Blending genomes: distributive conjugal transfer in mycobacteria,a sexier form of HGT

This review discusses a novel form of horizontal gene transfer (HGT) found in mycobacteria called Distributive Conjugal Transfer (DCT). While satisfying the criteria for conjugation, DCT occurs by a mechanism so distinct from oriT‐mediated conjugation that it could be considered a fourth category of HGT. DCT involves the transfer of chromosomal DNA between mycobacteria and, most significantly, generates transconjugants with mosaic genomes of the parental strains. Multiple segments of donor chromosomal DNA can be co‐transferred regardless of their location or the genetic selection and, as a result, the transconjugant genome contains many donor‐derived segments; hence the name DCT. This distinguishing feature of DCT separates it from the other known mechanisms of HGT, which generally result in the introduction of a single, defined segment of DNA into the recipient chromosome (Fig. 1 ). Moreover, these mosaic progeny are generated from a single conjugal event, which provides enormous capacity for rapid adaptation and evolution, again distinguishing it from the three classical modes of HGT. Unsurprisingly, the unusual mosaic products of DCT are generated by a conjugal mechanism that is also unusual. Here, we will describe the unique features of DCT and contrast those to other mechanisms of HGT, both from a mechanistic and an evolutionary perspective. Our focus will be on transfer of chromosomal DNA, as opposed to plasmid mobilization, because DCT mediates transfer of chromosomal DNA and is a chromosomally encoded process.     

The origins and early evolution of DNA mismatch repair genes--multiple horizontal gene transfers and co-evolution

To understand the evolutionary process of the DNA mismatch repair system, we conducted systematic phylogenetic analysis of its key components, the bacterial MutS and MutL genes and their eukaryotic homologs. Based on genome-wide homolog searches, we identified three new MutS subfamilies (MutS3-5) in addition to the previously studied MutS1 and MutS2 subfamilies. Detailed evolutionary analysis strongly suggests that frequent ancient horizontal gene transfer (HGT) occurred with both MutS and MutL genes from bacteria to eukaryotes and/or archaea. Our results further imply that the origins of mismatch repair system in eukaryotes and archaea are largely attributed to ancient HGT from bacteria instead of vertical evolution. Specifically, the eukaryotic MutS and MutL homologs likely originated from endosymbiotic ancestors of mitochondria or chloroplasts, indicating that not only archaea, but also bacteria are important sources of eukaryotic DNA metabolic genes. The archaeal MutS1 and MutL homologs were also acquired from bacteria simultaneously through HGT. Moreover, the distribution and evolution profiles of the MutS1 and MutL genes suggest that they have undergone long-term coevolution. Our work presents an overall portrait of the evolution of these important genes in DNA metabolism and also provides further understanding about the early evolution of cellular organisms.     

Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12

In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F’-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10^-12 CFU/recipient per hour.     

Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair–long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic “melting pot” where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.     

Codon Usage Methods for Horizontal Gene Transfer Detection Generate an Abundance of False Positive and False Negative Results

Bacteria acquire new DNA in a process known as horizontal gene transfer (HGT). To investigate the evolutionary impact of this transfer of DNA, various methods have been developed to detect past HGT events. For example, codon usage-based methods detect the presence of transferred genes by identifying atypical patterns of codon usage. However, some inherited genes exhibit atypical codon usage and some transferred genes have codon usage patterns similar to those of the inherited genes. In this study, we used a comparative phylogenetic approach with Methylobacterium and Caulobacter species to demonstrate that even well-designed codon usage methods fail to detect many HGT events and generate a high rate of false positives (60–75 %) and false negatives (23–61 %). Therefore, we recommend caution when employing codon usage methods to identify transferred genes and suggest that the rapidly increasing availability of bacterial genome sequences makes the phylogenetic approach the method of choice.     

The requirements for conjugal DNA synthesis in the donor strain during flac transfer.

