- Email: [email protected]
Concentration of molybdenum by extra-cellular material produced by rhizosphere bacteria
Soil Bid
Biochrm.
Vol. 8, pp. 461 to 464
Pergmmn
Press 1976.
Printed m GreatBrltaln.
CONCENTRATION OF MOLYBDENUM BY EXTRACELLULAR MATERIAL PRODUCED BY RHIZOSPHERE BACTERIA E. L. TAN and MARGARET W. LOCTIT Microbiology Department.
University of Otago. Dunedin. New Zealand (Actrptrtl 7 24pril 1976)
Summary--Rhizosphere bacteria have been found to concentrate molybdenum (MO). Investigations of where in the cell this MO is bound have shown that the MO is mainly bound by a passive process to the extra-cellular cell lavers. Some MO is bound to the cell walls but most is bound to the extra, cellular polysaccharide, probably to uranic acids.
INTRODUCTION
Following the observation that rhizosphere bacteria influence the concentration of molybdenum (MO) in plants, sometimes causing a lowered concentration in the plant (Loutit et al., 1968; Loutit and Brooks. 1970; Loutit et al.. 1972) various postulates have been advanced to explain this effect. An observation that rhizosphere bacteria were able to concentrate MO (Loutit et al., 1967) supports one postulate, but has led to the question as to where in the cells the MO is stored. This paper reports the results of experiments designed to answer this question. MATERIALS AND METHODS Bacteria
Isolates from the rhizospheres of plants shown to have a lowered concentration of MO due to the activity of rhizosphere bacteria (Loutit et al., 1972) were chosen for study. These were strains of Bacillus, Pseudomonas, Nocardiu, Achronwbacter (Alcaligenes), Flavobacterium, Arthrobacter and Streptomyces.
iment. the cells were deposited by centrifugation, washed twice in 0.85”” NaCl and resuspended in a minimal quantity of doubly-distilled water and placed in weighed silica crucibles. After drying for 20 h at 8o’C the cells were ashed at 500 C for 10 h and the ash prepared for atomic absorption.
The ash of each culture was dissolved in 0.4 ml 3 N HCl and to this was added 0.2 ml. 0.5 mg A13’.ml-’ from a stock solution of Al (N03)3 and 0.4 ml 100”” acetone. Solutions were filtered through cotton-wool plugged 1 ml syringes to remove any coarse particles of carbon matter. The filtered solutions were then sprayed into a Techtron AA6 atomic absorption spectrophotometer set at 313.3 nm (i). A series of standards for MO ranging from @lO/cg.ml- ’ was also sprayed in and a standard graph of absorption vs MO plotted. The technique for preparation of the cells was developed by Mr. R. Malthus of the MRC Biochemistry Research Group and was designed to prevent interference due to lead.
Media and solutions
Glucose peptone yeast extract medium (GPY); gelysate peptone (BBL) 20 g, glucose 10 g, NaCl 5 g, yeast extract 10 g, distilled water 1 l., pH 6.8. Liquid slime promoting medium (SP) (Goto et al., 1973). Molybdenum solution. a stock solution of NazMoO,. 2H20 (BDH) Analar grade containing 1 mg Mo.ml-‘. Preparation
of inocula
Cultures held in semi-solid medium (Loutit et al., 1972) were streaked on to GPY medium solidified with 1.5% agar (Davis) and single colonies picked off into 1Oml GPY medium and incubated for 40 h at 28°C. Uptake of Mo by cells
Ehrlenmeyer flasks (250 ml) containing lOOm1 of GPY medium with and without MO 10 pg.ml- 1 were incubated in a shaking water bath at 28°C. In some experiments incubation was at 4”C, in others at 20°C. After incubation, the time varying with the exper461
Cultures were grown as described for 96 h and after centrifuging and washing twice in 0.85”. saline the suspensions were resuspended in doubly-distilled water and the cell walls obtained by a modification of the method of Yamaguchi (1965). The cells were disrupted by sonication. and following centrifugation at 200g for 30 min the sediment was discarded. Further centrifugation at 650~ for 30min followed and the supernatant was discarded and the sediment repeatedly washed with 0.85”” NaCl and finally with doubly-distilled water. The cell wall material was then heated at 1WC for 10min. Isokftion
of‘ extra-crllulur
materitrl
