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At least 739 records · Page 41Linked to original sources
PubMed1994

Control of ColE2 plasmid replication: negative regulation of the expression of the plasmid-specified initiator protein, Rep, at a posttranscriptional step.

The incA gene of ColE2 is involved in the copy number control and incompatibility. Two promoters were identified around the incA gene. Transcription of the mRNA for the essential plasmid-coded initiator protein (Rep) mainly starts at a site about 140 bp upstream of the initiation codon of the Rep protein. The second transcript (RNA I) of about 115 nucleotides with two stem-and-loop structures is entirely complementary to the 5' untranslated region of the Rep mRNA. By using translational and transcriptional fusions of the rep gene of ColE2 and the lacZ gene of Escherichia coli, the incA gene product was shown to regulate expression of the rep gene at a posttranscriptional step. The results also suggest that the target of the incA gene product is the 5' untranslated region of the Rep mRNA. Deletion analyses reported here show that a region(s) about 17 to 70 bp upstream of the initiation codon of the Rep protein and another region inside the coding frame are important for efficient production of the Rep protein. This suggests that some additional sequence elements other than the initiation codon and the Shine-Dalgarno region and/or a secondary structure of the Rep mRNA are required for efficient production of the Rep protein. These results show that RNA I is an antisense RNA for the Rep mRNA and imply that it might regulate expression of the rep gene at the initiation step of translation by sequestering such additional sequence elements and/or by disrupting RNA secondary structure. We propose that RNA I represents the incA gene product.

Bacterial Proteins↗
PubMed1985

Plasmid-encoded initiation protein is required for activity at all three origins of plasmid R6K DNA replication in vitro.

DNA replication of plasmid R6K initiates at three unique sites, ori alpha, ori beta, and ori gamma. Replicating DNA molecules of a deletion derivative of R6K were synthesized in an in vitro system containing pi protein fraction from cells carrying a mini-R6K derivative that produced only this initiation protein as an R6K-encoded protein and analyzed by electron miscroscopy. Requirement of pi protein for the activity of all these three replication origins in vitro was verified. Frequencies of initiation at the three origins were almost equal.

DNA Replication↗
PubMed1988

Plasmid pColBM-Cl139 does not encode a colicin lysis protein but contains sequences highly homologous to the D protein (resolvase) and the oriV region of the miniF plasmid.

Colicins are usually released from producing cells by so-called lysis proteins. No sequence homologous to the structurally very similar colicin lysis genes was found in the gene cluster cmi cma cbi cba, which determines the activity and immunity proteins of colicin B and M on pColBM-Cl139. Instead, the region upstream of cmi contained sequences that showed 91% homology to the structural gene of protein D (resolvase) and 75.5% homology to the rfsF sequence of the Escherichia coli miniF plasmid. It is concluded that colicins B and M are not released via the activity of lysis proteins and that the highly homologous regions encode a resolvase and its target respectively.

Amino Acid Sequence↗
PubMed2000

Guanine-cytosine rich regions of plasmid DNA can be the target in anti-plasmid effect of phenothiazines.

The antiplasmid effects of promethazine on E. coli is the consequence of special complex formed with a covalently closed circular (ccc) form of plasmid DNA. The exact target in this macromolecule, however, was not clarified until recently. Caffeine and the chemically similar guanosine-5'- monophosphate (GMP) could compete with the antiplasmid effect of promethazine, showing that promethazine or other phenothiazines preferentially bind to xanthine type molecules. Among the xanthines, GMP was more effective at complex-forming than adenosine-5'-monophosphate (AMP). In addition, the Z-DNA was more susceptible than B-DNA. Therefore, one could suppose that guanine-cytosine (G-C) rich regions have higher affinity than adenine-thymine (A-T) rich region on phenothiazines. Because the G-C rich regions have a special role in the DNA stability via three hydrogen bonds, we suppose that these regions could have a key role in some biological effects such as antiplasmid and anticancer activity.

