NZ230039A

NZ230039A - Exopolysaccharides from pseudomonas paucimobilis, and the use of these as thickening agents and in agriculture

Info

Publication number: NZ230039A
Application number: NZ230039A
Authority: NZ
Inventors: Jacques Balandreau; Bernard Gueniot; Prakash Hebbar
Original assignee: Centre Nat Rech Scient
Priority date: 1988-07-25
Filing date: 1989-07-21
Publication date: 1991-04-26
Also published as: EP0353145A1; DK365289D0; ZA895493B; DK365289A; AU3884789A; FI893558A0; JPH02180902A; FR2634496A1; IL91026A0; BR8903664A; AU625278B2; NO893026L; FI893558A; PT91255A; NO893026D0; FR2634496B1; KR900001857A

Description

<div class="application article clearfix" id="description"> 23 0 0 39 Ptiority Date(s): . £r,v>'. 71.'.% Complete Specification Filed: Publication Date: ...APR jggj P.O. Journal, No: Patents Form No ■am)&*<■ NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION POLYSACCHARIDES, PROCESS FOR PREPARING THEM BY CULTURING PSEUDOMONAS PAUCIMOBILIS AND APPLICATION OF THE STRAINS IN AGRICULTURE i/We, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS)/ A French body corporate/ of 15 quai Anatole France/ 75007, Paris, FRANCE hereby declare the invention, for which ?/we pray that a patent may be granted to ir^/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - (followed by page la) jW' #**• IT C 25 ■<*!taswc. -la - The present invention relates to the preparation of new polysaccharides by Pseudomonas paucimobilis bacteria, as well as to the use of these bacteria in agriculture. 5 The present invention is based on the demonstra tion of especially advantageous properties of P. paucimobilis bacteria, in particular as polysaccharide-produc-ing organisms, but also as agents exhibiting substantial nitrogenase activity. 10 P. paucimobilis, especially strain RMDP17, produces a capsule polysaccharide (attached to the cell) and a non-capsule polysaccharide (released into the medium) in large amounts (1 to 2 g/1 of dry matter). This polysaccharide can optionally be used to 15 replace xanthan as a thickening agent in its many applications, in agri-foodstuffs, pharmacy and cosmetics in particular, in the light of the very special rheo-logical behaviour of the polysaccharide according to the invention, namely, in particular, that its viscosity is 20 rather insensitive to the ionic strength of the medium and to temperature. The present invention relates, in the first place, to an exopolysaccharide produced by P. paucimobilis . the saccharide chain of which is as follows: fi Glc 1 —3 Beta 1 Glc * 6 Alpha 1 Glc —4 Rha l| L f 6 n Beta 1 Glc The polysaccharide is a neutral compound. The polysaccharide is lightly acetylated but does not contain a pyruvyl or succinyl radical and generally contains less than 10 % of dry matter weight of uronic acid. Other characteristics of the polysaccharide according to the invention will be studied in greater detail in the examples. The present invention also relates to a process for preparing this polysaccharide containing less than 10 % amount of uronic acid, characterized in that a strain identified as P. paucimobilis is cultured on a culture medium and in that the exopolysaccharides are recovered from the culture medium. (followed by page 2) 23 0 0 3 - 2 - P. paucimobilis. especially strain RMDP17, has no special trophic requirements. It grows very well on most culture media, such as nutrient broth, nutrient agar, "Luria broth" and media for nitrogen-fixing bacteria such as Dobereiner and Day medium, Watanabe medium or RCV medium, the preparation of which is described in the examples. Studies of optimization of culture media showed that the most advantageous carbon sources for the preparation of exopolysaccharides were aldohexoses, in particular glucose, and that the glucose concentration was not a limiting factor. On the other hand, exopoly-saccharide production is substantially proportional to the availability of combined nitrogen. The other culture conditions do not constitute characteristic features of the present invention; the bacterium in question is aerobic and can grow at temperatures capable of varying from 18 to 37°C. A highly advantageous characteristic of the process according to the invention is the use of predators such as amoebae, predatory bacteria, myxobacteria, fungi, phages, protozoa, nematodes and leucocytes in order to increase exopolysaccharide production. It appears that the use of these predators enables mutants hyperproductive of polysaccharides to be selected. This operation may be conducted separately in order to isolate a hyperproductive mutant, or alternatively be used as a selection pressure in the culture medium. The polysaccharide in question may be used as a replacement for xanthan as a gelling and thickening agent, but it can also be used as a