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PubMed1988

Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination.

The heritable stability of the multicopy plasmid ColE1 and its natural relatives, requires the presence in the plasmid of a site (cer in ColE1) that acts as a substrate for site-specific recombination, thereby maintaining plasmids in the monomeric state. Multimerization, promoted by homologous recombination, leads to plasmid loss. Here we show that the Escherichia coli chromosome encodes at least two unlinked functions that act on cer and its analogous sites, to promote stabilizing site-specific recombination. One of these functions is encoded by a gene residing on a cosmid that also contains the argI and pyrB genes, mapping it to the 96-97 min region of the E. coli map.

Bacterial Proteins↗
PubMed1981

Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194.

We have constructed a hybrid plasmid pBC1, which consists of plasmid pC194 with an insert of B. subtilis DNA as its HindIII restriction site. This plasmid is stably maintained in B. subtilis. In contrast with pC194, monomeric ccc forms of pBC1 are active in transformation. Transformations with these monomeric molecules of pBC1 have a stringent requirement for recombination proficiency, as defined by recE in the recipient cell. The extent of dependence of the transforming activity of oligomeric pBC1 DNA on the recombination proficiency of the recipient cell decreases with increasing oligomer size. A model of DNA processing during plasmid transformation of B. subtilis is presented.

Bacillus subtilis↗
PubMed1985

New replication mutant pNH602 and its relationship to plasmid pAS3, another deletion derivative of plasmid R6K.

A stable deletion derivative pNH602 was obtained when the recently described higher-copy-number point mutant pNH601 of plasmid R6K was introduced to a minicells-producing strain of Escherichia coli. The size of plasmid pNH602 is 18.8 Mg/mol as determined by electron microscopy. The 7.2 Mg/mol fragment of R6K genome missing in pNH602 carries the Smr-determinant and the region finO and, according to the results of restriction analysis, it includes one EcoRI site. With its radioisotopically determined 33 copies of pNH602 per E. coli K-12 chromosome (npc), representing a 23% increase of the point mutant pNH601 and 150% enhancement of R6K npc, plasmid pNH602 differs from another closely related R6K deletion derivative pAS3 of the same size which exhibits only 20 npc. Both pNH602 and pAS3 plasmids are conjugative.

Chromosome Deletion↗
PubMed1985

Copy number of the broad host-range plasmid R1162 is determined by the amounts of essential plasmid-encoded proteins.

DNA of the broad host-range plasmid R1162 contains a 1700 base-pair segment essential for plasmid maintenance. This region, RepI, consists of two cotranscribed genes encoding polypeptides with molecular weights of 29,000 and 31,000. Fusion of RepI to the strong tac promoter results in greatly increased amounts of at least one of these polypeptides. In trans, this construction has two other properties: it can raise the copy number of R1162, and it can protect this plasmid from loss due to incompatibility. Both effects require intact RepI genes. These properties of the RepI region, along with those of an origin-linked region described earlier, are discussed with respect to current models for control of plasmid copy number.

Adenosine Triphosphatases↗
PubMed1988

Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III.

Soluble extracts of Escherichia coli capable of carrying out replication of the mini-RK2 derivative pCT461 have been prepared from cells carrying this plasmid or from plasmid-free bacteria. The latter are dependent upon exogenously added plasmid-encoded replication protein (TrfA) and require additional DnaA protein for optimum activity. This dependence upon DnaA was confirmed by the failure of DnaA-deficient cell extracts to support replication of pCT461 in the absence of added DnaA protein. Replication is unidirectional and begins at or near oriV, the vegetative replication origin of RK2. DNase I protection studies with purified TrfA indicate that this protein acts by binding to short (17 base-pairs) directly repeated DNA sequences present in oriV. The in vitro replication is resistant to rifampicin but can be abolished by antibodies against DnaG protein (E. coli primase) or DnaB protein (helicase) and by DNA gyrase inhibitors. Inhibition by arabinosyl-CTP suggests that DNA polymerase III is responsible for elongation of nascent DNA strands. These results are discussed in relation to the mechanism of RK2 replication and in the context of the host range of the plasmid.

