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Microbiology and Molecular Biology Reviews, June 2003, p. 277-301, Vol. 67, No. 2
1092-2172/03/$08.00+0 DOI: 10.1128/MMBR.67.2.277-301.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Microbial Ecology Group, University of Technology Berlin, D-10587 Berlin,1 Department Microbiology/Biotechnology, University Tuebingen, D-72076 Tuebingen, Germany,2 Consejo Superior de Investigaciones Cientificas, Centro de Investigaciones Biológicas, E-28006 Madrid, Spain3
SUMMARY INTRODUCTION Conjugative Transfer of Resistance Determinants from Antibiotic Producers into Pathogens Conjugative Transfer in Gram-Negative Bacteria as a Paradigm for Key Steps in Conjugative Plasmid Transfer CONJUGATIVE TRANSFER IN UNICELLULAR GRAM-POSITIVE BACTERIA Conservation of Conjugative DNA Relaxases Conservation of nic Regions Conjugative Transfer of Broad-Host-Range Plasmids The Transfer Regions of Plasmids pIP501, pRE25, pSK41, pGO1, and pMRC01 Homologies to Type IV Secretion Systems ATPases. Mating-channel proteins. Coupling proteins. Conjugative Transposons Sex Pheromone Plasmids Aggregation-Mediated Plasmid Transfer in Bacillus thuringienis and in Lactic Acid Bacteria RCR Mobilizable Plasmids: the pMV158 Family CONJUGATIVE PLASMID TRANSFER IN MYCELIUM- FORMING STREPTOMYCETES Different Types of Conjugative Streptomyces Plasmids Intermycelial Conjugative Transfer Mediated By a Septal DNA Translocator Protein Temporal and Spatial Regulation of Conjugative Transfer Pock Structures and Intramycelial Plasmid Spreading Experimental Evidence for the Transfer of a Double-Stranded Plasmid Molecule Model for the Conjugative Transfer of Streptomyces Plasmids CONJUGATIVE TRANSFER IN OTHER ACTINOBACTERIA SUMMARY AND FUTURE PERSPECTIVES Implications of Intergeneric Gene Transfer by Gram-Positive Transfer Systems Attempts to Elucidate the Role of the Type IV Components in Conjugative Plasmids from Gram-Positive Hosts Future Perspectives ACKNOWLEDGMENTS REFERENCES
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Most of the antimicrobial drugs currently in use are derived from metabolites of soil organisms, mainly fungi and actinomycetes. All resistance mechanisms that have been identified in pathogenic bacteria, including RNA methylases, ATP-binding cassette transporters, aminoglycoside phosphotransferases, and ß-lactamases, already exist in the respective antibiotic producers. In Streptomyces coelicolor (http://www.sanger.ac.uk/Projects/S_coelicolor/), as well as in the glycopeptide producers of the genus Amycolatopsis, even the vancomycin resistance determinants vanH (D-Ala dehydrogenase), vanA (D-Ala-D-Lac ligase), and vanX (D,D-dipeptidase) are present in the very same gene organization as found in the enterococcal conjugative transposon Tn1549 (79).
The resistance genes probably evolved in the antibiotic producers as part of the biosynthetic gene cluster to protect the producing organism from the detrimental action of its own antibiotic. Subsequent gene transfer events might have spread the resistance determinants to other bacteria. Whereas the ability of broad-host-range plasmids from gram-negative bacteria in intergeneric and transkingdom transfer is well documented (9, 53, 216), the role of the gram-positive transfer systems in the dissemination of resistance determinants needs further evaluation.
To facilitate homology studies with gram-negative systems and to develop a transfer model for gram-positive unicellular bacteria, the current model for conjugative transfer in gram-negative bacteria is briefly presented here. We restrict our overview to the fundamental findings of one of the best-studied conjugative systems, the IncP transfer (tra) system of the broad-host-range plasmid RP4. The IncP transfer system consists of two regions, Tra1 and Tra2, including 30 transfer functions, 20 of which are essential for intraspecies Escherichia coli matings. The central question in bacterial conjugation is how the DNA traverses the cell envelopes of the mating cells. The current model is that two protein complexes exist, namely, the relaxosome and the mating-pair formation (mpf) complex, which are connected via interaction with a TraG-like coupling protein. The relaxosome has been defined as a multiprotein-DNA complex that is generated at the plasmid origin of transfer, oriT. Plasmid-encoded and chromosomally encoded proteins participate in this complex (77, 120). The mpf complex is a plasmid-encoded multiprotein complex that is involved in the traffic of the donor DNA strand from the donor to the recipient cell (124).