soil conditioning agent, as will be described below in the examples. In addition, P. paucimobilis. especially RMDP17, possesses especially advantageous properties in the agricultural field of millet culture. In effect, the bacteria according to the invention possess a strong colonizing power for millet roots: after bacterization of the seeds, the bacterium is capable of growing and 23 0 0 39 - 3 - multiplying along the roots where, after 7 days, it can represent 10% of the total microflora (laboratory experiment in pots, on soil originating from Bambey station in Senegal). By inoculating millet seeds using these bacteria, it is possible to envisage increasing nitrogen fixation and hence improving growth. Similarly, this strong colonizing power and the production of siderophore should make it possible to control millet pathogens. The inoculation may be performed on the seed, for example by pelleting, but it is possible to envisage inoculating the bacteria into the soil with or without a carrier, according to known processes. As regards strain RMDP17, there should also be mentioned the property of giving, on medium devoid of combined nitrogen, both extremely highly raised shiny yellow colonies exhibiting strong nitrogenase activity, and shiny white colonies flat on the substrate and devoid of nitrogenase activity. Other characteristics and advantages of the invention will become apparent on reading the examples below, which refer to the attached drawings, wherein: Figure 1 shows the changes in relative viscosity in terms of the shear gradient, comparative behaviour of the product of strain RMD17 and xanthan (Rhodopol RAP 23-1976), measured in a "low shear" viscometer; Figure 2 is a diagram showing the influence of the presence of a predator (an amoeba) on exopolysac-charide production in terms of the buffer concentration; Figure 3 is a graph showing the influence, with time, of exopolysaccharides of strain RMDP17 compared with a succinoglycan produced by strain GM1848 on the percentage of stable aggregates. EXAMPLE 1 The P. paucimobilis strain designated RMDP17 was isolated from millet rhizosphere. The organism is an oxidase-negative, catalase-positive, mobile, rod-shaped Gram-negative bacterium; the API 20 NE profile is 0463 (3/7) (4/6) 4; the 20 EC m 23 0 0 39 - 4 - profile is 0 210 005; in the nitrogen-fixing condition, the colonies are yellow. An antiserum prepared in rabbits against this strain gives a strong cross-reaction with strain 5 Aj. 5 The latter, on DNA/rRNA hybridization has been shown to resemble closely Flavobacterium capsulatum ATCC 14 666 (Bally et al). This antiserum does not exhibit a cross-reaction with P. fluorescens strain W or P. cepacia. £**} 10 This strain, known as RMDP17, is very similar to P. paucimobilis. a taxon defined by Homes et al. It should be noted that it is a badly named taxon, remote from most Pseudomonas species which belong to De Ley's groups II and III; P. paucimobilis has links, in fact, 15 with group IV defined by De Ley. As such, this bacterium is seen to be related to Flavobacterium capsulatum, Azospirillum, Rhizobium. Aqrobacterium. This strain was cultivated on a medium, the preparation of which is described belows 20 Three solutions must be prepared: 1. "Elements" solution (mg/1 of distilled water) ZnS04. 7H20 430 MnS0A.H20 1300 NaMo0A.2H20 750 25 H3BO3 2800 CuSOi,. 7H20 26 CoS04.7H20 70 Distilled water 1000 ml 2. "Supersalts" solution (mg/1 of distilled water) 30 EDTA 400 MgS0A.7H20 2000 CaCl2. 2H20 2000 FeS0A.7H20 440 "Elements" solution 20 ml 35 Distilled water 1000 ml 3. Phosphate buffer KH2POA 40 g K2HP0A 60 g Distilled water 1000 ml 23 0 0 3 - 5 - Final medium; "Supersalts" solution 50 ml Phosphate buffer 15 ml Carbon substrate 1 to 10 g/1 5 Distilled water, qs 1000 ml Yeast extract 0.1 g/1 Notes The carbon substrate can be a sugar or malate. This medium is a modification of that used by-Weaver et al. for Rhodopseudomonas. 10 After culturing, the exopolysaccharides are separated as described below: The exopolysaccharides produced are precipitable with ethanol, introduced on the basis of 1.5 volumes per volume of must; they are then recoverable by centrifuga-15 tion (5000 g for 15 to 20 minutes) or by filtration on a series of Millipore filters decreasing in size (5, 2 and then 0.45 microns). They can then be dehydrated on the filter with increasingly concentrated solutions of ethanol (60, 75 and then 95° strength), and dried in an 20 oven at 35°C for 48 hours. EXAMPLE 2 Study of the properties of the polysaccharide Chemical analysis: Composition in terms of neutral and acid sugars 25 After partial hydrolysis (1 ml of molar sulphuric acid per 100 mg of exopolysaccharides), the composition of the hydrolysate was studied by GC of the trifluoro-acetylated methyl glycosides. Two monosaccharides were identified: glucose and rhamnose, in mole ratios of 3:1 30 or 4:1. On the same hydrolysate, the sugars and sub-stituents were identified and assayed by chromatography on an Aminex HPX-87 H ion exclusion column (300 x 7. 