Naphthalene and donor cell density influence field conjugation of naphthalene catabolism plasmids

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.     

Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.     

Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.     

Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches

Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9 incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway. A.O. and R.M. contributed equally to this work.     

Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site.

Horizontal transfer of genes responsible for pollutant biodegradation may play a key role in the evolution of bacterial populations and the adaptation of microbial communities to environmental contaminants. However, field evidence for horizontal gene transfer between microorganisms has traditionally been very difficult to obtain. In this study, the sequences of the 16S rRNA and naphthalene dioxygenase iron-sulfur protein (nahAc) genes of nine naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site, as well as a naphthalene-degrading bacterium from a contaminated site in Washington state and two archetypal naphthalene-degrading strains, were compared. Seven strains from the study site had a single nahAc allele, whereas the 16S rRNA gene sequences of the strains differed by as much as 7.9%. No nahAc alleles from the site were identical to those of the archetypal strains, although the predominant allele was closely related to that of Pseudomonas putida NCIB 9816-4, isolated in the British Isles. However, one site-derived nahAc allele was identical to that of the Washington state strain. Lack of phylogenetic congruence of the nahAc and 16S rRNA genes indicates that relatively recent in situ horizontal transfer of the nahAc gene has occurred, possibly as a direct or indirect consequence of pollutant contamination. Alkaline lysis plasmid preparations and pulsed-field gel electrophoresis have revealed the presence of plasmids ranging in size from 70 to 88 kb in all site isolates. Southern hybridizations with a 407-bp nahAc probe have suggested that the nahAc gene is plasmid borne in all the site isolates but one, a strain isolated from subsurface sediment 400 m upstream from the source of the other site isolates. In this strain and in the naphthalene-degrading strain from Washington state, nahAc appears to be chromosomally located. In addition, one site isolate may carry nahAc on both chromosome and plasmid. Within the group of bacteria with identical nahAc sequences the Southern hybridizations showed that the gene was distributed between plasmids of different sizes and a chromosome. This suggests that plasmid modification after transfer may have been effected by transposons. Horizontal transfer of catabolic genes may play a significant role in the acclimation of microbial communities to pollutants.     

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 IS Pre1 -like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 IS Pre2 -like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer – we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of IS Pre2 -like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.     

Isolation and characterization of altered plasmids in mutant strains of Pseudomonas putida NCIB 9816

The ability of P. putida NCIB 9816 to grow with naphthalene (Nah+) and salicylate (Sal+) is correlated with the presence of an 83 kilobase (kb) conjugative plasmid, pDTG1. Derivatives of pDTG1 were obtained from cells after exposure to halogenated analogs of naphthalene or salicylate. The selection of mutants having a Nah-Sal- or a Nah-Sal+ phenotype could be enhanced by the addition of triphenyltetrazolium chloride to the indicator medium. Structurally modified plasmids were characterized by restriction endonuclease digestion and Southern hybridization experiments. The region of pDTG1 DNA that encodes the enzymes responsible for the conversion of naphthalene to salicylate was identified. The structural changes in mutant plasmids were correlated with the absence of essential enzymatic activities.     

Horizontal Transfer of phnAc Dioxygenase Genes within One of Two Phenotypically and Genotypically Distinctive Naphthalene-Degrading Guilds from Adjacent Soil Environments

Several distinct naphthalene dioxygenases have been characterized to date, which provides the opportunity to investigate the ecological significance, relative distribution, and transmission modes of the different analogs. In this study, we showed that a group of naphthalene-degrading isolates from a polycyclic aromatic hydrocarbon (PAH)-contaminated hillside soil were phenotypically and genotypically distinct from naphthalene-degrading organisms isolated from adjacent, more highly contaminated seep sediments. Mineralization of ¹⁴C-labeled naphthalene by soil slurries suggested that the in situ seep community was more acclimated to PAHs than was the in situ hillside community. phnAc-like genes were present in diverse naphthalene-degrading isolates cultured from the hillside soil, while nahAc-like genes were found only among isolates cultured from the seep sediments. The presence of a highly conserved nahAc allele among gram-negative isolates from the coal tar-contaminated seep area provided evidence for in situ horizontal gene transfer and was reported previously (J. B. Herrick, K. G. Stuart-Keil, W. C. Ghiorse, and E. L. Madsen, Appl. Environ. Microbiol. 63:2330-2337, 1997). Natural horizontal transfer of the phnAc sequence was also suggested by a comparison of the phnAc and 16S ribosomal DNA sequences of the hillside isolates. Analysis of metabolites produced by cell suspensions and patterns of amplicons produced by PCR analysis suggested both genetic and metabolic diversity among the naphthalene-degrading isolates of the contaminated hillside. These results provide new insights into the distribution, diversity, and transfer of phnAc alleles and increase our understanding of the acclimation of microbial communities to pollutants.     

