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Amylase as an extracellular enzyme from plasmodia of myxomycetes
Myral. Res. 95 (7): 885-896 (1991)
Printed in Great Britain
885
SHORT COMMUNICATIONS
Amylase as an extracellular enzyme from plasmodia of myxomycetes
N. MUBARAK ALI AND INDIRA KAL YANASUNDARAM Cenlre for Advanced Siudy in Bolany, Universily of Madras, GUindy Campus, Madras 600 025, India
In the course of our studies on the nature of the relationship between plasmodia of myxomycetes and their bacterial associates, we found that the plasmodia of Physarum flavicomum and Slemonilis herbalica were able to degrade starch in starch agar plates, whereas their bacterial associates Flavobacterium breve and Paracaccus denilrificans in pure culture could not do so, suggesting a dependence of bacterial associate on the plasmodium for utilization of a complex substrate. Further, a wide zone of starch clearance around the plasmodium in P. flavicomum indicated that the enzyme was extracellular. The significance of this finding is that the production of extracellular amylase by myxomycetes is being reported for the first time, and it suggests that the myxomycetes may have an absorptive mode of nutrition besides the ingestive or holozoic mode generally ascribed to them.
During recent studies made in our laboratory, it was found that there was a certain specificity with regard to the bacterial species associated with plasmodia of different species of Myxomycetes, and the bacterial associates were transmitted to successive generations through the spores (Rahman, 1987). For this reason a symbiotic relationship was suggested, the bacteria being termed 'bacterial associates '. The aim of the present work was mainly to study the nature of the relationship between myxomycete plasmodia and their bacterial associates. Our first hypothesis was that the plasmodia and their bacterial associates might be interdependent for the enzymatic degradation of complex substrates. We have initially tested this with starch, since in culture Myxomycetes grow well on starch-containing media like oatmeal agar and com-meal agar. The cultures were taken from the culture collection being maintained by the second author, and included Physarum flavicomum Berk. (MUBL/IK/6) and Slemonitis herhatica Peck (MUBL/IK/5). The bacterial associates were isolated by inoculating I cm discs cut out from 7-day-old cultures of the plasmodia on carrot agar plates, into nutrient broth. The broth cultures after overnight incubation were streaked on nutrient agar plates and individual colonies picked out and pUrified. After various biochemical tests, the bacteria were identified using Bergey's manual (Buchanan & Gibbons, 1974). The bacterial associate of P. flavicomum was identified as Flavobacterium breve (Lustig) Homes & Owen and that of S. herbatica as Paracoccus denitrijicans (Beijerinck) Davis. As we were not able to get the plasmodia in axenic culture, we decided to test the monoxenic plasmodial culture and the pure culture of the bacterial associate separately for starch degradation, to find out which of the two organisms had this ability. This was done by growing the organism on plates of Czapek-Dox agar in which the sucrose was replaced by soluble starch (E. Merck) at the same concentration (30 g 1-1).
Discs of plasmodium cut out from carrot agar plates were inoculated in the centre, and parallel sets were streaked with bacterial inoculum from 24-h cultures on nutrient broth. Fresh plates in triplicate were inoculated daily up to 10 d, so as to have plasmodia of different ages for simultaneous testing. The cultures were incubated at 27°C. In addition, bacterial cultures were also incubated at 37°. Parallel bacterial inocula were also placed in tubes of starch fermentation broth for verification, as Czapek-Dox agar did not support good growth of bacteria, even with sucrose as a carbon source. After 24 h of bacterial growth, and after 24 h to 10 days of
A
.L
B
C
Fig. 1. Amylase production by 7-day-old plasmodia of Physarum [lavicamum. A, uninoculated control; B, C plasmodia on starch agar; both sets stained with iodine-potassium iodide. Note wide zones of clearance around the plasmodia.
886
Short Communications
Fig. 2. Amylase production by plasmodium of Siemoniiis herbatica at
different ages, starting from the 6th day. Starch degradation is complete by the loth day. The culture of bacterial associate (bottom left) looks no different from the controL indicating lack of enzyme.
