AU625278B2 - Polysaccharides, process for preparing them by culturing pseudomonas paucimobilis and application of the strains in agriculture - Google Patents

Description

t~"f 6% 78 S F Ref: 102208 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: o o Name and Address of Applicant: S Address for Service: Centre National de la Recherche Scientifique (CNRS) quai Anatole France 75007 Paris

FRANCE

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: adr:ao d0 O O Polysaccharides, Process for Preparing them by Culturing Pseudomonas paucimobilis and Application of the Strains in Agriculture The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/7

ABSTRACT

0 0 The present invention relates to a process for -0 0o0o0 preparing exopolysaccharides, characterized in that a strain of P. paucimobilis is cultured on a culture medium and in that the exopolysaccharides are recovered from the o o culture medium, and to the use of this bacterial species and/or its product in agricultural management and in the foodstuffs industry.

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O 0 i) 0 0 B 1A 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.

The present invention is based on the demonstration of especially advantageous properties of P. paucimobilis bacteria, in particular as polysaccharide-producing organisms, but also as agents exhibiting substantial nitrogenase activity.

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/l of dry matter).

This polysaccharide can optionally be used to 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 rheological behaviour of the polysaccharide according to the invention, namely, in particular, that its viscosity is rather insensitive to the ionic strength S 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: 4 Glc 1 3 Beta 1 Glc 6 Alpha 1 Glc 4 Rha l Beta 1 Glc o n represents the number of units although it is difficult to evaluate the molecular weight, generally n is 500<n<10 000 and preferably between 1 000<n<4 000.

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 ¢1culture medium.

IB According to a first embodiment of this invention, there is provided a process for preparing exopolysaccharides containing less than a 10% amount of uronic acid, characterized in that P.paucimobilis is cultured in a culture medium at 18 to 37°C and in that the exopolysaccharides containing less than a 10% amount of uronic acid are recovered from the culture medium.

According to a second embodiment of this invention, there is provided exopolysaccharide when prepared by the process according to the first embodiment.

According to a third embodiment of this invention, there is provided exopolysaccharide when produced by the process of the first embodiment, the polysaccharide chain of which is as follows: [4 Glc 1 3 Beta 1 Glc 6 Alpha 1 Glc 4 Rha 1]n S6 Beta 1 Glc wherein n is the number of units and has a value of 500 to 10,000.

According to a fourth embodiment of this invention, there is provided a method of thickening a substance or conditioning soil comprising applying to said substance or soil the exopolysaccharide according to the second or third embodiments.

S',i According to a fifth embodiment of this invention, there is 20 provided a nitrogen-fixing strain of Pseudomonas paucimobilis which gives, on solid medium devoid of combined nitrogen, extremely highly raised shiny yellow colonies, which strain is identified as RMDP17.

According to a sixth embodiment of this invention, there is provided a method of colonizing millet and related plants comprising inoculating the seeds of said millet or related plants with the bacterial strain according to the fifth embodiment.

According to a seventh embodiment of this invention, there is provided a biologically pure culture of Pseudomonas paucimobilis RMDP17 (CNCM 1-886).

GSA/856Z 2i 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 D6bereiner 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, exopolysaccharide production is substantially proportional to the availability of combined nitrogen.

o444 15 The other culture conditions do not constitute So characteristic features of the present invention; the 01, bacterium in question is aerobic and can grow at temperatures capable of varying from 18 to 37 0

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. 4 It appears that the use of these predators 25 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 I n -i i j/ n- -r 4 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.

Similaily, 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, a further property should also be mentioned: on a solid medium, devoid of combined nitrogen, it gives both S" extremely highly raised shiny yellow colonies exhibiting strong i' nitrogenase activity, and shiny white colonies flat on the substrate and devoid of nitrogenase activity.

Other characteristics and advantages of the invention will become S 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 RMDP17 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 expolysaccharide 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 Agrobacterium radiobacter 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 4; the 20 EC LMM/686Z S- 4 i 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.

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.

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, with group IV defined by De Ley. As such, this bacterium is seen to be related to Flavobacterium capsulatum, Azospirillum, Rhizobium, Agrobacterium.