Bacterial Proteins↗
PubMed1991

Regulation of the F plasmid traY promoter in Escherichia coli by host and plasmid factors.

F plasmid DNA transfer (tra) gene expression in Escherichia coli is regulated by chromosome- and F-encoded gene products. To study the relationship among these regulatory factors, we constructed low-copy plasmids containing a phi(traY'-'lacZ)hyb gene that couples beta-galactosidase and Lac permease synthesis to the F plasmid traY promoter. Wild-type transformants maintained high levels of beta-galactosidase over a broad range of culture densities. Primer extension analysis of tra mRNA from F'lac and phi(traY'-'lacZ)hyb strains indicated very similar, though not identical, transcription initiation sites. Moreover, phi(traY'-'lacZ)hyb gene expression required both TraJ and SfrA, as does tra gene expression in F+ strains. beta-Galactosidase activity was reduced approximately 30-fold in the absence of TraJ, which could be supplied in cis or in trans. In a two-plasmid system in which TraJ was supplied in trans by a lac-traJ operon fusion, phi(traY'-'lacZ)hyb expression was a linear, saturable function of traJ expression. Enzyme activity was reduced approximately tenfold in sfrA mutants. That reduction could not be attributed to an effect on the TraJ level. Several other cellular or environmental variables had only a modest effect on phi(traY'-'lacZ)hyb expression. Hyperexpression was observed at high cell density (twofold) and in anaerobic cultures (1.2- to 1.5-fold). In contrast, expression was reduced twofold in integration host factor mutants.

Aerobiosis↗
PubMed1984

Characterization of the basic replicons of the chimeric R/Ent plasmid pCG86 and the related Ent plasmid P307.

Restriction-enzyme fragments that can replicate autonomously after circularization were isolated from the chimeric R/Ent plasmid pCG86 and the Ent plasmid P307. Two such regions containing a basic replicon were located in each plasmid. One of the basic replicons of P307, RepFIB, is almost identical with one of the basic replicons of pCG86. The other basic replicon in P307, RepFIC, is partly homologous with the second basic replicon in pCG86, RepFIIA/RepFIC. The latter is a hybrid basic replicon and is in addition partly homologous with RepFIIA, a basic replicon present in IncFII R plasmids. By restriction-enzyme mapping and nucleotide-sequence analysis we have determined a site in the hybrid replicon where it ceases to be homologous with the RepFIIA basic replicon contained in the IncFII miniplasmid pSM1. The 2410-bp region of homology with pSM1 corresponds with a segment containing the origin of replication and all the genes responsible for replication control of pSM1.

Base Sequence↗
PubMed1985

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K.

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.

Base Sequence↗
PubMed1989

Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids.

An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19. The method of insertion involved both in vitro splicing and the natural transformation of D. radiodurans and yielded full-length clones in E. coli of pUE10 and pUE11. Both pS28 and pS19 replicated and expressed Kmr in E. coli and D. radiodurans. In both pS28 and pS19, D. radiodurans plasmid sequences were immediately upstream from the Kmr determinant. Transformation experiments suggested that Kmr expression in D. radiodurans was initiated in upstream D. radiodurans sequences. Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites. Both pS28 and pS19 transformed the MraI-producing D. radiodurans strain R1 at low frequencies. D. radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies. A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance.

Cloning, Molecular↗
PubMed1992

Dictyostelium giganteum plasmid Dgp1 is a member of the Ddp2 plasmid family.

