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At least 469 records · Page 26Linked to original sources
PubMed1994

Conjugal mobilization of plasmid DNA: termination frequency at the origin of transfer of plasmid R1162.

Conjugal transfer of plasmid R1162 is initiated and terminated at a 38-bp origin of transfer (oriT). Plasmids containing two directly repeated copies of oriT were used to determine the actual frequency of termination at this site. This frequency was calculated both for oriTnic, a mutated origin that cannot act as the initiation site of transfer, and for an unmutated oriT where transfer had been initiated. In both cases, the termination frequency decreased as the distance between the initiation and termination sites became greater and was significantly less than one for plasmids the size of R1162. A substantial proportion of recipient cells received more than one plasmid copy during transfer. Our results indicate that termination is inefficient but that this is partly compensated for by the transmission of multiple plasmid copies.

Base Sequence↗
PubMed2000

Mobilization of chimeric oriT plasmids by F and R100-1: role of relaxosome formation in defining plasmid specificity.

Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at nic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage at nic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriT allowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).

Bacterial Proteins↗
PubMed1991

[The ultrastructure of plasmid-containing and plasmid-free Salmonella derby cells].

The comparative electron-microscopic study of S. derby plasmid-containing and plasmid-free cells has revealed certain differences in their structures: These structural differences are always accompanied by changes in the form and size of the cells, the form of the cell wall with the appearance of fimbria-like processes, depending on the presence or absence of S. derby R-plasmid in the cells. These differences in the morphology and ultrastructure of S. derby cells, associated with the R-plasmid, are of interest in the study of molecular mechanisms of plasmid action on the development of various forms of whole cells and individual bacterial structures, which play an important role in the cell function.

Cell Wall↗
PubMedDate not supplied

Haemophilus parainfluenzae causing sexually transmitted urethritis. Report of a case and evidence for a beta-lactamase plasmid mobilizable to Escherichia coli by an Inc-W plasmid.

A multiple antibiotic resistance, beta-lactamase-producing strain of Haemophilus parainfluenzae was isolated from a patient with sexually transmitted urethritis that was contracted in Northwest Africa. The strain was found to harbor a small (3.2 megadaltons) plasmid encoding for beta-lactamase production, which was successfully mobilized to Escherichia coli in triparental mating experiments by means of a broad host-range Inc-W conjugative plasmid. Since H. parainfluenzae is believed to be a source and reservoir for the spread of beta-lactamase plasmids to other bacterial species, such a plasmid mobilization may suggest a new possible means for resistance plasmid dissemination.

Adult↗
PubMed1980

[Genetic control of plasmid F' formation. II. Role of recipient recA- in the process of plasmid F' formation].

We have chosen the process of F'-plasmid formation as a model for a study of the illegitimate, recA-independent recombination. Our genetic data showed that F'-formation was under genetic control of the Hfr donor cells. To determine the role of recipient cells in the process of F-prime formation we compared the pattern of transconjugant formation in the crosses of Hfr and F' donors with the recA-nalA+ and recA-nalA- recipients, nalA- mutation in the recipient lead to the sharp decrease of the total yield of transconjugants selected for proximal markers and decrease the fraction of conjugative plasmids in progeny. The plasmids inherited in the nalA- recipient in HfrxF- crosses possess only proximal selected markers while the appropriate distribution of proximal and more distal markers took place in the nalA+ recipient. The homogeneity for the sensitivity of nalA- progeny to the male specific phages in comparison with the nalA+ transconjugants was also observed. The data obtained show that the nalA- mutation has a strong influence on the process of final inheritance and probably formation of F'-plasmids in the recipient cells. The extent of the influence of the nalA- mutation on the pattern of F'-plasmid inheritance was dependent upon the primary F' structure formed (or existed) in the donor cell.

Conjugation, Genetic↗
PubMed1994

Requirements for mobilization of plasmids RSF1010 and ColE1 by the IncW plasmid R388: trwB and RP4 traG are interchangeable.

