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Small cryptic plasmids of multiplasmid, clinical Escherichia coli.

Clinical isolates of Escherichia coli were found to host a multiplicity of plasmids. These were resolved from plasmid gel profiles, from the properties of various transconjugants and transformants of E. coli DH1, by the topoisomerase I relaxation of covalently closed circle plasmid DNA, by electron microscopy, and by the determination of their compatibilities. The majority of these were unusually small, cryptic plasmids (SCPs). From one strain, KL4, 13 electrophoretic bands were resolved to five plasmids, three of which were SCPs. SCPs were phenotypically barren, and the smallest of these, pKL1, contained barely enough information for self-replication. A derivative of pKL1, pKL1Km, in which the transposon was restricted to a small 350-bp region, was stably maintained in Shigella, Salmonella, Serratia, and Citrobacter species and its replication was polA independent. pKL1 encoded only a single protein, RepA (Mr 17960), which specifically bound to pKL1 DNA. No apparent homologies with other RepA protein sequences could be detected. Thus the SCP, pKL1, is a novel minimal plasmid replicon encoding only enough information to ensure perpetuation. A hypothesis is presented describing SCPs as a class of selfish DNA that persists simply due to its ability to replicate and to its stability based on high copy number.

Conjugation, Genetic↗

Molecular analysis of a mobilizable theta-mode trimethoprim resistance plasmid from coagulase-negative staphylococci.

The Staphylococcus epidermidis plasmid pSK639 is the prototype of a newly described family of small plasmids identified in clinical staphylococcal isolates. pSK639 is 8 kb in length and possesses a composite structure consisting of an IS257-flanked segment mediating trimethoprim resistance (Tpr), and regions responsible for replication and mobilization of the plasmid. Comparative sequence analysis suggests that a pSK639-like plasmid may represent a progenitor of previously identified staphylococcal Tpr determinants related to the transposon-like structure, Tn4003. In contrast to the small staphylococcal plasmids characterized to date that all utilize a rolling circle mode of replication, the replication region of pSK639 was found to contain features typical of an iteron-controlled theta-mode replicon. pSK639 is the first small plasmid of this type to be identified in the staphylococci.

Amino Acid Sequence↗

Complete nucleotide sequence of the replicator region of Paracoccus (Thiobacillus) versutus pTAV1 plasmid and its correlation to several plasmids of Agrobacterium and Rhizobium species.

The complete nucleotide sequence of the replicator region of pTAV1, a cryptic, low copy number plasmid of Paracoccus versutus, was determined. The minimal replicon sequence (3149 bp) included in pTAV203/18 contains two open reading frames with coding capabilities for putative polypeptides of 23.8 (RepX) and 46 kDa (RepC'). The two genes have the same transcriptional polarity and both seem to be essential for replication of pTAV203. The predicted amino acid sequence of RepC' shows significant homology with the major replication-associated proteins of several Agrobacterium and Rhizobium plasmids. A probable origin of replication (oriV) was proposed to be localized at the 3' terminal end of the repC' gene.

Amino Acid Sequence↗

Cloning and characterization of the ori region of pSW1200 of Erwinia stewartii: similarity with plasmid P1.

The ori region of an Erwinia stewartii plasmid, pSW1200 (106 kb), has been cloned and sequenced. This region consists of a gene encoding a protein which has 91% similarity and 73% identity with the RepA protein of bacteriophage P1. The ori region also consists of eight copies of 19-bp iterons which are highly homologous to the iterons of P1. Similar to plasmid P1, pSW1200 replicon has a copy number of approximately 1. On the other hand, the copy number increases about ninefold if three of the iterons located downstream from repA gene are deleted. We also demonstrate that pGEM-5Z consisting of a copy of P1 iteron is incompatible with a pSW1200 derivative, pSW1201, suggesting that pSW1200 and P1 DNA are incompatible and both belong to the IncY group.

Base Sequence↗

New shuttle vectors for the introduction of cloned DNA in Desulfovibrio.

