[Cloning and expression of bacteriophage FMV lysocyme gene in cells of yeasts Saccharomyces cerevisiae and Pichia pastoris].

Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form.

[Using adaptive mutagenesis system for identification of early genes encoding de novo purine biosynthesis in methylotrophic yeast Pichia methanolica MH4].

A collection of spontaneous "Roman's mutants" (1654 mutants) for early genes of purine biosynthesis PUR1-PUR5 was obtained from 16 parental ade1(pur6) and ade2(pur7) strains of the methylotrophic yeast Pichia methanolica. Two genes, bifunctiional ADE7,4(PUR2,5) and ADES(PUR4), were identified earlier. For identification of the two remaining early genes (ADE3 and ADE8), a novel approach was used: a comparison of spectra of spontaneous Roman's mutants and relative sizes of genes (with regard to the length of polypeptides in amino acid residues). Significant correlation between relative sizes of genes and a proportion of mutants in the spectrum was shown in yeast Saccharomyces cerevisiae (according to analysis of data from the literature). Data on average polypeptide product lengths of genes encoding the first five steps of purine biosynthesis in 22 species of lower eucaryotes were obtained using the PubMed database. A correlation between the spectrum of spontaneous Roman's mutants and relative gene sizes was also shown experimentally in P. methanolica yeast. On the strength of this evidence, the gene ADE3 was identified as PUR1 and gene ADE8 as PUR3. Spontaneous mutagenesis producing Roman's mutants most probably is the result of transcription activity of early genes, which essentially agrees with experimental data on the correlation between the mutant spectrum and gene sizes. It was supposed that the adaptive mutagenesis, i.e., an enhancement of early genes transcription activity in response to stress, the accumulation of toxic metabolites (AIR or CAIR) as a result of the block of 6-7 stages of purine biosynthesis underlies the Roman's effect.

[Comparative analysis of genome sizes in Streptococcus thermophilus strains].

Twenty-five Streptococcus thermophilus isolates were analyzed using pulse-field gel electrophoresis (PFGE) and gene restriction profile analysis techniques. 16S rRNA gene sequences of the isolates were almost 100% homologous. However, genomic fingerprinting analysis has shown variability in both genome size and restriction fragments length. The genomes varied from 1417 to 2075 kb resulting in the difference between marginal genome sizes in about 600 kb. The results are indicative of Streptococcus thermophilus intraspecies genetic polymorphism, the origin of which requires further investigation.

[Genetic diversity of the natural strains of Streptococcus thermophilus].

The method of RAPD-PCR and comparative analysis of the PCR fingerprinting profiles similarity was used to characterize interspecific diversity of natural isolates of the lactic acid bacteria Streptococcus thermophilus. The strain genetic diversity was demonstrated using three primer variants, designed for different bacterial genome regions. The resolution of RAPD-PCR technique with different primers for identification at the species level and for certification at the strain level, was examined relative to the commercially important cultures of S. thermophilus. The results provided conclusion on preferable usage of RAPD-PCR with the primer ERIC-1 for specific identification of S. thermophilus, and with the primer M13 for certification of natural isolates of this species at the strain level.

[Comparison of genotypic and biochemical characteristics of Streptococcus thermophilus strains isolated from sour milk products].

Overall, 72 strains of lactic acid thermophilic streptococci isolated from sour milk products manufactured in various regions of Russia and European countries were analyzed using classical microbiological and molecular biological methods. Physiological and biochemical properties and genetic diversity of these Streptococcus thermophilus strains were studied, and a comparative analysis of the nucleotide sequences of the 16S rRNA gene was conducted. It has been demonstrated that the homology of proximal parts of the 16S rRNA gene of all the strains studied towards one another and towards the reference strain ATCC19258 amounts to 100%. As for the sugar fermentation, some strains display the characteristics untypical of the S. thermophilus members. The data obtained suggest that it is preferable to use gene 16S rRNA sequencing data for identification of natural isolates of closely related lactic acid bacterial species; moreover, this method is recommended for a precise species identification of industrial bacterial strains used in the food industry.

[Identification of industrial strains of lactic acid bacteria by methods of molecular genetic typing].

Various methods currently used in microbiology for determining taxonomic state of bacteria are discussed. The main focus is aimed at identifying and gene typing of lactic acid bacteria, used as starter cultures for industrial process of production of sour milk products, meat products, and probiotics.

[Bvg-negative regulation of repeated sequence transferring in Bordetella pertussis cells].

