FR2608624A1 - PROCESS FOR THE TRANSFORMATION OF BACILLUS THURINGIENSIS CELLS - Google Patents

Abstract

THE PROCESS OF TRANSFORMATION OF BACILLUS THURINGIENSIS CELLS INVOLVES THE PREPARATION OF COMPETENT CELLS BY GROWING THEM IN A HYPERTONIC AQUEOUS MEDIUM, THE TREATMENT OF THESE COMPETENT CELLS, POSSIBLY AFTER TREATMENT WITH LYSOZENE HYPERADIC, IN EXOGENIC MEDIUM, IN THE PRESENCE OF POLYETHYLENE-GLYCOL, WHILE MAINTAINING THE HYPERTONIC STATE, AND THE ISOLATION AND RESUSPENSION OF BACILLUS THURINGIENSIS CELLS THUS TREATED IN A HYPERTONIC ENVIRONMENT TO ALLOW EXPRESSION.

Description

The present invention relates to a process for transforming

  mation of Bacillus thuringiensis cells. The terms "transform" and "transformation", used

  in the present invention are intended to relate to a

  genetic transfer mechanism in which exogenous DNA is introduced into a recipient bacteria, by introducing

  thus genetic modifications in said bacteria re-

  ceveuse. Bacillus thuringiensis (BT) are Gram bacteria

  positive containing a crystalline protein, 8-endo-

  toxin (DET), which is toxic to the larvae of a cer-

  a lot of insects. Depending on the subspecies, BT is

  used as a selective biological pesticide against different

  harmful annuities. The subspecies thuringiensis, alesti and

  dendrolimus are, for example, pathogens against lepido-

  pterae; the subspecies israelensis, darmstadiensis 73-E-

  10-2, kyushuensis and morrisoni PG14 are against di-

  pterae; the tenebrionis subspecies is against coleo-

  pterae; the subspecies kurstaki HD-1, kenyae, aizawai and colmeri are against lepidoptera and diptera, while the subspecies dakota, indiana, tokokuensis and kumamotoensis are not known to be toxic to

all the nuisances.

  From an industrial and ecological point of view, it is often

  haitable to have additional biological pesticides with a different spectrum of activity, for example higher

or wider.

  We can, for example, achieve this goal by

  no new isolates from nature, by conju-

  gaison of bacteria or by transformation of bacteria.

  New strains of BT have recently been isolated

  having an interesting activity (for example var. tenebrio-

  nis with activity against beetles) and it also

  Recent successes regarding the economic

gaison of strains of BT.

  The transformation of bacteria offers the advantage that it

  allows it, if successful, to introduce a

  specific genetic information in bacteria.

  Thus, a gene encoding a DET was cloned into di-

  to microorganisms such as Escherichia coli, Bacillus

  subtilis and Pseudomonas fluorescens, and even in plan-

  your superiors (tobacco), by recombinant DNA techniques

  more specifically by transformation.

  These genetically engineered organisms produce this-

  during small amounts of DET compared to the amounts produced by natural BT strains. The commercial importance of these organisms is therefore questionable, at least

  as long as no means have been found to improve

  improve the expression of exogenous DNA encoding DET.

  It would therefore seem appropriate to try to obtain a better expression of exogenous genes (DNA) by using a BT bacterium as a recipient bacteria in techniques

of transformation.

  Known processing techniques are essential

  tially carried out using cells or prototypes

  plastids. The transformation of cells involves the presence of competent cells, that is to say cells in a precise physiological state allowing the binding and absorption of exogenous DNA. There is, however, no evidence of

  the existence of competent BT cells.

  Successful transformation of prototypes has been reported

  BT plastids by DNA with only very low yields, namely significantly lower than those obtained

  with the transformation of B. subtilis. The yields are low.

  These may be due in part to poor regeneration of protoplasts, including the transformed protoplast. Although Shall et al. (Fundamental and applied aspects of invertebrate Pathology - published by RA Samson, JM Vlak and D. Peters, 1986, page 402) announce that they have optimized the procedure using protoplasts and that '' they have developed improved regeneration media to transform BT or B. cereus with the DNA plasmid, they do not

  not specify the nature of the optimization or the improvement

  ration. The transformation frequencies indicated by Shall and

  coll. are therefore difficult to interpret.

  The present invention now relates to a pro-

  BT's improved transformation assignment. It is based on the discovery that BT microorganisms develop what

  a skill status is called when they are introduced

  added in a hypertonic aqueous medium.

  The term "hypertonic" as used in the present invention relates to a medium which is hypertonic

  vis-à-vis conventional LV environments (culture or growth

cell session).

