Conjugal transfer of plasmid pTF-FC2 from Agrobacterium to plant cells in the absence of T-DNA borders

The ability of the plasmid pTF-FC2 to transfer genes into plants was investigated. Using this plasmid as the backbone two plasmids were constructed namely pTD1 and pDER-bar. These plasmids contained, as plant selectable markers, the nptII and the bar genes, respectively. The nptII gene was flanked by the right and left borders and the bar gene was not. Transgenic plants were obtained through the co-cultivation of tobacco leaf discs with the Agrobacterium tumefaciens strain LBA4404(pAL4404)(pDER-bar). Molecular and genetic analysis indicated that the bar gene had been stably integrated into the plant genome and had been inherited in a Mendelian fashion. Integration was shown to be polar and unidirectional and in some cases the entire plasmid was found to have integrated into the plant genome. Interestingly, no plants were generated from tobacco leaf discs that were co-cultivated with the strain C58C1(pMP90)(pTD1).     

Exploring the evolutionary dynamics of plasmids: the <Emphasis Type="Italic">Acinetobacter</Emphasis> pan-plasmidome

Background  

Prokaryotic plasmids have a dual importance in the microbial world: first they have a great impact on the metabolic functions of the host cell, providing additional traits that can be accumulated in the cell without altering the gene content of the bacterial chromosome. Additionally and/or alternatively, from a genome perspective, plasmids can provide a basis for genomic rearrangements via homologous recombination and so they can facilitate the loss or acquisition of genes during these events, which eventually may lead to horizontal gene transfer (HGT). Given their importance for conferring adaptive traits to the host organisms, the interest in plasmid sequencing is growing and now many complete plasmid sequences are available online.     

The densovirus of Periplaneta fuliginosa (PfDNV) as an insect vector for persistent foreign gene expression in vivo

An infectious clone of the Periplaneta fuliginosa densovirus (PfDNV) has been constructed and the PfDNV genome can rescue from the plasmid and replicate as the wild-type virus in nymphs of P. fuliginosa. To investigate the ability of the cloned PfDNV genome to be used as a stable and persistent expression vector, we constructed seven recombinant plasmids in which the GFP reporter gene was inserted into the genome of PfDNV. When these recombinant constructs were transfected into hosts, the GFP was expressed efficiently in every clone. Southern blot analysis revealed that recombinant plasmids had integrated into host genome. Infectious recombinant virions could be produced from plasmids in which the GFP gene was downstream of and in frame with the NS3 and NS1 coding regions. These results indicate that PfDNV genome can be used as an insect vector for the transfer and persistent expression of an exogenous gene.     

Insertion of nonhomologous DNA into the yeast genome mediated by homologous recombination with a cotransforming plasmid

Bacterial plasmids containing no detectable homology with yeast DNA sequences were inserted into the yeast genome by cotransforming with a plasmid containing a yeast gene. Analysis of the yeast transformants confirmed that recombination events occurred between the prokaryotic sequences shared by the two plasmids and between the yeast sequences common to the cotransforming plasmid and to the genome. Multiple copies of the two plasmids, in both tandem and interspersed arrays, are inserted by this method. Populations of cells grown from individual transformants are heterogenous for the number of integrated sequences. The number of integrated bacterial sequences is greatly reduced after 100 generations of growth in the populations that initially contained large numbers of sequences, while it is stable in those populations that initially contained either a single or a small number of copies.     

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.     

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells.

An autonomously replicating shuttle vector was used to investigate enhancement of plasmid-chromosome recombination in mammalian host cells by gamma irradiation and UV light. Sequences homologous to the shuttle vector were stably inserted into the genome of African green monkey kidney cells to act as the target substrate for these recombination events. The shuttle vector molecules were irradiated at various doses before transfection into the mammalian host cells that contained the stable insertions. The homologous transfer of the bacterial ampicillin resistance gene from the inserted sequences to replace a mutant ampicillin sensitivity gene on the shuttle vector was identified by the recovery of ampicillin-resistant plasmids after Hirt extraction and transformation into Escherichia coli host cells. Gamma irradiation increased homologous shuttle vector-chromosome recombination, whereas UV light did not increase the frequency of recombinant plasmids detected. Introducing specific double-strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was increased by UV irradiation of the plasmid but did not change with time. The ampicillin-resistant recombinant plasmid molecules analyzed appeared to rise mostly from nonconservative exchanges that involved both homologous and possibly nonhomologous interactions with the host chromosome. The observation that similar recombinant structures were obtained from all the plasmid treatments and host cells used suggests a common mechanism for plasmid-chromosome recombination in these mammalian cells.     

