IE912898A1 - Restriction-deficient mutant - Google Patents

Abstract

The present invention relates to a mutant of Bacillus thuringiensis or B. cereus, which has a partial deficiency of the restriction barriers which are intrinsically present in the non-mutated starting strain and can therefore be transformed significantly better than said, non-mutated starting strain when vector DNA from a heterologous intermediate host which is naturally subject to restriction in B. thuringiensis and/or B. cereus is used. Also embraced are processes for the preparation of said mutants, which are based on a targeted enrichment of spontaneous mutants, and processes for the cloning of genes or other DNA sequences using said restriction-deficient mutants of Bacillus thuringiensis and/or Bacillus cereus.

Description

RESTRICTION-DEFICIENT MUTANT The present invention relates to partially restriction-deficient mutants of Bacillus thuringiensis and Bacillus cereus and to methods for the preparation of said mutants. The invention relates also to methods of overcoming restriction barriers in Bacillus thuringiensis and/or Bacillus cereus using said restriction-deficient mutants.

Restriction/modification systems are very common in microorganisms and have been known for a long time. More than 600 restriction enzymes and approximately 100 of the associated methylases have now been described in the scientific literature [Kessler & Holtke (1986)].

By far the greatest number of restriction/modification systems described hitherto belong to the so-called class Π systems. These class Π systems are characterised by their low degree of complexity and accordingly use relatively simple proteins. A class Π restriction enzyme, for example, is capable of recognising and of cleaving a specific DNA sequence, provided that the latter has not already been specifically methylated by the associated methylase. Neither enzyme requires ATP. The genes coding for these enzymes [methylases] frequently lie closely coupled adjacent to one another on the bacterial genome.

The few known restriction/modification systems of the more complex classes I and III originate from Escherichia coli, Salmonella and Haemophilus [Kessler & Holtke (1986)].

In addition to these classic systems there have been found in recent years, first in E. coli, and then in other species, restriction enzymes that, in a reversal of the situation with the class II systems, recognise and cleave a DNA sequence only when that system has been specifically methylated [Raleigh & Wilson (1986); Sladek et al (1986) and MacNeil (1988)]. -2The classic restriction enzymes of class II are one of the most important tools in rDNA technology and therefore constitute one of the basic essentials of modem molecular genetics. Their primary application is in the construction of recombinant DNA molecules.

Their natural function in the cell, however, is to defend against undesired foreign DNA, for example viral or bacterial DNA. Because of this natural function, which is based on the degradation of foreign DNA that has penetrated into the cell, some drastically reduced transformation frequencies have to be expected when working with recombinant DNA. This is referred to in terms of the presence of so-called restriction barriers [Matsushima et al (1987); Miller et al (1988) and McDonald & Burke, (1984)]. Even slight restriction barriers can be sufficient substantially to prevent the construction of a representative gene bank, since the restriction effect increases as the size of the recombinant plasmid increases. When there is restriction, therefore, fully intact genes can be isolated only with great difficulty. In the case of B. subtilis, for example, it has been shown that the restriction enzyme BsuM occurring in that strain [Uozumi et al (1977); Hoshino et al (1980) and Bron et al (1988)] is one of the main causes of the structural instability of recombinant plasmids in that organism [Haima etal (1987)].

Overcoming the restriction barriers brought about by the restriction enzymes of class II has therefore been, and continues to be, one of the main problems to be solved in molecular genetics. This can be achieved today in several ways.

One possible approach is to use non-restricting intermediate hosts, DNA of which can be transferred into the actual host without restriction. This approach is suitable, for example, for methylation-specific restriction systems. There may be used as the intermediate host, for example, a non-methylating mutant of E. coli or a naturally methylation-deficient strain [MacNeil (1988); European Patent No. 0 341 776 A2]. This approach is, however, rather complicated and has the additional disadvantage that these methylation-deficient E. coli strains are generally difficult to transform and are in no way ideal host strains.

In a further approach, heat-labile restriction enzymes are temporarily inactivated in vivo [Bailey & Winstanley (1986); Engel (1987)]. This approach is limited by the relative heat-sensitivities of the host and the restriction enzymes.

A further possible means of overcoming existing restriction barriers is the specific methylation of the DNA used. If the specific methylase for a certain restriction system is -3known and has been purified, the DNA can be specifically methylated in vitro before transformation and thus be protected against restriction (Vehmaanpera, 1988). In a situation where the corresponding methylase gene has been cloned, it is possible in principle for the DNA that is to be transformed to be methylated directly in vivo in a suitable host.

The solution that is probably the most satisfactory, and therefore the preferred solution, for the genetic engineer is, however, the isolation of mutants that do not synthesise the restriction enzymes responsible for the restriction barriers. Most of the E. coli K strains used as host strains carry, for example, a mutation in the gene for EcoK, an enzyme of class I. Since DNA of higher eukaryotes is generally strongly methylated, recently E. coli strains have been constructed that additionally carry mutations for the methylationspecific restriction systems mcrA, mcrB and miT [Kretz et al (1989)]. This approach is difficult in strains that have a relatively large number of restriction systems, for example Streptomyces fradiae [Matsushima etal (1987)].

Restriction barriers are ηοζ however, restricted to E.coli, but are to be found also in a large number of other microorganisms that are used for cloning experiments.

In B. thuringiensis and B. cereus, restriction/modification systems are very little known as yet, since it is only relatively recently that it has been possible to transform these Bacillus species efficiently (Schurter et al. 1989) and there has therefore been only a limited amount of interest in studying restriction/modification systems in those organisms. Accordingly, restriction barriers have hitherto been mentioned only in passing in connection with B. thuringiensis and B. cereus. Azizbekjan et al (1983), for example, describe an Avail isoschizomer of B. thuringiensis var. israelensis.

With the newly created means for the efficient transformation of B. thuringiensis and/or B. cereus and hence for the use of those organisms as cloning vehicles, however, these restriction barriers have become a problem also in the case of B. thuringiensis and B. cereus.

If, for example, a shuttle vector that is capable of replication both in B. thuringiensis and B. subtilis and in E. coli is used for the electroporation of B. thuringiensis strain HDlcryB [Stahly et al (1978)], a crystal-body-free derivative of B. thuringiensis var. kurstaki HD1, the transformation frequency achieved depends primarily on the strain from which the -4plasmid DNA was isolated. The absolute values obtained in each case may vary, but the relative frequencies remain substantially constant.

A representative example of these differences in the transformation frequencies is shown in Table 1. The results show that, for example, the transformation of the ’shuttle’ vector pHY300PLK is poorer by a factor of at least 103 when the plasmid is isolated from an E. coli host rather than a B. thuringiensis host. If the original strain is B. subtilis, the transformation frequency is reduced by a factor of approximately 10. If, on the other hand, the plasmid DNA is reisolated from the B. thuringiensis strain HDlcryB, it can be transformed back into HDlcryB at a high frequency.

This restriction effect undergoes a further increase if additional DNA is cloned into the shuttle vector, for example in the form of a protoxin gene. In this case the restriction barrier is found to have increased markedly again in the case of transformation into a B. thuringiensis host [see Tab. 4, pXI204, pXI93]. The reason for this is presumably that the DNA additionally integrated into the vector has further restriction cleavage sites, which represent an additional site of attack for the postulated host-specific [HDlcryB] enzymes. These restriction barriers can reach a value of approximately 104 - 105x, which makes the probability of a successful transformation of HDlcryB using these shuttle vectors very low.

In this case also the said plasmids can be transformed normally into HDlcryB only if they have previously been isolated from HDlcryB. Very often, however, it is desirable to clone DNA in a heterologous intermediate host, which, however, in many cases is incompatible or only slightly compatible with the restriction system present in B. thuringiensis and/or B. cereus. Transformation of B. thuringiensis and/or B. cereus with vector DNA that has previously been isolated from such a heterologous, non-compatible intermediate host then often leads to results that are not very satisfactory.

