Method for transduction of bacillus anthracis and closely related bacilli

FIELD: microbiology, genetics of microorganisms.

SUBSTANCE: invention relates to methods for transduction of anthrax pathogen and closely related bacilli. Method for transduction of Bacillus anthracis and closely related bacilli involves incubation a mixture of bacteriophage with recipient cells taken in the definite ratio. After the first bacteriophage generation in 20 min the preparation of recipient cell receptors is added to the transducing system and the transducing system is inoculated on selective media. Invention describes a method for preparing receptors from recipient cells B. anthracis, B. thuringiensis and B. cereus. Using the proposed method provides the full-value substitution of antiphage serum in the transducing system for a cheaper and simple in preparing component, namely, the preparation prepared from recipient cell receptors that neutralizes phage effectively and prevents excessive death of the recipient strain cells in the transducing system.

EFFECT: improved method for transduction.

9 dwg, 1 tbl, 2 ex

 

The invention relates to Microbiology and genetics of bacteria, in particular to methods of transduction anthrax and closely related bacilli.

On the territory of the Russian Federation, CIS and foreign countries is a significant amount affected with anthrax regions (1). In Russia every year there are cases of human and animal anthrax and, accordingly, are identified foci with different epidemiological and epizootic activity (2). Of particular relevance is the causative agent of anthrax acquired after use dispute Century anthracis as a weapon of bioterrorism (3).

So far not paid sufficient attention to the variability of Bacillus anthracis, making it difficult indication and identification. Especially sharply this question is in the context of possible changes in the genetic properties of the Century anthracis, when spontaneous genetic processes in B. anthracis and closely related bacilli occur in natural conditions (4).

Bacteriophages and plasmids - classical vectors carrying out horizontal distribution of genes among bacteria (5). Bacteriophages are able to introduce into the cells of the bacteria's genetic material that changes properties of the host and stably retained in the genome in cases when these changes for him field the us. Such transfer of genes from one bacterium to another via a bacteriophage is transduction. Plasmid transduction, i.e. the procedure for the introduction of plasmids into the new owners, which is a valuable tool in the study of many microorganisms, including bacillary species was first shown in Bacillus pumilis (6).

Development of interspecies transduction of plasmids and chromosomal genes between C. anthracis, B.cereus and B. thuringiensis most fully described by the American experimenters (7). As evidenced by the results of the experiments of these authors, the frequency of plasmid transduction largely depends on the multiplicity of infection (MOI), i.e. the ratio in transducible system phage - recipienta cell.

When a large plurality of phage, absorbirowawrzegosa on the recipient cells is cell death in transducible system and to achieve satisfactory output transductant impossible (Figure 1).

In their experiments (8, 9) we have seen in this pattern.

Transduction, in which the ratio of the phage - cell was less than 1 gave the highest frequency of transduction.

Transduction RVs of the Century anthracis to B. thuringiensis was the most convincing demonstration of this effect. When using phage BF (RVs) was necessary to prevent excessive cell death recipient strain in transducible system. It reached the camping by using a very low multiplicity of infection (1:100), or add antifirewall serum (APS) in transducers system for neutralization of transducers phage particles. In the latter case could be used a multiplicity of infection of 1:1, in which the frequency of transduction maximum.

However, obtaining the ASF with a satisfactory neutralizing activity of phage in transducible system - the problem is laborious, time consuming and expensive. First we must get the phage preparations with a concentration of 1010-1011petrobrazi units in 1 ml (10). Each drug phage should give at least three rabbits, because when applying the same method of immunization and antigen (phage) on different animals make different endpoint antibody titers (the difference is sometimes tenfold). In addition, you must have a guarantee in case of death of animals from disease or accidental causes. Duration immunization of rabbits to obtain vysokotirazhnyh ASF - 1.5-2.5 months.

Decided to find a replacement camera in transducible system other finalrelease component.

