Genes are able to replicate horizontally and vertically- a given gene may be more successful on horizontally mobile elements than others. This includes genes that exhibit a post-segregational killing (PSK) phenotype. PSK is generated by expression of a toxin and antitoxin from a mobile element, such that if a bacterium loses the element the toxin becomes active in the cell and the cell dies. All PSKs described to date involve a toxin and an antitoxin function, though within a given group of toxin and antitoxin gene pairs only some are likely to exhibit this phenotype. Here, I investigate what differentiates genes that induce PSK from biochemically similar genes that do not. One group of genes of which some are known to induce PSK is toxinantitoxin (TA) systems, composed of a stable toxin and an unstable antitoxin. I analyzed computational data on the distribution of type I TA systems (RNA antitoxin), which appear to be less mobile than type II TA systems (protein toxin). Data on validated TAs suggests a correlation between distribution, mobility and the PSK phenotype. Differences in phylogeny could be due to differences in tendency to exhibit PSK in different environments. This connection between distribution and PSK was explored by experimentally testing a computationally described operon, plasmid_Toxin-ptaRNA1, that exhibited structural and distributional similarities to a mobile type I TA system. Despite this, expression of the predicted toxin ORFs did not reduce growth (as measured by saturation density) in E. coli, and the operon did not induce PSK. The conditions of PSK were further tested with the toxin (barnase) and antitoxin (barstar), which are not known to have the phenotype. A number of heterologous expression systems were developed with these genes in E. coli to test their ability to exhibit PSK in a manner akin to both type II TA systems, with a cytoplasmic toxin, and bacteriocins,which have a secreted toxin. I used equations of logarithmic decay to model the necessary expression of the proteins in the cell and their rate of decay after plasmid loss to enable PSK. My results suggest there is likely to be an evolutionary trend toward TA systems with high expression levels of very unstable antitoxins. Secreted barnase was also tested experimentally for its ability to induce PSK similar to bacteriocins, which exhibit a PSK-like phenotype in monoculture by driving maintenance of the immunity encoding plasmid. Barnase did not induce PSK, possibly due to its inability to cause antibiosis in our test system. Structural similarities and biochemical similarities are not sufficient to determine whether a given system will act as a PSK because numerous contextual factors have an effect on whether the genes are addictive. A given set of genes may have the phenotype in one species but not another, under one set of environmental conditions but not another, or on one replicon but not another. This is consistent with the competition hypothesis, which states that genes will be selected for on mobile elements due to their ability to increase horizontal reproductive success, depending on the environmental conditions.

All bacteria initiate translation using formylated methionine, yet directly after translation, the formyl-group is removed. This sequence of addition and removal appears futile, yet every sequenced bacterial genome encodes the enzymes for formylation and deformylation, suggesting this process is essential. Puzzlingly, the process is absent from both Archaea and Eukaryotes, and moreover, bacterial mutants lacking both the formylase and deformylase activities are viable, albeit with a diminished growth rate. We created an Escherichia coli strain devoid of formylase and deformylase activity. This strain was then allowed to evolve over 1500 generations whereupon it reached wild-type growth rate, demonstrating that formylation can be completely dispensed with. This raises an additional question: if the formylation cycle is unnecessary, how did it emerge and why has it persisted? Our results show that the formylation-deformylation cycle could have evolved as a toxin-antitoxin pair (TA) with post-segregational killing (PSK) activity. TAs ‘addict’ cells to the plasmids that carry them by inducing PSK. We measured the stability of formylase-deformylase encoding plasmids and their ability to elicit PSK in our evolved E. coli strain. We report several lines of evidence consistent with the formylation-cycle having evolved from a plasmid-borne PSK element: 1) in the absence of deformylation, formyl-methionine on proteins is cytotoxic in bacteria 2) deformylation relieves the cytotoxicity of formyl-methionine, 3) the loss of a plasmid containing formylase and deformylase genes from evolved cells results in cessation of growth – a standard PSK phenotype. In addition, we introduced the E. coli formylase and deformylase genes into yeast and demonstrate that Met-tRNA formylation is not lethal, even in the absence of deformylation. This suggests PSK would be ineffectual in yeast, accounting for the absence of formylation from eukaryotic cytoplasmic translation. We also report the presence of formylase and deformylase genes in the two representative members of the archaeal Methanocopusculum genus. Moreover, we demonstrate that these genes have been acquired by a recent horizontal gene transfer from bacteria. Our results indicate that formylmethionine use in bacteria evolved, not through a direct functional benefit to cells, but through competition between infectious genetic elements.