Mechanisms of induction of conjugative DNA transfer

Bacterial conjugation is a major avenue of genetic exchange among bacteria. The genetic information required for conjugation is encoded by plasmids and transposons. Conjugative elements frequently carry, in addition, other genes which confer important properties to the host bacteria such as virulence or antibiotic resistance. The growing problem of antibiotic resistance observed in human and animal pathogens illustrates the significance of horizontal gene transfer for our society. The mechanisms of bacterial conjugation specified by conjugative plasmids remain poorly defined. In general for DNA to be transferred from one gram-negative bacterium to another, the cells must be in close physical contact. The conditions promoting intercellular pairing and the processes regulating the start of transfer remain obscure. To initiate transmission, the relaxosome, a specialized nucleoprotein structure of host and plasmid proteins at the plasmid transfer origin (oriT), cleaves specifically the DNA strand destined for transfer and unwinds the duplex DNA. The mechanisms of regulation of the these initial DNA processing reactions have not been fully elucidated for any conjugative system. This project proposes three lines of investigation designed to reveal details about the mechanisms that promote the initiation of conjugative DNA transfer for plasmids belonging to the IncF-IncW family of DNA mobilizing systems. Earlier studies of this laboratory have characterized in detail the genetic regulation of the "tra" (for transfer) genes of the antibiotic resistance factor R1. The first aim of this project is to investigate tra gene regulation at the cellular level using the genetic markers gfp and lacZ and light microscopy. These in situ studies performed in semi-natural environments will examine how R1 tra gene regulation is affected by the physiological status of the cell and by the micro-environment determined by the microbial community. The second aim relies on methodology developed in previous work to monitor the relaxosome-catalyzed origin cleavage activity of the broad host range IncW plasmid R388 in vivo. R388 plasmids possess relaxosomes in the absence of conjugation but these are inactive for origin cleavage. This study will identify conditions that relieve inhibition of the oriT cleaving activity of the R388 relaxosome in vivo. An extensive range of mutant derivatives of R388 is available to evaluate the contribution of specific pilus-forming and DNA processing plasmid gene products to the regulation of the R388 relaxosome. The third objective of this project period is to clarify the structure and activities of the relaxosome of plasmid R1. In contrast to R388, transfer initiation by the relaxosomes of R1drd19 and R1drd16 appears to be regulated first at a post-cleavage stage. This investigation will combine observations of the performance of mutant and wild type relaxosomal proteins in origin cleavage, self-transfer and mobilization in vivo with characterization of the in vitro properties of the purified proteins. Results of this project are expected to provide new insights into mechanisms of intracellular and intercellular regulation that control the initiation of conjugative DNA transfer

This project has contributed to our current understanding of the function and mechanisms of regulation of macromolecular transport systems utilized by Gram-negative bacteria. In this funding period we have focussed attention on the regulation of genes responsible for the production of elaborate membrane-spanning structures composed of several proteins which enable other proteins and DNA molecules to be transported outside of the cell, the cell surface filaments that enable bacterial cells to interact with each other and with surfaces, and the selection system and initiation mechanisms that determine which protein and DNA molecules are secreted by the bacterium. Bacterial cells use secretion processes to deliver molecules to other bacterial, plant or animal cells. Uptake of these secreted substances is often closely linked to infection and disease through direct and indirect effects unleashed in the recipient organisms (and their hosts). A particular class of secretion systems, the Type IV group, are specialized in transmitting DNA from cell to cell. Since DNA is involved, the consequences of uptake of genetic material, which can be stably maintained in the recipient cell and inherited by its progeny, affect diversity and evolution in bacteria. We investigate the role of Type IV secretion in bacterial virulence and the cell to cell transmission process known as bacterial conjugation. The molecular mechanisms of the conjugal secretion process are studied using biochemical and genetic approaches to gain insights to regulation of the initial cell contact, enzymatic processing of DNA molecules destined for transfer, and recognition of those molecules by the transport machinery. We also find that conjugation systems contribute to the development of biofilms - surface attached consortia of bacteria which develop into structured communities and encase themselves in a hydrated matrix of polymers. Organisms living in a differentiated biofilm community are resistant to antibiotics and difficult to remove or control. During the project we have characterized the effects of plasmids and conjugation systems on biofilm development. We also discover and study novel secretion systems that determine host and tissue specificities of pathogens. Insights gained into how these related systems promote the ability of bacteria to adhere and differentiate, to transfer genes including resistance to antibiotics, and to behave as pathogens are complemented by an active interest in discovery and development of inhibitory substances that disrupt these processes with a long term goal of intervention.