A method of monitoring the sequential events of IS481 transposition into the ctag site of bvg operon of Bordetella pertussis has been developed. Reproduction of virulent B. pertussis cells in vitro is accompanied by intrachromosomal site-specific IS481 transposition, which, in turn, results in inactivation of bvg operon of the causative agent and cell avirulent state. Avirulent bvg mutants of B. pertussis are incapable of intramolecular IS481 transposition. The frequency of the transposition increases when MgSO4 and nicotinic acid are present the culture medium. In the absence of these modulating factors. IS481 transposition along B. pertussis chromosome is inhibited but not arrested completely. Negative regulation of the bvg-repressed genes of B. pertussis seems to be a mechanism that controls bvg-dependent IS481 transposition.

[Use of methylotrophic bacteria Methylobacillus flagellatum KT for isolation of deuterated exogenous carbohydrates]. / Ispol'zovanie metilotrofnykh bakterii Methylobacillus flagellatum KT dlia polucheniia deiterirovannykh ékzogennykh uglevodov.

The cultivation conditions optimal for biosynthesis of exogenous polysaccharides by methylotrophic bacteria Methylobacillus flagellatum were evaluated. The mutant strain most active in accumulating exogenous polysaccharides was selected. Gradual adaptation of this strain to the deuterated medium containing 1 vol % CD3OD in deuterium oxide intensified biosynthesis of exogenous polysaccharides and inhibited bacterial growth (compared to the standard medium). The monomeric composition of exogenous polysaccharides obtained during cultivation on standard and deuterated media was estimated by high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. Nondeuterated exogenous polysaccharides contained only fructose, while deuterated exogenous polysaccharides included 98% fructose and 2% ribose. Paramagnetic resonance spectroscopy showed that the degree of deuterium incorporation into molecules of biosynthetic carbohydrates was 89%.

[Design of the human GM-CSF gene using the polymerase chain reaction and its expression in Pseudomonas putida cells]. / Konstruirovanie gena GM-CSF cheloveka s pomoshch'iu polimeraznoi tsepnoi reaktsii i ego ékspressiia v keltkakh Pseudomonas putida.

Construction of human GM-CSF gene was conducted by the PCR technique. Four exons of GM-CSF gene were synthesized on the basis of human blood DNA using thermostable Tth DNA polymerase. Synthetic oligonucleotides were used as primers. The oligonucleotides contained sequences complementary to the ends of exons. Joining of exons was conducted by reciprocal complementation of the terminal sequences, followed by filling and amplification of the joined products. In most cases the effective synthesis of exons and joined products was possible only after optimizing the polymerase reaction conditions for each primer pair. Determination of the nucleotide sequence of the synthetic gene showed complete identity with the natural one. The gene was introduced into an expression vector under control of the promoter tandem (tac+lac). Expression of the GM-CSF gene was obtained in Pseudomonas putida cells. The recombinant protein had biological activity.

[The effect of amino acids in the asparagine family on the aspartate kinase and homoserine dehydrogenase of ethionine-resistant mutants of Pseudomonas putida]. / Vliianie aminokislot asparaginovogo semeistva na aktivnost' aspartatkinazy i gomoserindegidrogenazy étioninrezistentnykh mutantov Pseudomonas putida.

The activity of aspartate kinase and homoserin dehydrogenase from ethionine resistant mutants Pseudomonas putida 25 and 6 have been studied as affected by amino acids from the family of asparagine. They are characterized by a capacity to the surplus synthesis of methionine. It is shown that mutants have negative regulation of the level of activity of the studied enzymes. It is supposed that the mutations (or mutation) could take place which affected properties of enzymes, which participated directly in the biosynthesis of methionine, in the analogue resistant clones 25 and 6.

[Localization of prophage SM of Pseudomonas aeruginosa in the chromosome of host cells]. / Lokalizatsiia profaga SM Pseudomonas aeruginosa v khromosome kletok-khoziaev.

Pseudomonas aeruginosa PAO SM-prophage was localized on the chromosome between thr-9001 and pur-66 locuses on 42-43 min of chromosomal genetic map. The location of prophage was identified on the basis of prophage linkage with the above-mentioned markers and confirmed by the purine, hypoxanthine and threonine deletions in course of thermoinduction of SM cts6 prophage from lysogens. The decrease for two orders in lysogenization frequency of thr mutants by SM bacteriophage suggests the integration of SM prophage in these cells into some other region of chromosome.

[Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria]. / Kharakteristika mutantov Methylobacillus flagellatum KT, defektnykh po fosfogliukoizomeraze, fermentu ribulozomonofosfatnogo tsikla obligatnykh metilotrofov.

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.

[Temperature-sensitive mutant of the obligate methylotroph Methylobacillus flagellatum KT deficient in glucose-6-phosphate dehydrogenase]. / Temperaturochuvstvitel'nyi mutant obligatnogo metilotrofa Methylobacillus flagellatum KT, defektnyi po gliukozo-6-fosfatdegidrogenaze.