The problem that was to be solved within the context of the present invention was therefore primarily to identify and to characterise restriction barriers in B. thuringiensis and/or B. cereus and to develop methods of overcoming those barriers. This problem has now been solved within the context of this invention, surprisingly, by making available a partially restriction-deficient mutant of B. thuringiensis, as can be seen in detail from the following detailed description. -5The present invention therefore relates especially to a mutant of B. thuringiensis and/or of B. cereus that has a partial deficiency of the restriction barriers inherent in the unmutated starting strain and that may therefore be transformed significantly better than the said non-mutated starting strain when vector DNA from a heterologous intermediate host that is naturally subject to restriction in B. thuringiensis and/or B. cereus is used.

Within the context of this invention, a heterologous intermediate host is to be understood as being a host organism that is suitable for the cloning of vector DNA and that is not identical at least with the B. thuringiensis and/or B. cereus strain from which the DNA to be cloned was originally isolated.

The invention relates especially to a partially restriction-deficient mutant of B. thuringiensis and/or B. cereus which, when shuttle vectors from a non-compatible intermediate host, such as E. coli and/or B. subtilis, are used, may be transformed significantly better than the non-mutated starting strain, preferably by a factor of > 102 and especially preferably by a factor of 10^104.

Within the context of this invention there is to be understood by a non-compatible intermediate host a host organism that has not developed any mechanisms for effectively protecting its DNA against restriction after transformation into a B. thuringiensis or B. cereus strain having a restriction system disclosed within the context of the present invention.

An especially preferred embodiment of the present invention is a partially restriction-deficient mutant of the B. thuringiensis strain HDlcryB which, when shuttle vectors from E. coli and/or B. subtilis are used, exhibits a transformability that is better than that of the non-mutated starting strain by a factor of >102, and especially by a factor of 102-104. depending on the particular transformation vector used.

Special preference is given within the context of the present invention to the partially restriction-deficient mutant B. thuringiensis var. kurstaki HDlcryB Res9, which is obtainable by means of spontaneous mutation from the B.t. strain HDlcryB and can be obtained by means of enrichment methods known per se, and to mutants and variants thereof that are derived directly from that strain and that still have the distinguishing restriction-reducing characteristics of the starting strain. -6The present invention relates also to the methods of preparing the said restriction-deficient mutants.

Preference is given to a method wherein, essentially, spontaneous mutants having a reduced restriction barrier are enriched by means of a series of several transformation and selection cycles, there preferably being used for the transformation shuttle vectors that code for different selection markers and that guarantee a sufficiently high rate of transformation, preferably a transformation rate of from 106 to 108 transformants, and those mutants are selected that, in addition to having a reduced restriction barrier, still exhibit a high degree of transformation efficiency.

The invention relates further to methods of reducing restriction barriers in B. thuringiensis and/or B. cereus using restriction-negative mutants, especially using the restrictionnegative mutant B. thuringiensis HDlcryB Res9.

The invention relates also to a method of reducing restriction barriers in B. thuringiensis and/or B. cereus, wherein a restriction-negative mutant, especially the restriction-negative mutant B. thuringiensis HDlcryB Res9, is used in combination with a specific methylase which, by methylating the inserted DNA, protects the latter from being digested by restriction enzymes inherent in B.thuringiensis and/or B. cereus and thus further increases the efficiency of the method.

As defined within the context of the present invention, a restriction-negative or restriction-deficient mutant is one that may be better transformed, by a factor of > 102, by a shuttle vector that has been isolated from E. coli than the unmutated starting strain. However, spontaneous mutations generally occur very rarely (106-108) in the case of B. thuringiensis and B. cereus, so that these rare mutants first have to be enriched by one of the known enriching methods. This can be achieved, preferably, by passing through several, preferably from 1 to 8, cycles consisting of transformation and selection, the most promising transformants being selected at the end of each cycle and used again in the next transformation/selection cycle. Once one of these rare mutants has been transformed, enrichment by a factor of 102 - 103 can be expected in each new cycle consisting of transformation/selection, that is to say, after only a few cycles the cell population should comprise a majority of mutated cells.

Since spontaneous mutants are rare (10'6 - 108), and therefore the probability that one of -7those mutants will be transformed is correspondingly low, the transformation conditions must be so selected that high rates of transformation, preferably transformation rates of at least 106-108, are achieved. This applies especially to the first two transformation cycles. These positive transformants can then be enriched, identified and finally isolated in further transformation/selection cycles.

The particular difficulty that the isolation of restriction-deficient mutants entails is avoiding the occurrence of undesired mutants.

Thus, for example, the enrichment of spontaneous, generally chromosomally coded resistance mutants can be avoided or at least kept as low as possible by using in each new transformation/selection cycle preferably plasmids having different selection markers.

In order to enrich restriction-deficient mutants, therefore, it is preferable to use efficiently transformable vectors that allow a specific and selective choice of the positive transformants. Suitable vectors contain preferably one or more marker genes that impart to the host cell a characteristic by which the cells transformed with the vector can be easily identified and subsequently selected. Preference is given to marker genes that code for antibiotics resistance. Some examples of suitable resistance genes are especially those that code for the antibiotics ampicillin, chloramphenicol, erythromycin, tetracycline, hygromycin, G418, kanamycin, bleomycin, neomycin or thiostrepton.

Preference is given also to marker genes that code for enzymes for which a chromogenic substrate is available. Examples of such marker genes are the lacZ gene [chromogenic substrate: X-gal —* 5-bromo-4-chloro-3-indolyl-B-D-galactoside]; the xylE gene [chromogenic substrate: catechol]; or the luxAluxB operon which, with long-chained aldehydes as substrate [for example n-decanal], generates the emission of light.

The transformed colonies can then be detected very easily by means of a specific colour reaction.

Also suitable for use in the method according to the invention are genes that impart a resistance to heavy metals, such as mercury.

Special preference is given within the context of this invention to a series of mutually compatible shuttle vectors that code for different markers and preferably have a certain -8instability in B. thuringiensis and/or B. cereus. Owing to this instability, it is relatively simple later to prepare plasmid-free derivatives from potentially res friction-negative mutants.

Owing to their specific characteristic, these vectors can also be used more than once for transformation within the context of mutant enrichment.

A further precondition for finding suitable mutants is initially high transformation rates in order to ensure a high degree of probability that one of those rarely occurring mutants will be transformed at that same time. Special preference is given to initial transformation rates of at least 106-108 or more transformants.

The transformation of B. thuringiensis and/or B. cereus using shuttle vectors suitable for the selection of transformants is preferably carried out by means of electroporation, as described, for example, in Schurter et al, 1989 or in EP-A 342 633.

In a specific embodiment preferred within the context of this invention, the B. thuringiensis and/or B. cereus cells are first of all incubated in a suitable nutrient medium with adequate ventilation and at a suitable temperature, preferably at from 20°C to 35°C, until an optical density (OD550) of from 0.1 to 1.0 is reached.

The age of the Bacillus cultures provided for the electroporation has a marked influence on the transformation frequency. Special preference is therefore given to an optical density of the Bacillus cultures of from 0.1 to 0.3, especially of 0.2. It should be pointed out, however, that it is possible to achieve good transformation frequencies also with Bacillus cultures from other growth phases, especially with overnight cultures.

Fresh cells or spores are generally used as starting material, but deep-frozen cell material can equally well be used. The deep-frozen cell material used is preferably in the form of suspensions of B. thuringiensis and/or B. cereus cells in suitable liquid media, to which a certain amount of antifreezing agent is advantageously added. Suitable antifreezing agents are especially mixtures of osmotically active components and DMSO in water or a suitable buffer solution. Further suitable components that may be considered for use in solutions of antifreezing agents include sugars, polyhydric alcohols, such as glycerol, sugar alcohols, amino acids and polymers, such as polyethylene glycol. -9If B. thuringiensis spores are used as starting material they are first inoculated in a suitable medium and incubated overnight at a suitable temperature, preferably at from 25°C to 28°C and with adequate ventilation. This batch is then diluted and treated further in the manner described above.

In order to induce sporulation in B. thuringiensis, any medium that induces such sporulation can be used. Preference is given within the context of this invention to a GYS medium according to Yousten AA and Rogoff MH (1969).

The introduction of oxygen into the culture medium is generally effected by agitation of the cultures, for example using a shaking device, speeds of from 50 rpm to 300 rpm being preferred.

The culturing of B. thuringiensis spores and of vegetative microorganism cells within the context of the present invention is effected in accordance with known, generally customary methods,' the use of liquid nutrient media being preferred for practical reasons.