(Fig. I). In the case of 5. herbafica, the plasmodium being an aphanoplasmodium, this was not very clear but the zone of clearance seemed to be restricted to areas of plasmodial growth (Fig. 2). From the foregoing results, two factors emerge as significant. In the plasmodial-bacterial associations tested by us, the plasmodia have the ability to break down the substrate, namely starch, whereas the bacteria lack this ability and apparently utilize the simpler degradation products. However, we cannot at this stage ascribe such a relationship with respect to all plasmodia, nor to all substrates. The second factor is that the plasmodia are capable of producing extra-cellular amylase. To the authors' knowledge, this is the first report of extra-cellular amylase from myxomycete plasmodia, even though starch-containing media have long been used in their culture. The taxonomic significance of this finding, in addition to earlier reports of extra-cellular enzymes (FaIT ef aI., 1974; Kilpatrick & Stirling, 1977), is to indicate the possibility of absorptive nutrition in the myxomycetes, besides the ingestive (holozoic) mode that is generally ascribed to them. Such a statement would put their systematic position once again in the melting pot, but it is too early at this stage to comment on their affinities. The authors thank the University Grants Commission, New Delhi, for the award of research project F3/44/86.
plasmodial growth, the plates were flooded with a freshly prepared solution of iodine-potassium iodide (iodine, I g; potassium iodide, 2 g; water, 300 ml). Zones of starch clearance were seen only in plasmodial cultures, indicating inability of bacteria to degrade starch. This was supported by the negative result in the broth tubes, where there was no colour change. In the case of P. flavicomum, starch degradation was observed from the 7th day and in the case of 5. herbafica, from the 6th day from the date of inoculation. A very interesting feature here was that the zone of clearance of starch extended far beyond the edges of the plasmodium in the case of P. flavicomum, indicating that the enzyme had diffused into the agar for a considerable distance
REFERENCES Buchanan. R. E. & Gibbons. N. E. (eds.) (1974). Bergey's Manual of Determinative Bacteriology. 8th ed. Baltimore, U.s.A.: Williams & Wilkins. Farr. D., Horisberger, M. & Jolles, P. (1974). An extracellular rennin-like enzyme produced by Physarum polycephalum. Biochimica eI Biophysica Acta 334, 410-416. Kilpatrick, D. C. & Stirling, J. L. (1977). Glycosidases from the culture medium of Physarum polycephalum. Biochemical Journal 161, 149-160. Rahman, Sanjida (1987). Laboratory cultivation and nutrition of Myxomycetes: association with bacteria. PhD. Thesis, University of Madras.
(Received for publication 22 November 1989 and in revised form 15 November 1990)
Printed in Great Britain
885
SHORT COMMUNICATIONS
Amylase as an extracellular enzyme from plasmodia of myxomycetes
N. MUBARAK ALI AND INDIRA KAL YANASUNDARAM Cenlre for Advanced Siudy in Bolany, Universily of Madras, GUindy Campus, Madras 600 025, India
In the course of our studies on the nature of the relationship between plasmodia of myxomycetes and their bacterial associates, we found that the plasmodia of Physarum flavicomum and Slemonilis herbalica were able to degrade starch in starch agar plates, whereas their bacterial associates Flavobacterium breve and Paracaccus denilrificans in pure culture could not do so, suggesting a dependence of bacterial associate on the plasmodium for utilization of a complex substrate. Further, a wide zone of starch clearance around the plasmodium in P. flavicomum indicated that the enzyme was extracellular. The significance of this finding is that the production of extracellular amylase by myxomycetes is being reported for the first time, and it suggests that the myxomycetes may have an absorptive mode of nutrition besides the ingestive or holozoic mode generally ascribed to them.
During recent studies made in our laboratory, it was found that there was a certain specificity with regard to the bacterial species associated with plasmodia of different species of Myxomycetes, and the bacterial associates were transmitted to successive generations through the spores (Rahman, 1987). For this reason a symbiotic relationship was suggested, the bacteria being termed 'bacterial associates '. The aim of the present work was mainly to study the nature of the relationship between myxomycete plasmodia and their bacterial associates. Our first hypothesis was that the plasmodia and their bacterial associates might be interdependent for the enzymatic degradation of complex substrates. We have initially tested this with starch, since in culture Myxomycetes grow well on starch-containing media like oatmeal agar and com-meal agar. The cultures were taken from the culture collection being maintained by the second author, and included Physarum flavicomum Berk. (MUBL/IK/6) and Slemonitis herhatica Peck (MUBL/IK/5). The bacterial associates were isolated by inoculating I cm discs cut out from 7-day-old cultures of the plasmodia on carrot agar plates, into nutrient broth. The broth cultures after overnight incubation were streaked on nutrient agar plates and individual colonies picked out and pUrified. After various biochemical tests, the bacteria were identified using Bergey's manual (Buchanan & Gibbons, 1974). The bacterial associate of P. flavicomum was identified as Flavobacterium breve (Lustig) Homes & Owen and that of S. herbatica as Paracoccus denitrijicans (Beijerinck) Davis. As we were not able to get the plasmodia in axenic culture, we decided to test the monoxenic plasmodial culture and the pure culture of the bacterial associate separately for starch degradation, to find out which of the two organisms had this ability. This was done by growing the organism on plates of Czapek-Dox agar in which the sucrose was replaced by soluble starch (E. Merck) at the same concentration (30 g 1-1).