A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis

Bacillus thuringiensis var. israelensis (BTI), serotype 14, which produces parasporal crystals toxic to certain dipteran larvae, was analyzed by agarose gel electrophoresis and found to contain a complex plasmid array. Eight plasmids were detected, with approximate sizes of 3.3, 4.2, 4.9, 10.6, 68, 75, 105, and 135 MDa, as well as a plasmidlike linear DNA element of ~10 MDa. Partially cured mutants of BTI implicated the 75-MDa plasmid in crystal production. Fifteen independently isolated acrystalliferous (Cry^?) mutants were found to lack this plasmid. In plasmid transfer experiments, several of the BTI plasmids transferred into a plasmid-free, Cry^? BTI recipient, but only transfer of the 75-MDa plasmid converted the recipient to crystal toxin production. The presence or absence of mosquito-toxic activity in all Cry⁺ and Cry^? variants of BTI was confirmed by bioassay of sporulated cultures against larvae of Aedes aegypti. Southern blot analyses revealed that in one unusual Cry⁺ variant in which no 75-MDa plasmid band was detectable, plasmid sequences were still present, possibly integrated into the chromosome. The 75-MDa plasmid could also apparently recombine with the 68-MDa plasmid, to which it was partially homologous.     

The integrated and free states of Streptomyces griseus plasmid pSG1

A 16.6-kb plasmid-pSG1-was isolated from Streptomyces griseus following transformation of protoplasts with unrelated plasmids. Southern hybridization experiments with radioactive probes prepared from pSG1 fragments and immobilized S. griseus DNA fragments indicated that the plasmid was present in the progenitor strain, in an integrated state. In the pSG1+ isolates plasmid sequences existed both as integrated sequences and as free plasmids. The integrated state of maintenance persisted in strains which have been cured of the free plasmid. The junction site on the plasmid was located on a 0.5-kb EcoRI-SalI fragment. The chromosomal integration site was demonstrated to be the same in all strains derived from S. griseus NRRL3851. The occurrence of both states of plasmid maintenance in the same clones indicates that an integrated pSG1 sequence does not interfere with free plasmid replication and partition. It suggests that the establishment of the free state may involve a replicative excision of pSG1 from the S. griseus chromosome.     

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101–pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1β) and RK2 (IncP-1α), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.     

Identification andin silico characterisation of putative conjugative transfer genes onGeobacillus stearothermophilus plasmids

We repor the first data demonstrating the presence of putative conjugative transfer genes on plasmids of the speciesGeobacillus stearothermophilus. Partial sequence analysis of the plasmid pGS18 fromG. stearothermophilus 18 was determined. It contained eleven complete open reading frames. Five of them encoded proteins which are homologous toBacillus megaterium pBM300 Mob/TraA,Lactococcus lactis pMRC01 TrsD and TrsE,Staphylococcus aureus pGO1 TrsG andS. aureus subsp.aureus pUSA03 TraL, the proteins that are associated with conjugative plasmid transfer. Southern hybridizations were performed on two other plasmids isolated fromG. stearothermophilus 3 andG. stearothermophilus 19 strains using the most homologous parts of those five genes as probes. Data from different hybridization patterns show a close homology of putative conjugative transfer genes between pGS18 and pGS3 hypothesizing a similar molecular organization of putative conjugative plasmid transfer region of both plasmids.     

Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4

The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature. Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence. The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments. Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species. Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis. Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.     