The common approach is developed for isolation of mutants deficient in key enzymes of ribulose monophosphate pathway for formaldehyde oxidation and assimilation by obligate methylotrophic bacteria. The approach is based on total isolation of temperature sensitive mutants and their biochemical characterization. A number of ts- mutants of obligate methylotroph M. flagellatum KT is isolated following nitrosoguanidine induced mutagenesis. The modified screening method was developed and used for identification of mutants deficient in the key enzymes of ribulose monophosphate pathway. The mutant deficient in glucose-6-phosphate dehydrogenase (zwf) was identified. The NAD-dependent activity of glucose-6-phosphate dehydrogenase was not measurable under nonpermissive temperature while the level of NADP-dependent activity was only four-fold less comparing with wild type strain. It was concluded that growth limitation of zwf mutant of M. flagellatum KT (designated T623) at 42 degrees C results from the absence of NAD-dependent activity of glucose-6-phosphate dehydrogenase.

[Genome organization of the plasmid of IncQ/P4 group and its vector derivatives]. / Organizatsiia genoma plazmid IncQ/P4-gruppu i vektory, skonstruirovannye na ikh osnove.

The genome organization and functioning of IncQ/P4 plasmids are reviewed. Based on these plasmids, cloning vectors have been constructed for broad host range of gram-negative bacteria. Together with one- and two-replicon vectors for cloning via insertion inactivation of markers, specialized plasmid vectors are described: cosmids, promoter-probe vectors, vectors for direct selection of recombinant molecules. Examples of using broad host range vectors for gene cloning and expression in non-enteric gram-negative bacteria are presented.

[Control of lysogeny and a genetic map of the temperate phage SM of Pseudomonas aeruginosa]. / Kontrol' lizogenizatsii i geneticheskaia karta umerennogo faga SM Pseudomonas aeruginosa.

76 mutants with impaired ability to lysogenize host cells were isolated in SM phage after mutagenesis using several chemical mutagens. By means of complementation test, these mutants were distributed into two groups, cI and cII. The mutants of the cI group were similar phenotypically to the cI mutants of phage lambda defective in synthesis of repressor. The mutants of the cII group establish and support the lysogenic state in infected cells with very low frequency. Temperature-sensitive mutants belonging to 13 complementation groups and nonlysogenizing mutants of the cI and cII groups were used in genetic mapping of SM phage. Mutual positions of markers and relative distances between them were determined by the method of two-factorial crosses. The greatest distance equal to 20 units of recombination was determined between ts 88 marker and one of early genes marked with ts 105 mutation. The genes cI and cII are closely linked to each other and also to ts 105 marker and are situated at one end of the genetic map.

[The effect of R-plasmids on the reproduction of Pseudomonas aeruginosa phage SM]. / Vliianie R-plazmid na reproduktsiiu faga SM Pseudomonas aeruginosa.

The inheritance of plasmids Rms163 and R74 by Pseudomonas aeruginosa strain PAO hs been shown to effect the reproduction of a temperature bacteriophage SM. The decrease in plating efficiency of bacteriophage on Pseudomonas aeruginosa PAO (rms163) lawn is explained by the high degree of cell lysogenization by bacteriophage. Plasmid R74 inhibits bacteriophage SM propagation ultimately, evidently due to interruption of definite stages in vegetative development of bacteriophage by the products of plasmid specific genes.

[Complementation and the physiological characteristics of the ts mutants of Pseudomonas aeruginosa phage SM]. / Komplementatsiia i fiziologicheskaia kharakteristika ts-mutantov faga SM Pseudomonas aeruginosa.

Seventy three temperature-sensitive mutants of Pseudomonas aeruginosa SM phage have been obtained using different mutagens and assigned to thirteen complementation groups. Representative mutants of each group have been studied with the aim of characterizing tentatively the time of genes expression in infected bacteria. Two genes appear to function during the first minutes after infection, whereas the remaining genes are needed for late functions. Most of the temperature-sensitive functions in the different mutants are reversible, i.e. they become active when the infected cells are shifted-down to the permissive temperature.

[Development of a system vector-host in methylotrophic bacteria]. / Razvitie sistemy vektor-khoziain u metilotrofnykh bakterii.

Cloning of methylotropic and other Gram negative bacteria's genes was performed using vectors derived from IncP4 plasmids. Plasmids, such s RSF1010 are 8.8 kb in length, have a high copy number and broad host range and can be mobilized efficiently by a number of conjugative plasmids. IncP4 plasmids have relatively few restriction enzyme's targets suitable for cloning. In this paper the construction of versatile and special purpose IncP4 vectors available for cloning DNA into broad range of bacterial species are described. The seria of versatile vectors involves the transposon containing plasmid and two-replicon vectors. In genetic construction of special vector for direct cloning of restriction fragments the genetic regulation elements of Tn 1 were used. On the base of IncP4 replicon special vectors for construction of bank genes (cosmids) and the vectors for cloning of regulation sequence were also constructed.