Conjugative plasmid transfer is the most important mechanism for bacteria to deliver and acquire genetic information to cope with rapidly changing environmental conditions. To transfer genetic information intercellularly mating cell-cell channels between donor and recipient bacteria have to be established. For plasmid transfer in Gram-negative bacteria subassemblies of these mating channels have been discovered, the order in which the transferred DNA contacts the transporter proteins has been determined and crystal structures of key components of the so-called conjugative type IV secretion systems have been solved. In contrast to this, knowledge on conjugative plasmid transfer of sex pheromone-inducible plasmids in Enterococcus faecalis is limited to molecular details on the complex regulation processes whereas for broad-host-range plasmids from Gram-positive bacteria investigations on the structure of the conjugative transfer apparatus and the interplay of the secretion components have recently started. The following chapter has the intention to give an overview of the state of the art on conjugative plasmid transfer in Gram-positive and Gram-negative bacteria.

Introduction

Bacterial conjugation is the most important means of gene delivery enabling adaptation of bacteria to changing environmental conditions including spread of antibiotic resistance genes, thereby generating multiply antibiotic resistant pathogens. Multiply resistant pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis represent a serious threat to antibiotic treatment of hospitalized and immuno-suppressed patients. Therefore, much effort has been and is still made towards elucidating the molecular mechanisms of conjugative plasmid transfer.

Bacterial conjugation systems are specialized types of type IV protein secretion systems (T4SS) dedicated to transport proteins (e.g., virulence factors, toxins) from bacterial pathogens to their mammalian hosts. The conjugative T4SS have evolved to transport DNA substrates in addition to proteins intercellularly.

Relevant progress has been made in deciphering the transport pathway of DNA and protein substrates through the Gram-negative (G-) cell envelope (see refs. 1, 2). Recently the Christie group² provided evidence for the order of transferred (T)-DNA contact with the T4SS proteins of the prototype T4SS of Agrobacterium tumefaciens during T-DNA export to the plant nucleus. The best characterized T4SS are the Agrobacterium T-DNA transfer system and the conjugative transfer systems of plasmids RP4, F and R388, all originating from G- bacteria. Intense investigations on the transfer mechanisms of plasmids from Gram-positive (G+) bacteria have started only a few years ago. An exception represent the well-studied sex-pheromone responsive plasmids of E. faecalis whose transfer underlies a complex regulatory mechanism exerted by small secreted signal molecules, the so-called pheromones (for recent comprehensive reviews see refs. 3, 4).

The chapter is divided into three parts summarizing the current knowledge of conjugative transfer mechanisms, mating cell-cell channel assembly and structure:

1. in G- bacteria;
2. of pheromone-responsive conjugative plasmids in E. faecalis; and
3. of nonpheromone-responsive plasmids in G+ bacteria.

Conjugative DNA Transfer in Gram-Negative Bacteria

Conjugative DNA transfer systems in G- bacteria, nowadays generally referred to as specialized T4SS, have been extensively studied for more than two decades. T4SS translocate DNA and protein substrates across the bacterial cell envelope. In general, T4SS transport their substrates to recipient cells via direct cell-to-cell contact. But there are also examples of contact-independent protein export and DNA release to and uptake from the extracellular milieu.^1,5,6

Considerable progress has been made towards the mechanistic understanding of intercellular DNA transport in the plasmid model systems RP4, R388 and F (for recent reviews see refs. 7-9) and in the T-DNA transport system of A. tumefaciens.^1,10 Several models for transenvelope DNA/protein transport have been proposed (for a summary see ref. 11) which match considerably well with the experimental data. Recently the order in which T-DNA contacts T4SS proteins on its way through the A. tumefaciens cell envelope has been determined by Christies´ group.^2,12,13 This discovery is a milestone towards the elucidation of the conjugative DNA/protein secretion mechanism.

On account of these very interesting results the state of the art of T4SS in G- bacteria will be presented on basis of the prototype T4SS, the A. tumefaciens T-DNA system (VirB/VirD4 transfer system).

