P. Tichý

Transfer of liposome-encapsulated plasmid DNA toBacillus subtilis protoplasts and calcium-treatedEscherichia coli cells

Protoplasts ofBacillus subtilis 168trpC2 str and cells ofEscherichia coli SK1590 after treatment with calcium chloride were transformed to tetracycline resistance with the recombinant plasmid pUN82 entrapped in the reverse phase evaporation hposomes. Frequency of transfer was 4×10-4 % inB. subtilis and 8×10-6 % inE. coli.

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Abstract Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.

Isolation of the pMI 10 plasmid from the α-amylase producing strain of Bacillus subtilis

SummaryB. subtilis A 18, a producer of exocellular amylase, was found to carry covalently closed DNA plasmid molecules (pMI 10). The pMI 10 was isolated and characterized by electron microscopy, electrophoretic mobility and restriction endonuclease cleavage pattern. The pMI 10 was absent in all α-amylase low productive or nonproductive clones. The pMI 10 DNA was transformed together with pUB 110 DNA into B. subtilis RM 125 arg-leu- recipient cells, and, hence, compatibility of these plasmids could be demonstrated.

Large-scale isolation and partial characterization of plasmid DNA from B. larvae.

Large-scale preparation of plasmid DNA from twoBacillus larvae strains 423 and 728, honey-bee pathogens, is described. The isolated plasmid DNAs were analyzed by restriction enzyme mapping. No difference in the resulting maps was found for six restriction enzymes. The plasmid DNAs were also compared by Southern blot hybridization and by electron microscopy. The results confirmed the identity of these plasmid DNAs. All these data suggest thatB. larvae strains harbor the same plasmid.

Transformation ofStreptomyces granaticolor with natural and recombinant plasmid vectors

Protoplasts ofStreptomyces granaticolor were found to be transformable by the broad-host-range plasmid pIJ350 but no transformants were detected when the narrow-host-range plasmid pIJ2 or the shuttle vector pPM66 (pIJ350 — pBR322) isolated fromE. coli cells were used. The onset of blue colour granaticin production byS. granaticolor cells was used as a marker to prepare protoplasts with a high transformation capcity. The presence of a restriction system is discussed.

Selective regeneration ofBacillus subtilis protoplasts transformed to kanamycin resistance

An HTY medium osmotically stabilized with 0.5 M D-glucitol was used for regeneration ofBacillus subtilis protoplasts. The application of glucitol as osmotic stabilizer allows simultaneous selection of cells resistant to kanamycin to be made since this antibiotic is not inactivated by glucitol when added to the regeneration medium.

Preparation of protoplasts and regeneration of intact cells of Streptomyces cinnamonensis

Protoplasts were prepared and intact cells were regenerated inStreptomyces cinnamonensis— a monensin producer— to make genetic manipulations with this strain possible. 70–80% of protoplasts were formed and up to 90% of them could regenerate into intact cells.

Effect of temperature on α-amylase formation and DNA replication inBacillus subtilis

The effect of temperature on extracellular α-amylase synthesis and chromosomal and plasrnid DNA replication inBacillus subtilis A18 carrying plasmid pMI 10 was studied. The specific growth rate μ increased with elevated temperature up to 42.5°C, while the activities of α-amylase per population dry mass decreased. No obvious quantitative changes of14C-thymidine incorporation per dry mass increase and no basic differences in plasmid copy number in the range of temperatures from 25 to 40°C were found.

Transformation of Streptomyces lincolnensis protoplasts with plasmid vectors

A method for the preparation and regeneration of protoplasts ofStreptomyces lincolnensis is described. Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25 % regenerated intact cells. Transformation ofS. lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replacons pIJ101, pSLG33 and SCP2. The efficiency of transformation was 3·103 transformants per μg plasmid DNA when (2–5)·107 recipient protoplasts were used. Interspecific transformations showed that there is no efficient restriction system inS. lincolnensis that would limit the transfer of genetic information fromS. lividans orE. coli.

Isolation and characterization of the pMI10 plasmid fromBacillus subtilis, an α-amylase producer

The plasmid DNApMI10 (5310 bp) was isolated from the α-amylase producing strainB. subtilis A18. Thirteen restriction endonucleases were used to digest pMI10 DNA and the restriction map of pMI10 DNA was constructed by mappingPstI (1),HindII (2),BglI (2),BspRI (3) andHindIII (3) sites.