The pBG1 replicon from the cryptic plasmid of Desulfovibrio desulfuricans G100A was inserted into pTZ18U derivatives to generate a new family of shuttle vectors. These plasmids are stable both in Escherichia coli and in Desulfovibrio, they present a large number of unique restriction sites, and colonies of recombinant clones can be identified by blue/white screening in E. coli. The pBMC, pBMK, and pBMS series carry the cat, npt, or strAB genes as selectable markers, respectively. The pBMC6, pBMK6, and pBMS6 plasmids can be introduced both in D. desulfuricans and in Desulfovibrio fructosovorans by electrotransformation, and the pBMC7, pBMK7, and pBMS7 plasmids contain additional mobilization functions which makes them suitable for conjugation.

Cloning, Molecular↗

Nucleotide sequence and genetic characterization of the novel IncQ-like plasmid pIE1107.

The analysis of the complete nucleotide sequence of the small resistance plasmid pIE1107 revealed a close similarity to the well-known IncQ plasmids. Highly conserved replication proteins and nearly identical origins of replication (oriV) suggest equivalent functions in the related replication systems. However, pIE1107 contains two copies of IncQ-oriV-like DNA which are slightly different regarding the iterons. Upon deletion of a silent copy of IncQ-oriV-like DNA the resulting plasmid is fully compatible with IncQ plasmids, indicating that there is no mutual communication between the replication control of the respective replicons. Experiments with cloned oriV DNA strongly suggest that the replication initiation protein of pIE1107 has specialized into the distinct target-iterons of its own oriV which differs only by a few nucleotides from the oriV of IncQ plasmids. Implications from the apparent highly specific protein-DNA recognition and from the incompatibility properties of pIE1107 for the evolution of a family of compatible, IncQ-like plasmids are discussed.

Amino Acid Sequence↗

Cloning of erythromycin-resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors.

Lactobacillus reuteri L1 and N16 strains contain a 7.0-kb plasmid (pTE80) and a 15-kb plasmid (pTE15), respectively, encoding resistance to erythromycin (Em(r)). Physical maps of both plasmids were established. Nucleotide sequences of the genetic determinants encoding Em(r) on pTE80 and pTE15 revealed the existence of a very similar (ca. 99% nucleotide sequence and ca. 98% amino acid sequence identity) open reading frame for an Em(r) transmethylase gene (erm) in both plasmids. These structural erm genes, 753 and 750 bp in length, respectively, were highly related (ca. 98% nucleotide sequence and ca. 97% amino acid sequence identity) to the erm gene of L. fermentum plasmid pLEM3. Sequence analysis showed that these two erm genes from pTE80 and pTE15 could be categorized under the ermB (ermAM) class. These are the first members of the ermB (ermAM) class of Em(r) determinant from L. reuteri to be characterized at the nucleotide sequence level. The Em(r) gene from pTE80 (erm80) was then ligated into pUC18/19 to construct replication origin (RO)-screening vectors pUE80(+) and pUE80(-) (pUE80(+/-)). These plasmids contain the pUC18/19-derived multiple cloning site, ampicillin-resistance trait, and the LacZ' gene, which enable direct screening for recombinants in Escherichia coli. Once the recombinant contains a RO from L. reuteri, the Em(r) trait of erm80 is used as a selection marker for the replication of the chimeric plasmid as it is transformed into L. reuteri using the cloned RO as a replicon. Replication regions from pTE80 and pTE15 were successfully cloned into the constructed vector pUE80(-). The RO cloned from pTE80 was further identified as being highly stable in L. reuteri and also bearing a relatively narrow host range compared with that of pTE15. The Em(r) determinant (erm80) and RO cloned from pTE80 could be used in the future construction of derivatives of cloning vectors for this microbe. Moreover, the pUE80(+/-) and pTE80-RO constructed in this study have the potential to be developed as a suicide vector and an E. coli-L. reuteri shuttle vector, respectively.

Amino Acid Sequence↗

A cell line that expresses a reporter gene in response to infection by Sindbis virus: a prototype for detection of positive strand RNA viruses.

