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The molecular biology behind the development ofAspergillus nidulans has been studied for many years. Although the physiology of mycelia can be altered by varying the growth medium, it has been shown that there is a very strong genetic contribution to the process. This study examined the role of three proteins in A. nidulalls hyphal growth. It was found that bemA (the orthologue of S. cerevisiae BEMl) is not essential, as previous studies had suggested, and that its deletion has only a moderate effect on cell growth. Deletion of the SH3-1 domain of bemA has no effect on cell growth, but its overexpression in the absence of native bemA results in a polarity defect. It was also shown that BemA has roles in asexual development and in the promotion of vacuolar fusion. BemA localises to the polarisome, not previously seen in A. Ilidulans, and shown to be distinct from the spitzenkorper. It is suggested that BemA is required for stable polarisome formation, similar to S. cerevisiae Bemlp. It was found that the polarisome is required for proper spitzenkorper formation, as well as an efficient secretory system. Depletion of HbrB, a filamentous fungi orphan, also affects cell morphology. The effect of depletion of HbrB on various elements of the secretory system was studied. Evidence presented suggests that HbrB may be involved in protein recycling at the late Golgi. It is known that repression of colA, the orthologue ofNeurospora crassa cal-I, causes a severe growth defect. In this study, the specific effect of CotA on growth regulation was studied. It was found that neither overexpression nor repression significantly affected the spitzenkorper, but that both had an effect on polarisome formation. CotA was found in punctate regions of fluorescence along the hyphae and occasionally at the tip (similar to Cotl).

Mycobacterium tuberculosis is one of the world’s most devastating pathogens. Despite completion of the genome sequence in 1998, research progress has been hampered by a lack of genetic tools and the difficulty of working with the organism. Existing genetic systems are limited by their lack of tight regulation or genetic instability. The aim of this study was to characterise and utilise a range of promoters to express mycobacterial genes in a controllable fashion by generating knockdown strains of a number of target genes, using both sense and antisense approaches. This would help to elucidate the function of a particular gene of interest and identify or validate new drug targets. Sets of genes shown to be inducible by certain stimuli such as tetracycline (Rv0277c, Rv0608, Rv0748, Rv1015c, Rv2487c and Rv3898c), streptomycin (whiB7), sodium dodecyl sulphate or ethanol (whiB6), hypoxia, nitric oxide and stationary phase (Rv2625c, Rv2626c, Rv2627c and hspX), or salicylate (Rv0560c) were selected from the literature. The upstream region of each gene was cloned in front of a reporter gene and activity was tested in M. smegmatis and/or M. tuberculosis. No inducible promoter activity was found for the upstream regions of the tetracycline, streptomycin, sodium dodecyl sulphate or ethanolresponsive genes. Inducible promoter activity was found for some of the hypoxiaresponsive genes and was monitored in relation to growth phase in M. tuberculosis wild type, a dosR deletion mutant and a Rv2625c deletion mutant. The upstream region of Rv0560c was found to contain a salicylateinducible promoter. Promoter elements of this promoter were identified and characterised in M. tuberculosis. Attempts to use the most promising promoters in an antisense setting using the reporter gene lacZ or the mycobacterial gene rpoB were unsuccessful.

Carotenoids are produced by all photosynthetic organisms and a large number of bacteria and fungi. They are responsible for a lot of pigmentation in nature, as well as often playing an essential role in the provision of light protection to cells and as precursors of vitamin A in higher organisms. Myxobacton is the primary carotenoid ester generated in the photoresistant bacterium Myxococcus xanthus. It is created through a complex light-regulated gene expression cascade and acts to protect the bacteria from blue light and the resultant generation of damaging singlet oxygen species in the presence of porphyrins. The final stage in its production is a ketonisation, and the enzyme responsible for this stage was unknown in M. xanthus. We propose a possible location for the gene encoding such a ketolase, crtW. The gene is found located within a four-gene operon separate from the other known carotenoid biosynthetic genes, and appears to have two alternative promoter regions. The additional genes in the operon were found to encode a putative MutT/Nudix family hydrolase and a periplasmic, molybdopterin and haeme-dependent oxidoreductase, YedYZ. It is also shown that crtW transcription is independent of cell exposure to blue light and that the product is an inner membrane, integral membrane protein probable ketolase. The evolutionary origins of crtW are considered in conjunction with a number of other carotenoid biosynthetic enzymes, suggesting that the gene was one of the last to be acquired by M. xanthus.

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.

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.