This component could serve as sodium dodecyl sulphate (SDS), with antiphagocytic properties to V phages (11).

We have determined the threshold neutralizing activity bacillary transducers phage - it was the concentration of SDS 0,175% defined neutralizing capacity threshold dose SDS - n×108the phage particles per milliliter (PC/ml). Such finalrelease parameters SDS could be quite suitable for transduction system, but it turned out that the recipient cells bacilli (.anthracis, .cereus, B. thuringiensis) in a vegetative state are more sensitive to detergent than their bacteriophages. This fact is described and phages of Escherichia coli (10).

Bacterial cell - host is not playing, apparently, any active (i.e. associated with its metabolism) role in the adsorption of most strains of phage (11). This is evidenced by the fact that phage particles irreversibly attached not only to killed by heating or poisoned by cyanide bacteria, but even the fragments of lysed or damaged cells. Therefore, in the membrane of bacterial cells must be some specific receptor sites that are able to engage in spontaneous irreversible chemical reaction with the bodies of adsorption, available phage particles. The understanding of the nature of these receptor sites engaged in Burnet in 1954 In extracts of sensitive bacteria to phage found them active receptors are able to bind phage particles and neutralize them. Burnet called these receptors in the extract of cells, agents that inhibit phage (phage-inhibiting agents) or ACE (13).

If gram-positive bacteria (bacilli) using lysozyme completely remove glue the internal shell, these protoplasts do not adsorb phages to which sensitive intact cells (14). Therefore, the bacterial receptor sites, serving for attachment of the phages are found in the cell wall (figure 2).

In the modern view (16, 17) surface layer (S - layer) bacteria (bacilli) is composed of protein or glycoprotein subunits, arranged in a hexagonal or tetragonal structures throughout the cell surface. They may function as phage receptors (16).

The bacilli S - layer completely covers the surface, forming an ordered lattice (18). Tiling protein crystal structures, which are the receptors of bacteriophages and bacteriocins, illustrated in Figure 3

For the first time in 1988 J.Ezzell and .Abshire (19) were extracted with SDS protein in the cell walls of C. anthracis, which is called EA1 (extractable antigen 1). Was demonstrated immunogenicity of the drug EA1, installed chromosomal determination of synthesis of this protein, however, its localization and function at the time were not studied. Only in 1995 J.Farchaus et al. (20) from the culture filtrates of cell C. anthracis allocated EA1, has defined its ultrastructure and localization on the cell surface. Has been proven affiliation EA1 to the S - layer of Bacillus anthracis and its close relatives. In subsequent studies the x was installed, that in B. anthracis and its close relatives the S - layer of cells is represented by two protein EA1 and Sap (surface array protein. Work J.Ezzell, T.Abshire (19) and J.Farchaus et al. (20) we use methods of obtaining protein - antigens surface structures of cells bacilli or extracts of the S - layer, which we regarded as the drugs of receptors for phages bacilli.

Several improved method (19) SDS extraction structures in the cell walls of C. anthracis, we have received preparations of receptors for infectious phages as follows: strains Century anthracis or B. thuringiensis or B. cereus, which we used as recipients in transducible system, were grown in BHI broth (DIFCO) for 20 hours in a shaker at 75 qual./min and a temperature of 35°C. Then broth culture of the recipients was centrifuged for 20 min at 10,000 rpm and 4°S, supernatant was removed. Cell mass resuspendable content of 0.1 g/ml in extracting SDS buffer containing 5 mm Tris HCl, 5 mm 2 mercaptoethanol and 0.25% SDS (pH 8,9). Further warming of the cell suspension at 70°C for 30 min, cooled at room temperature and centrifuged at 10,000 rpm for 30 min at 4°C. the Supernatant containing extracted protein (the receptor for phage), dialyzed over night at a temperature of 4°with periodic change of buffer 0,1M Tris - HCl solution (pH 80).