8 mm, Biorad): on the chromatogram, only two constituents (glucose and rhamnose) and traces of acetic acid were 35 seen. The detection was carried out by means of a dif ferential retractometer. Confirmation of the acid sub-stituents was carried out on a micro-Bondapak CI8 column modified by acetylation. Q 23 0 0 39 - 6 - Analysis of the monosaccharide arrangement by permethvlation The polysaccharide was permethylated according to the method of Paz-Parente et al. (1985), and the methyl ethers liberated by methanolysis analysed by GC according to the method of Fournet et al. (1981). The proposed structure is as follows: [4 Glc 1 >3 Beta 1 Glc >6 Alpha 1 Glc >4 Rha 1 L f 6 Jn Beta 1 Glc Acid substituents Their quantification was carried out by proton NMR. Only acetyl substituents could be detected. They are very few in number. There are no succinyl or pyruvyl substituents. Study of the physical properties Optical rotation and conformational transition temperature The product is soluble in the cold state in water. Its optical rotation is [alpha]"0 = -84. There is no conformational transition between 10 and 90°C. The optical rotation was established on exo-polysaccharide solutions at a concentration of 1 g/1 in 0.1 M NaCl, at 300 nm with a spectropolarimeter (FICA Spectrol 1). Rheoloqical properties This polysaccharide is endowed with a high viscosity, higher than that of xanthan (Figure 1). This property is relatively more stable than that of xanthan when the ionic strength increases or when the pH is acid; this viscosity is little affected by heating for 10 minutes to 90°C. m 230039 - 7 - O 5 ^ 10 v,-' Table 1 Comparison of the relative viscosities of the product of strain RMDP17 and xanthan (Solutions at a concentration of 1 g/1, viscosity measured using a "low shear" viscometer for a shear gradient gamma = 0.08 s"1) Medium Water RMDP17 1613 Xanthan 626 (Rhodopol RAP 23-1976) NaCl (0.1 2477 194 M) NaCl (1.7 M) 2440 223 CaCl? (0.3 M) 2558 164 HCl (0.5 N) 1186 32 Table 2 Changes in the relative viscosity at gamma = 0.94s"1 of the 15 product of strain RMDP17 in terms of the time of heating to 90"C Heating time 0 10 min 24 h 48 h Relative viscosity 472 406 58 9 The studies were carried out using a "low shear" 20 rheometer 30. Apart from the properties already men tioned, it should be noted that the polysaccharide has a viscosity which increases after heating to 90°C in NaCl; while it undergoes little decrease in an acid medium, in contrast it disappears completely in a basic medium. 25 EXAMPLE 3 The following studies enabled some advantageous features of the culture of the strain in question to be demonstrated. Carbon source 30 The table below summarizes the results obtained in a study of the influence of various carbon sources on growth and polysaccharide production, detected by fluorescence in the presence of "Calcofluor White". 23 0 0 3 - 8 - Table 3 Carbon source Growth Fluorescence Aldohexoses: Glucose ++ ++ Sucrose ++ ++ Fructose ++ ++ Alodpentoses: Arabinose + + Xylose + + Maltose + + Polyols: Mannitol (+) + Sorbitol (+) + Glycerol (+) + Organic acids: Citrate (+) + Malate + + Succinate + + Amino acids: L-Arginine 0 0 L-Glycine (+) 0 Methionine (+) 0 Glutamine (+) 0 L-Leucine + 0 Asparagine + 0 L-Alanine + 0 Tyrosine + 0 The production is maximal on aldohexoses. Studies of concentration show that the glucose concentration is not a limiting factor in the polysaccharide production, and that the production is substantially the same for 0.5 to 3 g/100 ml of glucose. A study was carried out of the influence of the phosphate buffer concentration in the RCV medium; it shows that even 40 ml/1 is an insufficient concentration. The most important factor for polysaccharide production is, without doubt, nitrogen nutrition; in contrast to the situation known to apply to many bacteria, exopolysaccharide production is, in this case, - 9 - 23 0 0 3 proportional to the availability of combined nitrogen. A study of a few spontaneous mutants for resistance to rifampicin shows that there is probably transcriptional control of the amount of exopolysaccharide synthesized. It is hence advantageous to use some rifam-picin-resistant mutants for preparing the exopolysaccharides . EXAMPLE 4 Cultures in the presence of predators, especially in the presence of a soil amoeba, enabled the advantages of this type of culture to be demonstrated. The presence of predators often modifies the level of bacterial populations, and can induce modifications, especially in respect of the envelopes, but it appears that this