Apparent fusion of the TOL plasmid with the R91 drug resistance plasmid in Pseudomonas aeruginosa.

The TOL catabolic plasmid was shown to be compatible with the R91 drug resistance plasmid. However, the TOL plasmid was extremely unstable in mutant PA03 of P. aeruginosa. By selecting for stabilization of the TOL plasmid in PA03 harbouring R91, it was possible to isolate a strain in which markers from both R91 and TOL appeared to exist in a single recombinant plasmid. This plasmid, pND3, encoded resistance to carbenicillin, was able to transfer at the same frequency as the R91 plasmid and encoded the ability to grow on m-toluate, p-toluate, m-xylene, p-xylene and toluene. In addition, it was shown to be incompatible with the NAH catabolic plasmid and it could be transferred by transduction. The TOL plasmid could stabilize in PA03 harbouring R91 without fusion with R91, and could stabilize in PA03 in the absence of R91. PA03 harbouring either the recombinant plasmid or the stable TOL plasmid in the absence of R91 could promote bacterial chromosome transfer between mutant derivatives of P. aeruginosa strain PA0.     

Design of a species-specific DNA probe based on the pFra plasmid of the plague pathogen]

The recombinant plasmid pBS1 carrying a 2 kb SalGI fragment of Yersinia pestis pFra plasmid was constructed by insertion of the fragment into a vector plasmid pBR327. SalGI-BspRI 400 bp subfragment was recloned into a pBR322 vector plasmid. Open reading frame was found in the fragment by DNA sequencing technique. The subfragment designated F1-probe permits one to identify specifically the Yersinia pestis strains harbouring pFra plasmid, thus, differing them from closely related Yersiniea and other representatives of Enterobacteriaceae family.     

Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64.

The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.     

Effects of the Recipient Strain and Ultraviolet Irradiation on Transduction Kinetics of the Penicillinase Plasmid of Staphylococcus aureus

When the penicillinase plasmid of Staphylococcus aureus PS 81(P(81))(T(81)) was transferred to its cured derivative of PS 81(N(P))(T(81)), there was a fivefold increase in the transduction frequency of penicillinase plasmid markers after ultraviolet (UV) irradiation of the phage instead of the expected decrease typical for plasmid-borne markers. These results were independent of the transducing phage, the donor, and the method of curing the recipient and were also obtained with a cured derivative of PS 80(PI(80)). With PS 52, a naturally occurring penicillin-sensitive strain, and a cured transductant of PS 52 as the recipients, typical plasmid kinetics were observed. The plasmid location of penicillinase plasmid markers in transductants was confirmed by their instability in ethidium bromide (EB). In a cross between isogenic plasmids (PI(258)penZ cad x PI(258)penI asa ero), transductants were doubly selected for cadmium and erythromycin resistances. There was a twofold increase in transduction frequency after UV irradiation of the transducing phage and an increase in the proportion of recombinant type transductants. CsCl-EB density centrifugation revealed that plasmid deoxyribonucleic acid (DNA) was present in PS 81(P(81))(N(T)) and its cured derivative [PS 81(N(P))(N(T))], but not in PS 52. Sucrose gradient analysis of plasmid DNA showed that the penicillinase plasmid of PS 81(P(81))(N(T)) was larger than the plasmid in its cured derivative. Thus, the cured derivative contains plasmid DNA which appears to recombine with the incoming plasmid, causing the rise in transduction frequency noted after UV irradiation of transducing phage.     

The use of the plasmid pTH10 for isolating the donor strains of Pseudomonas mallei

The plasmid pTH10 was transfered by conjugation into the Pseudomonas mallei strains. An attempt to construct the donor strains using the widely known technique employing the homology between the plasmid and chromosome due to the transposon Tn1 carried by the plasmid was unsuccessful. Among the clones resistant to bacteriophage PRD1 the variants were selected with the supposed integration of the plasmid into the chromosome. The latter clones required the ability to transfer the auxotrophic chromosomal markers in conjugation after the repeated conjugational transfer of the plasmid pTH10 into them.     

Investigation of the effect of growth environment on the stability of low-copy-number plasmids in Escherichia coli

The stability of a low-copy-number plasmid, pHSG415, in Escherichia coli, was investigated in batch and continuous culture. The plasmid was unstable in batch culture, but was significantly stabilized by growth in continuous culture with phosphate, nitrogen or potassium limitation. However, the plasmid was very unstable when grown in continuous culture with sulphate limitation. These results contrast with those obtained with multicopy plasmids such as pBR322, which is particularly unstable in carbon- or phosphate-limited continuous culture. The effect of growth rate on the stability of E. coli(pHSG415) grown in continuous culture with glucose limitation was also investigated. The plasmid was significantly more stable in cells grown at higher growth rates. The segregational instability (R) of the plasmid and the difference in growth rate between plasmid-free and plasmid-bearing cells (dmu) were calculated for each condition using the method of Cooper et al. (accompanying paper: Journal of General Microbiology 133, 1871-1880). It was found that the primary cause of the loss of pHSG415 from the cell population was the segregational instability of the plasmid.     

