The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. It was hypothesised that non-antibiotic producers that are niche competitors with producers will need to possess resistance to the antibiotic and will thus have acquired resistance genes. A detailed phylogenetic study, utilizing a novel multilocus sequence typing (MLST) scheme, was made of a collection of isolates and types strains with a Streptomyces griseus phenotype in addition to type strains from known producers of streptomycin and related compounds. strA and vph were found either within a biosynthetic gene cluster or independently. S. griseus strains possessing the streptomycin cluster formed part of a clonal complex and have been readily isolated from soil originating in every continent except Antarctica. Few copies of strA were detected in soil total community DNA, none of which were identical to the gene from the streptomycin cluster. All S. griseus strains possessing solely strA belonged to two clades and were closely related to streptomycin producers. The strA in the resistance-only strains is likely to have originated from the self-resistance gene of another aminoglycoside cluster and arrived in those S. griseus strains via horizontal gene transfer. S. griseus strains with only vph also formed two clades and were more distantly related to the producers than to one another. The high sequence divergence of the viomycin resistance genes also suggests that the vph homologue arrived in these two groups from another peptide antibiotic cluster via horizontal gene transfer. The expression of the strA gene was constitutive in resistance-only strains from both subgroups whereas streptomycin producers showed peak strA expression in late log phase which correlates with the switch on of streptomycin biosynthesis. One example of horizontal gene transfer of the streptomycin cluster was discovered, to a Streptomyces platensis strain, which contained a cluster with 84% sequence identity and almost identical gene structure and arrangement to that of the S. griseus cluster. Its expression pattern was also highly similar to that of S. griseus producers, but at a much lower level. Whilst there is evidence that antibiotics have diverse roles in nature, this work clearly supports the co-evolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.

Cyanobacteria are common components of the bacterioplankton in freshwater environments, where they play a key role as primary producers. Growth is limited by the availability of nutrients, particularly phosphate (Pi), and yet many species persist and flourish in environments with an unpredictable and constantly fluctuating supply of Pi. Genome analysis of the freshwater cyanobacterium Synechocystis sp. PCC6803 has identified that the membrane-bound transporter components of its Pho regulon consist of two high affinity (Pi) ABC transporters with multiple associated phosphate binding proteins (PBP), features in contrast to virtually all other known bacteria. Whilst the occurrence of duplicate ABC transporter mechanisms has been widely reported in freshwater cyanobacteria there are still very few reports that demonstrate the functional significance of individual, and apparently redundant, components of these ABC transporter systems. In previous work, disruption of one of the PBPs in Synechocystis sp. PCC6803, pstS1 (sll0680) led to an impairment in the expression of specific genes of the Pho regulon during Pi-deplete growth. This phenotype was not observed when the PBP from the second transporter was disrupted suggesting that each transporter could be functionally distinct. In this study 32Pi radiotracer uptake experiments using pst1 and pst2 deletion mutants showed Pst1 acts as a low affinity, high velocity transporter (Ks 3.7 ± 0.7 μM, Vmax 31.18 ± 3.96 fmol cell-1 min-1) and Pst2 a high affinity, low velocity system (Ks 0.07± 0.01 μM, Vmax 0.88 ± 0.11 fmol cell-1 min-1). Analysis of (qPCR) gene expression profiles and alkaline phosphatase activity also revealed how regulation of transporter abundance controls the nature of the Pi stress signal transduced by the SphS-SphR two component system. These Pi ABC transporters thus exhibit key differences in both their kinetic and regulatory properties, revealing a new strategy for the acquisition of phosphate that has potential implications for our understanding of the ecological success of this key microbial group.

