Escherichia coli strain with a deletion of the chromosomal ampC 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 of ampC gene (delta ampC) coding for beta-lactamase was constructed. Such introduction of TcR marker permits a fast and simple selection for the transfer of delta ampC by P1 transduction into industrial E. coli strains. This approach was used for constructing an E. coli strain suitable for penicillin acylase production.

Small cryptic plasmids of multiplasmid, clinical Escherichia coli.

Clinical isolates of Escherichia coli were found to host a multiplicity of plasmids. These were resolved from plasmid gel profiles, from the properties of various transconjugants and transformants of E. coli DH1, by the topoisomerase I relaxation of covalently closed circle plasmid DNA, by electron microscopy, and by the determination of their compatibilities. The majority of these were unusually small, cryptic plasmids (SCPs). From one strain, KL4, 13 electrophoretic bands were resolved to five plasmids, three of which were SCPs. SCPs were phenotypically barren, and the smallest of these, pKL1, contained barely enough information for self-replication. A derivative of pKL1, pKL1Km, in which the transposon was restricted to a small 350-bp region, was stably maintained in Shigella, Salmonella, Serratia, and Citrobacter species and its replication was polA independent. pKL1 encoded only a single protein, RepA (Mr 17960), which specifically bound to pKL1 DNA. No apparent homologies with other RepA protein sequences could be detected. Thus the SCP, pKL1, is a novel minimal plasmid replicon encoding only enough information to ensure perpetuation. A hypothesis is presented describing SCPs as a class of selfish DNA that persists simply due to its ability to replicate and to its stability based on high copy number.

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

The in 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 of Escherichia 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%.

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 on Salmonella typhimurium. The hereditary bleaching test on Euglena gracilis used for detecting extranuclear mutations, showed positive results for Kathon, triethanolamine and diamine silver tetraborate.

[Analysis of phenotype resistance of clinical strains of Escherichia coli]. / Analýza fenotypu rezistencie klinického kmena Escherichia coli.

The multiplasmid clinical E. coli KL4 strain isolated from urine of a patient was examined for resistance to antibacterial substances and the number of plasmids. The distribution of resistance genes to antibiotics in different plasmids was assessed. All assessed resistances were transmitted by the mechanism of bacterial conjugation. By means of conjugation of the clinical E. coli strain KL4 and E. coli DH1, transformation of the DH1 strain by plasmid DNA and electrophoretic analysis two R plasmids were identified. The largest plasmid carries the gene for resistance against tetracycline and it is conjugative. Another three plasmids were mobilized by this plasmid during conjugation. The largest among them codes resistance against ampicillin, azlocillin and ticarcillin. From the total of five plasmids of the KL4 strain three were cryptic with regard to the resistance phenotype.

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 chloroplasts from Euglena 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.

Hyperthermia and other factors increasing sensitivity of Euglena to mutagens and carcinogens.

The effects of different factors (temperature, light, enzymic activation) on the ability of selected mutagens and carcinogens to induce hereditary bleaching of Euglena gracilis were investigated. In the resting medium, the elevation of incubation temperature from 25 degrees C to 37 degrees C increased significantly the effect of all compounds tested on the frequency of bleached mutants of E. gracilis. The effect of light is not so unambiguous. While nitrosoguanidine (NG) exhibited practically the same bleaching activity both in the light and dark, the mutagenic effect of sodium azide (SA), nitrovin (NV), nitrosoethylurea (NEU), and benzo(a)pyrene (BP) was decreased in light. On the other hand, the light increased the bleaching activity of 5-nitro-2-furylacrylic acid (NFAA) significantly. The activation mixture S9 increased bleaching effect of NFAA and BP, whereas other mutagens were partially (NG and SA) or completely (NV and NEU) inactivated.