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The fluorescent dye, diamidinophenylindole-dihydrochloride (DAPI) can be added to CsCl gradients to enhance the density resolution of DNA species, independent of their topological configurations. When Proteus mirabilis and Escherichia coli strains carrying an RP4::Mucts plasmid were examined with the use of such a technique, it was found that after thermal induction of the prophage essentially al of the plasmid DNA became associated with the chromosome. This quantitative association is detergent-RNase- and pronase-resistant and dependent on the expression of Mu genes. The association is temporally, and probably functionally, correlated with the onset of Mu DNA replication. Genetic studies with F'::mini Mu plasmids indicate that some of the association results in stable Hfr formation, and does not require the product of Mu gene B.
Mol Gen Genet 1980
PMID:Fate of plasmids containing Mu DNA: chromosome association and mobilization. 645 Aug 74

The structural instability exhibited by IncP-1 plasmids in Pseudomonas aeruginosa strain PAT was shown to be Rec+ dependent and involved interaction with the resident plasmid pVS1. Structural instability resulted from deletion of plasmid deoxyribonucleic acid at a frequency of ca. 10(-2)/cell per generation. Deletants could be stabilized by transduction into P. aeruginosa strain PAO, but in strain PAT deletants had only a transient existence, as continued deletion led eventually to the loss of the entire plasmid. The patterns of markers lost in PAT were used to demonstrate a marker order for R68 similar to that published elsewhere for RP4 (Barth and Grinter, J. Mol. Biol. 113:455-474, 1977), except that only one Tra region was found. R68 also exhibited Rec+-dependent structural instability in PAO(pVS1) derivatives but, unlike the case in PAT, instability was not accompanied by chromosome mobilization. We isolated deletants of pVS1 which were unable to promote structural instability.
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PMID:Structural instability of IncP-1 plasmids in Pseudomonas aeruginosa PAT involves interaction with plasmid pVS1. 677 92

We examined expression of the megaplasmid pRme41b of Rhizobium meliloti in two different Rhizobium sp. Strains and in Agrobacterium tumefaciens. Transfer of pRme41b into these bacteria was facilitated by insertion of a recombinant plasmid coding for mobilization functions of RP4 into the nif region (Kondorosi, A., E. Kondorosi, C.E. Pankhurst, W. J. Broughton, and Z. Banfalvi, 1982, Mol. Gen. Genet., 188:433-439). In all cases, transconjugants formed nodule-like structures on the roots of Medicago sativa. These structures were largely composed of meristematic cells but they were not invaded by bacteria. Bacteria were found only within infection threads in root hairs, and within intercellular spaces of the outermost cells of the structures. The donor strain of R. meliloti containing pAK11 or pAK12 in pRme41b initially produced nodules on M. sativa that did not fix nitrogen (Fix-). In these nodules, bacteria were released from infection threads into the host cells but they did not multiply appreciably. Any bacteroids formed degenerated prematurely. In some cases, however, reversion to a Fix+ phenotype occurred after 4 to 6 wk. Bacteria released into newly infected cells in these nodules showed normal development into bacteriods.
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PMID:Morphology of root nodules and nodule-like structures formed by Rhizobium and Agrobacterium strains containing a Rhizobium meliloti megaplasmid. 688 19

The genes regulating resistance to potassium tellurite were cloned from plasmid pS221 with a broad host range. The studied DNA fragment encodes for the synthesis of two polypeptides with molecular mass of 38 and 43 kD. Metal tellurium crystals were localized in the periplasm of Pseudomonas aeruginosa ML4262 (pBS221) cells by electron microscopy. Results of colony hybridization permit us to propose that the cloned genes of potassium tellurite resistance have sites of homology with plasmids RP4(IncP), R27 (IncH), pBS38, pBS79, and R931(IncP-2).
Mol Gen Mikrobiol Virusol
PMID:[Determinant of plasmid pBS221 resistance to potassium tellurite]. 747 33

Bacterial plasmids are stabilized by a number of different mechanisms. Here we describe the molecular aspects of a group of plasmid-encoded gene systems called the proteic killer gene systems. These systems mediate plasmid maintenance by selectively killing plasmid-free cells (post-segregational killing or plasmid addiction). The group includes ccd of F, parD/pem of R1/R100, parDE of RP4/RK2, and phd/doc of P1. All of these systems encode a stable toxin and an unstable antidote. The antidotes prevent the lethal action of their cognate toxins by forming tight complexes with them. The antidotes are degraded by cellular proteases. Thus, the different decay rates of the toxins and antidotes seem to be the molecular basis of toxin activation in plasmid-free cells. The operons encoding the toxins and antidotes are autoregulated at the level of transcription either by a complex formed by the toxins and the cognate antidotes or by the antidote alone. The cellular targets of the killer proteins have been determined to be DNA gyrase in the case of ccd of F and DnaB in the case of parD of R1. Surprisingly, the Escherichia coli chromosome encodes at least two of these peculiar gene systems.
Mol Microbiol 1995 Jul
PMID:Programmed cell death in bacteria: proteic plasmid stabilization systems. 749 69

