Folia Microbiol. 22, 12--18 (1977)
Production and Characteristics of the Exocellular Polysaccharide in Mutant Strains of Xanthomonas fuscans J. KOh~iSEK, J. LASiK and M. WURST Departmeut of Molecular Biology and Genetics and JOcpartment of Soil ~licrobiology, Institute of Microbiology, Gzeehoslovak Academy of Sciences, Prague 4
Received June 3, 1976
ABSTRACT. Production of the exocellular polysaccharide of t h e p h y t o p a t h o g e n i c b a c t e r i u m Xanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes c o n t a i n i n g lactose. Six stable lee + m u t a n t s were obtained after the t r e a t m e n t with N-methyl-N-nitroso-2V'.nitroguanidine. T h e m u t a n t s were compared with the p a r e n t strain. Morphological a n d cultivation characteristics, as well as production of the exoeellular polysaceharide were compared. The production was f o u n d to be m a x i m a l during the s t a t i o n a r y phase of growth in strains cultivated under s u b m e r g e d conditions. Gas c h r o m a t o g r a p h y revealed t h a t t h e polysaecharide of the p a r e n t strain is formed b y r162a n d fl-D-glueose a n d 0~- a n d fl-D-mannose with a small a m o u n t of D-ribose a n d 6-deoxy-L-mannose. Composition of t h e polysaccharides produced b y the m u t a n t strains (lac +) does n o t qualitatively differ from t h a t of the p a r e n t strain. However, t h e y wore found to contain a higher q u a n t i t y of D-mannose, which is favourable for their industrial utilization.
Properties and compositioll of the exocellular polysaccharide produced by a strain of the phytopathogenic bacterium of the genus Xanthomonas causing bean fuscous blight and isolated from the surface of bean seeds (Lasik and Stan~k, 1963) were studied. With respect to a possible industrial use of the exocellular polysaccharide produced by this bacterium we looked for a cheap carbon source on which fermentation production of the polysaccharide could be performed. W h e y with a high caloric and nutrition value containing about 42~/o lactose in 9')~ dry weight is mostly considered at present. The strain of Xanthomonas fuscans used by us does not utilize lactose, so we attempted to isolate m u t a n t s which do ferment lactose. I n a previous paper (Lasik and KoniSek, 1976) we demonstrated t h a t it is possible to induce genetic changes of the strain of Xanthomonas fuscans by mutagenie processes. I t was found t h a t 2Y-methyl-N-nitroso-N'-nitroguanidine (nitrosoguanidine) exhibits mutagenic and lethal effects on this strain. A higher number of m u t a n t s with white colonies and m u t a n t s fermenting lactose was obtained. The parent strain of Xanthomonas fuscans can grow- on a complete medium forming smooth, glossy, mucous colonies of yellow colour; it utilizes glucose, sucrose, maltose and starch, it can grow" poorly on arabinose and D-xylose and does not ferment lactose. The m u t a n t s obtained usually reversed with a high frequency to the original parent state when subeultured on solid media. Therefore, in the present work we concent r a t e d on selection of stable lac + m u t a n t s induced with nitrosoguanidine and on their comparison with the parent strain, with respect to quality and q u a n t i t y of the exocellular polysaccharide produced.
