Mobilization of the pACYC184 plasmid by hybrid pAS8-121 delta plasmids in Escherichia coli cells

The plasmid pACYC184 is shown to be mobilized for conjugal transfer in Escherichia coli cells by the deleted (Tn7-TcR) derivatives of the hybrid conjugative plasmid pAS8-121 (RP4-Co1E1). Both the mobilized and mobilizing plasmids are autonomously inherited by the recipient cells when the mobilizing plasmid carries single copy of IS8 (the plasmid pAS8-121 delta 16). Cointegrates pAS8-121 delta 16D:: ::pACYC184 are found in the recipient cells with pACYC184 being inserted between two repeats of IS8 if the derivate plasmid pAS8-121 delta 16D having the duplication of IS8 is used to mobilize pACYC184 for conjugal transfer. The insertion of pACYC184 between IS8 repeats in the plasmid pAS8-121 delta 16D eliminates the plasmid ability to be inserted with high frequency into the chromosome of the phototrophic bacterium R. sphaeroides 2R. The cointegrate pAS8-121 delta 16D:: pACYC184 is stable but can be resolved during the transformation deriving the plasmid pACYC184:: IS8. The latter may be used as a probe for isolation and analysis of IS8 DNA sequences and for constructing the vectors on the basis of pACYC184.     

Formation of pAS8-1213 cointegrate with one of the plasmids of Azospirillum brasilense Sp245

Inheritance of the plasmid vector pAS8-1213 in Azospirillum brasilense Sp245 cells has been studied. The plasmid pAS8-1213 is shown to be uncapable of autonomous replication in the new host but able to integrate into the genetic structures of Azospirillum with high frequency. 90-95% of KmR-transconjugants of A. brasilense harbor pAS8-1213 cointegrated with the smaller host plasmid pAbSP245c(85Md). The formed cointegrate can be transferred into Azospirillum spp. 75 and RecA- strains of E. coli (HB101 and DH1) and stably maintained in these cells. The IS21 element inherent of the plasmid pAS8-1213 is supposed to participate in pAS8-1213::pAbSP245c cointegrate formation.     

High-frequency chromosome transfer in Rhodopseudomonas sphaeroides promoted by broad-host-range plasmid RP1 carrying mercury transposon Tn501.

Insertion of the mercury resistance transposon Tn501 into broad-host-range plasmid RP1 greatly enhanced the ability of this plasmid to promote chromosome transfer in the photosynthetic bacterium Rhodopseudomonas sphaeroides. Compared with the wild-type RP1, which produced less than 10(-8) recombinants per donor cell, RP1::Tn501 produced between 10(-3) and 10(-7) recombinants per donor cell depending upon the marker selected. Plasmid RP1::Tn501 promoted polarized transfer of the chromosome from one or perhaps two origins on the chromosome, giving rise to two linkage groups. All of the biosynthetic and antibiotic resistance genes that have been mapped, including those involved in photosynthesis, occur on one or another of these linkage groups.     

A new insertion sequence, IS121, is found on the Mu dI1 (Ap lac) bacteriophage and the Escherichia coli K-12 chromosome

We have discovered a new insertion sequence, now designated IS121, as a component of the Mu dI1 (Ap lac) phage. This sequence is 1.2 kilobases long and contains single recognition sites for the HincII, Bg1II, and HindIII restriction endonucleases. IS121 is present in at least three copies in the chromosome of several Escherichia coli K-12 strains. When present in the nonconjugative plasmid pBR322, IS121 can mediate cointegrate formation with an F' lac plasmid and transfer of pBR322 sequences to suitable recipients. IS121 is also capable of precise or nearly precise excision. As part of the study of IS121, we have determined the physical structure of the Mu dI1 (Ap lac) phage and established an extensive restriction endonuclease map of this phage. A revised schema for the formation of the Mu dI1 (Ap lac) phage is presented.     