Cultures were grown as described but in SP medium (Goto et al., 1973) for 96 h. The slimy extracellular material was removed by first subjecting the cultures to 30 s in a blender followed by centrifugation at 200 g for 2 h at 2O~C to remove the cells. The slime was precipitated with an equal volume of ethanol (Brown rt (11..19691. In other experiments the
E. L. TAN and MARGARET W. LOUTI’I
462
slime was purified by dissolving in water and insoluble material was removed by centrifugation. An equal volume of a lo”,,; aqueous solution of cetyltrimethyl-ammonium bromide was added to the solution (Srivastava et al., 1962). The acidic polysaccharide was precipitated as an insoluble complex which was collected by centrifugation, washed with water and dissolved in 2M NaCl (Kita rt [II., 1974). Insoluble material was removed by centrifugation and discarded. Ethanol was added to precipitate the slime which was washed with 90”,, ethanol. Hydrolysis and ,fiacactionutiott oj’ Pseudomonas cellular material
e.ww-
Slime obtained as described was hydrolysed by boiling in 1 N HCl with Dowex 50 ( x 4; H+ form) ion exchange resin 20&W mesh (Clamp and Putnam, 1964). To the hydrolysate was added an equal volume of a stock solution of MO 50 ILg.ml- ’ and the mixture left overnight at room temperature. The mixture (5 ml) was layered on to a Sephadex G-25 (fine) column (Pharmacia, Uppsala, Sweden) which had been equilibrated with citric acid-phosphate buffer at pH 3. The same buffer was used as elutant and 2.0ml fractions collected. MO in each fraction was determined by atomic absorption spectrophotometry but before this the absorption spectrum of each fraction was determined in a Shimadzu Double beam spectrophotometer U.V. 200 using 1 cm cuvettes and with citric acicLphosphate as the reference solution. The solution was scanned between 700 and 350nm and then from 45&200nm on U.V. scan. Total carbohydrate in each fraction was determined by anthrone reagent (Herbert et al.. 1971). Total protein in each fraction was estimated by the method of Schacterle and Pollack (1973). Uranic acid was determined with carbazole (Dische. 1955). To ensure that MO was bound to the carbohydrate fraction and not the protein fraction of the exopolysaccharide, the extracellular material was treated with cetyl methylammonium bromide (Srivastava et a/.. 1962) to precipitate out the acidic polysaccharide. The insoluble material obtained was washed three times with doubly-distilled water and then dissolved in 2~ NaCl (Kita et ~1.. 1974) and equal volumes of 50 pg MO. ml 1 added. The mixture was subjected to the treatment outlined above for the whole slime. The eluted fractions were subjected to the same procedures. RESULTS The various strains of bacteria were grown with and without MO in GPY medium which had been found to support the growth of all strains. Many of the isolates grew slowly and estimation of MO in the cells after varying periods of incubation up to 96 h, showed that MO was concentrated late in the growth cycle (Table 1). In other experiments strains were grown at 28 ‘C for 96 h and sets of flasks placed at 4’ C, 20 C and 28°C to equilibrate. MO was added to give a final concentration of 10 pg.ml- ‘. All flasks were then incubated with shaking at the stated temperature for a further 4 h. The cells were harvested and their MO estimated. The results in Table 2 indicate that the
Table 1. Molybdenum terial strains
grown
in pg.g-’ cell dry weight of bacin GPY medium after incubation for 30 or 96hr at 28 C 30h With MO*
96 h With MO
5.0 9.2
28.0 34.0 18.7
Bwillus Pseudomorws Nocurditr
12.0
Ackrottwhucter
15.5
ErwitG .Arthrohoclrr Slreptomwes
41.3 33.2
5.1 6.0 7.0
10.0 25.9
* 10.0 (~g MO. ml 1 added as sodium molybdate. No MO detected at the 0.01 pg MO. ml 1 concentration in controls where no MO was added. Table 2. Concentration incubated at varying
P.SelldotlW?lU.S Nocurdia Achromohucter
of MO in jLg.g-’ cell dry weight temperatures after addition of MO
28 C With MO*
20 ‘C With MO
4c With MO
32.5 20.5 42.5
24.6 21.4 39.5
10.0 23.4 34.8
* IO pg Mo.ml- ’ added as sodium molybdate. No MO detected at the 0.01 pg Mo.ml- 1 concentration in controls where no MO was added.