1977
EXOCELLULAR POLYSACCHARIDE OF X. FUSGANS
13
MATERIALS AND METHODS
The bacterial strain. The strain of Xanthomonas fuscans isolated from the surface of bean seeds of Orion variety (Lasik and Stan6k, 1963) was used throughout. Induction of lac + mutants. Bacterial suspension from the exponential phase of growth prepared b y cultivation in a liquid tryptose medium (Bacto tryptose Difco, 15.0; NaC1, 0.2; KeHPOd, 0.2; distilled water ad 1000 ml; p H 7.2; 10 ml of 500/o glucose added after sterilization) at 28 ~ The culture was centrifuged and washed with Tris-maleic acid buffer p H 6.0. The washed cell suspension was resuspended in buffer, the original number of cells was determined b y spraying different dilutions of the culture on a solid tryptose medium and nitrosoguanidine freshly prepared b y dissolution in buffer was added to aliquots of the culture. Nitrosoguanidine (Nmethyl-N-nitroso-N'-nitroguanidine, Koch-Light Laboratories L t d Colnbrook-BucksEngland) was applied at a final concentration of 1 mg per ml. The reaction mixture was incubated in a water bath at 28 ~ samples were taken at suitable time intervals and the treatment with nitrosoguanidine was terminated b y adding a ten-fold quantity of 4 ~ Tris-maleic acid buffer and centrifuging the culture (Adelberg et al., 1965; KoniSek and Ms 1970). The sedilnent was resuspended with a physiological saline to the original v o h m e and inoculated to a solid tryptose medium. The inoculated media were observed after a 2--3 d a y incubation at 28 ~ In order to allow for the phenotypic expression and selection of the induced lac + m u t a n t s samples were incubated for 48 h in a liquid tryptose medium, the culture was then centrifuged, washed with a physiological saline and used to inoculated the solid tryptose medium and a selective medium W H with 2% lactose (KzHPOd, 0.75; KNOa, 0.375; MgSOd. 7 H20, 0.15; NaC1, 0.075: CaC]2, 0.075; FeCls, 0.07; Bacto )'east extract Difco, 0.75; distilled water ad 850 ml; 20.0 g lactose dissolved in 150 lnl distilled water were added after sterilization). Determination of production of the exocelluIar polysaccharide under submerged conditions. Five ml of the bacterial suspension prepared from cultures taken from the exponential phase of growth were used to inoculate 80 ml of the liquid W H medium with 2 o/o glucose (parent strain of Xanthomonas fuscans) and with 2" o, 7o lactose (mutant strains). The cultivation was performed on a reciprocal shaker for 96 h at 28 ~ Samples were taken at indicated time intervals and growth of the culture was measured in a Prem nephelometer at 470 nm. Isolation and purification of the exocellular polysaccharide were performed according to Sevag (Sevag, 1934). The final product was dried under an infra-red lamp and weighed. Analysis of the exoceltular polysaccharide by gas chromatography. The used solutions of trimethylsilyl derivatives of individual monosaccharidcs, monosaccharides and silylation agents were described in a paper by Wurst et al. (1974). The isolated polysaccharide was hydrolyzed with l~~ H C1 in a sealed ampoule for 5 h at 105 ~ The method was modified in such a w a y that the hydrolysate was filtered through a bacteriological filter G3 prior to evaporation. Trimethylsilyl (TMS) derivatives of monosaccharides were prepared using a method suitable for silylation of sugars and related compounds (Sweeley et al., 1963). The evaporate of the sample of the hydroiyze4 polysacchaxide was first dissolved in warm pyridine and only then was p)~'idine with silylation agents added. The mixture of TMS derivatives of monosaccharides was analysed in an F 21 gas chromatograph (Perkin-Elmer, Norwalk, Conn., USA). Columns were filled with Chromosorb P of a grain size of 0.15--0.17 mm (Johns Manville, Denver, Col., USA) impregnated with 10% DC 200 silicone oil (Dow Corning, Midland, Michigan, USA). Separation proceeded isothermically at 190 ~ Carrier gas was nitrogen at a flow rate of 20 ml per min (Wurst et al., 1974).
14
J. K O N I ~ E K
Vol. 22
ET AL.