A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes >/=300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.     

Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns

Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.     

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids. This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid. Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies. These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA.     

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

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

A first global analysis of plasmid encoded proteins in the ACLAME database

Many plasmids are mobile genetic elements (MGEs) and, as other members of that group of DNA entities, their genomes display a mosaic and combinatorial structure, making their classification extremely difficult. As other MGEs, plasmids play a major role in horizontal transfer of genetic materials and genome reorganization. Yet, the full impact of such phenomenon on major properties of the host cell, such as pathogenicity, the ability to use new carbon sources or resistance to antibiotics, remains to be fully assessed. More and more complete plasmid genome sequences are available. However, in the absence of standards for storing plasmid sequence data and annotating genes and gene products on sequenced plasmid genomes, the resulting information remains rather limited. Using 503 sequenced plasmids organized in the ACLAME database, we discuss how, by structuring information on the genomes, their host and the proteins they code for, one can gain access to either global or more detailed analysis of the plasmid sequence information, as illustrated by a network representation of the relationships between plasmids.     

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

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

Genetic analysis of the mobilization of the non-conjugative plasmid Clo DF13

Summary Insertion of the transposon Tn901 within a region of almost one third of the Clo DF13 genome is compared with the loss of its transfer (indicated as Mob^-) by a conjugative plasmid. By use of both insertion and deletion mutants of Clo DF13, this region was located on the Clo DF13 physical map. Studies with transfer mutants of the F plasmid showed that, in contrast with the traG gene product, the gene products of traI, traD and traM do not play an essential role in the transfer process of Clo DF13. Because Clo DF13 can be transferred under conditions in which the coningative plasmid is not transferred at all, it is obvious that normally Clo DF13 is not transferred to recipient cells as a cointegrate of the conjugative plasmid and Clo DF13. Characterization of the Mob^- Clo DF13:: Tn901 plasmids showed that the absence or alteration of the Clo DF13 specified polypeptide B (molecular weight 61,000 daltons) is correlated with the transfer deficiency of these plasmids. The existence of transfer deficient Clo DF13:: Tn901 plasmids, which direct the synthesis of polypeptide B, showed that other Clo DF13 genetic information is also involved in the transfer of this plasmid. On basis of the site of the mutation in the genome, the synthesis of polypeptide B in the minicell system and the behaviour of the Mob^- mutants in complementation studies, we preliminarily divide the Mob^- Clo DF13:: Tn901 plasmids into three different classes. The possible role of Clo DF13 genetic information involved in the transfer process of this plasmid is discussed.     

In Rhizobium etli symbiotic plasmid transfer, nodulation competitivity and cellular growth require interaction among different replicons

Bacteria belonging to the genus Rhizobium are able to develop two different lifestyles, in symbiotic association with plant roots or through saprophytic growth. The genome of Rhizobium strains is constituted by a chromosome and several large plasmids, one of them containing most of the genes involved in symbiosis (symbiotic plasmid or pSym). Our model strain Rhizobium etli CFN42 contains six plasmids. We have constructed multiple plasmid-cured derivatives of this strain and used them to analyze the contribution of these plasmids to free-living cellular viability, competitivity for nodulation, plasmid transfer, and utilization of diverse carbon sources. Our results show that the transfer of the pSym is strictly dependent on the presence of another plasmid; consequently under conditions where pSym transfer is required, nodulation relies on the presence of a plasmid devoid of nodulation genes. We also found a drastic decrease in competitivity for nodulation in multiple plasmid-cured derivatives when compared with single plasmid-cured strains. Cellular growth and viability were greatly diminished in some multiple plasmid-cured strains. The utilization of a number of carbon sources depends on the presence of specific plasmids. The results presented in this work indicate that functional interactions among sequences scattered in the different plasmids are required for successful completion of both lifestyles.     

Origin and Evolution of Rickettsial Plasmids

Background

Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes.

Results

Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events.

Conclusion

Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as gene duplication and genesis. The plasmids are plastic and mosaic structures that may play biological roles similar to or distinct from their co-residing chromosomes in an obligate intracellular lifestyle.     

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.