PHAGE RESISTANT ICE NUCLEATING PSEUDOMONAS SYRINGAE

Abstract: There are disclosed mutant Pseudomonas syringae strains that are resistant to bacteriophage lytic infection. The microorganisms retain the ice nucleating properties of the parent microorganism. The microorganism is therefore useful in the making of snow, ice islands and in other processes for the making of ice.

Description: PHAGE RESISTANT ICE NUCLEATING PSEUDOMONAS SYRINGAE Technical Field The present invention relates to microorganisms that are useful for ice nucleating. More particularly, the microorganisms are bacteriophage-resistant mutants of Pseudomonas svringae. Background In US Patent 4,200,228 there is disclosed a method for the making of snow whereby microorganisms are included in droplets that are sprayed into the air. The microorganisms that are used are of the type which are known to promote ice nucleation. As a result, snow can be made at temperatures that are much higher than are ordinarily possible. A typical microorganism that is useful in this method is a Pseudomonad and particularly Pseudomonas syringae. In US Patent 4,637,217 there is disclosed a method for accelerating the freezing of sea water. Ice nucleating microorganisms are added to the water source, in this case sea water. The sea water is then distributed, such as by spraying, to make large ice structures. These ice structures are useful for oil drilling platforms in the polar regions. In this application of the ice nucleating microorganisms, the conditions of spraying are adjusted to promote the formation of ice on the surface rather than snow in the air. In addition to spraying, the patent also discloses other methods of distributing the ice nucleated sea water. For example, an area that is surrounded by a dam can be flooded by the nucleated sea water and allowed to freeze. It is apparent that there is a need for methods for the production of large amounts of the ice nucleating microorganisms. To meet this need, Pseudomonas syringae is grown in large scale fermentors. The microorganism is then recovered and used in a dry form. Unfortunately, Pseudomonas svringae bacteria are susceptible to lytic bacteriophage infection. These bacteriophage are readily found in the natural environment. When a large scale fermentation run is infected with bacteriophage, the growing cells lyse and therefore do not reproduce. Often, the entire run must be discarded. Contamination of industrial environments with virulent bacteriophage thus represents a serious problem for efficient and economical production of ice nucleating Pseudomonas syrjngae- Resistance to phage infection is usually accomplished by changes to the bacteriophage attachment receptors on the cell surface or to alterations in the cell wall and membrane composition. However, it is known that the ice nucleating capability of Pseudomonas syringae is also associated with cell surface proteins. Thus, it was anticipated that phage-resistant derivatives of Pseudomonas syringae would have altered ice nucleating capabilities. Thus, the problem to be solved was to produce a phage—resistant Pseudomonas syringae that retained its ice nucleating capability. PisciQsuτe of the Invention In accordance with the present invention there is provided an ice nucleating Pseudomonas syringae that is resistant to bacteriophage selected from the group consisting of ATCC 40491, ATCC 40492 and ATCC 40493. The following deposits are pertinent to the present invention. All of the deposits were made according to the Budapest Treaty with the American Type Culture Collection in Rockville Maryland, USA. ATCC 535 . 43 23 Sept. 1986, P. syringae (parent) ATCC 53810 1 Sept. 1988, P. syringae (phage 1 ) ATCC 53811 1 Sept. 1988, P. syringae (phage 1 ) ATCC 40491 1 Sept. 1988, bacteriophage ATCC 40492 1 Sept. 1988, bacteriophage ATCC 40493 1 Sept. 1988, bacteriophage ATCC 40494 1 Sept. 1988, bacteriophage Ice nucleating, bacteriophage-resistant strains of Pseudomonas svringae are made by challenging the parent with the bacteriophage. The method is described in detail below. The strain is considered to be bacteriophage-resistant if the bacterial isolate has the ability to grow and multiply under conditions in which the multiplicity of infection is greater than or equal to 1.0. The multiplicity of infection is defined as the number of plaque forming units of phage lysate divided by the number of bacterial cells in the starting culture. Mutant phage-resistant strains are then screened for ice nucleating activity. Preferred phage-resistant strains have ice nucleating activity and growth that are comparable to the parent strain, e.g. ATCC 53543, when both are grown under the conditions