R E S U L T S A N D DISCUSSION
Previous results (Lasik and KoniSek, 1976) indicated t h a t nitrosoguanidine at a concentration of 1 mg per ml at ]oH 6.0 exhibits a pronounced lethal effect on the strain of Xanthomonas fuscans from the beginning of the t r e a t m e n t ; after a 10 rain t r e a t m e n t about 60/o cells survive and after 20 min the lethality of cells is higher t h a n 99%. For the induction of lac + m u t a n t s of the strain Xanthomonas fuscans TABLE I. Yields o f p o l y s a c c h a r i d e s a f t e r a 96 h submerged cultivation of the parent strain of Xanthomonas fuscans in a liquid W H m e d i u m w i t h 2 ~ glucose a n d o f t h e lac + m u t a n t s in a liquid W H m e d i u m w i t h 2 % lactose (calculation p e r a d d e d sugar) Yield p e r a d d e d s u g a r
Xanthomcnas fuscan8
Parent Mutant Mutant Mutant Mutant Mutant Mutant
%
24.0 20.0 12.0 17.0 9.1 10.8 9.7
s t r a i n lay lac + No. 1 lac + No. 2 lac + No. 3 lac + No. 4 lac + No. 5 lac + No. 6
intervals of t r e a t m e n t with nitrosoguanidine yielding survival lower t h a n 10~/o were used in the present work. Nitrosoguanidine was also used at a final concentration of 1 mg per ml at pH 6.0. A total of 6 non-reverting lac + m u t a n t s were obtained, out of t h e m 3 were isolated at a 2~o survival and 3 at a survival lower t h a n 1%. M u t a n t strains growing on the medium containing tryptose form after a 3 d a y inTAlZI,E I I . C h r o m a t o g r a p h i c d a t a a n d c o m p o s i t i o n o f ' t h e e x o c e l l u l a r p o l y s a c c h a r i d e (~o) of t h e p a r e n t s t r a i n o f Xanthomonas fuecans a n d m u t a n t / a c + s t r a i n s . Xardhomonas fuscans
Monosacoharides (TMS d e r i v a t i v e s )
Elution ratio a)
lac+
lac
1 6-Deoxy-L-mannose D-Ribose ~-D-Manno8o ~-D-Galaotosc 0~-D- (]luoos0 ~-D-Manncse ~-D-Glucose D-Galacturonic a~id D-Gluouronic acid
0.49 0.53 1.00 1.22 1.36 1.45 1.90 1.68 2.14
V g (~-D-mannose)
305
1.1 1.0 35.7
26.1 6.9 29.2
1.8 0.6 41.6 1.0 20.0 9.8 25.2
I
2 0.9 0.8 41.9 1.0 20.3 10.6 24.5
I
3 0.3 2.3 47.7 1.3 18.5 9.4 20.5
I
4 0.7 2.8 32.2 2.1 20.1 8.8 32.9 0.4
r
5 1.0 0.8 43.8 1.5 19.1 9.9 23.9 0.1
I
6b 1.1 3.3 39.7 1.4 19.7 10.3 24.5
Production and Characteristics of the Exocellular Polysaccharide in Mutant Strains of Xanthomonas fuscans J. KOh~iSEK, J. LASiK and M. WURST Departmeut of Molecular Biology and Genetics and JOcpartment of Soil ~licrobiology, Institute of Microbiology, Gzeehoslovak Academy of Sciences, Prague 4
Received June 3, 1976
ABSTRACT. Production of the exocellular polysaccharide of t h e p h y t o p a t h o g e n i c b a c t e r i u m Xanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes c o n t a i n i n g lactose. Six stable lee + m u t a n t s were obtained after the t r e a t m e n t with N-methyl-N-nitroso-2V'.nitroguanidine. T h e m u t a n t s were compared with the p a r e n t strain. Morphological a n d cultivation characteristics, as well as production of the exoeellular polysaceharide were compared. The production was f o u n d to be m a x i m a l during the s t a t i o n a r y phase of growth in strains cultivated under s u b m e r g e d conditions. Gas c h r o m a t o g r a p h y revealed t h a t t h e polysaecharide of the p a r e n t strain is formed b y r162a n d fl-D-glueose a n d 0~- a n d fl-D-mannose with a small a m o u n t of D-ribose a n d 6-deoxy-L-mannose. Composition of t h e polysaccharides produced b y the m u t a n t strains (lac +) does n o t qualitatively differ from t h a t of the p a r e n t strain. However, t h e y wore found to contain a higher q u a n t i t y of D-mannose, which is favourable for their industrial utilization.
