Transformation of the coryneform bacterium cellulomonas flavigena with plasmid DNA via electroporation

Summary Using electroporation we have transformed Cellulomonas flavigena with a shuttle vector (pJA85) derived from the E. coli plasmid pUC8 and the Brevibacterium lactofermentum plasmid pULRS8. Upon transformation this plasmid was found to be stable, not to undergo detectable deletion, and to express antibiotic resistance markers originating in Brevibacterium.     

A rapid method for the isolation of plasmid DNA from bacteria

Summary A rapid method is described for the isolation of plasmid DNA from Escherichia coli and Pseudomonas putida. The effect of heating the cell preparation during plasmid extraction is discussed in relationship to the final plasmid yield.     

Transformation of Kluyveromyces lactis cells by plasmid DNA does not require alkaline cations

Summary A procedure for the transformation ofKluyveromyces lactis based on the Li salt method for introducing plasmid DNA into intact yeast cells is described. Contrary toSaccharomyces cerevisiae, lithium salts are dispensable for inducing competence inK. lactis. 2-Mercaptoethanol, a compound that stimulates transformation inS. cerevisiae, showed an opposite effect. inK. lactis. On the other hand, the presence of PEG 4000 and a heat shock were absolutely required to obtain high transformation efficiency.     

Expression of the threonine operon fromEscherichia coli inBrevibacterium flavum andCorynebacterium glutamicum

Summary The threonine operon fromEscherichia coli was cloned in plasmid pBR322, subcloned into the shuttle vector pCEM300 and the resulting recombinant plasmid was transferred intoBrevibacterium flavum andCorynebacterium glutamicum. The expression ofE. coli threonine genes in these coryneform bacteria was demonstrated by complementing thethrA andthrB mutations and by assaying homoserine dehydrogenase activity.     

Transformation ofBacillus subtilis with the treatment by alkali cations

Summary ExposingBacillus subtilis cultures to high concentrations of alkali cations, especially K⁺, allows efficient transformation by plasmids. The method allows transformation with unfractionated plasmid DNA, monomeric plasmid DNA as well as linear plasmid DNA.B. subtilis strains, not amenable to natural transformation, were also transformed by the present method.     

A new method for quantifying the probability of segregative plasmid loss inEscherichia coli B/r

Summary A new method for quantifying rates of segregative plasmid loss is described. The probability of plasmid loss is found from the decrease in the fraction of plasmid containing cells, following a synchronous division. Stability inEscherichia coli B/r increased with pH and dilution rate, but decreased with temperature.     

Molecular cloning of theEscherichia coli gene encoding xylose isomerase

Summary A 2.4 Kb DNA fragment restricted from a Clarke-Carbon ColEl plasmid, pLC32-9, containing the xylose isomerase gene has been inserted into the PstI site of pDB248, a shuttle plasmid between the bacteriumE. coli and the fission yeast,Schizosaccharomyces pombe. This recombinant plasmid, pDB248-XI, can genetically complement xylose isomerase deficientE. coli strains and xylose isomerase gene can be expressed inSchizosaccharomyces pombe.     

Introduction of foreign genes into Pharbitis nil calli using a vector derived from Agrobacterium pTi

Transgenic calli of Pharbitis nil which grow in the presence of kanamycin were obtained by introduction of plasmid pBI121, which carries kanamycin resistance and the gene for beta-glucuronidase. The calli were shown to have fragments of vector DNA in their genome and high levels of beta-glucuronidase activity. This is the first report of the introduction of T-DNA into P. nil and the T-DNA has been shown to be integrated without apparent rearrangement in its genome. The range of copy numbers was between 3 and 5. The beta-glucuronidase activities measured were about 10 times higher than those of transgenic tobacco by introduction of the same plasmid as previously described by Jefferson et al. (1987). Thus, the widely used CaMV 35S promoter also appears to be very active in P. nil.Abbreviations CaMV Cauliflower Mosaic Virus - NAA naphthylacetic acid - BA benzylaminopurine - NPT neomycinphosphotransferase - GUS beta-glucuronidase - CTX Cefotaxime - MS Murashige and Skoog - 4-MU 4-methylumbelliferone - NOS nopaline synthase - CTAB hexadecyltrimethylammonium bromide     

Plasmid maintenance and growth of recombinantSaccharomyces cerevisiae producing hepatitis B virus surface antigen

Summary The effects of host strains (Saccharomyces cerevisiae) and medium composition on the plasmid stability and expression level of hepatitis B virus surface antigen were investigated. Specific growth rates of cells carrying a plasmid, pMHBS, was found to be slower than those without the plasmid. It was also found that the plasmid was maintained in a more stable manner in the selective medium. The nonselective complex medium, however, was greatly favored for the growth of recombinant hosts as well as the HBsAg production.     

Instability of lactose and citrate metabolism of Leuconostoc strains

Summary The instability of Lac⁺ and Cit⁺ phenotypes was investigated inLeuconostoc mesenteroides subsp.cremoris ATCC 19245 and in four strains ofLeuconostoc mesenteroides subsp.dextranicum. The two phenotypes were linked respectively to a 14 Mdal and a 34 Mdal plasmid in Leuconostoc mesenteroides subsp.cremoris ATCC 19245. InLeuconostoc mesenteroides subsp.dextranicum the character Lac⁺ was linked to a 28 Mdal plasmid, while the Cit⁺ phenotype was stable.     

