Bacteria have been the causative agents of major infectious disease pandemics throughout human history. Over the past 4 decades, a combination of changing medical practices, industrialization, and globalisation have led to a number of emergences and re-emergences of bacterial pathogens. The design of rational control programs and bespoke therapies will require an enhanced understanding of the dynamics underpinning the emergence and transmission of pathogenic clones. The recent development of new technologies for sequencing bacterial genomes rapidly and economically has led to a greatly enhanced understanding of the diversity of bacterial populations. This thesis describes the application of whole genome sequencing of 2 bacterial pathogens, Staphylococcus aureus and Legionella pneumophila, in order to understand the dynamics of bacterial infections on different temporal and spatial scales. The first study involves the examination of S. aureus evolution during a chronic infection of a single patient over a period of 26 months revealing differences in antibiotic resistance profiles and virulence factor expression over time. The genetic variation identified correlated with differences in growth rate, haemolytic activity, and antibiotic sensitivity, implying a profound effect on the ecology of S. aureus. Importantly, polymorphisms were identified in global regulators of virulence, with a high frequency of polymorphisms within the SigB locus identified, suggesting this region may be under selection in this patient. The identification of genes under diversifying selection during long-term infection may inform the design of novel therapeutics for the control of refractory chronic infections. Secondly, the emergence and transmission of 3 pandemic lineages derived from S. aureus clonal complex 30 (CC30) were investigated. Independent origins for each pandemic lineage were identified, with striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements, such as bacteriophage and Staphylococcal pathogenicity islands, and non-synonymous mutations affecting antibiotic resistance and virulence. Hospitals in large cities were identified as hubs for the transmission of MRSA to regional health care centres. In addition, comparison of whole genome sequences revealed that at least 3 independent acquisitions of TSST-1 have occurred in CC30, but a single distinct clade of diverse community-associated CC30 strains was responsible for the TSS epidemic of the late 1970s, and for subsequent cases of TSS in the UK and USA. Finally, whole genome sequencing was used as a tool for investigating a recent outbreak of legionellosis in Edinburgh. An unexpectedly high level of genomic diversity was identified among the outbreak strains, with respect to core genome polymorphisms, and accessory genome content. The data indicate that affected individuals may be infected with heterogeneous strains. The findings highlight the complexities in identifying environmental sources and suggest possible differences in pathogenic potential among isolates from a single outbreak. Taken together, the findings demonstrate applications of bacterial genome sequencing leading to enhanced understanding of bacterial pathogen evolution, emergence, and transmission, which may ultimately inform appropriate infection control measures.

The rapid emergence of bacterial resistance to antibiotics has necessitated the development of alternative treatment strategies. Antimicrobial peptides (AMPs) are important immune system components that kill microbes rapidly and have broad activity-spectra, making them promising leads for new pharmaceuticals. Although the need for novel antimicrobials is great, we also need a better understanding of the mechanisms underlying resistance development to enable design of more efficient drugs and reduce the rate of resistance development. The focus of this thesis has been to examine development of bacterial resistance to AMPs and the resulting effects on bacterial physiology. The major model organism used was Salmonella enterica variant Typhimurium LT2. In Paper I, we observed that bacteria resistant to PR-39 appeared at a high rate, and that the underlying sbmA resistance mutations were low cost or even cost-free. Such mutants are more likely to rapidly appear in a population and, most importantly, will not disappear easily once the selective pressure is removed. In paper II, we isolated protamine-resistant hem- and cydC-mutants that had reduced growth rates and were cross-resistant to several other antimicrobials. These mutants were small colony variants (SCVs), a phenotype often associated with persistent infections. One SCV with a hemC-mutation reverted to faster growth when evolved in the absence of protamine. In paper III, the mechanism behind this fitness compensation was determined, and was found to occur through hemC gene amplification and subsequent point mutations. The study provides a novel mechanism for reversion of the SCV-phenotype and further evidence that gene amplification is a common adaptive mechanism in bacteria. In Paper IV, the antibacterial properties of cyclotides, cyclic mini-proteins from plants, were evaluated. Cycloviolacin O2 from violets was found to be bactericidal against Gram-negative bacteria. Cyclotides are very stable molecules and may be potential starting points for development of peptide antibiotics.

