Method of determining hexoses in supramolecular structures of cells in escherichia coli

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and molecular biology. Conservation of cells of Escherichia coli in presence of buffered 80-90% glycerol is performed. Cell envelopes are removed with 3% triton X-100. Cell supramolecular structures are successively extracted with increasing concentrations of salts: 0.14 M (bacterioplasm), 0.35 M (loosely linked with cell residue), 2 M NaCl (strongly linked with cell residue), and 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol (cell residue). Acid hydrolysis is carried out in said fractions. Anthrone method is carried out, with preliminary purification of anthrone preparation. Calibration graph is built and quantity of hexoses is determined by means of calculation formula.

EFFECT: invention makes it possible to determine quantity of hexoses in supramolecular structures of bacterial cell of Escherichia coli.

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The invention relates to biochemistry and molecular biology of prokaryotic cells and can be applied to the analysis of molecular-genetic mechanisms of formation of structure of prokaryotic cells and the role of carbohydrate components in their organization, which is necessary to reveal the ways regulation mechanisms of the interaction between macro - and microorganisms, as well as the search for new targets for drugs and the development of environmentally friendly medicinal drugs.

The known method of determination of hexoses [1], in which the acid hydrolysis and color reaction with astronom the products are colored. The drawback of this method is rather low sensitivity (100 µg) at the specified ratio acid-thiourea Andron.

The known method of determination of hexoses [2], based on the ability in the presence of concentrated sulfuric acid to give with astronom painted products. The drawback of this method is that as objects containing hexose, are chemically synthesized reagents - hexose with a fairly high concentration of 100-200 µg/ml.

There is a method to determine the total amount of carbohydrates in bacterial cells [3]. The disadvantage of this method is that bacterial cells do not have fractionalise molecular structure of the tours to ascertain the predominant localization uglevodnyh components inside the bacterial cell, and also that this method has low sensitivity because it allows you to determine the carbohydrate in a concentration of 10-100 μg/ml

The method for determination of carbohydrates [4] in the cell nuclei. The drawback of this method lies in the fact that the receipt of supramolecular structures is carried out from the cell nuclei of plants, and not from a nuclear-free prokaryotic cells, the shell of which can not be removed with 0.5% Triton X-100.

The above method of determining the carbohydrate components was adopted as a basis in which uglevodsoderzhashchie components extracted from cell nuclei, previously preserved in the tissues of seedlings of wheat glycerin environment, then isolated and purified in the density gradient of glycerol sequential extraction of nuclear fractions of 0.14 M (nucleoplasm), 0.35 M (chromatin (I) and 2 M (chromatin II) chloride, 6 M guanidine hydrochloride in the presence of 0.1% β-mercaptoethanol (nuclear matrix); 0,5 N. NaOH (nuclear-cytoplasmic residue) and the definition of contents of carbohydrates.

The disadvantage of this method is that the homogenization unacceptable for prokaryotes, because culture is composed of individual cells, not combined in the fabric, 0.5% Triton X-100 does not remove the cell wall prokaryotic cells, extraction fractions of 0.5 N. sodium hydroxide is not possible, the and, because the cellular residue is completely dissolved in 6 M guanidine-hydrochloride with 0.1% β-mercaptoethanol.

The aim of the invention is the expansion of the range of comparative methods to determine the intracellular localization hexoseaminidase component when the sensitivity of the method of 0.4-1 mg/ml

This goal is achieved by the fact that in the method of determination of hexoses in supramolecular structures of Escherichia coli cells initially preserved in the presence of buffered 80-90% glycerol cell off cell membrane with 3% Triton X-100, followed by the sequential extraction of cell supramolecular structures increasing salt concentrations: 0,14 M (bacteriology); 0.35 M (loosely associated with the cell balance); 2 M NaCl (strongly associated with cellular balance), 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol (cell balance) and the definition of contents of hexoses.

The invention is illustrated by the following example.