At the final stage extract cell wall bacilli, and in fact the drug receptor for phage was mixed (1:1) with rahovym buffer: 0.01 M Tris - HCl (1,21 g); 0.1 M NaCl (5,85 g); 0,001 MgSO4·7H2O (0.25 g) in 1 l distilled water (pH 7.5). Checked the neutralizing activity of drugs receptors recipient cells used in transducers system. When processing 1000 phage particles per milliliter (PC/ml) one ml of receptors of various bacilli within 20 minutes at a temperature of 33-35°is the neutralization of phage from 96.5-99,99%. Especially neutralization of phage receptors bacilli is evident in electron-microscopic controls (figure 4).

However, preparations of cell receptors for phages obtained by the method of SDS - extraction from different bacilli not always possessed a satisfactory neutralizing capacity when processing high (107-108FC/ml) concentrations of phage used in transducers system.

The closest analogue (main prototype) is the work (7), the contents of which are set out on page 1.

The aim of the invention is the search for less expensive, easy to manufacture component replacement in transducible system antifogov serum.

To increase the neutralizing capacity of the drug-receptor cells bacilli significantly changed the method of obtaining them.

4·7H2O (0.25 g) in 1 l distilled water (pH 7.5). The cell mass of each sample was treated with ultrasound (ULTRASCHALL - HOMOGENISATOR LABSONIC 1510) at 100 w twice for 3 minutes, leading to a creamy consistency. Disintegrated by ultrasound cells bacilli used as a drug receptor cells, neutralizing the phage.

Example No. 1. For comparative the ow characteristics of the neutralizing activity of ASF and cell receptors Century anthracis STI (K.R./V.A. STI) in relation to the phage BF received at the donor culture Century anthracis Davies (pBC16)S(8), to each sample of the phage BF/D (1 ml) with concentrations of 107and 103FC/ml add 1 ml of APS in a dilution of 1:10 and 1 ml K.R./V.A. The TIS. As a control phage samples with concentrations of 107and 103FC/ml was added to 1 ml of phage buffer. All samples of the test and control mixtures were incubated at 35°C for 30 min in a shaker at 75 oscillations per minute. Then by the method of Grazia was determined in each sample number is not neutralized phage on indicator culture Century anthracis S. Davies and ASF (1:10), and K.R./V.A. TIS possessed neutralizing activity: 100% against the phage with a concentration of 103FC/ml of 99.96-100% (respectively) relative to the phage 107FC/ml

Receptors of intact cells bacilli embedded in the cell wall, always are monovalent (figure 5), and "velomania" receptors from the cell surface bacilli - polyvalent against phage (Fig.6).

We used polivalentes receptor cells, sorbirovtsa phage to increase the capacity of drug receptors used in transducers system.

Example 2 Method of intraspecific and interspecific transduction with ASF and receptors of the cells of the recipient.

Material: 1) Century anthracis Davies (pBC16)S, TcRdonor RWs (TcR).

2) Century antracis STI R, TCS- recipient.

3) B. thuringiensis Pasteuer TCS- recipient.

4) Phage BF/D (RVs) with a concentration of 1.2·109FC/ml

5) Drug receptors recipient cells Century. anthracis STI R, TcS- (K.R./V.A. STI).

6) Drug receptors recipient cells of B. thuringiensis Pasteuer TcS- (K.R./B.t.P).

7) ASF to phage BF with neutralizing activity of 99.96% at a dilution of 1:10 to 107FC/ml

8) phage buffer (pH 7,5).

Method: 1 ml recipient cultures 2) and (3)grown in BHI broth (DIFCO) at 35°C for 3 hours, mixed with 1 ml of phage BF/D (RVs) with concentrations of 1.2·109, 1,2·108, 1,2·107, FC/ml, and control - 1 ml of recipient culture 2) and (3) with 1 ml of phage buffer. The mixture was incubated 20 min at 33°C in a shaker at 100 qual./minutes Then all the test and control samples were made and 1 ml ASF 1:10 or 1 ml 5) or 6) of the corresponding receptor and again incubated at 33°C. After 30 min was introduced into each sample another 1 ml of the ASF 1:10 or the corresponding receptor and was inoculated with 0.2 ml of each test and control samples on selective (Tc 20 μg/ml) environment Hottinger (3 cups per sample). Raised TcRcolony - transductant been taken into account within 48-72 hours of incubation of cultures at 35°With (see Table).