The bacterial twin-arginine translocation (Tat) pathway has the unique ability to export pre-folded proteins across the cytoplasmic membrane. Its name came from the almost invariant twin-arginine motif in the signal peptide of Tat substrates. Escherichia coli, a Gram-negative bacterium, is typically used to understand Tat function in bacteria. Until now, evidence has shown that TatA, TatB and TatC comprise the minimally functional unit, moreover, a quality control system exists to monitor the assembly of cofactors and the correctly folding state of proteins which avoids the futile export and initiates the degradation of rejected molecules. The research presented in this thesis sought to gain insight into the quality control mechanism of the Tat pathway in E. coli, and also study the relationship between transport and maturation of substrates. In the first place, a novel Tat substrate, YedY, was used to analyse the nature and variety of proofreading functions operating in conjunction with the Tat pathway. The single substitutions in three predicted ligands for the YedY molybdopterin centre led to complete inhibition of export and variable degradation of mutated YedY forms, indicating an effective proofreading activity. Circular dichroism spectroscopy and inductively coupled plasma mass spectrometry of purified proteins demonstrated the change of secondary structures between YedY and mutated variants, and also indicated the content of Mo in molybdopterin cofactor within proteins. The data suggested that the three mutated forms failed to correctly assemble cofactor which resulted in rejection by the Tat export pathway on the basis of the different changes of secondary structures. Further analysis shows that none of the known export chaperones for molybdenum cofactor-containing Tat substrates is required for YedY biogenesis; export is unaffected in cells lacking DmsD and TorD. In the second place, maturation of pre-YedY was blocked when an Ala>Leu substitution was made at the -1 position of the signal peptide, and a membrane-bound precursor form accumulated in the membrane. However, the mature domain had been transferred to the periplasm. The accumulation did not block transport of other Tat substrates, indicating the precursor exited from the translocation channel and integrated into the membrane bilayer. Since the precursor was not detected in the periplasm, it was suggested that the precursor has undergone lateral transfer into the bilayer during translocation. These results are discussed in relation to the overall mechanism of translocation and proofreading by the Tat pathway in E. coli.

The bacterial Tat pathway facilitates the translocation of pre-folded proteins over the cytoplasmic membrane. In Gram-negative bacteria, TatA, TatB and TatC (each an integral membrane protein) are the essential components. Most of our understanding of Tat function in bacteria has come from studies on Escherichia coli, a Gramnegative bacterium. Gram-positive bacteria have Tat systems that are composed of just a single TatA and TatC protein. The absence of TatB suggests a different organisation and translocation mechanism to the Tat systems of Gram-negative bacteria. Here the Tat pathway of Bacillus subtilis, a Gram-positive bacterium, was analysed in detail for the first time revealing important structural differences to the E. coli Tat pathway. Complementation experiments reveal the Tat pathway of B. subtilis is active in E. coli, pointing to functional conservation between Gram-negative and Gram-positive bacteria. The complexes formed by TatA and TatC in B. subtilis were investigated. TatA and TatC form a tight complex that is significantly smaller than its E. coli TatABC counterpart, possibly reflecting the presence of a different number of TatA and/or TatC units within this complex. TatA in B. subtilis like in E. coli also forms homooligomeric complexes separately from TatC. Unlike E. coli TatA complexes that vary enormously in size, the TatA complexes of Gram-positive bacteria are small and homogeneous in nature, suggesting an entirely different translocation mechanism involving a single defined translocon rather than a spectrum of size variants as proposed for E. coli. The TatA proteins from Gram-positive bacteria may be bifunctional and perform the roles of E. coli TatA and TatB. Here the first direct evidence to support this hypothesis is presented and domains important for both TatA and TatB roles identified. Finally a soluble population of TatA identified in B. subtilis was analysed and evidence is presented that suggests it maybe mis-localised.

Two-component signalling systems (TCSs) are found in most prokaryotic genomes. They typically comprise of two proteins, a histidine (or sensor) kinase (HK) and an associated response regulator (RR), containing transmitter and receiver domains respectively, which interact to achieve transfer of a phosphoryl group from a histidine residue (of the transmitter domain in the HK) to an aspartate residue (of the partner RR’s receiver domain). An automated analysis pipeline using the NCBI’s RPS-BLAST tool was developed to identify and classify all TCS genes from completed prokaryotic genomes using the PFAM and CDD protein domain databases. A large proportion of TCS genes were found to be simple hybrid kinases (HYs) containing both a transmitter domain and a receiver domain within a single protein, presumably the result of the fusion or combination of separate HK and RR genes. This propensity to consolidate functionality into a single protein was found to be limited in the presence of either a transmembrane sensory/input domain or a DNA binding domain – two spatially separated functions. While HK and RR genes are usually found together in the genome, in some species a large proportion of TCS domains are found as part of complex hybrid kinases (genes containing multiple TCS domains), in isolated or orphaned genes, or in complex gene clusters. In such organisms the lack of paired HK and RR genes makes it difficult to define genome-encoded signalling networks. Identifying paired transmitter and receiver domains from a pan-genomic survey of prokaryotes gives a database of amino acid sequences for thousands of interacting protein-protein complexes. Covariation between columns of multiple sequence alignments (MSAs) identifies particular pairs of residues representing interactions within the docked complex. Using numerical scores, these amino acids pairs were successfully used as explanatory variables in a generalised linear model (GLM) to predict the probabilities of interaction between transmitter and receiver domains.