Mobilization of plasmid RSF1010 by the IncW plasmid R388 requires the genes involved in W pilus synthesis plus trwB. traG of the IncP plasmid RP4 can substitute for trwB in RSF1010 mobilization by R388 but not in self-transfer of R388. This result suggests a dual specificity of TrwB-like proteins in conjugation. The same genetic requirements were found for R388 to mobilize the unrelated plasmid ColE1.

Amino Acid Sequence↗
PubMed2000

Construction of a large plasmid lacking linearizing single restriction sites by simultaneous in vivo recombination and plasmid shuffling in yeast.

Creation of large ( approximately 15 kb) recombinant plasmids can be done in a single step by co-transformation of yeast cells with a partial restriction digest of a plasmid vector and a linear insert whose ends overlap one of the vector restriction sites. This method is used to generate a plasmid expressing the Saccharomyces cerevisiae rRNA genes containing the Ca.LSU group I intron ribozyme from Candida albicans. This plasmid expresses functional rRNA and ribozyme.

Candida albicans↗
PubMed1994

Host-inhibitory functions encoded by promiscuous plasmids. Transient arrest of Escherichia coli segregants that fail to inherit plasmid RK2.

An effective strategy for a plasmid to be maintained in a growing population of bacteria is to prevent the proliferation of newly formed daughter cells that lack the plasmid. In this study, we determined that the promiscuous, incompatibility group P plasmid RK2 encodes such a system. To induce large numbers of RK2-free segregants of an RK2-containing host, we designed a genetic system that made RK2 replication temperature-sensitive. A double amber mutant of the trfA replication initiator gene was constructed to complement a trfA deletion mutant of an otherwise wild-type RK2 in an Escherichia coli supD(Ts) strain. At the permissive temperature (30 degrees C), RK2 was maintained stably in the strain. Shifting the cells to the non-permissive temperature (42 degrees C) resulted in the nearly synchronous appearance of cells lacking RK2. We found that the number of viable cells rose only slowly over a period of six hours, during which time the cells formed long filaments reaching 20 to 40 times the length of a normal E. coli cell. After six hours, the arrested cells regained the ability to divide and multiply exponentially, and the filaments were eventually reduced to normal-sized cells. Neither arrest nor filamentation required the host recA function. Inhibiting the replication of an RK2 mutant deleted for the par stability locus also induced the arrest of segregants lacking the plasmid, but the arrested cells were not filamented. Our results demonstrate that RK2 encodes at least two functions that are activated in a plasmidless segregant: (1) a filamentation-inducing function specified by the par stability locus, and (2) a post-segregational arrest function (psa) that inhibits the proliferation of plasmidless segregants independent of par. We discuss the possible roles of these functions in the stable maintenance of RK2.

Base Sequence↗
PubMed1997

The mobilization protein, MobM, of the streptococcal plasmid pMV158 specifically cleaves supercoiled DNA at the plasmid oriT.

The streptococcal plasmid pMV158 replicates by the rolling circle mechanism. It encodes a relaxase protein of 494 residues, termed MobM, involved in conjugative mobilization. MobM protein was overproduced, purified, and shown specifically to relax supercoiled pMV158 DNA. The 5'-end and the 3'-end of the nick site introduced by MobM have been determined by sequencing and by primer extension analysis. The nucleophilic attack exerted by MobM is in the 5'-GpT-3' dinucleotide, within the sequence 5'-TAGTGTG/TTA-3'. Upon cleavage, MobM protein remains tightly associated with its target DNA, probably through a covalent bond. The pMV158 oriT did not exhibit homologies with known origins of transfer of plasmids from Gram-negative bacteria. However, several plasmids from Gram-positive hosts have a region identical or very similar to the pMV158 oriT. To our knowledge, this is the first demonstration of a relaxase activity of a mobilization protein from a plasmid replicating by the rolling circle mechanism.

Amino Acid Sequence↗
PubMed2000

Antirestriction protein Ard (Type C) encoded by IncW plasmid pSa has a high similarity to the "protein transport" domain of TraC1 primase of promiscuous plasmid RP4.

