P1 Plasmid Research Articles

Evidence for positive regulation of plasmid prophage P1 replication: integrative suppression by copy mutants.

Like low-copy-number plasmids including P1 wild type, multicopy P1 mutants (P1 cop, maintained at five to eight copies per chromosome) can suppress the thermosensitive phenotype of an Escherichia coli dnaA host by forming a cointegrate. At 40 degrees C in a dnaA host suppressed by P1 cop, the only copy of P1 is the one in the host chromosome. Trivial explanations of the lack of extrachromosomal copies of P1 cop have been eliminated: (i) during integrative suppression, the P1 cop plasmid does not revert to cop+; (ii) the dnaA+ function of the host is not required to maintain P1 cop at a high copy number; and (iii) integrative recombination does not occur within the region of the plasmid involved in regulation of copy number. Since there are no more copies of the chromosomal origin (now located within the integrated P1 plasmid) than in a P1 cop+-suppressed strain, the extra initiation potential of the P1 cop is not used to provide multiple initiations of the chromosome. When a P1 cop-suppressed dnaA strain was grown at 30 degrees C so that replication could initiate from the chromosomal origin as well as from the P1 origin, multicopy supercoiled P1 DNA was found in the cells. This plasmid DNA was lost again when the temperature was shifted back to 40 degrees C.

Transfer of plasmid RP1 into chemolithotrophic Thiobacillus neapolitanus.

RP1, a broad-host-range incompatibility group P1 plasmid specifying multiple drug resistances, has been transferred into the chemolithotrophic bacterium Thiobacillus neapolitanus. The ability of T. neapolitanus to receive, express, and transmit RP1-encoded antibiotic resistances was examined. The data show that this obligate chemolithotroph can accept, replicate, and express heterologous plasmid DNA from a heterotrophic bacterium.

Partition of unit-copy miniplasmids to daughter cells: II. The partition region of miniplasmid P1 encodes an essential protein and a centromere-like site at which it acts

The stable maintenance of the unit-copy lambda-P1:5R miniplasmid is dependent on adjacent but separable replication (rep) and partition (par) regions of DNA derived from its P1 plasmid parent. The par region consists of an approximately 2.5 X 10(3) base-pair (kb) segment of DNA of which the terminal kb contains the plasmid incompatibility determinant incB. Two of the 14 lambda-P1:5R partition-defective point mutants isolated are amber (nonsense) mutants, showing that a plasmid-encoded protein is essential for proper partition. All of the Par- point mutants are complemented by the wild-type par region in trans. The complementing activity was shown to be an Mr 44,000 protein encoded by the end of the par region distal to incB. Deletion analysis showed that the incB sequence is essential in cis to the plasmid in order that the plasmid be receptive to the par protein. Thus incB appears to be the target site for par protein activity. We propose that the protein binds to incB, forming a complex that is recognized as a substrate for the cellular partition apparatus. The ability of a cloned incB sequence to compete for the par protein or for the cellular partition apparatus accounts for its activity as an incompatibility determinant. The existence of a plasmid-encoded par protein suggests a specific model for equipartition.

On the segregation of IS1-mediated cointegrates between bacteriophage P1 DNA and plasmid pBR322 derivatives

Considerable attention has been paid in recent years to the cointegration of two replicons mediated by mobile genetic elements in prokaryotes [1-3]. It is widely accepted that some transposons, namely Tn3 and its relatives including the IS element yd, form cointegrates as intermediates in their transposition [4-10]. These cointegrates are resolved by a site-specific recombinase encoded by the elements. Since this recombinase also acts as a repressor for its own biosynthesis [7,11], it is assumed to be present in low amounts in all cells harboring Tn3. Therefore, cointegrates are usually unstable, but stable cointegrates are obtained with mutants affected in the resolution process [4,6,7]. However, it is not yet clear whether cointegrates are also prerequisite intermediates in the transposition of other IS elements such as IS/, because cointegrates carrying IS/ as direct repeats at junctions of two replicons have been isolated without introducing a mutation in the elements [12-18]. This report describes stability and segregation processes of IS/ mediated cointegrates which had been formed between the bacteriophage P1 genome and plasmid pBR322 derivatives. 2.1. Strains and media

On the segregation of IS1-mediated cointegrates between bacteriophage P1 DNA and plasmid pBR322 derivatives

On the segregation of IS1-mediated cointegrates between bacteriophage P1 DNA and plasmid pBR322 derivatives

Mutants of plasmid prophage P1 and elevated copy number: isolation and characterization.