The aim of this study was to investigate the iron- and sulphur-oxidizing activities of thermophilic bacteria with reference to the possible use of such bacteria in the extraction of metals from mineral sulphides. The initial characterization of a range of isolates was based on growth studies with iron and sulphur substrates and on the comparison of whole cell protein electrophoresis patterns. Three groups of bacteria were isolated and studied: moderately thermophilic iron- and mineral sulphide-oxidizing bacteria, moderately thermophilic sulphur oxidizers and extremely thermophilic Sulfolobus-like organisms. Both moderately and extremely thermophilic acidophiles were isolated from hot spring and coal pile samples. The moderately thermophilic iron-oxidizing bacteria and the extreme thermophiles which were examined were sub-divided into three and four sub-groups respectively. In a comparative study of continuous flow iron-oxidation reactors, moderate thermophiles did not produce higher rates of ferric iron production than the mesophile T. ferrooxidans but iron oxidation was less sensitive to inhibition by chloride in a vessel containing a thermophile than in a vessel operating with the mesophile. Iron oxidation during autotrophic growth of moderately thermophilic acidophiles and the rapid dissolution of mineral sulphides during the autotrophic growth of both the moderate and the extreme thermophiles were demonstated, thus considerably increasing the potential industrial significance of these bacteria. The yield of soluble copper from a chalcopyrite concentrate was shown to increase with temperature from relatively low yields with the mesophile T. ferrooxidans, through moderate yields with the moderately thermophilic bacteria to almost complete mineral solubilization with the newly isolated Sulfolobus strains.

A study of the complete genome sequence of Streptomyces scabies 87.22, a common causative agent of scab disease of tubers including potato (Solanum tuberosum), is described. This work includes annotation of the genome and in-depth description of gene clusters likely to encode biosynthetic pathways for complex natural products and not also found in either “Streptomyces coelicolor” A3(2) or Streptomyces avermitilis MA-4680. Twenty-eight gene clusters were identified as likely to encode enzymes for the biosynthesis of complex natural products. Substances predicted by this work, not previously known to be made by S. scabies 87.22, were confirmed by collaborators as products - desferrioxamines, germicidins, and hopene. Of the clusters identified, fourteen gene clusters are not conserved in the other two streptomycete genome sequences for which comparisons have been undertaken. The Streptomyces genus is a reservoir of producer organisms from which many complex natural products of therapeutic importance have been isolated. These findings suggest that the cargo of cryptic and silent gene clusters amongst other members of this genus may add significantly to previous estimates of undiscovered bioactive natural products. Methods developed in this work could enable other researchers to rapidly identify gene clusters likely to encode enzymes involved in biosynthesis of complex natural products from complete genome sequences. De-replication is a problem for approaches to drug discovery based on activity screening and isolation of wild producer organisms. Computational methods in this work allow rapid de-replication of gene clusters following sequencing which may lead to discovery of many new natural products with therapeutic benefit. Sequences predicted to be involved in scab disease pathogenicity are not found in only one ‘pathogenicity island’ location as expected, but at several loci. Two possible mechanisms were identified from sequence data which it is suggested could be involved in regulation of pathogenicity traits: an MbtH-like protein family and an iron box sequence likely to be triggered response to low iron conditions.