Dgp1, a circular 4.4-kb plasmid found in the nuclei of Dictyostelium giganteum strain DG61, is a member of the same plasmid family as plasmids Ddp2 and pDG1. Dgp1 has sequence similarity to a conserved region of the Ddp2 and pDG1 open reading frames. As with Ddp2 and pDG1, a single large RNA is transcribed from Dgp1. This 3.3-kb transcript is present at about 350 copies per vegetative cell. The transcript abundance decreased about 10-fold in early aggregation and continued at this lower level until late culmination when it returned to the level seen in vegetative cells. Dgp1 has a repeat of several hundred base pairs in a location, relative to the transcribed region, similar to the inverted repeats found in Ddp2 and pDG1. Dgp1 cannot be maintained as a plasmid in Dictyostelium discoideum AX4 cells, suggesting that Dgp1 carries species-specific maintenance elements.

Amino Acid Sequence↗
PubMed1989

The nucleotide sequence of a small cryptic plasmid found in Staphylococcus aureus and its relationship to other plasmids.

The nucleotide sequence of a small (1273 bp) plasmid (pOX1000) of Staphylococcus aureus has been determined and compared with similar plasmids. The sequence includes a single open reading frame; two large palindromes and a 22 bp palindrome that is contiguously repeated three times upstream of the open reading frame. Composite plasmids of pE194ts and pOX1000 were constructed with pUC18 separately inserted into five different sites on pOX1000 and used to analyse the replication functions of the cryptic plasmid.

Amino Acid Sequence↗
PubMed1989

A new simple method of curing plasmids in lactic streptococci (Streptococcus cremoris; Streptococcus lactis, plasmids).

A simple method for curing plasmid DNA from lactic streptococci is described. When strains of lactic streptococci are grown overnight at 32 degrees C in an unbuffered medium (M17-) and held at the same temperature for an extended period (96 h), the acid environment induces loss of plasmid DNA of different sizes. If the process of growth in M17- broth followed by extended incubation at 32 degrees C is repeated, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. The method is simple and efficient in curing plasmids of lactic streptococci without use of any mutagenic chemical.

Culture Media↗
PubMed1986

Plasmid map: a microcomputer program for display and storage of plasmid data.

We describe a plasmid map program which runs on an IBM PC microcomputer and facilitates the drawing of circular plasmid maps. The user enters information from the keyboard in the form of restriction enzyme sites, genes and their locations, and other plasmid markers such as promoters, origins, or other sites. This information can then be stored in a file for future reference. The plasmid map can be displayed on the screen, printed on a dot-matrix printer, or plotted on a Hewlett Packard HP7475A plotter.

Base Sequence↗
PubMed1992

Nonidentity between plasmid and chromosomal copies of ISS1-like sequences in Lactococcus lactis subsp. lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes.

The nucleotide sequence of an insertion sequence (IS) observed during mating experiments using the lactose-protease plasmid, pUCL22, of Lactococcus (Lc.) lactis subsp. lactis CNRZ270, was found to be similar to that of ISS1 from Lc. lactis subsp. lactis ML3. The IS was named ISS1RS. The chromosome of this strain contains several copies of ISS1-like IS as assessed by hybridization. One of these copies was cloned and named ISS1CH. Its sequence differs from that of the plasmid-borne copy, and appears to be more closely related to ISS1N from Lc. lactis subsp. cremoris SK11. This suggests independent introduction of both ISS1 elements. Moreover, the observation of plasmid genes integrated in the CNRZ270 chromosome near ISS1CH suggests that their presence is the result of integration by a Campbell mechanism using both IS homologies. ISS1-like sequences were also found on plasmids of numerous Lc. lactis strains, as well as one out of seven Lactobacillus (Lb.) casei and one out of three Lb. plantarum strains examined.

Amino Acid Sequence↗
PubMed1987

Modulation of the low-calcium response in Yersinia pestis via plasmid-plasmid interaction.

