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PubMed1983

Formation of oligomeric structures from plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda heads.

Plasmids that carry cos lambda, the region necessary for lambda phage packaging and that are as small as four kilobases in size can be packaged into lambda phage heads in head-to-tail tandem oligomeric structures. Multimeric oligomers as large as undecamers have been detected. Oligomer formation depends upon the products of red and gam of lambda, and the general recombination occurs between different plasmids that share homologous DNA regions. The packaging efficiency of plasmids depends on its copy number in cells and its genome size. Upon injection into a cell, the DNA establishes itself as a plasmid in a tandem structure. When such a plasmid in a high oligomeric structure is used as the source of packaging DNA, the packaging efficiency of the plasmids is elevated. The oligomers are stable in recA cells, whereas they drift toward lower oligomers in recA+ cells.

Bacteriophage lambda↗
PubMed1986

Partitioning of plasmid R1. Structural and functional analysis of the parA locus.

The stability locus, parA+, of plasmid R1 is shown to be localized within a 1500 base-pair region of DNA on the largest EcoRI restriction fragment of plasmid R1. The nucleotide sequence of the region revealed the presence of two open reading frames, one of 320 codons, and another of 60 codons. The larger open reading frame encodes a polypeptide of 36,000 Mr. Deletions covering the promoter distal end of the 36,000 Mr reading frame give rise to synthesis of large amounts of truncated protein. Construction of promoter fusions between the parA+ promoter and the lacZ gene showed that the parA+ region encodes a factor that negatively regulates the expression of the 36,000 Mr protein. The locus exerting parA+-associated incompatibility, denoted incA+, was mapped to a 60 base-pair region covering the parA+ promoter. Most likely, this region is involved both in the negative regulation of the parA+ operon and in the parA+-associated incompatibility. Two explanations are suggested to explain this possible dual function of the parA+ promoter region. The parA+ region was cloned into an unstably inherited (par-) derivative of a mini-F derivative. The low copy number plasmid mini-F devoid of its own partition genes was stabilized more than 100-fold by carrying the parA+ genes. This observation is in accordance with the proposal that the parA+ locus specifies the true partition function of plasmid R1.

Base Sequence↗
PubMed2000

Atomic force microscopy studies of intercalation-induced changes in plasmid DNA tertiary structure.

Structural transitions in the tertiary structure of plasmid DNA have been investigated using atomic force microscopy. Changes in superhelical stress were induced by ethidium bromide intercalation, and conformational effects monitored by recording topographic images from DNA complexes of various ethidium bromide:base pair stoichiometry. Significant changes in the tertiary structure of individual DNA molecules were observed with increasing ethidium bromide concentration. The first distinct conformational transition was from a predominantly relaxed structure to one consisting solely of toroidal supercoils. A further increase in ethidium bromide concentration resulted in the formation of regions of plectonemic supercoiling. The ratio of plectonemic:toroidal supercoiling gradually increased until an extremely tightly interwound structure of solely plectonemic supercoiling was finally adopted. The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemic supercoiling is the predominant form adopted by plasmid DNA.

DNA, Superhelical↗
PubMed1993

kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus.

