This study examined the regulation of two genes, hcp and ogt, that are reported to be involved in protection from the mutagenic effects of reactive nitrogen species in Escherichia coli. Biochemical techniques were used to investigate promoter activity and the effects of the transcription factors that are reported to regulate expression of the hcp and ogt genes, namely NarL / NarP, FNR and NsrR. Transcription activation by NarL was then studied using semi-synthetic promoters. The hcp gene was found to be positively regulated by FNR and negatively regulated by NsrR. Contrary to previous reports, NarL and NarP were found to have little direct effect on expression of hcp, however, an indirect effect of NarL was detected. This study demonstrates that the indirect effect on hcp regulation of NarL is related to repression by NsrR and suggests that NarL is involved in the generation of reactive nitrogen species. The ogt gene was confirmed to be activated by NarL independently of FNR. Studies focused on characterising the different classes by which NarL / NarP can activate promoter activity independently of FNR. NarL was found to activate by class I, II and III mechanisms, whilst NarP was capable of class II activation only. Activation by NarL was studied and a library of alanine substitutions in the carboxyl terminal domain (CTD) of the α subunit of RNA polymerase was used to demonstrate direct interaction between NarL and RNA polymerase. NarL, the CTD of NarL, NarXL, NarP and the CTD of NarP were expressed from plasmids and transcription activation studied in cells lacking chromosomal narL and narP. Full-length NarL activated by Class I, II and III mechanisms, whilst the CTD of NarL only activated by class II mechanisms. Both the full-length NarP and the CTD of NarP only activated by class II mechanisms.

No description available.

No description available.

Staphylococcus aureus is a notorious human pathogen associated with severe nosocomial and community-acquired infections. In addition, S. aureus is a major cause of animal diseases including skeletal infections of poultry and bovine and ovine mastitis, which are a large economic burden on the broiler chicken and dairy farming industries. The population structure of S. aureus associated with humans has been well studied. However, despite the prevalence of S. aureus infections in broiler flocks, our understanding of the diversity of poultry S. aureus is very limited. In this study, multilocus sequence typing was performed on 48 strains of S. aureus isolated from broiler chickens on farms in 6 countries on 4 different continents, in addition to 9 isolates from different species of reared game and wild birds in Scotland. This was followed by fine scale population genetic analysis of a subset of strains by single nucleotide polymorphism discovery. These studies reveal that the majority of S. aureus isolates from broiler chickens are the descendants of a single human-to-poultry host jump by a subtype of the worldwide human clonal complex 5 (CC5) clonal lineage unique to Poland. In contrast to human subtypes of the CC5 radiation, which demonstrate strong geographic clustering, the poultry CC5 clade was distributed in different continents, consistent with wide dissemination via the global poultry industry distribution network. In order to establish the molecular basis for avian specificity in the CC5 poultry clade, whole genome sequences were determined for a sequence type 5 (ST5) poultry isolate from Ireland and a basal human associated ST5 MRSA strain from Poland. Sequence analysis revealed that the poultry CC5 clade has undergone genetic diversification from its human progenitor strain by acquisition of novel mobile genetic elements from an avian-specific accessory gene pool, and by the inactivation of several proteins important for human disease pathogenesis. In order to examine the importance of positive selection in the adaptation of S. aureus to poultry and for S. aureus evolution, in general, genome-wide analysis of the ratio of synonymous to non-synonymous substitutions was performed on 30 strains from 3 humans and other animals, from diverse lineages. Positive selection has affected proteins from the majority of functional categories, resulting in diversification of the proteome, metabolome and replication capacity, which may be associated with adaptation of S. aureus to diverse environments. For several proteins, an elevated rate of non-synonymous substitutions unique to animal-associated lineages is consistent with a role for these proteins in host adaptation. Taken together, the results of this study have determined the evolutionary history of a major new animal pathogen that has undergone rapid avian host adaptation and intercontinental dissemination. The data highlight the importance of gene acquisition and loss and positive selection in the adaptive evolution of S. aureus.

The time-dependence of molecular evolution, specifically over short timescales, has been shown to be a major confounding factor in the analysis of nucleotide changes between closely related strains or species. The assumption that selection works extremely quickly to purge all of the deleterious changes is at odds with the Nearly Neutral model of evolution, whereby the majority of changes are only mildly deleterious and therefore impose only a minor fitness cost so they are relatively rapidly purged only in populations with large effective population sizes. The aim of this project was to explore the patterns of nucleotide changes evident between the core genomes of nine E. coli and Shigella strains, with the latter having adopted a specific ecological niche in the recent evolutionary past. The Shigellae and E. coli show little difference in their extant genome compositions, in terms of nucleotide composition and genome size, however there are a markedly higher number of pseudogenes and insertion sequences present in the Shigella genomes. The polymorphism profiles of the core genomes reveal a time-dependency of dN/dS, Ti/Tv, +AT/+GC and the Metabolic cost of Amino acid changes, the nucleotide data showing a clear separation of the E. coli from the Shigellae, with the latter showing trends indicative of weaker purifying selection. Additionally these differences are evident when examining the nucleotide ratios (+AT/+GC & Ti/Tv) along the core genome, also revealing patterns of evolution associated with genome position. A simulation based approach reveals different projected nucleotide contents for the E. coli and Shigellae genomes further highlighting their different evolutionary paths as evident from the polymorphism profiles. The methods employed and developed in this study provide a useful and effective toolset for examining the evolution of bacterial genomes over short timescales, especially in light of the availability of multiple whole genome sequences for a given 'species'.