  The method of the invention involves the steps which consist:

  a) culturing BT cells in a hy- gous aqueous medium

  pertonic, b) to be introduced into the cell culture obtained in step a), and in the presence of polyethylene glycol, exogenous DNA while maintaining the hypertonic state, and c) to isolate and resuspend the BT cells

  thus treated in a hypertonic aqueous medium to per-

put the expression.

  In principle, any compound which does not work can be used.

  not pour the semi-permeable cell membrane which is not metabolized by, or toxic towards, the BT cell, to obtain the desired hypertonic state. In general, the desired hypertonic state is advantageously obtained at

  using saccharides, in particular mono- or di-

  saccharides, which are not metabolized by BT. Suitable examples of these saccharides are sucrose

and lactose.

  The concentration of saccharides to be used to obtain the desired hypertonic state is advantageously of the order of 0.4 M of saccharides per liter of aqueous medium, or higher. In general, we will get good results

  with essentially isotonic concentrations vis-à-vis

  screw of the BT cytoplasm. This osmotic state is generally obtained with a concentration of 0.4M to 0.5M of saccharides per liter of aqueous medium. Higher concentrations of saccharides can however be used, but they

  generally offer no benefit.

  The term "hypertonic" used below relates to a

  state or medium as defined above.

  It is important that the hypertonic conditions are essentially maintained from start to finish.

  different steps a) to c) of the process.

  Hypertonic aqueous media should be essen-

  tially neutral, that is to say they must advantageously-

  mentally have a pH of 7 2, better still 7 1.

  In addition to saccharides (used to maintain the hy-

  pertonic) and possible buffers (used to maintain an essentially neutral state of the medium) other ingredients can be and will be added, for example to allow the growth and development of the BT culture, if necessary, etc. These additional ingredients are conventional and known to those skilled in the art; they understand, for example,

nutrients and salts.

  Examples of suitable nutrients are, for example

  example, beef extract, yeast extract, pep-

  tones, tryptones, amino acids (e.g.

  tryptophan), nucleosides like thymidine and ana-

  logues. Examples of suitable salts are NaCl and MgCl2,6H20. A suitable hypertonic medium can contain from 0.05 to 0.1M of salts per liter. The salts will preferably include magnesium salts, such as MgC12.6H2O. The cell culture of BT (starting product) is advantageously prepared and cultivated under conventional conditions, that is to say under aeration and at room temperature, in an appropriate nutritive medium, for example in the minimal medium described by J Spizizen in Proc. natl. Acad. Sci. (Wash), 44, 171-175 (1958), optionally supplemented with amino acids, salts, for example catalytic amounts of manganese salt such as MnSO4,

  etc. It is advantageous to use, in step a), a cul-

  ture of BT cells which is in the growth phase

exponential growth.

  The freshly prepared BT cell culture is

  then diluted in a hypertonic medium to a concentration

  starting cell essentially less than 109 cells per ml, for example from 104 to 106 cells per ml,

  and the cell culture is cultured, in said hy-

  pertonic, until we get a midpoint of

  cell concentration slightly below 109 cells

  lules per ml, for example from 108 to 5,108 cells per ml.

  The hypertonic medium used to dilute the culture of freshly prepared BT cells is advantageously at -40 ° C., for example at 37 ° C. The culture is then allowed to take place at this temperature. It is quite obvious that it is necessary to guarantee a thorough ventilation. A slight amount of silicone is advantageously added to the culture medium

  cell to prevent foaming.

  When the desired final cell concentration is reached (slightly less than 109 cells per ml), the competent BT cells thus prepared can be treated with DNA, in the presence of polyethylene glycol (PEG), according to

  step b) of the process of the invention.

  It is however advantageous to treat the competent BT cells obtained according to step a) of the invention with

  moderate concentrations of lysozyme in a hy-

  pertonics and isolate and resuspend them

  lules of BT treated with lysozyme in a hypertensive medium

  before submitting them to process b). The quantity of

  lysozyme to be used must be less than that used

  read normally to prepare protoplasts. This quan-

  tity (concentration) will of course depend on various fac-

  such as the osmotic pressure of the medium, its temperature,

  the desired reaction time, etc. In general, a concen-

  suitable lysozyme tration is 20 to 300 gg, for example 200 gg, per ml of hypertonic aqueous medium (which is essentially less than the 2 to 15 mg per ml which would normally be necessary for applications

  concerning protoplasts). A distribution must be guaranteed.

  adequate lysozyme in the cell culture medium

  laire. The reaction time will therefore depend on the concentration

  tion and the quality of the lysozyme solution used.

  The optimal reaction time can be determined by

standard tests.