The composition of the nutrient media can vary slightly according to the strain of B. thuringiensis or B. cereus used. In general, complex media having poorly defined, readily assimilable carbon (C) and nitrogen (N) sources are preferred, such as are customarily used for culturing aerobic Bacillus species.

Apart from the LB medium preferably used within the context of the present invention, it is possible to use any other culture medium suitable for culturing B. thuringiensis and/or B. cereus, for example antibiotic medium 3, SCGY medium etc.. Sporulated B. thuringiensis cultures are preferably stored on GYS media (slant agar) at a temperature of 4°C. [The precise composition of the media referred to is given in the section Media and buffer solutions.] Once the cell culture has reached the desired cell density the cells are harvested by means of centrifugation and suspended in a suitable buffer solution that has preferably been cooled beforehand with ice. Especially suitable buffer solutions within the context of this invention are osmotically stabilised phosphate buffers that comprise as stabilising agent sugars, such as glucose or sucrose, or sugar alcohols, for example mannitol, and that have been adjusted to pH values of from 5.0 to 8.0. Special preference is given to phosphate buffers of the PBS type having a pH value of from 5.0 to 8.0, preferably from 5.5 to 6.5, - ίοthat contain sucrose as stabilising agent in a concentration of from 0.1M to 1.0M, preferably from 0.3M to 0.5M.

The incubation period for the Bacillus cells before and after electroporation is preferably from 0.1 to 30 minutes, especially 10 minutes. The temperature can be freely selected within a wide range. A temperature range of from 0°C to 35°C, preferably from 2°C to 15 °C, and most preferably a temperature of 4°C is preferred.

Aliquots of the suspended bacillus cells are then transferred to cuvettes or any other suitable vessels and incubated, together with the DNA to be transformed, for a suitable period, preferably for a period of from 0.1 to 30 minutes, especially from 5 to 15 minutes, and at a suitable temperature, preferably at a temperature of from 0°C to 35°C, especially at a temperature of from 2°C to 15°C and most preferably at a temperature of 4°C.

When working at low temperatures, it is advantageous to use precooled cuvettes or any other suitable precooled vessels.

The DNA concentration preferred forB. thuringiensis or B. cereus is in a range of from 1 ng to 20 pg. Especially preferred is a DNA concentration of from 10 ng to 2 pg.

The whole batch, comprising B. thuringiensis and/or B. cereus cells and the plasmid DNA to be transformed, is then introduced into an electroporation apparatus and subjected to electroporation, that is to say, exposed briefly to an electrical pulse.

Electroporation apparatuses that are suitable for use in the method according to the invention are now available from various manufacturers, such as Bio Rad (Richmond, CA, USA; ’Gene Pulser Apparatus’), Biotechnologies and Experimental Research, Inc. (San Diego, CA, USA; ’BTX Transfector 100’), Promega (Madison, WI, USA; ’X-Cell 2000 Electroporation System’), etc..

It is of course possible to use any other suitable apparatus in the method according to the invention.

The capacitance setting at the capacitor is advantageously from 1 pF to 250 pF, especially from 1 pF to 50 pF and most preferably 25 pF. The choice of starting voltage can be made freely within a wide range. Preference is given to a starting voltage Vo of from 0.2 kV to - 11 50 kV, especially from 0.2 kV to 2.5 kV and most preferably from 1.2 kV to 1.8 kV. The spacing of the electrode plates depends inter alia on the dimensions of the electroporation apparatus. It is advantageously from 0.1 cm to 1.0 cm, preferably from 0.2 cm to 1.0 cm. Especially preferred is a plate spacing of 0.4 cm. The spacing of the electrode plates and the starting voltage set at the capacitor produce the field strength values that act on the cell suspension. These values are advantageously in a range of from 100 V/cm to 50 000 V/cm. Especially preferred are field strengths of from 100 V/cm to 10 000 V/cm, especially from 3 000 V/cm to 4 500 V/cm.

The exponential decay time preferred within the context of the method according to the invention is from approximately 2 ms to approximately 50 ms, especially from approximately 8 ms to approximately 30 ms. Special preference is given to an exponential decay time of from approximately 14 ms to approximately 20 ms.

The fine adjustment of the freely selectable parameters, such as capacitance, starting voltage, plate spacing, etc., depends to a certain extent on the architecture of the apparatus used and can therefore vary from case to case, within certain limits. It is therefore possible in certain cases also to exceed or to fall below the limit values given, insofar as this might be necessary in order to achieve optimum field strength values.

The actual electroporation operation may be repeated once or several times, until an optimum transformation frequency for the particular system has been reached.

The electroporation can advantageously be followed by further incubation of the treated Bacillus cells, preferably for a period of from 0.1 to 30 minutes, at a temperature of from 0°C to 35°C, preferably from 2°C to 15°C. The electroporated cells are then diluted with a suitable medium and incubated again for a suitable period of time, preferably for from 2 to 3 hours, with adequate ventilation and at a suitable temperature, preferably at from 20°C to 35°C.

At the end of each new electroporation cycle with one of the vectors described in detail hereinbefore, the treated Bacillus thuringiensis and/or B. cereus cells are transferred to a selective medium and are incubated there at a temperature of from 10°C to 40°C, preferably at a temperature of from 20°C to 35°C and most preferably at a temperature of from 30°C to 33°C. The selective medium contains as selective substance preferably one of the above-mentioned antibiotics, depending on the vector used, and optionally a suitable solidifying agent, such as agar, agarose, gelatin, etc. - 12For further assessment of potentially restriction-negative mutants, first of all plasmid-free derivatives of the enriched mutants are prepared. For that purpose, individual colonies of the enrichment cultures obtainable from the most recent transformation/selection cycle in each case are selected and incubated on a suitable medium without selection.

The cultures obtainable in this manner are then diluted and plated out on suitable solid media, preferably on L agar, without selective markers. Suitable dilutions are then replicated on a suitable selective medium, preferably an L medium, that contains different selection markers in different concentrations. Special preference is given within the context of this invention to tetracycline, chloramphenicol and erythromycin in a concentration that inhibits the growth of sensitive cells but still allows the growth of the cells having the corresponding resistance plasmids. In this manner, it is relatively simple to find derivatives that exhibit no further growth in the presence of the specifically used selection markers.

In order to analyse the restriction barriers, the remaining isolates are then transformed using a suitable vector that may originate from E. coli on the one hand and from the unmutated starting strain of B. thuringiensis and/or B. cereus on the other. In this case also the transformation is preferably carried out as described above, via electroporation.

In the manner described hereinbefore, therefore, it is possible to select restriction-deficient mutants of B. thuringiensis and/or B. cereus that, when vector DNA from a heterologous intermediate host that is naturally subject to restriction in B. thuringiensis and/or B. cereus is used, may be transformed significandy better, preferably by a factor of > 102, than the said non-mutated starting strain.

Special preference is given to a partially restriction-deficient mutant of the B. thuringiensis strain HDlcryB which, when using shuttle vectors from a non-compatible intermediate host, such as E. coli and/or B. subtilis, has a transformability that is better by a factor of >102 than that of the non-mutated starting strain.

Special preference is given within the context of the present invention to the partially restriction-deficient mutant B. thuringiensis var. kurstaki HDlcryB Res9, which is obtainable by means of spontaneous mutation from the B.t. strain HDlcryB and can be obtained by means of enrichment methods that are known per se, and to mutants and • 13variants thereof that are derived directly from that strain and that still have the distinguishing restriction-reducing characteristics of the starting strain.

The restriction-deficient mutants described hereinbefore are outstandingly suitable, owing to their improved transformability, for use as host strains for the cloning and optionally the expression of genes or other useful DNA sequences, especially of protoxin genes.

In detail, the procedure is preferably as follows; first of all (a) the said genes or DNA sequences are isolated from a suitable source or are synthesised; (b) the isolated or synthesised genes or DNA sequences are operably linked to expression signals that are capable of functioning in Bacillus thuringiensis and/or Bacillus cereus and that may be of homologous or heterologous origin in relation to the genes or DNA sequences used; (c) the chimaeric genetic construction according to section (b) is transformed using suitable vectors, including those that are naturally subject to restriction in B. thuringiensis and/or B. cereus, into a restriction-deficient mutant of Bacillus thuringiensis and/or Bacillus cereus; and (d) a corresponding gene product is optionally expressed and, if desired, isolated.