Speciation and gene duplication are fundamental evolutionary processes that enable biological innovation. For over a decade, biologists have endeavored to distinguish orthology (homology caused by speciation) from paralogy (homology caused by duplication). Disentangling orthology and paralogy is useful to diverse fields such as phylogenetics, protein engineering, and genome content comparison. A common step in ortholog detection is the computation of Bidirectional Best Hits (BBH). However, we found this computation impractical for more than 24 Eukaryotic proteomes. Attempting to retrieve orthologs in less time than previous methods require, we developed a novel algorithm and implemented it as a suite of Perl scripts. This software, Selectable Taxon Ortholog Retrieval Iteratively (STORI), retrieves orthologous protein sequences for a set of user-defined proteomes and query sequences. While the time complexity of the BBH method is O(#taxa^2), we found that the average CPU time used by STORI may increase linearly with the number of taxa. To demonstrate one aspect of STORI’s usefulness, we used this software to infer the orthologous sequences of 26 ribosomal proteins (rProteins) from the large ribosomal subunit (LSU), for a set of 115 Bacterial and 94 Archaeal proteomes. Next, we used established tree-search methods to seek the most probable evolutionary explanation of these data. The current implementation of STORI runs on Red Hat Enterprise Linux 6.0 with installations of Moab 5.3.7, Perl 5 and several Perl modules. STORI is available at: <http://github.com/jgstern/STORI>.

Bacterial chromosomes are highly dynamic, continuously changing with respect to gene content and size via a number of processes, including deletions that result in gene loss. How deletions form and at what rates has been the focus of this thesis. In paper II we investigated how chromosomal location affects chromosomal deletion rates in S. typhimurium. Deletion rates varied more than 100-fold between different chromosomal locations and some large deletions significantly increased the exponential growth rate of the cells. Our results suggest that the chromosome is heterogeneous with respect to deletion rates and that deletions may be genetically fixed as a consequence of natural selection rather than by drift or mutational biases. In paper I we examined in a laboratory setting how rapidly reductive evolution, i.e. gene loss, could occur. Using a serial passage approach, we showed that extensive genome reduction potentially could occur on a very short evolutionary time scale. For most deletions we observed little or no homology at the deletion endpoints, indicating that spontaneous deletions often form through a RecA independent process. In paper III we examined further how large spontaneous deletions form and, unexpectedly, showed that 90% of all spontaneous chromosomal deletions required error-prone translesion DNA polymerases for their formation. We propose that the translesion polymerases stimulate deletion formation by allowing extension of misaligned single-strand DNA ends. In paper IV we investigated how the translesion DNA polymerase Pol IV, RpoS and different types of stresses affect mutation rates in bacteria. Derepression of the LexA regulon caused a small to moderate increase in mutation rates that was fully dependent on functional endonucleases but only partly dependent on translesion DNA polymerases. RpoS levels and growth stresses had only minor effects on mutation rates. Thus, mutation rates appear very robust and are only weakly affected by growth conditions and induction of translesion polymerases and RpoS.

Bacteria are many, old and varied; different bacterial species have been evolving for millions of years and show many disparate life-styles and types of metabolism. Nevertheless, some of the characteristics regarding how bacteria organize their chromosomes are relatively conserved, suggesting that they might be both ancient and important, and that selective pressures inhibit their modification. This thesis aims to study some of these characteristics experimentally, assessing how changes affect bacterial growth, and how, after changing conserved features, bacteria might evolve. First, we experimentally tested what are the constraints on the horizontal transfer of a gene highly important for bacterial growth. Second, we investigated the significance of the location and orientation of a highly expressed and essential operon; and we experimentally evolved strains with suboptimal locations and orientations to assess how bacteria could adapt to these changes. Thirdly, we sought to understand the accessibility of different regions of the bacterial chromosome to engage in homologous recombination. And lastly, we constructed bacterial strains with chromosomal inversions to assess what effect the inversions had on growth rate, and how bacteria carrying costly inversions could evolve to reduce these costs. The results provide evidence for different selective forces acting to conserve these chromosome organizational traits. Accordingly, we found that evolutionary distance, functional conservation, suboptimal expression and impaired network connectivity of a gene can affect the successful transfer of genes between bacterial species. We determined that relative location of an essential and highly expressed operon is critical for supporting fast growth rate, and that its location seems to be more important than its orientation. We also found that both the location, and relative orientation of separated duplicate sequences can affect recombination rates between these sequences in different regions of the chromosome. Finally, the data suggest that the importance of having the two arms of a circular bacterial chromosome approximately equal in size is a strong selective force acting against certain type of chromosomal inversions.

