The existence of transposable elements in the genomes of predominantly asexual species such as bacteria poses a problem for evolutionary biologists. Attempts to explain the persistence of such elements in terms of selfish spread are confounded by the fact that in an asexual species, there is no way for elements to infect new hosts. However, it is difficult to conceive of a mechanism by which transposable elements can increase their host's fitness.

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The insertion sequence ISY100 is a member of the IS630/Tc1/mariner superfamily of transposable elements. An in vivo transposition assay was set up in this study, confirming the TA target preference of ISY100 and showing that 30 bp from each end of ISY100 is sufficient for efficient transposition. Purified His-tagged transposase bound the transposon ends, protecting approximately 26 bp from cleavage by DNase I at each end. Two helix-turn-helix DNA binding motifs linked by an ‘AT-hook’-like sequence were predicted in the N-terminal domain of ISY100 transposase. Supercoiled plasmid containing ISY100 ends, and synthetic linear transposon ends were tested for cleavage by transposase in vitro. Cleavage products were observed and the cleavage sites were mapped. Linear DNA fragments containing single ISY100 ends were cleaved mainly one nucleotide inside the transposon end to produce a 3’ OH and one nucleotide outside the transposon end to produce a 5’ phosphate. Changes in the flanking TA dinucleotides at either one end or both ends of ISY100, reducing the efficiency of transposition in vivo. These changes also reduced the efficiency of cleavage in vitro. Changes at only one end inhibited cleavage at both ends implying communication between the two transposon ends. Synthetic pre-cut transposon ends were tested in an in vitro integration assay, and transposase catalysed the insertion of transposon 3’-OH ends into a target plasmid. Transposase mediated efficient integration of a mini-ISY100, pre-excised by transposase or restriction enzyme, into TA targets in vitro, confirming that excised transposon fragments are intermediates in the reaction. Target sequences of ISY100 from published data and this study were analyzed, yielding the consensus target sequence ADWTAWHT, in which the central TA is the duplicated target dinucleotide. When the Zif268 DNA-binding domain of Tn3 resolvase, transposition occurred into TA dinucleotides to one side of a Zif268 binding site with elevated frequency. This could be developed into a genetic tool for target-specific integration.

The scientific community has gathered an extremely detailed and sophisticated understanding of the genetic and molecular underpinnings of microbial communication. How these microbial communication systems arise and are maintained over evolutionary time-scales however has received relatively little attention. Some major questions remain unanswered such as; what is the function of small diffusible molecules? How does population structure affect the dynamics of social communication and what is the link between the ecology of communication and the virulence of a pathogenic population? Borrowing concepts from evolutionary theory can help to unravel these fundamental questions in the context of microbial communication as it has done in other taxa displaying social behaviours. In addition microbial model organisms in which molecular and genetic tools are abundant lend enormous power to empirical tests of evolutionary theory. This work combines both of these in an attempt to understand the evolution of bacterial communication using the model organism Pseudomonas aeruginosa and its well characterised Quorum Sensing systems. Specifically the focus is in three areas. Firstly this study reveals that the stability of bacterial signalling is vulnerable to perturbations in cost and benefit and genetic conflict. Secondly this study finds that spatial structure (biofilm vs planktonic) influences the outcome of social competition over signalling and reduces population viability. Thirdly this study finds that interspecific and intraspecific competition over public goods impose divergent selective pressures on communication.