Mechanisms and regulation of replication, the cell cycle, gene expression, and horizontal gene transfer in prokaryotes, focusing on Bacillus subtilis.

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Project Summary/Abstract Horizontal gene transfer (HGT) is a driving force in microbial evolution. It is largely mediated by mobile genetic elements, including viruses, conjugative plasmids, and integrative and conjugative elements (ICEs; aka conjugative transposons), and many bacterial genomes contain several mobile genetic elements, including ICEs and temperate phages. Conjugative elements are well known agents that contribute to the spread of genes for antibiotic resistances, virulence, symbiosis, metabolic functions, and more. ICEs were first discovered because they confer some of these phenotypes. However, potential phenotypes conferred to bacteria by the vast majority of ICEs are not known. Our recent work indicates that ICEs confer beneficial phenotypes that extend well beyond those of some of the previously characterized ICEs, and that some of these phenotypes involve functional interactions between ICEs and bacterial viruses. We are focusing on mechanisms controlling horizontal gene transfer, interactions between mobile genetic elements and their host cells, and the interplay between different mobile elements found in the same cell. Many of our studies are initiated in the bacterium Bacillus subtilis. It is easy to grow and manipulate, naturally contains a variety of mobile elements, including one ICE (ICEBs1) and one functional (SPß) and two defective (PBSX, skin) temperate phages, together comprising almost 6% of the genome. Our recent work indicates that there are beneficial phenotypes conferred by ICEBs1 to host cells that extend well beyond those conferred by previously characterized ICEs, including effects on the timing of sporulation and the activity of other resident elements. Despite the prevalence and importance of ICEs, there are major deficiencies in our understanding of these elements, especially in Gram positive bacteria. Notably, little is known about the interactions between ICEs and their host cells including with co-resident viruses, and the effects ICEs have on fitness of their bacterial hosts. Furthermore, little is known about the interactions between functions encoded by ICEs and those encoded by hosts, and how these interactions influence and determine the host range and efficiencies with which ICEs function in different species. Our work will continue to focus on the lifecycle of ICEBs1 and Tn916, an ICE that is naturally found in several bacterial pathogens and is involved in the spread of tetracycline resistance between them. The ability to experimentally induce ICEBs1 in ~25- 90% of cells in a population, to achieve relatively high conjugation frequencies, and to visualize events in single cells has allowed us to answer previously difficult or unstudied problems fundamental to the ICE lifecycle. Our expertise in chromosome dynamics, DNA replication, stress responses, and microbial development dovetails nicely with our studies of ICEs and phages, notably how these processes affect the lifecycles of mobile genetic elements and how mobile genetic elements affect these processes. We plan to pursue these interests, with particular focus on the connections between ICEs, phages, and cellular processes. Our findings should be relevant to the biology of many bacterial species, especially regarding the transfer of genes between bacteria growing in different environments, including the human microbiome.