In order further to increase the efficiency of the method according to the invention, it is possible to include in the method an additional step wherein the vector DNA is incubated in vitro in a suitable reaction mixture, together with a specific methylase that is capable of methylating one or more bases within the recognition sequence of a host-specific restriction-endonuclease, and the methylated vector DNA is then transformed into a restriction-deficient mutant of B. thuringiensis and/or B. cereus.

Special preference is given within the context of this invention to a method for the cloning and optionally the expression of genes or other useful DNA sequences, in which method vectors that originate from E. coli or B. subtilis are used.

In addition to structural genes, it is of course also possible for any other useful DNA sequences to be used, such as non-coding DNA sequences that have a regulatory function. There may be mentioned at this point by way of example an ’anti-sense’ DNA that is transcribed into RNA, but is not translated into protein. - 14Using the restriction-deficient mutants it is, moreover, possible for the first time routinely to set up representative gene banks in B. thuringiensis and/or Bacillus cereus, the procedure preferably being as follows: first of all (a) the total DNA of Bacillus thuringiensis is disintegrated into fragments mechanically or, preferably, using suitable restriction enzymes; (b) fragments of suitable size are isolated; (c) the said fragments are inserted into a suitable vector, including those that are naturally subject to restriction in B. thuringiensis and/or B. cereus·, (d) restriction-deficient Bacillus thuringiensis and/or Bacillus cereus cells are transformed with the said vector; and (e) there are selected from the transformants, using suitable screening methods, those that comprise the novel and desired DNA sequences.

In this case also the efficiency of the transformation can be increased further by means of additional methylation of the DNA to be inserted.

Special preference is given within the context of the present invention to a method wherein the said Bacillus thuringiensis is a strain that has a restriction/modification system comparable to that of the Bacillus thuringiensis strain HDlcryB.

This invention relates further to methods of reducing restriction barriers in B. thuringiensis and/or B. cereus using restriction-negative mutants, especially using the restrictionnegative mutant B. thuringiensis HDlcryB Res9.

A further increase in the efficiency of this method by repeating the reduction of the restriction barriers can be achieved by using a restriction-negative mutant, especially the restriction-negative mutant B. thuringiensis HDlcryB Res9, in combination with a specific methylase that, by methylating the inserted vector DNA, protects the latter from being digested by restriction enzymes inherent in B. thuringiensis and/or B. cereus and thus further increases the efficiency of the method.

In detail, it is possible to proceed as follows: the vector DNA, together with a specific methylase that is capable of methylating one or more bases within the recognition sequence of a host-specific restriction endonuclease, is incubated in vitro in a suitable -15reaction mixture and the methylated vector DNA is then transformed into a restrictiondeficient mutant of B. thuringiensis and/or B. cereus, especially into the restrictiondeficient mutant B. thuringiensis HDICryB Res9.

An example of such a methylase, which is in no way to be regarded as limiting, is the enzyme M-FnuDII, which methylates specifically the first cytosine within the recognition sequence of the restriction enzyme FnuDII or of its isoschizomers, such as BthKI [*CGCG] and therefore protects the vector DNA treated with the said methylase from being digested by that enzyme. In this manner it is possible significantly to reduce or completely to eliminate residual activity remaining after the exclusion of the Dam-specific restriction, which residual activity is nevertheless still responsible for reducing the transformation frequency of unmodified DNA by a factor of from 10 to 50.

In order to illustrate the rather general description and to provide a better understanding of the present invention, reference is made below to specific examples which are not, however, of a limiting nature, unless there is a specific indication to the contrary. The same applies to all lists given by way of example in the foregoing description. - 16NON-LIMITING EXAMPLES Example 1: Isolation of a restriction-negative mutant of the strain HDlcryB 1.1 Shuttle vectors used for the selection of mutants For the isolation of a restriction-deficient mutant of the Bacillus thuringiensis strain HDlcryB [Stahly DP et al (1978)], the following E. coli/B. thuringiensis shuttle vectors, all isolated from E. coli, are used, in the following order: Vector selection origin/reference (in B .thuringiensis) 1. pAM401 Cm 2. pHY300PLK Tc 3. pAM401 Cm 4. pHP13 Em, Cm Wirth ^raZ, 1986 Toyobo Co., Osaka, 530 Japan, Order No. PHY-001 Wirth et al, 1986 Bacillus Genetic Stock Center, Ohio State Univ., Columbus, Ohio 43210, USA, Strain No. 1P50 Since the plasmid pAM401 is extremely unstable and without selection is rapidly lost again, it can be used for transformation more than once. Since the other shuttle vectors also exhibit a certain instability in B. thuringiensis, plasmid-free derivatives can later be obtained relatively readily from potentially restriction-negative mutants. 1.2 Transformation of Bacillus thuringiensis HDlcryB The transformation of Bacillus thuringiensis using the shuttle vectors listed under 1.1 is carried out by means of electroporation. 1.2.1 Standard protocol for the transformation of R thuringiensis HDlcryB via electroporation ml of an LB medium (tryptone 10 g/1, yeast extract 5 g/1, NaCl 5 g/1) are inoculated with spores of B. thuringiensis var. kurstaki HDlcryB [Stahly DP et al (1978)], a - 17 plasmid-free variant of B. thuringiensis var. kurstaki HD1.

This batch is incubated overnight at a temperature of 27°C on a rotary shaker at 50 rpm. The B. thuringiensis culture is then diluted 100-fold in from 100 ml to 400 ml of LB medium and cultured further at a temperature of 30°C on a rotary shaker at 250 rpm until an optical density (OD550) of 0.2 has been achieved.

The cells are harvested by means of centrifugation and suspended in 1/40 volume of an ice-cooled PBS buffer (400 mM sucrose, 1 mM MgCl2, 7 mM phosphate buffer pH 6.0). The centrifugation and subsequent suspension of the harvested B. thuringiensis cells in PBS buffer is repeated once.

The cells thus pretreated can then be either electroporated directly or stored, after the addition of glycerol to the buffer solution [20% (w/v)], at from -20°C to -70°C and used at a later date. 400 μΐ aliquots of the ice-cooled cells are then transferred into precooled cuvettes and plasmid DNA is then added in a suitable concentration and the whole batch is incubated for from 0.1 to 10 minutes at 4°C.

When using deep-frozen cell material, first of all a suitable aliquot of frozen cells is thawed in ice or at room temperature. The subsequent treatment is effected analogously to the procedure for fresh cell material.

The cuvette is then introduced into an electroporation apparatus, where the B. thuringiensis cells present in the suspension undergo electroporation by being subjected, in a single discharge from a capacitor, to voltages of from 0.1 kV to 2.5 kV. A voltage of 1.3 kV is preferred.

The capacitor used in this case has a capacitance of 25 pF, the cuvettes have an electrode spacing of 0.4 cm, which, in the case of a discharge, depending on the setting, leads to an exponentially decreasing field strength having initial peak values of from 0.25 kV/cm to 6.25 kV/cm, especially of 3.25 kV/cm. The exponential decay time is in a range of from 10 ms to 20 ms.

For the electroporation experiments described, for example an electroporation apparatus -18supplied by Bio Rad may be used (’Gene Pulser Apparatus’, No. 165-2075, Bio Rad, 1414 Harbour Way South, Richmond, CA 94804, USA).

Of course any other suitable apparatus can be used in the electroporation method described hereinbefore.

After a further period of incubation of from 0.1 - 10 minutes at 4°C, the cell suspension is diluted with 1.6 ml of LB medium and incubated for 2 hours at a temperature of 30°C on a rotary shaker at 250 rpm.

Suitable dilutions are then plated out on L agar (LB medium solidified with agar, 15 g/1) that contains as additive an antibiotic suitable for the selection of the newly obtained plasmid. In the case of pHY300PLK, this is the antibiotic tetracycline, which is added to the medium in a concentration of 20 mg/1.

