DE3116648A1 - Plasmids with a gene for cobalt resistance, microorganisms which contain such a plasmid transformed, process for the production of cobalt-resistant microorganisms and process for removing cobalt from cobalt-containing solutions - Google Patents

Abstract

The invention relates to plasmids with a gene which codes for cobalt resistance. These plasmids confer on the microorganisms into which they are transformed resistance to cobalt in a concentration of at least 4 nmol/l. The invention also embraces a process for the production of cobalt-resistant microorganisms by transformation of a plasmid with a gene which codes for cobalt resistance. Furthermore, the invention also relates to the use of the cobalt-resistant microorganisms for removing cobalt from cobalt-containing solutions.

Description

The invention relates to plasmids which contain a gene

th, which codes for cobalt resistance, and microorganisms, which are a contain such a plasmid transformed. The invention also provides a process for the production of cobalt-resistant microorganisms, a process for separating cobalt from solutions containing cobalt and the use of cobalt-resistant ones Microorganisms for the separation of cobalt from solutions containing cobalt.

In general, microorganisms, such as bacteria, are in an environment which contains certain heavy metals or their ions, is not viable. This means, that the microorganisms do not grow in solutions containing such heavy metals can, but die. One of the heavy metals that fight the microorganisms in are usually sensitive, cobalt is at a concentration of = 1.5 mmol / l.

In contrast to this common property of microorganisms was found in a certain bacterial strain of the genus Klebsiella (Aerobacter aerogenes; Proc.

Soc. exp. Biol.-Med. 80 (1952), pp. 626-632) a resistance to Cobalt found; see K.L. Williams et al.

P.C. Newell, Genetics 82, pp. 287-307 (1976). This cobalt-resistant Klebsiella strain is still viable and capable of reproduction at cobalt concentrations of 4 mmol / l. In the following, it is always referred to as the "resistant Klebsiella strain". So far is not known what indicates the resistance of these bacteria to the presence of Cobalt is due.

The resistant Klebsiella strain is in the German Collection of Microorganisms deposited under the deposit designation DSM 2081.

Resistance to substances such as antibiotics or heavy metals in their The presence of microorganisms that are normally not viable can be chromosomal Being nature, i.e. going back to the main DNA of the microorganism. But it can a so-called plasmid, which contains a gene, may also be responsible for it coded for the corresponding resistance.

Plasmids are subcellular, extrachromosomal, structurally independent DNA-containing carriers of hereditary information, for example the resistance to a certain antibiotic.

Plasmids come of different types and with different functions in addition to the chromosomal DNA in numerous bacteria. A plasmid that contains a gene which codes for resistance to cobalt is not yet known.

The resistant Klebsiella strain was found to contain cobalt, which is dissolved in the solution surrounding it, causes precipitation. The precipitation takes place in the form of cobalt phosphate (CoP04) from solutions with a pH value of approx 6.50 to 8.50. In this context, the term "cobalt" is always used ionized form of the metal in its bivalent or trivalent oxidation state to understand. The cause and the mechanism of the precipitation of the cobalt ions is so far not known.

The invention is based on the object of providing plasmids, that contain a gene that codes for cobalt resistance.

Another object of the invention is to remove microorganisms to provide which contain a plasmid with a gene transformed into them, that codes for cobalt resistance.

Another object of the invention is to provide a method for Create production of cobalt-resistant microorganisms.

Finally, another object of the invention is to provide one Process for separating cobalt from solutions containing cobalt, it is simpler and economically the separation of this metal from technical, cobalt-containing Solutions, for example wastewater from cobalt-processing plants or from alkalis enables cobalt extraction from ores.

These objects are achieved by the invention. The invention relates to the subject matter characterized in the claims.

With the present invention plasmids are provided for the first time, that contain a gene that codes for cobalt resistance. These plasmids were made from the resistant Klebsiella strain isolated and obtained. The plasmids can after transformed into any other microorganisms common in cell biology methods will. These microorganisms also become resistant to cobalt. Further the gene that is responsible for cobalt resistance can be obtained from the plasmids using conventional genetic engineering methods encoded, cut out and cloned into another plasmid, which then is also able to attack a microorganism into which it is transformed, To give resistance to cobalt.