The study of bacterial evolution is fundamental for addressing current problems of antibiotic resistance and emerging infectious diseases and lays a solid foundation for successful and rational design in biotechnology and synthetic biology. The main aim of this thesis is to test evolutionary hypotheses, largely based on theoretical considerations and sequence analysis, by designing scenarios in a laboratory setting to obtain experimental data. Paper I examines how genomic GC-content can be reduced following a change in mutation rate and spectrum. Transcription-related biases in mutation location were found, but no replicative bias was detected. Paper II explores the distribution of fitness effects of random substitutions in two ribosomal protein genes using a highly sensitive fitness assay. The substitutions had a weakly deleterious effect, with low frequencies of both neutral and inactivating mutations. The surprising finding that synonymous and non-synonymous substitutions have very similar distribution of fitness effects suggests that, at least for these genes, fitness constraints are present mainly on the level of mRNA instead of protein. Paper III examines selective barriers to inter-species gene transfer by constructing mutants with a native gene replaced by an orthologue from another species. Results suggest that the fitness costs of these gene replacements are large enough to provide a barrier to this kind of horizontal gene transfer in nature. The paper also examines possible compensatory mechanisms that can reduce the cost of the poorly functioning alien genes and found that gene amplification acts as a first step to improve the selective contribution after transfer. Paper IV investigates the fitness constraints on horizontal gene transfer by inserting DNA from other species into the Salmonella chromosome. Results suggest that insertion of foreign DNA often is neutral and the manuscript provides new experimental data for theoretical analysis of interspecies genome variation and horizontal gene transfer between species.