Bacillus cereus cells can be transformed in the same way as B. thuringiensis cells in accordance with the above protocol. 1.2.2 Specific modifications within the individual transformation/selection cycles 1st electroporation: The Bacillus thuringiensis strain HDlcryB is cultured in accordance with the standard protocol [see Section 1.2.1] and concentrated 500-fold in order to achieve high transformation frequencies. [Detailed information on the electroporation of Bacillus thuringiensis cells can be found in European Patent Application EP-A 0 342 633].

After electroporation of the Bacillus thuringiensis HDlcryB cells with 5 gg of pAM401 plasmid DNA, the cells are diluted 5-fold in LB medium and incubated for 2 hours at 250 rpm and 30°C. The transformants are then selected using 20 gg/ml of chloramphenicol for 4 hours at 250 rpm and 30°C and are diluted 200-fold in LB medium for the next electroporation. 2nd electroporation: After 2 hours’ incubation at 150 rpm and 30°C in accordance with the standard protocol [see Section 1.2.1], the cells are washed and concentrated 400-fold.

After electroporation with 12 gg of pHY300PLK plasmid DNA, the cells are diluted -fold in LB medium and incubated for 1.5 hours at 250 rpm and 30°C. After a further -fold dilution, the transformants are selected overnight using 20 gg/ml of tetracycline at -19150 rpm and 30°C and are diluted 200-fold in LB medium for the next electroporation. 3rd electroporation: After incubation for 1.5 hours at 250 rpm and 30°C in accordance with the protocol, the cells are washed and concentrated 100-fold. After electroporation with 5 pg of pAM401 plasmid DNA, the cells, as described for the second electroporation, are diluted in LB, incubated and diluted again with LB and the transformants are selected using 10 pg of chloramphenicol for 2.5 hours at 250 rpm and 30°C. The culture thus selected is then stored at room temperature. 4th electroporation: The above culture is diluted 50-fold and in accordance with the standard protocol incubated overnight and then cultured further as a sub-culture, washed and concentrated. After electroporation with 5 pg of pHP13, the cells are diluted 5-fold and incubated for a period of 3.5 hours at 250 rpm and 30°C. The cells are then diluted again and plated out on L agar with 200 pg/ml of erythromycin.

The 3rd and 4th electroporations already exhibit markedly increased transformation frequencies, which is a first indication of the enrichment of restriction-deficient mutants. 1.3 Preparation of plasmid-free derivatives For further assessment of potentially restriction-deficient mutants first of all plasmid-free derivatives are prepared. For that purpose 10 individual colonies are picked from the above selection plates [after the 4th electroporation, see Section 1.2.2] and incubated in 10 ml of LB medium overnight at 50 rpm and 27 °C without selection.

The cultures are then diluted and plated out on L agar without selective markers. Suitable dilutions of these cultures are then replicated on L agar plates each containing one of the following selection markers: tetracycline [20 pg/ml], chloramphenicol [10 pg/ml] or erythromycin [200 pg/ml].

In this manner, of the original 10 colonies, it is relatively easy to find derivatives of 6 that exhibit no further growth in the presence of all three selection markers. In none of those derivatives can any of the previously inserted plasmids be detected. 1.4 Transformation with the shuttle vector pHY300PLK In order to analyse the restriction barriers, the remaining 6 isolates are transformed with pHY300PLK which originates on the one hand from E. coli and on the other from -20B. thuringiensis HDlcryB. The transformation is carried out by means of electroporation [see Example 1.2.1].

The results of Table 3 show that 4 of the 6 isolates tested exhibit markedly reduced restriction barriers as compared with the parent strain HDlcryB. Of those mutants, designated Res5, 7, 8 and 9, Res5, 7 and 9 have a restriction barrier that is approximately 50-100x smaller than that of HDlcryB. Res5 and 7, however, seen in absolute terms, are 10 times less transformable than HDlcryB. Although Res8 exhibits an extremely low restriction barrier, seen in absolute terms its transformability is approximately 500x poorer than that of HDlcryB, which cancels out the advantage of the reduced restriction barrier.

Only the restriction-negative mutant Res9 combines the advantages of a high degree of transformation efficiency with those of reduced restriction barriers.

The advantages of the mutant Res9 become even clearer in the case of transformation with recombinant plasmids. It is clear from Table 4 that shuttle vectors isolated from E. coli that contain a protoxin gene [pXI204, pXI93], can be transformed into B. thuringiensis HDlcryB only at a very low frequency, whereas the mutant Res9 allows efficient transformation using the same plasmids. 1.5 Taxonomic characterisation of the Bacillus thuringiensis strains used 1.5.1 Strain HDlcryB Identification of strain DSM 4574 Characteristics of the strain Bacillus Width /pm Length /pm 1.0-1.2 3.0-5.0 Mobility Spores ellipsoid + round - swollen sporangium - Gram reaction + Catalase + Anaerobic growth + VP reaction + pH in VP medium 4.9 Maximum temperature Growth positive at °C 45 Growth negative at °C 50 Growth in Medium pH 5.7 + NaCl 5 % + 7% - 10% - Acid from glucose + L-arabinose - xylose - mannitol - Gas from glucose - Lecithinase - Hydrolysis of starch + -22gelatin + casein + Utilisation of citrate + propionate Degradation of tyrosine NO2 from NO3 + indole phenylalanine desaminase arginine dihydrolase + unusual characteristic: no lecithinase activity 1.5.2 Strain HDlcryB Res9 Identification of strain DSM 5854 Characteristics of the strain Bacillus Width /pm 1.0-1.2 Length /pm 3.0-5.0 Mobility + Spores + ellipsoid + round - -23swollen sporangium Gram reaction + Catalase + Anaerobic growth + VP reaction + pH in VP medium 4.7 Maximum temperature Growth positive at °C 45 Growth negative at °C 50 Growth in Medium pH 5.7 + NaCl 5 % + 7 % - 10% - Acid from glucose + L-arabinose - xylose - mannitol - Gas from glucose - Lecithinase - Hydrolysis of starch + gelatin + casein + -24Utilisation of citrate + propionate Degradation of tyrosine NO2 from NO3 + indole phenylalanine desaminase arginine dihydrolase + unusual characteristic: no lecithinase activity Example 2: Characterisation of the restriction barriers of the strains HDlcryB and Res9 2.1 Restriction enzyme BthKl In crude extracts of the strain HDlcryB it is impossible to detect sequence-specific restriction enzymes, since the test DNA is totally degraded by non-specific nucleases. These interfering nucleases can to a large extent be separated off in a simple manner by means of a dextran/polyethylene glycol phase distribution [Schleif (1980)]. Depending on the characteristics of the nucleases and restriction endonucleases that are present, ionic strengths can be found at which the different enzymes prefer different phases. In the case of HDlcryB the non-specific nucleases can be removed at all ionic strengths. At NaCl concentrations lower than 10 mM or higher than 250 mM, a sequence-specific nuclease can be detected in the aqueous phase.

Further purification and separation of the restriction enzymes present in the strain HDlciyB have been carried out as follows by means of affinity chromatography on a heparin column [Bickle (1977)]. The cells are harvested from 5 litres of culture by means -25of centrifugation and disintegrated in a cell-disintegration apparatus [for example a French Pressure Cell] and the cell detritus is separated off by centrifugation. Nucleic acids are separated off by precipitation with polyethyleneimine (Pirrotta, 1980) and the proteins are precipitated with (NH^SC^ at 70 % saturation and taken up in a small volume of buffer. The proteins are then loaded onto a heparin column and eluted with an NaCl gradient [0M-1M]. Aliquots of the eluted fractions are then tested for the presence of restriction enzymes [Bickle (1977)]. The substrate used is pHY300PLK, a plasmid that is known to be subject to restriction and that is isolated from E. coli. In spite of incomplete separation of the non-specific nucleases, a sequence-specific restriction activity that generates a defined pattern of DNA fragments has been demonstrated. The restriction activity elutes at approximately 0.6M - 0.8M.

The enriched enzyme cleaves the plasmid pHY300PLK if it has previously been isolated from E. coli otB. subtilis, but not if it has been isolated from HDlcryB. Characteristically, therefore, in this case also the producer of the restriction enzyme is protected against digestion of its own DNA, probably as a result of sequence-specific methylation.