The integrity of all genomes is under constant threat, with DNA double strand breaks being particularly dangerous. Double strand breaks can be repaired by homologous recombination, a process catalysed by recombinase proteins of the RecA family. The archaeal recombinase, RadA, is homologous to eukaryotic and bacterial Rad51/RecA. Euryarchaea encode an additional Rad51/RecA homologue, RadB. RadB shares homology with the core domain of RadA and has been shown to bind both single and double stranded DNA, binds ATP and possesses a very weak ATPase activity. However, RadB does not catalyse strand exchange. RadB has been shown to interact with RadA, a Holliday junction resolvase (Hjc) and a DNA polymerase (PolD), suggesting a role in recombination. In this study, radB was deleted from the halophilic archaeon, Haloferax volcanii. 'delta' radB strains were slow growing, sensitive to mitomycin C and UV irradiation, and deficient for both crossover and non-crossover recombination. Deletion of radA results in similar phenotypic characteristics, and complete abrogation of recombination. Strains deleted for both radA and radB are equally defective as 'delta' radA strains, demonstrating that RadA is epistatic to RadB. A suppressor of 'delta' radB was isolated and identified as a mutation in the polymerisation domain of RadA (RadA-A196V). radA-A196V suppresses the slow growth, crossover and non-crossover recombination defects associated with 'delta' radB, as well as UV and mitomycin C sensitivity phenotypes. On account of the nature of this suppressor, the observed interaction between RadA and RadB, and the epistatic relationship between RadA and RadB, a role for RadB as a recombination mediator protein is proposed. Finally, strains were deleted for hjc. 'delta' hjc strains exhibit no growth, crossover and non-crossover recombination defects and no UV and mitomycin C sensitivity. This suggests that another, as yet unidentified, Holliday junction resolvase is encoded by Haloferax volcanii.

There is much debate about the evolutionary origins of diversity and many complex gene regulatory network features, such as global regulation. Using novel in silico models the evolutionary origins of complex features, heterogeneity and the role of stochastic molecular processes in gene regulatory network evolution are investigated. It is shown that: i) Repression is essential, even in constant environments, due to energetic constraints. ii) Stochastic basal gene expression forces shrinkage of genomes, whilst its absence leads to ‘bloating’. iii) Models evolved towards a biological goal have a very different network structure to nonadaptively evolved models. iv) Unstable mRNA, stable protein and rapid but robust replication times are strongly selected properties of evolved networks. v) Multiple network solutions within identical environmental conditions are observed with the presence of stochastic basal gene expression. vi) Two attractor states, one with high, and one with low stochastic basal gene expression, are observed in networks which can evolve their levels of basal expression. vii) Functional complexity of a gene regulatory network is dependent on environmental complexity. viii) Functional complexity evolves in hierarchical stages, requiring ‘core’ energy regulation mechanisms before environmental responses and adaptations for growth can be sustained and fixed. ix) Global gene regulation is strongly selected as an efficient energy regulation mechanism

Investigation of genomic diversity can provide insight into the evolutionary history of bacterial species. However, complete genome sequencing is not yet practical for large strain collections at the beginning of this project. In this project PCR-based methods to investigate the genomic diversity of non-sequenced strains were successfully developed. In \(Escherichia\) \(coli\), the distribution of two Type III secretion system, ETT2 (\(E.\) \(coli\) Type Three Secretion 2) and Flag-2 (\(E.\) \(coli\) Flagellar system 2), were surveyed among a collection of 79 strains. Remnants of both clusters were found to be present in most strains, suggesting that both have a long evolutionary history within \(E.\) \(coli\). The PCR-based methods were also developed for application as part of genome sequencing projects. They were used to explore the co-linear and variable regions between \(Campylobacter\) \(jejuni\) M1 and the genome sequenced strain \(C.\) \(jejuni\) strain 11168. The \(C.\) \(jejuni\) M1 genome was assembled into thirty-four genomic contigs relative to strain 11168, and the size and position of insertions/deletions were characterised. Similar methods were used to facilitate the finishing of the genome of \(Francisella\) \(tularensis\) strain FSC198, using sequence information from strain Schu S4. The completed genome sequence of FSC198 showed it to be almost identical to that of Schu S4. The two genomes differ at only 11 loci, eight SNPs (single nucleotide polymorphisms) and 3 VNTRs (variable number tandem repeats). This surprising finding suggested that the European isolate FSC198 may be derived from the US laboratory strain Schu S4. Two virulence factors, IglA and IglB, from a pathogenicity island of strain FSC198 were further investigated and found to interact at the protein level. These proteins are possibly involved in Type VI secretion, and may represent potential vaccine candidates.

The activity of the major pathogenicity determinant of enterohaemorrhagic Escherichia coli is primarily coordinated by expression of the LEE1 operon which is part of the locus of enterocyte effacement (LEE). The LEE1 operon regulatory region has been dissected. LEE-encoded transcription factor, GrlA, activates the LEE1 P1 promoter by binding to a target located within the 18 base pair spacer between the promoter 10 and 35 elements. Shortening this spacer to 17 base pairs increases P1 promoter activity and short-circuits GrlA dependent activation, suggesting that GrlA functions like MerR family transcription activators. It was also found that the LEE1 P1 promoter is overlapped by a cryptic promoter, designated P1A. A single base substitution in the P1 consensus -35 element unmasks P1A promoter activity. In contrast, P1A activity is much less when P1 is inactivated by a mutation in its -10 hexamer element. Hence, even when P1 is inactive, the consensus -35 element sequesters RNA polymerase and prevents its access to the P1A promoter. The LEE1 leader sequence also contains a mini-gene that encodes a dipeptide. Genetic studies showed that expression of this mini-gene is important for optimal expression of downstream genes.