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 .     

Dynamics of the IncW genetic backbone imply general trends in conjugative plasmid evolution

Plasmids cannot be understood as mere tools for genetic exchange: they are themselves subject to the forces of evolution. Their genomic and phylogenetic features have been less studied in this respect. Focusing on the IncW incompatibility group, which includes the smallest known conjugative plasmids, we attempt to unveil some common trends in plasmid evolution. The functional modules of IncW genetic backbone are described, with emphasis on their architecture and relationships to other plasmid groups. Some plasmid regions exhibit strong phylogenetic mosaicism, in striking contrast to others of unusual synteny conservation. The presence of genes of unknown function that are widely distributed in plasmid genomes is also emphasized, exposing the existence of ill-defined yet conserved plasmid functions. Conjugation is an essential hallmark of IncW plasmid biology and special attention is given to the organization and evolution of its transfer modules. Genetic exchange between plasmids and their hosts is analysed by following the evolution of the type IV secretion system. Adaptation of the trw conjugative machinery to pathogenicity functions in Bartonella is discussed as an example of how plasmids can change their host modus vivendi. Starting from the phage paradigm, our analysis articulates novel concepts that apply to plasmid evolution.     

A soil-based microbial biofilm exposed to 2,4-D: bacterial community development and establishment of conjugative plasmid pJP4

A soil suspension was used as a source to initiate the development of microbial communities in flow cells irrigated with 2,4-dichlorophenoxyacetic acid (2,4-D) (25 microg ml(-1)). Culturable bacterial members of the community were identified by 16S rRNA gene sequencing and found to be members of the genera Pseudomonas, Burkholderia, Collimonas and Rhodococcus. A 2,4-D degrading donor strain, Pseudomonas putida SM1443 (pJP4::gfp), was inoculated into flow cell chambers containing 2-day old biofilm communities. Transfer of pJP4::gfp from the donor to the bacterial community was detectable as GFP fluorescing cells and images were captured using confocal scanning laser microscopy (GFP fluorescence was repressed in the donor due to the presence of a chromosomally located lacI(q) repressor gene). Approximately 5-10 transconjugant microcolonies, 20-40 microm in diameter, could be seen to develop in each chamber. A 2,4-D degrading transconjugant strain was isolated from the flow cell system belonging to the genus Burkholderia.     

The putative Orf4 of broad-host-range conjugative plasmid R446 could Be related to the H-NS family of bacterial nucleoid-associated proteins

The iml determinant of IncM plasmid R446 was described initially as a short fragment of DNA causing (when cloned in multicopy plasmids) insensitivity to pilus-dependent bacteriophage lysis in bacteria harboring coresident IncM plasmids. We have performed a computational analysis of the iml determinant of IncM plasmid R446 and found that a potential polypeptide (Orf4) shows similarity to the H-NS family of nucleoid-associated modulators of gene expression. The predicted protein has a molecular weight of 16.3 kDa and an isoelectric point of 5.50. Orf4 is adjacent and upstream of Orf5, a potential polypeptide related to the Hha/YmoA/RmoA family of proteins which modulate bacterial virulence expression (Hha and YmoA) and R100 transfer (RmoA) in response to environmental conditions.     

Mobilization of R387 Inc K conjugative plasmid by the conjugative plasmid R64 Inc I

Plasmid aggregate (R387, R64) was constructed in E. coli K12 strain. Plasmid R387 Inc K was stimulated to conjugational transfer by plasmid R64 Inc I. This stimulation was caused neither by recombination between both plasmids nor by trans-complementation of R387 conjugational systems by gene(s) product(s) of R64 plasmid. The observed phenomenon resembled rather mobilization of nonconjugative plasmids by conjugative ones. As in mobilization, the observed increase in R387 transfer frequency could take place only when both interacting plasmids were present in donor cells. Moreover, the entry exclusion system functioning in recipient cells, toward stimulating R64 plasmid affected strongly the conjugational transfer of stimulated R387 plasmid. Analogous phenomenon was observed during mobilization of nonconjugative plasmids by conjugative ones.     

Isolation and characterization of a conjugative plasmid from Legionella pneumophila.

The conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legionella pneumophila were studied. To determine if pCH1 was transmissible by conjugation, mating experiments were performed between legionellae that harboured pCH1 and several plasmid-less recipients. Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCH1 as a probe. The results from these experiments showed that pCH1 could be conjugatively transferred into several strains of L. pneumophila serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia coli. Southern hybridization experiments in which pCH1 DNA was used as a probe showed that pCH1 does not share homology with other indigenous L. pneumophila plasmids. There was no detectable DNA homology between pCH1 and L. pneumophila chromosomal DNA. Additional mating experiments revealed that pCH1 was unable to mobilize the L. pneumophila chromosome. The conjugative transfer of pCH1 into plasmid-less avirulent or virulent serogroup 1 strains did not alter the intracellular growth characteristics of these strains in U937 cells, a human-monocyte-like cell line, or in the amoeba Hartmannella vermiformis. These results suggest that pCH1 does not contribute to the ability of L. pneumophila to enter or grow within eukaryotic cells.     

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.     