  The reaction temperature is advantageously

  taken between 20 and 40 C, preferably higher than the temperature

  ambient temperature, for example around 37 C.

  During treatment with lysozyme, you must maintain

  hypertonic state, as defined above.

  The lysozyme treatment is then terminated by centri-

  fugation of the cell suspension and resuspension of the aggregates in the hypertonic medium, advantageously at

Room temperature.

  The BT cell culture thus prepared - obtained

  according to step a), possibly followed by treatment at ly-

  sozyme - is then treated with DNA, for example the DNA plasmid, in the presence of polyethylene glycol (PEG). In

  this goal, DNA, as well as PEG, are used pending

  sions / solutions in hypertonic solutions, so

  that the osmotic pressure of the cell suspension of-

  essentially dies unchanged after the addition of DNA and PEG to this cell suspension.

  The quantity of PEG used will advantageously be chosen

  so that its concentration in the culture of BT cells is in the range of 100 to 400 g per liter, for example 300 g per liter of culture medium.

cellular.

  Transformation step b) can essentially be carried out under conditions known to be appropriate

  to conventional protoplast transformation processes.

  Consequently, the choice of the appropriate amount and type of PEG and the appropriate amount of DNA to be used can be properly made by those skilled in the art.

  profession of protoplast transformation.

  Thus, an example of PEG usable in this process is

PEG 6000.

  DNA quantities from 100 ng to 20 gg for 108 to 109

  BT cells will generally give good results.

  The incubation is advantageously carried out with shaking

  moderate at room temperature. Incubation time

  tion required is short, usually on the order of a few

minutes (see example).

  The suspension comprising the transformed cells is then treated using known methods, provided that the hypertonic state of the solvent of the cells is maintained (in the case where they are in solution / suspension). So,

  the suspension is, for example, diluted in a hy-

  pertonic, the suspension is mixed, centrifuged and

  aggregates resuspended in the hypertonic medium.

  The resulting suspension is then incubated at a time.

  temperature from 20 to 400C, for example at 37 C, to allow expression. The suspension is advantageously ventilated, by

  example using a stirred water bath. A time of incu-

  suitable ration is 30 minutes to 5 hours, better yet

  between 2 and 4 hours, for example 3 hours.

  Appropriate dilutions of the cell cultures thus obtained can then be placed in Petri dishes to determine colony forming units (CFU). The frequency of transformation can be determined by known methods employing standard techniques, such as petri dishes containing an antibiotic, visual observation, etc.

  The process of the present invention allows the transformation

  mation of BT cells with high yields. Transformation Clones Library Genes

  of genomes in BT cells, cloning and expression

  DET genes in BT, cloning and expression of

  DET genes modified in vitro and in vivo in BT, the syn-

  thesis of useful polypeptides, etc. When the transformed BT cells are intended to be used as biological pesticides, they are

  advantageously used in the form of insect compositions

  ticides, for example in the form of concentrated suspensions

  or in the form of powders. These compositions can be obtained

held in a classical manner.

  In the following nonlimiting example, the starting products (BT cells and DNA plasmid) are chosen so that the results are not ambiguous and do not

  may be due to interaction of the plasmid; those

  BT capsules used as starting material do not contain plasmids, the DNA plasmid used as a

  encoding transformation for resistance to tetracy-

  cline. Note that other BT and / or DNA cells

  exogenous, in particular a DNA plasmid, can be used

  read in the process of the invention with results

analogues.

  The temperatures are expressed in centigrade and the

  parts by weight, unless otherwise indicated.

Example

  Starting materials Strain: Bacillus thuringiensis, kurstaki subspecies

  HD1 cry B (obtained from M.-M. Lecadet, Institut Pas-

  (Paris,), not containing plasmids.

  DNA: pBC16.1 (Kraft. J. et al. (1978) Molec. Gen.

  Broom. 162; 59-67), extract from HDI cry B (pBC16.1), in the-

  which it was introduced by conjugation by cellular adaptation with B. subtilis BD224 (pBC16.1), coding for

resistance to tetracycline.

  SA Trp media: Spizizen minimal medium (Spizizen J. (1958) Proc. Natl. Acad. Sci. (Wash.) 44; 171-175) supplemented with 1% Casamino acids (Difco), 5 x 10-6M of MnSO4 and 20

ig / ml tryptophan.