The kil-kor regulon of IncP plasmid RK2 is a complex regulatory network that includes genes for replication and conjugal transfer, as well as for several potentially host-lethal proteins encoded by the kilA, kilB, and kilC loci. While kilB is known to be involved in conjugal transfer, the functions of kilA and kilC are unknown. The coregulation of kilA and kilC with replication and transfer genes indicates a possible role in the maintenance or broad host range of RK2. In this work, we found that a fourth kil locus, designated kilE, is located in the kb 2.4 to 4.5 region of RK2 and is regulated as part of the kil-kor regulon. The cloned kilE locus cannot be maintained in Escherichia coli host cells, unless korA or korC is also present in trans to control its expression. The nucleotide sequence of the kilE region revealed two potential multicistronic operons. The kleA operon consists of two genes, kleA and kleB, predicted to encode polypeptide products with molecular masses of 8.7 and 7.6 kDa, respectively. The kleC operon contains four genes, kleC, kleD, kleE, and kleF, with predicted products of 9.2, 8.0, 12.2, and 11.3 kDa, respectively. To identify the polypeptide products, each gene was cloned downstream of the phage T7 phi 10 promoter and expressed in vivo in the presence of T7 RNA polymerase. A polypeptide product of the expected size was observed for all six kle genes. In addition, kleF expressed a second polypeptide of 6 kDa that most likely results from the use of a predicted internal translational start site. The kleA and kleC genes are each preceded by sequences resembling strong sigma 70 promoters. Primer extension analysis revealed that the putative kleA and kleC promoters are functional in E. coli and that transcription is initiated at the expected nucleotides. The abundance of transcripts initiated in vivo from both the kleA and kleC promoters was reduced in cells containing korA or korC. When korA and korC were present together, they appeared to act synergistically in reducing the level of transcripts from both promoters. The kleA and kleC promoter regions are highly homologous and contain two palindromic sequences (A and C) that are the predicted targets for KorA and KorC proteins. DNA binding studies showed that protein extracts from korA-containing E. coli cells specifically retarded the electrophoretic mobility of DNA fragments containing palindrome A. Extracts from korC-containing cells altered the mobility of DNA fragments containing palindrome C. These results show that KorA and KorC both act as repressors of the kleAand kleC promoters. In the absence of korA and korC, expression of the cloned kleA operon was lethal to E.coli cells, whereas the cloned kleC operon gave rise to slowly growing, unhealthy colonies. Both phenotypes depended on at least one structural gene in each operon, suggesting that the operons encode genes whose products interact with critical host functions required for normal growth and viability. Thus, the kilA, kilC, and kilE loci of RK2 constitute a cluster of at least 10 genes that are coregulated with the plasmid replication initiator and the conjugal transfer system. Their potential toxicity to the host cell indicates that RK2 is able to establish a variety of intimate plasmid-host interactions that may be important to its survival in nature.

Amino Acid Sequence↗
PubMed1979

Reversible translocation of antibiotic resistance determinants in Salmonella ordonez.

Salmonella ordonez (BM 2000) codes for kanamycin (Km, aphA), ampicillin (Ap), streptomycin (SmSp:aadA and Sm:aphC), chloramphenicol (Cm), tetracycline (Tc) and sulfonamide (Su) resistances and for production of colicin Ib (Cib). Genetical analysis by incompatibility testing, conjugation, transformation and physical studies using electron microscopy, agarose gel electrophoresis, led us to associate the Km and Cib characters to a 98.7 kilobase (kb) IncI1 plasmid (pIP565), and the Sm (aphC) and Su determinants to a 8.3 kb plasmid (pIP605). The ApCmSmSp(aadA)SuTc determinants were not associated in BM2000 S. ordonez with a plasmid structure. Following conjugation of S. ordonez to E. coli, the ApCmSmSpSuTc determinants were found stably associated with a single plasmid structure (pIP173, 127.5 kb) belonging to IncI1 group. Agarose gel electrophoresis of plasmid DNA restriction endonuclease digests and electron microscopy heteroduplex analysis showed that the acquisition of the ApCmSmSpSuTc determinants resulted from the insertion into pIP565 of a 28.8 kb DNA sequence. This sequence coding for ApCmSmSpSuTu resistances in S. ordonez could be translocated either to pIP565 plasmid or to several IncI1 plasmids but never to plasmids belonging to IncW, IncP or IncFII, suggesting the existence of specific sequences on the IncI1 receptor plasmids. Moreover, R-determinants were translocated back "en bloc" from pIP173 to the chromosome of a susceptible S. ordanez. The results were consistent with the presence in BM2000 S. ordonez chromosomal DNA of an integrated translocatable sequence encoding ApCmSmSpSuTc resistances. Such a structural association could account for the stability of these resistances in the Salmonella ordonez serotype.

