Peanut agglutinin induced alterations in capsular and extracellular polysaccharide synthesis and ex -planta nitrogenase activity of cowpea rhizobia*

Transcription

1 J. Biosci., Vol. 12, Number 3, September 1987, pp Printed in India. Peanut agglutinin induced alterations in capsular and extracellular polysaccharide synthesis and ex -planta nitrogenase activity of cowpea rhizobia* BIΝΑ MODY and VINOD MODI Microbiology and Biotechnology Centre, Faculty of Science, Μ. S. University, Baroda , India MS received 18 May 1987; revised 29 July 1987 Abstract. The root exudate of Arachis hypogea (groundnut) and its seed lectin peanut agglutinin were found to stimulate the synthesis of exopolysaccharide and capsular polysaccharide of the microsymbiont cowpea Rhizobium strain JLn (c). The synthesis of capsular polysaccharide was enhanced 1 5-fold and 2-fold in the presence of peanut agglutinin and root exudate, respectively. The synthesis of capsular polysaccharide was suppressed in the presence of different forms of combined nitrogen. Quantitative differences were also detected between the exopolysaccharide of cells grown in the presence and absence of root exudate. Electron microscopic examination of negatively stained lectin-treated JLn (c) cells showed an increased deposition of capsular polysaccharide surrounding the cells. Hurthermore, ex planta nitrogenase activity of JLn(c) cells in the presence of lectin was found to be enhanced by 63% in correlation with the increased synthesis of polysaccharides. Keywords. Lectin; groundnut; root exudate; acetylene reduction; Rhizobium; combined nitrogen; gel filtration. Introduction Symbiotic nitrogen-fixing bacteria are specific in their ability to infect roots of their leguminous host plants. The specificity of infection is manifested by the carbohydrate constituents present on the rhizobial cell surface which recognize the corresponding legume lectin (Dazzo, 1980). In Rhizobium japonicum, the surface component involved in the specific binding of soybean lectin has been found to be either exopolysaccharide (EPS), capsular polysaccharide (CPS) (Bhuvaneswari et al., 1977; Tsien and Schmidt, 1977; Kamberger, 1979; Apte and Modi, 1983) or lipopolysaccharide (LPS) (Wolpert and Albersheim, 1976; Kato et al., 1979) or a combination of these. Recently, various workers have been investigating the effect of substances excreted by roots on the early events which mediate infection and nodulation (Bhagwat and Thomas, 1982, 1983, 1984; Halverson and Stacey, 1984a, b, 1985, 1986a). Halverson and Stacey (1986a) showed that Bradyrhizobium japonicum when treated with lectin or root exudate showed a prompt nodulation response, which led them to assume that the ability to nodulate faster may be a result of changes in the cell surface polysaccharides. The present study provides experimental evidences indicating *Part of this work was presented at the colloquium session of the 4th Hederation of Asian and Oceanian Biochemists Congress, held at Singapore, in November To whom all correspondence should be addressed, Abbreviations used: EPS, Exopolysaccharide; CPS, capsular polysaccharide; LPS, lipopolysaccharide; PNA, peanut agglutinin; YEM, yeast extract mannitol. 289