As evidenced by the result of the experiment (table), intraspecific and interspecific transduction frequencies the output of transductant in the experiments does not depend on applied transducible system components (API or drug cell receptors), neutralizing the phage. The frequency of transduction depends only on the concentration of phage in the choice of the recipient, i.e. the state in transducible system phage - recipient cell.

So, found a full replacement method is expensive, laborious and time consuming when receiving component (ASF) transducible system with a cheap, simple in the manufacture of component (drug-receptor cells of the recipient), which effectively neutralizes the phage, preventing excessive cell death recipient strain in transducible system.

18
Table
The result of intraspecific and interspecific transduction
Transducers mixtureThe concentration of phage BF/D (pBC16)no CupThe amount of CuRcoloniesTransducers mixtureThe concentration of phage BF/D (pBC16)no CupThe number of TcRcolonies
B. anthracis STI R, TcS+ BF/D (pBC16) + K.R./V.A. STI1,2·1091151B. anthracis STI R, TcS+ BF/D (pBC16) + ASF 1:101,2·1091132
2298
382386
1,2·1081641,2·108162
269258
352347
1,2·107181,2·10716
21228
3433
B. anthracis STI R, TcS+ phage buffer + K.R./V.A. STI10Century anthracis STI R, TcS+ phage buffer + AFS10
2020
3030
B. thuringiensis Pasteuer TcS+ BF/D (pBC16)+K.R./V.A. STI1,2·109123B. thuringiensis Pasteuer TcS+ BF/D (pBC16) + ASF 1:101,2·109123
2182
321324
1,2·1081501,2·108141
241233
353342
1,2·107131,2·107110
2627
3432
Century thuringiensis Pasteuer ThoseS+ phage buffer + K.R./V.A. STI10Century thuringiensis Pasteuer ThoseS+ phage buffer + AFS10
2020
3030

LITERATURE

1. Microbiological diagnosis of anthrax / Marinin LI, Onishchenko GG, Stepanov A.V. and others, - M: NERC's health Ministry, 1999. - 224 S.

2. Cherkasy B.L. laws of the territorial distribution and the activity permanently affected with anthrax points // Epidemiology and infectious diseases is I. - 1999, No. 2 - P.48-52.

3. Jngleshy So, O′'toole T., D. Henderson et al. Anthrax as a biological weapon // JAMA - 2002. - V.287. - P.2236-2252.

4. Bulantsev A.L., ALEXANDER Lipnitsky Spontaneous genetic processes in Bacillus anthracis and closely related bacilli. // Problems of especially dangerous infections. - Saratov, 2003. - S-86.

5. Ilina T.S. Mechanisms of horizontal gene transfer: Role of bacteriophages and integrons in the evolution of pathogenic bacteria. // Molecular genetics microbial. and virusol. - 2003. No. 4 - p.3-10.

6. Bramucci M.G., and Lovett P.S., Low - frequency. PBS1 - mediated plasmid transduction in Bacillus pumilis. // J. Bacteriol. - 1976, - V.127 - P.829-831.

7. Ruhfel R.E., Robillard N.J., Thome C.B. Interspecies Transduction of Plasmids among Bacillus anthracis, B. cereus and B. thuringiensis // J. Bacteriol. - 1984, - V.157, No. 3, - P.708-711.

8. Bulantsev A.P., ALEXANDER Lipnitsky, A. M. Barkov, Shelkovich A.I. possibility of genetic exchange between related bacilli using transduction. // Genetics and biochemistry virulence of the causative agents of especially dangerous infections. Materials of the Russian scientific conference. - Volgograd. - 1992. - P.56.