A Transenvelope Secretion Channel: The mpf Structure

All of the VirB proteins except for the VirB1 lytic transglycosylase are required for substrate export.³⁹ Thus far, no supramolecular organelles at cellular junctions of mating cells have been identified by high-resolution electron microscopy. However, several experimental findings support the existence of an envelope-spanning secretion apparatus. For example, the presumptive VirB/ VirD4 mating channel or subcomplexes thereof have been isolated by membrane solubilization with nonionic detergents. At least two large complexes were detected, one composed of the T-pilus associated proteins VirB2, VirB5, and VirB7, and the second consisting of several other VirB proteins and the VirD4 CP.⁴⁰ Independent studies have also reported the isolation of a VirB2/VirB5/VirB7 complex and subcomplexes of other VirB proteins, such as VirB7/VirB9, VirB6/VirB7/VirB9 and VirB7/VirB9/VirB10 by detergent solubilization and immunoprecipitation or GST-pull-down as-says (summarized in ref. 10).These results were confirmed by two-hybrid screens and further pairwise interactions were detected in vivo.^41,42

Taken the above mentioned results together with computer-based predictions and topology studies of individual VirB subunits, a general architecture for the VirB/VirD4 T4SS can be presented: The VirD4 CP and the two mpf ATPases, VirB4 and VirB11, are localized predominantly or exclusively at the cytoplasmic face of the inner membrane. VirB6 is a highly hydrophobic protein predicted to span the inner membrane several times. VirB8 and VirB10 are bitopic proteins with short N-terminal cytoplasmic domains, a transmembrane helix and large C-terminal periplasmic domains. VirB2 (the major pilin protein), VirB3 and VirB5 are located in the periplasm,VirB2, VirB5, and VirB7 assemble as the extracellular T pilus. VirB7 is a small lipoprotein, which forms together with VirB9 a covalently cross-linked dimer. This dimer or a higher-order VirB7-VirB9 multimer assemble at the outer membrane. VirB9 has nine possible β-sheet outer membrane-spanning segments according to the Schirmer-Cowan algorithm.⁴³ Therefore, it is the best candidate for forming an oligomeric secretion-like pore mediating secretion of substrates and/or protrusion of the T pilus across the outer membrane.¹⁰ A model for the general architecture of the T4SS is shown in Figure 1.

Figure 1

A model showing the A. tumefaciens VirB/D4 T4SS as a single, supramolecular organelle. The VirD4 CP is a homomultimeric integral membrane complex required for substrate transfer.TheVirB proteins assemble as a secretion channel and an extracellular T pilus. (more...)

Energy Supply-VirB4 and VirB11

VirB11 belongs to a large family of ATPases associated with macromolecule secretion systems.⁴⁴

Homologues are widely distributed among the G- bacteria, and they are also functional in secretion systems of G+ bacteria and in Archaea.¹⁰ VirB11 is highly insoluble and has been difficult to analyze biochemically. But soluble forms of VirB11 homologues have been characterized enzymatically and structurally. The VirB11-homologue of the Helicobacter pylori pathogenicity island, HP0525_Cag and several plasmid-encoded VirB11-homologues were shown to assemble as homohexameric rings as demonstrated by electron microscopy.⁴⁵ HP0525_Cag also presented as a homohexamer by X-ray crystallography. It is a double-stacked ring with a central cavity of about 50 Å in diameter.⁴⁶ The overall HP0525_Cag structure appears to be highly conserved, even among distantly related NTPases encoded by other transport or fimbrial biogenesis systems.¹⁰ HP0525_Cag is also structurally similar to members of the AAA ATPase superfamily.⁴⁷ Many AAA ATPases act as energy-dependent unfoldases in substrate remodelling. Considering that the T4SS are export systems, the VirB11-like ATPases might act as chaperones to unfold the protein substrates at the channel entrance.¹⁰ VirB11-like ATPases associate tightly but peripherally at the inner face af the inner membrane.^48,49 Genetic studies showed the coordinated action of VirB11 and the VirD4 CP for substrate transfer.¹⁰ Mutagenesis analyses of VirB11 proved that it participates both in pilus biogenesis and assembly or function of the secretion machine.⁵⁰ It is likely that VirD4 and VirB11 hexamers localize next to each other at the inner membrane as depicted in Figure 1. Llosa et al⁵¹ described a model that presents conjugation systems as two separately acting inner membrane translocases: the CP functions as a general recruitment factor for all T4SS substrates which is in agreement with all other prominent T4SS models, as shown in Figure 2. Although it is tempting to assume that the CP delivers the whole substrate to the mpf complex, they propose that the CP translocates the T-strand across the inner membrane while simultaneously transferring the relaxase to VirB11 and the other mpf proteins for secretion. The model of Llosa and coworkers explains very well most experimental data but it still remains difficult to envision how two transport proteins localized next to each other at the inner membrane coordinate their activities to mediate secretion of one substrate, the T-DNA-relaxase complex across the inner membrane.¹⁰

Figure 2

Possible architectures and substrate transloction routes for the T4SS. Numbers refer to VirB/VirD proteins of the Agrobacterium T-DNA transport system. Three working models describe the possible machine architectures and translocation routes: (1) a one-step (more...)