Construction of a shuttle vector betweenEscherichia coli andZymomonas anaerobia

Summary A 1.7-kb cryptic plasmid was isolated fromZymomonas anaerobia and used to construct a shuttle vector inserting useful parts of pUC9, pBR322, and pRK2501.Escherichia coli was employed to clone the new plasmid designated pSR12. The 7.7-kb plasmid pSR12 reisolated from the host cells could transform competent cells ofZ.anaerobia at 2×10^–7 frequency. This shuttle vector contains two antibiotic resistance markers, Kan^r and Tet^r, as well as restriction sites such as EcoRI, PstI, and XhoI, suitable for DNA recombinations.     

Transfer ofBacillus subtilis endo-β-1,4-glucanase gene intoZymomonas anaerobia

Summary The endo--1,4-glucanase gene ofBacillus subtilis origin cloned previously in a plasmid pBS1 was subcloned in a new plasmid pSCR815, and with the new plasmidZymomonas anaerobia was transformed. TheBacillus glucanase gene expressed in theZymomonas cells with efficiency much lower than inEscherichia coli.     

Plasmid-encoded ropiness production inLactobacillus casei SSP.casei

Summary Genetic determinants of the Muc⁺ character were investigated in two ropy strains,Lactobacillus delbrueckii ssp.bulgaricus 201 andL. casei ssp.casei NCIB 4114, which secrete a large amount of slime in culture media. Plasmid DNA analysis revealed the presence of two plasmids (4.5 and 2.3 Mdal) inL. casei ssp.casei, whileL. delbrueckii ssp.bulgaricus was plasmid free, suggesting a chromosomal location of Muc⁺ character in this strain. Curing experiments carried out onL. casei ssp.casei NCIB 4114 indicated a correlation between the Muc⁺ phenotype and the 4.5 Mdal plasmid.     

A newly isolatedBacillus stearotheromophilus K1041 and its transformation by electroporation

Summary Optimal conditions for the plasmid transformation of a newly isolatedBacillus stearothermophilus K1041 by electroporation were investigated. The optimal conditions allowed a transformation efficiency of 5.8×10⁵ transformants per μg plasmid pUB110.     

Cloning a mutatedtrp operon for the biosynthesis of an antibiotic agent

Summary Plasmid pCG8 containing a mutatedtrp operon was constructed and introduced into anEscherichia coli host for increasing tryptophan biosynthesis. Since tryptophan is a biosynthetic precursor of pyrrolnitrin, the mutated trp operon was inserted into aPseusdomonas vector pME290 to obtain the plasmid pCG12, and then was introduced intoPseudomonas pyrrocinia for enhancing the pyrrolnitrin synthesis. Data showed that the production of pyrrolnitrin of the transformed strain was five-times greater than that of the parental strain.     

Plasmid dna transformation of Lactobacillus strains by electropermeabilization

Summary Two thermophilic strains of Lactobacillus were transformed by electroporation; L.fermentum with a maximum of frequency of 1×10⁵/ug of plasmid vector pPSC₂₀DNA and 1.4×10³/ug pSA₃DNA. L.helveticus showed a very low frequency of transformation, from 9 to 26 transformants/ug DNA in all the experiments carried out with both the vectors. While L.fermentum transformants were very stable, in L.helveticus the acquired plasmid was lost after 30–50 generations.     

Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens

A chimaeric neomycin phosphotransferase II (NPT II) gene was introduced in Brassica oleracea using an oncogenic strain of Agrobacterium tumefaciens harbouring Ti plasmid which contains Nos/NPTII in its T-DNA. The transformation of B. oleracea with the oncogenic Ti plasmid, resulted in regeneration of shoots and roots without any exogenous requirement of phytohormones. The presence of NPT II gene was determined by hybridization of Tn5 encoded NPT II gene with DNA of kanamycin resistant regenerated plants. The expression of NPT II was demonstrated by kanamycin phosphorylation assay. Several regenerated plants were obtained, a few of them were found to be morphological variants and a chlorophyll deficient mutant plant was also obtained.     

Enhanced stability of a 2μ-based recombinant plasmid in diploid yeast

Summary The stability of a 2-based recombinant plasmid, pJDB219, has been compared in glucose-limited chemostat cultures of two haploid strains ofSaccharomyces cerevisiae and a diploid derived from them. The stability of the recombinant plasmid differed in the two haploid hosts but was greatest in the diploid. Enhanced stability in the diploid is probably a function of both the increased copy number and reduced selective burden of the plasmid.     

High level extracellular secretion of thermostable α-amylase ofBacillus stearothermophilus inEscherichia coli

Summary Bacillus stearothermophilus BR135 (ATCC 29609)amy gene was cloned in pBR322 from its plasmid DNA and was subcloned in a vector useful both forB. subtilis andE. coli.E.coli HB101 harboring the plasmid pSS099 when grown in L medium in presence of 5. g/ml chloramphenicol produces 70 units/ml of extracellular -amylase. This is nearly twice that ofE.coli cells harboring pSSO76, a plasmid havingamy ofB.stearothermophilus BR135 atHindIII site of pBR322. Characteristically the protein was a 58 kd protein and cross reacted with antiserum developed against purified -amylase of BR135.     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.