Although prosthecate bacteria have been observed in freshwater environments, questions concerning the function or survival value of these integral cellular extensions are unresolved. However, it is now becoming apparent that this group of microorganisms forms part of a highly speciallsed indigenous population. An electron microscope survey of the bacterial population from several oligotrophic water bodies has shown that Hyphomicrobiurn and Caulobacter species constitute up to 20% of such populations, the percentage being correlated with the nutrient status of the system. The multiappendaged genera, at best, approach 1% of the total microbial population. 'Enrichment' systems to which no nutrients have been added, select for the latter. Several isolates of multiappendaged bacteria have been obtained from such systems. Studies on one of these has shown that gross phenotypic variation can be brought about by varying the nutrient status of the culture medium. These studies have shown that the prosthecae can be induced or repressed by environmental stimull. This phenotypic variation consequently makes the estimation, simply on morphological grounds, of the incidence of multiappendaged bacteria in the environment difficult. It almost certainly leads to gross underestimates of numbers. Similarly, studies on Hyphomtcroblum have demonstrated that this prosthecate bacterbin not only exhibits differentiation which is obligate to the cell cycle, but is also capable of environmentally induced differentiation. These observations also highlight a fundamental split concerning prosthecal function: - (i) where prosthecal formation is obligate and intimately involved in the cell cycle. e.g. Caulobacter and Hyphomicroblum and (ii) where prosthecae are non-obligate and environmentally induced, e.g. the multiappendaged bacteria, Ancalomicrobium.

This project focuses on the Virus Infectivity Factor protein of HIV-1 and its relief of the block to virus replication exerted by the APOBEC3G component of the innate immune response. Virus Infectivity Factor (vif) is an accessory gene of HIV, deletion of which leads to large drops in virus infectivity. This decrease in infectivity was found to be due to APOBEC3G, an inhibitor of HIV replication which is constitutively expressed in peripheral blood mononuclear cells (PBMCs), the natural host cells for HIV in humans. Vif is indispensable to block the inhibitory effects of APOBEC3G thereby allowing normal viral replication to continue inside the host. This recognition of the critical role played by Vif in the viral replication cycle has centred studies on characterising the interactions of Vif with both APOBEC3G as well as other virus encoded proteins. Stimulation of proteasomal degradation of APOBEC3G is currently believed to be the primary anti-APOBEC3G effect induced by Vif. However recent reports, particularly those showing that Vif remains able to block the inhibitory actions of degradation resistant APOBEC3G, question the validity of this hypothesis. The recognition that both APOBEC3G and Vif become incorporated into HIV particles through an interaction with the precursor of the virion structural proteins, Pr55GAG has raised the possibility that they may compete with each other for this incorporation. Using techniques such as mammalian two-hybrid assays, sucrose gradient analysis of GAG virus-like particles (VLPs) and confocal imaging, the interactions of these proteins with Pr55GAG have been analyzed and the results obtained indicate that Vif competes with APOBEC3G for Pr55GAG binding leading to its displacement and exclusion from the budding HIV virions. This potentially important pathway for Vif activity and its significance in the development of novel antiretroviral drugs in the future will be discussed.

Based on the increase of 16S rRNA gene sequences in databases it is possible to design improved oligonucleotide primers for this gene. Primers were designed in silico to specifically amplify fragments of the gene from the Alpha, Beta and Gamma subgroups of the Proteobacteria, as well as from Bacteroidetes, Firmicutes, Cyanobacteria and Planctomycetes and tested in silico and in vitro. The aim was to investigate bacterioplankton diversity and reveal greater fingerprint diversity within these groups than is possible using primers specific for the entire domain Bacteria, and also to reduce clone library redundancy. It was then aimed to investigate the potential impacts of increased pCO2 and ocean fertilisation with iron (Fe) and phosphorus (P), on bacterioplankton diversity. Group-specific clone libraries representing contrasting marine regions were analysed, and the usefulness and specificity of the primers validated. The clone libraries showed members of the oligotrophic marine group (OMG) to be present in an in situ coastal mesocosm supplemented with nutrients. The newly-developed group-specific primers were used in combination with an improved method of denaturing gradient gel electrophoresis (DGGE) to profile in detail bacterial communities in mesocosms, which were maintained at 750 ppm of pCO2, the level projected for the global surface ocean in the year 3000, and 380 ppm of CO2, the present level. Increased pCO2 correlated with a decrease in abundance of some members of the Gammaproteobacteria. Otherwise there was little impact on diversity due to raised pCO2. The same DGGE protocol was applied to samples from an ocean Fe and P fertilisation experiment. Diversity change due to Fe was not evident. However in seawater amended with P there was an explosive growth of some cells with 16S rRNA genes similar to those of the SAR86 clade, and others with similarity to Gammaproteobacteria with large genomes such as Oceanospirillum sp. and Psychromonas sp.