The characteristics documented above thus all indicate a restriction enzyme of class II. In accordance with the accepted nomenclature [Szybalski et al (1988)], the enzyme isolated from B. thuringiensis HDlcryB receives the designation BthKl.

The present invention relates also to this restriction enzyme BthKl. 2.1.1 Specific recognition sequence of BthKl The specific recognition and cleavage sequence of the restriction enzyme isolated from HDlcryB can be determined as follows.

The DNA used as substrate is generally cut into many small fragments, which leads to the assumption that it is an enzyme the recognition sequence of which consists of only four nucleotides. Furthermore, DNA having an average to high GC content is cleaved frequently, DNA having a low GC content is cleaved rather less frequently, which in turn leads to the conclusion that the recognition sequence is rich in GC. In an enzyme that recognises a four sequence, this indicates that the sequence consists exclusively of GC. Using as substrate DNA of plasmids pC194 [Horinuchi and Weisblum (1982)] and pBC16 [Bernhard et al (1978)] isolated from B. subtilis and by means of a comparison with commercially available restriction enzymes, it has been shown unequivocally that the restriction enzyme BthKl is an isoschizomer of the commercially available enzymes Thai -26[GIBCO BRL, Gaithersburg, Maryland 20877, USA], Acc2 [Stratagene, La Jolla, CA 92037, USA], BstUl [New England Biolabs, Beverly, MA 01915-5510, USA] and Mvnl [Boehringer Mannheim, D-6800 Mannheim, FRG], that is to say, it recognises and cleaves the sequence CGCG if the latter has not been specifically methylated. The specific methylase M.BthKl postulated for the restriction enzyme BthKl provides protection not only against digestion by BthKl, but also against digestion by Thai or Acc2. Since the methylation of those two enzymes is known, it can be concluded that M.BthKl methylates at least the first C, and possibly even both Cs, in the sequence CGCG.

The restriction enzyme in the strain Res9 is analysed in the same manner as that described above for HDlcryB. The elution profile of a heparin/Sepharose affinity column in the case of Res9 is the same as that of HDlcryB, i.e. BthKl is still produced and is therefore not the cause of the reduced restriction barrier in the case of Res9. The mutation responsible therefor thus still remains unexplained. 2.2 Methylation-specific restriction Specific methylation of adenine or cytosine is effected in E. coli K by Dam or Dcm methylase. E. coli B strains are naturally dcm’. Restriction enzymes that recognise the same sequence as the Dam or the Dcm methylase, can, according to their type, be inhibited by this methylation. These restriction enzymes can therefore be used diagnostically to clarify the methylation status of a specific DNA. Using this method it is possible to show that both B. subtilis and B. thuringiensis exhibit neither Dam nor Dcm methylation.

Indications of the nature of the mutation in the strain Res9 can be obtained using DNA of the shuttle vector pHY300PLK isolated from strains having a different Dam/Dcm phenotype. Table 5 shows the transformation frequencies of that DNA into the strains HDlcryB and Res9. The Dam/Dcm phenotype of the DNA has in each case been carefully confirmed by digestion with the corresponding methylation-sensitive restriction enzymes.

The results from Table 5 can be summarised and interpreted as follows.

Dam methylation of the DNA is responsible for the majority of the restriction barriers observed in HDlcryB. Res9, on the other hand, is not influenced by the Dam phenotype and accepts both Dam-methylated and unmethylated DNA equally well. -27The Dcm methylation thus evidently has no influence on the transformability of B. thuringiensis HDlcryB. Since no isogenic strains were available, the relatively small, but reproducible, differences between the individual host strains cannot be explained by a single phenotype. There remains, however, the interesting observation that DNA from different host strains has different transformabilities.

By means of in vitro methylation with commercially available Dam methylase it is possible to confirm that Dam methylation is in fact the cause of some of the restriction barriers in HDlcryB (Tab. 6). In vitro methylation with three other methylases shows also that it is not a general methylation but specifically Dam methylation that causes the restriction barriers.

Example 3: Further reduction of the restriction barriers by means of specific methylation Of the two restriction systems present in the Bacillus thuringiensis strain HDlcryB, the main activity, which is based on a Dam-specific restriction, can be inactivated by a mutation in the derivative Res9. The residual activity that remains, which is still responsible for a reduction in the transformation frequency of unmodified DNA by a factor of 10-50 and is based on the restriction enzyme BthKI, can be significantly reduced by specific methylation with the methylase M-FnuDII. 3.1 Specific methylation of the shuttle vector pHY300PLK with M-FnuDII methylase The methylase M-FnuII supplied by New England Biolabs [No. 224S, New England Biolabs, 32 Tozer Road, Beverly, MA 01915-5599, USA] methylates specifically the sequence *CGCG and thus protects it against digestion with FnuDII and its isoschizomers, such as BthKI.

DNA of the shuttle vector pHY300PLK is isolated from Bacillus subtilis or E. coli and methylated in the following mixture: DNA pHY300PLK Tris-HCl [pH 7.5] EDTA β-mercaptoethanol S - adenosineme thionine M-FnuDII 0-3 Ug mM 10 mM 5 mM 0.08 mM 2 UN* -28*UN Methylation units. One unit corresponds to that amount of enzyme that is required fully to protect one pg of Lambda DNA [in one hour/37°C, in 10 pi of reaction volume] against digestion by FnuDIL The methylation is carried out over a period of one hour at a temperature of 37 °C. The methylation reaction is discontinued by inactivating the methylase. For that purpose the whole mixture is incubated once more, for 20 minutes at a temperature of 65°C. The DNA is then precipitated by the addition of ethanol and resuspended in 10 pi of TE buffer. Each mixture is reacted in triplicate, one aliquot in each case being used for the subsequent transformation, another for testing the plasmid DNA for the absence of damage. The latter test is carried out by means of agarose gel electrophoresis.

The effectiveness of the methylation is tested by digestion of the plasmid DNA with the FnuDII isoschizomer Thai. The results show that the DNA is protected against digestion by Thai, that is to say it is fully methylated, whereas the non-methylated controls are digested by the enzyme. 3.2 Combination of mutation and methylation The pHY300PLK plasmid DNA methylated hereinbefore in Section 3.1 is transformed in accordance with the protocol described in Example 1.2.1 into the restriction-deficient B. thuringiensis mutant Res9. The results are given in Table 7. The results show that a combination of the methylation of the plasmid DNA by Μ-FnuDII and the use of Res9 as host strain allows the restriction barriers that are to be found in the B. thuringiensis strain HDlcryB to be completely overcome. -29MEDIA AND BUFFERS LB medium [g/1] tryptone 10 yeast extract 5 NaCl 5 Lagar [g/1] LB medium solidified with AGAR 15 Antibiotic medium No. 3 (Difco Laboratories) [g/1] beef extract 1.5 yeast extract 1.5 peptone 5 glucose 1 NaCl 3.5 K2HPO4 3.68 KH2PO4 1.32 SCGY medium [g/1] casamino acids 1 yeast extract 0.1 glucose 5 K2HPO4 14 KH2PO4 6 sodium citrate 1 (NH4)2SO4 2 MgSO4 · 7 H2O 0.2 GYS medium (Yousten & Rogoff, 1969) [g/1] glucose 1 yeast extract 2 (NH4)2SO4 2 K2HPO4 0.5 -30MgSO4 · 7 H2O 0.2 CaCl2 · 2 H2O 0.08 MnSO4 · H2O 0.05 adjust pH to 7.3 before autoclaving.

