Structural studies of T4S systems by electron microscopy

Abstract: Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNA and/or proteins through the membranes of bacteria. Analysis of T4S system architecture is an extremely challenging task taking into account their multi protein organisation and lack of overall global symmetry. Nonetheless the last decade demonstrated an amazing progress achieved by X-ray crystallography and cryo-electron microscopy. In this review we present a structural analysis of this dynamic complex based on recent advances in biochemical, biophysical and structural studies

An Anomalous Type IV Secretion System in Rickettsia Is Evolutionarily Conserved

Bacterial type IV secretion systems (T4SSs) comprise a diverse transporter family functioning in conjugation, competence, and effector molecule (DNA and/or protein) translocation. Thirteen genome sequences from Rickettsia, obligate intracellular symbionts/pathogens of a wide range of eukaryotes, have revealed a reduced T4SS relative to the Agrobacterium tumefaciens archetype (vir). However, the Rickettsia T4SS has not been functionally characterized for its role in symbiosis/virulence, and none of its substrates are known.Superimposition of T4SS structural/functional information over previously identified Rickettsia components implicate a functional Rickettsia T4SS. virB4, virB8 and virB9 are duplicated, yet only one copy of each has the conserved features of similar genes in other T4SSs. An extraordinarily duplicated VirB6 gene encodes five hydrophobic proteins conserved only in a short region known to be involved in DNA transfer in A. tumefaciens. virB1, virB2 and virB7 are newly identified, revealing a Rickettsia T4SS lacking only virB5 relative to the vir archetype. Phylogeny estimation suggests vertical inheritance of all components, despite gene rearrangements into an archipelago of five islets. Similarities of Rickettsia VirB7/VirB9 to ComB7/ComB9 proteins of epsilon-proteobacteria, as well as phylogenetic affinities to the Legionella lvh T4SS, imply the Rickettsiales ancestor acquired a vir-like locus from distantly related bacteria, perhaps while residing in a protozoan host. Modern modifications of these systems likely reflect diversification with various eukaryotic host cells.We present the rvh (Rickettsiales vir homolog) T4SS, an evolutionary conserved transporter with an unknown role in rickettsial biology. This work lays the foundation for future laboratory characterization of this system, and also identifies the Legionella lvh T4SS as a suitable genetic model

Initiation of Agrobacterium tumefaciens T-DNA processing: purified proteins VirD1 and VirD2 catalyze site- and strand-specific cleavage of superhelical T-border DNA in vitro

T-DNA processing during agroinfection of plants is initiated by site- and strand-specific incision at the T-DNA border sequences of the Ti plasmid. Two proteins are required for this reaction: VirD2 (49.6 kDa), catalyzing a site-specific cleaving-joining reaction on single-stranded DNA in vitro (Pansegrau, W., Schoumacher, F., Hohn, B., and Lanka, E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11538-11542), and VirD1 (16.1 kDa), an accessory protein required for VirD2-mediated specific cleavage of double-stranded DNA. Following efficient overproduction, VirD1 was isolated in active form from inclusion bodies and purified to near homogeneity. The protein was applied together with purified VirD2 protein for specific cleavage of double-stranded T-DNA border sequences in vitro. The reaction proceeds on negative superhelical DNA and requires Mg2+ ions. Relaxed DNA is not cleaved. The 5' terminus of the broken DNA strand is covalently associated with protein, most probably VirD2, and the cleavage site is located at the same position that is found in vivo, indicating that the in vitro reaction mimics the one that takes place in induced agrobacteria. Relaxation of plasmid DNA occurs only upon addition of protein denaturants, suggesting that the DNA in the VirD1/VirD2 complex is topologically constrained by strong protein-DNA interactions. The characteristics of the VirD1/VirD2-mediated cleavage reaction strongly resemble those observed with relaxosomes of IncP plasmids involved in initiation of transfer DNA replication during bacterial conjugation