The invention relates to both those from the resistant Klebsiella strain obtained plasmids with the gene for cobalt resistance, as well as those mentioned above "other plasmids" that contain the cobalt resistance gene cloned.

The latter are explained in detail below using examples.

From the cobalt-resistant strain of the genus Klebsiella, two were found separate plasmids are isolated, both individually representing the microorganisms in which they are transformed, can impart cobalt resistance. This means that each of the two plasmids contains a gene that codes for cobalt resistance.

One of the two plasmids (hereinafter referred to as "large plasmid") has a molecular weight of approximately 50 x 106 Daltons.

Various restriction enzymes give a proportionate digestion high number of interfaces and a corresponding number of fragments. The below The values given for the number of fragments are minimum values, i.e. the stated Number of fragments is determined after digestion with the appropriate restriction enzyme observed with certainty on gel electrophoresis. But this cannot be ruled out that one or a few fragments that only appear weakly in each case available.

The large plasmid gives on digestion with the following restriction enzymes following minimum number of fragments Eco RI 11 Hind III 15 Pst I 18 BAM HI o Sal I 11 The second plasmid (hereinafter referred to as "small plasmid") has a Molecular weight of about 3.6 x 106 Daltons. It helps with digestion the abovementioned restriction enzymes the following minimum number of fragments: Eco RI o Hind III O Pst I 2 BAM HI 1 Sal I O The above characterized Plasmids of the invention can be isolated from the resistant Klebsiella strain be won. This isolation of the plasmids can be carried out according to the methods used in cell biology customary methods for this purpose can be carried out. For example, a Culture solution - the bacteria are centrifuged to separate the cells from the solution. The cells are suspended in buffered sucrose solution and then with lysozyme broken up. Then EDTA is added to intercept the magnesium ions and then, after an incubation period a buffered Triton solution (non-ionic detergent). The mixture is then centrifuged again and the supernatant solution is until decanted onto the very viscous material. This supernatant is then with crystalline CsCl and ethidium bromide added. The mixture is then centrifuged again. Which vary under the influence of acceleration as a function of their molecular density heavily migrated bands become visible through exposure to long-wave UV light made. The band that contains the desired DNA, for example the plasmid sought, can be removed from the side of the tube using an injection needle.

When the above characterized plasmids of the invention in a Microorganism, for example transformed into bacteria, then the the microorganism in question is resistant to cobalt. This phenomenon is the Transformation of each of the two plasmids described above individually, as well as observed during the transformation of the two plasmids together.

By transforming at least one of the plasmids of the invention With the gene that codes for cobalt resistance, microorganisms can be very diverse Species can be made resistant to cobalt, for example bacteria of the genera E. coli, Pseudomonas and Bacillus, and yeasts.

Resistance to cobalt means that the microorganisms that contain the plasmid transformed into them, at a cobalt concentration of at least 4 mmol / l are still viable and capable of reproduction.

Transformation of the plasmids of the invention, one for cobalt resistance The coding gene contained in a microorganism can be found in cell biology customary methods for this purpose can be carried out. For example, the cell wall of the microorganism can be made permeable by adding a calcium salt. The plasmid is then added to the culture solution of the microorganism. It wanders then through the permeable cell wall into the microorganism cells.

The method described above for making cobalt-resistant Microorganisms by transforming at least one of those characterized above Plasmids, as well as the microorganisms that a plasmid transformed into them with a gene that codes for cobalt resistance are also included the invention.

It was found that the properties of the resistant Klebsiella strain Cobalt from solutions containing cobalt ions from coincidences, including microorganisms of the invention occurs into which at least one of the plasmids of the invention is transformed and which in turn were made resistant to cobalt.

The property of cobalt-resistant microorganisms, cobalt to precipitate out of the solution surrounding them opens up a further aspect of the Invention of a new way of separating cobalt from solutions containing cobalt. A cobalt-resistant microorganism (either the resistant strain of Klebsiella or a microorganism into which at least one of the plasmids of the invention is transformed was) is in introduced a solution containing cobalt ions. The cobalt ions then fall out of the solution in the form of cobalt phosphate and can be separated off, for example by filtration.