PBS buffer [mM] sucrose 400 MgCl2 1 phosphate buffer, pH 6.0 7 TBST buffer [mM] Tween 20* * 0.05% (w/v) Tris/HCl* (pH 8.0) 10 NaCl 150 Buffer High [Maniatis et al (1982); page 104] [mM] NaCl 100 Tris/HCl* (pH 7.5) 50 MgCl2 10 dithiothreitol 1 Nick-translation buffer (lOx) [mM] Tris/HCl* (pH 7.2) 500 MgSO4 100 dithiothreitol 1 bovine serum albumin (BSA Pentax Fraction V) 500 gg/ml TE buffer [mM] Tris/HCl (pH 8.0) 10 EDTA 1 *T ween 20 polyethoxysorbitan laurate *Tris/HCl a,{X,tt-tris(hydroxymethyl)methylamino hydrochloride -31 DEPOSIT The microorganisms listed below, which are used within the context of the present invention, have been deposited with the ’Deutsche Sammlung von Mikroorganismen (German Collection of Microorganisms)’ in Brunswick, Federal Republic of Germany, a recognised international depository, in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Microorganism Date of deposit Deposit number Date of viability certificate Bacillus thuringiensis var. kurstaki HD1 4th March 1986 DSM 3667 5th March 1986 Bacillus thuringiensis var. kurstaki HDlcryB 4th May 1988 DSM 4574 4th May 1988 Bacillus thuringiensis var. kurstaki HD1 cryB Res9 21st March 1990 DSM 5854 21st March 1990 Bacillus thuringiensis var. kurstaki HDlcryB transf. with pK61 4th May 1988 DSM 4572 4th May 1988 Bacillus thuringiensis var. kurstaki HDlcryB transf. with pK93 4th May 1988 DSM 4571 4th May 1988 Since a B. thuringiensis can be distinguished from a B. cereus using the selected criteria only by its parasporal crystals, both the strain HDlcryB (DSM No. 4574) and the restriction-deficient strain Res9 (DSM No. 5854) can be classified as B. cereus.

This classification is possible because the strain HDlcryB is a plasmid-free, and therefore a crystal-free, derivative of HD1 (Stahly et al., 1978), which automatically results in its classification as B. cereus. Owing to the known differences between the three characterised strains, no taxonomic identity can therefore be expected. Since, however, the -32characteristics of the three strains shown in Section 1.5 do not conflict with their demonstrated related origin, the strains HDlcryB and Res9 continue to be described as B. thuringiensis within the context of the present invention. -33LITERATURE Asisbekjan, R. R. et al., Dokl. Akad. Nauk SSSR 274, 742-744,1984 Bailey, C. R. and Winstanley, D. J., J. Gen. Microbiol. 132, 2945-2947, 1986 Bernhard K et al., J. Bacteriol. 133, 897-903, 1978 Bickle, T. A. et al., Nucl. Acids Res. 4, 2561-2572, 1977 Bone, E. J. and Ellar, D. J., FEMS Microbiol. Letters 58,171-178, 1989 Bron., S. et al., Mol. Gen. Genet. 211,186-189,1988 Engel, P., Appl. Environm. Microbiol. 53, 1-3,1987 Haima, P. et al., Mol. Gen. Genet. 209, 335-342, 1987 Horinuchi and Weisblum, J. Bacteriol. 150, 815-825,1982 Hoshino, T. et al., Agric. Biol. Chem. 44, 621-623, 1980 Kessler, C. and Holtke, H.-J., Gene 47,1-153,1986 Kretz, P. L. et al., Nucl. Acids Research 17, 5409,1989 Lereclus, D. et al., FEMS Microbiol. Letters 60, 211-218, 1989 Macaluso, A., Abstr. Annu. Meet. Am. Soc. Microbiol., 309, 1989 MacNeil, D.J., J. Bacteriol. 170, 5607-5612,1988 MacNeil, D.J., Europ. Patent Appl., Case No. 8920 1140.4,1989 Matsushima, P. et al., Mol. Gen. Genet. 206, 393-400, 1987 MiUer, J. F. et al., Proc. Natl. Acad. Sci. USA 85, 856-860, 1988 Orzech, K. et al., J. Gen. Microbiol. 130, 203-208,1984 Pirrotta, V. and Bickle, T. A., in: Meth. Enzymol. 65, 89-95, 1980 Raleigh, E. A. and Wilson, G., Proc. Nad. Acad. Sci. USA 83,9070-9074,1986 Schleif, R., in: Meth. Enzymol. 65,19-23,1980 Schurter, W. et al., Molec. Genet. 218,177-181, 1989 Sladek, T. L. et al., J. Bacteriol. 165,219-225,1986 Stahly DP et al, Biochem. Biophys. Res. Comm., 84,581-588,1978 Szybalski, W. et al., Gene 74, 279-280,1988 Uozumi, T. et al., Mol. Gen. Genet 152, 65-69,1977 Vehmaanpera, J., FEMS, Microbiol. Letters 49, 101-105, 1988 Wirth R et al, J. Bacteriol. 165. 831-836,1986 -34TABLES Table 1. Restriction barriers in the transformation of Bacillus thuringiensis HDlcryB with shuttle vector pHY300PLK pHY300PLK isolated from: Transformation frequencies* into strain HDlcryB absolute relative B.thuringiensis HDlcryB 1.2 χ 107 1 E. coli HB101** 1.8 χ 103 1.5 χ Κλ4 B. subtilis ISW1214*** 7.6 χ 105 6.3 x 10'2 * for the relative value the frequency of the plasmid isolated from the strain HDlcryB is set at 1.

** [American Type Culture Collection (ATCC), Rockville, Maryland, USA, strain No. 33694] *** [Toyobo Co. LTD, Osaka, 530 Japan, Order No. PHY-001] Table 2. Shuttle vectors that have been used for the enrichment of restriction-deficient mutants of HDlcryB.

Vector Selection (in HDlcryB) gram* replicon 1. pAM401 2. pHY300PLK 3. pAM401 4. pHP13 Cm Tc Cm Em, Cm pIP501 pAMal pIP501 pTA1060 -35Table 3. Transformation behaviour of potential restriction-deficient derivatives of B. thuringiensis HD 1 cryB transformed strain Transformation frequencies* of pHY300PLK isolated from: B. thuring, HDlcryB E.coli BZ234 absolute relative absolute relative HDlcryB 9.5xl06 1 9.0x1ο3 9.5x1ο-4 Res5 1.3xl06 1 l.OxlO5 7.7xl02 Res7 1.5xl06 1 l.lxlO5 7.4xl0‘2 Res8 ό.ΟχΙΟ4 1 2.0x1ο4 3.3x1ο-1 Res9 l.lxlO7 1 7.0x10s 6.4xl0‘2 * for calculation of the relative value the frequency of the plasmid isolated from the strain HDlcryB is set at 1 Table 4. Characterisation of the restriction barriers of the strains HDlcryB and Res9.

I: Transformability of different recombinant plasmids isolated from E. coli.

Plasmid isolated from Transformation frequencies into the strains HDlcryB Res9 pXI204 * B.t. HDlcryB 1.3 χ 106 1.5 χ 106 pXI204 E.c. HB101 < 8 χ 101 6.4 χ 104 pXI93 ** B.t. HDlcryB 3.0 χ 105 8.3 χ 105 pXI93 E.c. HB101 4 1.3 χ 104 * described in EP-A 0 342 633; ** described in Schurter et al (1989) and EP-A 0 342 633; the internal reference pK entered in the deposit certificate issued by the DSM has now been amended to pXI in accordance with current international classification. -36Table 5. Characterisation of the restriction barriers of the strains HDlcryB and Res9. II: Influence of the host strain, especially its Dam/Dcm phenotype, on the transformability of the shuttle vector pHY300PLK.

Plasmid isolated froi phenotype relative* transformation frequency into B. thuringiensis, strain: HDlcryB Res9 i Dam Dcm B. th. HDlcryB . - 1 1 B. th. Res9 - - 1 1 B. subtilis ISW1214 - - 9.1xl0'2 8.9xl0'2 E. coli BZ103 +/- 1.3x1 O’2 0.7x1 O'2 E. coli 3225 - - 0.8xl0'2 0.4xl0'2 E. coli BL21 + . 2.4x10* 0.7x1 O’2 E. coli HB101 + + 8.2x10* 2.1xl0‘2 E. coli W3110 + + Ι.δχΙΟ-4 0.9xl0'2 * pHY300PLK isolated from HDlcryB or Res9 transformed with same frequency into strain HDlcryB and is set at 1. The transformation frequencies of the other isolates are given in relation to that value. The same applies also to transformations into strain Res9. - 37Table 6. Sequence-specific in vitro methylation of pHY300PLK-DNA isolated from the strain HDlcryB: Influence on the transformation behaviour of HDlcryB and Res9.