The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to induce its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. The experiments described in this thesis were carried out to test these possibilities. In cultures exposed to prolonged stationary phase, majority of the bacterial population dies and a few mutants that have the ability to scavenge the nutrients released by the dying cell mass survive. Bgl+ mutants were found to be enriched in twenty-eight-day-old Luria Broth grown cultures of E. coli that are wild type for bgl but carry the rpoS819 allele. Out of the five Bgl+ mutants that were isolated, four carried a mutation in the hns locus while one of them, ZK819-97, had an activating mutation linked to the bgl operon. Further analysis of ZK819-97 by DNA sequencing revealed the existence of a single C to T transition at the CAP binding site in the regulatory region. ZK819-97 was chosen for further analysis. Competition assays were carried out in which Bgl+ strain, ZK819-97 (Strr), and the parental Bgl- strain, ZK820 (Nalr), were grown independently for twenty-four hours in Luria Broth and then mixed in 1:1,000 (v/v) ratio reciprocally, without addition of fresh nutrients. ZK819-97, when present in minority, was found to increase in number and take over the parental strain, ZK820, i.e. ZK819-97 showed a Growth Advantage in Stationary Phase phenotype. To determine whether the GASP phenotype of ZK819-97 is associated with the bgl locus, the bgl allele from this strain was transferred by P1 transduction to its parental strain, ZK819. The resulting strain, ZK819-97T (Bgl+, Tetr), when competed with the parental strain, ZK819 Tn5 (Bgl-, Kanr), also showed a GASP phenotype when present in minority in the mixed cultures. To reconfirm this further, the bgl locus was deleted from ZK819-97T. The resulting strain, ZK819-97Δbgl, showed a loss of the GASP phenotype. When the bglB locus was disrupted in ZK819-97T, the resulting strain, ZK819-97ΔB, also failed to show a GASP phenotype, indicating that the phospho-β-glucosidase B activity is essential for this phenotype. The strain, ZK819-IS1, carrying an activating IS1 insertion within the bgl regulatory region also showed a GASP phenotype, confirming that this phenotype of the Bgl+ strain is independent of the nature of the activating mutation. All the above mentioned strains used in the competition assays carry a mutant allele of rpoS, rpoS819. Introduction of the wild type rpoS allele in these strains resulted in the loss of the GASP phenotype of the Bgl+ strain, suggesting that the two mutations work in a concerted manner. The Bgl+ strain was found to show the GASP phenotype only when present in minority of 1:1,000 or 1:10,000 in the mixed cultures and showed a slight disadvantage at higher ratios, indicating that the GASP phenotype of the Bgl+ strain is a frequency dependent phenomenon. In competition assays carried out between 24-hour-old cultures of Bgl+ and Bgl- strains resuspended in five-day-old spent medium prepared from a wild type E. coli strain, Bgl+ strain did not show any extra or early GASP phenotype. In addition, a reporter strain, which has a lacZ transcriptional fusion with the activated bgl promoter, was resuspended in spent medium prepared from a five-day-old culture of wild type strain of E. coli and bgl promoter activity was measured by β-galactosidase assay. The bgl promoter did not show any induction in this medium. These experiments suggest the absence of any β-glucoside like molecules in the spent medium within the sensitivity of these assays. A reporter strain that has a lacZ transcriptional fusion to the wild type bgl promoter was used to measure the expression level of this promoter during exponential and stationary phase of growth in LB. Expression of the wild type as well as various activated promoters of bgl was found to be enhanced in stationary phase. To investigate a possible role of the rpoS encoded stationary phase specific sigma factor, RpoS (σs), and another stationary phase factor, Crl, known to be important for the regulation of many genes of the σs regulon, the bgl promoter activity measurements were carried out in the presence or the absence of RpoS and/or Crl. RpoS along with Crl was found to negatively regulate the expression of wild type as well as activated promoters of bgl, both in exponential and stationary phase. In the absence of the negative regulation by RpoS and Crl, the increase in the bgl promoter activity was more pronounced as compared to that in its presence. rpoS and crl mutations are common in nature and it has been suggested that crl deletion gives a growth advantage to the strain in stationary phase. To test this possibility crl deletion was created in wild type as well as in attenuated rpoS allele background. The strain carrying the crl deletion was found to have a growth advantage in stationary phase over the wild type strain in the presence of wild type rpoS allele, while it shows a slight disadvantage in combination with mutant rpoS. Over expression of LeuO or BglJ is known to activate the bgl operon. To study a possible role of these factors in the regulation of the bgl expression in stationary phase, the bgl promoter activity was measured in strains that were deleted for leuO and/or bglJ, in the absence or presence of crl. These studies indicated that BglJ had a moderate effect on the bgl promoter activity in stationary phase in the absence of Crl but not in its presence. LeuO did not have a significant effect on the bgl promoter activity in either condition. Thus under the conditions tested, the physiological increase in the levels of LeuO and BglJ in stationary phase was insufficient to regulate the bgl expression. Preliminary results show that the bgl operon might be involved in the regulation of oppA, an oligopeptide transporter subunit, in stationary phase. Implications of these findings are discussed. The studies reported in this thesis highlight the involvement of the bgl operon of E. coli in stationary phase. This could be mediated by genetic as well as physiological mechanisms. This study also underscores the importance of observing organisms closer to their natural context and the need to reconsider the concept of ‘cryptic genes’.

In most bacterial pathogens, the genome is comprised within a single circular chromosome which is typically organized by the origin-to-terminus axis that divides the chromosome into equally-sized arms of replication (replichores). This similarity in length is presumed to be required for the synchronization of the two replication forks to meet at the terminus for efficient chromosome segregation. Transfer of genes between organisms, different from the route of parent to offspring, is called horizontal gene transfer (HGT). Acquiring foreign DNA through HGT is an important factor for the evolution of virulence in bacteria since it provides access to new features such as new toxins and antibiotic resistance genes. Chromosomes of many pathogenic bacteria such as Salmonella spp. carry such horizontally-transferred DNA fragments called pathogenicity islands. However, after such HGT events, the existing organization of chromosome can be disrupted and an imbalance between the two halves of the circular chromosome might occur. The predicted outcome of a replichore imbalance is the retardation of growth which in turn might result in the out-competition by other faster-growing bacteria in the environment. For that reason, we have investigated the association of the fitness cost and the replichore imbalance with isogenic strains with varying degrees of inter-replichore inversions. Our results showed that there is a correlation between the magnitude of replichore imbalance and fitness cost, for example 2.49-fold imbalance (one replichore 2.49-fold longer than the other) resulted in 11% reduction of fitness in comparison with balanced replichores. Therefore, our data suggest that the replichore imbalance could be utilized to predict the fitness cost of HGT events.