Generic interrelations of purple bacteria in the genus Rhodopseudomonas

The technique of DNA--DNA hybridization was used to study relations offween purple nonsulfur bacteria (the family Rhodospirillaceae). The level of homologies with Rhodopseudomonas sphaeroides 8259 was nearly the same for different species (8-17%) in the genus Rhodopseudomonas under the conditions optimal for hybridization. The same level of homologies was found for the DNA of Rhodospirillum rubrum, a species belonging to another genus of purple nonsulfur bacteria (13%). Rhodomicrobium vannielli was most remote from R. sphaeroides 8259 (3%). Similar results were obtained under other conditions of hybridization. The intraspecial heterogeneity of R. sphaeroides was studied in this work. The thermal stability of hybrid duplexes was analysed. The results are indicative of a considerable divergence of different R. sphaeroides strains (delta T50 = 2.1-11.6).     

Characterization of a methionine sulfoximine resistant strain of Rhodobacter capsulatus E1F1

The transposon-loaded plasmid pAS8-121, incapable of autonomous replication in Gram-negative bacteria of non-enteric group, was transferred to Methylobacillus flagellatum KT wild type strain MFK1. The transconjugants arose at a frequency of 10(-7) per donor cell. The majority of the transconjugants tested exhibited the transfer of all selected chromosomal markers at rather high (10(-4)-10(-6) per donor cell) but similar frequencies. Only one of the obtained donors, designated MFK 64, was capable of mobilizing M. flagellatum KT chromosome in a polarized manner. The integrated nature of the plasmid in this and other MFK1 (pAS8-121) derivatives was supported by the results of DNA-DNA hybridization.     

Transfer of chromosomal genes mediated by plasmid r68.45 in Rhodopseudomonas sphaeroides.

Plasmid R68.45 was transferred from Pseudomonas aeruginosa PAO25 to the photosynthetic species Rhodopseudomonas gelatinosa and Rhodopseudomonas sphaeroides by selection for resistance to antibiotics. R. sphaeroides strains carrying the plasmid could transfer the plasmid and also chromosomal genes to other strains of R. sphaeroides.     

Intermolecular Transposition of Is10 Causes Coupled Homologous Recombination at the Transposition Site

Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid. This was detected by the activity of the tet gene expressed from the phage 434 P(R) promoter. Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site. Cointegrate formation was abolished in δrecA strains, although simple insertions of IS10 were observed. This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor. Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event. Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome. This was accompanied by the formation of an additional copy of IS10 in the chromosome. Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10.     

Thermosensitive vector derived from RP1 plasmid for detection of transposable elements

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.     

Conjugation in Rhodopseudomonas sphaeroides mediated by R plasmids

Plasmids R68.45, RP4, RP4::Mu cts62, RP1ts::Tn10, RP1ts::Tn9, Rts1 and RP41 were transferred into cells of photosynthetic nitrogen-fixation bacterium Rhodopseudomonas sphaeroides from Escherichia coli and Pseudomonas aeruginosa. The transfer of plasmids occurred with high frequency of 10(-1) to 10(-2) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell. Bacteriophage Mu cts62 could be induced from the plasmid DNA in R. sphaeroides 2R cells and was capable of the lytic growth and producing phage progeny. It was demonstrated that an increase in the efficiency of donor chromosomal genes transfer into recipient cells could be achieved in crosses with the donor carrying RP4::Mcts62 plasmid.     

Isolation, characterization, and complementation of a paralyzed flagellar mutant of Rhodobacter sphaeroides WS8.