We describe a stably transformed cell line (BHKSINLuc2) that contains a defective Sindbis virus genome under the control of a Rous sarcoma virus promoter and the luciferase gene downstream of the viral subgenomic RNA promoter. This cell line expresses high levels of luciferase activity following infection with Sindbis virus and provides a sensitive assay for titering variants of Sindbis virus that lack the structural protein genes, in particular, Sindbis virus replicons that express heterologous proteins. Cell lines such as this may be of value for detection of positive-strand RNA viruses.

Alphavirus↗

Identification of mutations in a Sindbis virus variant able to establish persistent infection in BHK cells: the importance of a mutation in the nsP2 gene.

Sindbis virus is a positive strand RNA virus that has provided a valuable model for studying virus structure and replication. It is also being developed as a vector for the expression of heterologous proteins. Many studies with this virus are carried out in cultured BHK cells where infection is usually highly cytopathic and within 1 or 2 days after infection all of the cells are dead. Weiss et al. had established a persistently infected culture of BHK cells by infecting the cells with a virus preparation highly enriched in defective interfering (DI) particles and had isolated an attenuated virus, SIN-1 virus, from the culture [Weiss et al. (1980) J. Virol. 33, 463-474]. SIN-1 virus, free of DI particles, was able to establish a persistent infection in BHK cells. We initiated studies to determine what changes in the genome of the virus were responsible for this phenotype. We describe here the cDNA cloning and sequencing of the 5' terminus and the four nonstructural protein genes from SIN-1 virus. A single coding mutation in the nsP2 gene (a predicted change of Pro-726 --> Ser) produced a virus that was able to establish persistent infection in BHK cells. Additional mutations in the other genes were required to decrease the synthesis of viral RNA to a level similar to that found in cells infected with SIN-1 virus. Incorporation of the nsP2 mutation into a Sindbis virus expression vector led to a higher level of synthesis of the reporter protein, beta-galactosidase, than that obtained with the original Sindbis virus replicon.

Animals↗

Expression and detection of macrophage-tropic HIV-1 gp120 in the brain using conformation-dependent antibodies.

HIV-1 envelope proteins gp120 and gp41 are likely to play a role in the pathogenesis of HIV-associated neurocognitive disorders. While detection of gp120 in HIV-infected cell cultures is easy, it has not yet been possible to identify gp120 in human or animal brains in situ. The difficulty in detecting gp120 could be due to low expression levels of the protein, to the shedding of gp120 from infected macrophages/microglia, or to the use of inappropriate gp-specific antibodies. We addressed these questions by analyzing the subcellular localization, oligomeric structure, and shedding behavior of gp120 from a macrophage-tropic, CCR5-dependent primary isolate, BX08, expressed by a Semliki Forest virus replicon (SFVenvBX08) in vitro. We used the same SFV system injected in vivo into the rat brain in an attempt to detect gp120 in situ. Our results show that gp120/41 is expressed as monomers, dimers, and trimers in cell culture. Immunocytochemical analysis revealed that intracytoplasmic gp120 can be recognized by an anti-V3 antibody, whereas gp120 at the plasma membrane is detected exclusively by a conformation-dependent antibody. In the rat brain, the SFV vector allows gene expression in neurons from day 3 to day 9 after injection without any apparent brain damage nor reactive astrogliosis. In SFVenvBX08-infected neurons only conformation-dependent antibodies allowed gp120 labeling. These results suggest that previous difficulties in detecting gp120 in brain tissues may be due to the use of antibodies which were unable to recognize gp120 at the plasma membrane.

Animals↗

An infectious clone of the West Nile flavivirus.

West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent. We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones. This infectious clone was used to investigate effects of 5'- and 3'-nonrelated sequences on virus replication and infectivity of synthetic RNA. While a long nonrelated sequence at the 3'-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5'-end completely abrogated virus replication. Replacement of the conserved 5'-adenosine residue substantially delayed, but did not prevent, establishment of virus infection. In all cases, the recovered virus had restored its authentic 5'- and 3'-end genome sequences. However, the presence of extensive nonrelated sequences at both 5'- and 3'-ends could not be repaired.