The IncW plasmid pSa contains the gene ard encoding an antirestriction function that is specific for type I restriction and modification systems. The nucleotide sequence of ard was determined and an appropriate polypeptide of about 33 kDa was identified in Escherichia coli T7 expression system. Analysis of deduced amino acid sequence of Ard encoded by pSa revealed that this protein has no significant similarities with the known Ard proteins (ArdA and ArdB types) except the "antirestriction" motif (14 amino acid residues in length) conserved for all known Ard proteins. This finding suggests that pSa Ard may be classified as a new type of Ard proteins which we designated ArdC. The remarkable feature of ArdC is that it has a high degree of similarity (about 38 % identity) to the N-terminal region of RP4 TraC1 primase which includes about 300 amino acid residues and seems to be essential for binding to the single-stranded DNA and TraC1 protein transport to the recipient cells during the conjugal transfer of plasmid DNA. ArdC also binds to single-stranded DNA. In addition, this protein is able in vitro to protect the single-stranded but not double-stranded plasmid DNA against the activity of type II restriction endonuclease HhaI that cleaves both single and double-stranded DNA. We suggest that like TraC1, ArdC would be transported as a result of their interaction with the single-stranded DNA of transferred plasmid strand during conjugative passage through the cell envelope to the recipient bacterium. Such properties of ArdC protein might be useful to protect immediately the incoming single-stranded DNA from the host endonucleases.

Amino Acid Sequence↗
PubMed1994

Restriction map of the Salmonella enteritidis virulence plasmid and its homology with the plasmid of Salmonella typhimurium.

A restriction map of the 61 kb virulence plasmid of Salmonella enteritidis was constructed and compared with the Salmonella typhimurium virulence plasmid map. Polymerase chain reaction was used to detect a region homologous to rck gene in the S. enteritidis plasmid which was localized in the map by hybridization. Regions homologous to traT and vagD/vagC loci were not detected in this plasmid by this method.

Base Sequence↗
PubMed1995

Self-transmissible nif plasmid (pEA9) of Enterobacter agglomerans 339: molecular cloning and evidence for the existence of similar nif clusters on dissimilar plasmids in Enterobacter strains.

A cosmid library was generated to the 200-kb self-transmissible nif plasmid pEA9 isolated from Enterobacter agglomerans 339. The cosmid clone identified to contain the complete nif cluster was used to determine the nif gene organization and the physical map. The restriction pattern and nif gene organization of this nif cluster showed remarkable similarities to the nif cluster identified on the 110-kb plasmid pEA3 of Enterobacter agglomerans 333. Nucleotide sequence of several randomly selected regions of the nif cluster of pEA9 showed 96% similarity when compared to the known sequences of the nif cluster of pEA3. However, the homology ended abruptly at the flanking regions of the nif clusters and no similarity could be detected with the rest of the DNA of these plasmids. This reveals the existence of similar nif clusters on dissimilar plasmids, implying the horizontal transfer of the entire nif gene cluster.

Bacterial Proteins↗
PubMed1997

Identification of plasmids in the genus Marinococcus and complete nucleotide sequence of plasmid pPL1 from Marinococcus halophilus.

Several plasmids were detected in the Gram-positive halophilic eubacterium Marinococcus halophilus and in the related strain M52. The complete nucleotide sequence (3874 bp) of one of these plasmids, pPL1, was determined. Four major open reading frames were identified. Whereas orf3 and orf4 showed no sequence similarities to known proteins, rep displayed a high sequence similarity to replication proteins of rolling circle plasmids. Upstream of this ORF, a sequence resembling the double-strand origin was detected. A region probably constituting the single-strand origin was identified. The ORF mob showed sequence similarity with Mob proteins of rolling circle plasmids. The observed characteristics suggest that pPL1 replicates according to the rolling circle mechanism.

Amino Acid Sequence↗
PubMed1998

Characterization of a theta plasmid replicon with homology to all four large plasmids of Bacillus megaterium QM B1551.