Methylase methylated sequence relative4- transformation frequencies into strain: HDlcryB Res9 _ _ _ _ _ _ 1 1 M.Hhal GC* GC 0.9 0.8 M.Hpa II CC* GG 0.4 0.6 M.Pst I CTGCA5G 0.7 0.7 Dam GA$TC 3.7xl0'3 0.8 + the transformation frequencies into strain HDlcryB and Res9 were set at 1. * 5-methylcytosine $ N6-methyladenine Table 7. Transformation of B.thuringiensis HD lcryBRes9: Effect of methylation of pHY300PLK-DNA by the methylase M-FnuDII Origin of the plasmid Treatment of the DNA relative transformation frequency B.t. HDlcryB 1 B.s. ISW1214 0.06 B.s. ISW1214 control 0.02 B.s. ISW1214 M-FnuDII 0.58 E.coli TGI* 0.0031 E.coli TGI* control 0.0029 E.coli TGI* M-FnuDII 0.32 * Component of in vitro Mutagenesis Kit No. RPN1523 made by AMERSHAM, Buckinghamshire HP7 9NA, England

Claims (30)

What is claimed is:

1. A mutant of B. thuringiensis and/or B. cereus, that has a partial deficiency within the restriction barriers inherent in the unmutated starting strain and that may therefore be transformed significantly better than the said non-mutated starting strain when vector DNA from a heterologous intermediate host that is naturally subject to restriction in B. thuringiensis and/or B. cereus is used.

2. A partially restriction-deficient mutant according to claim 1 wherein the said heterologous intermediate host is a non-compatible intermediate host.

3. A partially restriction-deficient mutant according to claim 2 wherein the said non-compatible intermediate host is E. coli or Bacillus subtilis.

4. A partially restriction-deficient mutant according to claim 1 wherein the said unmutated starting strain is the B. thuringiensis strain HDlcryB.

5. A partially restriction-deficient mutant according to claim 1 wherein the said mutant has a transformability that is better by a factor of > 10 2 than that of the non-mutated starting strain.

6. A partially restriction-deficient mutant according to claim 1 that has the following distinguishing taxonomic characteristics: Bacillus Width /pm Length /pm 1.0-1.2 3.0-5.0 Mobility + Spores + ellipsoid + round - swollen sporangium - Gram reaction + Catalase + Anaerobic growth + VP reaction + pH in VP medium 4.7 Maximum temperature Growth positive at °C 45 Growth negative at °C 50 Growth in Medium pH 5.7 + NaCl 5 % + 7% - 10% - Acid from glucose + L-arabinose - xylose - mannitol - Gas from glucose - Lecithinase - Hydrolysis of starch + gelatin + casein + -40Utilisation of citrate + propionate Degradation of tyrosine NO 2 from NO3 + indole phenylalanine desaminase arginine dihydrolase + unusual characteristic: no lecithinase activity or a mutant or a variant thereof that originates directly or indirectly from that strain and that still has the distinguishing restriction-reducing characteristics of the starting strain.

7. The restriction-deficient mutant B. thuringiensis var. kurstaki HDlcryB Res9, which has the distinguishing characteristics of DSM 5854, or a mutant or a variant thereof that originates directly or indirectly from that strain and that still has the distinguishing restriction-reducing characteristics of the starting strain.

8. A method of preparing a restriction-deficient mutant of B. thuringiensis and/or B. cereus, wherein spontaneous mutants having a reduced restriction barrier are enriched by means of a series of several transformation and selection cycles, there preferably being used for the transformation shutde vectors that code for different selection markers and that guarantee a sufficiently high rate of transformation and those mutants are selected that, in addition to having a reduced restriction barrier, still exhibit a high degree of transformation efficiency.

9. A method according to claim 8 wherein the transformations at the start of the enrichment yield at least from

10 6 to 10 8 transformants. -41 10. A method according to claim 8 wherein the transformation is carried out by means of electroporation.

11. A method according to claim 8 wherein efficiently transformable vectors that allow a specific and selective choice of positive transformants are used in the different transformation/selection cycles.

12. A method according to claim 8 wherein the said vectors comprise one or more marker genes that impart to the host cell a characteristic that allows cells transformed with the vector to be recognised and subsequently to be selected.

13. A method according to claim 12 wherein the said marker genes (a) code for antibiotics resistance; (b) code for an enzyme for which a chromogenic substrate is available; or (c) impart a resistance to heavy metals.

14. A method according to claim 8 wherein in the course of the selection of suitable mutants first of all plasmid-free descendants are produced and these are then transformed with a shuttle vector that is known to undergo restriction.

15. A method of cloning a gene or another DNA sequence in Bacillus thuringiensis and/or Bacillus cereus using a restriction-deficient mutant according to any one of claims 1 to 7, wherein (a) the said gene or DNA sequence is isolated from a suitable source or is synthesised; (b) the isolated or synthesised gene or DNA sequence is operably linked to expression signals that are capable of functioning in Bacillus thuringiensis and/or Bacillus cereus and that may be of homologous or heterologous origin in relation to the gene or DNA sequence used; (c) the chimaeric genetic construction according to Section (b) is transformed using a suitable vector into a restriction-deficient mutant of Bacillus thuringiensis and/or Bacillus cereus according to any one of claims 1 to 6; and (d) a corresponding gene product is optionally expressed and, if desired, isolated.

16. A method according to claim 15 wherein an additional step is included wherein the vector DNA is incubated in vitro in a suitable reaction mixture together with a specific -42methylase that is capable of methylating one or more bases within the recognition sequence of a host-specific restriction endonuclease and the methylated vector DNA is then transformed into a restriction-deficient mutant of B. thuringiensis and/or B. cereus.

17. A method according to either claim 15 or claim 16 wherein the said vector DNA is naturally subject to restriction in B. thuringiensis and/or B. cereus.

18. A method according to either claim 15 or claim 16 wherein the said vector originates from E. coli or B. subtilis or has at least been isolated therefrom.

19. A method of establishing gene banks in B. thuringiensis and/or B. cereus, wherein (a) the total DNA of Bacillus thuringiensis is disintegrated into fragments mechanically or with the aid of suitable restriction enzymes; (b) fragments of suitable size are isolated; (c) the said fragments are inserted into a suitable vector; (d) restriction-deficient Bacillus thuringiensis and/or Bacillus cereus cells are transformed with the said vector, and (e) there are selected from the transformants, using suitable screening methods, those that comprise novel and desired DNA sequences.

20. A method as claim in claim 19 wherein an additional step is included wherein the vector DNA is incubated in vitro in a suitable reaction mixture together with a specific methylase that is capable of methylating one or more bases within the recognition sequence of a host-specific restriction endonuclease and the methylated vector DNA is then transformed into a restriction-deficient mutant of B. thuringiensis and/or B. cereus.

21. A method according to either claim 19 or claim 20 wherein the said Bacillus thuringiensis is a strain that has a restriction-modification system comparable to that of the Bacillus thuringiensis strain HDlcryB.

22. A method of reducing restriction barriers in B. thuringiensis and/or B. cereus, wherein a restriction-deficient mutant of B. thuringiensis and/or B. cereus, especially the restriction-negative mutant B. thuringiensis HDlcryB Res9, is used.

23. A method according to claim 22 wherein a restriction-deficient mutant of -43B. thuringiensis and/or B. cereus, especially the restriction-negative mutant B. thuringiensis HDlcryB Res9, is used in combination with a specific methylase which, by methylating the inserted DNA, protects the latter from being digested by restriction enzymes inherent in B. thuringiensis and/or B. cereus and thus further increases the efficiency of the method.

24. A partially restriction-deficient mutant according to claim 1, substantially as hereinbefore described and exemplified.

25. A restriction-deficient mutant according to claim 7, substantially as hereinbefore described and exemplified.

26. A method according to claim 8 of preparing a restrictiondeficient mutant, substantially as hereinbefore described and exemplified.

27. A restriction-deficient mutant, whenever prepared by a method claimed in any one of claims 8-14 or 26.

28. A method according to claim 15 of cloning a gene or another DNA sequence, substantially as hereinbefore described

29. A method according to claim 19 of establishing a gene bank, substantially as hereinbefore described.

30. A method according to claim 22 of reducing restriction barriers, substantially as hereinbefore described and exemplified.