A paralyzed Rhodobacter sphaeroides mutant strain (PARA1) was isolated by a motility screening procedure following mutagenesis of wild-type R. sphaeroides WS8-N with the transposable element TnphoA (Tn5 IS50L::phoA). PARA1 synthesized a wild-type level of flagellin, as detected by Western immunoblotting with antiflagellar antiserum. Flagellar staining showed that flagellin was assembled into apparently normal external flagellar filaments. Electron micrographs of basal body structures from PARA1 showed that some ring structures that were present were similar to those in wild-type R. sphaeroides WS8-N. PARA1 cells were nonmotile under all growth conditions. No pseudorevertants to motility were seen when PARA1 was grown in the presence of kanamycin to select for the presence of the transposon. The presence of the single copy of TnphoA in the PARA1 chromosome was demonstrated by Southern blotting. Western blotting of cytoplasmic, periplasmic, and membrane fractions of PARA1 with anti-alkaline phosphatase antiserum showed that the transposon had been inserted in-frame into a gene encoding a membrane protein. A SalI restriction endonuclease fragment was cloned from the chromosome of PARA1; this fragment contained a portion of the transposon and R. sphaeroides DNA sequence 5' of the site of insertion. This flanking R. sphaeroides DNA sequence was used to probe an R. sphaeroides WS8 cosmid library. A cosmid designated c19 hybridized to the probe, and a SalI restriction endonuclease fragment derived from this cosmid restored wild-type motility to PARA1 when introduced into this mutant strain by conjugation. The significance of this finding in a bacterium with unidirectionally rotating flagella is discussed.     

Broad-host-range plasmid vector for the in vitro construction of transcriptional/translational lac fusions

A broad-host-range plasmid was constructed that allows the in vitro formation of beta-galactosidase fusions. DNA from the photosynthetic bacterium Rhodopseudomonas sphaeroides was cloned into this plasmid and a number of R. sphaeroides isolates were recovered that had varying levels of beta-galactosidase activity. beta-galactosidase antigenic activity from the fusion strains could be localized immunologically in polypeptides with an Mr of 120 000 or greater. Expression of beta-galactosidase activity under control of fusion derivatives was either very low or nonexistent in Escherichia coli relative to R. sphaeroides, indicating that R. sphaeroides promoters or translational start signals function poorly in E. coli.     

Physical characterization of a plasmid cointegrate containing an F''his gnd element and the Salmonella typhimurium LT2 cryptic plasmid.

A recombinant plasmid (pAS19) isolated from a derivative of Salmonella typhimurium LT2, containing the strain LT2 cryptic plasmid and an F'his gnd element, has been physically characterized. The pAS19 plasmid contour length equals the sum of the contour lengths of the cryptic plasmid and F'his gnd element. Deoxyribonucleic acid (DNA)-DNA hybridization experiments demonstrated that whereas the pAS19 plasmid exhibits extensive DNA homology with both the cryptic plasmid and the F'his gnd element, there is little DNA homology between these latter two plasmids. The DNA fragmentation pattern of the pAS19 plasmid produced by the restriction endonuclease R-EcoRI is consistent with that expected for a composite plasmid cointegrate containing most, if not all, of the DNA sequences present in its two component plasmids.     

IS-like element IS8 in RP4 plasmid and its involvement in cointegration

The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi: :RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasmids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence.     

New tools for the physical and genetic mapping of Lactococcus strains.

Tools for the genetic and physical analysis of the Lactococcus lactis subsp. lactis genome were developed. Plasmid pRC1 does not replicate in Gram+ bacteria; it contains unique ApaI, NotI and SmaI restriction sites and an erythromycin-resistance (ErR) encoding gene, ermAM, functional in L. lactis subsp. lactis. When a chromosomal L. lactis subsp. lactis DNA fragment was cloned into this vector, the resulting plasmid became integrated, after transformation, into the bacterial chromosome by homologous recombination in a Campbell-like manner. The integration lead to the generation of new rare restriction sites near to the host fragment. This procedure allows precise mapping of cloned genes onto the chromosomal restriction map. The mapping of the his operon of L. lactis subsp. lactis provides an illustration. The cloning into pRC1 of an IS element able to transpose into the chromosome of the target cell, gave rise to an integration plasmid able to insert randomly rare restriction sites onto the bacterial chromosome. The L. lactis IS element, ISS1RS, was cloned into pRC1, yielding pRL1. Pulsed-field gel electrophoresis analysis of ErR clones obtained after transformation with pRL1, showed that this plasmid was stably integrated at a number of different sites in the L. lactis subsp. lactis chromosome, via transposition. Plasmids pRC1 and pRL1 can greatly facilitate the construction of the physical and genetic map of the chromosome of lactococcal strains.     