Example. Experiments were performed during the growth of a population of cells of E. coli strain HB101 (F-ara14 galK2 hsdS20(rB-mB-) leuB6 lacY1 mtl-1 proA2 recA13 rpsL20(StrR) supE44,9,14 thi-1 xyl-5 Δ(mcrC-mrr), obtained from the all-Union collection of microorganisms (Pushchino) and provided Markusevi T.V. (Institute of biology, USC RAS, group genetics of microorganisms, strain E. coli JC-158 (Hfr PO1, thi1, serA6, lacI22, relA1), PR is delivered Stupak .. and Stupak AA (Institute of biology, Ufa branch of RAS, laboratory of mathematical and molecular genetics). Cultivation of the strain E. coli HB101 was carried out in a liquid nutrient medium LB and M9. Bacterial cells used in the experiment was originally in the columns with agar LB medium (Luria-Bertani): in 1 liter of distilled water was dissolved with stirring (magnetic stirrer type MM 2A) bacto-tripton (Difco, USA), 10 g yeast extract (Difco, USA), 5 g sodium chloride (reagent-grade, "Rahim") - 10, bringing the pH to 7.5 was carried out using 1 M of sodium hydroxide (reagent-grade, "Rahim"). The medium was sterilized at 120-123°C at vapor pressure of 1 ATM in a standard autoclave. To 1 liter of medium was added 1.5 g agar-agar (Difco, USA). The culture was stored at 4°C. From the agar column in the sterile environment of the cells transferred using loops in liquid LB medium in an amount of 5 ml, in chemical beaker with a volume of 20 ml, closed with a cotton-gauze tube and incubated at 37°C, 120 rpm on a controlled setting (WVMT-12-250) for 16 hours. Of the 16 hour liquid culture of strain HB101 was rasiwasia "dash" to separate colonies on the agar LB medium with the addition of the antibiotic streptomycin (Str) at a concentration of 25 μg/ml in Petri dishes and were incubated at 37°C for 16 hours.

Grown well-formed individual p is I a bacterial colony using a bacteriological loop was one case replanted in liquid LB medium with the addition of the antibiotic Str at a concentration of 25 μg/ml in a volume of 5 ml and were incubated at 37°C, 120 rpm for 16 hours. 100 µl of the culture were made in liquid minimal medium M9 in an amount of 5 ml and were incubated at 25°C, 120 rpm for 16 hours. The composition of the medium M9: Na2HPO4(CHP, "Rahim") 6 g, KH2PO4(CHP, "Rahim") 3 g, NaCl (reagent-grade, "Rahim") - 0.5 g, NH4Cl (CHP, "Rahim") - 1 g per 1 l of distilled water. The medium was sterilized at 120-123°C, vapor pressure of 1 ATM in a standard autoclave. After cooling, 1 l of medium was added to the solution, sterilized separately: 0.01 M CaCl210 ml, 1 M MgSO4·7H2O - 1 ml 20% glucose solution (Biorad), vitamins to a final concentration of 1 µg/ml of amino acids to a final concentration of 40 μg/ml and antibiotic Str at a concentration of 25 μg/ml

In fresh M9 liquid medium in a volume of 50 ml in a 300 ml flask was inoculated with 600 ál 16 hour culture and incubation was carried out at 25°C, 120 rpm for 6 days.

The first sample was taken in a day in a volume of 1.5 ml of the Subsequent samples were taken on the second, fourth and sixth day. Control the growth of the cultures was performed using the photocolorimeter CPK-2 by the change in optical density of cell suspension OD590at a wavelength of 590 nm and a sensitivity equal to 2. The supernatant was removed, the precipitate to dry a little. To precipitation was added 50 μl of medium of the following composition: 80-90% glycerol in 0.01 M Tris-HCl buffer pH 6.8 with the addition of 0.005 M MgCl2; 0.025 M KCl; 0.00 M CaCl 2; 0.005 M NaCl for preservation of cells at minus 25°C.

Further precipitation cells were washed with 3% Triton X-100 in the medium of the following composition: M triethanolamine (tea)·HCl pH 6.8; 0.005 M MgCl2; 0.025 M KCl; 0.003 M CaCl2; 0.005 M NaCl pH 6,8; was shaken for 30 min on microsecure (Micro-shaker type 326 m, Poland), followed by centrifugation at 4000 rpm (K-23, East Germany) for 20 min to remove cell membrane, after which the precipitate washed twice in the medium of the following composition: 0.005 M MgCl2; 0.025 M KCl; 0.003 M CaCl2; 0.005 M NaCl; 0.01 M Tris-HCl pH 6.8, followed by centrifugation under the above conditions. Lower concentrations of Triton X-100 is not removed by the membrane of E. coli cells.

Cytoplasmic proteins were extracted to 0.14 M NaCl, 0.01 M Tris-HCl pH 6.8 buffer. Faction, loosely associated with the cell residue was isolated by extraction of sediment 0.35 M NaCl, 0.01 M Tris-HCl pH 6.8 buffer. Next, the precipitate was fractionally suspendirovanie in Tris-HCl buffer with 2 M NaCl. In the sediment remained a fraction containing the cell balance with the cell membrane. Subsequent extraction was performed 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol in Tris-HCl buffer. In the above buffer precipitate was completely dissolved. Cell fractions were stored at -196° C in nitrogen.