Binding Sites↗
PubMed1997

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene.

The 26 to 28kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse-tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.

Antigens, Bacterial↗
PubMed2006

Crystal structure of pi initiator protein-iteron complex of plasmid R6K: implications for initiation of plasmid DNA replication.

We have determined the crystal structure of a monomeric biologically active form of the pi initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-A resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal alpha4' and the N-terminal alpha4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in alpha4', whereas the alpha4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely alpha2 and alpha5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of pi revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.

Amino Acid Sequence↗
PubMed1984

Virus-plasmid interactions: mutants of bacteriophage T3 that abortively infect plasmid F-containing (F+) strains of Escherichia coli.

Bacteriophage T7 and many closely related phages abortively infect plasmid F-containing (F+) strains of Escherichia coli. However phage T3, which is also closely related to T7, grows normally in F+ hosts. Mutants of phage T3 that, like T7, are subject to F-mediated restriction have been isolated. These T3 mutants lack or are defective in one or both of two genes that are nonessential for phage growth in F-, wild-type strains. Our results show that the products of phage T3 gene 1.1 or 1.2, or both, are essential for growth and suggest that the comparable phage T7 genes are naturally defective in their ability to counteract the inhibitory effects of F-encoded proteins.

DNA Replication↗
PubMed1992

Crosstalk between plasmid vegetative replication and conjugative transfer: repression of the trfA operon by trbA of broad host range plasmid RK2.

Previous deletion and complementation analysis has indicated that the region between trfA and kilBI (trbB) encodes trans-acting factor, designated trbA, required for conjugative transfer of broad host range plasmid RK2. In analysing the nucleotide sequence of this region we have discovered a gene encoding a 12 kDa polypeptide. The predicted amino acid sequence of this protein shows similarity at its C-terminal to KorA from the central control operon of RK2 and at its N-terminal to immunity repressor protein from phage phi 105 of Bacillus subtilis as well as the Sin protein of B. subtilis which regulates alternate developmental processes including sporulation, motility and competence. We show that TrbA represses transcription of both trfA (vegetative replication) and kilBI (trbB) (required for conjugative transfer and whose product has similarity to ComG, required for competence of B. subtilis) and may help to coordinate expression of both sets of functions. This region has similarities to some temperate bacteriophage immunity regions in modulating divergent transcription required for alternative means of propagation.

Amino Acid Sequence↗
PubMed1988

Transfer of beta-lactamase plasmids from Neisseria gonorrhoeae to Neisseria meningitidis and commensal Neisseria species by the 25.2-megadalton conjugative plasmid.

Two highly tetracycline-resistant, beta-lactamase-producing Neisseria gonorrhoeae strains were used as donors for conjugation with Neisseria meningitidis and commensal Neisseria species. We found that both strains were able to transfer the 4.4- and 3.2-megadalton beta-lactamase plasmids to the recipients tested, with frequencies between 10(-1) and 10(-9).

Conjugation, Genetic↗
PubMed2004

Isolation of a minireplicon of the virulence plasmid pXO2 of Bacillus anthracis and characterization of the plasmid-encoded RepS replication protein.

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.

Amino Acid Sequence↗
PubMed1997

Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators.