The invention therefore also relates to a method for separating of cobalt from solutions containing cobalt, which is characterized in that one a microorganism that contains a plasmid that codes for cobalt resistance, introduced into the solution, precipitating the cobalt, and the precipitated cobalt compound separates from the solution. Both the resistant Klebsiella strain, as well as another microorganism of the invention can be used in the at least one of the plasmids of the invention containing a gene for cobalt resistance encoded, transformed.

All solutions are to be understood as 'cobalt-containing solutions' from which cobalt extraction may be of technical interest. In particular Of course, solutions that contain cobalt only in low concentrations are possible -possibly in addition to other metals in larger quantities -contained, so that the Extraction using conventional methods is uneconomical. Examples of such Solutions are the wastewater from cobalt-processing plants, for example-der Glass industry, as well as solutions obtained by leaching ores that only have a low cobalt content.

In this method of the invention, the cobalt-resistant microorganism is used in the cobalt-containing solution, for example the cobalt-containing wastewater from a glass factory, brought in. The cobalt ions are released from the solution as trivalent cobalt phosphate selectively failed. The cobalt phosphate can be separated off, for example, by filtration and reconditioned for the extraction of the metal or put to another use will. The precipitation of the cobalt from the cobalt-containing solutions can take place at a pH value from about 3.50 to 8.50, preferably 6.50 to 7.50. The temperature is not particularly critical. Preferred is a temperature at which the used Microorganism has good growth conditions, i.e. a temperature of around 15 up to 400C, the cobalt concentration of the solution that makes up the cobalt through a cobalt-resistant The microorganism to be precipitated depends on the viability of the Microorganism. It can usually be at least about 4 mmol / l.

Within the method according to the invention for separating cobalt from solutions containing cobalt is for certain reasons discussed below the use of certain microorganisms are more preferred than the use from other microorganisms. For example, the use of the resistant Klebsiella strain economically not particularly favorable, since bacteria of the genus Klebsiella not by particularly excellent viability and growth ability in technical Identify solutions that contain heavy metal ions. Is preferred from this Reason the use of a microorganism whose good growth ability in such Solutions, for example in wastewater, is known. Specific examples of such Microorganisms are non-pathogenic strains of the genus Pseudomonas.

For this reason, an embodiment of the invention is therefore preferred Process for the separation of cobalt from solutions containing cobalt, in which an under microorganism suitable for the aforementioned point of view is used, transformed into at least one of the plasmids of the invention with the cobalt resistance gene became.

The inventive method for separating cobalt from cobalt-containing Solutions can be further developed in a further embodiment and under can be improved from the following point of view.

The two plasmids with the gene that codes for cobalt resistance, those isolated from the resistant Klebsiella strain come only in proportion low copy number in the microorganisms. The large plasmid has a copy number from about 2 t that the small one from about 4 to 5. As a result of these low copies number is the capacity of the microorganisms that contain these plasmids to precipitate of cobalt not particularly large. Although their use for separation. of cobalt possible from solutions containing cobalt, urid cobalt is thus obtained from solutions can be worked up to remove the cobalt using conventional methods is not worthwhile, from an economic point of view, a greater efficiency would be of the microorganisms in the separation of cobalt from cobalt-containing solutions is desirable.

The invention therefore also provides a method in which the Gene coding for cobalt resistance from the plasmids described above of the invention and cloned into another suitable plasmid, and this new plasmid obtained in this way into a suitable microorganism. transformed will.

In this embodiment of the invention, one of the plasmids that contain the gene coding for cobalt resistance, with a restriction enzyme in cut in a suitable manner and the fragment which has the desired gene, obtained using methods commonly used in genetic engineering. Then the fragment in cloned another suitable plasmid, again using the. in genetic engineering methods customary for this purpose can be used.

In this embodiment of the invention, the gene for cobalt resistance is derived encoded from one of the two plasmids of the invention derived from the resistant Xlebsiella strain were won. Because of its smaller size, it becomes practical establish preferably the small plasmid is used.

