Conjugative plasmid transfer in gram-positive bacteria.

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions

The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1-3 and 7-9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4-6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli.     

Conjugative Plasmid Transfer in Gram-Positive Bacteria

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

A classification scheme for mobilization regions of bacterial plasmids

Transmissible plasmids can be classified according to their mobilization ability, as being conjugative (self-transmissible) or mobilizable (transmissible only in the presence of additional conjugative functions). Naturally occurring mobilizable plasmids carry the genetic information necessary for relaxosome formation and processing, but lack the functions required for mating pair formation. Mobilizable plasmids have a tremendous impact in horizontal gene transfer in nature, including the spread of antibiotic resistance. However, analysis of their promiscuity and diversity has attracted less attention than that of conjugative plasmids. This review will focus on the analysis of the diversity of mobilizable plasmids. For this purpose, we primarily compared the amino acid sequences of their relaxases and, when pertinent, we compared these enzymes with conjugative plasmid relaxases. In this way, we established phylogenetic relationships among the members of each superfamily. We conducted a database and literature analysis that led us to propose a classification system for small mobilizable plasmids in families and superfamilies according to their mobilization regions. This review outlines the genetic organization of each family of mobilization regions, as well as the most relevant properties and relationships among their constituent encoded proteins. In this respect, the present review constitutes a first approach to the characterization of the global gene pool of mobilization regions of small mobilizable plasmids.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.     

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.     

The Acquisition of Indigenous Plasmids by a Genetically Marked Pseudomonad Population Colonizing the Sugar Beet Phytosphere Is Related to Local Environmental Conditions

The transfer of naturally occurring conjugative plasmids from the indigenous microflora to a genetically modified population of bacteria colonizing the phytospheres of plants has been observed. The marked strain (Pseudomonas fluorescens SBW25EeZY6KX) was introduced as a seed dressing to sugar beets (Beta vulgaris var. Amethyst) as part of a field experiment to assess the ecology and genetic stability of deliberately released bacterial inocula. The sustained populations of the introduced strain, which colonized the phytosphere, were assessed throughout the growing season for the acquisition of plasmids conferring mercury resistance (Hg(supr)). Transconjugants were isolated only from root and leaf samples collected within a narrow temporal window coincident with the midseason maturation of the crop. Conjugal-transfer events were recorded during this defined period in two separate field release experiments conducted over consecutive years. On one occasion seven of nine individual plants sampled supported transconjugant P. fluorescens SBW25EeZY6KX, demonstrating that conjugative gene transfer between bacterial populations in the phytosphere may be a common event under specific environmental conditions. The plasmids acquired in situ by the colonizing inocula were identified as natural variants of restriction digest pattern group I, III, or IV plasmids from five genetically distinct groups of large, conjugative mercury resistance plasmids known to persist in the phytospheres of sugar beets at the field site. These data demonstrate not only that gene transfer may be a common event but also that the genetic and phenotypic stability of inocula released into the natural environment cannot be predicted.     

F factor conjugation is a true type IV secretion system

The F sex factor of Escherichia coli is a paradigm for bacterial conjugation and its transfer (tra) region represents a subset of the type IV secretion system (T4SS) family. The F tra region encodes eight of the 10 highly conserved (core) gene products of T4SS including TraAF (pilin), the TraBF, -KF (secretin-like), -VF (lipoprotein) and TraCF (NTPase), -EF, -LF and TraGF (N-terminal region) which correspond to TrbCP, -IP, -GP, -HP, -EP, -JP, DP and TrbLP, respectively, of the P-type T4SS exemplified by the IncP plasmid RP4. F lacks homologs of TrbBP (NTPase) and TrbFP but contains a cluster of genes encoding proteins essential for F conjugation (TraFF, -HF, -UF, -WF, the C-terminal region of TraGF, and TrbCF) that are hallmarks of F-like T4SS. These extra genes have been implicated in phenotypes that are characteristic of F-like systems including pilus retraction and mating pair stabilization. F-like T4SS systems have been found on many conjugative plasmids and in genetic islands on bacterial chromosomes. Although few systems have been studied in detail, F-like T4SS appear to be involved in the transfer of DNA only whereas P- and I-type systems appear to transport protein or nucleoprotein complexes. This review examines the similarities and differences among the T4SS, especially F- and P-like systems, and summarizes the properties of the F transfer region gene products.     

TraT lipoprotein, a plasmid-specified mediator of interactions between gram-negative bacteria and their environment.

