Cut and move: protein machinery for DNA processing in bacterial conjugation

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Conjugation is a paradigmatic example of horizontal or lateral gene transfer, whereby DNA is translocated between bacterial cells. It provides a route for the rapid acquisition of new genetic information. Increased antibiotic resistance among pathogens is a troubling consequence of this microbial capacity. DNA transfer across cell membranes requires a sophisticated molecular machinery that involves the participation of several proteins in DNA processing and replication, cell recruitment, and the transport of DNA and proteins from donor to recipient cells. Although bacterial conjugation was first reported in the 1940s, only now are we beginning to unravel the molecular mechanisms behind this process. In particular, structural biology is revealing the detailed molecular architecture of several of the pieces involved.

Introduction

Mechanisms leading to lateral gene transfer in bacteria are classically categorized as transduction, transformation or conjugation [1, 2, 3]. Transduction occurs via bacteriophages, which can incorporate portions of the host bacterial DNA and introduce them into newly infected hosts. Transformation consists of the uptake of naked DNA from the environment. Finally, conjugation is the unidirectional transfer of single-stranded (ss) DNA (known as the T-strand) of conjugative plasmids (or chromosome-integrated conjugative elements) from a donor to a recipient cell by intimate cell-to-cell contact [3, 4, 5, 6]. After transfer, the recipient becomes a transconjugant, possessing the capacity to start new rounds of conjugation. Through this highly efficient mechanism, a few conjugative-plasmid-harbouring cells within a strain can spread this information among the whole population within short timescales, thus enabling rapid dissemination of adaptive genes and infectious or antibiotic resistance factors. Studies of Escherichia coli strain K12 plasmid F led to the discovery of bacterial conjugation in the 1940s; this plasmid has since become a model for plasmid-encoded conjugation systems in Gram-negative bacteria [7, 8]. Another example is the enterobacterial plasmid R388, which confers resistance to the antibiotics sulphonamide and trimethoprim [9]. Conjugative plasmids have also been found in several Gram-positive bacterial genera, such as Streptococcus, Enterococcus and Staphylococcus [10, 11, 12]. Conjugative-like DNA delivery further occurs between bacteria and eukaryotic plant and fungi cells. A well-known example is Agrobacterium tumefaciens, the etiological agent of crown gall disease, which transfers the tumour-causing plasmid pTi to plants [13].

Most of the proteins engaged in conjugation are encoded by plasmid genes located in the tra (transfer) region, which includes the mpf (mating-pair formation) and dtr (DNA processing and transport) genes [14, 15, 16•]. Dtr encodes proteins responsible for the process in which the T-strand is prepared for transfer. It includes the formation of the relaxosome [17], a multicomponent nucleoprotein complex comprising an ATP-dependent relaxase/helicase, the T-strand, a transcriptional regulator and the host-encoded integration host factor (IHF), and its recruitment to the membrane transport pore (see Figure 1). Mpf encodes proteins that participate in pilus formation and assembly of a type IV secretion system (T4SS), a multiprotein organelle required for horizontal transfer through membranes in Gram-negative bacteria (for recent reviews, see [15, 18, 19, 20, 21, 22]). Conjugation initiates when the pilus, anchored on the donor cell surface, binds to the surface of the recipient cell through its distal end and subsequently retracts to enable stable intercellular wall-to-wall contact. An unknown mating signal then triggers mobilisation of donor DNA, which leads to a site-specific nick in the plasmid T-strand. The relaxosome is subsequently coupled to the T4SS by T4CP, a dtr-encoded receptor or coupling protein ([22, 23]; see Figure 1).

In recent years, structural biology has revealed the detailed molecular architecture of several of the pieces involved in the intricate scenario of conjugation. Here, we review the structures of proteins that participate in the first two stages of DNA transfer, namely processing and recruitment to the cell membrane.

Section snippets

Relaxase/helicase

A key player in the generation of the transferable T-strand is the relaxase/helicase, TrwC in the R388 plasmid system and TraI in plasmid F. Initially described as helicase I in E. coli, this endonuclease has been termed ‘relaxase’ because it relaxes supercoiled plasmid double-stranded (ds) DNA by cleaving one of the strands. It participates in DNA mobilisation by nicking within the origin-of-transfer region (oriT) of the T-strand [23]. Subsequently, a catalytic tyrosine remains covalently

Transcriptional regulator

A protein named TrwA in plasmid R388 has a dual role: it enhances the relaxase/helicase activity of TrwC and represses the operon that jointly encodes TrwA, TrwB and TrwC within dtr [42]. The equivalent protein in the F system, TraY, has been shown to likewise enhance the relaxase/helicase activity of TraI and to regulate the expression of tra genes [43]. TrwA/TraY displays an N-terminal DBD, which recognises two palindromic sites near oriT, and a C-terminal dimerisation domain. In the absence

Integration host factor

IHF is a heterodimeric E. coli protein made up of two structurally equivalent ∼10 kDa subunits that are ∼30% identical in sequence. It recognises a specific DNA sequence and assists in many prokaryotic processes that require special DNA axis distortion, such as replication, transcriptional regulation and several site-specific recombination events. The protein dimerises through an N-terminal segment consisting of two consecutive α helices and a three-stranded antiparallel β sheet (Figure 1).

Coupling protein

Transport of the relaxosome to the T4SS pore requires the assistance of a T4CP — an indispensable homohexameric nucleoside triphosphate (NTP)-dependent integral membrane protein [49••, 50, 51, 52, 53]. T4CPs include TrwB from R388, TraD from F, TraG from several other Gram-negative plasmids, archetypal VirD4 from A. tumefaciens pTi and related proteins [22, 54]. The first 70 residues of TrwB form its transmembrane domain. The structure of the remaining 48 kDa cytosolic domain resembles an

Ancillary transfer protein

Several plasmids, such as F and R1, contain within their tra region a gene encoding protein TraM, which is required for T-strand processing complementing relaxase/helicase, IHF and the transcriptional regulator. Other plasmids, such as R388, lack a TraM orthologue [59]. TraM is a 14 kDa cytoplasmic DNA-binding protein that binds to three sites within oriT and, in this manner, represses its own gene promoter. It might be a constituent of the relaxosome and/or could signal to the cell that the

Conclusions

Bacterial conjugation, an early-discovered pathway for lateral gene transfer and the main process responsible for the spread of antibiotic resistance, has been a ‘black box’. Structural biology is now making a dramatic contribution to unveiling the molecular machinery underlying this complicated protein–DNA transfer mechanism. Structural analyses of the components of the T4SS transport apparatus are currently underway (reviewed in [65]). Also, the structures of the main players in DNA

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by the Ministerio de Educación y Ciencia of Spain (grants GEN2003-20642, BIO2003-00132 and BFU2005-06758/BMC) and the Generalitat de Catalunya (grant 2005SGR-00280 and the Centre de Referència en Biotecnologia). AG Blanco is acknowledged for assistance with Figure 1 and Jan Löwe for providing the FtsK coordinates.

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