Genetic study of plasmid integration into yeast chromosomes. IV. Integration of the plasmid pYF91 into different yeast chromosomes

Integration of the episomic chimeric plasmid pYF91 into yeast chromosomes has been studied. Plasmid insertion into the chromosomes was observed to occur with the frequency of 4 X 10(-8). 379 integrants were selected from the highly unstable (cir0) transformants. The fact of plasmid integration into particular chromosomes was confirmed for 318 integrants. Genetic analysis showed that the plasmid can integrate into the region of LEU2 gene or into another arm of chromosome III (227 integrants), and also into other chromosomes: I, II, IV, V, VI, VII, VIII, IX, XII, XV (91 integrants). It is suggested that integration is the result of recombination between yeast chromosomes and homologous plasmid regions carrying LEU2 gene or Ty element and "delta" sequence.     

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 .     

Control of plasmid incompatibility: characteristics of the bireplicon hybrid pAS8 and its deletion mutants]

The phenomenon of incompatibility has been investigated using deletion mutants of hybrid bireplicon plasmid pAS8. The hybrid pAS8 displays incompatibility specific for both components of its structure. In contrast to P-specificity of pAS8, functions of ColE1-specificity are not effectively expressed. Expression of ColE1-specificity in pAS8 plasmid and its derivatives is characterized by different directions and this is due to the presence or absence of genes of RP4 replication machinery in the plasmid DNA. Mutant plasmids show different efficiency of P-specificity depending on the extension of deletion in the region of essential genes of the RP4 component. Some of the mutants, in spite of the loss of replication genes, including origin of vegetative replication, are incompatible with the representatives of the Inc P group in both directions of testing. Different character and the level of expression of ColE1- and P-specificity in the pAS8 hybrid and its deletion derivatives are not associated with change in the number of plasmid DNA copies, for all of them are subjects to stringent control of replication. The data suggest the existence of incompatibility functions control mechanism which does not seem to include replication genes. Possible ways of realization of the inc genes functions are discussed.     

A host-dependent hybrid plasmid suitable as a suicidal carrier for transposable elements

Plasmid pAS8Tc^s rep-1::Tn7 (abbreviated pAS8Rep-1), a derivative of the RP4-ColE1 hybrid plasmid pAS8 displaying ColE1-dependent replication/maintenance, was found capable of the introduction of transposon Tn7 into the genome of phytopathogenic Pseudomonas. The plasmid is potentially useful as a general purpose suicidal Tn carrier for bacteria that do not support stable replication/maintenance of ColE1 but are within the conjugational host range of RP4.     

Site-directed mutagenesis in Escherichia coli of a stable R772::Ti cointegrate plasmid from Agrobacterium tumefaciens

The host range of an octopine Ti plasmid is limited to Rhizobiaceae. This has been extended also to Escherichia coli in the form of a stable cointegrate with the wide-host-range plasmid R772. Its structure was studied by constructing a physical map of R772 and of the R772::pTiB6 cointegrate. An insertion sequence present in R772, called IS70, turned out to be involved in cointegrate formation. We found one intact copy of IS70 and a small segment of IS70, respectively, at the junctions of R772 and Ti DNA. The absence of a complete second copy of IS70 is a likely explanation for the stability of the cointegrate plasmid. A procedure for site-directed mutagenesis of this cointegrate plasmid in E. coli is described. The effect of mutations in the Ti plasmid part can be studied subsequently by transferring the cointegrate into Agrobacterium tumefaciens. The advantage of this procedure for Ti plasmids over other methods used at present is discussed.