2 290 Bina Mody and Vinod Modi changes in the cell surface polysaccharides of cowpea rhizobia in response to peanut agglutinin (PNA) and host root exudate. Materials and methods Cultures Cowpea Rhizobium strains JLn(c) and RA-1 (Modi et al., 1985) were our laboratory isolates and strain NC-92 was obtained from ICRISAT, Hyderabad. The cultures were grown in modified yeast extract mannitol (YEM) broth (Vincent, 1970) on a rotary shaker (116 rpm) at 29 C (± 1 C) till they attained late log phase. PNA PNA was extracted from the seeds of Arachis hypogea and purified by (NH 4 ) 2 SO 4 precipitation followed by DEAE-cellulose and Sephadex G-150 column chromatography according to the method of Lotan et al. (1975). The purified PNA was used for further experiments. CPS CPS was extracted from the cell pellet of late log phase cultures by suspending it in 0 5 Μ NaCl and putting it on a shaker for 2 h at room temperature. Supernatant was collected after centrifugation at 20,000 g for 20 min. CPS was precipitated from the supernatant by adding 3 volumes of cold ethanol (70%), dissolved in distilled water, dialysed overnight and estimated by the phenol-sulphuric acid method (Ashwell, 1966). Chromatographic fractionation of EPS EPS was extracted by acetone precipitation of the supernatant obtained from centrifuged late log phase cultures. This precipitated EPS was dissolved in distilled water and fractionated on a Sepharose-4B gel column ( cm). Samples containing 0 9 and 1 5 mg of EPS fro cells grown in the presence and absence of root exudate respectively were loaded on the column. Elution was performed with 10 mm phosphate buffer, ph 7 2, at a flow rate of 3 ml in 9 min. All the fractions were analysed by the phenol-sulphuric acid method for estimation of EPS using glucose as a standard (Ashwell, 1966). Collection of root exudates A. hypogea seeds were first surface sterilized and allowed to germinate in agar petri plates for 5 days under sterile conditions. Seedlings were then suspended on a perforated steel stand specially devised for collection of root exudate in 250 ml beakers containing Jensen's solution (Jensen, 1942). Plants were grown under sterile conditions for a period of 10 days after germination. Root exudate was collected by centrifugation for 20 min at 12,000 g and concentrated approximately 50-fold by

3 Alterations in polysaccharide synthesis and nitrogenase activity 291 lyophilization at 40 C to give 56 μg/ml of protein. This concentrated root exudate was used for further experiments. Treatment of cowpea rhizobia with PNA and root exudates Cowpea rhizobia were grown in Vincent's modified YEM broth until early log phase (12 h) and centrifuged at 20,000 g for 20 min. Cells were resuspended in 15 ml of fresh YEM broth to give a cell density of 10 9 cells/ml. This suspension was then divided into 3 equal fractions. To one of the fractions, 2 5 ml of PNA (40 μg protein/ml) was added, while another was supplemented with 2 5 ml of root exudate (56 μg protein/ml). To the third fraction, 2 5 ml of fresh YEM broth was added to serve as an unsupplemented control. PNA and root exudate solutions were filter-sterilized prior to addition and their protein content was estimated by the method of Bradford (1976). The cultures were further incubated at 29 C for 10 h, till they reached the stationary phase. CPS and EPS were estimated after separating them by high speed centrifugation at 40,000 g for 40 min. Assay for nitrogenase activity Nitrogenase activity was measured by the acetylene reduction assay procedure. JLn(c) cells were grown microaerobically in LNB-5 medium at 29 C (± 1 C). Microaerobic condition was created by flushing sealed tubes containing 2 ml of LNB-5 broth with nitrogen gas. After 20 h of growth, 1 ml of acetylene gas was injected into the tubes and incubation continued for 1 h at room temperature. From the headspace, 1 ml of gas was withdrawn and analysed by gas chromatography using a Chromosorb 105 column. Electron microscopy Negative staining of late log phase JLn (c) cells was carried out with 2% ammonium molybdate by the drop method for whole cells placed on an electron microscopic grid. In brief, a drop of bacterial suspension was deposited onto a carbon and Formvar-coated grid, cells were allowed to adsorb for 5 min and the free-standing suspension was subsequently removed by blotting with filter paper. Adsorbed cells were stained with 2% ammonium molybdate for 60 s. Samples were examined in a JEOL 100 SX electron microscope at 60 or 80 kv. Specimens were examined at field magnifications varying from X3,000 to X40,000. Results and discussion Two independent findings (Bhuvaneswari and Bauer, 1978; Dazzo et al., 1981) have suggested the possible influence of host root environment on the synthesis or modification of polysaccharides which serve as lectin receptors on the rhizobial cell surface. We observed that the root exudate of peanut plants stimulates EPS synthesis in cowpea Rhizobium strains JLn(c),.RA-1 and NC-92 by about 2 36-, and 2 45-fold, respectively (table 1). These findings support the earlier observation of Bhagwat and Thomas (1982) that cowpea root exudate elicits a faster nodulation response, increased polysaccharide synthesis and greater infectivity of cowpea rhizobia. When cells were grown in the presence of root exudate, qualitative changes in EPS were observed by chromatographic fractionation on a Sepharose-4B column