In one embodiment, the plasmid is in a digestion buffer dissolved and digested with Bam HI. This restriction enzyme cuts the small plasmid only in one place, i.e. the plasmid ring is opened and the plasmid receives a linear shape. Then the opened plasmid is in alloy buffer with a suitable alloyed with another plasmid, which was also digested with Bam HI. In another In the embodiment, the small plasmid can also be cut with Pst I. Included two fragments arise that are necessary for cloning into the desired other plasmid be used. In an analogous manner, the large plasmid can also be treated with suitable restriction enzymes are digested and the fragments obtained are cloned into another suitable plasmid will.

In the last two embodiments mentioned, in which a mixture is obtained from cloning products, these are converted into a suitable microorganism transformed.

The cobalt-resistant transformants are then used in gene technology usual procedures selected.

A plasmid is selected as "suitable other plasmid" which Has properties that make it particularly suitable for the determination according to the invention appear, i.e. that after transforming the new, cloned plasmid in a microorganism a high capacity of this microorganism to precipitate of cobalt is achieved from solutions containing cobalt. With that in mind in particular, the other plasmid should be a high copy number plasmid. this means that the plasmid is present in the microorganism cells into which it is transformed is available in large numbers of copies. A higher copy number of the plasmid leads namely to a higher capacity of the microorganism in the precipitation of the cobalt. this is an indication that the plasmi-d with the cobalt resistance gene plays a role at the exit precipitation of the cobalt from the cobalt-containing solutions plays.

A high copy number plasmid is, for example, the well-known pBR 322.

Another beneficial property of the other plasmid is that that it is itself transferable. This means that the plasmid in which the fragment is cloned with the gene coding for cobalt resistance, should be able to to migrate out of one cell and into another cell.

A specific example of a plasmid containing the above Has properties and thus for cloning the fragment with the cobalt resistance The gene generating the gene is particularly suitable, the plasmid RP 4 is also suitable are its derivatives; See M. Bagdasarian et al, Plasmids of Medical, Environmental and Commercial Importance, K.N. Timmis and A. Puhler, editors1 Elsevier / North-Holland Biomedical Press 1979.

In this preferred embodiment of the invention, then that "Another plasmid", which now contains the gene coding for cobalt resistance, into one transformed into a suitable microorganism. Here is under "suitable microorganism" in turn to understand a microorganism, which due to its properties for particularly suitable for the purpose sought in this embodiment of the invention is. Preferably used microorganisms should therefore, as already mentioned, in be able to work in the cobalt-containing technical solutions that make up the precipitation and extraction of cobalt is intended to be viable and capable of reproduction provided that they have at least one plasmid with one for cobalt resistance coding gene included. A specific example of microorganisms that this Meet requirements are non-pathogenic strains of the genus Pseudomonas.

These transformed microorganisms can be used in the above-described Way to be used for the separation of cobalt from cobalt-containing solutions.

The accompanying figure shows the results of gel electrophoresis the digestion products of mixtures of the large and small plasmids of the invention with various restriction enzymes. The application chutes shown on the figure contain from right to left: the first application chute an uncut sample; the upper band corresponds to the large one and the lower one, roughly in the middle of the The band in the figure corresponds to the small plasmid, in the supercoiled form.

The second and third jobs dig up the bands of digestive products with Eco RI; only the large plasmid is cut.

The fourth and fifth jobs get rid of the digestive product bands of T5-DNA, digested with Hind III or of M13-DNA, digested with Hae III as molecular weight markers.

The sixth and seventh order chutes the bands of digestive products with Hind III; only the large plasmid is cut.

The eighth and ninth jobs chute the bands of digestive products with Pst I; both the large and the small plasmid are cut.

The tenth hopper again uses the same molecular weight marker like the fourth tube.

The eleventh and twelfth jobs get rid of the gangs of digestive products with Bam HI; only the small plasmid is cut once.

The thirteenth and fourteenth orders clear the bands of digestive products with Sal I; only the large plasmid is cut.

The examples illustrate the invention.