9. Bulantsev A.L., Lipnitsky A.V., Barcov A.M. Genetic exchenge between bacilli by transduction. // The 2nd international workshop on the molecular biology of Bacillus cereus. Bacillus anthracis, and Bacillus thuringiensis - Taos, New Mexico, USA. August 11-13, 1999. - R.31.

10. Adams M Bacteriophages // Ed. Jn. literature - M.I 961, - P.52-53; 94-98; 406-410.

11. Kurochkin NA, A.K. Adams, Agapova SM Antirakovye properties of media containing sodium dodecyl sulphate. // Problems of especially dangerous infections. - Saratov, 1977, VIP - P.44-47.

12. Stent, Molecular biology of bacterial viruses. // Ed. "Peace", M., 1965. - S-117.

13. Burnet F.M. The phage - inactivating agent of bacterial extracts. // J.Pathol. Bacteriol., - 1934. V.38. - P.285.

14. Weihull C. The isolation of protoplasts from B. megaterium Bacillus by controlled treatment with lisozyme. // J.Bacteriol. - 1953, - V.55 - P.688.

15. Bulantsev A.L. the Influence of bacteriophage on the electrophoretic mobility of some species of bacteria of the family Enterobacteriaceae. // Thesis. Kida. the honey. Sciences. - Rostov n/D - 1970, - 170 C.

16. Sleytr U.B., Sara M., Kupcu Z., P. Messner Ultrafiltration membranes with uniform porese from crystalline bacterial cell wall layers: Application of S - layers of selected strains ofBaccillus stearothermofilus and Desulfotomaculum nigrificans. // Arch. Environ. - 1986, - V.146, - P.19-24.

17. Bacilli (Genetics and biotechnology) / under the editorship of Harvard K. // M., Mir - 1992. - S-318.

18. Sara M., U.B. Sleytr, S - Layer Proteins // J. Bacteriol. - 2000 - V.182 - No. 4 - R-868.

19. Ezzell j, Abshire So, Immunological Analysis of Cell - Associated Antigens of Bacillus anthracis. // Infect. immun. - 1988. - V.55. No. 2. - P.349-356.

20. Fachaus J., Ribot, W., Downs, M., J. Ezzell Purification and Characterization of the Major Surface Array Protein from the Aviruent Bacillus anthracis Delta Sterne - 1. // J. Bacteriol. - 1995 - V.177 - No. 9 - R-2489.

The way transduction in Bacillus anthracis and closely related bacilli, including incubation of bacteriophage and recipient cells, adding in the received transducers drug mixture, preventing the death of the recipient cells, characterized in that for incubation 1 ml of the bacteriophage and recipient cells, the incubation is carried out at a temperature of 33°who for 20 min, after incubation for transducers mixture was added 1 ml of the drug receptor recipient cells capable of neutralizing the bacteriophage and representing disintegrated by ultrasound dead cells from germinated spores of Bacillus anthracis or a closely related bacilli from germinated spores, and 0.2 ml of the mixture plated on selective medium, cultures are incubated at 35°C for 48-72 h take into account the increased transductant.



 

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11 cl, 1 tbl, 6 ex

FIELD: biotechnology, microbiology, genetic engineering.

SUBSTANCE: invention relates to microorganism E. coli showing deficiency by chromosomal genes degP and prc that encode proteases DegP and Prc, respectively, and comprising mutant gene spr containing the E. coli suppressor sequence prc with the point mutation at position 148 that results to substitution of tryptophan for arginine at position 148. The heterologous polypeptide is prepared by culturing this microorganism. Invention provides preparing heterologous polypeptides showing the high degree of effectiveness.

EFFECT: valuable biological properties of microorganism.

23 cl, 13 dwg, 6 tbl, 2 ex

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