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Applied Biochemistry and Microbiology (v.46, #7)

Modern methods of genome sequencing and their application for deciphering genomes of microorganisms by N. V. Ravin (pp. 663-670).

Development of new methods for genome sequencing allows rapid and relatively inexpensive determination of the large volumes of nucleotide sequences and opens new possibilities to conduct fundamental and applied research in biology, medicine, and biotechnology. The traditional and the newest methods of genome sequencing of microorganisms including the Sanger sequencing method, parallel pyrosequencing (454 Life Sciences/Roche), Solexa/Illumina technologies, and ligation-based sequencing (SOLiD System) are discussed in the current review. The main areas and prospects of applying various sequencing methods to achieve diverse scientific goals are presented. The possibilities of applying methods of parallel pyrosequencing for deciphering genomes of microorganisms are reviewed, using as an example the genome projects completed at the Bioengineering Center, Russian Academy of Sciences.

Keywords: genome; microorganism; DNA sequencing

Metabolic engineering in silico by V. A. Likhoshvai; T. M. Khlebodarova; M. T. Ree; N. A. Kolchanov (pp. 671-687).

This review briefs on the main directions in the field of mathematical modeling of metabolic processes aimed at a rational design of genetically modified organisms. The class of generalized Hill functions is described, and their application to modeling of nonlinear processes in Escherichia coli metabolic systems is illustrated by several examples. A model for the pyrimidine biosynthesis in E. coli, taking into account the nonlinear effects of a negative allosteric regulation of enzyme activities involved in the control of the subsequent stages by the end products of synthesis, is considered. It has been shown that the model displays its own continuous oscillation mode of functioning with a period of approximately 50 min, which is close to the duration of E. coli cell cycle. The need in considering the nonlinear effects in the models as essential elements in the function of metabolic systems far from equilibrium is discussed.

Keywords: mathematical modeling; metabolic engineering; generalized Hill functions; regulation; Escherichia coli

Development of a technique based on the affinity resins for evaluation of DNA methylation status in vertebrates by V. M. Pekhov; A. M. Mazur; E. B. Prokhorchuk (pp. 688-696).

The methods for synthesis and application of resins based on the functional domains of Kaiso and CpG-binding protein (CGBP), which can bind methylated and unmethylated CpG-dinucleotides, respectively, are shown. Kaiso resin was obtained by the affinity interaction of glutathione-sepharose with a chimeric protein, which is expressed in Escherichia coli and contain glutathione S-transferase (GST) and zinc finger domain of methyl-DNA-binding Kaiso protein within the same translation frame. Kaiso resin, like MBD-domain based resin, has an ability to bind methylated DNA. Experiments with the short DNA fragments demonstrated that methylated DNA is eluted from the resin by 0.7 M KCl, whereas unmethylated DNA is washed out by 0.2–0.5 M KCl after binding. Quantitative PCR showed that the enrichment with methylated p16 promoter region and the absence of accumulation of γ-actin unmethylated promoter were observed due to the binding of genomic DNA, isolated from the colo 320 cell line (human colorectal adenocarcinoma), with the Kaiso resin. The CGBP resin based on the CxxC domain of CGBP protein binds to the sequences which contain unmethylated CpG-dinucleotides. Our experiments also showed no effect of MBD3L1 protein on MBD2-resin capacity of binding with methylated DNA. The obtained resins can be applied to study methylation status of both specific DNA sequences and the whole genome.

Keywords: DNA; Kaiso; methylation; microarray; CGBP; MBD2.

Investigation on macrokinetics of heterogeneous process of monosaccharide isomerization using non-growing cells of a glucoisomerase producer Arthrobacter nicotianae immobilized inside SiO₂-xerogel by G. A. Kovalenko; L. V. Perminova; E. I. Chernyak; L. I. Sapunova (pp. 697-705).

Macrokinetic peculiarites of heterogeneous process of monosaccharide (glucose/fructose) isomerization using biocatalysts prepared by incorporation of non-growing cells of a glucose isomerase-producing strain Arthobacter nicotianae inside SiO₂-xerogel have been investigated. It was shown that the process proceeds in kinetic regime without diffusion limitation and biocatalyst activities at 60 and 75°C were 19 and 32 U/g, respectively. Time of equilibrium in the reaction of monosaccharide isomerization was a function of starting (“triggering”) glucose isomerase activity in a unit of reaction volume. When the activity exceeds 10 U/ml, equilibrium equimolar mixture of glucose and fructose was produced within a few hours. It was established that a continuous process carried out in a plug-flow packed-bed reactor is more efficient than a batch process accompanied with recycling, first of all, to significant improvement of operation stability of the designed biocatalysts. Under model conditions of industrial heterogeneous process of producing glucose-fructose syrups, the half-life time of inactivation of the biocatalysts was more than 500 h at (65 ± 5)°C.

