The Bacillus subtilis conjugative transposon ICEBs1 mobilizes plasmids lacking dedicated mobilization functions

Integrative and conjugative elements (ICEs, also known as conjugative transposons) are mobile elements that are found integrated in a host genome and can excise and transfer to recipient cells via conjugation. ICEs and conjugative plasmids are found in many bacteria and are important agents of horizontal gene transfer and microbial evolution. Conjugative elements are capable of self-transfer and also capable of mobilizing other DNA elements that are not able to self-transfer. Plasmids that can be mobilized by conjugative elements are generally thought to contain an origin of transfer (oriT), from which mobilization initiates, and to encode a mobilization protein (Mob, a relaxase) that nicks a site in oriT and covalently attaches to the DNA to be transferred. Plasmids that do not have both an oriT and a cognate mob are thought to be nonmobilizable. We found that Bacillus subtilis carrying the integrative and conjugative element ICEBs1 can transfer three different plasmids to recipient bacteria at high frequencies. Strikingly, these plasmids do not have dedicated mobilization-oriT functions. Plasmid mobilization required conjugation proteins of ICEBs1, including the putative coupling protein. In contrast, plasmid mobilization did not require the ICEBs1 conjugative relaxase or cotransfer of ICEBs1, indicating that the putative coupling protein likely interacts with the plasmid replicative relaxase and directly targets the plasmid DNA to the ICEBs1 conjugation apparatus. These results blur the current categorization of mobilizable and nonmobilizable plasmids and indicate that conjugative elements play a role in horizontal gene transfer even more significant than previously recognized.     

Examination of an inverted repeat within the F factor origin of transfer: context dependence of F TraI relaxase DNA specificity

Prior to conjugative transfer of plasmids, one plasmid strand is cleaved in a site- and strand-specific manner by an enzyme called a relaxase or nickase. In F and related plasmids, an inverted repeat is located near the plasmid strand cleavage site, and others have proposed that the ability of this sequence to form a hairpin when in single-stranded form is important for transfer. Substitutions were introduced into a cloned F oriT region and their effects on plasmid transfer were assessed. For those substitutions that substantially reduced transfer, the results generally correlated with effects on in vitro binding of oligonucleotides to the F TraI relaxase domain rather than with predicted effects on hairpin formation. One substitution shown previously to dramatically reduce both plasmid transfer and in vitro binding to a 17-base oligonucleotide had little apparent effect on binding to a 30-base oligonucleotide that contained the hairpin region. Results from subsequent experiments strongly suggest that the relaxase domain can bind to hairpin oligonucleotides in two distinct manners with different sequence specificities, and that the protein binds the oligonucleotides at the same or overlapping sites.     

The relaxase of the Rhizobium etli symbiotic plasmid shows nic site cis-acting preference

Genetic and biochemical characterization of TraA, the relaxase of symbiotic plasmid pRetCFN42d from Rhizobium etli, is described. After purifying the relaxase domain (N265TraA), we demonstrated nic binding and cleavage activity in vitro and thus characterized for the first time the nick site (nic) of a plasmid in the family Rhizobiaceae. We studied the range of N265TraA relaxase specificity in vitro by testing different oligonucleotides in binding and nicking assays. In addition, the ability of pRetCFN42d to mobilize different Rhizobiaceae plasmid origins of transfer (oriT) was examined. Data obtained with these approaches allowed us to establish functional and phylogenetic relationships between different plasmids of this family. Our results suggest novel characteristics of the R. etli pSym relaxase for previously described conjugative systems, with emphasis on the oriT cis-acting preference of this enzyme and its possible biological relevance.     

The diversity of conjugative relaxases and its application in plasmid classification

Bacterial conjugation is an efficient and sophisticated mechanism of DNA transfer among bacteria. While mobilizable plasmids only encode a minimal MOB machinery that allows them to be transported by other plasmids, conjugative plasmids encode a complete set of transfer genes (MOB+T4SS). The only essential ingredient of the MOB machinery is the relaxase, the protein that initiates and terminates conjugative DNA processing. In this review we compared the sequences and properties of the relaxase proteins contained in gene sequence databases. Proteins were arranged in families and phylogenetic trees constructed from the family alignments. This allowed the classification of conjugative transfer systems in six MOB families: MOB_F, MOB_H, MOB_Q, MOB_C, MOB_P and MOB_V . The main characteristics of each family were reviewed. The phylogenetic relationships of the coupling proteins were also analysed and resulted in phylogenies congruent to those of the cognate relaxases. We propose that the sequences of plasmid relaxases can be used for plasmid classification. We hope our effort will provide researchers with a useful tool for further mining and analysing the plasmid universe both experimentally and in silico .     