property has never been used for selecting mutants hyperproductive of capsular material; the diagram in Figure 2 shows that strain RMDP17 strongly increases its production of exopolysaccharides when placed in the presence of a soil amoeba IAcanthamoeba sp.). The use of predators as a means of selecting mutants hyperproductive of polysaccharides constitutes an important feature of the present invention. The term "predator" is to be understood in the broadest sense, and includes all predatory bacteria, myxobacteria, fungi, phages, protozoa, nematodes and leucocytes. EXAMPLE 5 Strain RMDP17 was isolated from the rhizosphere of a millet growing on a ferralitic soil of Andhra Pradesh in India. The colonizing power on this plant was assessed by means of a very simple experiment: millet, cv. Souna, was inoculated on the seeds by soaking in a dense culture of the strain, and grown for three weeks in the laboratory on Dior soil from Bambey agricultural research station in Senegal. At the end of this period, the bacterium had well colonized the root system of the millet, where it represented approximately 12% of the total bacterial microflora. This bacterial species hence has some potential as an inoculum for millet for a 23 - 10 - variety of purposes, which can range from an increase in its mineral nutrition and its growth to the control of major and minor pathogens or harmful bacteria. It would also be possible to envisage using the bacterium after f*"s 5 genetic manipulation for introducing genes useful to man into the millet rhizosphere. Finally, the production of large amounts of hydrophilic polysaccharides by Pseudo-monas paucimobilis could, if desired, be turned to good account in order to increase the available water reserve 10 in the rhizosphere and to enable the resistance of millet ■"Soot*' or other plants to water stress to be improved, A study was carried out of the effect of adding the polysaccharide produced by Pseudomonas paucimobilis RMDP17 to soil. Figure 3 shows a comparison of the effect 15 of the product of strain RMDP17 with that of a conventional type succinoglycan produced by a strain of Aaro-bacterium radiobacter. strain GM 1848. The percentage of benzene-stable aggregates was measured by the traditional method of H6nin. Soil samples (clayey-loamy soil from the 20 Nancy region) were watered to the field capacity at the beginning of the experiment, after adding the polysaccharides, and then incubated in the air. They gradually dehydrated during incubation and, on day 20, the moisture content was readjusted to the field capacity; the samples 25 treated with the product of strain RMDP17 then show remarkable behaviour: their structuring increases and returns to a state equal to that observed at the beginning of the experiment, if not better (Figure 3). This property is very unusual, and could be 30 turned to good account in order to use the P. paucimobilis polysaccharides as soil conditioners or as an additive in all situations where the soil is used as an ingredient in the production of a product whose mechanical properties are an essential feature. Applications could 35 be envisaged in different fields, from agriculture (improvement of capping soils) to civil engineering (consolidation of embankments). 23 0 0 39 - li - The Pseudomonas paucimobilis RMDP17 strain has been registered on 3uly 7th, 1989, at the "Collection Nationale de Cultures de Microorganismes de 1'Institut Pasteur", 28 rue du Docteur-Roux - 7572^ Paris Cedex 15, under n° 1-886, in accordance with the Budapest Treaty provisions. 230039 - 12-BIBLIOGRAPHY Bally R., Thomas-Bauzon D. , Heulin T., Balandreau J., Richard C. and De Ley J., 1983, Determination of the most frequent N2-fixing bacteria in a rice rhizosphere. Can. J. Microbiol., 29, 881-887. De Ley J., 1981. Evolution des cistrons codant pour l'ARNr bact^rien [Evolution of the cistrons coding for bacterial rRNA] . Symbioses, JL3, 109-121. Dobereiner J. and Day J.M., 1976, Associative symbioses in tropical grasses: characterization of microorganisms and dinitrogen fixing sites. In: Nitrogen fixation, edited by W.E. Newton and C.J. Nyman, Washington State University Press, Pullman, W.A., pp. 518-538, Holmes B., Owen R.J., Evans A., Malnick H. and Willcox W.R., 1977. Pseudomonas paucimobilis. a new species isolated from human clinical specimens, the hospital environment and other sources. Intern. J. Syst. Bacterid. 27, 133-146. Watanabe I. and Barraquio W.L., 1979. Low levels of fixed nitrogen required for isolation of free-living N2-fixing organisms from rice roots. Nature (London), 277:565-566. Weaver P.K., Wall J.D. and Gest H., 1975. Characterization of Rhodopseudomonas capsulata. Arch Microbiol., 105,207-216. 25 WHAT fiWB CLAIM fSc- </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS

1. Process for preparing exopolysaccharides containing less than 10 % ) amount of uronic acid, characterized in that a strain identified as P. paucimobilis is cultured on a culture medium and in that the exopoly-5 saccharides containing less than 10 96 amount of uronic acid are recovered .from the culture medium.

2. Process according to Claim 1, characterized in C*^) that the carbon source. in the culture medium is an aldohexose. 10

3. Process according to one of Claims 1 and 2, characterized in that culturing is performed in the presence of a predator.

4. Process according to Claim 3, characterized in that the predator is a soil amoeba. 15

5. Process according to one of Claims 1 to 4, characterized in that the strain is strain RMDP17.

6. Process according to one of Claims 1 to 5, characterized in that the exopolysaccharide is recovered by precipitation from the culture medium. 20

7. Exopolysaccharide as it can be obtained by carrying out the process according to one of Claims 1 to 6.

8. Exopolysaccharide as it can be obtained by carrying out the process according to claim 5.

9. Exopolysaccharide, the saccharide chain of which is as follows: [4 Glc 1 >3 Beta 1 Glc *6 Alpha 1 Glc *4 Rha l) L t 6 "n Beta 1 Glc

10. Application of the exopolysaccharides according 30 to one of Claims 7 ~ 9 , by way of a thickening agent and/or soil conditioning agent. 11, A method for growing millet which involves the use of a nitrogen-fixijia^strai^BBI Pseudomonas paucimobilis. CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) By Their Attorneys _ BALDWIN, SON & </div>