Cloning and characterization of the replicon of the Nocardia italica plasmid, pNI100

A 19-kb plasmid, pNI100, was isolated from Nocardia italica CCRC12359; its replicon was cloned and characterized as having a single open reading frame (ORF) of 1188 bp specifying 396 amino acids (aa). Analyses of the deduced aa sequence of the Rep protein indicated that characteristics of three consensus sequences and a P-loop-like motif in the Rep protein of plasmid pSG5, a conjugative plasmid involving a rolling-circle replication mechanism, were conserved in those of plasmid pNI100. Phenotypically, a pock structure was produced in the regenerated mycelium by introducing pNI100 DNA into the Streptomyces lividans protoplast. This result strongly suggests that pNI100 is a conjugative plasmid and probably replicates by a rolling-circle replication mechanism. By using the replicon of pNI100, a bifunctional plasmid pNI105 that could replicate in both Escherichia coli and S. lividans was constructed and found to be a useful cloning shuttle vector.     

Requirements for the formation of plasmid-transducing particles of Bacillus subtilis bacteriophage SPP1.

We had previously proposed that the production of concatemeric plasmid DNA in plasmid-transducing SPP1 particles is a consequence of phage-directed rolling-circle-type replication of plasmid DNA. The production of such DNA was greatly enhanced when DNA/DNA homology was provided between phage and plasmid DNAs (facilitation of transduction). Here we present evidence that synthesis of concatemeric plasmid DNA can proceed after phage infection under conditions non-permissive for plasmid replication. We also propose that the naturally occurring homology between plasmid and phage is sufficient to account for the frequency of transduction observed in the absence of facilitating homology. Homology of greater than 47 bp gives the maximal facilitation of plasmid transduction. Recombination is not an essential part in the synthesis of concatemeric plasmid DNA.     

Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.     

Mutations in the recD gene of Escherichia coli that raise the copy number of certain plasmids.

Chromosomal mutants were isolated in which, for several small plasmids, there was an increased amount of either covalently closed circular plasmid DNA or total plasmid DNA or both. The mutations were mapped to recD, which has been shown to affect exonuclease V activity and a variety of plasmid maintenance and replication functions. Our results suggest that rolling-circle plasmid replication can occur in recD mutants and that site-specific recombination can resolve the resulting linear multimers into covalently closed circular plasmid forms.     

Inactivation of the replication-termination system affects the replication mode and causes unstable maintenance of plasmid R1

Two so-called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini-derivative pOU47 despite the presence of a functional partition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2 bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA. Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild-type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling-circle structures with double-stranded tails. Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling-circle replication.     

大肠杆菌中整合状态的F′质粒复制起点的克隆和分析

用标记获救法克隆了整合状态的F′质粒的复制起点,证明了由这一复制起点构成的mini-F质粒在不亲和性和对吖啶橙的敏感性方面和自主状态的F′质粒都没有不同。对这一复制起点和来自自主状态的F质粒的复制起点进行了亚克隆,并作限制性内切酶酶切分析比较,没有发现两者在结构上有差异。本文的结果提示,F质粒和F′质粒在发动染色体复制中对recA基因的依赖性的不同,可能与质粒整合在染色体上的位置不同有关。     

Translocation of the chloramphenicol-resistance determinant in Staphylococcus aureus

pC658: a plasmid determining resistance to chloramphenicol in the hospital strain of Staphylococcus aureus JM658 was transduced after irradiation of phage lysate with high doses of UV. The localization of determinants causing resistance to chloramphenicol in the obtained transductants was investigated by modified Arber's method and variants resistant to chloramphenicol, but suspected of absence of chloramphenicolase plasmid were selected. Additionally the absence of plasmid DNA was demonstrated in the selected strains. The possibility of plasmid and chromosomal localization of the same gene indicates its translocable nature. The obtained results suggests transposomal character of the genes determining resistance to chloramphenicol in the pC658 plasmid, occurring in the hospital strain of S. aureus.     

Expression of the gene for tetracycline resistance of plasmids R6 and RP4 in bacteria of the family Enterobacteriaceae]

It was found that manifestation of the tetracycline resistance gene depended on the type of the plasmid containing the gene. The tetracycline resistance gene was subject to less repression in plasmid R6 than in plasmid RP4. Sensitivity of the initial plasmid-free bacteria varied within lower dose ranges than that of the plasmid-carrying strains. Regulation of the tetracycline resistance gene manifestation in the given plasmid may change in different bacterial hosts, i.e. in different cytoplasmic environment at different gene background.     