The RP4 relaxosome was localized in the cytoplasm and found to be associated with the cytoplasmic membrane independent of the membrane-spanning mpf complex (89, 123). DNA relaxases are the key enzymes in the initiation of conjugative transfer and operate by catalyzing the cleavage of a specific phosphodiester bond in the nic site within oriT in a strand- and site-specific manner. In all systems encoded by self-transmissible and mobilizable plasmids studied so far, the DNA cleavage reaction is a strand transfer reaction involving a covalent DNA-relaxase adduct as an intermediate. This intermediate is proposed to be a prerequisite for the recircularization of the cleaved plasmid after completion of transfer by a joining reaction between the free 3' hydroxyl and the 5' terminus of the covalently bound relaxase. An exception is plasmid CloDF13, for which data suggest that nic cleavage possibly results in a free nicked-DNA intermediate (152).
IncP-type relaxases seem to be the most widely distributed among different gram-positive and gram-negative conjugative plasmids, conjugative transposons, mobilizable elements, and the agrobacterial T-DNA transfer system (226). All conjugative DNA relaxases have common domains in which the N-terminal moiety seems to contain the catalytic activity whereas the C-terminal moiety may be involved in interactions with other components of the transfer machinery. The enzymatic properties of DNA relaxases are discussed in more detail below.
Biochemical, genetic, and electron microscopic data imply the existence of complicated structures of the mpf complex. Eleven mpf components (trbB to trbL) and traF are required for IncP pilus formation in the absence of any DNA-processing factors (92), and these components are also required to establish conjugative junctions (181). The mpf system of RP4 was localized in the cell membrane (89) and was suggested to form a complex that connects the cytoplasmic and the outer membrane. These data agree with a role of the mpf complex in protein transport. Experimental evidence for interaction of the complex with DNA has been recently obtained, since nonspecific DNA binding activity of TrbE was shown (11).
The tra1-encoded TraG protein is also associated with the cytoplasmic membrane independent of the presence of the Tra2 region. The results also suggest a connection of TraG with the mpf complex, thereby supporting its proposed role as a potential interface between the mpf system and the relaxosome (89).
Gram-negative bacteria possess two very efficient barriers which have to be traversed by macromolecules during export from and import into the cell: the outer membrane and the inner membrane, which are separated by a cellular compartment, the periplasm. From this point of view, it is evident that macromolecules such as plasmid DNA and prepilin subunits (the building blocks of the pili) need a transport channel to cross the two membranes and the periplasmic space.
Conjugative plasmids have evolved systems of regulation that minimize the metabolic and phenotypic load exerted by the maintenance of a conjugative transfer apparatus while optimizing the adaptive advantages of self-transmission. For instance, IncP plasmids transfer at high frequencies under optimal conditions, so that the transfer frequencies can approach one transfer event during a 5-min mating on nutrient agar. However, IncP transfer genes are not expressed constitutively. In fact, their expression is regulated by complex local autoregulatory circuits as well as by global regulators, resulting in the coordinated expression of transfer genes with other plasmid functions (52, 225).
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Three conserved motifs (I to III) were first identified in IncP-like relaxases (160, 163). Motif I contains a conserved Tyr residue (Tyr-22 in Tra1 from RP4) that reversibly attacks the DNA backbone in the relaxase-catalyzed reaction. A Ser residue within motif II was shown to be involved in tight binding of the 3' terminus generated in the DNA cleavage reaction. Motif III contains two His residues thought to be involved in activating Tyr-22 for its nucleophilic attack at the nic site (161, 163). Interestingly, these conserved His residue are found not only in conjugative relaxases but also in several rolling-circle replicating (RCR) initiator proteins, and they have been proposed to participate in the binding and coordination of the metal cation (Mg2+ or Mn2+) needed for cleavage of the DNA substrate (101, 114). Motifs I and III are found in all conjugative DNA relaxases, which were divided into four distinct DNA relaxase families, the IncP, the IncF/IncW, the IncQ, and the RCR (pMV158)-type family, on the basis of to overall homology (226).
The relaxases of conjugative and mobilizable plasmids from gram-positive bacteria mainly belong to two families, the IncQ-type family and the pMV158-type family (91, 226). The relaxases encoded by pIP501, pRE25, pSK41, pMRC01, and pGO1 belong to the IncQ-type family. An alignment of the IncQ-type relaxases, including three proteins of plasmids from gram-negative bacteria (RSF1010, pTF1, and pSC101) and five relaxases of gram-positive bacterial origin, is shown in Fig. 1. It shows conservation of motif I, characterized by the Tyr residue (Tyr-26 in pIP501-encoded TraA), and of motif III, specified by the two His residues (His-134 and -136 in TraA) in all members of the family. Mutation of these His residues in TraI of RP4 resulted in strong reduction of relaxase-mediated cleavage activity (162, 163).
