I. Holubová

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.

Curing effect of clorobiocin on Escherichia coli plasmids

SummaryClorobiocin, an inhibitor of the gyrB subunit of DNA gyrase, was used for the curing of some Escherichia coli plasmids. Of the plasmids studied, ampicillin resistant R28K and a miniplasmid derived from R1 drd-19 were effectively eliminated. We also succeeded in eliminating the ColA factor from E. coli strain B834 (pBS103), which was resistant to the effect of currently used curing agents. Although a derivative of ColE1-pBR322 was effectively cured by clorobiocin, the ColE1-plasmid was resistant to its effect. The ColV plasmid determining virulence was effectively eliminated.

Transfer of plasmids from Escherichia coli and Pseudomonas aeruginosa to methylotrophic bacteria and their detection in the new hosts.

Several plasmids of incompatibility group P were transferred fromEscherichia coli andPseudomonas aeruginosa strains toMethylophilus methylotrophus and two other methylotrophs to test their recipient ability. The presence of plasmids in transconjugants was confirmed by electrophoretic analysis. Optimal conditions for detection of plasmid DNA in the strains tested based on alkaline lysis of cells at elevated temperature were established. Special behaviour of plasmids carrying the Mu phage in methylotrophic hosts is described.

An efficient method for isolation of plasmid DNA from methylotrophic bacteria

A method, suitable for the isolation of closed circular plasmid DNA from methylotrophic bacteria is described. Improvement of cell lysis was achieved by butanol extraction of cells before application of the lytic agent. Using this method, cryptic plasmids of 7.8, 14, 36 and 200 kb were purified from soil-isolated methylotrophs.

Genetic transfers in Brevibacterium sp.

A positive genetic transfer by protoplast fusion was obtained in auxotrophic mutantsBrevibacterium sp. M27his andBrevibacterium sp. M27arg. Transformation and protoplast fusion with liposomes (as genetic transfers in intact cells and their protoplasts by both the chromosomal and plasmid DNA) did not lead to transfer of the markers followed.

Membrane mutation affecting energy-linked functions in Escherichia coli K 12.

A small-colony forming variant ofEscherichia coli with a mutation in thenof gene was analysed. The alternation of the protein composition in the cytoplasmic membrane and the interaction with K and E group colicina indicated a membrane mutation. The effect of this mutation on some membrane-bound processes, the activity of Mg2+-activated ATPase, the growth on different carbon sources and the active transport of amino acids, is described. This mutation does not exert any effect on the electron transport system.

Characterization of new plasmids from methylotrophic bacteria

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotrophMethylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genusMethylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and theE. coli plasmid pK19 Kmr, which were checked for conjugative transfer fromE. coli into the methylotrophic host.

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.

Isolation and characterization of mutants of the RP4 plasmid coding for increased resistance to ampicillin

E. coli strain J53(RP4) was mutagenized with ethyl methanesulfonate and N-methyl-N′-nitro-N-nitrosoguanidine. Clones showing a two- to threefold increase in resistance to ampicillin were produced. This increase was not due to an increased number of RP4 copies per chromosome. The level of penicillinase activity was twice higher in comparison with the parental strain. No detectable changes were found in the region coding for the resistance to ampicillin on the plasmid by restriction analysis.

The mechanism of resistance to streptomycin inEscherichia coli. Functional analysis of the permeability barrier of cells harbouring the Rldrd-19Km plasmid

The mechanism of phenotypically altered SM resistance in mutants ofEscherichia coli JC5455 (Rldrd-19Km-)lrs and JC5455 (pON5300) was compared with that of the standard strain JC5455(@#@ Rldrd-19Km-). On analyzing the membrane polypeptides in polyacrylamide gel both mutants were found to possess a protein spectrum different from that of the standard strain. Transport of D-xylose and L-arginine was the same in all strains, transport of L-proline was decreased in J05455 (pON5300) which may indicate a mutational interference with energy metabolism. The basic uptake of dihydrostreptomycin was the same in all strains but there were differences after preincubation of cells with streptomycin or glucose. The increased resistance of JC5455 (Rldrd-19Km-)lrs may be due to observed quantitative differences in membrane polypeptides that might play a role in the binding and functional expression of aminoglycoside-3’-adenylyl transferase which modifies streptomycin. The increased sensitivity toward streptomycin in JC5455 (pON5300) can be explained by a mutation due to N-methyl-N’-nitro-N-nitrosoguanidine in the host cell since this change of sensitivity to streptomycin could not be transferred by transformation into a nonmutagenized strain. The coincidence of inducibility of increased transport of streptomycin by this antibiotic and the altered frequency of reversion to high levels of streptomycin resistance in JC5455 (pON5300) and in the transformant JC5455 (pON5302) may indicate that the altered reversibility toward phenotypically high resistance to streptomycin is a property of pON5300 and is transferred by transformation.