Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

Synthesis of ovalbumin and globin during simultaneous translation of their mRNA in the presence of tRNA of different origin in the cell-free protein-synthesizing system from wheat germ

Competition among ovalbumin and globin mRNA under their simultaneous translation in the tRNA-dependent cell-free system from wheat germ was studied. One of the mRNAs was added to samples in a constant amount, that provided 50% protein synthesis level of the maximum, other--in increasing amount (from 0 to the maximum). The ration of ovalbumin and globin synthesis rates has been shown to depend essentially (1.5-2 fold) on the nature of tRNA, being added to the system. The obtained data suggest that functional adaptation of tRNA is a part of the mechanism of protein biosynthesis regulation and it is possible to modulate some mRNAs translation selectivity on the elongation by different sets of tRNA.     

Long-term survival of and plasmid stability inPseudomonas andKlebsiella species and appearance of nonculturable cells in agricultural drainage water

One year after introduction into agricultural drainage waterPseudomonas fluorescens R2f (RP4),Pseudomonas putida CYM318 (pRK2501), andKlebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air.In contrast toK. aerogenes NCTC418 (pBR322), bothPseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with³²P-labelled plasmid RP4 DNA was found in several drainage water samples that originally receivedP. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 andK. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts.     

Occurrence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment

Eleven hundred Bacillus strains isolated from marine sediment from the Minas Basin, Nova Scotia, Canada, were purified on LB agar supplemented with ampicillin, chloramphenicol, erythromycin, streptomycin, tetracycline, or mercuric chloride. Seventy-seven isolates were examined for plasmid DNA, and for resistance to 11 antibiotics, HgCl2, and phenylmercuric acetate. Minimum inhibitory concentrations of Ag, Cd, Co, Cu, and Zn were also determined. Forty-three percent of antibiotic- and mercury-resistant strains contained one or more plasmids ranging from 1.9 to 210 MDa. Fifty-four percent carried plasmids greater than 20 MDa, and 97% were resistant to two or more metals. There was no correlation between plasmid content and resistance either to antibiotics or to mercurial compounds in these strains. Mercury-resistant isolates were unable to transform Hg2+ to volatile Hg0 by virtue of a mercuric reductase enzyme system (mer). Strains resistant to Hg2+ were investigated for their ability to produce H2S and intracellular acid-labile sulfide when grown in the absence and presence of HgCl2. Lower levels of H2S and intracellular sulfide were detected only in metal-resistant strains grown in the presence of HgCl2, suggesting that cellular sulfides complexed with Hg2+ in these strains.     

Oxygen binding of myoglobins of diving animals]

When studying oxygen binding by myoglobins of diving animals it is shown that half-saturation of myoglobins obtained from the muscles of animals who can stop the external respiration for a long period of time occurs at oxygen strength of 0.62-0.67 mm Hg. Such indices of oxygenation of respiratory hemoprotein of muscles are characteristic of most mammals. Temperature being decreased from 37 to 15 degrees C, myoglobin affinity to oxygen considerably increases.     

Protein immunoblot and immunodot in biochemical studies

General notions on the protein immunoblot and immunodot are reported. Possibilities of their wide application in biochemical studies are discussed. The employed materials, the most important operations when performing different variants and modifications of these methods as well as resolution and advisability of their application are comprehensively analyzed.     

Immune sensor for determining myoglobin level

For the first time, the photoluminescence of porous silicon was used to create an immune sensor capable of detection of low concentrations of myoglobin important for the diagnostics of heart failure diseases which in both model solutions and human serum, being. It is shown that the sensitivity of the sensor was 10 ng/ml, time of 1 measurement--15-30 min. Duration of the analysis can be even shorter if measurements are performed in the kinetic regime. The comparison of the characteristics of the immune sensor and standard method ELISA is also given in the paper.     

Express control of toxic substances and pathogenic microorganisms. Immune analysis and immune sensors

In the review the overall characteristics of biological warfare components, which represent danger to people in the case of their application by military or terrorist groups are discussed. The main part of the review is devoted to modern approaches of antibody obtaining and, in particular, preparation of specific recombinant immunoglobulins as well as to different immune chemical methods of determination of individual toxins and pathogenic microorganisms. A special attention is paid to existing data about the development of rapid, selective, high sensitive, simple and fully automated instrumental methods on the basis of biosensor technology which is designed for the control of components of biological warfare in environmental objects. Additionally industrially manufactured biosensors and their characteristics are given and analyzed.     

Immobilization of biocomponents for immune optical sensors

Immobilisation of both human immunoglobulin(IgG) and antiimmunoglobulin (anti-IgG) was performed by means of polyelectrolyte self-assembly. This technique was compared with direct immobilisation of the immune components on bare gold and their covalent binding via glutaraldehyde as a bifunctional reagent. Additionally, the immune components were properly oriented during their immobilisation by using a predeposited layer of the protein A. Methods of the surface plasmon resonance (SPR) and planar interferometry were employed for monitoring the immobilisation as well as specific immune reaction. It was shown that in case of the use of polyelectrolyte self-assembly it is possible to achieve the sensitivity of the analysis up to 30 ng/ml for SPR and up to 1 ng/ml for planar interferometer based immune sensors.