Plasmid-mediated suppression of the mutational activation of the bgl operon in Shigella sonnei

SSOR, a clinical isolate of Shigella sonnei which exhibits a Salicin-negative phenotype, is unable to mutate to give rise to Sal+ derivatives although a homolog of the Escherichia coli bgl operon is retained by the strain. This was correlated to the presence of an endogenous plasmid in the strain. A plasmid-cured derivative, AK711, could give rise to Sal+ mutants in two steps. Introduction of the plasmid DNA, extracted from SSOR, into various strains of E. coli and S. sonnei, resulted in ampicillin resistant transformants. Interestingly, the presence of the plasmid suppressed the mutational activation of the bgl operon in the transformants. This was further substantiated by the observation that, transformants that have lost the plasmid regained the ability for mutational activation of the bgl operon. Preliminary characterisation of the plasmid indicated a size of 3.8 kb with an origin of replication resembling that of ColE1 replicons and the bla gene homolog of Tn3. Observations of the mutation frequency at the srl and lac loci in the presence of the plasmid indicate that there is a reduction in the mutation frequency, suggesting an antimutator activity associated with the plasmid.     

Sequence analysis of the family of penicillinase-producing plasmids of Neisseria gonorrhoeae

The exact nature of the sequence differences between the medically important family of gonococcal penicillinase-producing plasmids has been ascertained. The entire DNA sequence of the Asia-type plasmid, pJD4, demonstrated that it is 7426 bp and contains two direct repeats (DR30) that are implicated in the formation of deletion variant plasmids, such as the Africa-type plasmid. We have identified putative DnaA and IHF binding sites, various open reading frames that are thought to specify functional proteins, and some important DNA sequences involved with conjugative transfer of gonococcal beta-lactamase plasmids. The deletion in the Africa-type plasmid is 1827 bp and one of the DR30 repeats is also missing. The deletion in the Rio-type plasmid and several Toronto-type plasmids was determined to be 2273 bp and the sequence spanning the deletion was identical irrespective of geographic or temporal origin. The &Ncirc;imes-type plasmid is an Africa-type plasmid and also contains an IS5 insertion sequence. Since IS5 has not been identified in gonococcal isolates, we suggest that this sequence may have been inserted after the original gonococcal plasmid was transformed into Escherichia coli. The New Zealand plasmid is an Asia-type plasmid that contains an endogenous tandem duplication of 1883 bp and the direct DR2 is implicated in this duplication. The nature of the defined truncation of Tn2 present in the various plasmids is also discussed.     

Transposition of Tn904 encoding streptomycin resistance into the octopine Ti plasmid of Agrobacterium tumefaciens.

A transfer-deficient derivative of plasmid RP1-pMG1 was isolated after insertion of Mu cts62. The Tra- R plasmid was used to donate Tn904, encoding streptomycin resistance, to Ti plasmid pAL102 harbored by Agrobacterium tumefaciens Ach5. Under conditions promoting high Ti transfer frequencies, 155 strains were isolated in which the streptomycin marker coupled with Ti plasmid in further transfer experiments. These isolates represent stable insertions of Tn904 into the Ti plasmid. In addition, 19 strains were isolated in which the insertion of Tn904 was apparently unstable. The frequency of stable Tn904 transpositions was estimated to be 3 x 10(4-) per transferred Ti plasmid. Evidence was obtained that Tn904 readily may transpose from the Ti plasmid into the bacterial chromosome. The strains carrying Ti plasmids with stable insertions were characterized with respect to virulence, octopine degradation, octopine synthesis in induced tumors, and Ti plasmid transfer. Thirteen of the strains were found to be affected in tumor-inducing ability.     

Cloning and Characterization of the Replicon of the Nocardia italica Plasmid, pNI100

A 19-kb plasmid, pNI100, was isolated from Nocardia italica CCRC12359; its replicon was cloned and characterized as having a single open reading frame (ORF) of 1188 bp specifying 396 amino acids (aa). Analyses of the deduced aa sequence of the Rep protein indicated that characteristics of three consensus sequences and a P-loop-like motif in the Rep protein of plasmid pSG5, a conjugative plasmid involving a rolling-circle replication mechanism, were conserved in those of plasmid pNI100. Phenotypically, a pock structure was produced in the regenerated mycelium by introducing pNI100 DNA into the Streptomyces lividans protoplast. This result strongly suggests that pNI100 is a conjugative plasmid and probably replicates by a rolling-circle replication mechanism. By using the replicon of pNI100, a bifunctional plasmid pNI105 that could replicate in both Escherichia coli and S. lividans was constructed and found to be a useful cloning shuttle vector.     

含四个串联核酶和GFP基因的转基因质粒的构建

以含绿色荧光蛋白（GFP）基因的质粒pSK100-DS、含切割对虾杆状病毒基因的核酶Rz1的质粒pRGRzl、含核酶Rz2的质粒pRGRz2和转基因空质粒pcDNA3为基础,把绿色荧光蛋白GFP基因克隆于pcDNA3的SV40启动了下面,由SV40启动子控制,含四个两种核酸基因的四联体克隆于pcDNA3的多克隆位点区,由T7启动子控制,构建成含两个Rz1、两个Rz2和GFP基因的转基因质粒pGTR,以用于转基因抗病毒对虾的研究。