold and 1.7-fold, respectively). On the other hand, histone deacetylase (HDA) inhibitors enhanced the activity of amphotericin B against biofilms of all three Candida species. Sodium butyrate and sodium valproate, for example, when added concurrently with amphotericin B, completely eliminated biofilm populations of C. albicans. Overall, these results demonstrate an apoptotic process in amphotericin-treated biofilms of three Candida species. They also indicate that HDA inhibitors can enhance the action of the drug and in some cases even eradicate persister subpopulations, suggesting that histone acetylation might activate apoptosis in these cells.

The findings of laboratory and field investigations into various aspects of the effects of helminth parasites on the ecology and behaviour of minnows Phoxinus phoxinus and three spined sticklebacks Gazsterosteus aculeatus are reported. A sampling programme was implemented at Loch Maragan over a 16 month period between October 1992 and January 1994 (Chapter 2). The overall prevalence of L intestinalis in minnows from the loch was 17.8%: plerocercoids were found to be weakly overdispersed within the minnow population, although one plerocercoid generally dominated multiple infections whenever they occurred. Both total parasite weight, and the weight of the largest plerocercoid present in an infection were found to be positively and highly significantly correlated with host length, with older, larger fish exhibiting higher parasite burdens. This suggests that fish become infected with L. intestinalis at Loch Maragan during a temporally-limited period during early life. No significant effect of the parasite on host body condition was detected. A qualitative model for transmission of L. intestinalis at the site, based on the available epidemiological data and the prevailing ecological conditions, is proposed. The shoaling and investigative behaviours of minnows from two sites in central Scotland were found to differ, and possible reasons for this variation, based on the ecological disparity between the two sites are suggested (Chapter 3). Both small and medium-sized fish formed more cohesive shoals and schooled more frequently when water depth was reduced, but small fish polarised more frequently than medium-sized fish for any given water depth. The effects of visual oddity on individual shoaling behaviour were investigated (Chapter 3). Size-oddity had little influence on shoaling behaviour in the experimental trials, since the non-uniformity of individual small minnows, when placed in a tank with a group of medium-sized minnows, did not affect their shoaling tendency.