Physical characterization of plasmids isolated fromStreptosporangium

Plasmids were isolated from two species ofStreptosporangium by CsCl-ethidium bromide equilibrium density gradient centrifugation. A plasmid isolated fromS. brasiliense, designated pSgB-1, was characterized by electron microscopy and agarose gel electrophoresis. The pSgB-1 plasmid is a closed circular DNA molecule of 9.4 × 10⁶ Da. A restriction endonuclease map was generated and unique cleavage sites were found forEcoRI, ClaI, XbaI, and MstII. Another plasmid, pSgV-1, isolated fromS. viriodogriseum, has an estimatedM_r of 54 × 10⁶. The pSgB-1 plasmid is phenotypically cryptic but an unusual phenotypic trait, resembling phage plaques, may be associated with theS. viridogriseum plasmid pSgV-1.     

A novel high-copy plasmid, pEC, compatible with commonly used Escherichia coli cloning and expression vectors

A 66164-bp cryptic plasmid, pEIB1, was isolated from strain Vibrio anguillarum MVM425 and sequenced. A plasmid carrying a 1089-bp fragment, containing the minimal replication region of pEIB1, a kanamycin-resistance marker and an l-arabinose promoter, designated pEC, was maintained as a high copy plasmid in E. coli and stably inherited in the absence of antibiotic selection. Significantly, pEC was compatible with the widely used ColE1, pSC101 and p15A replicons making it a useful tool for a dual-plasmid expression system.     

Cloning and expression of the ilvB gene of Escherichia coli K-12

Summary A plasmid containing theilvB operon, which codes for acetohydroxy acid synthase I ofEscherichia coli K-12, was isolated using a ligated mixture of DNA from plasmid pBR322 and F'ilvB4 treated with endonucleaseSalI. A shortened derivative of this plasmid was isolated by cloning a 3.4 kb bacterial fragment into plasmid pKEN005 to yield plasmid pTCN12. The orientation of theilvB operon relative to plasmid genes was determined by restriction enzyme mapping. Measurement of the level of the product of theilvB gene, acetohydroxy acid synthase I, indicated that plasmid pTCN12 contained a functionalilvB promoter and control region. The DNA from this plasmid was used as a probe to show that the rate of synthesis ofilvB mRNA was proportional to the levels of acetohydroxy acid synthase I.     

Molecular cloning and genetic mapping of the PET494 gene of Saccharomyces cerevisiae

Summary The activity of the nuclear gene PET494 is required to allow expression of the yeast mitochondrial gene oxi2. To aid the study of the mechanism of action of PET494 we have isolated this gene from yeast DNA. A clone bank of yeast DNA fragments in a yeast-E. coli shuttle vector was screened by transformation for a plasmid able to complement the pet494-1 amber mutation. A complementing plasmid was obtained that contained a unique 4.4 kb yeast sequence. This 4.4 kb sequence contains the PET494 gene. Integration of a plasmid containing it into chromosomal DNA by homologous recombination, and subsequent genetic analysis, demonstrated that the 4.4 kb fragment was tightly linked to the pet494-1 mutation. In addition, the corresponding 4.4 kb sequence isolated from a pet494-1 mutant failed to complement the mutation. A 2 kb fragment, subcloned from the original plasmid retained the ability to complement the mutation. The pet494-1 mutation maps to chromosome XIV between rna2 and lys9, approximately 2.4 cm from lys9.     

Assessment of virulence determinants in Yersinia enterocolitica 1A,O:5 strains isolated from chicken carcasses

The virulence potential associated with 42–45 MDa plasmid pYV, of three Yersinia enterocolitica, 1A, O:5, strains isolated previously from chicken carcasses was tested by plasmid extraction, and by four in vitro tests: (1) Auto-agglutination; (2) Calcium dependence for growth at 37 °C; (3) Congo Red and Crystal Violet binding; and (4) Resistance to the bactericidal effect of serum. The in vitro tests were negative in all strains tested, but from strain PP131 it was possible to extract a plasmid which was quite similar in size to the wild type pYV, but heavier and not observed in any other isolated strains. These contradictions between the presence of a plasmid of approximately 42–45 MDa and the in vitro tests negative results may suggest that there is no homology between the PP131 plasmid and pYV. From these results we conclude that the plasmid found in PP131 is not pYV and that the three Y. enterocolitica 1A, O:5, strains are non-pathogenic on pYV classical mechanism basis, which do not exclude the possibility of them becoming infectious by a different mechanism, especially in children or immune-depressed individuals.     

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.     

Interactions between two heterogenic R plasmids: Cointegrative suppression of the thermosensitive replication of Rts1 by a nonconjugative derivative of NR1

A recombinant plasmid, R428, between a temperature-sensitive R plasmid, Rts1, and a transfer-deficient mutant of NR1, NR1–4, was isolated. Experiments on stability at higher temperature, transfer frequency, and density profiles of DNAs suggested that R428 is a cointegrate and that the thermosensitivity of Rts1 replication was suppressed.     

<Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> is Able to Gain and Maintain a Plasmid Harbouring In35 Found in <Emphasis Type="Italic">Enterobacteriaceae</Emphasis> Isolates From Argentina

The aim of this study was to determine the presence of bla _CTX-M-2 in our A. baumannii population and their putative role as an alternative mechanism of resistance to third-generation cephalosporins in this species. The bla _CTX-M-2 gene is widespread among the Enterobacteriaceae isolates from our country; however, it was not found in 76 isolates A. baumannii non-epidemiologically related clinical isolates resistant to third-generation cephalosporins isolated since 1982 in hospitals from Buenos Aires City. A plasmid isolated from Proteus mirabilis that possesses the complex class 1 integron In35::ISCR1::bla _CTX-M-2 was used to transform the natural competent A. baumannii clinical strain A118. PCR, plasmid extraction, DNA restriction, and susceptibility test confirmed that A118 could gain and maintain the plasmid possessing In35::ISCR1::bla _CTX-M-2, the genetic platform where the bla _CTX-M-2 gene is dispersing in Argentina.     

Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621 bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.     

Characterization of a cryptic plasmid (p0M1) inButyrivibrio fibrisolvens by restriction endonuclease analysis and its cloning inEscherichia coli

A small cryptic plasmid has been identified in a strain of the ruminal bacteriumButyrivibrio fibrisolvens. This plasmid has been isolated and purified. It is approximately 2.8 kbp in length and contains restriction sites for a number of common endonucleases including single sites for EcoRI, PvuII, and PstI. A map of the plasmid restriction sites has been constructed. This plasmid, designated p0M1, has been ligated to pBR325, pAT153, and pHV33 and transformed intoEscherichia coli, and the resulting hybrid plasmids have been mapped. The possible uses of such hybrid plasmids for gene cloning inB. fibrisolvens are discussed.     

Plasmids of serogroup 1 strains ofLegionella pneumophila

Twelve strains ofLegionella pneumophila were tested for the presence of plasmid DNA. Three strains, belonging to serogroup 1, had large plasmids of 83.8×10⁶ daltons, as determined by electron microscopy. A fourth strain, also from serogroup 1, had a similar large plasmid in addition to a smaller plasmid. Restriction analysis of plasmid DNA isolated from the strains with a single size plasmid indicated that the plasmids were structurally very similar. The biologic functions of these plasmids are yet to be determined.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

Isolation and Characterization of a ColE1-like Plasmid fromEnterobacter agglomeranswith a Novel Variant ofromGene

Complete nucleotide sequence of a plasmid isolated fromEnterobacter agglomeranshas been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring thecatgene (pBW4), which could be introduced intoEscherichia colicells, and demonstrating that pBW4 cannot replicate in the absence of thepolAfunction and that its copy number is significantly decreased in thepcnBmutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starvedrelAmutants but not inrelA⁺bacteria. Inactivation of the putativeromgene by insertion of an ampicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number inE. colidespite the fact that amino acid sequence of the putative RNA I modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derivedrom-like coding sequence is also homologous to therom-like gene present in theProteus vulgarisplasmid pPvu1. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) inE. coli,one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the newrom-like gene coding for a protein useful in investigation of RNA–protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.     

Properties of plasmids from Methylomonas clara

Three independently isolated clones of the obligate methylotrophic bacterium Methylomonas clara (ATCC 31226) were tested for their plasmid content. Strains B45-BE-2 and B45-BE-3 were shown to harbor one class of plasmid molecules each, with molecular sizes of 28 MDa (46 kb) and 10 MDa (16 kb), respectively. Strain B45-BE-1 does not contain extrachromosomal DNA. Restriction maps of plasmid pBE-2 from strain B45-BE-2 and of plasmid pBE-3 from strain B45-BE-3 were established and the smaller plasmid pBE-3 shown to be a linear deletion derivative of plasmid pBE-2. Two other deletions were characterized. When subcloned into the Escherichia coli vector pBR322, plasmid pBE-3 was unable to complement for the polymerase A dependence of the pBR322 replicon. Additional experiments indicate a general failure of the M. clara specific replicon to function in E. coli.     

