Isolation of plasmid DNA from Bifidobacterium

Conditions for accumulation of the biomass and a procedure for isolation of plasmid DNA from bifidobacteria in microquantities were developed. It was shown that all the strains tested had 1 to 3 plasmids of different molecular weights electrophoretically detected. Relation between the detected plasmid DNA and bifidobacteria resistance to tetracycline and fusidin as well as utilization of some carbohydrates is discussed.     

Isolation and characterization of replication-deficient mutants of ColE1 plasmids.

Replication-defective mutants of plasmid ColE1 were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1, with a ColE1-Tn3-containing plasmid. The replication-defective ColE1 mutants isolated were all spontaneous deletion mutants that had lost the ColE1 replication origin and regions adjacent to it. The extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the ColE1 plasmid component of the chimeras by both agarose and polyacrylamide gel electrophoresis. None of the chimeras containing the replication-defective ColE1 mutants was able to replicate in the presence of chloramphenicol. The expression of ColE1 incompatibility was either markedly reduced or not detectable in the replication mutants isolated.     

Isolation and characterization of pLS 1 plasmid mutants with increased copy numbers

Abstract Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids.     

Isolation of plasmid deletion mutants and study of their instability

We describe a method which allows isolation of deletions within hybrid plasmids. It is based on the fact that the tetracycline resistance (Tc^R) gene of pBR322 can be inactivated by inserting foreign DNA into its HindIII site, and that the easily selectable Tc^R mutants of such plasmids are generally (>90%) due to deletions of certain hybrid plasmid sequences. We have found that Tc^R mutants are usually maintained within the cell recombined with the parental Tc^S plasmids. Such heterodimers dissociate in both Rec⁺ and in recA hosts. Parental rather than mutant plasmids are then retained by the host cell.     

pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors

Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66. pIJ101 was found to be self-transmissible by conjugation, to elicit "lethal zygosis" and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed. Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.     

Transduction of multi-copy plasmid pBR322 by bacteriophage Mu

Summary The temperate bacteriophage Mu transduces the 4363 bp multi-copy plasmid pBR322 at frequencies similar to those of chromosomal markers. Plasmid transducing particles contain DNA molecules of Mu DNA length. Plasmid DNA is transduced as a head-to-tail oligomer that becomes circularized in the recipient cell. The rec system of the donor strain participates in oligomer formation and the rec system of the recipient strain is required for oligomer circularization. Possible mechanisms that may explain the origin of plasmid transducing particles are discussed.     

Isolation and characterization of plasmid DNA fromRuminococcus

A method has been developed, utilizing mutanolysin and proteinase K, for the rapid lysis of strains of the rumen cellulolytic bacteriumRuminococcus. This has enabled bacterial chromosomal and plasmid DNA to be isolated. A small cryptic plasmid has been identified inRuminococcus flavefaciens strain 186. It is 5.2 kb long, contains a singleBamHI site, and two sites forBglII,EcoRI, andHindIII. This plasmid has potential in the development of genetic vectors for rumen bacteria.     

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.     

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.     

Analysis of linear plasmids isolated from Streptomyces: association of protein with the ends of the plasmid DNA

Linear DNA plasmids, designated pSLA1 and pSLA2, which were isolated from two strains of Streptomyces sp. producing lankacidin group antibiotics, were analyzed by using restriction endonucleases. Cleavage patterns of these plasmids were very similar, indicating that they are closely related. Cleavage maps of pSLA2 were constructed with BamHI, SalI, BglII, and EcoRI. A protein was associated with the restriction fragments of both ends of pSLA2 and this was not removed by sodium dodecyl sulfate-phenol treatment. pSLA2 was senstive to a 3′-exonuclease, exonuclease III, but was resistant to the 5′-exonuclease, λ-exonuclease. These results suggest that the protein is associated with the 5′ termini of pSLA2. The same terminal structure was also found on pSLA1. The approximate copy number of the plasmid was estimated by a brief method using agarose gel electrophoresis.     

Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids.

The 2,647-bp nucleotide sequence of cryptic plasmid pTD1, isolated from the oral spirochete Treponema denticola, was determined. The sequence revealed two open reading frames, A and B, which encode polypeptides of 335 and 235 amino acids, respectively. Open reading frame A shows sequence similarity to genes that encode replication proteins from a group of plasmids common in gram-positive bacteria, which replicate via a single-stranded intermediate.     

pIJ101, a multi-copy broad host-range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors

Summary Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66.pIJ101 was found to be self-transmissible by conjugation, to elicit lethal zygosis and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed.Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.     

Isolation of plasmid DNA sequences that complement Rhodobacter sphaeroides mutants deficient in the capacity for CO2-dependent growth

Mutants deficient in the proper regulation and derepression of ribulose-1.5-bisphosphate carboxylase oxygenase (RuBPC/O) in Rhodobacter sphaeroides were isolated by ethyl methanesulphonate (EMS) and Tn5 mutagenesis of a recA parental strain. Mutants were identified by their ability to grow under conditions where the organism requires basal levels of RuBPC C/O for growth yet fail to grow under conditions which require derepression of the enzyme (Aut-). The newly isolated Aut- mutants exhibited phenotypes distinguishable from the previously isolated Aut- mutant, strain KW25/11. Rocket immunoelectrophoretic examination of RuBPC/O levels revealed marked variance in the ability of mutants to derepress form I and form II RuBPC/O in the absence of exogenous carbon. Evidence that some of the mutants possessed different mutations was substantiated by complementation of the EMS-generated mutants by entirely different genes isolated from a genomic library of R. sphaeroides constructed in the broad-host-range cosmid vector pVK102. Southern hybridization analysis of the complementing library isolates showed the complementing genes to be normally carried on the endogenous plasmids of R. sphaeroides. The gene complementing mutant strain KW25/11 was mapped by Tn5 insertional inactivation and the complementing region found to reside on a 1.5 kb PstJ. BamHI fragment. Complemented strains were unable to match wild-type levels of RuBPC/O under conditions requiring derepression of the enzyme, except for mutant strain EMS45. The Aut- phenotype, represented by the mutants isolated in this study, stems from a deficiency in some aspect of photoautotrophic growth.     

Isolation and preliminary characterization of R6K plasmid deletion mutants.

Several deletion mutants of R6K have been isolated by mutagen treatment of bacterial host carrying wild type of the plasmid and search for clones that lost ampicillin or streptomycin resistance. The molecular weight of the mutants as estimated by agarose gel electrophoresis was 15 times 10(6) minus 23 times 10(6) compared to 26 times 10(6) for the parental plasmid. The mutants were characterized in respect of the level of resistance to ampicillin and frequency of conjugational transfer. Some of the mutants were found to differ in Eco RI digestion pattern from the wild type.     

Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids.

Staphylococcus aureus plasmid pE194 manifests a natural thermosensitivity for replication and can be established in several species, both gram positive and gram negative, thus making it attractive for use as a delivery vector. Like most characterized plasmids of gram-positive bacteria, pE194 generates single-stranded DNA. The direction of pE194 replication is clockwise, as determined by the strandedness of free single-stranded DNA. Significant homology exists between a 50-base-pair sequence in the origin of pE194 and sequences present in plasmids pMV158 (Streptococcus agalactiae), pADB201 (Mycoplasma mycoides), and pSH71 (Lactococcus lactis). We used an initiation-termination reaction, in which pE194 initiates replication at its own origin and is induced to terminate at the related pMV158 sequence, to demonstrate that pE194 replicates by a rolling-circle mechanism; the initiation nick site was localized to an 8-base-pair sequence.