[Modeling the Propagation of Microbial Cells and Phage Particles from the Sites of Permafrost Thawing.]

A method is proposed for integral assessment of the propagation of microbial cells and viral parti- cles during seasonal thawing of relic ice wedge layers. The results of on-site and laboratory investigation car- ried out in the upper part of permafrost exposure at Mamontova Gora (Yakutiya, Russia) are presented. To increase reliability of the results, suspensions of two microbial species and two coliphage species were intro- duced as biomarkers directly on the surface of thaing ice and in the meltwater flow. Each of the four different model biological objects was shown to possess unique parameters of movement in the meltwater flow and is able to move 132 m in 25-35 min with the water flow.

Plate acoustic wave sensor for detection of small amounts of bacterial cells in micro-litre liquid samples.

Ultrasonic acoustic waves propagating in thin piezoelectric plates with free faces are used for bacteria detection in micro-litre liquid samples deposited on one of the plate surface. The limits of the detection at normal conditions are as low as 0.04% for highly diluted rich cultural Luria-Bertani broth (LB-media) in distillate water, 0.07% for bacterial cells in distillate water, and 0.6% for bacterial cells in LB-media. For all analytes the most probable detection mechanism is the change in liquid conductivity. Because of no using any sorbent film the long-term stability of the detection is expected as very high.

[Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon].

The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions.

[Microbiological synthesis of [2H]-inosine with a high degree of isotopic enrichment by the gram-positive chemoheterotrophic bacterium Bacillus subtilis].

A 2H-labeled purine ribonucleoside inosine was microbiologically synthesized (yield, 3.9 g/L of culture liquid) using a deuterium-adapted strain ofthe gram-positive chemoheterotrophic bacterium Bacillus subtilis, cultivated in a heavy water medium with a high degree of deuteration (99.8 at % 2H) containing 2% hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum as a source of 2H-labeled growth substrate produced in an M9 minimal medium with 98% 2H20 and 2% [2H]-methanol. The inosine extracted from the culture liquid of the producer strain was fractionated by adsorption (desorption) on an activated carbon surface, extraction with 0.3 M ammonium-formate buffer (pH 8.9), subsequent crystallization in 80% ethanol, and ion exchange chromatography on a column with AG50WX 4 cation exchange resin equilibrated with 0.3 M ammonium-formate buffer containing 0.045 M NH4Cl. Fast atom bombardment (FAB) mass spectrometry demonstrated incorporation of five deuterium atoms in the inosine molecule (62.5% 2H), three of which were contained in the ribose moiety and two in the hypoxanthine moiety.

A cell population structuring model to estimate recombinant strain growth in a closed system for subsequent search of the mode to increase protein accumulation during protealysin producer cultivation.

In this paper we have proposed a new structured population growth model, further developing a model previously proposed by the authors. Based on this model, optimal growth characteristics of the recombinant strain Escherichia coli BL-21 (DE3) [pProPlnHis(6)] were determined, which allowed us to increase the output of metalloproteinase by 300%. We have experimentally demonstrated the applicability of the new model to cell cultures with implanted plasmids and the potential practical use for an output increase of a wide variety of biosynthesis processes.

[Synthesis of spyropyran derivatives of retinal and study of their interaction with bacterioopsin from Halobacterium salinarum].

The synthesis of retinal analogue series that contain a spyropyran moiety instead of a trimethylcyclohexene ring was proposed. The process of the retinal analogue interaction with bacterioopsin from apomembranes of Halobacterium salinarum and the spectral properties of the newly formed pigments were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed.

Interactions between (15)N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortuantely, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in (13)C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C(alpha) signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the (31)P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The (13)C-peptide peaks were selectively detected in a (13)C-detected (1)H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The (13)C-MAOSS results thus independently confirms previous findings from (15)N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O'Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428-9437]. In summary, application of house hold foil opens the possibility of measuring high resolution (13)C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and supramolecular structures of membrane active peptides, proteins and aggregates.

[Deuterium oxide as a stress factor to the methylotrophic bacterium Methylophilus sp]. / Oksid deiteriia kak stressovyi faktor u metilotrofnykh bakterii Methylophilus sp.