A series of mutations of the P1 plasmid prophage that lead to increased copy number was isolated and analyzed. The copy number of the mutants was elevated at least five- to eightfold relative to wild-type P1, as determined by single-cell resistance to antibiotics, activity of enzymes, content of superhelical DNA, and reassociation kinetics. The copy number of two of the mutants was temperature dependent. Based on dominance tests, the mutants fell into two classes, cis specific and recessive. The latter class included a temperature-sensitive copy mutant. The existence of a class of recessive mutants suggests that the replication of the P1 plasmid is negatively regulated.

Cointegrates between bacteriophage P1 DNA and plasmid pBR322 derivatives suggest molecular mechanisms for P1-mediated transduction of small plasmids.

We characterized cointegrates formed in an Escherichia coli rec+ strain between bacteriophage P1 genomes and small plasmids related to pBR322. The partners were, on the one hand, either phage P1 DNA, which carries one copy of IS1, or phage P1-15 DNA, a derivative which lacks the IS1, and, on the other hand, plasmids containing either a split IS1 or no. In the presence of IS1 sequences on both partners, cointegrates were usually formed by reciprocal recombination between SI1 sequences. Cointegrates between P1 and a plasmid carrying no IS1 sequence were formed by transpositional cointegration mediated by IS1 of P1. Cointegrates between P1-15 and small plasmid containing a split IS1 were formed by one of three ways: (a) acquisition of an IS1 by P1-15 followed by reciprocal recombination between IS1 sequences, (b) transpositional cointegration mediated by the split IS1 element, Tn2657, or (c) involvement of the invertible segment carried on P1-15 DNA. Most cointegrates segregated into the small plasmids and phage P1 derivatives. A comparison of the phenomenon studied and of their frequencies allowed us to conclude that cointegrate formation is a molecular mechanism involved in the transduction of plasmids smaller than those packageable into P1 virions, although it does not seem to be the only process used.

A novel role for site-specific recombination in maintenance of bacterial replicons

If daughter copies of unit-copy replicons recombine with each other, a replicon dimer results that cannot be partitioned equally to daughter cells at cell division. We present evidence that dimer formation interferes with plasmid equipartition in the case of a miniplasmid derived from the unit-copy plasmid prophage of bacteriophage P1. Asymmetric partition occurs, leading to a relatively high rate of loss of the plasmid from the growing population. In contrast, the wild-type P1 plasmid is maintained very efficiently in host cells. We show that this efficient maintenance is due to the presence of the loxP-cre site-specific recombination system present on the intact P1 plasmid. This system promotes rapid recombination between two IoxP sites on dimer molecules, resolving them into monomeric substrates for proper partition. We suggest that bacterial replicons that are maintained with great accuracy in recombination-proficient cells might also encode high-efficiency recombination systems.

Lytic cycle replicative forms of bacteriophages P1 and P1 dl: Concatemer forms

The permutedness of bacteriophage P1 chromosomes has implied that concatemers must be lytic cycle chromosomal forms (LCF). We have partially characterized LCF of P1 and a P1 dl, employing isopycnic fractionation in CsCl-ethidium bromide gradients and alkaline sucrose gradient centrifugation. The genetic identity of LCF was established by DNA filter annealings of radioactive LCF. In alkaline sucrose gradients, concatemer strands of LCF between 2 and 10 genome units long were recognized as DNA sedimenting slower than monomer supercoils and faster than single-strand monomers. Fractionation of pulse-labeled LCF showed that by 8 min after induction, most or all of the DNA synthesis proceeds by extension of concatemers. Pulse-chase experiments showed that the concatemer strands are part of persisting LCF. The P1 dl plasmid of M. E. Rae and M. Stodolsky ( Virology, 58, 32–54, 1974 ) was found to contain 133kb, as contrasted with 90 kb for the P1 plasmid. Concatemer strands were generated in thermally induced P1 dl lysogens, and had a higher average sedimentation rate than those of P1. In the P1-infected P1 dl lysogen, the sedimentation patterns of P1 and P1 dl strands were distinct. This fact indicated that recombination between P1 and P1 dl was not the major contributor to the formation of concatemer strands.