VirB4 possesses two putative membrane-spanning domains, one close to the N terminus and another located more centrally near the Walker A nucleoside triphosphate binding motif.⁴⁸ VirB4 self-interacts as shown by Dang et al⁵² and Ward et al.⁴² ATP hydrolysis has not been convincingly shown for VirB4-like proteins, but these proteins require intact Walker A motifs to mediate substrate export.^53,54 Bohne et al⁵⁵ reported that the presence of a subset of VirB proteins including VirB4 in Agrobacterium recipient cells increases the efficiency of plasmid uptake in intraspecies matings significantly. These data led to the proposal that these VirB proteins assemble as a complex that stabilizes mating junctions or perhaps they directly facilitate DNA transport across the recipient cell envelope.⁵⁶ However, it was also demonstrated that a Walker A mutation does not decrease the capacity of VirB4 to stimulate plasmid DNA acquisition by recipient cells. Dang et al⁵² argued that VirB4 probably contributes structural information required for substrate transfer in either direction across the cell envelope. But an intact ATP-binding motif is necessary for configuring this T4SS specifically for substrate export. Christie concludes that the VirD4 CP, VirB11, and VirB4 must interact in complex and dynamic ways—probably through ATP-powered conformational changes— to energize substrate transfer to and across the inner membrane (ref. 10 and Fig. 1).

Pheromone-Responsive Conjugative Plasmids in E. faecalis

Though the enterococcal pheromone-inducible conjugative plasmids such as pCF10, pAD1, and pPD1 represent a unique class of mobile genetic elements spreading virulence traits readily with high transfer efficiency even in aqueous systems, the mechanism of their conjugative DNA secretion system has not been studied intensively. However, the complex regulatory mechanisms underlying specific efficient plasmid exchange has been investigated in great detail. The hereafter presented state of the art will focus on the regulatory machinery that interacts specifically with the pheromone peptides thereby controlling plasmid acquisition of plasmid-free enterococcal recipient cells. The data were derived mainly from a comprehensive review on enterococcal peptide sex pheromones by Chandler and Dunny.⁴

The pheromone plasmids are induced to transfer from donor cells by mating pheromones that are produced by potential recipient cells. The pheromone plasmids have evolved a fascinating and complex regulatory system to ensure their maintenance and stable existence in a population. Their transfer genes are induced by small (7-8 amino acids (aa)) peptides chromosomally encoded by all known enterococcal strains. Each peptide is highly specific for a cognate plasmid or for a family of closely related plasmids. All pheromones analyzed so far are produced by proteolytic processing of the cleaved signal sequences of secreted lipoproteins. The processed peptides are secreted into the growth medium and are utilized by the plasmid-containing donor cells to sense the presence of a nearby recipient cell. The pheromone-responsive plasmids use an interesting combination of host and plasmid encoded proteins to sense exogenous pheromone in order to activate the expression of transfer genes and to avoid self-induction by pheromone that is encoded on the chromosome of the host cell.⁴ An overview of the main steps involved in pheromone-induced conjugation in enterococci will be given based upon data obtained from plasmid pCF10, the model plasmid of the Dunny group. pCF10-transfer is induced when the recipient-produced pheromone is detected by the donor ell at the cell surface by the plasmid-encoded lipoprotein PrgZ.⁵⁸ PrgZ acts together with the chromosomally encoded oligopeptide permease system (Opp) to import the pheromone into the cytoplasm of the recipient cell.⁵⁹ Import of the pheromone is necessary for pheromone response. The interaction process that likely initiates the induction in donor cells is binding of the imported pheromone to the plasmid-encoded cytoplasmic protein PrgX. PrgX is a negative regulator of expression of conjugative functions. Binding of pheromone to PrgX abolishes its repression so that transfer genes are synthesized. Two additional pCF10-encoded proteins, PrgY and iCF10, are required to keep the transfer system off in pCF10-harbouring cells grown in the absence of exogenous pheromone. PrgY and iCF10 are supposed to block self-induction of donor cells by endogenous pheromone.