The adaptation of the methylotrophic bacterium Methylophilus sp. B-7741 to growth in highly deuterated media was studied. For the first time, we showed the cross adaptation of bacterial cells to deuterated media and oxidative and osmotic stresses. The activity at catalase in deuterated cells was higher than in the control cells. Deuterated cell-free culture liquids showed protective effects on the growth of Methylophilus sp. B-7741 in deuterated media, which was manifested as an increase in the deuterated biomass yield. These data and the data available in the literature suggest that the mechanisms of bacterial cell adaptation to heavy water and to oxidative and osmotic stresses are similar.

[Effect of deuteration on methanol dehydrogenase activity in Methylophilus sp. B-7741]. / Vliianie deiterirovaniia na aktivnost' metanoldegidrogenazy Methylophilus sp. B-7741.

We studied the effect of deuterium oxide present in the medium on the activity of methanol dehydrogenase (EC 1.1.99.8) from methylotrophic bacteria Methylophilus sp. B-7741. Methanol dehydrogenase activity in extracts of the biomass obtained in a highly deuterated medium (2H-enzyme) was 34-47% of enzyme activity in the control biomass, which depended on reaction conditions. The isotopic effects of substrate deuterium (methanol) for 1H-enzyme and 2H-enzyme were 1.37 +/- 0.05 and 1.38 +/- 0.01, respectively. We revealed for the first time the reverse isotopic effect of solvent deuterium in the reaction catalyzed by methanol dehydrogenase (0.80 +/- 0.02 and 0.60 +/- 0.01 for 1H-enzyme and 2H-enzyme, respectively).

[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%.

15N and 31P solid-state NMR investigations on the orientation of zervamicin II and alamethicin in phosphatidylcholine membranes.

The topologies of zervamicin II and alamethicin, labeled with (15)N uniformly, selectively, or specifically, have been investigated by oriented proton-decoupled (15)N solid-state NMR spectroscopy. Whereas at lipid-to-peptide (L/P) ratios of 50 (wt/wt) zervamicin II exhibits transmembrane alignments in 1,2-dicapryl (di-C10:0-PC) and 1,2-dilauroyl (di-C12:0-PC) phosphatidylcholine bilayers, it adopts orientations predominantly parallel to the membrane surface when the lengths of the fatty acyl chains are extended. The orientational order of zervamicin II increases with higher phospholipid concentrations, and considerable line narrowing is obtained in di-C10:0-PC/zervamicin II membranes at L/P ratios of 100 (wt/wt). In contrast to zervamicin, alamethicin is transmembrane throughout most, if not all, of its length when reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. The (31)P solid-state NMR spectra of all phospholipid/peptaibol samples investigated show a high degree of headgroup order, indicating that the peptides do not distort the bilayer structure. The observed differences in peptide orientation between zervamicin and alamethicin are discussed with reference to differences in their lengths, helical conformations, distribution of (hydroxy)proline residues, and hydrophobic moments. Possible implications for peptaibol voltage-gating are also described.

[The biosynthesis of 2H-labelled inosine by Bacillus subtilis bacteria in a heavy-hydrogen medium]. / Biosintez 2H-mechenogo inozina bakteriei Bacillus subtilis v tiazhelovodorodnoi srede.

We studied biosynthesis of the purine ribonucleoside inosine by the strain of Bacillus subtilis, which was adapted to deuterium through plating to individual colonies on 2% agar with 2H2O and subsequent selection. For growing the strain, a special heavy hydrogen medium with a high level of deuteration (89-90 at. % 2H) based on 2% hydrolyzate of the biomass of the methylotrophic Brevibacterium methylicum as a source of 2H-labeled growth substrates was obtained on 98 vol % 2H2O and 2 vol % [U-2H] methanol. We provide experimental data on the strain growth and accumulation of inosine in the culture medium. A study of the level of inosine deuteration using mass-spectroscopy of fast atoms has shown a polymorphism of deuterium incorporation in the molecule (isotope composition of inosine: 4 at. 2H, 20%; 5 at. 2H, 38%; 6 at. 2H, 28%; 7 at. 2H, 14%), deuterium being incorporated in the ribose and hypoxanthine fragments of the molecules.

Biosynthesis of (2)H-Labeled Phenylalanine by a New Methylotrophic Mutant Brevibacterium methylicum.