Keywords: Arthobacter nicotianae cells immobilized inside SiO₂-xerogel; glucose/fructose isomerization; a plug-flow packed-bed reactor

Study of the structures of biofilms formed by Salmonella typhimurium bacteria on abiotic surfaces by the methods of light and transmission electron microscopy by T. A. Smirnov; L. V. Didenko; I. G. Tiganova; S. G. Andreevskaya; N. V. Alekseeva; T. V. Stepanova; Yu. M. Romanov (pp. 706-711).

Process of biofilm formation by different Salmonella strains on abiotic surfaces has been studied. Differences in the structural organization were revealed by the analysis of the fine cell structure in the planktonic and biofilm cultures. It was shown, by the methods of light and electron microscopy and also by cytochemistry, that the two strains share similarities in structure and have individual features. Differences in the density of the extracellular matrix and the sizes of cell aggregates were established. The stages in the processes of growth and death of biofilms were demonstrated by the investigation of the process dynamics. The signs of aging in biofilms were disorganization of extracellular matrix and appearance of the planktonic cells. Microscopic and cytochemical methods used in this work were recommended for the investigation of the effects of various biocide agents on biofilms.

Keywords: biofilm; light microscopy; cytochemistry; electron microscopy; Salmonella typhimurium

Kinetics of anaerobic biodegradation of glycerol by sulfate-reducing bacteria by V. G. Dinkel; F. B. Frechen; A. V. Dinkel; Yu. Yu. Smirnov; S. V. Kalyuzhnyi (pp. 712-718).

A kinetic model has been developed and kinetic parameters of anaerobic degradation of glycerol, an abundant by-product of biofuel manufacturing, by a consortium of sulfate reducing bacteria (SRB) in a closed system have been determined. The following main species of SRB has been identified in the consortium: Desulfovibrio baarsii, Desulfomicrobium sp., and Desufatomaculum sp. The proposed model included processes of glycerol degradation, sulfate reduction, and inhibition by metabolic products, as well as effects of pH and temperature. The suggested equation for the anaerobic glycerol degradation was based on Edward and Andrew’s equation. The following kinetic parameters of the anaerobic glycerol degradation were obtained for the initial glycerol concentration from 0.15 to 4 ml/l and sulfate concentration of 2760 mg/l at 22°C: maximum specific growth rate of SRB μ_max = 0.56 day⁻¹, economic coefficient of ashless biomass from glycerol of 0.08 mol SRB/mol COC, and yield of ashless biomass from sulfate of 0.020 mol SRB/mol SO₄. It was shown that the optimum molar ratio of $$ {{C_{Gl} } mathord{left/ {vphantom {{C_{Gl} } {C_{SO_4 } }}} ight. kern- ulldelimiterspace} {C_{SO_4 } }} $$ for SRB growth was 0.8. Initial boundary concentration of inhibition by undissociated hydrogen sulfide was 70 mg/l. Dependence of the specific growth rate of bacteria on the temperature was approximated by the Arrhenius equation in the temperature range of 20–30°C with the goodness of fit R² = 0.99.

Keywords: anaerobic biodegradation; degradation; glycerol; kinetics; waste water decontamination; sulfates; sulfate reducing bacteria; heavy metals

An enhanced biodegradation of crude oil by Psedomonas plasmid-bearing strains in model soil systems by A. A. Vetrova; A. A. Ovchinnikova; I. F. Puntus; A. E. Filonov; A. M. Boronin (pp. 719-725).

Oil biodegradation in sterile and nonsterile model soil systems has been studied. A comparison of process efficiency using indigenous microorganisms, introduced plasmid-free bacteria, and strains bearing various plasmids of naphthalene degradation was carried out. It was shown, in almost all variants of the experiment, that the total oil reduction in the nonsterile soil was higher than in the sterile soil. The highest level of degradation was observed in soil systems containing plasmid-bearing strains.

Keywords: bioremediation; plasmids of biodegradation; Pseudomonas

Development and optimization of several stages of the technological process of filgrastim substance production by N. V. Kononova; A. I. Bobruskin; T. I. Kostromina; T. D. Melikhova; A. A. Vainson; E. V. Sveshnikova; A. A. Zinchenko; A. V. Demin; V. A. Martyanov; A. M. Shuster; D. I. Bairamashvili (pp. 726-732).

Technology of industrial production of an active pharmaceutical substance (APS) of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) involved the use of a highly productive E. coli strain capable of biosynthesis of rhG-CSF in the form of inclusion bodies (IB). A method of strain cultivation has been described, and methods of IB isolation, industrial-scale purification, filgrastim APS production, and quality control have been developed. Clinical trials of the preparation, carried out in the leading Russian clinics, were successful. Efficiency and safety of the preparation have been confirmed. A ready pharmaceutical form Neupomax® been produced in Russia since 2008 according to the technology developed.

Keywords: isolation; granulocyte colony-stimulating factor; purification; production; filgrastim

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Applied Biochemistry and Microbiology (v.46, #7)

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Applied Biochemistry and Microbiology (v.46, #7)