This thesis is a genetic study of the cytoplasmically- inherited determinant [psi] of Saccharomyces cerevisiae . [psi] is a potentiator of ochre suppression. The molecular basis of [psi] was investigated using mutagenesis as a probe. The psi⁺ phenotype (efficient suppression) can be mutated to psi^- phenotype (loss of suppression) by ultra-violet light (UV) and nitrosoguanidine (NTG) . The UV-induced mutation was a single-hit event and the pre-mutational lesion was partly photoreactivable . Repair or expression of UV-induced mutation to the [psi] determinant was under the same genetic control as for nuclear mutation. It was concluded that [psi] has a DNA genome. The 'extrachromosomal mutagens' thymidylate starvation, 5-fluorouracil, manganese chloride and cycloheximide failed to induce psi^- mutants whilst guanidine hydrochloride, dimethyl sulphoxide and potassium chloride were shown to induce this mutation at frequencies up to 100%. Several other physical and chemical agents caused a high frequency of loss of the psi⁺ phenotype. A new class of recessive nuclear mutation (pnm) was shown to cause a loss of the psi⁺ phenotype. A simple comple- mentation test was devised to distinguish them from cytoplasmic psi^- mutants. The dominant PNM^- mutation was shown not to cause a physical loss of the [psi] genome. Two mutants with a modified PNM^- phenotype were analysed. Attempts to demon- strate genetically the involvement of [psi] with the 80S ribosome were unsuccessful. The psi⁺ phenotype was conclusively demonstrated to be inherited independently' of the nucleus using a 'heterokaryon test'. Two models for the [psi] phenomena were proposed; one postulating the presence of a DMA 'plasmid' and one postulating the involvement of a stable, self-perpetuating metabolic state.

Resistance to β-lactam antibiotics in Campylobacter is often associated with the production of a β-lactamase; to date the genomically encoded bla\(_{OXA-61}\) and the closely related cj0299 are the only described β-lactamase genes of Campylobacter. Cj0299 of C. jejuni NCTC11168 was assigned a novel oxacillinase number OXA-193. Previously, a novel β-lactamase (CjBla2)was identified in two bla\(_{OXA-193}\) negative isolates,P843 and P854. Southern blotting with a probe for bla\(_{OXA-193}\) confirmed that Bla2 is not the product of a mutated bla\(_{OXA-193}\) gene. A further thirteen veterinary isolates of Campylobacter have been identified that have the same phenotype as P843 and P854. Whole genome sequencing of P854 revealed four putative beta-lactamase genes, one of which, P854_1490, encodes a completely novel oxacillinase (OXA-184). PCR screening and sequencing of the other putative CjBla2 producers revealed six to contain the novel oxacillinase. A further five encode a variant of this novel OXA in which a point mutation has led to an amino acid coding change from leucine to isoleucine (OXA-185). These isolates represent a selection of flaA types and were isolated from two separate locations at different times, therefore it is unlikely that they are a clonal population. Two conjugative plasmids, each approximately 45Kb in size, have been identified from two veterinary isolates of Campylobacter. These plasmids have been shown to horizontally transfer resistance to tetracycline and to the β-lactams penicillin, ampicillin and oxacillin, between Campylobacter, a process never previously described. The two β-lactam resistance encoding plasmids thought to contain a β-lactamase gene(s) have been named pBla1 and pBla2. The role of efflux in beta-lactam resistance has also been investigated; inactivation of the efflux pump gene cmeB in the reference strain NCTC11168 resulted in increased susceptibility to a range of beta-lactams including the cephalosporins which Campylobacter are reported to be innately resistant to and have been included in some Campylobacter selective media. The work completed as part of this PhD program has furthered the understanding of beta-lactam resistance in Campylobacter and has demonstrated that it is clearly a multi-faceted mechanism incorporating various chromosomally encoded beta-lactamases, putative transferable beta-lactamases and efflux.