In the above fractions was performed acid hydrolysis and color reaction with astronom. Method [1] is one of the common the United. The essence of the method is that by boiling in the presence of concentrated sulfuric acid from peptides hatshepsuts glucosamine groups, easily splits into monosaccharides. Under the action of monosaccharides compounds containing H-group, in an acidic medium are formed thioacetal that when interacting with astronom give products, painted blue, with optimal absorption (especially for hexoses), when a620. Direct application of the original method [1] in our conditions did not produce results. So they had done experiments that identify the optimal conditions of the reaction. According to literature data net Andron has a melting temperature of 163°C (colorless needles), 154°C [5,6].

Sales drug antrona ("Rahim", 1987), probably due to the large number of impurities instead of white needles had yellow crystals with a melting point of 105-110°C. Thin-layer chromatography on Silufol (Silufol UW 254, "Reanal") gave two bands. Clearance sales of the drug antrona was achieved in several ways (table 1). We managed to clear the sales of the drug only to tPL145°C. the Crystals were stable when stored.

Subsequent production reaction were performed with purified astronom tPL145°C and recrystallized thiourea tPL180°C. the Concentration of sulfuric acid and timoci the ins was selected empirically (table 2), guided by the fact that Andronova reaction is selective for hexoses with a maximum absorption at A620and the fact that aromatic amino acids, proteins, pentoses (ribose, xylose), hexosamine, uronic acids do not show maximum absorption at A620[2].

Based on the data shown in table 2, for the production method were selected concentration of thiourea - 2 g (%) and sulfuric acid - 74,2%. Sulfuric acid should be sufficiently concentrated, as in the case of further dilution rolled crystals of thiourea. For ideal sales sulfuric acid 92% with a density of 1.83.

To determine microanatomical of hexoses (0.4 to 1 μg/ml) astronomy method, constructed of standard calibration curve for D-glucose ("Serva", 1988) (figure 1).

Preparation antropofago reagent: 72 ml of sulfuric acid (by volume) carefully added to 25 ml of distilled water. The mixture is cooled to 35°C and add 50 mg anthrone, stirred to dissolve, then add 2 g of thiourea, mix to dissolve. The reagent is prepared prior experience.

The course of the reaction

To 0.2 ml of the investigated solution add 1 ml antropofago reagent, quickly stirred and cooled in an ice bath to 0°C. Then placed in a boiling water bath for 15 minutes, cooled Rapidly to room temperature t-20°C. Developing blue is paint colorimetrate on SF-26 (LOMO, USSR) against distilled water, And620.

Control 1: 0.2 ml 0.05 M Tris-HCl pH 8.0+1 ml antropofago reagent

Control 2: 0.2 ml sample+1 ml (74,2% H2SO4+2% thiourea; without antrona).

The content of hexoses in the analyzed samples was evaluated based on 1 cell according to the formula:

C=(C1-K1-K2)V1,2A,

where

C - the number of hexoses (ICG) in supramolecular structures on 1 cage;

1,2 - volume of reaction mixture (ml)containing 0.2 ml of sample taken for analysis;

C1- the number of hexoses (ICG), was found in the standard curve (figure 1) in the total volume of the reaction mixture;

V is the total volume of the analyzed extract (ml);

A - the number of cells taken in the analysis.

K1control 1; K2control 2 - readings of optical density translated into evidence was found in the standard curve (figure 1).

The method is stable, does not give sharp fluctuations. The colour intensity of the sample is maintained for 1.5 h, and then decreases.

Table. 1 illustrates the cleaning methods of the sales of the drug antrona (Reachim).

On table 2 shows the effectiveness of the concentrations of sulfuric acid and thiourea on Andronovo reaction.

Figure 1 shows the standard calibration curve for D-glucose. The sensitivity of the method is 0.4-1 mg/ml

Figure 2 presents the growth curve of E. coli strain HB101. On the ordinate axis shows the optical density of the periodic culture. On the x-axis shows the age of the periodical culture of the day.

Figure 3 shows the dynamics of the content of protein, carbohydrate components, DNA and RNA during growth of a population of cells of E. coli HB101. The amount of protein was determined by the binding protein with Kumasi bright blue G (Loba, Austria) [7, 8]. To determine DNA and RNA used method A.S. Spirin [9]. Since this methodological approach was developed for the cell nuclei of plants and methods, its components, ostentatious to determine nanotechnologist, we found it appropriate to provide information on protein, DNA and RNA during growth of a population of E. coli HB101 to give a full description of the obtained suprastructures. For a more accurate interpretation of the us schedule table 3 presents quantitative indicators of Bioorganic composition of the bacterial cells of E. coli in periodical culture.