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Another family of DNA relaxases is made up of the mobilization (Mob) proteins encoded by many RCR plasmids isolated from a variety of gram-positive bacteria. Interestingly, these proteins were first described as participating in the generation of cointegrates between staphylococcal plasmids, so that they were termed plasmid recombination enzymes (Pre) (151). Indications that these proteins were involved in mobilization were provided later by methods showing that the mobM gene of the streptococcal plasmid pMV158 was required for its mobilization by pIP501 between strains of Streptococcus pneumoniae (180), and that two regions of the staphylococcal plasmid pC221 are involved in transfer (174), as well as by sequence similarity analyses (159, 209). Comparative studies of staphylococcal plasmids related to pT181 showed that these plasmids also carry a Pre function (175). In addition, the region where the plasmid cointegration occurred (named recombination site a [RSa]) was identified as the plasmid oriT (91, 173). The only Mob protein of this category of plasmids that has been characterized so far is the pMV158-MobM protein (91), but there are nearly 50 Mob proteins that show a high degree of similarity to MobM, and they include Mob proteins from well-characterized staphylococcal plasmids like pUB110, pE194, pT181, and pC221; curiously enough, staphylococcal plasmid closely related to pUB110, pC194, does not appear to carry a mobilization cassette (91).
Using the Pfam algorithms from the Sanger Institute (http://www.sanger.ac.uk/cgi-bin) and from the Swiss Institute for Experimental Cancer Research (http://hits.isb-sib.ch/cgi-bin/PFSCAN), all these proteins, especially those from RCR plasmids, can be grouped within a single family, termed the Mob-Pre family of proteins, and they have been found in a wide variety of bacteria from the family Bacteroidaceae to firmicutes (Bacillus-Clostridium-Staphylococcus groups) and proteobacteria. The family can be extended to other proteins that show homology to the Mob proteins at the C-terminal end, like two hypothetical proteins from Lactobacillus lactis (36.1 kDa; accession number Q9L973) and from Moraxella catharralis (80.7 kDa; accession number Q9L973).
The RSF1010-oriT family (118, 226) includes the prototype IncQ plasmids RSF1010 (55) and R1162 (25), the Thiobacillus ferrooxidans plasmid pTF1 (60), the A. tumefaciens Ti plasmid pTiC58 (49), the Salmonella plasmid pSC101 (138), and four plasmids from gram-positive hosts, namely, pRE25, pIP501, pGO1, and pMRC01. The 5' end of the nick site was mapped for five of these nine plasmids and showed identical nic sites for RSF1010 and R1162 (55, 60) and for pTF1 and pIP501 (60, 215), respectively, while the dinucleotide cleaved in oriTpGO1 (48) is different.
A consensus sequence for the RSF1010-oriT family was deduced: 5'-NcgtNtaAgtGCGCcCTta-3 (Fig. 2). An additional similarity is the presence of inverted repeats directly adjacent to the nic site. These inverted repeats have the potential to generate hairpin structures, so that their generation would allow the specific recognition of the oriT region by the cognate DNA relaxase and the cleavage reaction, which would take place in an unpaired region. Although all conjugative and mobilizable plasmids analyzed thus far show different inverted repeats within their oriT regions, their location relative to the nick is similar. Experimentally determined nick sites in the DNAs of plasmids of the RSF1010-oriT family mapped between 7 and 11 nucleotides upstream of the inverted repeats. In addition, the inverted repeats of RSF1010, pTF1, R1162, pIP501, pRE25, and pGO1 are all centered on the nucleotide sequence GAA.
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Most of the broad-host-range conjugative streptococcal plasmids encode resistance to macrolides, lincosamides, and the streptogramin B antibiotics (MLSr). This resistance determinant (erm) is found in a wide variety of gram-positive cocci and bacilli and has also been found in species of the gram-negative genus Bacteroides (153). Some streptococcal plasmids, like pIP501, also carry a resistance determinant against chloramphenicol. Broad-host-range plasmids of the MLSr type have been found in various clinically important streptococci worldwide (reference 132 and references therein). Comparisons of the nucleotide and amino acid sequence of the streptococcal MLSr gene with those of different gram-positive bacteria suggest that the MLSr determinants are ancestrally related (61, 135, 219). Transfer of the streptococcal broad-host-range plasmids to a wide range of gram-positive species, including Enterococcus, Lactococcus, Staphylococcus, Clostridium, Pediococcus, and Listeria, has been demonstrated.
The appearance of conjugative plasmids in staphylococci coincided with reports of the emergence of gentamicin resistance in U.S. hospitals in the mid-1970s. However, the first demonstrations of true conjugative transfer of antibiotic resistance plasmids in staphylococci were made much later as a consequence of new outbreaks of infections due to gentamicin-resistant staphylococci in several hospitals (6, 73, 136). In these early reports, interspecies conjugative transfer between Staphylococcus epidermidis and S. aureus was demonstrated to occur on human skin (102, 208). The presence of conjugative resistance plasmids with identical restriction patterns in hospital isolates of S. aureus and S. epidermidis from the same patient (6) confirmed the epidemiological importance of bacterial conjugation.
Staphylococcal plasmids seem to be remarkably stable, since the plasmids which were detected in hospitals in the early 1980s were still the main carriers of gentamicin resistance genes in S. aureus 10 years later (7).