Klebsiella spp. are opportunistic pathogens that cause hospital and community acquired infections such as pneumonia, urinary tract infection, septicaemia, soft tissue infections, liver abscess, and meningitis. Multidrug-resistant strains possessing extended-spectrum β-lactamases (ESBLs) has become an increasing problem worldwide. The over use and, in some cases, misuse of antibiotics in humans and in animal husbandry has been cited as a responsible factor in the development of drug resistance in all bacterial species. The advancing age; female gender, hospital crossinfection, the food chain trade and human migrations have contributed to increase the risk for community-acquired ESBL. A total of 223 isolates collected in 2006 and 2007 at Royal Infirmary of Edinburgh, Scotland, 219 K. pneumoniae, 2 K. oxytoca, 1 Enterobacter cloacae, and one isolate Salmonella enterica were identified by API 20E and confirmed genotypically with gyrA PCR-RFLP method. The antimicrobial susceptibility results showed that 34 (15.2%), 36 (16.1%), 35 (15.7%), 45 (20.2%), 30 (13.5%) and 55 (24.7%) of these strains were found to be resistant to cefotaxime, ceftazidime, ceftriaxone, naladixic acid, ciprofloxacin and cefoxitin. None of the isolates were found resistant to meropenem keeping carbapenems the drug of choice for the treatment of multiresistant isolates. The overall frequency of ESBL producers observed in this study was 35 (15.7%) most of them 32/35 (91.4%) were from K. pneumoniae. The genetic analysis showed that SHV β-lactamases were detected in 32, whereas TEM and CTX-M were detected in 24 and 16 respectively. From the ESBL-producing isolates, molecular methods identified nine strains possessing ESBL-SHV genes (1 strain blaSHV-5, 1 strain blaSHV-80 and 8 strains blaSHV-12), whereas the remaining were from the “non-ESBL” producing strains. Conjugation methods demonstrated that 29/32 isolates harboured transferable blaSHV genes. The large SHV transposon-borne promoters were amplified from only one non-transferable blaSHV-11, 15 isolates produced the small SHV transposon-borne promoters. Furthermore, the IS26 was found 73bp upstream of the blaSHV gene in all small SHV transposon-borne promoters. A new blaLEN gene was identified from K. pneumoniae (KpII) phylogenetic group but remained susceptible to all cephalosporins. Sixteen (7.3%) of K. pneumoniae isolates were found to be producers of the CTX-M- 15 ESBL, of which two isolates (12.5%) were reported to be from communityacquired infections. The insertion sequence ISEcp1 was detected by sequencing 48 nucleotides upstream of blaCTX-M-15 in all isolates but one. Five different clones of CTX-M-15-producing isolates were identified by PFGE. The findings indicated a higher prevalence of qnr genes than in previous studies but still low in general. By PCR, 18 (8%) (11 qnrB1, 2 qnrB6 and 5 qnrA1) genes were identified from K. pneumoniae isolates. Also, the findings indicated the frequent coexpression of fluoroquinolones and ESBLs resistance in the same isolate. Two K. oxytoca strains were isolated from urine and blood specimens of hospitalized patients. Both strains were positive for the blaOXY-2 gene. One strain showed resistance to pencillins, monbactams, cephalosporins including cefotaxime and ceftazidime but was not inhibited by clavulanic acid. It differed by an amino acid substitution Ala237→Thr, which enhances the binding of cefotaxime. S1-nuclease plasmid profiles were obtained for some isolates. A total of one to two plasmids, ranging in size from approximately 40 to 210 kb, were observed per strain. The plasmids from 24 ESBL K. pneumoniae strains were assigned to be IncN or IncFII replicons. Analysis of phylogenetic groups showed that the majority of K. pneumoniae isolates were belonged to KpI-type. Both K. oxytoca strains were assigned to be KoII phylogenetic group based on rpoB and gyrA sequencing. Integrons are capable of capturing and mobilizing genes called gene cassettes which play an important role in the dissemination of antimicrobial resistance through horizontal transmission. In fact, the present study indicated a high frequency of occurrence of class 1 integrons among ESBL-positive K pneumoniae. Three isolates positive for class 1 integrons were found positive for class 2 integrons as well. Class 1 integrons including dfr, aadA and ereA2 gene cassettes have been identified by sequencing, which confer resistance to trimethoprim, streptomycin/spectinomycin and erythromycin respectively. In conclusion, the results from this thesis report the emergence of hospital and community-acquired highly resistant CTX-15 β-lactamase in the Edinburgh, Scotland. The prevalence of ESBL-producing isolates in Scotland is still much lower than in many other European countries. The dissemination of SHV- and TEM- β- lactamase types in this study is more predominate than CTX-M-15.

