Restriction mapping of plasmid pSa1, isolated from Streptomyces albus G strain

A novel plasmid designated pSa1 has been isolated from Streptomyces albus G strain producing SalGI restriction endonuclease. Molecular weight of the plasmid is 3.4 +/- 0.2 mD. The action of 12 restriction endonucleases on the plasmid DNA was studied. Restriction map of pSa1 DNA was established for SmaI, HindII, XbaI and KpnI endonucleases.     

One-kilobase direct repeats of plasmid pSa

One-kilobase, direct repeats were found on either side of the chloramphenicol resistance gene of plasmid pSa. The right repeat corresponded to the region coding for sulfanilamide resistance. The repeats were not identical as judged by distances between restriction enzyme sites, hybridization, and by the ability to confer resistance to sulfanilamide.     

Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/molBam HI pSa fragment carrying determinants of resistance to four antibiotics in the uniqueBam HI site of pNH602. The resultingin vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 perE. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in theBam HI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymesBam HI andEco RI and its physical and genetic map was constructed.     

Genetic map of the crown gall suppressive IncW plasmid pSa

Summary A genetic map of the W incompatibility group plasmid pSa has been prepared through the construction of deletion derivatives of pSa and the cloning of various fragments of pSa in pBR322. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to chloramphenicol, sulfonamides, spectinomycin, streptomycin, kanamycin, gentamycin, and tobramycin. Information sufficient for the replication of the plasmid in both Escherichia coli and Agrobacterium tumefaciens is contained within a 4 kilobase pair region. Two regions have been identified as involved in the transfer of the plasmid; one of these regions is also involved in the inhibition of oncogenesis by pSa when it is present in an oncogenic strain of A. tumefaciens. Certain of the deletion derivatives of pSa are potential vectors for the cloning and analysis of A. tumefaciens Ti plasmid DNA.     

Plasmid-mediated chloramphenicol resistance in Staphylococcus hyicus

A small plasmid of 3.95 kb, encoding resistance to chloramphenicol (Cm) was detected in three of 33 Staphylococcus hyicus strains. The plasmid in each of the three strains was indistinguishable by Southern-blot hybridization and restriction enzyme analysis. It was shown by curing and by transformation to specify resistance to Cm. A preliminary restriction map of the plasmid, designated pSC2, is presented. Chloramphenicol acetyltransferase was demonstrated by enzyme assay and by SDS-PAGE of cell-free lysates of pSC2 transformants.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Suppression of tumorigenicity by the IncW R plasmid pSa in Agrobacterium tumefaciens

Summary The effect of the IncW R plasmid, pSa, on tumorigenicity and on the expression and maintenance of the Ti plasmid in tumorigenic strains of A. tumefaciens was determined. Plasmid pSa could be transferred into and stably maintained by both octopine-and nopaline-utilizing A. tumefaciens strains. The R plasmid had no effect on Ti plasmid maintenance or on Ti plasmid functions, such as octopine utilization or conjugal bacterial transfer. However, A. tumefaciens strains harboring both the R plasmid and the Ti plasmid in most instances failed to induce tumors on a number of plant species. This effect on tumorigenicity is specific to pSa. When pSa is cured from the A. tumefaciens transconjugants or when their Ti plasmids are genetically transferred to an appropriate recipient, the resultant strains lacking the R plasmid regain tumorigenicity. Restriction endonuclease analysis of plasmid DNA isolated from transconjugants harboring pSa showed no difference in Ti plasmid cleavage patterns when compared to plasmid DNA isolated from the tumorigenic parent strain. These results indicate that pSa does not induce detectable permanent genetic alteration of the Ti plasmid. Rather, it appears that the R plasmid suppresses some Ti plasmid function(s) necessary for tumorigenicity.     

Long-term starvation-induced loss of antibiotic resistance in bacteria

Escherichia coli, Pseudomonas fluorescens, and aPseudomonas sp. strain 133B containing the pSa plasmid were starved in well water for up to 523 days. There were two patterns of apparent antibiotic resistance loss observed. InPseudomonas sp. strain 133B, there was no apparent loss of antibiotic resistance even after starvation for 340 days. InE. coli, by day 49 there was a ten-fold difference between the number of cells that would grow on antibiotic- and nonantibiotic-containing plates. However, over 76% of the cells that apparently lost their antibiotic resistance were able to express antibiotic resistance after first being resuscitated on non-selective media. By day 523, only 12% of these cells were able to express their antibiotic resistance after being resuscitated. After starvation for 49 days, cells that could not grow on antibiotic medium even after resuscitation, showed a permanent loss of chloramphenicol (Cm) resistance but retained resistance to kanamycin (Km) and streptomycin (Sm). Restriction enzyme digests show that a 2.5 to 3.0 Kb region from map location 12.5 to 15.5 Kb was deleted. This coincides with the 2.5 Kb reduction in plasmid size observed in 3 isolates that had lost antibiotic resistance after starvation for 49 days.Published as Technical Paper #9224, Oregon Agricultural Experiment Station.     

Genetic organization of the broad-host-range IncP-1 plasmid R751.

We have identified regions encoding conjugal transfer, plasmid maintenance, and trimethoprim resistance on the IncP-1 plasmid R751 by complementation tests with cloned deoxyribonucleic acid fragments and self-replicating derivatives constructed in vitro. The genes for replication and transfer show a scattered organization similar to that previously determined for RK2, another IncP-1 plasmid. Derivatives of RK2 are able to complement R751 derivatives defective in these functions. Restriction enzyme cleavage sites in R751 deoxyribonucleic acid are clustered in regions of the plasmid physical map. Neither region is required for plasmid maintenance or transfer, although one determines resistance to trimethoprim. A similar clustering of cleavage sites is seen with RK2, which nevertheless has a very different restriction map.     

Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478.

A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, SalI, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb SalI fragment of R478.     

Isolation and sequencing of the replication region of plasmid pBFp1 isolated from a marine biofilm

A 24 kb plasmid, pBFp1, encoding mercury resistance was previously isolated from a marine biofilm. Isolation and sequencing of a 4280 bp DNA fragment containing the plasmid replicon (rep-pBFp1) revealed a putative open reading frame encoding a RepA protein and an oriV-like region containing an A+T rich sub-region, iterons, and DnaA boxes. Sequence comparisons showed significant similarities to the incW plasmid pSa both for the RepA amino acid sequence and in the iteron DNA sequence. Plasmid pBFp1 was also shown to be incompatible with pSa in standard incompatibility testing. A probe from the repA gene of pBFp1 was further made and tested on a collection of plasmids exogenously isolated from marine habitats in a previous study.     

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.     

Construction of a new shuttle vector for Lactobacillus.

To clone the malolactic enzyme gene from Lactobacillus sp. 89, construction of a shuttle vector able to express itself in Lactobacillus sp. 89 and Escherichia coli was undertaken. The shuttle plasmid pLE16 resulted from the union of pBR328 and of the pLB10 plasmid extracted from Lactobacillus bulgaricus 10. The bacterial transformation in Lactobacillus sp. 89 was performed by electroporation, and the clones were selected on MRS medium with 30 micrograms.mL-1 chloramphenicol added. Fifty percent of the clones from Lactobacillus sp. 89 lost their resistance to chloramphenicol following 28 generations when the selection pressure was not maintained. The restriction map of pLE16 (7600 bp) was established using several restriction enzymes.     

Conjugative plasmids in Bordetella avium.

Three of five antibiotic-resistant plasmids isolated from virulent strains of Bordetella avium were found to be conjugative. A physical and genetic map of one of these plasmids, the 51.5-kb plasmid p4093, revealed that the area of p4093 responsible for streptomycin and tetracycline resistance was located in a region consisting of a cluster of restriction enzyme recognition sites, whereas the remainder of p4093 contained relatively few restriction sites. Additionally, the genes involved in the conjugative ability of p4093 were clustered in at least two widely separated regions of the plasmid.     

A physical map of plasmid pDU1 from the cyanobacterium Nostoc PCC 7524

Nostoc 7524 contains three different plasmids of molecular weight, 4, 8, and 28 Mdal. The smallest plasmid, designated pDU1, because of its size and ease of isolation, may prove to be useful as a cloning vector. Plasmid pDU1 was incubated separately with 26 different restriction enzymes and only 8 of the enzymes tested cut pDU1. A composite restriction enzyme map consisting of a total of 17 restriction sites was constructed for BglI, HindIII, HpaI, and XbaI. The sites of restriction enzyme cleavage were determined by single, double, and partial digests of plasmid DNA or redigestion of isolated restriction fragments. All the restriction sites were aligned relative to the single BglI site. This is the first restriction enzyme map of a plasmid from a filamentous cyanobacterium.     

Plasmid shuttle vector with two insertionally inactivable markers for coryneform bacteria

A new shuttle vector pCEM500 replicating inEscherichia coli and inBrevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Km^r/Gm^r from plasmid pSa of Gram-negative bacteria and Sm^r/Sp^r from plasmid pCG4 ofCorynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained inB. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.     

Isolation of the replication region of an indigenous plasmid of Rhodobacter sphaeroides

Abstract The isolation of the replication region of an indigenous plasmid of 42 kb of the phototrophic bacterium Rhodobacter sphaeroides is described. This plasmid was digested with the Bgl II restriction enzyme, ligated to the 2.7 Bgl II fragment of transposon Tn 10 , which contains the tet genes conferring tetracycline resistance, and the mixture was transformed into the Escherichia coli MC1061 strain. One of several chimeric plasmids harboring the replication region of the 42-kb plasmid obtained by this process was named pUA33 and further characterized. Plasmid pUA33 is approx. 8.3 kb. A partial restriction map has been constructed. Plasmid pUA33 is stable in E. coli cells growing under non-selective conditions and is non-self-transmissible. All these data suggest that the pUA33 plasmid may be a very useful tool for gene cloning in R. spheroides .     

Conjugative plasmids inBordetella avium

Three of five antibiotic-resistant plasmids isolated from virulent strains ofBordetella avium were found to be conjugative. A physical and genetic map of one of these plasmids, the 51.5-kb plasmid p4093, revealed that the area of p4093 responsible for streptomycin and tetracycline resistance was located in a region consisting of a cluster of restriction enzyme recognition sites, whereas the remainder of p4093 contained relatively few restriction sites. Additionally, the genes involved in the conjugative ability of p4093 were clustered in at least two widely separated regions of the plasmid.     

Isolation and preliminary characterization of plasmid in Bacillus licheniformis strain MP3

A restriction analysis of a single covalently closed circular plasmid isolated from Bacillus licheniformis strain MP3 was performed. A restriction map of the 6·44 kb plasmid is presented. The existence of genetic markers such as antibiotics and heavy metal ions resistance was investigated. The availability of two single restriction sites and the stability of this plasmid make it ideal for development as a cloning vector.