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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We previously have shown that a chromosomally integrated copy of the tra gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates transcription of tra as well as its own synthesis, can promote the conjugal transfer of both chromosomal and plasmid genes in Streptomyces lividans. Using an antibody generated against a fusion protein containing the C-terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface-grown S. lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found. Expression of Tra during the S. lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites. Comparison of the membrane concentration of Tra protein with tra mRNA concentration during the S. lividans life cycle indicated that the disappearance of Tra is post-transcriptionally controlled and thus is not mediated by KorA. The results of ‘interrupted mating’ experiments, together with the time of appearance of Tra in S. lividans membranes, indicate that the intermycelial transfer of pIJ101 in S. lividans is complete by the onset of cellular differentiation.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra+ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.
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  • 3
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Ferrihsemie acid, a ferric-porphyrin complex resulting from the oxidation of haem, was prepared by the method of Morrison and Williams2. Throughout a wide pH range, this compound has an intense absorption band near 400 m(ji, characteristic of the hsematins (the Soret band- ^max ~ 105), as well as ...
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  • 4
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possibly other replicons occur more frequently than has hitherto been appreciated. The sequence changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existed in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous,and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.
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  • 5
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The relationship between replication control and plasmid incompatibility has been investigated using a composite replicon, pPM1, which consists of the pSC101 plasmid ligated to another small multicopy plasmid, RSF1050. Since pPM1 can utilise the replication system of either of the two functionally distinct components, propagation of the composite plasmid can occur in the presence of a mutation of one of its moieties. Such mutants are detected by their inability to rescue the composite plasmid under conditions not permissive for replication of the other moiety. Mutations in incompatibility functions can be detected by the failure of the composite replicon to exclude co-existing plasmids carrying a replication system identical to the one on pPM1. The inability of the composite plasmid to replicate at 42° in a host synthesizing temperature-sensitive DNA polymerase I, which is required by the RSF1050 replication system, was used to isolate pPM1 mutants defective in replication of the pSC101 component. Mutants defective in the incompatibility functions of pSC101 were obtained by selecting derivatives that allow the stable coexistence of a second pSC101 replicon in the same cell. Analysis of these two classes of mutants indicates that plasmids selected for defective pSC101 replication ability nevertheless retain pSC101 incompatibility. In contrast, plasmid mutants that have lost incompatibility functions were found always to be defective in replication ability.
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  • 6
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Promoter-probe plasmid vectors were constructed for Streptomyces lividans using expression of the Escherichia coli chloramphenicol acetyltransferase gene as an indicator of promoter activity. These vectors have been used to isolate and to study the activity of DNA sequences that contain transcriptional control signals from Streptomyces, Bacillus licheniformis, E. coli, and Serratia marcescens. Studies of these promoter regions in heterospecific hosts indicate that genus or species-specific factors may present barriers to the expression of bacterial genetic material in certain heterologous cellular environments. While promoter regions isolated from E. coli, S. marcescens and B. licheniformis all appear to be recognized by the RNA polymerase of S. lividans, the Streptomyces transcriptional control signals isolated do not appear to function normally in E. coli.
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  • 7
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We present data showing that the SLP1 plasmids found in Streptomyces lividans after mating with S. coelicolor strain A3(2) orginate as deletion mutants of a 17 kb segment of the S. coelicolor chromosome. Excision of the entire 17 kb segment yields a transiently existing plasmid containing a site for integration into the chromosome of recipient SLP1- S. lividans strains at a unique locus that corresponds to the original chromosomal location of SLP1 in S. coelicolor. The deletion mutants of SLP1 lack the attachment site and/or other regions required for its integration, and thus persist in the recipient as autonomously replicating plasmids. Plasmids that contain the complete 17 kb sequence of the chromosomally integrated SLP1 segment were constructed in vitro by circularization of restriction endonuclease-generated fragements of chromosomal DNA carrying a tandemly-duplicated integrant of SLP1. Transformation of an SLP1- S. lividans strain with such plasmids results in chromosomal integration of the SLP1 sequence at the same site at which it is integrated in S. lividans cells that acquire the sequence by mating with S. coelicolor. A model for the site-specific excision and integration of SLP1 is presented.
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  • 8
    ISSN: 1617-4623
    Keywords: Plasmid replication ; Incompatibility ; Helix-turn-helix motifs ; Superinfection immunity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We report here the existence and initial characterization of a genetic locus (imp) that inhibits maintenance of SLP1-derived plasmids as extrachromosomal replicons in a manner distinct from normal incompatibility between autonomous SLP1 replicons. Thetrans-actingimp function has been localized to a 1.8 kbEco47III restriction fragment present on integrated SLP1 elements. At least part of this DNA segment is absent from SLP1-derived plasmids. DNA sequence analysis of theimp region indicates that it contains three overlapping open reading frames (ORFs) that may constitute a polycistronic operon. The effects of insertions within theimp region indicate that uninterrupted transcription through all three ORFs is necessary forimp activity.
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  • 9
    ISSN: 1617-4623
    Keywords: Plasmid transfer ; Repressor ; Transcription control ; lac genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary ThekorA andkorB loci ofStreptomyces lividans plasmid pIJ101 have previously been shown to control expression of the pIJ101tra (formerlykilA) andkilB genes at the transcriptional level. We show here that mutations in translational open reading frames (ORFs) that map within the kor loci abolish repression of theS. lividans lac gene directed by thetra andkilB promoters. Introduction of thekorA andkorB ORFs intoEscherichia coli maxicells under control of anE. coli promoter gave rise to 31 kDa and 10 kDa proteins that correspond in size to the products expected from the sequences of the respective ORFs; these proteins controlled transcription from the pIJ101tra andkilB promoters in theE. coli host. Mutations that affected the KorA or KorB phenotype altered the structure of, or eliminated, the protein products of thekorA andkorB ORFs, further demonstrating that these ORFs encode the functional repressors of the pIJl01kil/kor gene system.
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  • 10
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A highly efficient method for transformation of Bacillus subtilis by plasmid DNA is reported. The procedure, which involves polyethylene glycolinduced DNA uptake by protoplasts and subsequent regeneration of the bacterial cell wall, yields up to 80% transformants with an efficiency of 4x107 transformants per μg of supercoiled DNA. Plasmids constructed by in vitro ligation or endonuclease-generated fragments of linear plasmid DNA can also transform PEG-treated protoplasts, but at a lower frequency.
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