described in European patent application 88103346.8, filed 4 March 1988. The currently preferred medium for the comparison described above contains: 90 g/L sucrose; 45 g/L monosodium glutamate; 4 g/L magnesium sulfate; 2.75 g/L potassium sulfate; .112 g/L iron sulfate; and .0024 g/L zinc sulfate. By comparable, we mean that the mutant is about the same as the parent or better. Once the phage-resistant strain is isolated, it can be grown in a conventional manner. Particularly preferred methods and media are described in European patent applications 87113771.7 and 87113772.5, filed 21 September 1987; 87118942.9, filed 21 December 1987 and 88103346.8, filed 4 March 1988. The ice nucleating microorganism is re¬ covered in a dry form. The microorganism from the fermentation can be prepared in dried form in a number of ways. Spray drying and freeze drying are typical examples. Any drying process will reduce the INA to a certain extent. One preferred method that preserves a large amount of the INA that is produced in the fermentor is the process that is described in United States Patent 4,706,463. In this process, the medium is cooled, concentrated, run into a cryo¬ genic liquid to form pellets and then the pellets are freeze dried at relatively low temperature. A process for spray drying ice nucleating microorganisms is described in European patent application 89103483.7, filed 28 February 1989. Methods; A. Growth of Bacterial Cultures Cultures of Pseudomonas syringae strain ATCC 53543 and all phage-resistant derivatives of this strain were grown in RK medium containing 80 g/1 mannitol, 20 g/1 yeast extract and 1 g/1 MgS0 4 «7H 2 0). Cultures (25ml) were incubated in 250 ml erlenmeyer flasks with aeration at 24 β C. B. Pescription of Bacteriophage Phage-resistant derivatives of Pseudomonas syringae strain ATCC 53543 were isolated following exposure to three bacteriophage types isolated from the fermentation environment. These bacteriophage have been designated as types ATCC 40491, ATCC 40492 and ATCC 40493. The phage differ on a molecular level as determined by restriction endonuclease digestion of their double—stranded DNA. The pattern of DNA fragments generated following restriction digestion indicates that phage types ATCC 40491 and ATCC 40493 are similar, however phage ATCC 40493 is capable of lytic infection on a phage ATCC 40491— resistant derivative of strain ATCC 53543. This observation indicates that minor differences in the genomes of these two bacteriophage isolates result in variations in host infectivity. Phage types ATCC 40492 and ATCC 40494 (a fourth isolate from the fermentation environment) differ greatly in molecular organization compared to phage types ATCC 40491 and ATCC 40493 based on restriction patterns. Southern hybridization of phage DNA from all four phage types 32 with P-labeled DNA probes from phage ATCC 40493 indicated a high degree of homology between phage types ATCC 40491, ATCC 40492 and ATCC 40493. No homology of phage ATCC40493 DNA to phage ATCC 40494 DNA was observed. C. Isolation of Bacteriophage Pseudomonas syringae bacteriophage were initially isolated from fermentation broths exhibiting a rapid loss of cell density during the fermentation process. Bacteriophage are separated from cells and cell debris by centrifugation at 10,000 RPM for 15 minutes at 4 β C. To further purify the phage particles, the phage lysate from the centrifugation step is filtered through a low protein binding 0.2 μm filter. Bacteriophage suspensions are stored as liquid stocks in sterile test tubes at 4 β C. The titer of phage particles is determined by plating 0.1 ml of serially diluted phage lysate suspensions (10 , 10 , 10 , 10 , and 10 —8) with 0.1 ml of an overnight phage-sensitive Pseudomonas syringae culture (e.g. strain ATCC 53543) onto Pseudomonas agar F plates containing 38 g/1 Pseudomonas agar F (Medium B from Gibco Laboratories USA) and 10 g/1 glycerol. Plates are incubated overnight at 24°C. Lytic infection is evidenced by the formation of plaques or circular clearing zones on a lawn of non—infected bacterial growth. Phage titer (the number of plaque forming units per ml of lysate) is determined by counting the plaques and computing the number of phage per ml in the original undiluted lysate. The appearance of 100 plaques on a o plate from the 10 dilution tube of lysate thus indicates a phage titer of 1.0 x 10 plaque forming units in the undiluted lysate. To increase phage titers, bacteriophage were added to an exponentially growing bacterial culture of strain ATCC 53543 at a multiplicity of infection of 1.0 (i.e., one plaque forming unit of phage lysate to every bacterial cell in the culture). Incubation was continued