Virulent cells of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are known to exhibit a low-calcium response in vitro characterized by restriction of growth and induction of V antigen at 37 degrees C in Ca2+-deficient media (Lcr+). A shared Lcr plasmid mediates these properties and encodes yersiniae outer membrane peptides (Yops) that are expressed in Lcr+ Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis. We present direct evidence here verifying that synthesis of major Yops in the former two species is, like V, repressed by Ca2+ and that these structures are located primarily in the outer membrane. We also verified that rabbits infected with live Lcr+ Y. pestis can raise antibodies against V and Yops. Similar antisera, however, were recovered after immunization with sterile extracts of Ca2+-starved Lcr+ cells of Y. pestis. Results of immunoblots obtained with these antisera showed that restricted Y. pestis produced Yops of about 46 kDa (YopB) and 44 kDa (YopC) after shiftup by addition of Ca2+ at 37 degrees C or reduction of temperature to 26 degrees C. It is established that virulent cells of Y. pestis also possess a unique plasmid known to mediate pesticinogeny (Pst+). Restricted Lcr+, Pst- Y. pestis expressed YopB and YopC plus additional 76 kDa (YopF), 48 kDa (YopH), 36 kDa (YopD), 32.5 kDa (YopJ), and 27 kDa (YopE) outer membrane structures at concentrations comparable to those in Ca2+-starved Y. pseudotuberculosis and Y. enterocolitica. These findings indicate that carriage of the Pst plasmid prevents expression of the Lcr plasmid-mediated Yops in wild type Y. pestis.

Bacterial Outer Membrane Proteins↗
PubMed1987

A new method for the physical and genetic mapping of large plasmids: application to the localisation of the virulence determinants on the 90 kb plasmid of Salmonella typhimurium.

A new method based on transposon-promoted deletions was used to generate a set of deletions in the 90 kb virulence plasmid of Salmonella typhimurium. The analysis of 16 deletion mutants allowed: (1) construction of the restriction map of the plasmid for HindIII, BamHI and BglII; (2) localisation of the plasmid region involved in virulence; (3) identification of two functional replicons on the plasmid.

DNA Mutational Analysis↗
PubMed2004

Plasmid transfer in bacilli by a self-transmissible plasmid p19 from a Bacillus subtilis soil strain.

The cryptic 95-kb plasmid p19 of the Bacillus subtilis 19 soil strain promotes the transfer of a small kanamycin resistance plasmid pUB110. To facilitate direct selection for p19 transfer, a plasmid derivative carrying the chloramphenicol resistance gene was constructed. The frequency of transfer of the large plasmid between cells of B. subtilis 19 approached 100% but was more than two orders of magnitude lower when the strain B. subtilis 168 was a recipient. However, when the restriction-deficient strain B. subtilis 168 was a recipient, the transfer efficiency was almost completely recovered. The effectiveness of pUB110 mobilization was virtually not altered in all these cases. pC194 was not mobilized by p19. The kinetics of p19 conjugative transfer is also presented.

Bacillus subtilis↗
PubMed2005

Sequence analysis of the plasmid genome of the probiotic strain Lactobacillus paracasei NFBC338 which includes the plasmids pCD01 and pCD02.

Lactobacillus paracasei NFBC338 is a probiotic strain that was isolated from the human gastrointestinal tract (GIT) and contains a plasmid genome of 80kb. Using a shotgun sequencing approach, two of the plasmids, pCD01 (19,882bp) and pCD02 (8554bp) have been completely sequenced, and four contiguous sequences (Contigs) have been assembled. Bioinformatic analysis of pCD01 revealed that it contains 23 putative open reading frames (ORFs) and that it contains regions characterised by potential replication functions and multidrug resistance (MDR). In contrast, the content of pCD02 is mainly cryptic, although, it does contain two insertion sequence (IS) elements. Indeed, up to 17% of the entire plasmid genome encodes putative transposable elements. In addition, there are a number of interesting ORFs distributed over the four Contigs that show significant homology to genes such as those involved in adherence and biotin metabolism, which may prove beneficial to Lb. paracasei NFBC338 under certain environmental conditions. This study provides a novel insight into the rich plasmid complement of this probiotic Lactobacillus strain, which may potentially be exploited as the basis for development of improved genetic tools for probiotic lactobacilli.

Adhesins, Bacterial↗