  Hypertonic medium (HD): Beef extract 1.50 g / 1 Peptone 5.00 g / 1 NaCl 3.50 g / 1 Sucrose 171.15 g / 1 Maleic acid 2.32 g / 1 MgCl2.6H20 4.07 g / l pH 6.7 Luria medium (LA): Tryptone 10 g / 1 Yeast extract 5 g / 1 NaCl 10 g / 1 Agar (Difco Bacto) 15 g / 1 Thymidine 20 mg / 1 Antibiotics: Tetracycline, 10- 100 wg / ml on boxes in the middle Luria Solutions

SMM:

  Sucrose 171.15 g / 1 Maleic acid 2.32 g / l

MgCl2,6H20 4.07 g / l-

pH 6.5

PEG:

PEG 6000 40 g SMM qs 100 ml

  Lysozyme: 2 mg / ml in HD, freshly prepared.

  Process An overnight culture of HD1 cry B is prepared in 15

  ml of SA Trp and it is grown under aeration at 20 C. The slow

  tomorrow morning, the culture is diluted 50-100 times in a preheated HD medium, to a cell concentration

  of 7.5 x 105 per milliliter. We add 2 il de si-

  licone to prevent foaming. The culture believes 370C under moderate aeration for three and a half hours, namely up to a cell concentration of 2.5 x 108 - 3

  x 108 per ml. Lysozyme is added to a concentration

  final tion of 200 gg / ml and 1 ml of cell suspension is incubated for 30 minutes at 37 ° C. in a stirred water bath

  (150 rpm). The cell suspension is then centered

  trifugated for one minute under 10,000 G and the aggregates

  are resuspended in 1 ml of fresh MH at room temperature.

ambient ture.

  0.5 ml of cell suspension is added to 50 l of

  SMM, to which were added 100 ng-10 lig of DNA plasmid.

  The cells are transformed by adding 1.5 ml of so-

  PEG lution, moderate shaking and 2 min incubation. at room temperature. 5 ml of MH are added to the cell suspension, which is mixed gently but thoroughly and which is centrifuged for 20 minutes under 3000 G. The aggregates are resuspended in 0.6 ml of MH and incubated for 3 hours at 37 ° C. in a stirred water bath (150 rpm: ip) to allow expression. We put the dilutions

  suitable in boxes in the middle Luria to de-

  complete CFUs and in long-acting boxes

  containing tetracycline for the selection of the sub-

transforming stance.

  1-2 x 103 transforming substances are obtained per μg of intact DNA plasmid, with a frequency of 5 × 10 -5 - 10 -4.

Claims (12)

R E V E N D I C A T I ONS   1.- Process for transforming Bacillus thu- cells   ringiensis, characterized in that it comprises the stages which consist: a) in cultivating Bacillus thuringiensis cells in a hypertonic aqueous medium, b) in introducing into the cell culture obtained in stage a), and in the presence of polyethylene -glycol, exogenous DNA while maintaining the hypertonic state, and   c) isolating and resuspending the Ba- cells   cillus thuringiensis thus treated in an aqueous medium   hypertonic to allow expression.   2.- Method according to claim 1, characterized in that the culture of Bacillus thuringiensis cells of step a) is treated with moderate concentrations of lysozyme, while maintaining the hypertonic conditions, and in that the the Bacillus thuringiensis cells thus treated are isolated and resuspended in an aqueous medium   hypertonic before treatment according to steps b) and c).   3.- Method according to claim 1 or 2, characterized in that one obtains the hypertonic state by using   saccharides that are not metabolized by Bacillus thu-   ringiensis. 4.- Method according to claim 3, characterized in that   at least 0.4M saccharides are used per liter of mid watery place.   5.- Method according to any one of claims 1 to   4, characterized in that the initial concentration in this   lules of Bacillus thuringiensis introduced in step a) is 104 to 106 cells per ml of medium and in that the cells are cultured up to a concentration of 108 to   slightly less than 109 cells per ml of medium.   6.- Method according to any one of claims 2 to   , characterized in that the lysozyme concentration is   20 to 300 micrograms per ml of aqueous medium.   7.- Method according to any one of claims 1 to   6, characterized in that the hypertonic medium has a pH in the interval from 6 to 8.   8.- Method according to any one of claims 1 to   7, characterized in that the hypertonic medium comprises a magnesium salt.   9.- Method according to any one of claims 1 to   8, characterized in that it is produced at a temperature between 20 and 40 C.   10.- Method according to any one of claims 1 to   9, characterized in that 100 nanograms are used at 20   micrograms of DNA for 108 to 109 Bacillus thu- cells ringiensis.   11.- Method according to any one of claims 1 to   10, characterized in that 100 to 400 g of poly-   ethylene glycol per liter of cell culture.   12.- Method according to any one of claims 1 to   11, characterized in that the hypertonic state is maintained   from step c) for 30 minutes to 5 hours.