Example 1 Isolation and recovery of the plasmids The resistant Klebsiella strain is in LB medium (Luria broth; composition: 5 g NaCl, 5 g yeast extract and 10 g tryptone in 1 liter of water). Then 100 ml of the cell culture are 5 minutes at a temperature of OOC and 5000 r.p.m. to separate the cells from the culture solution centrifuged. After separation, the cells are in 3 ml of a at room temperature 25 percent sucrose solution dissolved in 0.05 mol / l Tris / HCl buffer with a pH of 7.5. The solution obtained is then mixed with 0.3 ml of lysozyme solution (10 mg / ml in 0.25 mol / l Tris / HCl buffer with a pH of 7.5). The solution is now taking the occasional Incubated on ice for 5 minutes gently shaking. Then 1.2 ml 0.25 mol / l EDTA of pH 8.0 added and incubated again for 5 minutes on ice. After that, the Solution with 4.8 ml of Triton solution (nonionic detergent) added, the following Composition comprises: 10 ml 10% Triton X-100, 50 ml 0.25 mol / l EDTA of the pH value 8.0, 10 ml of 1 mol / l Tris / HCl buffer with a pH of 8.0 and 135 ml of water. The received Mixture is now 30 minutes at 17,000 r.p.m. centrifuged. The supernatant will decanted by pouring into a cylinder until it reaches the very viscous material is. This remains in the vessel. Then the supernatant solution is per ml with 1 g crystalline cesium chloride and 0.1 ml ethidium bromide solution (10 mg / ml) offset. The resulting mixture is again heated to 55,000 r.p.m. for 20 hours. centrifuged. Those in the cesium chloride gradient differ depending on their molecular density Migrated bands are created by irradiating the tube with long-wave UV light made visible. The lower band contains the supercoiled DNA. You can with a The injection needle can be removed from the side of the tube.

The removed fraction (band) is then three times with the same Volume of butanol saturated with water, gently shaken. The aqueous phase is then dialyzed against Tris / EDTA buffer of pH 6.7. Then will the solution once with the same volume of a buffered phenol solution (Tris / EDTA of pH 7.6) and then dialyzed again against the same buffer.

After these purification steps, the plasmids are obtained which are caused by Gel electrophoresis or can be separated in a sucrose gradient. Included 0.05 mg of the large and 0.02 mg of the small plasmid are obtained. The purity is> 95%.

Example 2 Transformation of the plasmids into E. coli The transformation is after a modification of the procedure of Cohen et al., Proc. Natl. Acad. Sci.

USA 69 (1972), pp. 2110-2114.

Transformable, calcium-treated cells are made by culture of E. coli in LB medium at 370C up to a cell density of 6 × 10 8 cells / ml (optical density 650 = 0; 6). The culture is 10 minutes at OOC and 8000 r.p.m. centrifuged. The cells are separated from the supernatant solution, Washed once with 0.5 volume of 0.01 mol / l saline solution and then in 0.5 volume suspended in a 0.03 mol / l calcium chloride solution. The resulting solution turns 20 Incubated minutes at OOC. Thereafter, the cells are centrifuged for 10 minutes at 8000 r.p.m. and a temperature of OOC from the solution and again suspended in 0.1 volume of 0.03 mol / l calcium chloride solution. This solution becomes the Transformation used.

The transformation mixture with a volume of 0.3 ml is through Mix 1.0 ßg DNA (plasmids) in 0.03 mol / l calcium chloride solution and 0.2 ml of transformable calcium treated cells are obtained. The mixture will Incubated for 60 minutes at OOC and then warmed to 420C for 5 minutes. Afterward the transformation mixture is diluted with 10 times the volume of LB medium and Incubated for 3 hours at 370C.

Afterwards the samples are selective on an agar medium with or without Pressure applied. The plates are incubated at 37 ° C for at least 18 hours. Cobalt-resistant E. coli cultures are obtained which are in a culture solution with a content of 4 mmol / l cobalt chloride are viable and capable of reproduction.

In a further experiment the procedure given above is used only the small plasmid separated from the large plasmid by gel electrophoresis was transformed into the transformable E. coli cells. There will be too Cultures are obtained which are normal for life and in the presence of 4 mmol / l cobalt chloride are capable of multiplying.