Rule-based modelling of conjugative plasmid transfer and incompatibility

COSMIC-rules, an individual-based model for bacterial adaptation and evolution, has been used to study virtual transmission of plasmids within bacterial populations, in an environment varying between supportive and inhibitory. The simulations demonstrate spread of antibiotic resistance (R) plasmids, both compatible and incompatible, by the bacterial gene transfer process of conjugation. This paper describes the behaviour of virtual plasmids, their modes of exchange within bacterial populations and the impact of antibiotics, together with the rules governing plasmid transfer. Three case studies are examined: transfer of an R plasmid within an antibiotic-susceptible population, transfer of two incompatible R plasmids and transfer of two compatible R plasmids. R plasmid transfer confers antibiotic resistance on recipients. For incompatible plasmids, one or other plasmid could be maintained in bacterial cells and only that portion of the population acquiring the appropriate plasmid-encoded resistance survives exposure to the antibiotics. By contrast, the compatible plasmids transfer and mix freely within the bacterial population that survives in its entirety in the presence of the antibiotics. These studies are intended to inform models for examining adaptive evolution in bacteria. They provide proof of principle in simple systems as a platform for predicting the behaviour of bacterial populations in more complex situations, for example in response to changing environments or in multi-species bacterial assemblages.     

Characterization of conjugative plasmid EDP208.

EDP208 is a conjugative plasmid belonging to incompatibility group IncF0 lac, A restriction endonuclease map of this plasmid was constructed using five restriction enzymes: BamHI, HindIII, PvuI, SstI, and XhoI. On the basis of these mapping studies, the plasmid was found to be 90 kilobases in length. Clones were constructed from four large HindIII fragments of plasmid EDP208. One fragment, HindIII-20.5, was found to contain the lac genes and the origin of vegetative replication (oriV). Another fragment, HindIII-27.5, was found to contain all of the genes necessary for sex pilus formation, but it was nontransmissible. However, when used to complement a plasmid carrying an adjacent fragment, HindIII-23, the transfer of the latter occurred, suggesting that HindIII-23 contains the origin of transfer (oriT). The further localization of genes concerned with pilus biosynthesis was achieved by transposon mutagenesis. Six EDP208::Tn1 and thirty-seven EDP208::Tn5 mutants were isolated on the basis of their resistance to f1, a filamentous phage which adheres to intact pilus tips. The positions of the inserted transposons were determined on the restriction map and a 16.5-kilobase region was found to be required for pilus synthesis.     

Identification of cell wall antigens associated with a large conjugative plasmid encoding phage resistance and lactose fermentation ability in lactic streptococci

In order to begin to analyze the gene products encoded by phage resistance plasmids in lactic streptococci, we identified phage-resistance plasmids by screening resistant strains from commercial starter cultures for the ability to carry out unselected cotransfer, by conjugation, of phage resistance with lactose fermentation ability (lac+). In this fashion, we identified a large (90 kilobases) plasmid, pCLP51R, that encodes the lac+ marker, resistance to a lytic phage called LP10G (1pr+), high-frequency conjugal donor ability (hft+), and clumpy growth of host bacteria in broth culture (clu+). The mechanism of resistance conferred by this plasmid appears to involve interference with a step in the phage replication cycle that occurs after the initial attachment of the phage. Polyacrylamide gel electrophoresis of cell surface extracts of isogenic strains, carrying or lacking pCLP51R, combined with immunoblotting analysis, showed that there were several plasmid-related differences in the banding pattern of low molecular weight proteins and that the plasmid resulted in production of several unique antigenic polypeptides in the size range of 15-30 kd, as well as modification of chromosomally encoded antigens to different molecular weight forms.     

NIC, a conjugative nicotine-nicotinate degradative plasmid in Pseudomonas convexa

The plasmid nature of genes specifying degradation of nicotine and nicotinate in Pseudomas convexa strain 1 (Pc1) is indicated by mitomycin curing and conjugational transfer to other strains. The NIC plasmid appears to be compatible with other metabolic plasmids in Pseudomonas putida.     

Nucleotide sequence and analysis of conjugative plasmid pVT745

The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.     

Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages

Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation.     

The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule

The SLP2 plasmid had previously been demonstrated genetically to exist In Streptomyces lividans by its ability to promote conjugation and to elicit‘pocks’on recipient (SLP2^?) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50kb linear DNA was isolated from SLP2⁺ but not SLP2^? strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44 bp imperfect terminal inverted repeat. The terminal 12 bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by λ exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a tree form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.     

Isolation of a conjugative plasmid in Escherichia coli determining formaldehyde resistance

A clinical isolate of Escherichia coli which was resistant to the disinfectant formaldehyde was investigated. The strain harboured a plasmid of 62 MDa size. It was shown by conjugation, transformation and plasmid-curing experiments that the formaldehyde resistance is plasmid-mediated and transferable to other strains.     

A large conjugative plasmid from a soil strain of Bacillus subtilis

A large plasmid 35.5 kb in size was found in the soil Bacillus subtilis strain. This plasmids was shown to be capable of conjugal mobilization of small plasmids.     

The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages.

Streptomyces ambofaciens ATCC23877 and derivatives contain the 11-kb element pSAM2 present in an integrated state or as a free and integrated plasmid. This element, able to integrate site-specifically in the genome of different Streptomyces species, is conjugative and mobilizes chromosomal markers. Besides these plasmid functions, we have shown that the site-specific recombination system of pSAM2 presents strong similarities with that of several temperate phages. The integration event is promoted by a site-specific recombinase of the integrase family. The int gene encoding this integrase is closely linked to the plasmid attachment site (attP). A small open reading frame (ORF) overlaps the int gene and the predicted protein exhibits similarities with Xis proteins involved in phages excision. The integrated copy of pSAM2 in strain ATCC23877 is flanked by att sequences (attL and attR). Another att sequence (attX) is present in this strain and attX and attL are the boundaries of a 42-kb fragment (xSAM1) absent, as well as pSAM2, from S.ambofaciens DSM40697. Sequences partially similar to pSAM2 int gene are found near the chromosomal integration zone in both S.ambofaciens strains. The possible origin of pSAM2, an element carrying plasmid as well as phage features, is discussed.