A replicon from one of an array of seven indigenous compatible plasmids of Bacillus megaterium QM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bp HindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3' end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act in trans. A small region with similarity to the B. subtilis chromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressed rep gene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of a B. megaterium replicon.

Amino Acid Sequence↗
PubMed1999

Dgp1 and Dfp1 are closely related plasmids in the Dictyostelium Ddp2 plasmid family.

Dictyostelium plasmids Dgp1 and Dfp1, two members of the Ddp2 plasmid family, are 86% identical in nucleotide sequence. These small (4481 and 5015 bp), high copy number, nuclear plasmids carry both a gene homologous to the Ddp2 rep gene and a long 0.47- to 0. 48-kb inverted repeat region. Their Rep proteins are 82.8% identical in amino acid sequence and carry all 10 of the conserved peptide sequence motifs found in the Ddp2 family Rep proteins. Unlike other members of this family, Dgp1 carries two copies and Dfp1 carries four copies of a 162- to 166-bp direct repeat element. Both the direct and inverted repeat elements, as well as the promoter of the rep gene, are highly conserved (81 to 90% identical) between Dgp1 and Dfp1. In contrast, these regions are not highly conserved and the Rep proteins are only about 40% identical among the other known members of the plasmid family.

Amino Acid Sequence↗
PubMed1992

An expression vector system providing plasmid stability and conditional suicide of plasmid-containing cells.

A cloning vector system was constructed on the basis of the pBR322 derivative pEG1 by introducing the whole parB locus of plasmid R1 cloned behind the promoter of the alkaline phosphatase gene (phoA) of Escherichia coli. The parB locus in combination with the phoA promoter ensures both (i) plasmid stabilization due to the post-segregational killing of plasmid-free cells during growth and (ii) killing of the cells induced by the potential environmental signal phosphate limitation. This vector, therefore, appears to be a model system for increasing the stability of recombinant plasmids and for decreasing the potential risks in the application of recombinant bacteria in industrial fermentations.

Alkaline Phosphatase↗
PubMed1995

Plasmid and host functions required for lambda plasmid replication carried out by the inherited replication complex.

We have shown previously that in amino acid-starved, relaxed (rel-) mutants of Escherichia coli replication of the lambda plasmid occurs via the lambda O-containing replication complex (RC) that was assembled prior to the onset of amino acid starvation and is inherited by one of the two daughter plasmid circles in each replication cycle. This replication is regulated neither by binding of the lambda O initiator to ori lambda, nor by the lambda Cro-mediated repression. Here we show that it is dependent on both RNA polymerase and DnaA functions, which is consistent with our recent finding that transcriptional activation of ori lambda is under the control of DnaA. In the system studied, DnaA-regulated transcriptional activation of ori lambda seems to be the only rate-limiting process. The lambda plasmid replication mediated by the inherited RC appeared to be independent of the functions of lambda P and DnaJ required in RC assembly In vitro experiments performed by others suggest that DnaJ first binds to the ori lambda-bound lambda O-lambda P-DnaB pre-primosome and subsequently lambda P complexed with DnaJ is preferentially recognized by DnaK-GrpE; chaperone-mediated rearrangement of this structure relieves DnaB helicase of lambda P inhibition. Recently we proposed that this process is directly coupled to the insertion of the pre-primosome between DNA strands transiently separated by transcription. This last-mentioned process may be required in lambda plasmid replication mediated by the inherited RC, which appeared in turn to be dependent on DnaK and GrpE functions.

Bacterial Proteins↗
PubMed1984

Plasmids containing one inverted repeat of Tn21 can fuse with other plasmids in the presence of Tn21 transposase.

In the presence of the Tn21 transposase, plasmids that contain a single Tn21 inverted repeat sequence fuse efficiently with other plasmids. This reaction occurs in recA strains, is independent of the transposon-encoded resolution system, and results in insertions into different sites in the recipient plasmid. All fusion products studied contained at least one complete copy of the donor plasmid; most also contained some duplication of it as well. The data are consistent with processive models of transposition.

Base Sequence↗