Characterization of a Haemophilus ducreyi mobilizing plasmid.

The OriV site of Haemophilus ducreyi mobilizing plasmid pHD147, determined by replication in Escherichia coli polA, is located close to the OriT site. The OriT site, located by recombination-proficient and -deficient cells, and the OriV site map in a region of pHD147 homologous to the beta-lactamase-specifying plasmids of H. ducreyi and Neisseria gonorrhoeae.     

Characterization of plasmid pRT1 from Pyrococcus sp. strain JT1

We discovered a 3,373-bp plasmid (pRT1) in the hyperthermophilic archaeon Pyrococcus sp. strain JT1. Two major open reading frames were identified, and analysis of the sequence revealed some resemblance to motifs typically found in plasmids that replicate via a rolling-circle mechanism. The presence of single-stranded DNA replication intermediates of pRT1 was detected, confirming this mode of replication.     

Characterization of a plasmid from moderately halophilic eubacteria.

A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonas halmophila, Deleya halophila and Vibrio costicola were found to harbour an 11.5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coli JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirmed by Southern hybridization. The plasmid carries genetic determinants that mediate resistance to kanamycin, tetracycline, and neomycin. This property, together with its relatively small size, its stability in E. coli cells, and the presence of unique restriction sites, makes pMH1 a good candidate for the development of a cloning vector for moderate halophiles.     

Characterization of pGA1, a new plasmid from Corynebacterium glutamicum LP-6.

A new plasmid, pGA1, has been isolated from Corynebacterium glutamicum LP-6, and its detailed restriction map has been prepared. The 4.9-kb plasmid has a G + C content of 57%. It replicates in C. glutamicum ATCC13032 and is compatible with the three other plasmids, pCC1, pBL1 and pHM1519, commonly used for vector construction for amino acid-producing corynebacteria. Fusions of pGA1 with different Escherichia coli replicons (transferred from E. coli to Corynebacterium via transformation of spheroplasts or by filter mating experiments with intact cells) are shown to be suitable as shuttle plasmids; some of them are highly stable in C. glutamicum, even when propagated without any selection pressure.     

Characterization of a cryptic plasmid from Bacillus sphaericus strain LP1-G

A cryptic plasmid from Bacillus sphaericus strain LP1-G, designated as pLG, was sequenced and characterized. It was an 11,066bp circular molecule, with G+C content of 37%. The plasmid pLG was predicted to encode 23 putative ORFs, and ORF 21 shared the highest identity with Rep of pGI1 and pBMB9741, members of rolling-circle replication (RCR) pC194-family. Sequence analysis revealed a pC194-type double strand origin (dso) and a single strand origin (sso) like sequence located upstream and downstream of ORF 21, respectively. Moreover, Mung bean nuclease analysis and Southern hybridization confirmed the existence of single stranded DNA (ssDNA) intermediates, indicating that pLG belongs to the RCR pC194-family. Accumulation of multiple ssDNA intermediates in native strain LP1-G and decline of ssDNA and supercoiled DNA in rifampicin-treated strain implied that a special mechanism might be employed by pLG. Furthermore, the copy number of pLG in its original host was determined and about 58 copies of the plasmid exist in each cell. Subcloning and transformation experiments proved that the minimal replicon of pLG was within a 1.6-kb fragment, which was composed of rep gene and dso. These data are a good basis for the understanding of replication mechanisms and genetics of this B. sphaericus plasmid.     

Characterization of conjugative plasmid EDP208.

EDP208 is a conjugative plasmid belonging to incompatibility group IncF0 lac, A restriction endonuclease map of this plasmid was constructed using five restriction enzymes: BamHI, HindIII, PvuI, SstI, and XhoI. On the basis of these mapping studies, the plasmid was found to be 90 kilobases in length. Clones were constructed from four large HindIII fragments of plasmid EDP208. One fragment, HindIII-20.5, was found to contain the lac genes and the origin of vegetative replication (oriV). Another fragment, HindIII-27.5, was found to contain all of the genes necessary for sex pilus formation, but it was nontransmissible. However, when used to complement a plasmid carrying an adjacent fragment, HindIII-23, the transfer of the latter occurred, suggesting that HindIII-23 contains the origin of transfer (oriT). The further localization of genes concerned with pilus biosynthesis was achieved by transposon mutagenesis. Six EDP208::Tn1 and thirty-seven EDP208::Tn5 mutants were isolated on the basis of their resistance to f1, a filamentous phage which adheres to intact pilus tips. The positions of the inserted transposons were determined on the restriction map and a 16.5-kilobase region was found to be required for pilus synthesis.     