Denaturation map of bacteriophage P1 DNA

Electron microscopy of bacteriophage P1 plasmid DNA partially denatured by high pH in the presence of formaldehyde and mounted by the protein monolayer technique revealed a unique denaturation pattern. Alignment with the restriction cleavage map was obtained by comparing the melting pattern of plasmid DNA linearized by a single cut introduced by the restriction enzyme PstI with those of the large HindIII, BglII, and BamHI restriction fragments. Major A + T-rich segments center around map units 3, 21, 46, and 52. Other denatured regions are located around map units 29, 32 (within the invertible segment), 62, 88, and 96. The invertible segment of P1 DNA was precisely mapped within the BamHI-5 and the BglII-5 DNA restriction fragments. The DNA segment melting first and therefore having the highest A + T content spans the P1-specific restriction modification region between map units 0 and 6 of the P1 genome. The Pl-specific recombination site, loxP, is also located in a region of low DNA helix stability. The denaturation pattern of the DNA of a hybrid P1-R plasmid revealed IS 1-mediated transposition of the r-determinant into a site adjacent to the most A + T-rich region on Pl.

Group Y incompatibility and copy control of P1 prophage

We have identified a restriction fragment ( EcoRI-5) of bacteriophage P1 that, when cloned in a λ prophage, expresses incompatibility characteristic of the unit copy P1 plasmid prophage. Lysogens of λ-P1 chimeras in which the P1 fragment is EcoRI-5 fail to maintain P1 or P7 plasmids. In order to study the nature of this incompatibility, we isolated P1 mutants that overcome it. These mutants exhibit an elevated copy number. We provide evidence that the increased copy number results from a defect in a repressor of replication that can be furnished in trans by a chromosomally integrated P1, but not by EcoRI-5 itself. We, therefore, suggest that the incompatibility exerted by EcoRI-5 is not attributable to the represser of replication involved in the above copy control defect. Instead, it could be attributed to the presence of a DNA site required for proper plasmid partition at cell division. The elevated copy number of the P1 mutants would then enable them to compete favorably with the single copy of the cloned EcoRI fragment for a cellular component of the partition apparatus. Thus, incompatibility could be overcome.

Curing of P1 prophage from Escherichia coli K-12 recA(P1) lysogens superinfected with P1 bacteriophage.

Curing of the P1 plasmid prophage in recA(P1) lysogens by superinfection with another P1 phage was specific and independent of immunity and incompatibility expression.

The maintenance of the P1 plasmid prophage

The maintenance of the P1 plasmid prophage

Direct repetition of a 1.2 Md DNA sequence is involved in site-specific recombination by the P1 plasmid R68

R68.45, a mutant R68 plasmid, carries a 1.5 Md DNA insertion near its kanamycin-resistance region. This DNA consists of a 1.2 Md DNA repetition of neighbouring R68-DNA and a 0.3 Md “foreign” DNA fragment that is flanked by this direct DNA repeat. This fragment seems to be involved in the formation of R'68.45 plasmids. Duplication of the 1.2 Md DNA sequence is also involved in site-specific recombination events of RP4. This 1.2 Md DNA fragment has the properties of an IS sequence and is denoted IS8.

Escherichia coli heat-labile enterotoxin: DNA-directed in vitro synthesis and structure.