The entire transfer region of the staphylococcal self-transmissible plasmid pGO1 has also been sequenced (143), and very recently we determined the 3' part of the pIP501 tra region encompassing orf7-15 (accession number AJ505823).
Sequence comparisons revealed interesting similarities extending the known homologies of the first six orf genes carried on pSK41, pMRC01, pGO1, pIP501, and pRE25 (15, 59, 68, 186). The modular organization of these tra regions is shown in Fig. 3. The arrangement of the first seven genes is well conserved among all 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. In pSK41 and pGO1, the nicking activity is encoded not by the first gene of the tra region but by the nes gene (for pSK41, nes/oriT are located approximately 11 kb upstream of the tra region, [15]). The pMRC01 tra region is the most distantly related and contains seven unique genes. Interestingly, the traG gene in pMRC01 is missing, while traK and traL homologues are present in all five plasmids (Fig. 3).
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The operon organization of the major part of the pIP501 transfer genes was elucidated recently. Reverse transcription-PCR studies of mRNA isolated from Enterococcus faecalis JH2-2 cultures harboring pIP501 revealed cotranscription of the first 11 genes of the pIP501 tra region. The tra genes orf1 to orf11 are transcribed as a single operon of 11.3 kb (118). The compact organization of the pIP501 oriT region makes autoregulation of the tra operon by the TraA protein likely. The -10 region of the Ptra promoter overlaps half of an inverted repeat structure, proposed to represent the binding site for the TraA relaxase, the product of orf1 (118). This assumption is currently under investigation (B. Kurenbach and E. Grohmann, unpublished data).
Type IV systems include conjugative transfer apparatus, filamentous bacteriophage secretion, protein secretion systems of several pathogens, and natural transformation systems. Several reviews of type IV secretion in gram-negative bacteria have been published recently (30, 33, 36, 37, 51, 119, 193). A list of sequenced members of the type IV secretory pathway (IVSP) family is available at http://www-biology.ucsd.edu/
msaier/align/align_table/VirB_Table_S4_.html. We briefly describe homologues found on conjugative elements of gram-positive bacterial origin (Table 2).
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VirB11, TrbB (RP4), TrwD (R388), and HPO525 of the Helicobacter pylori cag pathogenicity island belong to a family of ATPases with members present among all type II and IV secretion systems characterized so far (37). ATP hydrolysis activity of purified VirB11 has been demonstrated (35). These ATPases generally associate tightly, but peripherally, with the cytoplasmic membrane. Genetic and biochemical studies have supplied evidence for the formation of homo-oligomers of these ATPases. Recently, the TrbB, TrwD, and HPO525 ATPases have been shown to assemble as homohexameric rings with a 12-nm diameter, as visualized by electron microscopy (116, 117). The rings were stabilized by the addition of ATP. ATP hydrolysis was increased by the addition of phospholipids, thus indicating that interaction of these proteins with the cell membrane is likely. The crystal structure of a binary complex of HPO525 bound to ADP has been solved at a resolution of 2.5 Å (223). In the hexamer, the N- and C-terminal domains build two rings, which together form a chamber open on one side and closed on the other. The crystal structure led to a model in which the VirB11-type ATPases function as GroEL-like chaperones in translocation of unfolded proteins across the cytoplasmic membrane (117, 223). For the TrwD ATPase of R388, association with membrane vesicles that was independent of ATP hydrolysis was demonstrated, so that the protein could indeed act as a chaperone involved in the translocation of transfer components across the membranous system (131).
Three putative gene products with homologies to type IV secretion systems are encoded by plasmids pIP501, pRE25, pSK41, pGO1, and pMRC01 (Table 2). pIP501-Orf5, pRE25-Orf28, pGO1-TrsE, pMRC01-TraE, and pSK41-TraE have homologies, albeit weak, to the IncP/TrbE IncF/TraC Ti/VirB4 family of conjugative ATPases (sequenced VirB4 homologuesat http://www-biology.ucsd.edu/msaier/align/align_table/VirB_Table_S4_.html). Orf5 (pIP501) shows a score of 71.2 and E value of 3 x 10-13 as a member of the VirB4 family of intracellular trafficking and secretion proteins (COG3451). Orf20, encoded by Tn1549, a VanB-type conjugative transposon of the Tn916 family, has significant similarity to TrsE, the VirB4 homologue encoded by pGO1 (27% identity in 437 of a total of 800 amino acids [79]).
VirB4-type proteins are ubiquitous among the type IV systems and are sometimes present in two or more copies. Experimental evidence for VirB4 self-association and a structural contribution to channel formation that is independent of the VirB4 ATPase activity has been provided (for a review, see reference 37). Based on these features, this family of ATPases might transduce information, possibly in the form of ATP-induced conformational changes, across the cytoplasmic membrane to extracytoplasmic subunits (52).