Properties and compositioll of the exocellular polysaccharide produced by a strain of the phytopathogenic bacterium of the genus Xanthomonas causing bean fuscous blight and isolated from the surface of bean seeds (Lasik and Stan~k, 1963) were studied. With respect to a possible industrial use of the exocellular polysaccharide produced by this bacterium we looked for a cheap carbon source on which fermentation production of the polysaccharide could be performed. W h e y with a high caloric and nutrition value containing about 42~/o lactose in 9')~ dry weight is mostly considered at present. The strain of Xanthomonas fuscans used by us does not utilize lactose, so we attempted to isolate m u t a n t s which do ferment lactose. I n a previous paper (Lasik and KoniSek, 1976) we demonstrated t h a t it is possible to induce genetic changes of the strain of Xanthomonas fuscans by mutagenie processes. I t was found t h a t 2Y-methyl-N-nitroso-N'-nitroguanidine (nitrosoguanidine) exhibits mutagenic and lethal effects on this strain. A higher number of m u t a n t s with white colonies and m u t a n t s fermenting lactose was obtained. The parent strain of Xanthomonas fuscans can grow- on a complete medium forming smooth, glossy, mucous colonies of yellow colour; it utilizes glucose, sucrose, maltose and starch, it can grow" poorly on arabinose and D-xylose and does not ferment lactose. The m u t a n t s obtained usually reversed with a high frequency to the original parent state when subeultured on solid media. Therefore, in the present work we concent r a t e d on selection of stable lac + m u t a n t s induced with nitrosoguanidine and on their comparison with the parent strain, with respect to quality and q u a n t i t y of the exocellular polysaccharide produced.
1977
EXOCELLULAR POLYSACCHARIDE OF X. FUSGANS
13
MATERIALS AND METHODS
The bacterial strain. The strain of Xanthomonas fuscans isolated from the surface of bean seeds of Orion variety (Lasik and Stan6k, 1963) was used throughout. Induction of lac + mutants. Bacterial suspension from the exponential phase of growth prepared b y cultivation in a liquid tryptose medium (Bacto tryptose Difco, 15.0; NaC1, 0.2; KeHPOd, 0.2; distilled water ad 1000 ml; p H 7.2; 10 ml of 500/o glucose added after sterilization) at 28 ~ The culture was centrifuged and washed with Tris-maleic acid buffer p H 6.0. The washed cell suspension was resuspended in buffer, the original number of cells was determined b y spraying different dilutions of the culture on a solid tryptose medium and nitrosoguanidine freshly prepared b y dissolution in buffer was added to aliquots of the culture. Nitrosoguanidine (Nmethyl-N-nitroso-N'-nitroguanidine, Koch-Light Laboratories L t d Colnbrook-BucksEngland) was applied at a final concentration of 1 mg per ml. The reaction mixture was incubated in a water bath at 28 ~ samples were taken at suitable time intervals and the treatment with nitrosoguanidine was terminated b y adding a ten-fold quantity of 4 ~ Tris-maleic acid buffer and centrifuging the culture (Adelberg et al., 1965; KoniSek and Ms 1970). The sedilnent was resuspended with a physiological saline to the original v o h m e and inoculated to a solid tryptose medium. The inoculated media were observed after a 2--3 d a y incubation at 28 ~ In order to allow for the phenotypic expression and selection of the induced lac + m u t a n t s samples were incubated for 48 h in a liquid tryptose medium, the culture was then centrifuged, washed with a physiological saline and used to inoculated the solid tryptose medium and a selective medium W H with 2% lactose (KzHPOd, 0.75; KNOa, 0.375; MgSOd. 7 H20, 0.15; NaC1, 0.075: CaC]2, 0.075; FeCls, 0.07; Bacto )'east extract Difco, 0.75; distilled water ad 850 ml; 20.0 g lactose dissolved in 150 lnl distilled water were added after sterilization). Determination of production of the exocelluIar polysaccharide under submerged conditions. Five ml of the bacterial suspension prepared from cultures taken from the exponential phase of growth were used to inoculate 80 ml of the liquid W H medium with 2 o/o glucose (parent strain of Xanthomonas fuscans) and with 2" o, 7o lactose (mutant strains). The cultivation was performed on a reciprocal shaker for 96 h at 28 ~ Samples were taken at indicated time intervals and growth of the culture was measured in a Prem nephelometer at 470 nm. Isolation and purification of the exocellular polysaccharide were performed according to Sevag (Sevag, 1934). The final product was dried under an infra-red lamp and weighed. Analysis of the exoceltular polysaccharide by gas chromatography. The used solutions of trimethylsilyl derivatives of individual monosaccharidcs, monosaccharides and silylation agents were described in a paper by Wurst et al. (1974). The isolated polysaccharide was hydrolyzed with l~~ H C1 in a sealed ampoule for 5 h at 105 ~ The method was modified in such a w a y that the hydrolysate was filtered through a bacteriological filter G3 prior to evaporation. Trimethylsilyl (TMS) derivatives of monosaccharides were prepared using a method suitable for silylation of sugars and related compounds (Sweeley et al., 1963). The evaporate of the sample of the hydroiyze4 polysacchaxide was first dissolved in warm pyridine and only then was p)~'idine with silylation agents added. The mixture of TMS derivatives of monosaccharides was analysed in an F 21 gas chromatograph (Perkin-Elmer, Norwalk, Conn., USA). Columns were filled with Chromosorb P of a grain size of 0.15--0.17 mm (Johns Manville, Denver, Col., USA) impregnated with 10% DC 200 silicone oil (Dow Corning, Midland, Michigan, USA). Separation proceeded isothermically at 190 ~ Carrier gas was nitrogen at a flow rate of 20 ml per min (Wurst et al., 1974).