4 292 Bina Mody and Vinod Modi Table 1. Effect of PNA and root exudate on the synthesis of EPS by different cowpea rhizobia. Values in parantheses indicate relative synthesis of EPS, per cent of control. Values are means of 3 replications. a Amounts of PNA and root exudate added to the cell suspensions were equivalent to 100 and 140 µg protein, respectively. Figure 1. Purification of EPS of Rhizobium sp. JLn (c) on Sepharose-4B column. Acidic EPS of JLn (c) cells grown in the absence (O) and presence ( ) of root exudate. (figure 1). The elution patterns obtained showed an additional peak (C) of high molecular weight polysaccharide in the sample treated with root exudate as compared to the EPS from control cells which showed two peaks (A and B). Enhanced EPS synthesis was also observed for different cowpea Rhizobium strains grown in the presence of PNA (40 µg/ml) (table 1). CPS synthesis in strain JLn(c) was also found to be enhanced about 1 5 and 2-fold in the presence of PNA and root exudate, respectively (table 2). Since the lectin elicited an activation response analogous to that by the root exudate, it is likely that the active component present in the root exudate may be a lectin. The observation regarding the presence of lectin trifoliin A in root exudate by Dazzo et al. (1982) lends further support to this assumeption. These results provide direct evidence to support Halverson and Stacey's (1986a) prediction regarding the alteration of CPS in B.japonicum by soybean lectin. CPS synthesis was considerably reduced in the presence of combined nitrogen in the form of KNO 3 (20 mm), NH 4 C1 (20 mm), NH 4 NO 3 (10 mm) and glutamine (20 mm). In the absence of root exudate or PNA, these nitrogen compounds caused different degrees of inhibition in CPS synthesis (table 2). This inhibitory effect of combined nitrogen on CPS synthesis was partly overcome by the addition of PNA or root exudate. Soybean lectin was unable to cause significant increase in the

5 Alterations in polysaccharide synthesis and nitrogenase activity 293 Table 2. Effect of PNA, root exudate and combined nitrogen on the synthesis of CPS by Rhizobium sp. JLn(c). Numbers in parantheses indicate relative synthesis of CPS, per cent control. Values are the means of 3 replications. a Amounts of PNA and root exudate added to the cell suspensions were equivalent to 100 and 140 µg protein, respectively. ND, Not determined. Table 3. Effect of lectin on capsular and extracellular polysaccharide production by cowpea Rhizobium sp. JLn (c). Numbers in parantneses indicate relative syntiiesis of EPS, per cent of control. a Amounts of PNA and SBL added to the cell suspensions were equivalent to 100 and 140 µg protein, respectively. synthesis of either CPS or EPS, emphasizing the specificity exhibited by PNA towards its homologouss symbiont (table 3). These findings along with the reported effect of combined nitrogen on the symbiotic properties of legume root secretion (Bhagwat and Thomas, 1984) suggest that the nitrogen status of the rhizosphere regulates the infectivity of the symbiont by controlling its polysaccharide synthesis. Previous work of Dazzo and Brill (1978) has ndicated a similar inhibitory effect of combined nitrogen (when added to the rooting medium) on the levels of clover lectin trifoliin A present on the root surface which determines host specificity to infection. Similarly, Halverson and Stacey (1986b) have shown an inhibitory effect of combined nitrogen on the activity of soybean root exudate to prompt nodulation. Both these observations point to a common characteristic of combined nitrogen in indirectly reducing host susceptibility to infection by altering root exudate properties. Close on these observations, the present study suggests a direct influence of combined nitrogen on polysaccharide synthesis on the microsymbiont, as shown in table 2.

6 294 Bina Mody and Vinod Modi Figure 2. Transmission electron micrograph of negatively stained JLn(c) cells. (A) Untreated cells and (B) cells incubated in the presence of PNA for 16 h. ( 18,000). Biochemical observations regarding the effects of lectin on EPS and CPS synthesis were further confirmed by electron microscopic studies. When JLn(c) cells were incubated m the presence of PNA, increased amounts of EPS were observed in the