3' Untranslated Regions↗

Vaccine potential of Ebola virus VP24, VP30, VP35, and VP40 proteins.

Previous vaccine efforts with Ebola virus Zaire (EBOV-Z) emphasized the potential protective efficacies of immune responses to the surface glycoprotein and the nucleoprotein. To determine whether the VP24, VP30, VP35, and VP40 proteins are also capable of eliciting protective immune responses, these genes were expressed from alphavirus replicons and used to vaccinate BALB/c and C57BL/6 mice. Although all of the VP proteins were capable of inducing protective immune responses, no single VP protein protected both strains of mice tested. VP24, VP30, and VP40 induced protective immune responses in BALB/c mice, whereas C57BL/6 mice survived challenge only after vaccination with VP35. Passive transfer of immune sera to the VP proteins did not protect unvaccinated mice from lethal disease. The demonstration that the VP proteins are capable of eliciting protective immune responses to EBOV-Z indicates that they may be important components of a vaccine designed to protect humans from Ebola hemorrhagic fever.

Animals↗

Reverse genetics of coronaviruses using vaccinia virus vectors.

In this article, we describe the reverse genetic system that is based on the use of vaccinia virus cloning vectors. This system represents a generic approach to coronavirus reverse genetics and was first described for the generation of recombinant human coronavirus 229E representing a group I coronavirus. Subsequently, the same approach has been used to generate recombinant avian infectious bronchitis coronavirus and, recently, recombinant mouse hepatitis virus, representing group III and group II coronaviruses, respectively. We describe how vaccinia virus-mediated homologous recombination can be used to introduce specific mutations into the coronavirus genomic cDNA during its propagation in vaccinia virus and how recombinant coronaviruses can be isolated. Finally, we describe how the coronavirus reverse genetic system has now been extended to the generation of coronavirus replicon RNAs.

Cloning, Molecular↗

Experimental and theoretical definition of geminivirus origin of replication.

Geminiviruses are plant pathogens that replicate by a rolling-circle mechanism, analogous to that used by several prokaryotic ssDNA replicons. Recent reports provide important progress in understanding the structure and functioning of replication origin from these viruses. We have used these data to propose models for the initiation of replication in dicot- and monocot-infecting geminiviruses.

Base Sequence↗

Early replication signals in nuclei of Chinese hamster ovary cells.

DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescence microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time.

Animals↗

Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells.

The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-phenylindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.

Animals↗

Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis.

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (orf) [alpha and beta]. Inactivation or deletion of orf beta results in SegA- plasmids. Better than random segregation requires an active segB region. The segB region contains two orfs (orf epsilon and orf zeta). Inactivation of either of the orfs does not lead to an increase in cell death, but orf zeta- plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.

Bacillus subtilis↗

Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWW0.

Toluene degrading (xyl) genes on a Pseudomonas TOL plasmid pWW0 are located within a 39-kb DNA portion. The 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pACYC184 and R388 in Escherichia coli recA strains. Thus the 56- and 17-kb regions were designated Tn4651 and Tn4652, respectively. Genetic analysis of Tn4652 demonstrated that its transposition occurs by a two-step process, namely, cointegrate formation and its subsequent resolution. The presence in cis of DNA sequences of no more than 150 bp at both ends of Tn4652 was prerequisite for cointegrate formation, and this step was mediated by a trans-acting factor, transposase, which was encoded in a 3.0-kb segment at one end of the transposon. Cointegrate resolution took place site-specifically within a 200-bp fragment, which was situated 10 kb away from the transposase gene. Based on the stability of cointegrates formed by various mini-Tn4652 derivatives, it was shown that the cointegrate resolution requires two trans-acting factors encoded within 1.0- and 1.2-kb fragments that encompass the recombination site involved in the resolution.

Chromosome Deletion↗