DNA inversion on conjugative plasmid pVT745

Plasmid pVT745 from Actinobacillus actinomycetemcomitans strain VT745 can be transferred to other A. actinomycetemcomitans strains at a frequency of 10(-6). Screening of transconjugants revealed that the DNA of pDMG21A, a pVT745 derivative containing a kanamycin resistance gene, displayed a structural rearrangement after transfer. A 9-kb segment on the plasmid had switched orientation. The inversion was independent of RecA and required the activity of the pVT745-encoded site-specific recombinase. This recombinase, termed Inv, was highly homologous to invertases of the Din family. Two recombination sites of 22 bp, which are arranged in opposite orientation and which function as DNA crossover sequences, were identified on pVT745. One of the sites was located adjacent to the 5' end of the invertase gene, inv. Inversion of the 9-kb segment on pVT745 derivatives has been observed in all A. actinomycetemcomitans strains tested except for the original host, VT745. This would suggest that a host factor that is either inactive or absent in VT745 is required for efficient recombination. Inactivation of the invertase in the donor strain resulted in a 1,000-fold increase in the number of transconjugants upon plasmid transfer. It is proposed that an activated invertase causes the immediate loss of the plasmid in most recipient cells after mating. No biological role has been associated with the invertase as of yet.     

Nucleotide sequence and analysis of conjugative plasmid pVT745

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

Promiscuous stimulation of ParF protein polymerization by heterogeneous centromere binding factors

The segrosome is the nucleoprotein complex that mediates accurate segregation of bacterial plasmids. The segrosome of plasmid TP228 comprises ParF and ParG proteins that assemble on the parH centromere. ParF, which exemplifies one clade of the ubiquitous ParA superfamily of segregation proteins, polymerizes extensively in response to ATP binding. Polymerization is modulated by the ParG centromere binding factor (CBF). The segrosomes of plasmids pTAR, pVT745 and pB171 include ParA homologues of the ParF subgroup, as well as diverse homodimeric CBFs with no primary sequence similarity to ParG, or each other. Centromere binding by these analogues is largely specific. Here, we establish that the ParF homologues of pTAR and pB171 filament modestly with ATP, and that nucleotide hydrolysis is not required for this polymerization, which is more prodigious when the cognate CBF is also present. By contrast, the ParF homologue of plasmid pVT745 did not respond appreciably to ATP alone, but polymerized extensively in the presence of both its cognate CBF and ATP. The co-factors also stimulated nucleotide-independent polymerization of cognate ParF proteins. Moreover, apart from the CBF of pTAR, the disparate ParG analogues promoted polymerization of non-cognate ParF proteins suggesting that filamentation of the ParF proteins is enhanced by a common mechanism. Like ParG, the co-factors may be modular, possessing a centromere-specific interaction domain linked to a flexible region containing determinants that promiscuously stimulate ParF polymerization. The CBFs appear to function as bacterial analogues of formins, microtubule-associated proteins or related ancillary factors that regulate eucaryotic cytoskeletal dynamics.     

A cryptic plasmid from Pasteurella multocida has a predicted protein nearly identical to a transport protein from Actinobacillus actinomycetemcomitans

Several plasmids from Pasteurella multocida have been shown to carry antibiotic resistance genes but no other genes possibly related to the organism's pathogenesis. We report here that sequence from the plasmid pLEM from a fowl isolate of P. multocida, strain 1059, contained one open reading frame that had significant identity with a predicted protein from pVT745, a plasmid that was isolated from a human oral isolate of Actinobacillus actinomycetemcomitans. This predicted protein had significant homology at the amino acid level to cation transport proteins.     

Why is entry exclusion an essential feature of conjugative plasmids?

Entry exclusion is a property of plasmids by which the cells that contain them become bad recipients in additional conjugation rounds. This work reviews entry exclusion essential features and analyzes the mechanisms of action of the best studied systems. We searched for homologs of the proteins responsible for experimentally known exclusion systems. Results were used to classify exclusion systems in families of related elements. We arrive to the conclusion that all conjugative plasmids contain at least one entry exclusion gene. Although entry exclusion genes seem to be part of the plasmid conjugative machinery, they are systematically absent in phylogenetically related type IV protein exporting machines involved in virulence for plants and animals. We infer from this fact that entry exclusion is an essential feature of conjugative plasmid biology. Mathematical models suggest that plasmids expressing entry exclusion selectively eliminate plasmids lacking it, reinforcing its essential character and suggesting that entry exclusion plays a direct role in plasmid survival. Other experimental results confirm that entry exclusion is essential for the stability of a conjugative plasmid. We suggest that entry exclusion limits the damage of lethal zygosis (bacterial death produced by excessive rounds of conjugation). Additionally, it avoids competition in a host among identical plasmid backbones. Conversely, the lack of entry exclusion in conjugative transposons can be understood as a means of generating rapid evolutionary change.     

Evidence for the role of horizontal transfer in generating pVT1, a large mosaic conjugative plasmid from the clam pathogen, Vibrio tapetis

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600(T) reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae.     