Example 3 Cloning of the BAM HI digest of the small plasmid in plasmid pBR 322 digested with BAM HI. The DNA of both the small plasmid and of the plasmid pBR 322 are reacted with the restriction enzyme under the following conditions Bam HI digests DNA 10 iil (DNA concentration: 0.1 μg / ml) 10-fold more concentrated Bam HI buffer * 5 Wl H2 0 33 Rl Bam HI enzyme 2 Al (activity: 0.5 U / l) * Bam HI buffer contains: Tris / HCl 10 mmol / l MgCl2 6 mmol / l NaCl 100 mmol / l B-mercaptoethanol 6 mmol / l bovine serum albumin 200 mmol / l pH value 7.5 0.5 μg each of the Bam HI digestion of the small plasmid and of pBR 322. are mixed with 0.5 U of T4 ligase in alloying buffer from 20 mmol / l Tris / HCl, 6 mmol / l.! gC12, 6 mmol / l ßi-mercaptoethanol and 1 mmol / l Adenosine triphosphate, pH 7.5, alloyed. The resulting mixture is 2 hours incubated at room temperature.

The transformation of the alloyed DNA into E. coli is carried out according to the S.N. Cohen op cit. The transformants obtained are selected in agar plates with a content of about 2.5 mmol / l Coli2.

The cobalt-resistant colonies are removed and stored at 40C.

Example 4 Separation of cobalt from a solution containing cobalt A solution is prepared which, in addition to the components of the LB medium, includes the following Metal salts contains: Com12 4 mmol / l FeCl2 1.5 mmol / l NiCl2 1.5 mmol / l in 1 1 of these Solution is at a temperature of 370C an E. coli transformant introduced and allowed to grow for 14 hours. The cell density amounts to then about 4 x 109 cells / ml.

During this time, pink cobalt phosphate separates out of the solution which is separated from the supernatant solution together with the cells. the Analysis of the clear supernatant shows that the cobalt content is below 0.2 mmol / l has sunk.

Claims (14)

"Plasmids with a gene for cobalt resistance, microorganisms that Such a plasm d contain transformed, a process for the production of cobalt-resistant Microorganisms and processes for separating cobalt from solutions containing cobalt P a t e n t a n s p r ii c h e 1. plasmid, d a d u r c h e k e n n n z e i c h n e t that it contains a gene that codes for cobalt resistance. 2. Plasmid according to claim 1, characterized by the following additional Properties: Molecular weight: about 50 x 10 6 Daltons minimum number of fragments after digestion with restriction enzymes: Eco RI 11 Hind III 15 Pst I 18 Bam HI O Sal I 11 3. Plasmid according to claim 1, characterized by the following Additional properties: Molecular weight: about 3.6 x 10 Daltons minimum number of the fragments after digestion with restriction enzymes: Eco RI o Hind III O Pst I 2 Bam HI 1 Sal I 0 4. Microorganism, characterized in that it has one in it Contains transformed plasmid with a gene coding for cobalt resistance. 5. Process for the production of cobalt-resistant microorganisms, characterized in that at least one plasmid according to claims 1 to 3 in. transforms the microorganism. 6. The method according to claim 5, characterized in that the gene coding for the cobalt resist z from one of the plasmids according to claim 1 to 3 cloned into another plasmid and the resulting plasmid into the microorganism transformed. 7. The method according to claim 6, characterized in that as another plasmid uses a high copy number plasmid. 8. The method according to claim 6 and 7, characterized in that one a self-transferable plasmid was used as the other plasmid. 9. The method according to claim 6 to 8, characterized in that one the plasmid RP4 or a derivative thereof is used. 10. The method according to claim 5 to 9, characterized in that one a microorganism is used for transformation, which is transformed into technical, metal ions containing solutions is viable and capable of multiplying. 11. The method according to claim 10, characterized in that one Microorganism of the genus Pseudomonas is used. 12. Process for separating cobalt from solutions containing cobalt, characterized in that a microorganism containing a plasmid is which codes for cobalt resistance, introduces it into the solution and the precipitated cobalt compound separates from the solution. 13. The method according to claim 12, characterized in that one Microorganism used, which according to the method according to any one of claims 5 to 11 was obtained. 14. Use of cobalt-resistant microorganisms for separation of cobalt from solutions containing cobalt.