for 24 hours at 24 β C. Isolation of bacteriophage from the lysate and determination of phage titer were accomplished as described earlier. This process was continued until phage titers reached a concentration of 1.0 x 10 plaque forming units per ml. D. The INA (ice nucleating activity) is calculated using conventional techniques. The INA is determined by placing a plurality of microorganism containing water droplets (10 μl) on paraffin coated aluminum foil. The foil is maintained at -5 β C by placing it on a constant temperature bath. Details regarding this procedure are found in the literature, for example, Vali, Quantitative Evalua¬ tion of Experimental Results on the Heterogeneous Freezing of Supercooled Liquids, J. Atoms Sci., 28. 402-409 (1971). Examples 1 &.2: Isolation of Bacteriophage- Resistant Derivatives of ATCC 53543 Spontaneous phage-resistant derivatives of strain ATCC 53543 were isolated following exposure of the bacteria to phage types ATCC 40491,-ATCC 40492 o and ATCC 40493. Approximately 2.5 x 10 plaque forming units of each of the phage types and 1 x o 10 bacteria were spread onto a Pseudomonas agar F plate (previously described). The plates were incubated at 24°C for 24 to 36 hours. Colonies resistant to phage infection were picked from the cleared lawn of lysed phage-sensitive bacteria. Resistant colonies typically were observed after 36 hours of growth. Colonies resistant to infection by phage types ATCC 40491, ATCC 40492 and ATCC 40493 were screened in RK medium (previously described) for the ability to express at high levels the ice nucleation phenotype associated with the phage-sensitive ATCC 53543 parent. Ice nucleation assays were performed using the standard drop freezing method (described in Methods section D). All isolates were re-tested for bacteriophage ATCC 40491-, ATCC 40492-, ATCC 40493—resistance during this screening process. One of the isolates, designated as strain ATCC 53811 was chosen for further analysis based on its combined resistance to phage types ATCC 40491, ATCC 40492, and ATCC 40493, its fermentation performance and ability to express the ice nucleation phenotype. A second phage-resistant strain designated ATCC 53810 was isolated in a similar manner as described above except that a phage ATCC 40491-resistant strain was used as the starting strain. It also expressed the ice nucleating phenotype. Both phage-resistant microorganisms have been shown to be resistant to a fourth phage type (phage ATCC 40494) isolated from the local environment. It is important to note that resistance to this fourth bacteriophage was obtained without prior exposure of the microorganisms to phage ATCC 40494. Resistance of strains ATCC 53811 and ATCC 53810 to infection by other broad host range Pseudomonas bacteriophage has also been demonstrated. These data suggest that alterations occurring in the original ATCC 53543 strain following exposure to phage types ATCC 40491, ATCC 40492, and ATCC 40493 have resulted in infection resistance to a broad range of other Pseudomonas phage types in strains ATCC 53811 and ATCC 53810. The novel alterations to the cell surface responsible for the resistance to lytic infection by phage types previously unseen by these organisms has not negatively effected ice nucleation capabilities. Several other strains of Pseudomonas syringae were made in a similar manner. Each of the strains were phage-resistant and had ice nucleating activity. The previously mentioned ATCC 53810 and 53811 are preferred since they exhibit ice nucleating activity and growth that are comparable to the parent ATCC 53543 strain. Thus, the present invention provides Pseudomonas syringae which can be used in a fermentation environment which may contain lytic bacteriophage. In preferred embodiments, ice nucleating activity and growth are comparable to the parent strain. Industrial Applicability The present invention can be used in the large scale manufacture of ice nucleating microorganisms which can be used in making snow and ice.

Claims: Claim : 1. An ice nucleating Pseudomonas syringae that is resistant to bacteriophage selected from the group consisting of ATCC 40491, ATCC 40492 and ATCC 40493. 2. An ice nucleating Pseudomonas syringae according to claim 1 wherein the parent is Pseudomonas svringae ATCC 53543. 3. ATCC 53810. 4. ATCC 53811. 5. An ice nucleating Pseudomonas syringae according to claim 1 which is also resistant to ATCC 40494.

Inventors: LADUCA RICHARD JOSEPH, LIEVENSE LISA ROSEN, SWEETING KIMBERLY BLACK, LADUCA, RICHARD, JOSEPH, LIEVENSE, LISA, ROSEN, SWEETING, KIMBERLY, BLACK
Applicants: EASTMAN KODAK CO, EASTMAN KODAK COMPANY
Application year: 1989
Main classification: C12N1
IPC codes: C12N1