concentration of MO by the cells was predominantly a passive process except in the case of Pseudomonas where some MO apparently entered by an active process. A chance observation that the amount of MO concentrated by Nocardia increased approximately two and a half fold when grown in GPY medium minus glucose and the fact that the culture appeared slimy led us to examine the cells under the electron microscope. The cells were seen to be surrounded by a diffuse layer but when grown in the presence of glucose no such layer was evident. These observations coupled with the finding that uptake of MO occurred late in the growth cycle. that the process was largely passive and the report that extracellular polysaccharide accumulates late in the growth cycle (Sutherland, 1972) led us to speculate that MO was being concentrated in the extra-cellular polysaccharide layers. The possibility that MO was being concentrated in the cell wall could not be excluded. A series of experiments was begun therefore, to test these ideas. Cells were grown in the presence or absence of MO and their Table 3. Molybdenum in ccg.g- ’ dry weight in whole cells and cell walls of bacteria grown in GPY media after incubation for 96 h at 38 C
Pseud0motru.s Nocurdicr Achrorrwhwter
Whole cells With MO*
Walls With MO
34.0 18.7 41.3
10.5 14.6 23.2
* 10 LLgMo.ml- 1 added as sodium molybdate. No MO detected at the 0.01 pg MO .ml~ 1 concentration in controls to which no MO was added.
463
Molybdenum binding to extracellular polysaccharide
3.0
2.0
-
k is 9
1.0
----
CAR3OtMlRAlE MO
0 fmctlon number Fig. 1. Distribution
of molybdenum
and carbohydrate
in fractionated
extracellular
material from
Pseudormnas.
cell walls harvested. Although some MO was bound to the walls the amount was not equal to that bound to whole cells (Table 3). A Pseudomonas strain was then grown in the presence and absence of MO, in SP medium, and the extra-cellular layers of polysaccharide extracted and MO estimated. MO was also estimated in the cells from which polysaccharide had been removed. Two points emerge from these experiments. When grown in SP medium the amount of MO bound to the cells was greater, 65.8 pg Mo.g-’ dry weight, than when grown in GPY medium, 34 pg Mo.g-‘. Most of the MO was bound to the slime, 358.06 pg Mo.g-’ dry weight, compared with 32.1 pg Mo.g-’ in dry weight of cells from which slime had been removed. The SP medium increased the amount of 3.0
slime produced by the cells and’most of the MO was bound to this slime. As the separation of slime and cells was purely mechanical it is unlikely that leaching of MO from the cells to the slime would account for the high concentrations of MO in the slime. In a further series of experiments the extracellular slime was extracted from cells grown with and without MO and the slime fractionated as described. MO was estimated in the fractions as was protein and carbohydrate. From Fig. 1 it is clear that MO was bound to a particular fraction of the slime and the fraction which contained the highest concentration of MO also showed the highest carbohydrate concentration. Trace amounts of protein were detected in each fraction but there was no correlation between protein and MO concentration. To make quite sure that MO was binding to carbo-
r
2.0 I 2 r”
31
9 ._ 14 -
-----
CARBOHYDRATE MO
120 fraction number
Fig. 2. Distribution of molybdenum and carbohydrate in acidic polysaccharide fraction of extracellular material from Pseudomonas.
E. L. TAN and MARGARI:T W. LOUTIT
464
hydrate, acidic polysaccharide was precipitated and MO added. The complex was then hydrolysed and fractionated and MO estimated in each fraction. Again the largest amount of MO was found in the fraction containing the largest amount of polysaccharide (Fig. 2). No protein was detected in any fraction. In both experiments involving isolation and fractionation of extra-cellular polysaccharide the fraction in which most MO was found also gave the strongest reaction for uranic acid. No uranic acid was detected in fractions lacking MO. DISCL’SSION The observation that bacterial cells could concentrate large amounts of MO posed a puzzle as to where in the cells such large amounts of MO could be stored. It has been known for some time that some sheathed bacteria stored Mn and Fe in their sheaths (Bergey, 1974). Dugan and Parson (1971) reported that Zoogalore rurnigcrtr II5 formed a capsular matrix which concentrated Cu. Co, Fe, Ni and Zn. Tornabere (1972) found that the effectiveness of Azotohac~rr in immobilizing Pb could be related to the amount of capsular material surrounding the cells. Many bacteria however. do not form definitive capsules or sheaths but a number do appear to produce extracellular slimes as the present report shows. That these slimes concentrate MO and probably other metals is of interest particularly in relation to the finding that bacteria affect entry of MO to plants (Loutit et al., 1968; Loutit and Brooks. 1970). As bacteria on the root surface appear to be surrounded by mucilage (Rovira. 1974) it is of interest to investigate whether binding to the extracellular polysaccharide produced by rhizosphere bacteria is a means by which MO is prevented from entering the root. The role of uranic acid in this binding requires further investigation.