Ampicillin↗
PubMed2001

Change of plasmid DNA structure, hypermethylation, and Lon-proteolysis as steps in a replicative cascade.

I have defined conditions under which RepFIC plasmid DNA can be maintained in a state of lowered helical density. In exponentially growing cultures, the DNA of lowered helical density is present in small amounts but never totally absent, suggesting that it is a normal variant of plasmid maintenance. It is fully methylated at frequent sites by dam-methyltransferase, some not previously recognized, further suggesting that the variant is a precursor of replication. The low-helical density plasmid is present in dam hosts, indicating that methylation is not essential for the change in helical density. The lowered helical density is stabilized in lon hosts, suggesting that Lon-protease may remove both the protein(s) that lower the helical density and the dam-methyltransferase after each round of replication.

ATP-Dependent Proteases↗
PubMed1983

DNA restriction--modification genes of phage P1 and plasmid p15B. Structure and in vitro transcription.

The EcoP1 and EcoP15 DNA restriction-modification systems are coded by the related P1 prophage and p15B plasmid. We have examined the organization of the genes for these systems using P1 itself, "P1-P15" hybrid phages expressing the EcoP15 restriction specificity of p15B and cloned restriction fragments derived from these phage DNAs. The results of transposon mutagenesis, restriction cleavage analysis. DNA heteroduplex analysis and in vitro transcription mapping allow the following conclusions to be drawn concerning the structural genes. (1) All of the genetic information necessary to specify either system is contained within a contiguous DNA segment of 5 x 10(3) bases which encodes two genes. One of them, necessary for both restriction and modification, we call mod and the other, required only for restriction (together with mod), we call res. (2) The res gene is about 2.8 x 10(3) bases long and at the heteroduplex level is largely identical for P1 and P15: it shows a small region of partial nonhomology and some restriction cleavage site differences. The mod gene is about 2.2 x 10(3) bases long and contains a 1.2 x 10(3) base long region of non-homology between P1 and P15 toward the N-terminus of the gene. The rest of the gene at this level of analysis is identical for the two systems. (3) Each of the genes is transcribed in vitro from its own promoter. It is possible that the res gene is also transcribed by readthrough from the mod promoter.

Coliphages↗
PubMed1988

The S1-sensitive form of d(C-T)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids.

Homopurine-homopyrimidine sequences that flank certain actively transcribed genes are hypersensitive to single strand-specific nucleases such as S1. This has raised the possibility that an unusual structure exists in these regions that might be involved in recognition or regulation. Several of these sequences, including d(C-T)n.d(A-G)n, are known to undergo a transition in plasmids to an underwound state that is hypersensitive to single strand-specific nucleases; this transition occurs under conditions of moderately acid pH and negative supercoiling. Chemical probes were used to examine the reactivity of a restriction fragment from a human U1 gene containing the sequence d(C-T)18.d(A-G)18 as a function of supercoiling and pH, and thus analyze the structure in this region. Hyperreactivity was seen in the center and at one end of the (C-T)n tract, and continuously from the center to the same end of the (A-G)n tract, in the presence of supercoiling and pH less than or equal to 6.0. These results provide strong support for a triple-helical model recently proposed for these sequences and are inconsistent with other proposed structures.

Base Sequence↗
PubMed1979

Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function.

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.

Conjugation, Genetic↗
PubMed1991

Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7.

Detailed restriction and nucleotide sequence analysis of the Pseudomonas putida TOL plasmid pDK1 xylE gene revealed significant homology with isofunctional xylE (81.5%) and nahH (78.0%) genes from the TOL pWW0 and NAH7 plasmids. The highest degrees of nucleotide and apparent amino acid conservation (82.2 and 86.4%, respectively) among all three genes were found to exist within a region comprising 264 nucleotides encoding the C terminus. A comparison of localized regions revealed significantly greater homology between xylEpWW0 and xylEpDK1 within the C-terminal region, whereas xylEpWW0 and nahH showed greater similarity at the N terminus. The possibility that xylEpWW0 may represent a genetic hybrid of xylEpDK1 and nahH is discussed.