The TraT protein is a cell-surface-exposed, outer membrane lipoprotein specified by large, usually conjugative, F-like plasmids. Two biological activities have been associated with the protein: (i) prevention of self-mating of cells carrying identical or closely related conjugative plasmids, by blocking the formation of stable mating aggregates; and (ii) resistance to the bactericidal activities of serum, possibly by inhibiting the correct assembly or efficient functioning of the terminal membrane attack complex of complement. The protein therefore interacts not only with components of the outer membrane but also with specific external agents. In conjugative plasmids the traT gene lies within the region necessary for the conjugal transfer of DNA (tra), although its expression is not necessarily dependent on the expression of other tra genes. Recently, however, the gene has been discovered in isolation from other tra genes in nonconjugative virulence-associated plasmids, providing further evidence that the TraT protein may have a role in pathogenesis. The nucleotide sequences of several traT genes have been determined, and comparison of the corresponding amino acid sequences suggests that a central region of five amino acid residues flanked by hydrophobic domains determines the specificity of the protein in surface exclusion. Additionally, studies of mutants with different amino acid alterations within the hydrophobic domains have shown that insertion of charged residues disrupts normal outer membrane integrity. This review considers our current knowledge of the distribution, structure, and biological role(s) of the protein. Recent applications of the protein in studies of the unusual permeability properties of the outer membrane and for the transport of foreign antigenic determinants to the bacterial cell surface are also discussed.     

The complete nucleotide sequence of the resistance plasmid R478: defining the backbone components of incompatibility group H conjugative plasmids through comparative genomics

Horizontal transfer of resistance determinants amongst bacteria can be achieved by conjugative plasmid DNA elements. We have determined the complete 274,762 bp sequence of the incompatibility group H (IncH) plasmid R478, originally isolated from the Gram negative opportunistic pathogen Serratia marcescens. This self-transferable extrachromosomal genetic element contains 295 predicted genes, of which 144 are highly similar to coding sequences of IncH plasmids R27 and pHCM1. The regions of similarity among these three IncH plasmids principally encode core plasmid determinants (i.e., replication, partitioning and stability, and conjugative transfer) and we conducted a comparative analysis to define the minimal IncHI plasmid backbone determinants. No resistance determinants are included in the backbone and most of the sequences unique to R478 were contained in a large contiguous region between the two transfer regions. These findings indicate that plasmid evolution occurs through gene acquisition/loss predominantly in regions outside of the core determinants. Furthermore, a modular evolution for R478 was signified by the presence of gene neighbors or operons that were highly related to sequences from a wide range of chromosomal, transposon, and plasmid elements. The conjugative transfer regions are most similar to sequences encoded on SXT, Rts1, pCAR1, R391, and pRS241d. The dual partitioning modules encoded on R478 resemble numerous sequences; including pMT1, pCTX-M3, pCP301, P1, P7, and pB171. R478 also codes for resistance to tetracycline (Tn10), chloramphenicol (cat), kanamycin (aphA), mercury (similar to Tn21), silver (similar to pMG101), copper (similar to pRJ1004), arsenic (similar to pYV), and tellurite (two separate regions similar to IncHI2 ter determinants and IncP kla determinants). Other R478-encoded sequences are related to Tn7, IS26, tus, mucAB, and hok, where the latter is surrounded by insLKJ, and could potentially be involved in post-segregation killing. The similarity to a diverse set of bacterial sequences highlights the ability of horizontally transferable DNA elements to acquire and disseminate genetic traits through the bacterial gene pool.     

Genetic analysis of bacterial plasmid promiscuity

The genetic basis of the promiscuous behaviour of bacterial plasmids has been investigated by study of the incompatibility P-1 group of conjugative plasmids of gram-negative bacteria. Both transposon mutagenesis and the construction of minireplicons linking varying combinations of the plasmid genome have shown that specific genomic regions control the conjugational transfer and vegetative replication of the plasmid in specific bacterial hosts. These include the plasmid DNA primase gene, the origin of plasmid transfer, a region near the origin of transfer, the origin of plasmid vegetative replication, thetrans- acting gene essential for the initiation of plasmid replication and a region involved in its regulation. DNA sequence analysis has identified the requirement of specific direct repeats within the origin of replication for plasmid replication in some but not in other hosts. The cloning of some of the trans-acting genes onto multicopy cloning vectors and complementation tests have shown that the requirements of these gene products vary in different hosts and that the plasmid has evolved genetic strategies for their optimal expression.     

When phage,plasmids, and transposons collide: genomic islands,and conjugative- and mobilizable-transposons as a mosaic continuum

Plasmids and bacteriophage represent the classical vectors for gene transfer within the horizontal gene pool. However, the more recent discovery of an increasing array of other mobile genetic elements (MGE) including genomic islands (GIs), conjugative transposons (CTns), and mobilizable transposons (MTns) which each integrate within the chromosome, offer an increasingly diverse assemblage contributing to bacterial adaptation and evolution. Molecular characterisation of these elements has revealed that they are comprised of functional modules derived from phage, plasmids, and transposons, and further that these modules are combined to generate a continuum of mosaic MGE. In particular, they are comprised of any one of three distinct types of recombinase, together with plasmid-derived transfer and mobilisation gene functions. This review highlights both the similarities and distinctions between these integrating transferable elements resulting from combination of the MGE toolbox.     