Characterization of a plasmid from the ruminal bacterium Selenomonas ruminantium.

A 4.8-kilobase-pair plasmid was isolated from the ruminal bacterium selenomonas ruminantium HD4 by using a sodium carbonate-EDTA washing buffer to improve cell lysis (R.G. Dean, S.A. Martin, and C. Carver, Lett. Appl. Microbiol. 8:45-48, 1989). This plasmid, designated pSR1, appears to be quite stable. No evidence of plasmid DNA was detected in S. ruminantium D or GA192. All three strains were tested for antibiotic resistance, and all were kanamycin resistant (MIC, 25 to 50 micrograms/ml). Only strain D was tetracycline resistant (MIC, 25 micrograms/ml), and all strains were sensitive to ampicillin (MIC, 1 microgram/ml). pSR1 was cloned into pBR322, and a map of pSR1 was constructed by using HindIII, ClAI, BamHI, and PvuII. Although ClaI, BamHI, ScaI, and EcoRV digested recombined plasmid isolated from Escherichia coli, these restriction endonucleases were not effective in digesting plasmid isolated directly from S. ruminantium HD4.     

Characterization of a circular plasmid from the yeast Kluyveromyces waltii.

A new plasmid was found in the yeast Kluyveromyces waltii. This high-copy-number plasmid, named pKW1, is a double-stranded circular DNA plasmid of 5619 bp. It has several features characteristic of the 2 mu-type plasmids: presence of two inverted repeats and four open reading frames, as well as the interconversion of two isomeric forms. However, the nucleotide sequence shows little homology with known yeast plasmids. An ARS function was localized within a segment of 545 bp near one of the inverted repeats. Chimeric plasmids carrying this segment efficiently transformed K. waltii. A strain of K. waltii cured of the plasmid (cir degree) was also obtained. In the pKW1 sequence, a functionally neutral region was found at which foreign DNA can be inserted with little effect on plasmid stability. Such constructions carrying the full sequence of pKW1 replicated autonomously in a cir degree host and were particularly stable. pKW1-derived full-sequence plasmids also transformed K. thermotolerans, but not K. lactis.     

Characterization of the Streptomyces plasmid pVE1

pVE1 (11.0 kb) was isolated from Streptomyces venezuelae ATCC 14585 and characterized. pVE1 has a broad host range and an apparent copy number of about 100 during exponential growth, rising to 1500 during stationary phase. It is a pock-forming, self-transmissible fertility factor which promotes chromosome recombination in S. lividans at frequencies of about 0.1% per total parents. A detailed restriction map for 15 enzymes was determined. Genes for ThioR (thiostrepton resistance), NeoR (neomycin resistance), and pBR322 derivatives were inserted into pVE1 and the resulting plasmids were analyzed for self-transmissibility and stability. The plasmid has an essential region of approximately 2.5 kb and a region of 1.0-3.6 kb required for conjugation. NeoR and ThioR vectors were constructed with unique HindIII, PvuI, BamHI, EcoRI, BglII, and EcoRV sites available for insertion of foreign DNA.     

Characterization of the partitioning system of Myxococcus plasmid pMF1

pMF1 is the only autonomously replicating plasmid that has been recently identified in myxobacteria. This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMF1.21-pMF1.23 (parCAB). The pMF1.22 ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that the pMF1.23 (parB) product is a DNA-binding protein of iteron DNA sequences, while the product of pMF1.21 (parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ib par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1 par system has novel structural and functional characteristics.     