.4clirlo~clcti~er,l~/l/-This worh was supported in part by a grant from the DSIR through the Applied Biochemistry
Division.
REFERENCES BERCEY (1974) Brrqey’s Mumrtrl of’ Drtrrruincrtiw Bwfrriology 8th edn. (R. E. Buchanan and N. E. Gibbons. Eds.) Williams & Wilkins Co.. Baltimore. BROWN M. R. W.. FOSTER J. H. S. and CI.AMP J. R. (1969) Composition of P.sru4lonwru1.s trrruginosu slime. Bidwm. J. 112, 521 515.
CLAMP J. R. and POSTMANF. W. (1964) The carbohydrate J. hiol. Cl~r?n. prosthetic group of human y-globulin. 239, 3233-3243. DIS~HF Z. (1955) Sugars in polysaccharides. In Mcfk0ds of Biochcwicul .Am~l!xi~ (D. Glich Ed.) 2. 3 I3 358. Interscience. New York. DVC;AN P. R. and PARRSONA. B. (1971) Production of extracellular polysaccharides matrix in Zoog/loen mmiyrru. .Appl. ,2lic,ohio/. 21. 657 661. Go~o S.. MURAKAU’A T. and KUMHARA S. (1973) Slime production by P.\c,rrt/on~or~as trt~uc/inostr. J. Microhid. JUtwi. 17. 45 51. HERBERT D.. PHIPPS P. J. and STRANGER. E. (1971) Chemical analvsis of microbial cells. In ~Vrthods irl Microhioloy!, 5 (J. Norris and D. W. Robbins Eds.) Academic Press. New York. KITA Y.. SEIZI I. and ISONO M. (1974) Chemical structure of an acidic polysaccharide produced by Serrtrtia pisctr~WWJI. Ctrrhoh,u/. Res. 38, 239m 346. LOUTIT M. W. and BROOKS R. R. (1967) Differences in molybdenum uptake by microorganisms from the rhizosphere of Rtrp/w~us .mrirus L grown in two soils of similar origin. PI. Soil 27. 335 34%. LOUTIT M. W. and BROOKS R. R. (1970) Rhizosphere organisms and MO concentration in plants. .Soi/‘Bio/. LOUTIT M. W. and BROOKS R. R. (1970) Rhizosohere organisms and MO concentration in plants. Soil’Biol. LOUT‘IT M. W.. HILLAS H. and SPEAKS G. F. S. (1972) Studies on rhizosphere organisms and MO concent;ation in plants. Identification of rhizosphere isolates to generic level. SC/ Bid. Biocht. 4, 267-270. LOUTII M. W.. MAL~IIUS R. S. and LOUTIT J. S. (196X) The cfiect of soil microorganisms on the concentration of molybdenum in the radish (RupI~ttm~ ,sutims L) variety “White Icicle”. J. ,4yric,. Res. N.Z. 11, 42W34. ROREM E. S. (1955) Uptake of rubidium and phosphorus ions by polysaccharide producing bacteria. J. Burt. 70, 691-701. ROVIRA A. D. (1974) Scanning electron micrographs of microorganisms on the roots of wheat. b4icrohiol. Ed. 1. 15 13. SCHA(.~RLE G. R. and POLLAC~: R. L. (1973) A simplified method for the quantitative assay of small amounts of protein in biological material. .4nn/. &o&z. 51, 654656. SKIVASIAVA H. C.. BR~( NIUG~R L.. GRUECH H. J. and ADAMS G. A. (1962) Preparation and properties of polysaccharlde~ lipid complex from Scrrtrti~r I~IU~~‘LW~VI.S.Cm. J. hioc/wr~~. P/l?siol. 40, 905 91X. SUTHERLAND I. W. (1972) Bacterial exopolysaccharide. At/r. hliuo. Physiol. 8, 143-713. TORNAHERI:T. G. (1972) Microbial uptake of lead. Science 176, 13341335. YAMAGUCIII T. (1965) Comparison of the cell wall composition of morphological distinct Actinomycete. .I. Butt. 89, 444453.
