M. Mačor

Comparative study of two forms of aro A CP4 gene in Escherichia coli

The enzyme CP4 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) from Agrobacterium tumefaciens CP4, encoded by the aroA gene, has been used for the construction of genetically modified crops resistant to total herbicide glyphosate. During the study of possible horizontal gene transfer of aroA CP4 gene from genetically modified food in gastrointestinal tract to bacterial community living in the animal gut, we have discovered and characterized truncated form of aroA CP4 within the cloning experiments in Escherichia coli. We have compared properties of the recombinant E. coli strains with both CP4 EPSPS enzyme forms.

Euglena gracilis as a supplementary test organism for detecting biologically active compounds

The mutagenic activity of more than 120 antimicrobial agents and protective components was investigated. Only Kathon showed a consistent increase in revertant counts in the Ames test onSalmonella typhimurium. The hereditary bleaching test onEuglena gracilis used for detecting extranuclear mutations, showed positive results for Kathon, triethanolamine and diamine silver tetraborate.

Escherichia coli strain with a deletion of the chromosomalampC gene marked with TcR, suitable for production of penicillin G acylase

Escherichia coli strain which contains a marker of tetracycline resistance gene (TcR) placed by P1 transduction beside the chromosomal deletion, ofampC gene (ΔampC) coding for β-lactamase was constructed. Such introduction of TcR marker permits a fast and simple selection for the transfer of ΔampC by P1 transduction into industrialE. coli strains. This approach was used for constructing anE. coli strain suitable for penicillin acylase production.

Hereditary bleaching ofEuglena gracilis affected by 3-(5-nitro-2-furyl)acrylic acid

Light and elevated incubation temperature were found to be involved in the ability of four nitrofurans to cause the hereditary bleaching effect inEuglena gracilis. The enzyme activation by S9 mix also played a significant role in the case of 3-(5-nitro-2-furyl)acrylic acid. When using the combination of the above factors the most pronounced hereditary bleaching was reached.

Streptomycin and N-Methyl-N’-nitro-N-nitroso-guanidine inhibit incorporation of14C-precursors of macromolecule synthesis inEuglena gracillis

Streptomycin and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) cause a 100% hereditary bleaching ofEuglena gracilis. In spite of the outcomes being the same, the corresponding mechanisms differ. By investigating the incorporation of the14C-labelled precursors of macromolecule synthesis we showed streptomycin to inhibit the synthesis of nucleic acids and proteins to the same extent (56%), whereas in the case of MNNG a 66% inhibition of nucleic acid synthesis and a 24% one of protein synthesis were observed.

Effect of elevated temperature on genotoxicity of chemotherapeuticals toward Euglena gracilis

A collection of 20 compounds was tested for their ability to induce a permanent loss of ohloroplasts fromEuglena gracilis cells under conditions increasing the sensitivity of the flagellate to genotoxic compounds,viz. in the resting medium and at an elevated temperature. Streptomycin, dihydrostreptomycin, gentamicin, kanamycin and partially chloramphenicol exhibited mutagenic effects. Eight antibiotics eliminated chloroplasts only from growing cultures and seven antibiotics did not induce the mutation at all.

Cloning and transfer of sucrase operon in Escherichia coli

The sucrase operon was transferred from the donor Escherichia coli MM23 strain by the phage P1 into the Escherichia coli TG1, mini-Mu phage into the strain Escherichia coli JT4 and subsequently to the basic cloning vectors. We have prepared strains of Escherichia coli XL1 Blue, Escherichia coli JM109 that are able to grow on various concentrations of sucrose. We also transferred the sucrase operon by conjugation of mobile plasmid pUR400 into the strain Escherichia coli MC4100.

Cloning of sucrase operon with mini-mu and plasmid-mediated metabolism of sucrose

Thein vivo cloning system based on mini-Mu derivatives was used for cloning the sucrase operon. We constructed several recombinant plasmids pJT21, pMM2324, pMM2325 with a complete sucrase operon. Two strains ofEscherichia coli containing this plasmid replicon were able to grow on different concentrations of sucrose. The stability of recombinant plasmids was determined after cultivation under nonselective conditions. The stability after 5-d cultivation was higher than 75%.

Influence onEuglena gracilis of three types of inhibitors

Three groups of antibiotics were established according to their mechanism of action on the incorporation of14C-labelled precursors inEuglena gracilis:1. antibiotics markedly inhibiting nucleic acid synthesis and negligibly affecting protein synthesis and inducing permanently bleachedE. gracilis; 2. antibiotics markedly inhibiting protein synthesis and only moderately nucleic acid synthesis and not causing permanent bleaching ofE. gracilis; 3. compounds inhibiting both protein synthesis and nucleic acid synthesis to a similar degree, some of them bringing about permanent bleaching ofE. gracilis. Nitrofurantoin was comparable to compounds of the first group, sodium azide to those of the third group. Both exhibited 100 % bleaching ofE. gracilis, although with the latter this occurred only after a short-term increase of the incubation temperature from 25 to 37 °C.