Mating-channel proteins. Interestingly, Orf7 encoded by pIP501 and its homologues (Table 2) show weak similarities to the family of lytic transglycosylases (pfam01464; score for Orf7, 36.1; E value, 0.007; http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid = pfam01464&version = v1.54) encoded by bacteriophages and type III and type IV secretion systems. For Orf7, the membrane localization was predicted by the PSORT program. The family of lytic transglycosylases includes the pilT gene of conjugative plasmids R64 and ColIb-P9 (AB021078 [178]), the p19 gene of the conjugative resistance plasmid R1 (P14499 [12]), trbN of RP4 (M93696), the traL gene of pKM101 (AAA86448), and virB1 of the T-DNA transfer machinery of A. tumefaciens (P17791). Although the contribution of a functional lytic transglycosylase to pathogenicity could be established only for VirB1 (17, 127, 144), it is tempting to speculate that all these transglycosylases aid the DNA and/or protein(s) to cross the cell envelope by locally opening the peptidoglycan (12, 57).
Recently, determination of the nucleotide sequence of the E. faecalis conjugative sex pheromone plasmid pAD1 was completed (75). By sequence analysis, two Orf proteins with significant similarity to lytic transglycosylases were identified: (i) Orf41, which has 61% similarity to TraG of S. aureus plasmid pSK41, and (ii) Orf50, another potential VirB1 homologue, which has 243% similarity to Orf16 of Tn916.
Coupling proteins. Coupling proteins are thought to link the DNA transfer intermediate to, and perhaps lead it through, the mating channel. This family of proteins includes TraG (RP4 and Ti), TrwB (R388), TraD (F), and VirD4 of the T-DNA transfer system.
Gomis-Rüth et al. (85, 86) proposed an elegant model based on the crystal structure of the coupling protein TrwB of plasmid R388. TrwB was shown to be a large multimeric DNA-binding integral membrane protein that participates in the transfer of the single DNA strand during the mating process. The three-dimensional structure of TrwB was shown to be a homohexamer. TrwB revealed an almost spherical quaternary structure with striking similarity to F1-ATPase. A central channel with a diameter of 20 Å traverses the hexamer, although this channel may be too narrow in the cytoplasmic extreme to accommodate a single DNA strand appropriately if there is no further modification (86). The TrwB structure also shows high similarity to DNA helicases, which use the energy from nucleoside triphosphate (NTP) hydrolysis to unwind double-stranded DNA. The strong structural resemblance of TrwB to ring helicases suggests that the transferred DNA single strand might pass through the central channel of the TrwB hexamer, thereby entering the translocation apparatus connecting the donor and recipient cells. ATP hydrolysis would provide the energy to pump the single-stranded DNA through the TrwB channel, in much the same way as it does in helicases for their processive movement along the DNA (86). In fact, conformational changes have been observed in TrwB crystals after binding and putative ATP hydrolysis (85).
Topology analysis of TraG (the coupling protein of plasmid RP4) revealed that it is a multimeric transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. It has been suggested that TraG forms a pore and that the relaxosome binds to the TraG pore via a TraG-DNA complex and that TraG interactions with the TraI relaxase (185).
Orf10 of the pIP501 tra region belongs to the pfam02534 TraG/TraD family of coupling proteins (score, 291; E value, 1e - 79). These proteins contain a P-loop and a Walker B site for nucleotide binding. For pIP501, the most closely related putative type IV secretion proteins are all encoded by pRE25 (e.g., Orf10 is 99% identical to Orf33 of pRE25). The pIP501 orf10 product is also 27% identical to the orf16 product encoded by the E. faecalis conjugative transposon Tn1549 (79). On the recently completed E. faecalis V583 genome sequence, another putative VirD4 homologue (20% identity) was detected. Putative homologues of coupling proteins have been detected on the chromosomes of many recently sequenced gram-positive and gram-negative bacteria as well as on transposons harbored by them.
In summary, the broad-host-range plasmids, pIP501 and pRE25, as well as pSK41, pGO1, and pMRC01, encode at least one protein homologue of most of the protein families involved in T-DNA transfer and in gram-negative bacterial plasmid transfer (37). These provide substrate presentation (VirD4 homologue), energetics of the translocation process (VirB4 homologue), and formation of the mating channel (putative VirB1 homologue). Homologues for contact formation between donor and recipient cells and for the major components of the mating channel (VirB1 is not an essential transfer protein) were not yet detected. These homologies to type IV secretion systems make a similar mechanism perhaps simpler, because only one membrane per mating partner has to be crossed for the conjugative DNA transport of plasmids from gram-positive hosts to be likely. However, the most important questions still remain unanswered: how the cell-cell contact between donor and recipient cells is established and how the DNA-protein complex is transported through the cell envelope.