Amino Acid Sequence↗
PubMed1994

Gene cloning in Clostridium difficile using Tn916 as a shuttle conjugative transposon.

A pBR322-based vector, pCI195, containing a 4.2-kb region of the conjugative transposon Tn919 was used as a vector for gene cloning in Clostridium difficile. The plasmid was found to integrate into the chromosome of a Bacillus subtilis strain that contained Tn916 delta E. Southern blot analysis of the recombinant demonstrated that pCI195 had inserted into Tn916 delta E by a recombination event. The transposon::plasmid structure could be transferred, by filter mating, from B. subtilis to C. difficile (at a frequency of 10(-8) per donor), where it entered the chromosome at a specific site. Segregation of plasmid and transposon markers was observed on transfer, although the Tn916 delta E::pCI195 was stably maintained in C. difficile. To demonstrate that pCI195 could be used for gene cloning in C. difficile, a 1.1-kb fragment of the C. difficile toxin B gene was cloned into pCI195 to generate pPPM100. Tn916 delta E::pPPM100 was transferred into a nontoxigenic C. difficile strain by filter mating, where it entered the genome at a specific site. pCI195 should be useful as a general cloning vector for C. difficile, as the transposon::plasmid structure could be transferred to different C. difficile strains. This is the first report of gene cloning in C. difficile.

Bacillus subtilis↗
PubMed1999

Improved immunogenicity of HIV-1 epitopes in HBsAg chimeric DNA vaccine plasmids by structural mutations of HBsAg.

To improve the immunogenicity of epitopes from the envelope protein of HIV-1, we have developed gene gun-delivered subunit DNA vaccines by inserting the sequences encoding the V3 region into the hepatitis B virus (HBV) envelope gene, often called the surface antigen (HBsAg). We have examined the possibility of modifying the immune response to V3 by introducing modifications into the carrier HBsAg in gene gun DNA immunization of mice. In some plasmid constructions, the V3 sequence was introduced into the preS2 region of the HBsAg. Although this region is not present in all protein subunits of the HBsAg particles produced, abolishing the internal translational initiation site for the S protein had no effect on the immune response to V3. Expression of V3 at the N-terminal or C-terminal part of the HBsAg protein resulted in equal anti-V3 antibody and cytotoxic T-lymphocyte (CTL) responses. However, elimination of secretion by single amino-acid mutations in the HBsAg decreased the anti-HBsAg antibody response but enhanced the anti-V3 antibody response. In contrast, the CTL response to V3 was independent of the structural mutations but could be improved by a total deletion of the HBsAg sequence part. Thus, the immune response to heterologous epitopes can be altered by modifications in the carrier HBsAg protein. Modifications of the HBsAg carrier might interfere with the dominant immune response to the HBsAg epitopes, allowing better antibody induction to less immunogenic foreign epitopes. However, for induction of CTL responses, the expression of minimal epitopes may be advantageous.

Animals↗
PubMed1990

Agrobacterium plasmids encode structurally and functionally different loci for catabolism of agrocinopine-type opines.

Agrobacterium tumefaciens strains C58, T37, K827 and J73, A. rhizogenes strains A4 and 15834, and A. radiobacter strain K299 were all susceptible to agrocin 84 and this sensitivity was enhanced in each case by addition of agrocinopines A and B. Analysis of transconjugants showed that sensitivity of strain A4 to agrocin 84 was encoded by pArA4a and not by the rhizogenic plasmid, pRiA4. The acc region of the A. tumefaciens nopaline-type Ti plasmid pTiC58, contained on the recombinant plasmid pTHH206, hybridized strongly to restriction fragments of plasmids from strains T37, K827, J73 and K299. Hybridizing fragment patterns generated with BamHI and EcoRI were identical among the four Ti plasmids while pAtK299 showed restriction fragment length polymorphisms at acc with the two enzymes. At moderate stringency, the pTiC58 acc region hybridized weakly to a single restriction fragment from the Ar plasmid of A. rhizogenes strain A4, but not to pTiBo542, which encodes catabolism of the closely related opines agrocinopines C and D. Plasmid pAtK84b of A. radiobacter strain K84 is induced for conjugal transfer by agrocinopines A and B. However, no hybridization was detected between this plasmid and acc from pTiC58 under conditions of moderate stringency. Like pTiC58, pAtK84b conferred transport of agrocinopines A and B on its host bacteria despite the absence of detectable sequence homology with the pTiC58-derived acc probe. However, unlike pTiC58, pAtK84b failed to confer sensitivity to or uptake of agrocin 84 on its bacterial host. These results indicate that at least four distinguishable systems exist for catabolism of the two agrocinopine opine families with the prototype locus, exemplified by acc from pTiC58, being strongly conserved among nopaline-type Ti plasmids.