Genetic Characterization of a Novel Rhizobial Plasmid Conjugation System in Rhizobium leguminosarum bv. viciae Strain VF39SM

Rhizobium leguminosarum strain VF39SM contains two plasmids that have previously been shown to be self-transmissible by conjugation. One of these plasmids, pRleVF39b, is shown in this study to carry a set of plasmid transfer genes that differs significantly from conjugation systems previously studied in the rhizobia but is similar to an uncharacterized set of genes found in R. leguminosarum bv. trifolii strain WSM2304. The entire sequence of the transfer region on pRleVF39b was determined as part of a genome sequencing project, and the roles of the various genes were examined by mutagenesis. The transfer region contains a complete set of mating pair formation (Mpf) genes, a traG gene, and a relaxase gene, traA, all of which appear to be necessary for plasmid transfer. Experimental evidence suggested the presence of two putative origins of transfer within the gene cluster. A regulatory gene, trbR, was identified in the region between traA and traG and was mutated. TrbR was shown to function as a repressor of both trb gene expression and plasmid transfer.     

Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria

The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost.     

TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens

Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild‐type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase‐mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site‐specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.     

Nicking by transesterification: the reaction catalysed by a relaxase

DNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site- and strand-specific manner by catalysing a transesterification. The product of the reaction is a nicked double-stranded DNA molecule with a sequestered 3'-OH and the relaxase covalently bound to the 5' end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase-catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.     

Elements in the co-evolution of relaxases and their origins of transfer

The central elements in the conjugative mobilization of most plasmids are the relaxase and its cognate origin of transfer (oriT). The relaxase of the plasmid R1162, together with its oriT, belong to a large and widely distributed family of related relaxase/oriT pairs. Several of the properties of these elements are considered for R1162 and for other members of this family with a view to understanding how systems for mobilization might have evolved.     

Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4.

Two essential transfer genes of the conjugative plasmid RP4 were altered by site-directed mutagenesis: traG of the primase operon and traI of the relaxase operon. To evaluate effects on the transfer phenotype of the point mutations, we have reconstituted the RP4 transfer system by fusion of the transfer regions Tra1 and Tra2 to the small multicopy replicon ColD. Deletions in traG or traI served to determine the Tra phenotype of mutant plasmids by trans complementation. Two motifs of TraG which are highly conserved among TraG-like proteins in several other conjugative DNA transfer systems were found to be essential for TraG function. One of the motifs resembles that of a nucleotide binding fold of type B. The relaxase (TraI) catalyzes the specific cleaving-joining reaction at the transfer origin needed to initiate and terminate conjugative DNA transfer (W. Pansegrau, W. Schröder, and E. Lanka, Proc. Natl. Acad. Sci. USA 90:2925-2929, 1993). Phenotypes of mutations in three motifs that belong to the active center of the relaxase confirmed previously obtained biochemical evidence for the contributions of the motifs to the catalytic activity of TraI. Expression of the relaxase operon is greatly increased in the absence of an intact TraI protein. This finding suggests that the relaxosome which assembles only in the presence of the TraI in addition to its enzymatic activity plays a role in gene regulation.     

Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated

Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal ∼300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.Transfer of conjugative plasmids between bacteria contributes to genome diversification and acquisition of new traits. Conjugative plasmids encode most proteins required for transfer of one plasmid strand from the donor to the recipient cell (reviewed in references 11, 24, and 43). In preparation for transfer, a complex of proteins assembles at the plasmid origin of transfer (oriT). Within this complex, called the relaxosome, a plasmid-encoded relaxase or nickase binds and cleaves one plasmid strand at a specific oriT site (nic). As part of the cleavage reaction, the relaxase forms a covalent linkage between an active-site tyrosyl hydroxyl oxygen and a single-stranded DNA (ssDNA) phosphate, yielding a 3′ ssDNA hydroxyl (19, 30). Upon initiation of transfer, the plasmid strands are separated, and the cut strand is transported into the recipient. The relaxase is likely transferred into the recipient (12, 31) while still physically attached to plasmid DNA. The transferred relaxase may then join the ends of the ssDNA plasmid copy in the final step of plasmid transfer. Complementary strand synthesis in the donor and the recipient generates a double-stranded plasmid that is competent for further transfer. Successful conjugation requires effective temporal regulation, yet the mechanisms governing this regulation are poorly understood.The F plasmid oriT is ∼500 bp long and includes multiple binding sites for integration host factor (IHF), TraY, and TraM and a single site for TraI, the F relaxase (11). IHF, TraY, and TraM, participants in the relaxosome, bind double-stranded DNA to facilitate the action of TraI, perhaps by creating or stabilizing the ssDNA conformation around nic required for TraI recognition. The F TraI minimal high-affinity binding site includes ∼15 nucleotides around nic (39), and throughout the text, we refer to oligonucleotides that contain the TraI wild-type (wt) or variant binding site as oriT oligonucleotides. F TraI is 192 kDa (42), and in addition to its relaxase activity, TraI has a 5′-to-3′ helicase activity (4). These activities must be physically joined to allow efficient plasmid transfer (29), yet how the two activities are coordinated is a mystery. The relaxase region of F TraI has been defined as the N-terminal ∼300 amino acids (aa) (6, 40). Conserved helicase motifs, including those associated with an ATPase, lie between amino acids 990 and 1450. The C-terminal region (positions 1450 to 1756) plays an important role in bacterial conjugation, possibly involving protein-protein interactions with TraM (32) and/or inner membrane protein TraD (28).The 70-kDa central region of TraI that lies between the relaxase and helicase domains has been implicated in two functions. Haft and colleagues described TraI variants with 31-amino-acid insertions in this TraI region that facilitated plasmid transfer with greater efficiency than that afforded by the wild-type protein when these proteins are expressed at high levels (16). On the basis of this observation, the authors proposed that the region participated in a negative regulation of transfer. Matson and Ragonese demonstrated that this central region is required for TraI helicase function, likely due to participation in ssDNA recognition essential for the helicase activity (28). We wondered whether the proposed regulatory and ssDNA binding roles of the central region are linked and whether this region might help modulate TraI helicase and relaxase activities. Our objectives in this study were to confirm the role of the central region in ssDNA recognition, to assess the affinity and specificity of the ssDNA recognition by the central region, and to determine whether the relaxase and central domain ssDNA binding sites demonstrate cooperativity in binding. Our work yielded two significant and surprising results. First, the binding site within the TraI central region binds ssDNA with high affinity and significant sequence specificity, both unusual characteristics for a helicase. Second, the central region and relaxase ssDNA binding sites show an apparent strong negative cooperativity of binding, possibly explaining the role of the central region as a negative regulator and providing clues about how the timing of conjugative transfer might be regulated.     

Sequence homology between Inc N group plasmids

DNA-DNA hybridization combined with "Southern blotting" was used to analyse the genetic organization and the nucleotide sequence homology between different regions of a previously characterized Inc N group plasmid pCUI and nine other Inc N group plasmids. The following conclusions could be reached: (1) N plasmids isolated from different parts of the world share substantial DNA sequence homology and also some similarity of overall genetic organization, (2) the majority of the N plasmids used in this study showed conservation of distribution of BglII and KpnI cleavage sites. Often, restriction endonuclease fragments of similar electrophoretic mobility encoded the same genetic function, (3) in one case, the N-specific properties appear to be integrated into the bacterial chromosome. (4) the plasmid DNA in strains carrying two Inc N plasmids, R199 and R113 were each composed of two molecular species only one of which constituted an N group plasmid.     

Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR.

Primer systems for PCR amplification of different replicon-specific DNA regions were designed on the basis of published sequences for plasmids belonging to the incompatibility (Inc) groups IncP, IncN, IncW, and IncQ. The specificities of these primer systems for the respective Inc groups were tested with a collection of reference plasmids belonging to 21 different Inc groups. Almost all primer systems were found to be highly specific for the reference plasmid for which they were designed. In addition, the primers were tested with plasmids which had previously been grouped by traditional incompatibility testing to the IncN, IncW, IncP, or IncQ group. All IncQ plasmids gave PCR products with the IncQ primer systems tested. However, PCR products were obtained for only some of the IncN, IncP, and IncW group plasmids. Dot blot and Southern blot analyses of the plasmids revealed that PCR-negative plasmids also failed to hybridize with probes derived from the reference plasmids. The results indicated that plasmids assigned to the same Inc group by traditional methods might be partially or completely different from their respective reference plasmids at the DNA level. With a few exceptions, all plasmids related to the reference plasmid at the DNA level also reacted with the primer systems tested. PCR amplification of total DNA extracted directly from different soil and manure slurry samples revealed the prevalence of IncQ- and IncP-specific sequences in several of these samples. In contrast, IncN- and IncW-specific sequences were detected mainly in DNA obtained from manure slurries.     

Conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies

Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single-chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single-chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high-throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the recipient cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr-26 and involved in the conjugative DNA-processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the recipient cell and open a new approach to identify specific inhibitors of bacterial conjugation.     

