Identification and Characterization of a Pseudomonas Megaplasmid Family and Its Sensitivity to an Inhibitory Compound Conserved Across Pseudomonas spp.

Abstract

Genetic exchange across organisms that do not undergo sexual recombination occurs via a process known as horizontal gene transfer (HGT) and is facilitated by various entities including plasmids. HGT occurs frequently throughout the bacterial tree of life and accounts for the spread of genes associated with antibiotic resistance, symbiosis, virulence, and metabolism allowing bacteria to colonize niches that were once unobtainable. Plasmids are extrachromosomal secondary replicons that rely on host replication machinery and can come in various sizes from the extremely small (< 1kb), to the very large (nearly 2Mb). Megaplasmids are classified as plasmids > 350kb, and the majority of their predicted gene annotations have an unknown function. Many megaplasmids do encode selective traits such as metal resistances, but it is still unclear what remaining functions are critical for the persistence of large mobile elements within bacteria when the majority of encoded genes have unknown function. Are plasmids providing direct benefits to the host, are they neutral, are they an expendable evolutionary test bed, or are they selfish elements with negative affects to the host? To add to this complexity, conjugation and acquisition of megaplasmids is costly to the host cell due to host resource depletion of tRNA, ATP, and replication and transcription machinery (among others costs) resulting in reduced fitness, which could allow other bacterial cells to outcompete cells with fitness costs. By understanding the evolutionary histories of megaplasmids and building megaplasmid model systems through the identification of new megaplasmids, we may begin to understand what role these large and mobile secondary replicons play in bacterial populations. Furthermore, bacteria competing for resources will be forced to limit fitness costs and maximize benefits of when to produce a toxin, as constitutive approaches tend to be less cost effective unless under constant pressure from competitors susceptible to said toxin. It is still unclear how bacteria manage this selective process, but stress responses and quorum sensing are thought to modulate this process by recognizing alterations in the environment or non-self cells. When bacteria undergo large scale HGT with megaplasmids it is possible such a large genomic change could alter a bacterial cell to the point of no longer being recognized as self. Within this dissertation, I address these issues by focusing on the Pseudomonas syringae megaplasmid pMPPla107. In Appendix A, my coauthors and I utilize computational and molecular approaches to identify and determine that the megaplasmid pBASL58 in P. putida Leaf58 shares a plasmid ancestor with pMPPla107. This work provides a methodology to identify and compare megaplasmids for relatedness and also gives context to global megaplasmid evolution. In Appendix B, my coauthors and I characterize a conserved, bacteriostatic agent that specifically inhibits the growth of cells carrying pMPPla107. Our work here also provides the biochemical data necessary to continue towards molecular structure identification using NMR. Finally, in Appendix C we perform a long-term evolutionary experiment of P. stutzeri carrying pMPPla107, utilize genetics and comparative genomic approaches to identify two compensatory mutations that result in resistance to the inhibitory agent described in Appendix B. The first is a pMPPla107 SNP in skaA (Supernatant Killing Activity), and the second is a 369kb deletion in pMPPla107 revealing two mechanisms of susceptibility to the inhibitory agent.