The biosynthesis of (2)H-labeled phenylalanine was done by converse of low molecular weight substrates ([U-(2)H]methanol and (2)H2O) in a new RuMP facultative methylotrophic mutant Brevibacterium methylicum. To make the process work, adapted cells with improved growth characteristics were used on minimal medium M9 with the maximum content of (2)H-labeled substrates. Alanine, valine, and leucine/isoleucine were produced and accumulated exogeneously in addition to the main product of biosynthesis. Electron impact mass spectrometry of methyl esters of the N-Dns-amino acid mixture obtained after the chemical derivatization of growth medium with dansyl chloride and diazomethane, was done to calculate the deuterium enrichment of the amino acids synthesized. The experimental data testified to the character of labeling of amino acid molecules as heterogeneous; however, high levels of deuterium enrichment were detected in all presented molecules-for phenylalanine the enrichment was six, leucine/isoleucine-five, valine-five, and alanine-three deuterium atoms.

(2)H- and(13)C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring.

Biosynthetic preparation of(2)H- and(13)C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing(13)C- or(2)H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of(2)H- and(13)C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.

Growth and enzymological characteristics of a pink-pigmented facultative methylotroph Methylobacterium sp. MB1.

Growth characteristics of batch and continuous cultures of the pink facultative methylotroph Methylobacterium sp. MB1 were determined. The response of a chemostat culture to a pulse increase of methanol concentration was studied. Malate, succinate and oxaloacetate additions to the methanol-supplemented medium decreased batch culture growth inhibition by methanol. The carotenoid content in cells grown in a chemostat decreased with increasing growth rate. The key enzyme activities of C1-metabolism were measured in a chemostat culture at different dilution rates.

[Mutations in the purine nucleoside phosphorylase (pup) gene of Escherichia coli K-12 characterized by leaky damage to enzymatic activity and a pleiotropic effect]. / Mutatsii po purinnukleozidfosforilaznomu (pup) genu Escherichia coli K-12, kharakterizuiushchiesia nepolnym povrezhdeniem aktivnosti fermenta i pleiotropnym éffektom.

The phenotype of earlier obtained mutants AIR38 and AIR6 is caused by leaky mutations of the structural gene for purine nucleoside phosphorylase (pup). These mutants are unable to grow on the medium with inosine as the only carbon source in the presence of thymidine. In contrast to ordinary leaky mutations, AIR38 and AIR6 are dominant in heterozygotes. When the strain F' with mutations AIR38 or AIR6 on episomes was used for conjugational matings with F- recA (pup+), recombinants with unexpected phenotype were observed: about 20% of recombinants F' became pup+ and thus the convertion of AIR38 and AIR6 alleles to pup+ took place. AIR38 mutation, unlike AIR6, is mapped in the proximal region of the pup gene and is characterized by pleyotropic effect on deo-genes: the inosine-induction of purine nucleoside phosphorylase in AIR38 mutant is absent and the induction of thymidine phosphorylase by exogenous thymidine is decreased. A speculation was made that the mutation AIR38 altered the structure of purine nucleoside phosphorylase in the site responsible for its interaction with membrane.

[Regulation of the activity of Escherichia coli deo-operon structural genes: the mutation mapped within the operon boundaries and affecting drm and pup gene activity]. / Reguliatsiia aktivnosti strukturnykh genov deo-operona Eschirchia coli: mutatsiia, kartiruiushchaiasia v predelakh operona i vliiaiushchaia na aktivnost' genov drm i pup.

The mutant AIR38 is isolated from Escherichia coli K-12 strain deficient in thymidilate synthetase and deoxyriboaldolase (HfrH, thy, dra)--by selection for low thymine requirement on the medium containing inosine as the carbon source. Under the conditions mentioned the mutant AIR38 (thy, dra) grows at low thymine concentration (2 mkg/ml), and is uncapable to grow in the presence of thymidine (40 mkg/ml). Dra+ derivatives of the AIR38 do no catabolize inozine in the presence of thymidine as well. The mutation AIR38 is mapped within the deo-operon between drm and pup mutation markers. The levels of phosphodeoxyribomutase and purine nucleoside phosphorylase in cell extracts of AIR38 are 2.5-6-fold decreased. In transductional experiments with phage P1 and the mutant AIR38 as recipient the delayed haploidization of merozygotes dra+, AIR+/dra, AIR38, thy and the dominant expression of the sensitivity to thymidine in the presence of inosine as the carbon source are observed. It is supposed that the mutation AIR38 affects the structural gene of purine nucleoside phosphorilase by altering the mode of interaction of this enzyme with the membrane under the conditions of thymine starvation.