This strain was cultivated a medium, the preparation of which is described below: Three solutions must be prepared: 1. "Elements" solution (mg/l of distilled water) ZnSO 4 .7H 2 0 430 MnSO 4

.H

2 0 1300 NaMoO 4 2HzO 750

H

3

BO

3 2800 CuS0. 7H 2 0 26 CoSO. 7H 2 0 Distilled water 1000 ml 2. "Supersalts" solution (mg/l of distilled water) A 30 EDTA 400 MgSO. 7H 2 0 2000 CaCl 2 .2H.O 2000 FeSO 4 .7H 2 0 440 "Elements" solution 20 ml Distilled water 1000 ml 3. Phosphate buffer

KH

2

PO

4 40 g

K

2 HPO 60 g Distilled water 1000 ml r 5 Final medium: "Supersalts" solution 50 ml Phosphate buffer 15 ml Carbon substrate 1 to 10 g/l Distilled water, qs 1000 ml Yeast extract 0.1 g/l Note: The carbon substrate can be a sugar or malate.

This medium is a modification of that used by Weaver et al. for Rhodopseudomonas.

After culturing, the exopolysaccharides are separated as described below: S' The exopolysaccharides produced are precipitable with ethanol, introduced on the basis of 1.5 volumes per volume ofzmat; they are then recoverable by centrifugation (5000 g for 15 to 20 minutes) or by filtration on a series of Millipore filters decreasing in size 2 and S. 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 oven at 35°C for 48 hours.

SEXAMPLE 2 Study of the properties of the polysaccharide Chemical analysis: 'Composition in terms of neutral and acid sugars 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 trifluoroacetylated methyl glycosides. Two monosaccharides were Sidentified: glucose and rhamnose, in mole ratios of 3:1 or 4:1. On the same hydrolysate, the sugars and substituents were identified and assayed by chromatography cn 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 seen. The detection was carried out by means of a differential retractometer. Confirmation of the acid substituents was carried out on a micro-Bondapak C18 column modified by acetylation.

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311~ 6 Analysis of the monosaccharide arrangement by permethylation 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 11 S6 Beta 1 Glc wherein n is the number of units and has a value of approximately 500 to 10,000.

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 25 optical rotation is [alpha] 300 -84.

There is no conformational transition between 10 and 90°C. The optical rotation was established on exopolysaccharide solutions at a concentration of 1 g/l in 0.1 M NaCl, at 300 nm with a spectropolarimeter (FICA Spectrol 1).

Rheological properties This polysaccharide is endowed with a high viscosity, higher than that of xanthan (Figure 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 4 C r£^y LMM/686Z I "m-runrr;r--~7?=~ 7 Table 1 Comparison of the relative viscosities of the product of strain RMDP17 and xanthan (Solutions at a concentration of 1 g/l, viscosity measured using a "low shear" viscometer for a shear gradient gamma 0.08 s Medium Water NaCI NaCl CaCl HC (0.1 M) (1.7 M) (0.3 M) (0.

RMDP17 1613 2477 2440 2558 11 Xanthan 626 194 223 164 1 5 N) 86 32 (Rhodopol RAP 23-1976) Table 2 Changes in the relative viscosity at qarmia 0.94s of the product of strain RMDP17 in terms of the time of Heating time Relative viscosity heating to 0 10 min 2 472 406 5 ;4 h 8 48 h 9 The studies were carried out using a "low shear" rheometer 30. Apart from the properties already mentioned, it should be noted tnat the polysaccharide has a viscosity which increases after heating to 90 0 C in NaCl; while it undergoes little decrease in an acid medium, in contrast it disappears completely in a basic medium.

EXAMPLE 3 The following studies enabled some advantageous features of the culture of the strain in question to be demonstrated.

Carbon source 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".

-8- Table 3 Carbon source Aldohexoses: Alodpentoses: Polyols: Organic acids: Amino acids: Glucose Sucrose Fructose Arabinose Xylose Maltose Mannitol Sorbitol Glycerol Citrate Malate Succinate L-Arginine L-Glycine Methionine Glutamine L-Leucine Asparagine L-Alanine Tyrosine Growth Fluorescence a o os

J

a o 00 0 r a <t oo 0 0 0 0 6 0 9~ means means means 0 means growth abundant significant slight none fluorescence abundant production production none none 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/100ml of glucose.

A s"dy was carried out on the influence of the phosphate buffer concentration in the RCV medium; it showed that exopolysaccharide production was adequate with a minimum of 10 ml/l buffer in the RCV medium (see Fig. 2) but production did increase with increasing buffer concentrations, even up to 40 ml/l.