Exposure of pCF10-containing cells to exogenous pheromone, cCF10, is phenotypically visible by aggregation of the culture resulting from upregulation of the expression of aggregation substance PrgB from the pCF10-encoded prgB gene.⁶⁰ The aggregation results in close contact between donor- and recipient cells probably enabling effective plasmid transfer, even in liquid medium. Approximately 15 additional genes are encoded 3´ from prgB. Data from the Dunny group suggest that many of these genes are upregulated by pheromone.⁴

The PrgZ pheromone binding protein is critical in the first step of pheromone induction: recognition of pheromone and import into the cytoplasm. All pheromone-responsive conjugative plasmids encode a PrgZ-like protein. They are homologues of the peptide-binding OppA proteins found in a wide range of bacterial species.⁶¹ The PrgZ-type proteins are cell surface proteins anchored to a lipid moiety on the outer surface of the cytoplasmic membrane.⁵⁸ The PrgZ family of pheromone-binding proteins have been shown to increase the sensitivity of each plasmid system to its cognate pheromone.^58,62

The pheromones themselves are encoded within the chromosome of E. faecalis, processed by host proteins and secreted inlextremely small amounts into the culture medium. In the case of pCF10, cCF10 is released at ˜10^-11 M and can induce a donor cell at concentrations of 2 × 10^-12 M corresponding to less than five molecules per cell under the conditions tested.⁶³ Despite multiple pheromones with different spectra of activities secreted by a single cell, each plasmid responds specifically to its cognate pheromone with surprising sensitivity.^64,65 The nucleotide sequence of the E. faecalis strain V583⁶⁶ showed that the pheromone precursors lie within the N-terminal signal sequences of predicted surface lipoproteins.⁶⁷ Proteolytic processing of the pheromone precursor is required prior to release of the mature pheromone into the culture medium. Signal peptidase II cleaves at a specific cysteine residue liberating the signal peptide from the lipoprotein.⁶⁸

How does the plasmid prevent a response to its own host´s endogenously produced pheromone? The pheromone plasmids have evolved two independent mechanisms of avoiding this self-induction such that the transfer response is only induced if a nearby recipient is detected. One mechanism is exerted by the synthesis of a plasmid encoded inhibitor peptide, iCF10 in the case of pCF10, which neutralizes endogenously produced pheromone in the culture medium.⁴ The inhibitor peptides are proposed to compete with the pheromone for binding to the surface binding protein PrgZ.^69,70 The level of iCF10 in donor cultures has been found to be 10-100-fold above the pheromone level. This molar ratio is just sufficient to neutralize the cCF10 activity released by the same cells. The inhibitor peptides are very similar to each other and to the inducing pheromones. They are 7-8 aa hydrophobic peptides likely being processed from 22 to 23 aa precursors. Despite the apparent homology between the inhibitors and their peptides, their functions are very specific. Data on the pPD1 and the pCF10 plasmid imply that the function of the inhibitor may include more than just competitive inhibition of PrgZ and may instead involve specificity at some other level, since the response of the systems to their cognate pheromones and inhibitors is highly specific.^64,65 This specificity determinant/mechanism remains to be unravelled.

PrgY is the other pCF10-encoded element involved in control of endogenous pheromone. While the inhibitors control endogenous pheromone in the culture medium, the membrane protein PrgY controls endogenous pheromone activity that remains associated with the cell.⁴ Buttaro et al⁷¹ showed that a significant amount of pheromone in cCF10 producing cells remains associated to the cell wall. The average plasmid-free recipient cell appears to have twice as much cCF10 in its cell wall than the amount that is secreted into the supernatant. When pCF10 is acquired, the concentration of cCF10 in the supernatant is not affected⁷⁰ whereas the cell wall-associated cCF10 is decreased

8-fold from that of plasmid-free recipient cells.⁷¹ PrgY was shown to be involved in this reduction of envelope-associated cCF10 after acquisition of the plasmid, but its mechanism of action is not clear. Other PrgY-like proteins have been identified in recent genome sequencing projects. Curiously, the bacterial species that have been found to encode a PrgY-type protein, do not have characterized peptide-signal systems and are quite distantly related to E. faecalis. This finding further deepens the mystery surrounding the role of this protein in the regulation of the pheromone induction process.⁴