Although neither rifampicin nor spectinomycin had any effect on the frequency of Flac transfer by a sensitive donor, rifampicin but not spectinomycin prevented donor conjugal DNA synthesis as measured in matings between a dnaB donor and a tdk recipient. An untranslated RNA species is therefore probably required for this synthesis, although transfer took place even in its absence. Donor conjugal DNA synthesis was abolished in a dnaE donor, showing that DNA polymerase III is responsible for this process; again, plasmid DNA transfer was not affected.Flac mutants lacking the F pilus gave neither donor conjugal DNA synthesis nor plasmid DNA transfer, probably because they could not receive a “mating signal” to activate the transfer process. The products of traI and traM were also required both for donor conjugal DNA synthesis and for physical transfer of plasmid DNA, probably being involved in the conversion of covalently closed circular plasmid DNA into the open circular form that is the substrate for the independent although normally simultaneous synthesis and transfer steps. In contrast, donor conjugal DNA synthesis took place at a normal rate in both piliated traG and traN mutants, and at a reduced rate in traD mutants, although in no case was there physical transfer of plasmid DNA. These gene products are therefore required for DNA transfer to the recipient, and in addition, the absence of the traD product may hinder DNA synthesis.Based upon these results, a scheme for the processing of DNA during conjugation is presented.     

Differential Regulation of Horizontally Acquired and Core Genome Genes by the Bacterial Modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.     

Horizontal gene transfers link a human MRSA pathogen to contagious bovine mastitis bacteria

Background

Acquisition of virulence factors and antibiotic resistance by many clinically important bacteria can be traced to horizontal gene transfer (HGT) between related or evolutionarily distant microflora. Comparative genomic analysis has become an important tool for identifying HGT DNA in emerging pathogens. We have adapted the multi-genome alignment tool EvoPrinter to facilitate discovery of HGT DNA sequences within bacterial genomes and within their mobile genetic elements.

Principal Findings

EvoPrinter analysis of 13 different Staphylococcus aureus genomes revealed that one of the human isolates, the hospital epidemic methicillin-resistant MRSA252 strain, uniquely shares multiple putative HGT DNA sequences with different causative agents of bovine mastitis that are not found in the other human S. aureus isolates. MRSA252 shares over 14 different DNA sequence blocks with the bovine mastitis ET3 S. aureus strain RF122, and many of the HGT DNAs encode virulence factors. EvoPrinter analysis of the MRSA252 chromosome also uncovered virulence-factor encoding HGT events with the genome of Listeria monocytogenes and a Staphylococcus saprophyticus associated plasmid. Both bacteria are also causal agents of contagious bovine mastitis.

Conclusions

EvoPrinter analysis reveals that the human MRSA252 strain uniquely shares multiple DNA sequence blocks with different causative agents of bovine mastitis, suggesting that HGT events may be occurring between these pathogens. These findings have important implications with regard to animal husbandry practices that inadvertently enhance the contact of human and livestock bacterial pathogens.     

Bacterial Hha-like proteins facilitate incorporation of horizontally transferred DNA

Horizontal gene transfer (HGT), non-hereditary transfer of genetic material between organisms, accounts for a significant proportion of the genetic variability in bacteria. In Gram negative bacteria, the nucleoid-associated protein H-NS silences unwanted expression of recently acquired foreign DNA. This, in turn, facilitates integration of the incoming genes into the regulatory networks of the recipient cell. Bacteria belonging to the family Enterobacteriaceae express an additional protein, the Hha protein that, by binding to H-NS, potentiates silencing of HGT DNA. We provide here an overview of Hha-like proteins, including their structure and function, as well as their evolutionary relationship. We finally present available information suggesting that, by expressing Hha-like proteins, bacteria such as Escherichia coli facilitate HGT incorporation and hence, the impact of HGT in their genetic diversity.     