A direct-selection vector derived from pColE3-CA38 and adapted for foreign gene expression

The construction of a plasmid vector, pVT25, which allows an efficient and direct selection for transformed cells carrying recombinant plasmids is described. In this vector, the replicon and Ap^R gene from plasmid pBR327 are fused to the colE3 gene of pColE3-CA38, whereby positive selection is based on the inactivation of the lethal colicin E3 by the insertion of a foreign DNA fragment. However, pVT25 can be maintained within the Escherichia coli cells when complemented with another plasmid, pVT26, which expresses the colicin E3 immunity (imm) and the Tc^R phenotypes. Furthermore, pVT25 was used to regulate the expression of the synthetic human proinsulin gene fused to the colE3 gene at the single ClaI site. The production of the characteristic C-peptide of proinsulin, monitored by radioimmunoassay, was shown to be under the control of the inducible promoter of the colE3 gene.     

Secretory expression of α single-chain insulin precursor in yeast and its conversion into human insulin

A synthetic single-chain porcine insulin precursor (PIP) gene and an α-mating factor leader sequence (αMFL) gene obtained by the PCR method are inserted between the promoter and 3'-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/α MFL-PIP. The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cererisiae transformed by pVT102-U/αMFL-PIP. The precursor is purified and converted into human insulin by tryptic transpeptidation. The purified human insulin is fully active and can be crystallized. The overall yield of human insulin is 25 mg per liter of culture medium.     

A ColE1-encoded gene directs entry exclusion of the plasmid.

To detect entry exclusion of the ColE1 plasmid, we established an assay system that was not influenced by incompatibility of extant plasmids in the recipient cells or by the viability of the cells due to the killing action of colicin E1 protein. The assay revealed that exc1 and exc2, assigned as genes directing entry exclusion, had no exclusion activity. Instead, mbeD, which had been characterized as a gene for plasmid mobilization, directed the exclusion activity. MbeD was overexpressed and identified as a 35S-labeled protein, which was recovered in both the soluble and membrane fractions, particularly in the inner membrane fraction. An amphipathic helical structure was predicted in the N-terminal region of MbeD as well as in the corresponding homologous proteins of ColA and ColK. These proteins may bind to the inner membrane via the N-terminal amphipathic helix and function in entry exclusion.     

TrbK, a small cytoplasmic membrane lipoprotein, functions in entry exclusion of the IncP alpha plasmid RP4.

TrbK is the only plasmid-encoded gene product involved in entry exclusion of the broad-host-range plasmid RP4. The corresponding gene, trbK, coding for a protein of 69 amino acid residues maps in the Tra2 region within the mating pair formation genes. TrbK carries a lipid moiety at the N-terminal cysteine of the mature 47-residue polypeptide. The mutant protein TrbKC23G cannot be modified or proteolytically processed but still acts in entry exclusion with reduced efficiency. An 8-amino-acid truncation at the C terminus of TrbK results in a complete loss of the entry exclusion activity but still allows the protein to be processed. TrbK localizes predominately to the cytoplasmic membrane. Its function depends on presence in the recipient cell but not in the donor cell. TrbK excludes plasmids of homologous systems of the P complex; it is inert towards the IncI system. The likely target for TrbK action is the mating pair formation system, because DNA or any of the components of the relaxosome were excluded as possible targets.     

Identification of a maintenance system on rolling circle replicating plasmid pVT736-1

Distribution of plasmid molecules to the two daughter cells at cell division is of major importance for their stable inheritance. Several mechanisms that control equipartitioning of low-copy-number plasmids have been described in molecular terms. However, no homologous or analogous systems have been identified for intermediate or high-copy-number plasmids, including rolling circle replicating (RCR) plasmids. It has been suggested that distribution of such plasmids at cell division relies solely on random segregation. Plasmid pVT736-1 is a 2 kb RCR plasmid that was isolated from the Gram-negative capnophilic coccobacillus Actinobacillus actinomycetemcomitans . The plasmid contains a DNA region of approximately 0.8 kb that is associated with its segregational stability. An operon that consists of two genes ( orf3 and orf2 ) is followed by a putative cis -acting site that contains an integration host factor (IHF) binding site, flanked by several repeats. Mutations in orf 2 resulted in plasmid instability. In addition, this DNA region was able to stabilize partially a heterologous replicon, p15A. Homologues or analogues of the pVT736-1 stabilization system have been detected on numerous plasmid and bacterial genomes.     

RP1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids.

Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.     

Protein diversity confers specificity in plasmid segregation

The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.     

General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids.

The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. The fertility of the cloned region could still be inhibited by a coresident IncP plasmid. The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW). They have been separately cloned. PILW also contains the genes involved in entry exclusion. MOBW contains oriT and the gene products required for efficient mobilization by PILW. MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1.     

Nucleotide sequence and gene organization of ColE1 DNA

The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein).     

Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum.

A Chesapeake Bay water isolate of Mycobacterium scrofulaceum containing a 115-megadalton plasmid (pVT1) grew in the presence of 100 microM HgCl2 and converted soluble 203Hg2+ to volatile mercury at a rate of 50 pmol/10(8) cells per min. Cell extracts contained a soluble mercuric reductase whose activity was not dependent on exogenously supplied thiol compounds. The enzyme displayed nearly identical activity when either NADH or NADPH served as the electron donor. A spontaneously cured derivative lacking pVT1 failed to grow in the presence of 100 microM HgCl2 and possessed no detectable mercuric reductase activity.     

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

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

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.