Biochrm.
Vol. 8, pp. 461 to 464
Pergmmn
Press 1976.
Printed m GreatBrltaln.
CONCENTRATION OF MOLYBDENUM BY EXTRACELLULAR MATERIAL PRODUCED BY RHIZOSPHERE BACTERIA E. L. TAN and MARGARET W. LOCTIT Microbiology Department.
University of Otago. Dunedin. New Zealand (Actrptrtl 7 24pril 1976)
Summary--Rhizosphere bacteria have been found to concentrate molybdenum (MO). Investigations of where in the cell this MO is bound have shown that the MO is mainly bound by a passive process to the extra-cellular cell lavers. Some MO is bound to the cell walls but most is bound to the extra, cellular polysaccharide, probably to uranic acids.
INTRODUCTION
Following the observation that rhizosphere bacteria influence the concentration of molybdenum (MO) in plants, sometimes causing a lowered concentration in the plant (Loutit et al., 1968; Loutit and Brooks. 1970; Loutit et al.. 1972) various postulates have been advanced to explain this effect. An observation that rhizosphere bacteria were able to concentrate MO (Loutit et al., 1967) supports one postulate, but has led to the question as to where in the cells the MO is stored. This paper reports the results of experiments designed to answer this question. MATERIALS AND METHODS Bacteria
Isolates from the rhizospheres of plants shown to have a lowered concentration of MO due to the activity of rhizosphere bacteria (Loutit et al., 1972) were chosen for study. These were strains of Bacillus, Pseudomonas, Nocardiu, Achronwbacter (Alcaligenes), Flavobacterium, Arthrobacter and Streptomyces.
iment. the cells were deposited by centrifugation, washed twice in 0.85”” NaCl and resuspended in a minimal quantity of doubly-distilled water and placed in weighed silica crucibles. After drying for 20 h at 8o’C the cells were ashed at 500 C for 10 h and the ash prepared for atomic absorption.
The ash of each culture was dissolved in 0.4 ml 3 N HCl and to this was added 0.2 ml. 0.5 mg A13’.ml-’ from a stock solution of Al (N03)3 and 0.4 ml 100”” acetone. Solutions were filtered through cotton-wool plugged 1 ml syringes to remove any coarse particles of carbon matter. The filtered solutions were then sprayed into a Techtron AA6 atomic absorption spectrophotometer set at 313.3 nm (i). A series of standards for MO ranging from @lO/cg.ml- ’ was also sprayed in and a standard graph of absorption vs MO plotted. The technique for preparation of the cells was developed by Mr. R. Malthus of the MRC Biochemistry Research Group and was designed to prevent interference due to lead.
Media and solutions
Glucose peptone yeast extract medium (GPY); gelysate peptone (BBL) 20 g, glucose 10 g, NaCl 5 g, yeast extract 10 g, distilled water 1 l., pH 6.8. Liquid slime promoting medium (SP) (Goto et al., 1973). Molybdenum solution. a stock solution of NazMoO,. 2H20 (BDH) Analar grade containing 1 mg Mo.ml-‘. Preparation
of inocula
Cultures held in semi-solid medium (Loutit et al., 1972) were streaked on to GPY medium solidified with 1.5% agar (Davis) and single colonies picked off into 1Oml GPY medium and incubated for 40 h at 28°C. Uptake of Mo by cells
Ehrlenmeyer flasks (250 ml) containing lOOm1 of GPY medium with and without MO 10 pg.ml- 1 were incubated in a shaking water bath at 28°C. In some experiments incubation was at 4”C, in others at 20°C. After incubation, the time varying with the exper461
Cultures were grown as described for 96 h and after centrifuging and washing twice in 0.85”. saline the suspensions were resuspended in doubly-distilled water and the cell walls obtained by a modification of the method of Yamaguchi (1965). The cells were disrupted by sonication. and following centrifugation at 200g for 30 min the sediment was discarded. Further centrifugation at 650~ for 30min followed and the supernatant was discarded and the sediment repeatedly washed with 0.85”” NaCl and finally with doubly-distilled water. The cell wall material was then heated at 1WC for 10min. Isokftion
of‘ extra-crllulur
materitrl