Molecular identification of Acetobacter isolates from submerged vinegar production, sequence analysis of plasmid pJK2-1 and application in the development of a cloning vector

Three new Acetobacter strains were isolated from vinegar. By plasmid profiling they were recognized as genotypically different from each other. Sequencing of the genes for 16S and 23S rRNA and DNA–DNA hybridization of total DNA against DNA of all type strains of Acetobacter identified Acetobacter strains JK2 and V3 as A. europaeus, and Acetobacter strain JK3 as A. intermedius. In contrast to the type strain of A. europaeus (DSM 6160), A. europaeus JK2 and V3 do not require acetic acid for growth and can be successfully transferred between media with and without acetic acid. This phenotypic characteristic enables convenient handling of both strains in genetic studies. Plasmid pJK2-1 from A. europaeus JK2 was used as the basis for shuttle plasmid construction with the aim of developing an efficient vector system for these strains. The entire nucleotide sequence of pJK2-1 was determined. High amino acid identities were found for three open reading frames: Rep (replication protein); Dinj1 (DNA damage inducible enzyme); and Dinj2 proteins. A recombinant plasmid pUCJK2-1 (5.6 kb) consisting of the entire plasmid pJK2-1 and the entire plasmid pUC18 was successfully used in transformation experiments. Plasmid pJT2 (5.8 kb) was constructed from pUCJK2-1 with the aim of reactivating the lacZ′ gene. Received: 14 June 1999 / Revision received: 27 August 1999 / Accepted: 17 September 1999     

Molecular analysis of plasmid pAP4 from Acetobacter pasteurianus

The complete nucleotide sequence of plasmid pAP4 isolated from Acetobacter pasteurianus 2374^T has been determined. Plasmid pAP4 was analysed and found to be 3,870 bp in size with a G+C content of 50.1%. Computer assisted analysis of sequence data revealed 2 possible ORFs with typical promoter regions. ORF1 codes for a protein responsible for kanamycin resistance similar with Tn5 transposone, ORF2 encodes a resistance to ampicillin identical with Tn3 transposone. Plasmid has in A. pasteurianus five copies and in E. coli DH1 about 30 copies per chromosome and it segregation stability in both strains is very high. Based on the data on replication region, plasmid does not code for a replication protein and origin region is similar with ColE1-like plasmid.     

Cloning of the Genes That Control Formation of the Fimbrial Colonization Factor Antigen III (CFA/III) from an Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli strain 260-1 produces colonization factor antigen III and heat-labile enterotoxin. A 55-kb plasmid controlling the expression of the colonization factor antigen was isolated from this strain after it was labeled with ampicillin resistance transposon, Tn3. When this plasmid was introduced into E. coli K-12 strains, it induced the formation of pili that were morphologically and immunologically identical to those on the surface of 260-1 cells, as examined by electron microscopic observation and with the specific antiserum. The physical map of the plasmid was constructed, and the 17.4-kb region was found to be responsible for the expression of the pili.     

First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene

Aims: Isolation and full sequence analysis of ColE‐type plasmid, which carries the qnrS2 gene. Methods and Results: Quinolone resistance (qnrS2) gene‐carrying plasmids were isolated from Aeromonas sobria and Aeromonas hydrophila strains, and plasmid sequencing was achieved by a primer‐walking approach. The total sizes of these plasmids (pAQ2‐1 and pAQ2‐2) were 6900 bp and 6903 bp, respectively, and they were 99·1% identical to each other. The genes (oriV and repA) for plasmid replication were organized similar to the corresponding genes in the ColE2‐type plasmids, pAsa3 and pAsa1, isolated from Aeromonas salmonicida subsp. salmonicida, but the gene (mobA) for mobilization was homologue to ColE1‐type plasmid (pAsa2) from Aer. salmonicida subsp. salmonicida. Additionally, the qnrS2 gene was part of a mobile insertion cassette element in the plasmid. Conclusions: Two plasmids were assumed to be the same plasmid, and this identification of a plasmid‐mediated qnrS2 gene from the two different strains underlines a possible diffusion of these resistance determinants in an aquaculture system. Significance and Impact of the Study: This is the first finding of the ColE‐type plasmid carrying the qnrS2 gene.     

Expression of an Erwinia sp. gene encoding diphenyl ether cleavage in Escherichia coli and an isolated Acinetobacter strain PE7

Summary An Acinetobacter strain PE7 with the ability to grow on salicylic acid and to degrade diphenyl ethers was isolated from a petroleum waste pit in Louisiana. A cloned Erwinia sp. dpe gene encoding diphenyl ether cleavage was introduced into PE7 in order to enhance its degradative ability. A broad-host-range expression plasmid, pDPE2388, was constructed by inserting an SspI-HpaI fragment from a dpe gene-containing plasmid, pDPE7321, into the kanamycin resistance gene of plasmid pKT230. The DNA fragment contained the dpe gene flanked between sp6 and T7 promoters. Transconjugants of pDPE2388 plasmid into PE7 were isolated. Expression of the dpe gene in Escherichia coli or PE7 displayed a degradative ability to cleave the following diphenyl ethers: 4-chlorodiphenyl ether, 4-nitrodiphenyl ether, and 4-hydroxydiphenyl ether.Offprint requests to: V. R. Srinivasan