Characterization of a theta-replicating plasmid from Streptococcus thermophilus

Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology, into at least four groups (A-D). Here, we report the characterization of plasmids of group B and D. The sequence analysis of pSMQ173b (group D) indicates that this plasmid contains 4449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of the pGI3 family. The plasmid pSMQ308 (group B) contains 8144 bp and six ORFs. Two ORFs likely encode a primase/helicase and an integrase. Northern blot experiments demonstrate that these two orfs are transcribed within the three strains containing plasmids of group B. Two-dimensional agarose gel electrophoresis shows that pSMQ308 replicates via a theta mechanism. To our knowledge, this is the first report of a plasmid replicating via a theta mode in S. thermophilus. Finally, a classification of 20 sequenced S. thermophilus plasmids into six groups based on their mode of replication is proposed.     

Rolling-circle replication of a high-copy BPV-1 plasmid.

We investigated the replicating form of a bovine papillomavirus type 1 (BPV-1) deletion mutant by direct electron-microscopic analysis of low molecular weight cellular DNA fractions. The detection of viral plasmid DNA replication intermediates was facilitated by the isolation of a spontaneously transformed mouse cell subclone containing an unusually high viral genome copy number (approx. 1000 per cell), and by employing a slight modification of the Hirt fractionation procedure to reduce the level of contaminating linear chromosomal DNA fragments. We observed exclusively rolling-circle-type viral DNA replication intermediates, at a frequency of detection of approximately one replication intermediate per 200 monomeric circular viral DNA molecules. The demonstration of rolling-circles with longer-than-genome-length tails indicated that this high-copy viral plasmid was not subject to a strict once-per-cell-cycle mode of DNA replication. Our observations provide further evidence in favour of an alternative replication mode of the BPV-1 genome, and may help to explain earlier conflicting findings concerning the mechanism of stable BPV-1 plasmid copy-number-control.     

Characterization and sequence analysis of a small plasmid from Bacillus thuringiensis var. kurstaki strain HD1-DIPEL

The complete nucleotide sequence of a small (2.055 kb) plasmid pHD2 from Bacillus thuringiensis var. kurstaki strain HD1-DIPEL was obtained. The sequence encoded two open reading frames (ORFs) which corresponded to polypeptides of Mr 26,447 and 9122. Comparison of the sequence with those obtained for other plasmids from Gram-positive organisms suggested that pHD2 may belong to the extensive and highly interrelated family of plasmids exhibiting replication via a ssDNA intermediate: a putative nick site was proposed on the basis of sequence homology and one ORF exhibited distant homology with the site-specific topoisomerases encoded by the pT181 family of staphylococcal plasmids, while the other ORF exhibited considerable similarity to a small polypeptide (RepA) encoded by plasmid pLS1. Constructs consisting of pHD2, pBR322, and the chloramphenicol resistance gene from pC194 were capable of stable maintenance in B. thuringiensis var. israelensis, but were subject to apparently specific deletions in the heterologous host. The same constructs could not be established in Bacillus subtilis.     

Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1

The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.     

Characterization of a plasmid in isolates of Corynebacterium sepedonicum

Lysis of mid-log phase cells of the Gram-positive bacterium, Corynebacterium sepedonicum, by a combination of lysozyme treatment and incubation with alkaline sodium dodecyl sulfate at 56 degrees C led to the recovery of a single plasmid. The plasmid was purified in CsCl density gradients, and its molecular weight estimated to be 31 megadaltons (46 kilobases), as determined from its relative mobility in agarose gels, from its contour dimensions in electron micrographs, and from the size of the fragments generated when it was cleaved with various restriction endonucleases. Thirteen widely divergent isolates of C. sepedonicum were screened for the presence of plasmid, and of these, 11 were shown to harbour a single plasmid at a level of about 30 copies per cell. Cleavage of the plasmid with PstI gave an identical banding pattern in agarose gels for the fragments from all of the plasmid-carrying isolates. The relevance of plasmid incidence and distribution in C. sepedonicum is discussed in relation to the possibility of developing a test for the detection of bacterial ring rot by using plasmid DNA as a hybridization probe.