Conjugative transfer of Tn916 requires a series of genes located at the right end of the transposon (189). A map of Tn916 with open reading frames thought to be related to conjugation is shown in Fig. 4. Genetic data suggest that a single DNA strand is transferred from the donor cell to a recipient cell during conjugative transfer of Tn916 (187). Tn916-oriT was identified as a segment of DNA that, when cloned onto a plasmid, causes mobilization of the plasmid by Tn916 (103). Tn916-oriT is presumably the site where the DNA is nicked to initiate the transfer of a single-stranded DNA molecule, although this has not been demonstrated directly. Furthermore, definite experimental evidence for a DNA relaxase encoded by Tn916 exerting this site- and strand-specific nick at oriT has not yet been obtained.
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The best-studied pheromone-induced plasmid transfer systems are pAD1, pCF10, and pPD1. pAD1 is a 59.3-kb hemolysin/bacteriocin plasmid that responds to the pheromone cAD1. The 65-kb pCF10 encodes tetraycline resistance and confers response to the pheromone cCF10. pPD1 is a 56-kb plasmid encoding bacteriocin production (Bac21), and its conjugative response depends on the pheromone cPD1. For pAD1, two oriT sequences have been identified (1, 75), with oriT1 being located within the repA determinant whereas the more efficiently utilized oriT2 is located between orf53 and orf57, two genes found to be essential for conjugation (74). oriT2 of pAD1 contains a large inverted repeat (about 140 nucleotides) adjacent to a series of short direct repeats. The orf57 gene product, the TraX relaxase, nicks within the inverted repeat (46, 74). Orf53 exhibits certain structural similarities to TraG-like proteins, although there is little overall homology (74).
Pheromone internalization is essential for induction of the pheromone response (122). This import is achieved by pheromone-binding lipoproteins, the products of traC for pAD1 and pPD1 and of prgZ for pCF10, which act as surface receptors that bind to the exogenous pheromone peptide. The pheromone is then internalized, making use of a host-encoded peptide transport system (for a recent detailed review, see reference 44). A functional analysis of TraA, the intracellular sex pheromone receptor encoded by pPD1, was recently performed (99). When cPD1 is taken up by a pPD1 donor cell, it binds to an intracellular receptor, TraA. Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells. Horii et al. (99) discussed how TraA transduces the signal of cPD1 to the mating response. For pAD1, Fujimoto and Clewell (78) presented evidence that after transport into the bacterial cell, the primary target of pheromone is the pAD1-encoded TraA protein and that a conformational shift leads to induction of conjugation functions via an alteration of TraA DNA-binding activity at the iad promoter. The available information relating to the complex regulation of the pheromone response has been generated most extensively with pAD1 and pCF10 (for recent reviews, see references 43, 44, and 64).
The response system developed by conjugative pheromone plasmids to sense whether a potential host harbors the same plasmid is unique among plasmids studied so far. However, parallels to the Ti plasmids appear to exist, insofar as many of the components of the pheromone-mediated conjugation system also seem to be involved in host-parasite interactions of enterococci (95, 217, 226). Hirt et al. (95) demonstrated that E. faecalis cells harboring pCF10 showed significantly increased virulence in a rabbit endocarditis model. Their results confirmed in vivo induction of the normally highly controlled plasmid-encoded aggregation substance. Host plasma induction was dependent on the presence of the pCF10-encoded pheromone receptor protein PrgZ, indicating the requirement of the pheromone-sensing system in the induction process (95).
Interestingly, the traH gene of the conjugative S. aureus plasmid pSK41 has been reported to encode a lipoprotein precursor bearing a signal sequence whose carboxyl-terminal region consists of seven or eight contiguous amino acid residues identical to cAD1 (16, 69). A cAD1 activity could even be detected in supernatants of pSK41-carrying staphylococci but not in plasmid-free cells. Thus far, the involvement of recipient-produced pheromones as mating signals related to plasmid transfer has been observed only in E. faecalis. However, a few other bacterial species secrete peptides with a cAM373-like activity, the pAM373-specific pheromone. These include Enterococcus hirae, S. aureus, and Streptococcus gordonii (41).
A 65.1-kb conjugative plasmid, pMG1, that transfers efficiently in broth matings from E. faecalis to E. faecium strains and vice versa was isolated from a gentamicin-resistant E. faecium clinical isolate (100). Interspecies transfer of pMG1 occurs at a frequency of approximately 10-4 per donor cell in broth matings and appears to proceed independently of the presence of pheromone-like signal molecules in the culture supernatants. Interestingly, Southern hybridization of pMG1 DNA did not show any homology to pheromone-responsive plasmids and the broad-host-range plasmids pAMß1 and pIP501. These results indicate that another efficient broth transfer system might exist in E. faecium which differs from the sex pheromone-mediated transfer system in E. faecalis (100).