Discs of plasmodium cut out from carrot agar plates were inoculated in the centre, and parallel sets were streaked with bacterial inoculum from 24-h cultures on nutrient broth. Fresh plates in triplicate were inoculated daily up to 10 d, so as to have plasmodia of different ages for simultaneous testing. The cultures were incubated at 27°C. In addition, bacterial cultures were also incubated at 37°. Parallel bacterial inocula were also placed in tubes of starch fermentation broth for verification, as Czapek-Dox agar did not support good growth of bacteria, even with sucrose as a carbon source. After 24 h of bacterial growth, and after 24 h to 10 days of
A
.L
B
C
Fig. 1. Amylase production by 7-day-old plasmodia of Physarum [lavicamum. A, uninoculated control; B, C plasmodia on starch agar; both sets stained with iodine-potassium iodide. Note wide zones of clearance around the plasmodia.
886
Short Communications
Fig. 2. Amylase production by plasmodium of Siemoniiis herbatica at
different ages, starting from the 6th day. Starch degradation is complete by the loth day. The culture of bacterial associate (bottom left) looks no different from the controL indicating lack of enzyme.
(Fig. I). In the case of 5. herbafica, the plasmodium being an aphanoplasmodium, this was not very clear but the zone of clearance seemed to be restricted to areas of plasmodial growth (Fig. 2). From the foregoing results, two factors emerge as significant. In the plasmodial-bacterial associations tested by us, the plasmodia have the ability to break down the substrate, namely starch, whereas the bacteria lack this ability and apparently utilize the simpler degradation products. However, we cannot at this stage ascribe such a relationship with respect to all plasmodia, nor to all substrates. The second factor is that the plasmodia are capable of producing extra-cellular amylase. To the authors' knowledge, this is the first report of extra-cellular amylase from myxomycete plasmodia, even though starch-containing media have long been used in their culture. The taxonomic significance of this finding, in addition to earlier reports of extra-cellular enzymes (FaIT ef aI., 1974; Kilpatrick & Stirling, 1977), is to indicate the possibility of absorptive nutrition in the myxomycetes, besides the ingestive (holozoic) mode that is generally ascribed to them. Such a statement would put their systematic position once again in the melting pot, but it is too early at this stage to comment on their affinities. The authors thank the University Grants Commission, New Delhi, for the award of research project F3/44/86.
plasmodial growth, the plates were flooded with a freshly prepared solution of iodine-potassium iodide (iodine, I g; potassium iodide, 2 g; water, 300 ml). Zones of starch clearance were seen only in plasmodial cultures, indicating inability of bacteria to degrade starch. This was supported by the negative result in the broth tubes, where there was no colour change. In the case of P. flavicomum, starch degradation was observed from the 7th day and in the case of 5. herbafica, from the 6th day from the date of inoculation. A very interesting feature here was that the zone of clearance of starch extended far beyond the edges of the plasmodium in the case of P. flavicomum, indicating that the enzyme had diffused into the agar for a considerable distance
REFERENCES Buchanan. R. E. & Gibbons. N. E. (eds.) (1974). Bergey's Manual of Determinative Bacteriology. 8th ed. Baltimore, U.s.A.: Williams & Wilkins. Farr. D., Horisberger, M. & Jolles, P. (1974). An extracellular rennin-like enzyme produced by Physarum polycephalum. Biochimica eI Biophysica Acta 334, 410-416. Kilpatrick, D. C. & Stirling, J. L. (1977). Glycosidases from the culture medium of Physarum polycephalum. Biochemical Journal 161, 149-160. Rahman, Sanjida (1987). Laboratory cultivation and nutrition of Myxomycetes: association with bacteria. PhD. Thesis, University of Madras.
(Received for publication 22 November 1989 and in revised form 15 November 1990)