Adenine Nucleotides↗
PubMed1995

The influence of a metastable structure in plasmid primer RNA on antisense RNA binding kinetics.

Replication of the ColE1 group plasmids is kinetically regulated by the interaction between plasmid-encoded primer RNA II and antisense RNA I. The binding is dependent on alternative RNA II conformations, formed during the transcription, and effectively inhibits the primer function within some time interval. In this paper, the folding pathways for the wild type and copy number mutants of ColE1 RNA II are studied using simulations by a genetic algorithm. The simulated pathways reveal a transient formation of a metastable structure, which is stabilized by copy number mutations. The folding kinetics of the proposed conformational transitions is calculated using a model of a multistep refolding process with elementary steps of double-helical stem formation or disruption. The approximation shows that the lifetime of the metastable structure is relatively long and is considerably increased in the mutants, resulting in a delay of the formation of the stable RNA II structure, which is the most sensitive to the inhibition by the antisense RNA I. Thus the effect of copy number mutations can be interpreted as a compression of the time window of effective inhibition due to an increased time spent by the RNA II in the metastable state. The implications of metastable foldings in RNA functioning are discussed.

Algorithms↗
PubMed1990

Expression of plasmid-encoded structural proteins permits engineering of bacteriophage T4 assembly.

A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48. The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage. The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage. Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates. Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate. The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein. Tests with a deleted variant of gp48 were inconclusive because the protein was inactive. A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system. This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length. The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction.

Chromosome Deletion↗
PubMedDate not supplied

Novel method for covalent fluorescent labeling of plasmid DNA that maintains structural integrity of the plasmid.

We have developed a chemical strategy for covalent coupling of fluorophores to plasmid DNA. A p-azido-tetrafluoro-benzyl-lissamine conjugate was synthesized and purified. This conjugate was used to covalently associate fluorescent molecules to plasmid DNA by photoactivation. In contrast to nick-translated plasmid DNA, plasmid-lissamine conjugates appeared on gel as supercoiled DNA. Reporter gene was expressed after transfection of the plasmid-lissamine conjugates in NIH 3T3 cells, although gene transfer efficiency was decreased by 60% as compared with unlabeled DNA. Intracellular traffic of plasmid-lissamine conjugates was studied in transfected cells. After cytoplasmic microinjection, fluorescent plasmid did not diffuse from the site of injection and appeared to be progressively degraded in the cytoplasm.

3T3 Cells↗
PubMed2003

Effect of phenol on ultra structure and plasmid DNA of Xanthomonas oryzae pv. oryzae.

Most phenolic substances of plant origin are toxic to microorganisms and they confer some degree of protection to plants against phytopathogens. Xanthomonas oryzae pv. oryzae, bacterial blight pathogen of rice (Oryza sativa) was treated with phenol (monohydroxy benzene) and its effects on the morphology and cytological changes of the bacterium were studied. Total lysis of cells occurred with 5 mM conc of phenol while at 2 mM conc, the cell walls became rough and cell contents started shrinking. Plasmids isolated from both treated (2 mM) and control cells did not show any marked difference under electron microscope except that they differed in their quantity and might influence pathogenicity.

Chromosomes, Bacterial↗