Mobilization of small plasmids between strains of B. thuringiensis subsp. israelensis is accompanied by non-pheromone-induced and protease-sensitive coaggregation between donor and recipient cells (4). Two aggregation phenotypes (Agr+ and Agr-) were identified. They are characterized by macroscopically visible aggregates when exponentially growing cells of the Agr+ and the Agr- types are mixed in broth. The mobilization of small plasmids was found to occur unidirectionally, from Agr+ to Agr- cells. The Agr+ phenotype is transferred at a high frequency (100%) to Agr- cells in broth matings (4). Loci essential for the Agr+ phenotype have been localized on plasmid pXO16 (104), and it is supposed that the pXO16-mediated B. thuringiensis subsp. israelensis plasmid transfer system mobilizes plasmids of distinct replication types independent of the presence of oriT and mob functions on the mobilized plasmids (5). The fact that all plasmids tested so far (theta-replicating plasmid pAMß1; ori43-, ori44-, and ori60-containing plasmids; and Bacillus cereus plasmid pBC16) could be mobilized by the pXO16-encoded conjugation system suggests that pXO16 possesses an exceptional and, so far, unique system (5), whose molecular basis remains to be elucidated.
Transfer of plasmid-encoded genes for lactose catabolism by a conjugation-like mechanism in lactic acid bacteria was described early (80, 109). Subsequently, conjugal transfer of lactose utilization genes has been reported for various Lactococcus lactis strains and for a lactose plasmid in Lactobacillus casei (210). Cell aggregation is mediated by the interaction of two cell surface components. One seems to be active only after molecular rearrangements of the lactose plasmid with the sex factor, and its clu gene(s) is encoded by the sex factor on the enlarged lactose plasmid. The second component is constitutively expressed and is encoded by a chromosomal agg gene(s). High-frequency transfer and cell aggregation occur only when a pair of strains includes both the agg and clu genes. These genes can be present in the donor, in which case it aggregates, or clu can be in the donor with agg in the recipient, in which case the mating mixture aggregates but the individual strains do not (210).
The conjugative transfer systems encoded by the sex factor of L. lactis subsp. lactis 712 and the conjugative plasmid pRS01 of L. lactis subsp. lactis ML3 (3, 81) have features in common with the aggregation-mediated plasmid transfer system in E. faecalis. In both systems, donor-recipient aggregation is associated with efficient plasmid transfer, but there is no evidence for a sex-pheromone-like induction system in L. lactis (210). Plasmid pRS01 and the sex factor from L. lactis subsp. lactis 712 are prototype mobile elements in lactococci (140). Both elements mediate high-frequency transfer of genes encoding lactose utilization (Lac+) by insertion sequence-directed cointegration with nonconjugative Lac+ plasmids (82, 171). The clu genes have been shown to be associated within an inversion region (3, 83; J.-J. Godon, C. Pillidge, K. Jury, C. A. Shearman, and M. J. Gasson, Proc. 4th Int. Conf. Streptococcal Genet., p. 43, 1994). Mapping of pRS01 identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in conjugative transfer. Sequence analysis of the Tra1 region revealed a gene, ltrB, with extensive homology to replicative and conjugative relaxases (139). oriT of pRS01 was localized and shown to reside within the Tra1 region upstream of the ltrB gene (141). Conjugative transfer of pRS01 requires splicing of a group II intron, LI.ltrB, for accurate translation of the mRNA for the exon gene ltrB (140). The protein product of ltrB was shown to be a conjugative relaxase, essential for pRS01 transfer. A functional promoter within LI.ltrB was identified upstream from the ltrA gene. LtrA is required for efficient splicing of LI.ltrB in vivo. Zhou et al. (227) showed that the major source of ltrA mRNA in the LI.ltrB system is from this promoter within the intron and that the promoter activity is essential for normal expression of LtrA protein, LI.ltrB splicing, and pRS01 conjugation functions in L. lactis.
It has been shown that an autoaggregating strain of L. plantarum was able to act as a donor of, or recipient for, the broad-host-range Inc18 plasmid pAMß1 with high efficiency of plasmid transfer when mated on solid surfaces and at a low rate in broth matings. It was suggested that cell aggregation and high frequency of conjugation are associated with a secreted protein of 32 kDa, which recognizes and specifically binds to lipoteichoic acids or substitutions in teichoic acids in the cell membrane (176). The molecular mechanism of this plasmid transfer system remains to be elucidated.