The study of supramolecular structures on the carbohydrate content of the component includes a preliminary preservation of cells in glycerol medium, removing the cell membrane and the allocation of these cellular fractions of increasing the concentrations of salts. Figure 2 illustrates the dynamics of the periodic growth of culture, the analysis of figure 3 (table 3) shows the ratio of protein/hexose during the phase of active growth, the deceleration phase and stationary phase of growth of a population of bacterial cells in the periodical culture. Shows the change in the ratio of protein/hexose at the level of all abutments cell populations of E. coli. Interest is the dynamics of the ratio of protein/hexose in genetic and transcriptional suprastructures of E. coli cells.

The proposed method is recommended in the study of molecular-genetic mechanisms of formation of prokaryotic cell structure and role of carbohydrate components in their organization. The obtained data can be useful in the modeling of the structure (3D-folding and dynamics of biomacromolecules. Knowledge of the biochemical processes that occur at different phases of growth of a population of bacteria, allows to reveal the regulatory mechanisms of interaction between macro - and microorganisms, which allows controlling the bacterial communities inhabiting the human body.

Table 2
Color and optical characteristics of the control samples when applying RA is personal concentrations of sulfuric acid and thiourea
Sulfuric acid (% by volume)The concentration of thiourea (g %)Color reactionAnd620
861dark green2
2brown-green2
3maroon2
74,21blue0,091±0,004
2the yellowish-blue0,032±0,004
3yellow-blue0,029±0,004

Protein
Table 3
Bioorganic composition of bacterial cells of Escherichia coli in periodical culture
The time of growth (days)FactionThe total content in the fractions
HexoseDNARNA
FG/cell
1Bacteriology140150,280
21991,070,23
56141,140,35
693190,051,16
1Loosely associated with the cell balance5850,830,16
2170,80,030,32
51150,72 0,36
62710,51,16
1Strongly associated with cell balance1371,110,89
2720,850,36
520130,860,54
63731,920
1Cell balance with the cell membrane15080,080,38
2189120,440
518690,2/td> 0,6
6267130,650,77

The method of determination of hexoses in supramolecular structures of Escherichia coli cells, including the preservation of cells in the presence of buffered 80-90% glycerol, removing cell membranes with 3% Triton X-100, followed by the sequential extraction of cell supramolecular structures increasing salt concentrations: 0,14 M (bacteriology); 0.35 M (loosely associated with the cell balance); 2 M NaCl (strongly associated with cellular balance), 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol (cell balance), to be held in the above fractions of acid hydrolysis and determination in the amount of hexoses using antropofago method with preliminary clearance of the drug antrona, the construction of the calibration graph and applying computational formulas

where
With the number of hexoses (ICG) in supramolecular structures by 1;
1,2 - volume of reaction mixture (ml)containing 0.2 ml of sample taken for analysis;
C1 is the number of hexoses (ICG), was found in the standard curve (figure 1) in the total volume of the reaction mixture;
V is the total volume of the analyzed extract (ml);
And the number of cells taken analis.



 

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FIELD: biotechnologies.

SUBSTANCE: method includes the following stages: contact of a sample with a source of nutrition for cells, containing antioxidant, representing pyroracemic acid or its salt, and an inhibitor of cell proliferation, which is selected from ciprofloxacin and cefalexin; contact of the specified sample with fluorescent-marked oligonucleotide probes, capable of specific hybridisation at least with one section of ribosomal nucleic acids, which belong to microorganisms of Legionella pneumophila kind and type; and detection and quantitative determination of a fluorescent signal.

EFFECT: provided method and set make it possible to more accurately and reliably detect and calculate viable microorganisms of Legionella pneumophila type, having excluded natural fluorescence of microorganisms from calculation.

6 cl, 2 dwg, 6 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of determining maximum concentration of living bacteria. A suspension of bacteria with turbidity standard of 5 units is brought to concentration of 500000 mc per 1 ml. Electrical resistance of the suspension is then determined in a direct current field with maximum voltage across open ends of electrodes of 2.8 V. Maximum resistance in the range of 900-1500 kOhm indicates maximum concentration of living bacteria in suspensions of different types of microorganisms.

EFFECT: method provides quality estimation of concentration of living bacteria, which is sufficient for further identification of said bacteria, and cuts analysis time.

1 tbl, 4 ex

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