Synonymous codon usage bias has been extensively studied in Bacteria and Eukaryotes and yet there has been little investigation in the third domain of life, the Archaea. In this thesis I therefore examine the coding sequences of nearly 70 species of Archaea to explore patterns of codon bias. Heterogeneity in codon usage among genes was initially explored for a single species, Methanococcus maripaludis, where patterns were explained by a single major trend associated with expression level and attributed to natural selection. Unlike the bacterium Escherichia coli, selection was largely restricted to two-fold degenerate sites. Analyses of patterns of codon usage bias within genomes were extended to the other species of Archaea, where variation was more commonly explained by heterogeneity in G+C content and asymmetric base composition. By comparison with bacterial genomes, far fewer trends were found to be associated with expression level, implying a reduced prevalence of translational selection among Archaea. The strength of selected codon usage bias (S) was estimated for 67 species of Archaea, and revealed that natural selection has had less impact in shaping patterns of codon usage across Archaea than across many species of Bacteria. Variation in S was explained by the combined effects of growth rate and optimal growth temperature, with species growing at high temperatures exhibiting weaker than expected selection given growth rate. Such a relationship is expected if temperature kinetically modulates growth rate via its impact upon translation elongation, since rapid elongation rates at high temperatures reduce the selective benefit of optimal codon usage for the efficiency of translation. Consistent with this, growth temperature is negatively correlated with minimal generation time, and numbers of rRNA operons and tRNA genes are reduced at high growth temperatures. The large fraction of thermophilic Archaea relative to Bacteria account for the lower values of S observed. Two major trends were found to describe variation in codon usage among archaeal genomes; the first was attributed to GC3s and the second was associated with arginine codon usage and was linked both with growth temperature and the genome-wide excess of G over C content. The latter is unlikely to reflect thermophilic adaptation since the codon primarily underlying the trend appears to be selectively disfavoured. No correlations were observed with genome wide GC3s and optimal growth temperature and neither was GC3s associated with aerobiosis. The identities of optimal codons were explored and found to be invariant across U and C-ending two-fold degenerate amino acid groups. The identity of optimal codons and anticodons across four and six-fold degenerate amino acid groups was found to vary with mutational bias. As was first observed in M. maripaludis, selected codon usage bias was consistently greater across two-fold relative to four-fold degenerate amino acid groups across Archaea. This broad pattern could reflect ancestral patterns of optimal codon divergence, prevalent among four-fold but not two-fold degenerate amino acid groups. Consistent with this, the strength of selected codon usage bias was found to be reduced following the divergence of optimal codons, and implies that optimal codon divergence typically proceeds following the relaxation of selection. Finally, a method was developed to partition the strength of selection (S) into separate components reflecting selection for translational efficiency (Seff) and selection for translational accuracy (Sacc) by comparing the codon usage across conserved and nonconserved amino acid residues. While estimates of Sacc are somewhat sensitive to the designation of conserved sites, a general pattern emerged whereby accuracy-selected codon usage bias was consistently strongest across a subset of the most highly conserved sites. Several estimates of Sacc were consistently higher than the 95% range of null values regardless of the dataset, providing evidence for accuracy-selected codon usage bias in these species.

The Saccharomyces cerevisiae Rmi1 protein is a component of the highly conserved Sgs1-Top3-Rmi1 complex, which is required for the maintenance of genome stability. The rmi1Δ deletion mutant has proven difficult to study because it exhibits very poor growth, and rapidly accumulates second site suppressor mutations. Furthermore, deletion of the putative HJ resolvase genes, MUS81-MUS81 or SLX1-SLX4 in rmi1Δ mutants causes synthetic lethality. In order to study phenotypes caused by loss of functional Rmi1, and to explore the genetic interactions between RMI1 and the MUS81, MUS81, SLX1 and SLX4 genes, a temperature sensitive mutant of RMI1 was isolated, named rmi1-1. Similar to rmi1Δ deletion mutants, rmi1-1 cells are highly sensitive to the DNA damaging agent, MMS and the replication inhibitor, HU. In addition, rmi1-1 mutants accumulate replication-associated branched DNA structures, and arrest in G₂/M after a transient exposure to MMS. These cells are proficient in DNA damage checkpoint activation. Deletion of SLX1, SLX4, MUS81 or MUS81 in the rmi1-1 strain causes synthetic lethality, which is associated with cell cycle defects. Following a transient exposure to MMS, rmi1-1 mutants accumulate homologous recombination intermediates. These intermediates are slowly resolved at the restrictive temperature, revealing a redundant resolution activity in the absence of functional Rmi1. This resolution depends upon Mus81-Mms4, but not on Slx1-Slx4 or Yen1. I propose that while the Sgs1-Top3-Rmi1 complex constitutes the main pathway for removal of homologous recombination intermediates following a perturbed S-phase, Mus81-Mms4 can act as a back up for resolution of these intermediates, which most likely represent double Holliday junctions. In this study, I also present screens for high copy suppressors of rmi1-1 phenotypes, and for novel Rmi1 interaction partners.