So far, no data are available on the DNA transport mechanism of pCF10 and the other pheromone-inducible enterococcal plasmids. However, it seems reasonable to argue that the DNA secretion process of these plasmids might proceed via a T4S-like mechanism as proved for all known G-systems and currently being studied for the broad-host-range conjugative plasmids from G+ bacteria (see below): The following findings are in favour of a T4S-like mechanism: On two pheromone plasmids, namely pAD1 and pAM373, oriTs have been found. pAD1 has two oriTs, oriT1 and oriT2 and encodes a relaxase, TraX, which has been demonstrated to specifically nick in oriT2. oriT_pAM373 has been shown to be similar to oriT2_pAD1. Both plasmids are able to mobilize the non conjugative plasmid pAMα1, which encodes two relaxases that are involved in transfer.⁷² ORF53 encoded by pAD1 is a protein essential for conjugation, which exhibits structural similarities to TraG-like CPs.⁷³ Recently nucleotide sequencing of the 67,673-bp pheromone plasmid pCF10 has been completed. pCF10 contains 57 orfs, orf35 encodes a relaxase, pcfG⁷⁴ with highest homology to LtrB, the relaxase of the Lactococcus lactis conjugative plasmid pRS01.⁷⁵

Nonpheromone-Responsive Plasmids in G+ Bacteria

Enterococci harbour also a pheromone-independent conjugative plasmid, namely the 65.1-kb pMG1, that transfers efficiently in broth matings. Interestingly, Southern hybridization of pMG1 DNA showed no homology to pheromone-responsive plasmids and the broad-host-range conjugative plasmids pAMß1 and pIP501.⁷⁶

Aggregation-mediated plasmid transfer in Bacillus thuringiensis and in lactic acid bacteria has been summarized in Grohmann et al⁷⁷ and will not be discussed further here. Recently Belhocine et al⁷⁸ demonstrated that conjugation is one of the mechanisms by which group II introns, originally discovered on a L. lactis conjugative plasmid (pRS01) and within a chromosomally located sex factor in L. lactis 712, are broadly disseminated between widely diverged G+ organisms.

Conjugative Transfer of Broad-Host-Range Plasmids

Transfer of broad-host-range G+ plasmids occurs at a variable frequency (generally in the range of 10^-3 to 10^-6) depending on the plasmids and the mating-pair genotype, and mating requires cocultivation of donor and recipient cells on a solid surface.⁷⁷ Most conjugative plasmids identified so far in streptococci and enterococci actually show a broad host range (and hence are referred to as broad-host-range plasmids,^79,80 while those found in staphylococci seem to be limited to the genus Staphylococcus.

The complete nucleotide sequences of the staphylococcal plasmid pSK41,^81,82 the lactococcal plasmid pMRC01,⁸³ the enterococcal plasmid pRE25, the streptococcal plasmid pIP501^84,85 and the complete tra region of the staphylococcal plasmid pGO1⁸⁶ have been determined. Sequence comparisons revealed interesting similarities of the tra regions of these self-transmissible plasmids.⁷⁷

All of the tra regions show a highly modular organization so that the arrangement of the first seven genes is well conserved among the compared tra regions, with the exception of an insertion of two genes of unknown function between the putative relaxase gene traA and gene traB in pMRC01. The pMRC01 tra region is the most distantly related and contains seven unique genes. Interestingly, a traG gene homologue coding for a putative lytic transglycosylase is present in all plasmids except for pMRC01, while traK homologues coding for a putative CP are present in all five plasmids (Fig. 3).

Figure 3

Comparison of the tra regions of pIP501, pRE25, pGO1, pSK41, and pMRC01. Similar gene products are shown in the same colour. Cream-colored boxes represent tra genes unique to pMRC01. The putative transfer proteins of pRE25 and pIP501 show a high degree (more...)

Information about the regulatory processes involved in gene transfer of nonpheromone-responsive plasmids in G+ bacteria is scarce: TrsN, a 7.2-kDa protein encoded by pGO1, was shown to repress the synthesis of essential tra genes by binding to promoter-like sequences upstream of trsA, the first gene of the conjugative gene cluster trs.⁸⁷

Tanimoto and Ike⁸⁸ detected a gene, traA, in the unrelated plasmid pMG1, which is upregulated during conjugation. They found that the traA gene product is associated with the formation or stabilization of mating aggregates during broth mating.

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