Plasmids are key vectors of horizontal gene transfer and essential genetic engineering tools. They code for genes involved in many aspects of microbial biology, including detoxication, virulence, ecological interactions, and antibiotic resistance. While many studies have decorticated the mechanisms of mobility in model plasmids, the identification and characterization of plasmid mobility from genome data are unexplored. By reviewing the available data and literature, we established a computational protocol to identify and classify conjugation and mobilization genetic modules in 1,730 plasmids. This allowed the accurate classification of proteobacterial conjugative or mobilizable systems in a combination of four mating pair formation and six relaxase families. The available evidence suggests that half of the plasmids are nonmobilizable and that half of the remaining plasmids are conjugative. Some conjugative systems are much more abundant than others and preferably associated with some clades or plasmid sizes. Most very large plasmids are nonmobilizable, with evidence of ongoing domestication into secondary chromosomes. The evolution of conjugation elements shows ancient divergence between mobility systems, with relaxases and type IV coupling proteins (T4CPs) often following separate paths from type IV secretion systems. Phylogenetic patterns of mobility proteins are consistent with the phylogeny of the host prokaryotes, suggesting that plasmid mobility is in general circumscribed within large clades. Our survey suggests the existence of unsuspected new relaxases in archaea and new conjugation systems in cyanobacteria and actinobacteria. Few genes, e.g., T4CPs, relaxases, and VirB4, are at the core of plasmid conjugation, and together with accessory genes, they have evolved into specific systems adapted to specific physiological and ecological contexts.

Mobility of Plasmids

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea . Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Biological Diversity of Prokaryotic Type IV Secretion Systems

Type IV secretion (T4S) systems are ancestrally related to bacterial conjugation machines These systems assemble as a translocation channel, and often also as a surface filament or protein adhesin, at the envelopes of Gram-negative and Gram-positive bacteria These organelles mediate the transfer of DNA and protein substrates to phylogenetically diverse prokaryotic and eukaryotic target cells Many basic features of T4S are known, including structures of machine subunits, steps of machine assembly, substrates and substrate recognition mechanisms, and cellular consequences of substrate translocation A recent advancement also has enabled definition of the translocation route for a DNA substrate through a T4S system of a Gram-negative bacterium This review emphasizes the dynamics of assembly and function of model conjugation systems and the Agrobacterium tumefaciens VirB/D4 T4S system We also summarize salient features of the increasingly studied effector translocator systems of mammalian pathogens

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Biogenesis, architecture, and function of bacterial type iv secretion systems

Type IV secretion systems (T4SSs) are important virulence factors used by Gram-negative bacterial pathogens to inject effectors into host cells or to spread plasmids harboring antibiotic resistance genes We report the 15 angstrom resolution cryo-electron microscopy structure of the core complex of a T4SS The core complex is composed of three proteins, each present in 14 copies and forming a approximately 11-megadalton two-chambered, double membrane-spanning channel The structure is double-walled, with each component apparently spanning a large part of the channel The complex is open on the cytoplasmic side and constricted on the extracellular side Overall, the T4SS core complex structure is different in both architecture and composition from the other known double membrane-spanning secretion system that has been structurally characterized

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Structure of a type IV secretion system core complex.

Bacterial conjugation in Gram-negative bacteria is triggered by a signal that connects the relaxosome to the coupling protein (T4CP) and transferosome, a type IV secretion system. The relaxosome, a nucleoprotein complex formed at the origin of transfer (oriT), consists of a relaxase, directed to the nic site by auxiliary DNA-binding proteins. The nic site undergoes cleavage and religation during vegetative growth, but this is converted to a cleavage and unwinding reaction when a competent mating pair has formed. Here, we review the biochemistry of relaxosomes and ponder some of the remaining questions about the nature of the signal that begins the process.

Conjugative DNA metabolism in Gram-negative bacteria.

Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 (DeltaN155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 x 10(7) liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP.

Thirty-Eight C-Terminal Amino Acids of the Coupling Protein TraD of the F-Like Conjugative Resistance Plasmid R1 Are Required and Sufficient To Confer Binding to the Substrate Selector Protein TraM

Klaus Lorenzoni

Papers

Thirty-Eight C-Terminal Amino Acids of the Coupling Protein TraD of the F-Like Conjugative Resistance Plasmid R1 Are Required and Sufficient To Confer Binding to the Substrate Selector Protein TraM