-helices as determined by circular dichroism experiments (C. de Antonio, M. García de Lacoba, M. E. Farías, and M. Espinosa, unpublished results). Gel retardation assays showed that the protein was specifically bound to a linear double-stranded DNA segment containing two inverted repeat sequences, termed IR-1 and IR-2, which partially overlap (Fig. 5A). IR-2 has the potential to generate a secondary structure that would leave the MobM nic site unpaired and exposed in a single-stranded configuration. This would explain why MobM is able to cleave supercoiled DNA and single-stranded oligonucleotides harboring the IR but not linear double-stranded DNA because, in the latter case, the nic site would be buried within the DNA helix (90, 91). DNase I footprinting assays showed that purified MobM protein protected the IR-2 sequence (90). Since the -10 extended region of the promoter that directs synthesis of the mobM mRNA (at least in lactococcal cells) is also included within the IR-2 (66), it would appear that the protein regulates its own synthesis, similarly to the Mob protein of plasmid pBBR1 (205), a hypothesis that is currently under investigation (C. de Antonio and M. Espinosa, personal communication). Consequently, this pMV158-DNA region was defined as the oriT plasmid in vitro (90, 91) and was later shown to promote transfer of pSC101 when MobM was provided in trans (65). An identical, or nearly identical, sequence and structure of the oriT region of pMV158 can be found in various RCR plasmids from different origins (Fig. 5A); curiously, oriT-like sequences were found in plasmids like pCI411 and pA1, which lack a mob gene (91). Whether this genetic situation reflects a remnant of an ancient mobilization cassette present in these plasmids and/or, in addition to mobilization, these sequences play a role in plasmid cointegration when incompatibility processes take place (e.g., a plasmid-bearing host being colonized by an incompatible replicon under selection conditions) is presently unkown.
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Based on homology analyses, it has been proposed that the DNA-binding domain of the Mob proteins is located within their C-terminal moieties, a region that in MobM contains a putative coiled-coil region that could be involved in protein dimerization (de Antonio, personal communication). No indication of DNA-binding motifs, such as helix-turn-helix or ribbon-helix-helix motifs, has been obtained.
| CONJUGATIVE PLASMID TRANSFER IN MYCELIUM- FORMING STREPTOMYCETES |
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The Streptomyces plasmids also include integrative plasmids, such as pSAM2 (21, 156, 166) or the Amycolatopsis methanolica plasmid pMEA300 (213). These elements can be excised from the chromosome and also exist as autonomous molecules, replicating either by the RCR mechanism (e.g., pSAM2) or by a theta mechanism (e.g., pMEA300). Integration occurs by site-specific recombination mediated by a plasmid-encoded integrase via an attachment site that overlaps with a chromosomal tRNA gene (22, 157). The different plasmids integrate into different tRNA genes. Since the tRNAs are quite highly conserved, the host range of the integration system is broader than the host range for autonomous replication (133). For pSAM2, it has been demonstrated that conjugative transfer requires excision of the integrated pSAM2 molecule and its autonomous replication in the donor strain (172).
Another type of plasmid includes the large low-copy-number plasmids that replicate very stably. Only a very few low-copy-number plasmids have been characterized. The best-studied representative is SCP2, the first Streptomyces plasmid that has been physically isolated (184). SCP2 is 31,317 bp in size, replicates very stably, and accepts the cloning of large fragments encoding whole antibiotic biosynthetic pathways (129). The availability of the complete nucleotide sequence allowed the identification of two resident transposable elements, IS1648 and Tn1547 (AL645771). A derivative, SCP2*, was isolated and shown to mobilize chromosomal markers with enhanced frequency (19). The transfer features of SCP2* have been characterized by transposon mutagenesis (26).
The Streptomyces plasmids also include small multicopy number plasmids. These plasmids have a molecular size of 8 to 13 kb and replicate by the rolling-circle mechanism via a single-stranded plasmid intermediate (108, 111, 147, 191, 220). All actinomycete RCR plasmids sequenced so far encode replication initiator proteins similar to pC194 RepA (146) and can be phylogenetically grouped within a single cluster of the RCR group III of the Database of Plasmid Replicons (http://www.essex.ac.uk/bs/staff/osborn/DPR/DPR_RCRIIIphylo.htm).Despite their small size, most of them are conjugative and are transferred to a plasmid-free recipient with the same efficiency as are larger plasmids (111). This class of plasmids shows a modular architecture; e.g., plasmids pSN22 and pIJ101 have a nearly identical replication region, whereas the transfer and spread genes show little to no similarity (106). The transfer and spread genes of pSN22 are very similar to those of pJV1 (191). Plasmids pSG5 and pSVH1 have a similar transfer region (60 to 70% identity), while the regulator TraR and the replication region are different (145). Very small nonconjugative plasmids such as pSB24.2 (23) and pSL33 (67) have also been isolated; they are probably deletion derivatives of larger plasmids and do not represent a typical Streptomyces plasmids.
All these different types of plasmids are conjugative, and they transferred to a plasmid-free recipient with an efficiency of nearly 100% (111). The plasmid transfer is associated with the mobilization of chromosomal markers (Cma) at a frequency ranging between 0.1 and 1% (100).
The complete nucleotide sequences of some representatives of all plasmid types have been determined. This allows the comparative analysis of the plasmid-encoded functions and the detailed characterization of the loci involved in the conjugative transfer. This comparison reveals that although the Streptomyces plasmids are not closely related to each other and have only very limited sequence similarity, most of them carry the same set of functionally homologous genes (Fig. 7).
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