Cultures were grown as described but in SP medium (Goto et al., 1973) for 96 h. The slimy extracellular material was removed by first subjecting the cultures to 30 s in a blender followed by centrifugation at 200 g for 2 h at 2O~C to remove the cells. The slime was precipitated with an equal volume of ethanol (Brown rt (11..19691. In other experiments the
E. L. TAN and MARGARET W. LOUTI’I
462
slime was purified by dissolving in water and insoluble material was removed by centrifugation. An equal volume of a lo”,,; aqueous solution of cetyltrimethyl-ammonium bromide was added to the solution (Srivastava et al., 1962). The acidic polysaccharide was precipitated as an insoluble complex which was collected by centrifugation, washed with water and dissolved in 2M NaCl (Kita rt [II., 1974). Insoluble material was removed by centrifugation and discarded. Ethanol was added to precipitate the slime which was washed with 90”,, ethanol. Hydrolysis and ,fiacactionutiott oj’ Pseudomonas cellular material
e.ww-
Slime obtained as described was hydrolysed by boiling in 1 N HCl with Dowex 50 ( x 4; H+ form) ion exchange resin 20&W mesh (Clamp and Putnam, 1964). To the hydrolysate was added an equal volume of a stock solution of MO 50 ILg.ml- ’ and the mixture left overnight at room temperature. The mixture (5 ml) was layered on to a Sephadex G-25 (fine) column (Pharmacia, Uppsala, Sweden) which had been equilibrated with citric acid-phosphate buffer at pH 3. The same buffer was used as elutant and 2.0ml fractions collected. MO in each fraction was determined by atomic absorption spectrophotometry but before this the absorption spectrum of each fraction was determined in a Shimadzu Double beam spectrophotometer U.V. 200 using 1 cm cuvettes and with citric acicLphosphate as the reference solution. The solution was scanned between 700 and 350nm and then from 45&200nm on U.V. scan. Total carbohydrate in each fraction was determined by anthrone reagent (Herbert et al.. 1971). Total protein in each fraction was estimated by the method of Schacterle and Pollack (1973). Uranic acid was determined with carbazole (Dische. 1955). To ensure that MO was bound to the carbohydrate fraction and not the protein fraction of the exopolysaccharide, the extracellular material was treated with cetyl methylammonium bromide (Srivastava et a/.. 1962) to precipitate out the acidic polysaccharide. The insoluble material obtained was washed three times with doubly-distilled water and then dissolved in 2~ NaCl (Kita et ~1.. 1974) and equal volumes of 50 pg MO. ml 1 added. The mixture was subjected to the treatment outlined above for the whole slime. The eluted fractions were subjected to the same procedures. RESULTS The various strains of bacteria were grown with and without MO in GPY medium which had been found to support the growth of all strains. Many of the isolates grew slowly and estimation of MO in the cells after varying periods of incubation up to 96 h, showed that MO was concentrated late in the growth cycle (Table 1). In other experiments strains were grown at 28 ‘C for 96 h and sets of flasks placed at 4’ C, 20 C and 28°C to equilibrate. MO was added to give a final concentration of 10 pg.ml- ‘. All flasks were then incubated with shaking at the stated temperature for a further 4 h. The cells were harvested and their MO estimated. The results in Table 2 indicate that the
Table 1. Molybdenum terial strains
grown
in pg.g-’ cell dry weight of bacin GPY medium after incubation for 30 or 96hr at 28 C 30h With MO*
96 h With MO
5.0 9.2
28.0 34.0 18.7
Bwillus Pseudomorws Nocurditr
12.0
Ackrottwhucter
15.5
ErwitG .Arthrohoclrr Slreptomwes
41.3 33.2
5.1 6.0 7.0
10.0 25.9
* 10.0 (~g MO. ml 1 added as sodium molybdate. No MO detected at the 0.01 pg MO. ml 1 concentration in controls where no MO was added. Table 2. Concentration incubated at varying
P.SelldotlW?lU.S Nocurdia Achromohucter
of MO in jLg.g-’ cell dry weight temperatures after addition of MO
28 C With MO*
20 ‘C With MO
4c With MO
32.5 20.5 42.5
24.6 21.4 39.5
10.0 23.4 34.8
* IO pg Mo.ml- ’ added as sodium molybdate. No MO detected at the 0.01 pg Mo.ml- 1 concentration in controls where no MO was added.