The most important factor for polysaccharide production is, without doubt, nitrogen nutrition; in contrast to the situation known to apply to zmny bacteria, exopolysaccharide production is, in this case, LMM/686Z 9 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 r~a.epicin-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 (Acanthamoeba 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 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 cl, 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 genetic manipulation for introducing genes useful to man into the millet rhizosphere. Finally, the production of large amounts of hydrophilic polysaccharides by Pseudomonas paucimobilis could, if desired, be turned to good account in order to increase the available water reserve in the rhizosphere and to enable the resistance of millet 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 of the product of strain RMDP17 with that of a conventional type succinoglycan produced by a strain of Agrobacterium radiobacter, strain GM 1848. The percentage of benzene-stable aggregates was measured by the traditional method of Henin. Soil samples (clayey-loamy soil from the 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 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 A 30 turned to good account in order to use the P. pauc.mobilis 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 be envisaged in different fields, from agriculture (improvement of capping soils) to civil engineering (consolidation of embankments).

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1 :i 11 The Pseudomonas paucimobilis RMDP17 strain has been registered on July 7th, 1989, at the "Collection Nationale de Cultures de Microorganismes de 1'Institut Pasteur", 28 rue du Docteur-Roux 75724 Paris Cedex 15, under n° 1-886, in accordance with the Budapest Treaty provisions.

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BIBLIOGRAPHY

Bally Thomas-Bauzon Heulin Balandreau J., Richard C. and De Ley 1983, Determination of the most frequent N 2 -fixing bacteria in a rice rhizosphere. Can.

J. Microbiol., 29, 881-887.

De Ley 1981. Evolution des cistrons codant pour 1'ARNr bact6rien [Evolution the cistrons coding for bacterial rRNA]. Symbioses, 13, 109-121.

D6bereiner J. and Day 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, pp. 518-538, Holmes Owen Evans Malnick H. and Willcox S '.977. Pseudomonas paucimobilis, a new species isolated from human clinical specimens, the hospital environment and other sources. Intern. J. Syst.

00. Bacteriol. 27, 133-146.

Watanabe I. and Barraquio 1979. Low levels of fixed nitrogen required for isolation of free-living N 2 -fixing organisms from rice r-ots. Nature (London), 277:565-566.

Weaver Wall J.D. and Gest 1975. Characterization of Rhodopseudomonas capsulata. Arch Microbiol., 105,207-216.

Claims (14)

1. A process for preparing exopolysaccharides containing less than a 10% amount of uronic acid, characterized in that P.paucimob11 s is cultured in a culture medium at 18 to 37°C and in that said exopoly- saccharides containing less than a 10% amount of uronic acid are recovered from the culture medium.

2. The process according to claim 1, characterized in that the carbon source in the culture medium is an aldohexose.

3. The process according to claim 1 or claim 2, characterized in that the culturing is performed in the presence of a predator.

4. The process according to claim 3, characterized in that the predator is a soil amoeba.

The process according to any one of claims 1 to 4, characterized in that said P. .paucimobilis is strain RMDP17.

6. The process according to any one of claims 1 to characterized in that said exopolysaccharide is recovered by precipitation from said culture medium.

7. Exopolysaccharide when prepared by the process according to any one of claims 1 to 6.

8. Exopolysaccharide when produced by the process according to any one of claims 1 to 6, the polysaccharide chain of which is as follows: 7.i [4 Glc 1 3 Beta 1 Glc -4 6 Alpha 1 Glc 4 Rha 1] 6i Beta 1 Gk wherein n is the number of units and has a value of 500 to 10,000.

9. The exopolysaccharide of claim 8, wherein n has a value of 1,000 to 4,000.

10. A method of thickening a substance or conditioning soil eB, comprising applying to said substance or soil the exopolysaccharide according to any one of claims 7 to 9.

11. A nitrogen-fixing strain of Pseudomonas paucimobilis which gives, on solid medium devoid of combined nitrogen, extremely highly raised shiny yellow colonies, which strain is Identified as RMDP17.

12. A method of colonizing millet and related plants comprising v v o- inoculating the seeds of said millet or related plants with the bacterial cc gt strain according to claim 11. GSA/856Z -u~ 14

13. A biologically pure culture of Pseudomonas paucimobilis RMDP17 (CNCM 1-886).

14. A process for preparing exopolysaccharide3 containing less than a 10% amount of uronic acid, which process is substantially as hereinbefore described with reference to any one of Examples 1, 3 or 4. Exopolysaccharide, the polysaccharide chain of which is as follows: [4 G1c 1 3 Beta 1 Glc 6 Alpha 1 Glc 4 Rha 1] T 6 Beta 1 Glc wherein n is as defined in claim 8; which exopolysaccharide is substantially as hereinbefore described with reference to Example 2. DATED this NINTH day of DECEMBER 1991 Centre National de la Recherche Scientifique (CNRS) Patent Attorneys for the Applicant SPRUSON FERGUSON Irrersr er~a r t p~\qIAN i 0 9. L:I Cc '4.C L Kt O T" LMM/686Z i