Isolation of plasmids present in thermophilic strains from hot springs in Jordan

The plasmid profile of two thermophilic bacterial strains isolated from recreation thermal springs in Jordan has been investigated. These strains are Streptococcus thermophilus and Bacillus sp1, which have been isolated from Zerka – Maeen and Himma hot springs respectively. Supercoiled and circular plasmid forms were detected, explaining the effect of DNA conformation on the mobility of the plasmid in the agarose gel electrophoresis. Two plasmids have been isolated and characterized by restriction endonucleases to facilitate their use as cloning vectors in thermophilic strains. The sizes of the plasmids were approximately 3 kb (from Streptococcus thermophilus) and 7 kb (from Bacillus spl). These plasmids were then digested with three different restriction enzymes (EcoRI, Bam HI, and HindIII), one of which was found to possess a single site for both plasmids, this enzyme is EcoRI.     

Drug Resistance and R Plasmids in Vibrio anguillarum Isolated in Cultured Ayu (Plecoglossus altivelis)

Two hundred twenty-six strains of Vibrio anguillarum collected from cultured ayu (Plecoglossus altivelis) between 1978 and 1980 were studied for their sensitivities to 10 chemotherapeutics. In order to determine whether the drug-resistant strains possessed transferable R plasmids, they were conjugated with Escherichia coli. Almost all the strains isolated during the 3 years showed resistance to nalidixic acid (NA) and/or furazolidone (NF). NA and NF resistance were not transferred to Escherichia coli from any of the strains. Chloramphenicol-resistant strains were isolated in every year and almost all of them carried transferable R plasmids. Only one strain with tetracycline resistance was found among the strains tested. Strains resistant to sulfonamides, streptomycin, ampicillin (ABP), and trimethoprim (TMP) increased rapidly in 1980, and a large number of them carried transferable R plasmids. Transferable R plasmids encoded with resistance to ABP and TMP were detected for the first time in V. anguillarum strains. The R plasmids detected in the strains isolated in 1980 were classified into incompatibility groups E, A, and an untypable group. The R plasmid DNAs were cleaved by EcoRI to yield 11 to 13 fragments. The estimated molecular weights of the R plasmids from the five strains ranged from 97 to 104 M daltons.     

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10⁶ was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.     

An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

The structures and fidelity of replication of mouse mitochondrial DNA-pSC101 EcoRI recombinant plasmids grown in E. coli K12

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (LA9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs.Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments.A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165±10 nucleotide pairs.     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Mapping of the resistance genes of the R plasmid NR1.

Summary The drug resistance genes on the r-determinants component of the composite R plasmid NR1 were mapped on the EcoRI restriction endonuclease fragments of the R plasmid by cloning the fragments using the plasmid RSF2124 as a vector. The sulfonamide (Su) and streptomycin/spectinomycin (Sm/Sp) resistance genes are located on EcoRI fragment G of NR1. The expression of resistance to mercuric ions (Mer) requires both EcoRI fragment H and I of NR1. The expression of chloramphenicol (Cm) and fusidic acid (Fus) resistance requires EcoRI fragments A and J of NR1. The kan fragment of the related R plasmid R6-5 can substitute for EcoRI fragment J of NR1 in the expression of Cm and Fus resistance. The structural genes for Cm and Fus resistance appear to be a part of an operon whose expression is controlled by the same promoter.     

DNA Hybridization Probe for Use in Determining Restricted Nodulation among Bradyrhizobium japonicum Serocluster 123 Field Isolates

Several soybean plant introduction (PI) genotypes have recently been described which restrict nodulation of Bradyrhizobium japonicum serocluster 123 in an apparently serogroup-specific manner. While PI 371607 restricts nodulation of strains in serogroup 123 and some in serogroup 127, those in serogroup 129 are not restricted. When DNA regions within and around the B. japonicum I-110 common nodulation genes were used as probes to genomic DNA from the serogroup strains USDA 123, USDA 127, and USDA 129, several of the probes differentially hybridized to the nodulation-restricted and -unrestricted strains. One of the gene regions, cloned in plasmid pMJS12, was subsequently shown to hybridize to 4.6-kilobase EcoRI fragments from DNAs from nodulation-restricted strains and to larger fragments in nodulation-unrestricted strains. To determine if the different hybridization patterns could be used to predict nodulation restriction, we hybridized pMJS12 to EcoRI-digested genomic DNAs from uncharacterized serocluster 123 field isolates. Of the 36 strains examined, 15 were found to have single, major, 4.6-kilobase hybridizing EcoRI fragments. When tested for nodulation, 80% (12 of 15) of the strains were correctly predicted to be restricted for nodulation of the PI genotypes. In addition, hybridization patterns obtained with pMJS12 and nodulation phenotypes on PI 371607 indicated that there are at least three types of serogroup 127 strains. Our results suggest that the pMJS12 gene probe may be useful in selecting compatible host-strain combinations and in determining the suitability of field sites for the placement of soybean genotypes containing restrictive nodulation alleles.     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Mutagenesis of plasmid DNA with hydroxylamine: Isolation of mutants of multi-copy plasmids

Summary An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their -lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33° C, but carrier strains grow well at 28° C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.     

Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids

Summary Wild-type strain A454 (Streptococcus pyogenes) transferred en bloc its erythromycin (Em) and tetracycline (Tc) resistance markers into several plasmid-free streptococcal recipients. No plasmid DNA was detected in either the wild-type or the transconjugant strains. Crosses were performed between A454 and S. faecalis Rec⁺ or Rec^- recipients carrying hemolysin-bacteriocin plasmids, pIP964 or pAD1. The Em Tc-resistant transconjugants obtained harbored either the parental plasmid or an Em Tc resistance plasmid derived from pIP964 or pAD1. The restriction endonuclease analysis of 12 derivative plasmids showed insertions of various sizes into different fragments of pIP964 or pAD1. A454 and the Em Tc-resistant plasmid-free transconjugants were found to contain two EcoRI DNA fragments, that shared homology with ³²P-labeled pIP1077, one of the Em Tc resistance derivative plasmids, but not with ³²P-labeled pIP964. No homology was detected between pIP1077 and the cellular DNA of the antibiotic-susceptible recipients.Previously Thea Horodniceanu     

Altered tetracycline resistance in pSC101 recombinant plasmids

Summary Investigation of tetracycline resistance genetically determined by the plasmid pSC101 and several recombinants of pSC101 containing of EcoRI generated DNA fragments inserted at the EcoRI site has revealed significant differences in the phenotypic expression of that resistance. The altered phenotypes of the recombinant plasmids may be the result of the location of the EcoRI site of pSC101, which has been determined to be near the genetic elements involved with tetracycline resistance.     

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus with high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site And two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).     

Transposon Mutagenesis and Excision of R′ Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10⁻⁷ to 10⁻⁹. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg^r transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with ³²P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg^r both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg^r transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R′ plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

New cloning vectors using benomyl resistance as a dominant marker for selection inNeurospora crassa and in other filamentous fungi

We describe five new plasmid vectors derived from pBR322 that carry theNeurospora crassa β-tubulin gene conferring resistance to benomyl. The benomyl resistance gene has been modified to eliminate an internalEcoRI site to facilitate the cloning ofEcoRI restriction fragments. These plasmids allow rapid subcloning of fragments from one replicon to another without insert fragment purification due to the presence of different drug resistance markers (resistance to kanamycin, tetracycline, or chloramphenicol) carried on the plasmids. These vectors will allow rapid transformation ofN. crassa and other filamentous fungi to allow phenotypic characterization of subcloned fragments.     

Regulation of the L-arabinose operon in strains of Escherichia coli containing ColE1-ara hybrid plasmids.

Summary Hybrid plasmids were constructed from fragments of F'ara episomes formed by the restriction endonuclease EcoRI and a linear form of the plasmid ColE1 created by cleavage with EcoRI. Hybrid plasmids were constructed containing the entire ara region or the ara region with various parts deleted. E. coli K12 host strains were constructed which contained different deletions of the ara region. The hybrid plasmids were transferred to those strains whose ara deletion complemented that of the plasmid. The initial differential rates of synthesis of L-arabinose isomerase, the product of the araA gene, were determined for the Ara⁺, plasmid containing strains. These studies demonstrated that strains containing (araO1BA)718 produce elevated levels of araC protein, suggesting the araC promoter has been altered by this deletion. Evidence is also presented which suggests that araC protein activates the ara-BAD operon to higher levels when it is present in cis rather than trans. Amplification of the products of the cloned genes is observed when compared to haploid levels in some cases.     

Analysis of DNA,lipopolysaccharide structure,and some cultural and morphological properties in closely related strains of Azospirillum brasilense

We studied closely related Azospirillum brasilense strains Sp7 and Cd. For probing of their genomes, the fragments of 85 MDa (p85) and 120 MDa (p120) from A. brasilense Sp245 plasmids were hybridized with 115-MDa (p115) and 90-MDa (p90) plasmids of strain Sp7, respectively. Strain Cd was found to lose the 115-MDa plasmid and one of the two EcoRI restriction fragments of the total DNA (localized within p115 and the chromosome) that was homologous to an EcoRI-generated p85 fragment of 2.4 kb. On the contrary, in the total DNA of strain Sp7-S, in spite of the previously established disappearance of the 115-MDa replicon, two fragments homologous to p85 were revealed, as with strain Sp7. It is suggested that the Sp7-S genome contains the total p115 DNA or at least a certain part of it. Strains Sp7 and Cd were found to differ in size and morphology of colonies on solid and semisolid media, in the levels of resistance to the cation surfactant cetavlon, and in the antigen structure of lipopolysaccharides.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 224–230.Original Russian Text Copyright © 2005 by Petrova, Matora, Burygin, Borisov, Katsy.     

Molecular cloning of restriction fragments and construction of a physical and genetic map of the Escherichia coli plasmid R538-1.

A genetic and physical map of Escherichia coli plasmid R538-1 was constructed using restriction endonucleases and molecular cloning techniques. R538-1 DNA was cleaved into 12 fragments by endonuclease · R · EcoRI, 6 fragments by endonuclease R · HindIII, and 3 fragments by endonuclease R · BamHI. The order of these fragments was determined by standard restriction fragment mapping techniques. Endo · R · EcoRI, endo · R · HindIII, endo · R · BamHI, and endo · R · PstI fragments obtained from R538-1 and ColE1-derived plasmids (pMB9, ColE1Ap^r, and pBR322) were ligated in vitro and used to transform E. coli C600. Transformants were selected for antibiotic resistance markers carried by R538-1. Analysis of the R538-1 fragments contained in these hybrid plasmids permitted the construction of a genetic map of the R538-1 plasmid. The genetic map of this plasmid is very similar to that of plasmid R100.     

In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions

Summary The construction of a plasmid carrying the right end PstI·B fragment of bacteriophage Mu DNA and of plasmids containing in addition the left end EcoRI·C fragment of Mu DNA into the vector pBR322 is described. Inversion of the G segment still occurs in all these plasmids. By marker rescue and complementation experiments the right PstI cleavage site was located to the left of gene Q. The composite plasmids inheriting also the left end EcoRI fragment of Mu DNA express both the immunity and killing functions of Mu and direct the in vitro synthesis of presumably Mu-specific polypeptides. These results demonstrate that Mu-specific functions can be analyzed from cloned fragments.