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.
Mol Microbiol 1995 Jul
PMID:Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1. 749 70

Comparative analysis of recipient activity of Pseudomonas mallei, Pseudomonas pseudomallei, and Pseudomonas cepacia strains towards naturally occurring and recombinant plasmid replicons was carried out. Autonomic broad host range vector plasmids based on RSF1010(IncQ) and pSa(IncW) replicons as well as integrative vectors based on pSUP202(Co1E1) replicon have been constructed. The study has shown that naturally occurring plasmids RSF1010(IncQ), pSa(IncW), R15(IncN), and RP4(IncP) are being efficiently transferred and stably maintained in investigated Pseudomonas strains. However, recombinant plasmids with the mini-replicon pSa which are stable in Escherichia coli have shown segregative instability in Pseudomonas strains, whereas derivatives of plasmid RSF1010 demonstrated different stability depending on the type of insertion. Plasmid pSUP202 derivative integrative vector pSM525 is efficiently introduced and stably maintained in P. mallei C-5 strain. Two vector systems for genetic manipulations in P.mallei and P.pseudomallei cells have been developed.
Mol Gen Mikrobiol Virusol
PMID:[Pseudomonas mallei and Pseudomonas pseudomallei: introduction and maintenance of natural and recombinant plasmid replicons]. 754 12

Conjugative transfer of DNA that occurs between bacteria also operates between bacteria and higher organisms. The transfer of DNA between Gram-negative bacteria requires initial contact by a sex pilus followed by DNA traversing four membranes (donor plus recipient) using a transmembrane pore. Accumulating evidence suggests that transfer of the T-DNA from Agrobacterium tumefaciens to plants may also occur via a conjugative mechanism. The virB operon of the Ti plasmid exhibits close homologies to genes that are known to encode the pilin subunits and pilin assembly proteins. The proteins encoded by the PilW operon of IncW plasmid R388 share strong similarities (average similarity = 50.8%) with VirB proteins. Similarly, the TraA, TraL and TraC proteins of IncF plasmid F have similarities to VirB2, VirB3 and VirB4 respectively (average similarity = 45.3%). VirB2 protein (12.3 kDa) contains a signal peptidase-I cleavage sequence that generates a polypeptide of 7.2 kDa. Likewise, the 12.8 kDa propilin protein TraA of plasmid F also possesses a peptidase-I cleavage site that generates the 7.2 kDa pilin structural protein. Similar amino acid sequences of the conjugative transfer genes of F, R388 as well as plasmid RP4 and the genes of the ptI operon of Bortedella pertussis suggest the existence of a superfamily of transmembrane proteins adapted to the promiscuous transfer of DNA-protein complexes.
Mol Microbiol 1994 Apr
PMID:Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis. 791 64

The IncP alpha promiscuous plasmid (R18, R68, RK2, RP1 and RP4) comprises 60,099 bp of nucleotide sequence, encoding at least 74 genes. About 40 kb of the genome, designated the IncP core and including all essential replication and transfer functions, can be aligned with equivalent sequences in the IncP beta plasmid R751. The compiled IncP alpha sequence revealed several previously unidentified reading frames that are potential genes. IncP alpha plasmids carry genetic information very efficiently: the coding sequences of the genes are closely packed but rarely overlap, and occupy almost 86% of the genome's nucleotide sequence. All of the 74 genes should be expressed, although there is as yet experimental evidence for expression of only 60 of them. Six examples of tandem-in-frame initiation sites specifying two gene products each are known. Two overlapping gene arrangements occupy different reading frames of the same region. Intergenic regions include most of the 25 promoters; transcripts are usually polycistronic. Translation of most of the open reading frames seems to be initiated independently, each from its own ribosomal binding and initiation site, although, a few cases of coupled translation have been reported. The most frequently used initiation codon is AUG but translation for a few open reading frames begins at GUG or UUG. The most common stop-codon is UGA followed by UAA and then UAG. Regulatory circuits are complex and largely dependent on two components of the central control operon. KorA and KorB are transcriptional repressors controlling at least seven operons. KorA and KorB act synergistically in several cases by recognizing and binding to conserved nucleotide sequences. Twelve KorB binding sites were found around the IncP alpha sequence and these are conserved in R751 (IncP beta) with respect to both sequence and location. Replication of IncP alpha plasmids requires oriV and the plasmid-encoded initiator protein TrfA in combination with the host-encoded replication machinery. Conjugative plasmid transfer depends on two separate regions occupying about half of the genome. The primary segregational stability system designated Par/Mrs consists of a putative site-specific recombinase, a possible partitioning apparatus and a post-segregational lethality mechanism, all encoded in two divergent operons. Proteins related to the products of F sop and P1 par partitioning genes are separately encoded in the central control operon.
J Mol Biol 1994 Jun 24
PMID:Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis. 801 87

The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.
Mol Microbiol 1994 Apr
PMID:Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4. 805 33


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