7 Alterations in polysaccharide synthesis and nitrogenase activity 295 medium and an electron dense capsule surrounding the cells was evident (figure 2). A direct correlation has been implicated between nitrogenase activity and the synthesis of EPS (Kurz and LaRue, 1975; Pagan et al., 1975). It is possible therefore that PNA may affect both these parameters simultaneously. When JLn (c) cells were grown in the presence of different concentrations of PNA, 30 µg/ml of PNA was found to be the optimum concentration; this caused 63% increase in nitrogenase activity and 68% increase in EPS synthesis control (figure 3). Figure 3. Effect of PNA on EPS production ( ) and acetylene reduction (O) by JLn(c) cells. Cells were grown in the presence of different concentrations of PNA. Control of C 2 H 2 reducing activity under microaerobic conditions, but in the absence of PNA, is presented as 0 µg/ml of PNA. On the basis of the above observations, we suggest that lectin probably determines early events in the infection process by selectively enhancing the synthesis of specific sugars on the bacterial cells. The presence of these sugars is a pre-requisite for establishment of successful infection. The nitrogen status of the rhizosphere soil has also been found to affect the synthesis of rhizobial polysaccharides, the presence of combined nitrogen masking the possible effect lectin and root exudate. Thus, the root environment of the host has a critical role to play during the initial phase of symbiosis by inducing some alteration on the rhizobial cell surface, thereby dictating pre-nodulation events. Acknowledgement One of the authors (B. M.) wishes to thank the Council of Scientific and Industrial Research, New Delhi, for the award of a fellowship. References Apte, R. and Modi, V. (1983) Experientia, 39, 509. Ashwell, G. (1966) Methods Enzymol, 8, 85. Bhagwat, A. A. and Thomas, J. (1982) Appl. Environ. Microbiol., 43, 800. Bhagwat, A. A. and Thomas, J. (1983) Arch. Microbiol., 136, 102. Bhagwat, A. A. and Thomas, J. (1984) Arch. Microbiol., 140, 260.

8 296 Bina Mody and Vinod Modi Bhuvaneswari, T. V., Pueppke, S. G. and Bauer, W. D. (1977) Plant Physiol, 60, 486. Bhuvaneswari, T. V. and Bauer, W. D. (1978) Plant Physiol., 62, 71. Bradford, M. M. (1976) Anal. Biochem., 72, 248. Dazzo, F. B. (1980) in The cell surface: mediator of development processes (eds S. Subtelny and N. Wessells) (New York: Academic Press) p Dazzo, F. B., Hrabak, E., Urbano, M., Sherwood, J. and Truchet, G. (1981) in Current Perspectives in Nitrogen Fixation (eds W., Newton and A. Gibson) (Canberra: Australian Academy of Science) p Dazzo, F. B. and Brill, W. J. (1978) Plant Physiol,, 62, 18. Dazzo, F. B., Truchet, G. L, Sherwood, J. Ε., Hrabak, Ε. Μ. and Gardiol, Α. Ε. (1982) Appl. Environ. Microbiol., 44, 478. Halverson, L. J. and Stacey, G. (1984a) Plant Physiol., 74, 84. Halverson, L. J. and Stacey, G. (1984b) in Advances in nitrogen fixation research (eds R. Veegar and W. Newton) (Hague: Nyhoff Publishers) p Halverson, L. J. and Stacey, G. (1985) Plant Physiol, 77, 621. Halverson, L. J. and Stacey, G. (1986a) Appl. Environ. Microbiol, 51, 753. Halverson, L. J. and Stacey, G. (1986b) Microbiol. Rev., 50, 193. Jensen, Η. L. (1942) Proc. Linn. Soc. Ν. S. W., 66, 98. Kamberger, W. (1979) Arch. Microbiol, 121, 83. Kato, G., Maruyama, Υ. and Nakamura, Μ. (1979) Agric. Biol Chem., 43, Kurz, W. G. W. and LaRue, Τ. Α. (1975) Nature (London) 256, 407. Lotan, R., Skutelsky, E., Danon, D. and Sharon, N. (1975) J. Biol. Chem., 250, Modi, M., Shah, K. and Modi, V. (1985) Arch. Microbiol, 141, 156. Pagan, J. D., Child, J. J., Scowcroft, W. R. and Gileson, A. H. (1975) Nature (London) 256, 406. Tsien, H. C. and Schmidt, E. L. (1977) Can. J. Microbiol, 23, Wolpert, J. S. and Albersheim, P. (1976) Biochem. Biophys. Res. Commun., 70, 729. Vincent, J. Μ. (1970) A manual for the practical study of the root nodule bacteria (Oxford: Blackwell Scientific Publications).