concentration of MO by the cells was predominantly a passive process except in the case of Pseudomonas where some MO apparently entered by an active process. A chance observation that the amount of MO concentrated by Nocardia increased approximately two and a half fold when grown in GPY medium minus glucose and the fact that the culture appeared slimy led us to examine the cells under the electron microscope. The cells were seen to be surrounded by a diffuse layer but when grown in the presence of glucose no such layer was evident. These observations coupled with the finding that uptake of MO occurred late in the growth cycle. that the process was largely passive and the report that extracellular polysaccharide accumulates late in the growth cycle (Sutherland, 1972) led us to speculate that MO was being concentrated in the extra-cellular polysaccharide layers. The possibility that MO was being concentrated in the cell wall could not be excluded. A series of experiments was begun therefore, to test these ideas. Cells were grown in the presence or absence of MO and their Table 3. Molybdenum in ccg.g- ’ dry weight in whole cells and cell walls of bacteria grown in GPY media after incubation for 96 h at 38 C
Pseud0motru.s Nocurdicr Achrorrwhwter
Whole cells With MO*
Walls With MO
34.0 18.7 41.3
10.5 14.6 23.2
* 10 LLgMo.ml- 1 added as sodium molybdate. No MO detected at the 0.01 pg MO .ml~ 1 concentration in controls to which no MO was added.
463
Molybdenum binding to extracellular polysaccharide
3.0
2.0
-
k is 9
1.0
----
CAR3OtMlRAlE MO
0 fmctlon number Fig. 1. Distribution
of molybdenum
and carbohydrate
in fractionated
extracellular
material from
Pseudormnas.
cell walls harvested. Although some MO was bound to the walls the amount was not equal to that bound to whole cells (Table 3). A Pseudomonas strain was then grown in the presence and absence of MO, in SP medium, and the extra-cellular layers of polysaccharide extracted and MO estimated. MO was also estimated in the cells from which polysaccharide had been removed. Two points emerge from these experiments. When grown in SP medium the amount of MO bound to the cells was greater, 65.8 pg Mo.g-’ dry weight, than when grown in GPY medium, 34 pg Mo.g-‘. Most of the MO was bound to the slime, 358.06 pg Mo.g-’ dry weight, compared with 32.1 pg Mo.g-’ in dry weight of cells from which slime had been removed. The SP medium increased the amount of 3.0
slime produced by the cells and’most of the MO was bound to this slime. As the separation of slime and cells was purely mechanical it is unlikely that leaching of MO from the cells to the slime would account for the high concentrations of MO in the slime. In a further series of experiments the extracellular slime was extracted from cells grown with and without MO and the slime fractionated as described. MO was estimated in the fractions as was protein and carbohydrate. From Fig. 1 it is clear that MO was bound to a particular fraction of the slime and the fraction which contained the highest concentration of MO also showed the highest carbohydrate concentration. Trace amounts of protein were detected in each fraction but there was no correlation between protein and MO concentration. To make quite sure that MO was binding to carbo-
r
2.0 I 2 r”
31
9 ._ 14 -
-----
CARBOHYDRATE MO
120 fraction number
Fig. 2. Distribution of molybdenum and carbohydrate in acidic polysaccharide fraction of extracellular material from Pseudomonas.
E. L. TAN and MARGARI:T W. LOUTIT
464
hydrate, acidic polysaccharide was precipitated and MO added. The complex was then hydrolysed and fractionated and MO estimated in each fraction. Again the largest amount of MO was found in the fraction containing the largest amount of polysaccharide (Fig. 2). No protein was detected in any fraction. In both experiments involving isolation and fractionation of extra-cellular polysaccharide the fraction in which most MO was found also gave the strongest reaction for uranic acid. No uranic acid was detected in fractions lacking MO. DISCL’SSION The observation that bacterial cells could concentrate large amounts of MO posed a puzzle as to where in the cells such large amounts of MO could be stored. It has been known for some time that some sheathed bacteria stored Mn and Fe in their sheaths (Bergey, 1974). Dugan and Parson (1971) reported that Zoogalore rurnigcrtr II5 formed a capsular matrix which concentrated Cu. Co, Fe, Ni and Zn. Tornabere (1972) found that the effectiveness of Azotohac~rr in immobilizing Pb could be related to the amount of capsular material surrounding the cells. Many bacteria however. do not form definitive capsules or sheaths but a number do appear to produce extracellular slimes as the present report shows. That these slimes concentrate MO and probably other metals is of interest particularly in relation to the finding that bacteria affect entry of MO to plants (Loutit et al., 1968; Loutit and Brooks. 1970). As bacteria on the root surface appear to be surrounded by mucilage (Rovira. 1974) it is of interest to investigate whether binding to the extracellular polysaccharide produced by rhizosphere bacteria is a means by which MO is prevented from entering the root. The role of uranic acid in this binding requires further investigation.
.4clirlo~clcti~er,l~/l/-This worh was supported in part by a grant from the DSIR through the Applied Biochemistry
Division.
REFERENCES BERCEY (1974) Brrqey’s Mumrtrl of’ Drtrrruincrtiw Bwfrriology 8th edn. (R. E. Buchanan and N. E. Gibbons. Eds.) Williams & Wilkins Co.. Baltimore. BROWN M. R. W.. FOSTER J. H. S. and CI.AMP J. R. (1969) Composition of P.sru4lonwru1.s trrruginosu slime. Bidwm. J. 112, 521 515.
CLAMP J. R. and POSTMANF. W. (1964) The carbohydrate J. hiol. Cl~r?n. prosthetic group of human y-globulin. 239, 3233-3243. DIS~HF Z. (1955) Sugars in polysaccharides. In Mcfk0ds of Biochcwicul .Am~l!xi~ (D. Glich Ed.) 2. 3 I3 358. Interscience. New York. DVC;AN P. R. and PARRSONA. B. (1971) Production of extracellular polysaccharides matrix in Zoog/loen mmiyrru. .Appl. ,2lic,ohio/. 21. 657 661. Go~o S.. MURAKAU’A T. and KUMHARA S. (1973) Slime production by P.\c,rrt/on~or~as trt~uc/inostr. J. Microhid. JUtwi. 17. 45 51. HERBERT D.. PHIPPS P. J. and STRANGER. E. (1971) Chemical analvsis of microbial cells. In ~Vrthods irl Microhioloy!, 5 (J. Norris and D. W. Robbins Eds.) Academic Press. New York. KITA Y.. SEIZI I. and ISONO M. (1974) Chemical structure of an acidic polysaccharide produced by Serrtrtia pisctr~WWJI. Ctrrhoh,u/. Res. 38, 239m 346. LOUTIT M. W. and BROOKS R. R. (1967) Differences in molybdenum uptake by microorganisms from the rhizosphere of Rtrp/w~us .mrirus L grown in two soils of similar origin. PI. Soil 27. 335 34%. LOUTIT M. W. and BROOKS R. R. (1970) Rhizosphere organisms and MO concentration in plants. .Soi/‘Bio/. LOUTIT M. W. and BROOKS R. R. (1970) Rhizosohere organisms and MO concentration in plants. Soil’Biol. LOUT‘IT M. W.. HILLAS H. and SPEAKS G. F. S. (1972) Studies on rhizosphere organisms and MO concent;ation in plants. Identification of rhizosphere isolates to generic level. SC/ Bid. Biocht. 4, 267-270. LOUTII M. W.. MAL~IIUS R. S. and LOUTIT J. S. (196X) The cfiect of soil microorganisms on the concentration of molybdenum in the radish (RupI~ttm~ ,sutims L) variety “White Icicle”. J. ,4yric,. Res. N.Z. 11, 42W34. ROREM E. S. (1955) Uptake of rubidium and phosphorus ions by polysaccharide producing bacteria. J. Burt. 70, 691-701. ROVIRA A. D. (1974) Scanning electron micrographs of microorganisms on the roots of wheat. b4icrohiol. Ed. 1. 15 13. SCHA(.~RLE G. R. and POLLAC~: R. L. (1973) A simplified method for the quantitative assay of small amounts of protein in biological material. .4nn/. &o&z. 51, 654656. SKIVASIAVA H. C.. BR~( NIUG~R L.. GRUECH H. J. and ADAMS G. A. (1962) Preparation and properties of polysaccharlde~ lipid complex from Scrrtrti~r I~IU~~‘LW~VI.S.Cm. J. hioc/wr~~. P/l?siol. 40, 905 91X. SUTHERLAND I. W. (1972) Bacterial exopolysaccharide. At/r. hliuo. Physiol. 8, 143-713. TORNAHERI:T. G. (1972) Microbial uptake of lead. Science 176, 13341335. YAMAGUCIII T. (1965) Comparison of the cell wall composition of morphological distinct Actinomycete. .I. Butt. 89, 444453.