Comparative analysis of eight Arthrobacter plasmids

Despite the prevalence of Arthrobacter in the environment little is known about their plasmids, or the capacity of Arthrobacter plasmids to mediate horizontal gene transfer. In this study, we compared eight plasmids from five Arthrobacter strains in order to identify putative core maintenance genes for replication, segregation, and conjugation. Iteron like sequences were identified on some of the plasmids; however, no genes with obvious similarity to known replication sequences such as an origin of replication, or rep genes were identified. All eight plasmids contained a putative conjugation system. Genes with similarity to a relaxase, coupling protein, and various components of a type IV secretion system were identified on each plasmid; it appears that three different systems may be present. Putative parA partitioning genes were found in all of the plasmids. Each of the Arthrobacter strains examined contained a putative parB gene; however, of the three plasmids in Arthrobacter strain FB24 only one plasmid had a putative parB gene. Cluster analysis of many of the Arthrobacter genes suggested that they often formed branches within existing families of plasmid maintenance genes. Comparison of a concatenation of all the maintenance genes from each plasmid suggests that the eight Arthrobacter plasmids represent multiple evolutionary pathways.     

Broad-host-range plasmids from agricultural soils have IncP-1 backbones with diverse accessory genes

Broad-host-range plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to antibiotics and heavy metals or degradation of pollutants. Although some broad-host-range plasmids have been extensively studied, their evolutionary history and genetic diversity remain largely unknown. The goal of this study was to analyze and compare the genomes of 12 broad-host-range plasmids that were previously isolated from Norwegian soils by exogenous plasmid isolation and that encode mercury resistance. Complete nucleotide sequencing followed by phylogenetic analyses based on the relaxase gene traI showed that all the plasmids belong to one of two subgroups (β and ε) of the well-studied incompatibility group IncP-1. A diverse array of accessory genes was found to be involved in resistance to antimicrobials (streptomycin, spectinomycin, and sulfonamides), degradation of herbicides (2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenoxypropionic acid), and a putative new catabolic pathway. Intramolecular transposition of insertion sequences followed by deletion was found to contribute to the diversity of some of these plasmids. The previous observation that the insertion sites of a Tn501-related element are identical in four IncP-1β plasmids (pJP4, pB10, R906, and R772) was further extended to three more IncP-1β plasmids (pAKD15, pAKD18, and pAKD29). We proposed a hypothesis for the evolution of these Tn501-bearing IncP-1β plasmids that predicts recent diversification followed by worldwide spread. Our study increases the available collection of complete IncP-1 plasmid genome sequences by 50% and will aid future studies to enhance our understanding of the evolution and function of this important plasmid family.     

An in vivo assay for conjugation-mediated recombination yields novel results for Streptomyces plasmid pIJ101

Efficient transmission of circular plasmids in Streptomyces spp. proceeds by an uncharacterized mechanism that requires a cis-acting locus of transfer (clt) and often only a single plasmid-encoded protein. For circular plasmids from other bacteria, site- and strand-specific nicking takes place at the cis-acting oriT locus via the plasmid-encoded relaxase protein prior to single-strand transfer. Using an assay originally designed to demonstrate that conjugative transfer of plasmids containing tandem oriT loci results in the formation of a single composite oriT locus, we show here that an analogous construct involving the pIJ101 clt locus apparently does not undergo such a conjugation-mediated event during plasmid transfer. Our results, which imply that streptomycete plasmids are transferred by a functionally distinct mechanism compared to oriT-containing plasmids, are complementary to other recent evidences that support a novel double-stranded model for streptomycete circular plasmid transfer.     

Cryptic plasmids isolated from Campylobacter strains represent multiple, novel incompatibility groups

Three small, cryptic plasmids from the multi-drug-resistant (MDR) Campylobacter coli strain RM2228 and one small, cryptic plasmid from the MDR Campylobacter jejuni strain RM1170 were sequenced and characterized. pCC2228-1 has some similarity to Firmicutes RepL family plasmids that replicate via a rolling-circle mechanism. pCC2228-2 is a theta-replicating, iteron-containing plasmid (ICP) that is a member of the same incompatibility (Inc) group as previously described Campylobacter shuttle vectors. The other two ICPs, pCC2228-3 and pCJ1170, represent a second novel Inc group. Comparison of the four plasmids described in this study with other characterized plasmids from C. jejuni, C. coli, C. lari, and C. hyointestinalis suggests that cryptic plasmids in Campylobacter may be classified into as many as nine Inc groups. The plasmids characterized in this study have several unique features suitable for the construction of novel Campylobacter shuttle vectors, e.g., small size, absence of many common multiple-cloning site restriction sites, and Inc groups not represented by current Campylobacter shuttle plasmids. Thus, these plasmids may be used to construct a new generation of Campylobacter shuttle vectors that would permit transformation of environmental Campylobacter isolates with an existing repertoire of native plasmids.     

General mutagenesis of F plasmid TraI reveals its role in conjugative regulation

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies approximately 100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.