14
J. K O N I ~ E K
Vol. 22
ET AL.
R E S U L T S A N D DISCUSSION
Previous results (Lasik and KoniSek, 1976) indicated t h a t nitrosoguanidine at a concentration of 1 mg per ml at ]oH 6.0 exhibits a pronounced lethal effect on the strain of Xanthomonas fuscans from the beginning of the t r e a t m e n t ; after a 10 rain t r e a t m e n t about 60/o cells survive and after 20 min the lethality of cells is higher t h a n 99%. For the induction of lac + m u t a n t s of the strain Xanthomonas fuscans TABLE I. Yields o f p o l y s a c c h a r i d e s a f t e r a 96 h submerged cultivation of the parent strain of Xanthomonas fuscans in a liquid W H m e d i u m w i t h 2 ~ glucose a n d o f t h e lac + m u t a n t s in a liquid W H m e d i u m w i t h 2 % lactose (calculation p e r a d d e d sugar) Yield p e r a d d e d s u g a r
Xanthomcnas fuscan8
Parent Mutant Mutant Mutant Mutant Mutant Mutant
%
24.0 20.0 12.0 17.0 9.1 10.8 9.7
s t r a i n lay lac + No. 1 lac + No. 2 lac + No. 3 lac + No. 4 lac + No. 5 lac + No. 6
intervals of t r e a t m e n t with nitrosoguanidine yielding survival lower t h a n 10~/o were used in the present work. Nitrosoguanidine was also used at a final concentration of 1 mg per ml at pH 6.0. A total of 6 non-reverting lac + m u t a n t s were obtained, out of t h e m 3 were isolated at a 2~o survival and 3 at a survival lower t h a n 1%. M u t a n t strains growing on the medium containing tryptose form after a 3 d a y inTAlZI,E I I . C h r o m a t o g r a p h i c d a t a a n d c o m p o s i t i o n o f ' t h e e x o c e l l u l a r p o l y s a c c h a r i d e (~o) of t h e p a r e n t s t r a i n o f Xanthomonas fuecans a n d m u t a n t / a c + s t r a i n s . Xardhomonas fuscans
Monosacoharides (TMS d e r i v a t i v e s )
Elution ratio a)
lac+
lac
1 6-Deoxy-L-mannose D-Ribose ~-D-Manno8o ~-D-Galaotosc 0~-D- (]luoos0 ~-D-Manncse ~-D-Glucose D-Galacturonic a~id D-Gluouronic acid
0.49 0.53 1.00 1.22 1.36 1.45 1.90 1.68 2.14
V g (~-D-mannose)
305
1.1 1.0 35.7
26.1 6.9 29.2
1.8 0.6 41.6 1.0 20.0 9.8 25.2
I
2 0.9 0.8 41.9 1.0 20.3 10.6 24.5
I
3 0.3 2.3 47.7 1.3 18.5 9.4 20.5
I
4 0.7 2.8 32.2 2.1 20.1 8.8 32.9 0.4
r
5 1.0 0.8 43.8 1.5 19.1 9.9 23.9 0.1
I
6b 1.1 3.3 39.7 1.4 19.7 10.3 24.5