Plasmid Donor Affects Host Range of Promiscuous IncP-1β Plasmid pB10 in an Activated-Sludge Microbial Community

Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression was repressed in the donors by chromosomal lacI^q. The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the α- and γ-Proteobacteria. The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443, Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.     

Comparative genomics and phylogeny of the IncI1 plasmids: a common plasmid type among porcine enterotoxigenic Escherichia coli

Increasing reports of multidrug resistance conferred by conjugative plasmids of Enterobacteriaceae necessitate a better understanding of their evolution. One such group is the narrow-host-range IncI1 plasmid type, known for their ability to carry genes encoding resistance to extended-spectrum beta lactamases. The focus of this study was to perform comparative sequencing of IncI1 plasmids from porcine enterotoxigenic Escherichia coli (ETEC), isolated irrespective of antimicrobial susceptibility phenotype. Five IncI1 plasmids of porcine ETEC origin and one IncI1 plasmid from a Salmonella enterica serovar Kentucky isolate from a healthy broiler chicken were sequenced and compared to existing IncI1 plasmid sequences in an effort to better understand the overall genetic composition of the IncI1 plasmid lineages. Overall, the sequenced porcine ETEC IncI1 plasmids were divergent from other sequenced IncI1 plasmids based upon multiple means of inferred phylogeny. High occurrences of IncI1 and IncA/C plasmid-associated genes and the bla_TEM and bla_CMY-2 beta lactamase genes were observed among porcine ETEC. However, the presence of bla_TEM and bla_CMY-2 did not strongly correlate with IncI1 plasmid possession, suggesting that these plasmids in porcine ETEC are not primarily associated with the carriage of such resistance genes. Overall, this work suggests a conservation of the IncI1 plasmid backbone among sequenced plasmids with a single locus for the acquisition of accessory genes, such as those associated with antimicrobial resistance. Furthermore, the high occurrence of IncI1 and IncA/C plasmids among clinical E. coli from commercial swine facilities is indicative of extensive horizontal gene transfer among porcine ETEC.     

DNA Sequence Analysis of Plasmids from Multidrug Resistant Salmonella enterica Serotype Heidelberg Isolates

Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc) A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.     

Origin and Evolution of Rickettsial Plasmids

Background

Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes.

Results

Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events.

Conclusion

Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as gene duplication and genesis. The plasmids are plastic and mosaic structures that may play biological roles similar to or distinct from their co-residing chromosomes in an obligate intracellular lifestyle.     

Environmentally co‐occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context‐dependent fitness effects

Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co‐occurring pQBR family environmental mercury resistance plasmids and measured their effects on competitive fitness of a Pseudomonas fluorescens SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid‐borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus‐encoding cluster and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolize sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity among co‐occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity.     

Fate and structure of DNA microinjected into mouse TK L cells

Co-microinjection of single linearized molecules of plasmids containing the human β-globin gene (pRK1) and the herpes simplex virus (HSV) type I thymidine kinase gene (pX1) into the mouse TK^? L cell nucleus results in covalent linkage between these (or derived) molecules within the nucleus as revealed by Southern blotting, plasmid rescue, and recovery of plasmid-derived DNA from a Charon 4A phage library of cellular DNA. The microinjected DNA is predominantly found as high molecular weight DNA as determined by Hirt fractionation. Southern blotting data and recombinants from the Charon 4A library suggest that the plasmid DNA is in the form of a head-to-tail linear concatamer of up to 80 copies. Passage of these microinjected cells in selective medium (HAT) results in coordinate amplification of both plasmids, which are maintained in an approx. 3:1 molar ratio of pRK1 to pX1-derived molecules. Hybridization in situ shows the DNA to be integrated on a translocation chromosome, t(4;4). Integration does not appear to be site-specific, since plasmid DNA from another microinjected cell line, C2B, appears on a different translocation chromosome, t(8?;14). Plasmid rescue experiments confirm a previous finding that passage of pBR322 DNA through eukaryotic cells may result in deletions of normally stable plasmid DNA upon subsequent transformation of E. coli. These deletions appear to occur in the bacteria, and originate in a 128 bp region between the Sal I and Hae II sites of pBR322.     

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.