Transkingdom Genetic Transfer from Escherichia coli to Saccharomyces cerevisiae as a Simple Gene Introduction Tool

Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP and IncQ groups. We also investigated forms of transfer of these vectors from Escherichia coli into Saccharomyces cerevisiae to develop TKC as a simple gene introduction method. Both types of vectors were transferred precisely, conserving the origin of transfer (oriT) sequences, but IncP-based vectors appeared to be more efficient than an IncQ-based vector. Interestingly, unlike in agrobacterial T-DNA (transfer DNA) transfer, the efficiency of TKC transfer was similar between a wild-type yeast strain and DNA repair mutants defective in homologous recombination (rad51Δ and rad52Δ) or nonhomologous end joining (rad50Δ, yku70Δ, and lig4Δ). Lastly, a shuttle vector with two repeats of IncP-type oriT (oriT^P) sequences flanking a marker gene was constructed. TKC transfer of this vector resulted in precise excision of both the oriT^P loci as well as the marker gene, albeit at a low frequency of 17% of all transconjugants. This feature would be attractive in biotechnological applications of TKC. Taken together, these results strongly suggest that in contrast to agrobacterial T-DNA transfer, the circularization of vector single-stranded DNA occurs either before or after transfer but requires a factor(s) from the donor. TKC is a simple method of gene transfer with possible applications in yeast genetics and biotechnology.     

细菌中基因组岛的转移与调控机制研究进展

基因水平转移可导致细菌不同种属间个体DNA的交换,从而使细菌对环境的适应性增强,是细菌进化的重要途径之一。基因组岛是基因水平转移的重要载体,可移动的基因组岛能够整合到宿主的染色体上,并在特定的条件下切除,进而通过转化、接合或转导等方式转移到新的宿主中。基因组岛具有多种生物学功能,如抗生素抗性、致病性、异源物质降解、重金属抗性等。基因组岛的转移造成可变基因在不同种属细菌间的广泛传播,例如毒力和耐药基因的传播导致了多重耐药细菌的产生,威胁人类健康。基因组岛由整合酶介导转移,同时在转移的过程受到多种不同转录因子的调控。本文对细菌中基因组岛的结构特点、转移和调控机制以及预测等方面进行了综述,并最终阐明基因组岛的转移及其调控机制是遏制基因组岛传播的重要策略。     

Evidence of horizontal gene transfer between land plant plastids has surprising conservation implications

Background and AimsHorizontal gene transfer (HGT) is an important evolutionary mechanism because it transfers genetic material that may code for traits or functions between species or genomes. It is frequent in mitochondrial and nuclear genomes but has not been demonstrated between plastid genomes of different green land plant species.MethodsWe Sanger-sequenced the nuclear internal transcribed spacers (ITS1 and 2) and the plastid rpl16 G2 intron (rpl16). In five individuals with foreign rpl16 we also sequenced atpB-rbcL and trnL_UAA-trnF_GAA.Key ResultsWe discovered 14 individuals of a moss species with typical nuclear ITSs but foreign plastid rpl16 from a species of a distant lineage. None of the individuals with three plastid markers sequenced contained all foreign markers, demonstrating the transfer of plastid fragments rather than the entire plastid genome, i.e. entire plastids were not transferred. The two lineages diverged 165–185 Myr BP. The extended time interval since lineage divergence suggests that the foreign rpl16 is more likely explained by HGT than by hybridization or incomplete lineage sorting.ConclusionsWe provide the first conclusive evidence of interspecific plastid-to-plastid HGT among land plants. Two aspects are critical: it occurred at several localities during the massive colonization of recently disturbed open habitats that were created by large-scale liming as a freshwater biodiversity conservation measure; and it involved mosses whose unique life cycle includes spores that first develop a filamentous protonema phase. We hypothesize that gene transfer is facilitated when protonema filaments of different species intermix intimately when colonizing disturbed early succession habitats.     

First description of natural and experimental conjugation between Mycobacteria mediated by a linear plasmid

Background

In a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. The transfer mechanism of this insertion sequence to M. kansasii was investigated here.

Methodology/Principal Findings

A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. The linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau.

Conclusions/Significance

Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.     

The Pseudomonas aeruginosa Pathogenicity Island PAPI-1 Is Transferred via a Novel Type IV Pilus

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. The notable ability of P. aeruginosa to inhabit a broad range of environments, including humans, is in part due to its large and diverse genomic repertoire. The genomes of most strains contain a significant number of large and small genomic islands, including those carrying virulence determinants (pathogenicity islands). The pathogenicity island PAPI-1 of strain PA14 is a cluster of 115 genes, and some have been shown to be responsible for virulence phenotypes in a number of infection models. We have previously demonstrated that PAPI-1 can be transferred to other P. aeruginosa strains following excision from the chromosome of the donor. Here we show that PAPI-1 is transferred into recipient P. aeruginosa by a conjugative mechanism, via a type IV pilus, encoded in PAPI-1 by a 10-gene cluster which is closely related to the genes in the enterobacterial plasmid R64. We also demonstrate that the precursor of the major pilus subunit, PilS2, is processed by the chromosomally encoded prepillin peptidase PilD but not its paralog FppA. Our results suggest that the pathogenicity island PAPI-1 may have evolved by acquisition of a conjugation system but that because of its dependence on an essential chromosomal determinant, its transfer is restricted to P. aeruginosa or other species capable of providing a functional prepilin peptidase.The genomes of a number of microorganisms, primarily those that have a capability of changing and adapting to a wide range of environments, evolve by acquisition of novel genetic information in blocks of genes via a process referred to as horizontal gene transfer (HGT). Other bacterial species change their genetic repertoire minimally, principally those that have adapted to a particular environment and, in the case of pathogenic bacteria, to a specific host. For HGT-mediated acquisition of genes to occur, a recipient has to be in an environment where donor genetic material is available, such as different strains of the same species cohabitating a shared niche or growing in a large and diverse community of several hundred different microorganisms. Moreover, for bacteria to become successful recipients of foreign genetic material, they have to posses one of three mechanisms of HGT: natural competence for uptake of foreign DNA (transformation), the ability to be infected by transducing bacteriophages (transduction), or serving as recipients during conjugation of plasmids or mobilized chromosomal DNA (conjugation). Acquired genetic material can consist of individual genes, where they recombine into homologous sequences in the recipient genome and thus increase the genetic diversity. However, large blocks of hundreds of contiguous genes in elements called genomic islands can be also transferred between bacteria, allowing the recipient microorganisms to acquire a number of new traits by a single HGT event.Previous studies comparing genomes of the opportunistic pathogen Pseudomonas aeruginosa pointed toward HGT as an important factor in its evolution (23). The genomes of all strains sequenced to date contain a significant fraction of horizontally acquired genes, in genomic islands and prophages, consisting of a few to several hundred. These islands can be recognized by the presence of certain signature features, such as an atypical nucleotide composition relative to the rest of the genome, location within predicted sites of chromosomal integration (att sites), and the presence of genes encoding bacteriophages and conjugation machineries. We have recently demonstrated that PAPI-1, a large P. aeruginosa genomic (pathogenicity) island, can be excised from its tRNA att site and that a copy can be transferred into a recipient, where it integrates into the same tRNA gene (27). Inspection of the genes in PAPI-1 and features of the transfer process, namely, an integrase-dependent excision and formation of a circular intermediate, suggested that PAPI-1 is an integrative and conjugative element and that it is likely transferred by a conjugative mechanism.Here we extended our analysis of PAPI-1 by testing its transfer from a preselected group of P. aeruginosa PA14 mutants with insertions in each of the genes on the island. Among those mutants that were defective in PAPI-1 transfer, one group of genes encode homologs of type IV pilus proteins. While type IV pili have been found to be involved primarily in bacterial adhesion and twitching motility (24), the PAPI-1-encoded pilus is closely related to the conjugative apparatus of plasmid R64 (14). Moreover, we show that an essential posttranslational modification reaction, converting the precursor of the major pilin subunit encoded in PAPI-1 into a mature protein, is carried out by an enzyme encoded in the chromosome of the donor cells. The acquisition and adaptation of groups of genes and subsequent loss of an essential function may represent a novel evolutionary strategy, limiting horizontal transfer to a specific bacterial species.     

Generation of Genic Diversity among Streptococcus pneumoniae Strains via Horizontal Gene Transfer during a Chronic Polyclonal Pediatric Infection

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.     

Contribution of horizontally acquired genomic islands to the evolution of the tubercle bacilli

The contribution of horizontal gene transfer (HGT) to the evolution of Mycobacterium tuberculosis -- the main causal agent of tuberculosis in humans -- and closely related members of the M. tuberculosis complex remains poorly understood. Using a combination of genome-wide parametric analyses, we have identified 48 M. tuberculosis chromosomal regions with atypical characteristics, potentially due to HGT. These specific regions account for 4.5% of the genome (199 kb) and include 256 genes. Many display features typical of the genomic islands found in other bacteria, including residual material from mobile genetic elements, flanking direct repeats, insertion in the vicinity of tRNA sequences, and genes with putative or documented virulence functions. Southern blotting analysis of nine of these 48 regions confirmed their presence in "Mycobacterium prototuberculosis," the ancestral species of the M. tuberculosis complex. Finally, our results strongly suggest that the ancestor of the tubercle bacilli was an environmental bacillus that exchanged genetic material with other bacterial species, including Proteobacteria in particular, present in its surroundings. This study describes a rational approach to searching for mycobacterial virulence genes, and highlights the importance of dissecting gene transfer networks to improve our understanding of mycobacterial pathogenicity and evolution.     

Speciation in <Emphasis Type="Italic">Chlamydia</Emphasis>: Genomewide Phylogenetic Analyses Identified a Reliable Set of Acquired Genes

Horizontal gene transfer (HGT), a process through which genomes acquire sequences from distantly related organisms, is believed to be a major source of genetic diversity in bacteria. A central question concerning the impact of HGT on bacterial genome evolution is the proportion of horizontally transferred sequences within genomes. This issue, however, remains unresolved because the various methods developed to detect potential HGT events identify different sets of genes. The present-day consensus is that phylogenetic analysis of individual genes is still the most objective and accurate approach for determining the occurrence and directionality of HGT. Here we present a genome-scale phylogenetic analysis of protein-encoding genes from five closely related Chlamydia, identifying a reliable set of sequences that have arisen via HGT since the divergence of the Chlamydia lineage. According to our knowledge, this is the first systematic phylogenetic inference-based attempt to establish a reliable set of acquired genes in a bacterial genome. Although Chlamydia are obligate intracellular parasites of higher eukaryotes, and thus suspected to be isolated from HGT more than the free-living species, our results show that their diversification has involved the introduction of foreign sequences into their genome. Furthermore, we also identified a complete set of genes that have undergone deletion, duplication, or rearrangement during this evolutionary period leading to the radiation of Chlamydia species. Our analysis may provide a deeper insight into how these medically important pathogens emerged and evolved from a common ancestor.     

Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase

Background

Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.

Methodology/Principal Findings

We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.

Conclusions/Significance

The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.     

Phylogenomic Analysis Demonstrates a Pattern of Rare and Ancient Horizontal Gene Transfer between Plants and Fungi

Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries and is an important evolutionary phenomenon in the ancestry of many microbes. The role of HGT in plant evolutionary history is, however, largely unexplored. Here, we compare the genomes of six plant species with those of 159 prokaryotic and eukaryotic species and identify 1689 genes that show the highest similarity to corresponding genes from fungi. We constructed a phylogeny for all 1689 genes identified and all homolog groups available from the rice (Oryza sativa) genome (3177 gene families) and used these to define 14 candidate plant-fungi HGT events. Comprehensive phylogenetic analyses of these 14 data sets, using methods that account for site rate heterogeneity, demonstrated support for nine HGT events, demonstrating an infrequent pattern of HGT between plants and fungi. Five HGTs were fungi-to-plant transfers and four were plant-to-fungi HGTs. None of the fungal-to-plant HGTs involved angiosperm recipients. These results alter the current view of organismal barriers to HGT, suggesting that phagotrophy, the consumption of a whole cell by another, is not necessarily a prerequisite for HGT between eukaryotes. Putative functional annotation of the HGT candidate genes suggests that two fungi-to-plant transfers have added phenotypes important for life in a soil environment. Our study suggests that genetic exchange between plants and fungi is exceedingly rare, particularly among the angiosperms, but has occurred during their evolutionary history and added important metabolic traits to plant lineages.