Escherichia coli heat-labile enterotoxin was synthesized in a cell-free system directed by DNA of the plasmid P307. Synthesis of the toxin, assayed by the elongation induced in Chinese hamster ovary cells, was strongly stimulated by cyclic AMP and occurred at physiological levels of Mg2+ only when the polyamine spermidine was present. Activity was abolished by heat and antisera prepared against the enterotoxins of both E. coli P263 and Vibrio cholera. Tritium-labeled enterotoxin was purified by immunoprecipitation and examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. When gel slices were assayed for the ability to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in erythrocyte ghosts, two peaks were found, one at Mr 26,000 and frequently, but not always, another at Mr 23,000. Detection of radiolabeled protein by fluorography and scintillation counting of gel slices revealed three prominent polypeptides, two corresponding to the peaks having adenylate cyclase-stimulating activity and a further one of Mr 11,500, identical to that of the cholera subunit B. The data suggest that the E. coli heat-labile enterotoxin synthesized in the cell-free system has a subunit structure.

Cistrons encoding Escherichia coli heat-labile toxin

The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied. The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313. Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences. Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons. Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region. The functions of these LT gene products were investigated. The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers. The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity. The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region. Both cistrons are probably transcribed from the same promoter.

Identification of the P1 compatibility and plasmid maintenance locus by a mini P1 lac+-plasmid

pIH1972 is a 23.6-Mdal lac + plasmid derived from P1 dlw. This plasmid is stably maintained and it exhibits absolute incompatibility with P1 in RecA bacteria. A cluster of P1- EcoRI DNA fragments 15, 17, 18, 21, and 23 and part of fragments 14 and 5 are identified in the PI DNA region carried by pIH1972. It is suggested that P1 compatibility and plasmid maintenance genes are carried on the identified 10% segment of the P1 genome.

Rearrangements of Genetic Material in Escherichia coli As Observed on the Bacteriophage P1 Plasmid

IS elements (Starlinger and Saedler 1976; Bukhari et al. 1977) play an important role in structural rearrangements of genetic materials, particularly because of their ability to transpose, either alone or together with linked genetic materials, and to generate deletions and inversions. Possibly, IS elements and transposons (transposable, or Tn, elements) are widespread in nature, and in addition to being driving forces in evolution, they may fulfill yet unknown important functions.

Mechanism of Defective Lysogenization by Phage P1 in a lon− Mutant of Escherichia coli K‐12

Phage P1 cannot lysogenize a lon- mutant of Escherichia coli K-12, which is defective in the regulation of cellular division cycle to result in snake formation (14). P1 mutants, called P1pla, can lysogenize the lon- host. These mutations have been classified into two complementation groups: one is cis-dominant; the other is trans-dominant. A temperature-sensitive lon- mutant was isolated, which exhibited the lon- phenotype at 42 C but not at 33 C. A temperature-shift experiment of the P1-lysogenic derivative of the lon- ts mutant showed lysis of the culture and induction of the phage production. It is proposed that P1 plasmid may be under a certain regulatory circuit of the division cycle of the host bacterium by indirectly regulating the production of P1 immune repressor, or alternatively by directly derepressing the functions of P1 prophage.

Bipartite control of immunity conferred by the related heteroimmune plasmid prophages, P1 and P7 (formerly φAmp)

Coliphages P1 and P7 (formerly known as φAmp) exhibit a number of similarities including sensitivity to a repressor of lytic functions specified by the c1 gene of either prophage. The ability of bacteria harboring one of these plasmid prophages to allow the growth of the other phage is shown to depend upon the product of a viral gene ant that is not closely linked to c1 and that antagonizes repression. The ant gene of P1 and P7 prophage is normally repressed by an immunity-specific repressor, but ant is expressed constitutively in members of a class of virulent mutants. In one such vir mutant of P7, a nonsense ant mutation was obtained. The ant mutation has been separated by recombination from the closely linked cis-dominant vir mutation. The properties of this mutant, among other P1 and P7 mutants affecting immunity, conform to the bipartite scheme of immunity control established in Salmonella phage P22 and independently concluded by J. R. Scott and her collaborators to apply to P1 and P7. The essentially superfluous ant circuit may possibly permit an enlarged scope for parasitism.