Plasmid-mediated horizontal gene transfer is a coevolutionary process

Conjugative plasmids are key agents of horizontal gene transfer (HGT) that accelerate bacterial adaptation by vectoring ecologically important traits between strains and species. However, although many conjugative plasmids carry beneficial traits, all plasmids exert physiological costs-of-carriage on bacteria. The existence of conjugative plasmids, therefore, presents a paradox because non-beneficial plasmids should be lost to purifying selection, whereas beneficial genes carried on plasmids should be integrated into the bacterial chromosome. Several ecological solutions to the paradox have been proposed, but none account for co-adaptation of bacteria and conjugative plasmids. Drawing upon evidence from experimental evolution, we argue that HGT via conjugation can only be fully understood in a coevolutionary framework.     

Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430

The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.     

Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport.

The DNA transfer stage of conjugation requires the products of the F sex factor genes traMYDIZ and the cis-acting site oriT. Previous interpretation of genetic and protein analyses suggested that traD, traI, and traZ mapped as contiguous genes at the distal end of the transfer operon and saturated this portion of the F transfer region (which ends with an IS3 element). Using antibodies prepared against the purified TraD and TraI proteins, we analyzed the products encoded by a collection of chimeric plasmids constructed with various segments of traDIZ DNA. We found the traI gene to be located 1 kilobase to the right of the position suggested on previous maps. This creates an unsaturated space between traD and traI where unidentified tra genes may be located and leaves insufficient space between traI and IS3 for coding the 94-kilodalton protein previously thought to be the product of traZ. We found that the 94-kilodalton protein arose from a translational restart and corresponds to the carboxy terminus of traI; we named it TraI*. The precise physical location of the traZ gene and the identity of its product are unknown. The oriT nicking activity known as TraZ may stem from unassigned regions between traD and traI and between traI and IS3, but a more interesting possibility is that it is actually a function of traI. On our revised map, the position of a previously detected RNA polymerase-binding site corresponds to a site at the amino terminus of traI rather than a location 1 kilobase into the coding region of the gene. Furthermore, the physical and genetic comparison of the F traD and traI genes with those of the closely related F-like conjugative plasmids R1 and R100 is greatly simplified. The translational organization we found for traI, together with its identity as the structural gene for DNA helicase I, suggests a possible functional link to several other genes from which translational restart polypeptides are expressed. These include the primases of the conjugative plasmids ColI and R16, the primase-helicase of bacteriophage T7, and the cisA product (nickase) of phage phi X174.     

Genetic organization of plasmid pXF51 from the plant pathogen Xylella fastidiosa.

The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed. This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer. Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria. A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin. One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event. A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids. An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins. None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog. These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium. This property may be of great importance for future development of transformation techniques in X. fastidiosa.     

Peptide pheromone-induced transfer of plasmid pCF10 in Enterococcus faecalis: probing the genetic and molecular basis for specificity of the pheromone response

The tetracycline resistance plasmid pCF10 represents a class of unique mobile genetic elements of the bacterial genus Enterococcus, whose conjugative transfer functions are inducible by peptide sex pheromones excreted by potential recipient cells. These plasmids play a significant role in the dissemination of virulence and antibiotic resistance genes among the enterococci, which have become major nosocomial pathogens. Pheromone response by plasmid-carrying donor cells involves specific import of the peptide signal molecule, and subsequent interaction of the signal with one or more intracellular regulatory gene products. The pheromones are chromosomally encoded hydrophobic octa- or hepta-peptides, and different families of homologous plasmids encode the ability to respond to each pheromone. Among the four pheromone-responsive plasmids that have been characterized in some detail, there is considerable conservation in the genes encoding pheromone sensing and regulatory functions, and the peptides themselves show considerable similarity. In spite of this, there is extremely high specificity of response to each peptide, with virtually no "cross-induction" of transfer of non-cognate pheromone plasmids by the pheromones. This communication reviews the evidence for this specificity and discusses current molecular and genetic approaches to defining the basis for specificity.     

Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae.

Cytophaga johnsonae displays many features that make it an excellent model of bacterial gliding motility. Unfortunately, genetic analyses of C. johnsonae, or any related gliding bacteria, were not possible because of a complete lack of selectable markers, cloning vectors, transposons, and convenient methods of gene transfer. As a first step toward a molecular analysis of gliding motility of C. johnsonae, we developed these genetic techniques and tools. Common broad-host-range plasmids and transposons did not function in C. johnsonae. We identified one Bacteroides transposon, Tn4351, that could be introduced into C. johnsonae on plasmid R751 by conjugation from Escherichia coli. Tn4351 inserted in the C. johnsonae genome and conferred erythromycin resistance. Tn-4351 insertions resulted in auxotrophic mutations and motility mutations. We constructed novel plasmids and cosmids for genetic analyses of C. johnsonae. These cloning vectors are derived from a small cryptic plasmid (pCP1) that we identified in the fish pathogen Cytophaga psychrophila D12. These plasmids contain the ermF (erythromycin resistance) gene from Tn4351 and a variety of features that facilitate propagation and selection in E. coli and conjugative transfer from E. coli to C. johnsonae.     

Plasmids spread very fast in heterogeneous bacterial communities

Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes.