Comparative phylogenetics of ICEHin1056 family reveals deep evolutionary associations of mobile genetic elements responsible for transfer of antibiotic resistance genes

BackgroundIntegrating and conjugating elements (ICEs) are self-transmissible mobile genetic elements. ICEs are composed of modules of conserved genes, with accessory genes at hotspots. Antibiotic resistance genes are often encoded on ICEs, leading to rapid intraspecific and interspecific spread of resistance. Our aim was to study ICEs with homology to ICEHin1056 in Haemophilus influenzae using the large number of whole genome sequences now available.MethodsMembers of the ICEHin1056 family were identified with tBLASTx searches on the National Center for Biotechnology Information genome database. The query sequences were concatenated core genes from ICEHin1056. Alignments were performed with the Artemis Comparison Tool. Sequences were stored in a BIGS (Bacterial Isolate Genome Sequence) database and homologues of core genes identified. Alignments were performed in ClustalW and phylogenetic trees drawn with MEGA (Molecular Evolutionary Genetics Analysis). Ancestral sequences were predicted with GASP (Gapped Ancestral Sequence Prediction). Predicted ancestral sequences were used as BLAST inputs to find further possible members of the ICE family and more distant relatives.FindingsWe identified over 100 whole or partial sequences in the ICEHin1056 family in a-proteobacteria, b-proteobacteria, and g-proteobacteria. This is the largest comparative phylogenetic study of ICEs performed to date and demonstrates extensive lateral gene transfer across the whole phylum. The three core ICE modules encode replication, type IV secretion, and excision/integration. The conservation of synteny implies a powerful selective advantage of the ICE. GC content of the core modules mirrors that of the host chromosome, suggesting coexistence deep in evolutionary history. Absence of core genes or modules represents lifestyle adaptations of the mobile genetic element. Absence of an integrase and presence of a replicative DNA helicase are markers of a plasmid lifestyle. A variety of accessory genes are found at hotspots; they confer a survival advantage in the ecological niche of the organism, which ranges from eukaryotic pathogens to extreme environments.InterpretationThis large comparative phylogenetic study of ICEs allows inference about evolutionary associations within the ICEHin1056 family. This evolutionary history is so ancient that it may link all mobile genetic elements transferred by conjugation in proteobacteria. This provides important insights into the mobile gene pool and may have implications for prediction of spread of antibiotic resistance and pathogenicity.FundingUK National Health Service.     

A globally distributed mobile genetic element inhibits natural transformation of Vibrio cholerae

Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA⁺ ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence decreased to ∼40% from 2006 to 2011. Thus, IdeA⁺ ICEs may have limited the role of natural transformation in V. cholerae. However, the rise in prevalence of strains lacking IdeA may now increase the role of this conserved mechanism of HGT in the evolution of this pathogen.The causative agent of the diarrheal disease cholera, Vibrio cholerae, is annually responsible for 3.5 million infections worldwide (1). This facultative pathogen naturally resides in temperate aquatic environments and causes disease when ingested in contaminated food or water. A critical nutrient for Vibrio species in the aquatic environment is the chitinous exoskeleton of crustacean zooplankton (2–4). Chitin is an insoluble polysaccharide composed of β-1,4-linked GlcNAc. In addition to serving as a carbon and nitrogen source, chitin also induces a physiological state in V. cholerae known as natural competence (5). In this state, bacteria can take up DNA from the extracellular environment and integrate this DNA into their chromosomes by homologous recombination. This cumulative process of DNA uptake and integration is known as natural transformation and is one mechanism for horizontal gene transfer (HGT) in V. cholerae. HGT by natural transformation is used by pathogenic microbes to evolve in the face of clinical intervention and immune pressure. Indeed, in V. cholerae, this mechanism of HGT is hypothesized to have generated an antigenic variant, the O139 outbreak strain, through homologous recombination and replacement of the locus responsible for O-antigen biosynthesis (6–9).Another mechanism of HGT in V. cholerae is integrating conjugative elements (ICEs) of the SXT/R391 family. These elements can range from ∼80 to 110 Kb in size and contain all of the genes required for conjugative transfer into naive hosts (10, 11); they integrate in a site-specific manner into the 5′ end of the highly conserved prfC (peptide-chain-release factor C) gene (10–12). The first natural transfer of an ICE into V. cholerae likely occurred between 1980 and 1985 (10, 13) and, by the 1990s, virtually all clinical isolates of V. cholerae contained an ICE (13). These elements confer resistance to multiple antibiotics, and it is likely that widespread use of antibiotics has rapidly selected for strains containing ICEs. There are at least 10 genetically distinct ICEs circulating in the V. cholerae population (11). These ICEs share a core set of genes, but have varied gene content at distinct sites. The most common ICE in V. cholerae is VchInd5, which is present in ∼77% of currently sequenced clinical isolates (10, 11). It is hypothesized that the current (seventh) pandemic of cholera originated in the Bay of Bengal, and strains have spread globally from this region in three overlapping waves of transmission (13, 14). Strains containing VchInd5 are globally distributed, indicating that the original transfer of VchInd5 into V. cholerae may have occurred in this region.In 2010, cholera spread to Haiti, a region that previously lacked this disease (15, 16). Phylogenetic and Bayesian analyses indicate that all strains in Haiti share a common ancestor, which was introduced into the region at the outset of the epidemic (16, 17). Consistent with this finding, strains from Haiti ubiquitously harbor a VchInd5 ICE. Throughout the epidemic, strains have acquired mutations that are likely generated intrinsically, and there is no evidence of horizontal gene transfer among these isolates (16). Consistent with this finding, strains from the Haiti outbreak were found to be poorly transformed by chitin-induced natural competence (16).In this study, we identify and characterize an ICE-encoded DNase present on VchInd5 that inhibits HGT by natural transformation in V. cholerae. We also assess the role and prevalence of this DNase in limiting transformation among clinical isolates from Haiti and Bangladesh.     

Biased gene transfer mimics patterns created through shared ancestry

In phylogenetic reconstruction, two types of bacterial tyrosyl-tRNA synthetases (TyrRS) form distinct clades with many bacterial phyla represented in both clades. Very few taxa possess both forms, and maximum likelihood analysis of the distribution of TyrRS types suggests horizontal gene transfer (HGT), rather than an ancient duplication followed by differential gene loss, as the contributor to the evolutionary history of TyrRS in bacteria. However, for each TyrRS type, phylogenetic reconstruction yields phylogenies similar to the ribosomal phylogeny, revealing that frequent gene transfer has not destroyed the expected phylogeny; rather, the expected phylogenetic signal was reinforced or even created by HGT. We show that biased HGT can mimic patterns created through shared ancestry by in silico simulation. Furthermore, in cases where genomic synteny is sufficient to allow comparisons of relative gene positions, both tyrRS types occupy equivalent positions in closely related genomes, rejecting the loss hypothesis. Although the two types of bacterial TyrRS are only distantly related and only rarely coexist in a single genome, they have many features in common with alleles that are swapped between related lineages. We propose to label these functionally similar homologs as homeoalleles. We conclude that the observed phylogenetic pattern reflects both vertical inheritance and biased HGT and that the signal caused by common organismal descent is difficult to distinguish from the signal due to biased gene transfer.     

Cell-to-cell movement of mitochondria in plants

We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer from Nicotiana sylvestris into Nicotiana tabacum cells. The alloplasmic N. tabacum line we used carries Nicotiana undulata cytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer from N. sylvestris to N. tabacum could be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility to orf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.Horizontal gene transfer (HGT), the acquisition of gene(s) across species mating boundaries, results in a phylogeny of the transferred gene(s) that is incongruent with the phylogeny of the organism. In flowering plants, HGT is relatively rare in the nucleus, but frequently involves mitochondrial DNA (mtDNA); for reviews see refs. 1–3. Pioneering papers described HGT of several mitochondrial genes (4–6) and, in an extreme case, incorporation of six genome equivalents in the 3.9-Mb Amborella trichopoda mtDNA (7). These findings imply that mechanisms exist for DNA delivery between unrelated species. Parasitic plants are frequent participants in HGT, either as donors (8, 9) or recipients (5) of foreign DNA; the DNA exchange between the host and parasite is probably facilitated by the physical connection (for review, see ref. 3). HGT between nonparasites, however, necessitates alternative modes of DNA transfer. Transfer via vectoring agents such as viruses, bacteria, fungi, insects, and pollen; transformational uptake of plant DNA released into the soil; and occasional grafting of unrelated species were proposed (6). The first experimental evidence in support of grafting as a potential mechanism of HGT came from demonstrating exchange of plant DNA in tobacco tissue grafts (10). Movement of entire chloroplast genomes was subsequently demonstrated through tobacco graft junctions, interpreted as evidence for cell-to-cell movement of the organelles (11, 12). However, evidence for cell-to-cell movement of mitochondrial DNA is missing in plants despite the fact that the majority of horizontal gene transfer events involve mitochondrial sequences.We report here an experimental system for the successful identification of a rare mitochondrial HGT event. Replacing the cytoplasm of Nicotiana tabacum with the cytoplasm of Nicotiana undulata makes the flowers of N. tabacum male sterile due to conversion of anthers to stigmatoid petals (Fig. 1 D–G). Such N. tabacum plants are called alloplasmic substitution lines for carrying an alien cytoplasm and are cytoplasmic male sterile (CMS) because they inherit male sterility only from the maternal parent (13). We reasoned that movement of Nicotiana sylvestris mitochondria into CMS cells should restore anther morphology and pollen production, a change that is easy to detect in plants even if restricted to a few flowers.Open in a separate windowFig. 1.Restoration of fertile flower anatomy facilitates identification of mitochondrial graft transmission event. (A) N. tabacum Nt-CMS and fertile N. sylvestris Ns-F graft partners and GT19-C seed progeny. (B) Grafting tobacco in culture. The scion is Nt-CMS, which carries the nuclear gentamycin resistance marker; and the rootstock is Ns-F, which carries the plastid spectinomycin resistance (aadA) and aurea bar^au genes. Arrow points to graft junction. (C) Selection of gentamycin and spectinomycin double-resistant clones. (Right) Stem slices from the graft region; (Left) from above and below. Arrow points to double-resistant clone. (D) One isolated anther from a wild-type N. tabacum flower (above) and the anther after homeotic conversion of the N. tabacum alloplasmic substitution line (below). (E) Flower morphology of the graft partners and mixed flower anatomy on the GT19-C graft transmission plant. (Right) Flowers are shown with corolla; (Left) with corolla removed. Note homeotic transformation of anthers into stigmatoid petals in Nt-CMS graft partner and the GT-CMS flowers. GT-F and N. sylvestris Ns-F flowers are fertile. The flowers of Nt-CMS graft partner and GT19-C plant (GT-CMS and GT-F) are pink, a nuclear trait; those of the N. sylvestris graft partner are white. A close-up of (F) GT-CMS, (G) GT-intermediate, and (H) GT-F flowers. (Scale bars in Lower Right corners, 10 mm.)We looked for cell-to-cell movement of mitochondria in stem grafts of two species, N. tabacum and N. sylvestris. We first selected for the nuclear marker from N. tabacum and the chloroplast marker in N. sylvestris and regenerated plants from double-resistant tissue derived from the graft junction. We identified branches with fertile flowers on one of the regenerated plants, indicating presence of fertile mtDNA in the otherwise CMS plant, and analyzed the mtDNA of its fertile and CMS seed progeny. Recombination at alternative sites in the mitochondrial genome facilitated the identification of a candidate mitochondrial gene responsible for homeotic transformation of anthers resulting in CMS.     

Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes

Horizontal gene transfer (HGT) can radically alter the genomes of microorganisms, providing the capacity to adapt to new lifestyles, environments, and hosts. However, the extent of HGT between eukaryotes is unclear. Using whole-genome, gene-by-gene phylogenetic analysis we demonstrate an extensive pattern of cross-kingdom HGT between fungi and oomycetes. Comparative genomics, including the de novo genome sequence of Hyphochytrium catenoides, a free-living sister of the oomycetes, shows that these transfers largely converge within the radiation of oomycetes that colonize plant tissues. The repertoire of HGTs includes a large number of putatively secreted proteins; for example, 7.6% of the secreted proteome of the sudden oak death parasite Phytophthora ramorum has been acquired from fungi by HGT. Transfers include gene products with the capacity to break down plant cell walls and acquire sugars, nucleic acids, nitrogen, and phosphate sources from the environment. Predicted HGTs also include proteins implicated in resisting plant defense mechanisms and effector proteins for attacking plant cells. These data are consistent with the hypothesis that some oomycetes became successful plant parasites by multiple acquisitions of genes from fungi.     

Global extent of horizontal gene transfer

Horizontal gene transfer (HGT) is thought to play an important role in the evolution of species and innovation of genomes. There have been many convincing evidences for HGT for specific genes or gene families, but there has been no estimate of the global extent of HGT. Here, we present a method of identifying HGT events within a given protein family and estimate the global extent of HGT in all curated protein domain families ( approximately 8,000) listed in the Pfam database. The results suggest four conclusions: (i) for all protein domain families in Pfam, the fixation of genes horizontally transferred is not a rampant phenomenon between organisms with substantial phylogenetic separations (1.1-9.7% of Pfam families surveyed at three taxonomic ranges studied show indication of HGT); (ii) however, at the level of domains, >50% of Archaea have one or more protein domains acquired by HGT, and nearly 30-50% of Bacteria did the same when examined at three taxonomic ranges. But, the equivalent value for Eukarya is <10%; (iii) HGT will have very little impact in the construction of organism phylogeny, when the construction methods use whole genomes, large numbers of common genes, or SSU rRNAs; and (iv) there appears to be no strong preference of HGT for protein families of particular cellular or molecular functions.     

Soj (ParA) DNA binding is mediated by conserved arginines and is essential for plasmid segregation

Soj is a member of the ParA family of ATPases involved in plasmid and chromosomal segregation. It binds nonspecifically and cooperatively to DNA although the function of this binding is unknown. Here, we show that mutation of conserved arginine residues that map to the surface of Bacillus subtilis Soj caused only minimal effects on nucleotide-dependent dimerization but had dramatic effects on DNA binding. Using a model plasmid partitioning system in Escherichia coli, we find that Soj DNA-binding mutants are deficient in plasmid segregation. The location of the arginines on the Soj structure explains why DNA binding depends on dimerization and was used to orient the Soj dimer on the DNA, revealing the axis of Soj polymerization. The arginine residues are conserved among other chromosomal homologues, including the ParAs from Caulobacter crescentus, Pseudomonas aeruginosa, Pseudomonas putida, Streptomyces coelicolor, and chromosome I of Vibrio cholerae indicating that DNA binding is a common feature of members of this family.     

Regulation of genetic flux between bacteria by restriction–modification systems

Restriction–modification (R-M) systems are often regarded as bacteria''s innate immune systems, protecting cells from infection by mobile genetic elements (MGEs). Their diversification has been recently associated with the emergence of particularly virulent lineages. However, we have previously found more R-M systems in genomes carrying more MGEs. Furthermore, it has been suggested that R-M systems might favor genetic transfer by producing recombinogenic double-stranded DNA ends. To test whether R-M systems favor or disfavor genetic exchanges, we analyzed their frequency with respect to the inferred events of homologous recombination and horizontal gene transfer within 79 bacterial species. Genetic exchanges were more frequent in bacteria with larger genomes and in those encoding more R-M systems. We created a recognition target motif predictor for Type II R-M systems that identifies genomes encoding systems with similar restriction sites. We found more genetic exchanges between these genomes, independently of their evolutionary distance. Our results reconcile previous studies by showing that R-M systems are more abundant in promiscuous species, wherein they establish preferential paths of genetic exchange within and between lineages with cognate R-M systems. Because the repertoire and/or specificity of R-M systems in bacterial lineages vary quickly, the preferential fluxes of genetic transfer within species are expected to constantly change, producing time-dependent networks of gene transfer.Prokaryotes evolve rapidly by acquiring genetic information from other individuals, often through the action of mobile genetic elements (MGEs) such as plasmids or phages (1). In bacterial population genetics, the events of gene transfer are usually termed horizontal gene transfer (HGT) when they result in the acquisition of new genes and homologous recombination (HR) when they result in allelic replacements. The distinction between the two evolutionary mechanisms (HGT and HR) is not always straightforward: incoming DNA may integrate the host genome by double crossovers at homologous regions, leading to allelic replacements in these regions and to the acquisition of novel genes in the intervening ones. HR takes place only between highly similar sequences, typically within species (2). As a result, it usually involves the exchange of few polymorphisms, eventually in multiple regions, between cells (3). It may also result in no change if the recombining sequences are identical, which leaves no traces and cannot be detected by sequence analysis. HGT may occur between distant species, resulting in the acquisition of many genes in a single event. The replication and maintenance of MGEs have fitness costs to the bacterial host and have led to the evolution of cellular defense systems. These systems can sometimes be counteracted by MGEs, leading to evolutionary arms races.Restriction–modification (R-M) systems are some of the best known and the most widespread bacterial defense systems (4). They encode a methyltransferase (MTase) function that modifies particular DNA sequences in function of the presence of target recognition sites and a restriction endonuclease (REase) function that cleaves them when they are unmethylated (5). R-M systems are traditionally classified into three main types. Type II systems are by far the most abundant and the best studied (6). With the exception of the subType IIC, they comprise MTase and REase functions encoded on separate genes and are able to operate independently from each other. R-M systems severely diminish the infection rate by MGEs and have been traditionally seen as bacteria''s innate immune systems (7). However, successful infection of a few cells generates methylated MGEs immune to restriction that can invade the bacterial population (8). Hence, R-M systems are effective as defense systems during short periods of time and especially when they are diverse across a population (9, 10). In particular, it has been suggested that they might facilitate colonization of new niches (11). Type II R-M systems are also addictive modules that can propagate selfishly in populations (12). Both roles of R-M systems, as defense or selfish systems, may explain why they are very diverse within species (13, 14). Accordingly, R-M systems endure selection for diversification and are rapidly replaced (15, 16).Several recent large-scale studies of population genomics have observed more frequent HR within than between lineages (17, 18). This suggests that HR might favor the generation of cohesive population structures within bacterial species (19). Specific lineages of important pathogens that have recently changed their R-M repertoires show higher sexual isolation, such as Neisseria meningitidis, Streptococcus pneumoniae, Burkholderia pseudomallei, and Staphylococcus aureus (20–22). For example, a Type I R-M system decreased transfer to and from a major methicillin-resistant S. aureus lineage (23). Diversification of R-M target recognition sites could thus reduce transfer between lineages with different systems while establishing preferential gene fluxes between those with R-M systems recognizing the same target motifs (cognate R-M). However, these results can be confounded by evolutionary distance: closely related genomes are more likely to encode similar R-M systems, inhabit the same environments (facilitating transfer between cells), and have similar sequences (that recombine at higher rates). The advantages conferred by new genes might be higher when transfer takes place between more similar genetic backgrounds.Here, we aimed at testing the effect of R-M systems on the genetic flux in bacterial populations. We concentrated on Type II R-M systems because they are the best studied, very frequent, and those for which we could predict sequence specificity. We inferred genome-wide counts of HR and HGT and tested their association with the frequency of R-M systems encoded in the genomes. We then made a more precise test of the key hypothesis that bacteria carrying similar R-M systems establish highways of gene transfer, independently of phylogenetic proximity and clade-specific traits.     

Adaptive horizontal transfer of a bacterial gene to an invasive insect pest of coffee

Horizontal gene transfer (HGT) involves the nonsexual transmission of genetic material across species boundaries. Although often detected in prokaryotes, examples of HGT involving animals are relatively rare, and any evolutionary advantage conferred to the recipient is typically obscure. We identified a gene (HhMAN1) from the coffee berry borer beetle, Hypothenemus hampei, a devastating pest of coffee, which shows clear evidence of HGT from bacteria. HhMAN1 encodes a mannanase, representing a class of glycosyl hydrolases that has not previously been reported in insects. Recombinant HhMAN1 protein hydrolyzes coffee berry galactomannan, the major storage polysaccharide in this species and the presumed food of H. hampei. HhMAN1 was found to be widespread in a broad biogeographic survey of H. hampei accessions, indicating that the HGT event occurred before radiation of the insect from West Africa to Asia and South America. However, the gene was not detected in the closely related species H. obscurus (the tropical nut borer or "false berry borer"), which does not colonize coffee beans. Thus, HGT of HhMAN1 from bacteria represents a likely adaptation to a specific ecological niche and may have been promoted by intensive agricultural practices.     

The archaeal Ced system imports DNA

The intercellular transfer of DNA is a phenomenon that occurs in all domains of life and is a major driving force of evolution. Upon UV-light treatment, cells of the crenarchaeal genus Sulfolobus express Ups pili, which initiate cell aggregate formation. Within these aggregates, chromosomal DNA, which is used for the repair of DNA double-strand breaks, is exchanged. Because so far no clear homologs of bacterial DNA transporters have been identified among the genomes of Archaea, the mechanisms of archaeal DNA transport have remained a puzzling and underinvestigated topic. Here we identify saci_0568 and saci_0748, two genes from Sulfolobus acidocaldarius that are highly induced upon UV treatment, encoding a transmembrane protein and a membrane-bound VirB4/HerA homolog, respectively. DNA transfer assays showed that both proteins are essential for DNA transfer between Sulfolobus cells and act downstream of the Ups pili system. Our results moreover revealed that the system is involved in the import of DNA rather than the export. We therefore propose that both Saci_0568 and Saci_0748 are part of a previously unidentified DNA importer. Given the fact that we found this transporter system to be widely spread among the Crenarchaeota, we propose to name it the Crenarchaeal system for exchange of DNA (Ced). In this study we have for the first time to our knowledge described an archaeal DNA transporter.Upon UV treatment, Sulfolobales species induce the expression of Ups pili (UV-inducible pili of Sulfolobus) (1–3). These are type-IV pili (T4P) that are essential for cellular aggregation and chromosomal DNA exchange (3, 4). The ability of Sulfolobales to exchange DNA was shown to increase cellular fitness under UV stress (4). Because other DNA-damaging agents such as bleomycin and mitomycin C also induce Ups pili and cellular aggregation, the transfer of DNA is thought to play a role in repair of double-strand breaks via homologous recombination (4).Not much is known about DNA transfer among archaea; only a few examples of competence and conjugation systems have been described. Four archaeal species were shown to be naturally competent: Pyrococcus furiosus, Thermococcus kodakarensis, Methanobacterium thermoautotrophicum, and Methanococcus voltae (5–8). However, these natural transformation mechanisms have not been studied on a molecular level and in none of these archaeal species homologs from bacterial competence systems could be identified. Distinct machineries must therefore be present in archaea. Because bacterial natural transformation often involves T4P (9), one could hypothesize that Sulfolobales also exchange DNA via an uptake and release mechanism in which the Ups pili play a vital role similar to that in bacterial competence systems. However, the exchange of DNA among Sulfolobus species was shown to be insensitive to DNase treatment, and recombinants could not be obtained by mixing the cells with lysate or purified DNA (10). This demonstrates that exchange of DNA requires cellular contact and transfer occurs directly from one cell to another without passing through the surrounding medium. A conjugation-like mechanism or cellular fusion therefore seems more likely.DNA transfer among archaea via direct cellular contact was first described for the euryarchaeon Haloferax volcanii. Similar to the UV-inducible transfer of DNA among Sulfolobales, Haloferax species exchange chromosomal DNA between cells connected by bridges (11). This transfer is thought to occur in a bidirectional manner via cell fusion leading to the formation of diploid cells with mixed chromosomes (12). Interestingly, this type of DNA transfer was shown to occur between different Haloferax species and involved DNA fragments of up to 500 kbp DNA (13). Nevertheless, the mechanism of DNA transfer is so far not understood. Other described archaeal conjugative systems include self-transmissible plasmids, which have so far only been studied for Sulfolobus species. These plasmids are grouped into the so-called pKEF and pARN plasmids (14, 15) and only a few of their genes encode homologs of bacterial conjugation proteins, including the so-far-unstudied ATPases VirD4 and VirB4. It is unknown how cellular contact is initiated to achieve plasmid transfer. During plasmid conjugation, Sulfolobus islandicus cells form aggregates, similar to those observed upon UV stress (16). One could therefore imagine that cells make use of the genomically encoded Ups system to initiate cell contact. Many other conjugation proteins such as relaxases can also not be identified in archaea based on homology, indicating that again distinct mechanisms must be present that differ significantly from their bacterial counterparts. Hence, archaeal DNA transfer remains a poorly investigated topic.Previously performed microarray studies on Sulfolobus solfataricus and Sulfolobus acidocaldarius revealed in addition to an up-regulation of the ups operon several other up-regulated genes (1, 2), including genes involved in, for instance, DNA repair, such as the operon encoding helicase HerA, nuclease NurA, Rad50, and Mre11 (17, 18). Because we were interested in the mechanism of DNA transfer between Sulfolobus cells, we searched for up-regulated genes putatively involved in DNA transport. We focused on three clustered genes encoding one larger and two smaller membrane proteins. Additionally, we looked at a virB4/herA homolog. Homologs of these genes are present in the genomes of all Sulfolobales and several Desulfurococcales and Acidilobales, in which they are predicted to form an operon. In deletion mutants of either the larger membrane protein or the VirB4/HerA homolog, DNA transfer was completely abolished, showing that they are indeed involved in DNA transfer. Using PCR on genomic markers we could moreover show that, unlike other prokaryotic cell-to-cell contact-dependent DNA transfer systems, this system functions as a DNA importer. We have therefore for the first time to our knowledge given insights into an archaeal DNA transporter and showed that it functions very differently from bacterial conjugation systems. Because this system is present in many members of the Crenarchaeota, we propose the name Crenarchaeal system for exchange of DNA (Ced).     

mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p

Cdc7p is a protein kinase that is required for G₁/S transition and initiation of DNA replication in Saccharomyces cerevisiae. The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown. We report here the characterization in S. cerevisiae of a recessive mutation in a member of the MCM family, MCM5/CDC46, which bypasses the requirement for Cdc7p and its interacting factor Dbf4p. Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, our studies suggest that Cdc7p is required late in G₁ because in its absence the Mcm5p/Cdc46p blocks the initiation of DNA replication. Moreover, Mcm5p/Cdc46p may have both positive and negative effects on the ability of cell to initiate replication.     

Stable genetic transformation of intact Nicotiana cells by the particle bombardment process

We show that the genetic transformation of Nicotiana tabacum can be achieved by bombarding intact cells and tissues with DNA-coated particles. Leaves or suspension culture cells were treated with tungsten microprojectiles carrying plasmid DNA containing a neomycin phosphotransferase gene. Callus harboring the foreign gene was recovered from the bombarded tissue by selection on medium containing kanamycin. Kanamycin-resistant plants have subsequently been regenerated from the callus derived from leaves. Transient expression of an introduced β-glucuronidase gene was used to assess the efficiency of DNA delivery by microprojectiles. The frequency of cells that were stably transformed with the neomycin phosphotransferase gene was a few percent of the cells that transiently expressed the β-glucuronidase gene. These results show that gene transfer by high-velocity microprojectiles is a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium.     

DNA-uptake machinery of naturally competent Vibrio cholerae

Natural competence for transformation is a mode of horizontal gene transfer that is commonly used by bacteria to take up DNA from their environment. As part of this developmental program, so-called competence genes, which encode the components of a DNA-uptake machinery, are expressed. Several models have been proposed for the DNA-uptake complexes of competent bacteria, and most include a type IV (pseudo)pilus as a core component. However, cell-biology–based approaches to visualizing competence proteins have so far been restricted to Gram-positive bacteria. Here, we report the visualization of a competence-induced pilus in the Gram-negative bacterium Vibrio cholerae. We show that piliated cells mostly contain a single pilus that is not biased toward a polar localization and that this pilus colocalizes with the outer membrane secretin PilQ. PilQ, on the other hand, forms several foci around the cell and occasionally colocalizes with the dynamic cytoplasmic-traffic ATPase PilB, which is required for pilus extension. We also determined the minimum competence regulon of V. cholerae, which includes at least 19 genes. Bacteria with mutations in those genes were characterized with respect to the presence of surface-exposed pili, DNA uptake, and natural transformability. Based on these phenotypes, we propose that DNA uptake in naturally competent V. cholerae cells occurs in at least two steps: a pilus-dependent translocation of the incoming DNA across the outer membrane and a pilus-independent shuttling of the DNA through the periplasm and into the cytoplasm.Natural competence for genetic transformation is one of three modes of horizontal gene transfer (HGT) in prokaryotes and is often tightly regulated (1–3). Large pieces of DNA containing a series of genes can be transferred by natural transformation without the need for direct interaction with other microbes or mobile genetic elements. This process can foster rapid evolution, and HGT is known to be involved in the spread of antibiotic resistance, adaptation to new environmental niches, and the emergence of new pathogens.Many bacterial species are able to enter a state of natural competence, including the human pathogen Vibrio cholerae. In this bacterium, competence is induced upon growth on chitinous surfaces (3, 4), the natural habitat of V. cholerae (5). Although we have gained a reasonably clear understanding of the regulatory network driving competence induction in this organism (for a review, see ref. 3), almost nothing is known about its DNA-uptake machinery. Indeed, the sophisticated DNA-uptake complexes used by naturally competent bacteria during transformation are still poorly characterized (6), especially in Gram-negative bacteria in which the transforming DNA (tDNA) must cross two membranes and the periplasmic space (including the peptidoglycan layer) to enter the cytoplasm and recombine with the chromosome (the latter step is not required if the tDNA consists of plasmid DNA). Interestingly, the majority of competence-protein localization studies using cellular microbiology approaches are based on studies performed with the Gram-positive bacterium Bacillus subtilis. For B. subtilis, a multicomponent protein machine may be responsible for DNA uptake (1, 7, 8), as many transformation proteins colocalize to the pole(s) of the cell (9–12). Furthermore, using single-molecule experiments with laser tweezers, Hahn et al. showed that DNA binding and uptake also occur preferentially at the cell pole (9). It is unknown whether a polar localization pattern of the DNA-uptake machinery is universal for all naturally competent bacteria and essential for its functionality. We addressed this question and demonstrate that upon competence induction, V. cholerae cells produce a type IV pilus (Tfp)-like appendage that extends beyond the outer membrane. We also visualized other components of the DNA-uptake complex, using fluorescently labeled fusion proteins, and showed that those components and the pilus are not strictly associated with the cell poles of V. cholerae. Furthermore, we identified a minimal set of competence genes required for efficient transformation of V. cholerae. We show that most gene products within this competence regulon contribute to DNA uptake and efficient transformation, even though the Tfp-related competence proteins are not entirely essential for transformation. These data provide unique insight into the function of the competence proteins with respect to DNA transfer across the outer membrane, the periplasm, or the inner membrane and suggest an at least two-step DNA-uptake process in the Gram-negative bacterium V. cholerae.     

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.     

Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms

Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of “Marseille” virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are “melting pots” of microbial evolution where diverse forms emerge, including giant viruses with complex gene repertoires of various origins.     

Horizontal gene transfer: a critical view

It has been suggested that horizontal gene transfer (HGT) is the "essence of phylogeny." In contrast, much data suggest that this is an exaggeration resulting in part from a reliance on inadequate methods to identify HGT events. In addition, the assumption that HGT is a ubiquitous influence throughout evolution is questionable. Instead, rampant global HGT is likely to have been relevant only to primitive genomes. In modern organisms we suggest that both the range and frequencies of HGT are constrained most often by selective barriers. As a consequence those HGT events that do occur most often have little influence on genome phylogeny. Although HGT does occur with important evolutionary consequences, classical Darwinian lineages seem to be the dominant mode of evolution for modern organisms.