The xyl genes of Pseudomonas putida TOL plasmid that specify catabolism of toluene and xylenes are organized in four transcriptional units: the upper-operon xylUWCAMBN for conversion of toluene/xylenes into benzoate/alkylbenzoates; the meta-operon xylXYZLTEGFJQKIH, which encodes the enzymes for further conversion of these compounds into Krebs cycle intermediates; and xylS and xylR, which are involved in transcriptional control. The XylS and XylR proteins are members of the XylS/AraC and NtrC families, respectively, of transcriptional regulators. The xylS gene is constitutively expressed at a low level from the Ps2 promoter. The XylS protein is activated by interaction with alkylbenzoates, and this active form stimulates transcription from Pm by sigma70- or sigmaS-containing RNA polymerase (the meta loop). The xylR gene is also expressed constitutively. The XylR protein, which in the absence of effectors binds in a nonactive form to target DNA sequences, is activated by aromatic hydrocarbons and ATP; it subsequently undergoes multimerization and structural changes that result in stimulation of transcription from Pu of the upper operon. This latter process is assisted by the IHF protein and mediated by sigma54-containing RNA polymerase. Once activated, the XylR protein also stimulates transcription from the Ps1 promoter of xylS without interfering with expression from Ps2. This process is assisted by the HU protein and is mediated by sigma54-containing RNA polymerase. As a consequence of hyperexpression of the xylS gene, the XylS protein is hyperproduced and stimulates transcription from Pm even in the absence of effectors (the cascade loop). The two sigma54-dependent promoters are additionally subject to global (catabolite repression) control.

Bacterial Proteins↗
PubMed1975

Relaxation complexes of plasmid DNA and protein. II. Characterization of the proteins associated with the unrelaxed and relaxed complexes of plasmid ColE1.

The proteins of the DNA-protein relaxation complex of plasmid ColE1 were labeled with [3H]leucine by growth of ColE1 containing Escherichia coli cells in the presence of this radioactive labeled amino acid. Three major [3H]leucine-labeled proteins are found associated with the supercoiled DNA in the ColE1 relaxation complex. The molecular weights of these proteins, determined by sodium dodecyl sulfate-acrylamide gel electrophoresis, are 60,000, 16,000, and 11,000, respectively. Induction of relaxation of the supercoiled DNA by treatment of the complex with sodium dodecyl sulfate results in a dissociation of the two smaller proteins from the DNA. The 60,000 protein, however, remains associated specifically with the nicked strand of the open circular DNA. The strand-specific association of this protein with the relaxed DNA resists heat denaturation of the DNA, sedimentation through an alkaline (pH 12.5) sucrose gradient, and centrifugation to equilibrium in an alkaline (pH 12.5) CsCl gradient.

Bacterial Proteins↗
PubMed1987

Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid.

We provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA- and ParB- miniplasmid of R1 pKN1562, without increasing its copy number. The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two proteins whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system determines in cis a basal but detectable stability.

Amino Acid Sequence↗
PubMed1984

The use of mini-Gal plasmids for rapid incompatibility grouping of conjugative R plasmids.

The galactose operon of Escherichia coli K-12 has been used as a phenotypic marker for miniplasmids derived in vitro from R plasmids representing six incompatibility groups. This has enabled the development of a rapid incompatibility typing scheme in which the miniplasmids are used as incompatibility exemplars, their presence in strains being monitored on galactose fermentation indicator media.

Bacteriological Techniques↗
PubMed2005

I. Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine: evaluation of plasmid DNA biodistribution/persistence and integration.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

Animals↗
PubMed1986

Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins.

The minimal beta-replicon of plasmid R6K contains an open reading frame for a 151-amino acid protein in addition to the seven 22-base pair direct nucleotide sequence repeats, the structural gene (pir) for the pi initiation protein, and the beta-origin sequence that have been shown to be required for replication activity. In this work, a site-specific mutation by linker insertion in this putative coding sequence, designated bis, resulted in a nonfunctional beta-replicon. The nonfunctional beta-replicon was complementable in trans and the protein coded by the bis sequence was detected in an immunoblot assay as a hybrid product from a bis-lac z fused gene. The bis gene is not required for a functional alpha or gamma origin replication origin of R6K. A site-specific mutation in the upstream pir gene was shown to lead to a loss of synthesis of the bis product and inactivation of the beta-replicon. Trans-complementation of this mutation for beta-replicon activity required the wild-type sequence of the pir gene joined to the intact bis sequence. These results indicate that the bis product is required for activity specifically of the beta-origin, and its synthesis is coupled in cis to the expression of pi protein from an unaltered pir gene.

Bacterial Proteins↗