Soil Bid. Biochem. Vol. 28, No. 7, pp. 917-921, 1996 Copyright 0 19% Elsevier science Ltd Pergamon Printed in Great Britain. Au rights resewed PII: !30038-0717(%)ooos9-4 0038-0717/96$15.00 + 0.00 CELL-BOUND CELLULASE AND POLYGALACTURONASE PRODUCTION BY RHIZOBIUM AND BRAD YRHIZOBIUM SPECIES JOSE I. JIMENEZ-ZURDO,’ PEDRQ F. MATEOS,‘* FRANK B. DAZZO” and E. MARTINEZ-MOLINA’ ‘Departamento de Microbiologia y Genetica, Facultad de Farmacia, Fkiificio Departamental, Universidad de Salamanca, Salamanca 37007, Spain and 2Department of Microbiology and Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, U.S.A. (Accepted 12 February 1996) Summary-Carboxymethyl cellulase and polygalacturonase activities were evaluated in wild-type strains from different taxonomic groups of rhizobia which nodulate a specific range of legume hosts that do not normally overlap (R. meliloti, R. loti, R. leguminosarumbiovar trifolii, R. leguminosarumbiovar phaseoli, R. leguminosarum biovar viceae, and Bradyrhizobium japonicum). To detect these enzymatic activities we used various methods, a double-layer plate assay, quantitation of reducing sugar products with a bicinchoninate reagent, and viscometric assay. CM-cellulase activity was found in extracts from all the rhizobia tested. In contrast, polygalacturonase activity was only found in R. leguminosarumbiovar trifolii and R. meliloti. CM-cellulase and polygalacturonase enzymes were cell-bound and not detected in the extracellular culture medium. CM-cellulase isozymes were examined using activity gel electrophoresis. The results obtained shown a heterogeneity of CM-cellulase isozymes among the strains tested. These findings are consistent with the hypothesis of an involvement of rhizobial cellulolytic enzymes in the invasion of legumes by Rhizobium. Copyright 0 1996 Elsevier Science Ltd INTRODUCTION locally induced in the plant by components of the bacteria. The precise mechanism whereby Rhizobium success- The infection of legume roots by Rhizobium is a fully infects temperate legumes remains unknown. delicately balanced process (Dazzo and Hubbell, Nutman (1956) proposed that rhizobia invade root 1982); the infected root hair must remain intact and hairs by an invagination process of the plant cell wall, viable, otherwise infection would abort. An involve- which implies that there is no real penetration of the ment of wall-degrading enzymes would require that bacteria into the root cell. Ljunggren and Fghraeus their activity be regulated to remain very low and (1961) proposed that polygalacturonase production localized at the site of infection in order to avoid by plant root cells, induced by homologous strains destruction of the root hair. Although there is of Rhizobium, would result in an increase in plant indirect evidence for the involvement of hydrolytic cell wall softening at the infection site on the root enzymes in the infection process (Martinez-Molina hair; thus allowing the bacteria to penetrate the cell and Olivares, 1982; Al-Mallah et al., 1987; Chaliflour membrane and initiate an infection thread. The and Benhamou, 1989; Al-Mallah et al., 1989), little is ability of Rhizobium meliloti to induce polygalactur- known about these enzymes in Rhizobium. Some onase production by alfalfa roots is linked to a studies failed to demonstrate the production of plasmid carried by the bacteria (Palomares et al., these enzymes by different species of Rhizobium 1978). (McCoy, 1932; Hunter and Elkan, 1975), but Electron microscopic studies of the infection several studies have detected pectinolytic (Hubbell process have showed a localized degradation of the et al., 1978; Martinez-Molina and Olivares, 1982; root hair wall, suggesting the involvement of cell wall Plazinski and Rolfe, 1985; Angle, 1986; Prasuna and hydrolytic enzymes in the penetration process Ali, 1987; Saleh-Rastin et al., 1991), cellulolytic (Higashi and Abe, 1980; Callaham and Torrey, (Martinez-Molina et al., 1979; Morales et al., 1984; 1981; Ridge and Rolfe, 1985; Turgeon and Bauer, Saleh-Rastin et al., 199 1) and hemicellulolytic 1985). The main point of divergence would be the (Martinez-Molina et al., 1979; Lopez and Signer, possibility that the wall-degrading enzymes involved 1987) enzyme activities from pure cultures of in the process are associated with the bacteria or rhizobia. In general, the activity of these rhizobial enzymes is very low and at the limit of sensitivity of conventional reducing sugars and plate assays; this *Author for correspondence. has hampered biochemical study of these polysaccha- 917 918 Jose I. Jimenez-Zurdo et al. ride-degrading enzymes and research aimed at Mateos et al. (1992). In addition, the BCA reducing determining their role in the establishment of the sugar assay was modified as follows. The reaction symbiotic interaction. mixtures contained 0.4 ml of 1% carboxymethyl Mateos et al. (1992) developed assays with cellulose (CMC) or 0.5% sodium polygalacturonate improved sensitivity and reliability to detect and (NaPp) solution, 0.4 ml of the enzyme sample and 0.8 measure cellulolytic and pectinolytic activities; these ml of 200 mM PCA buffer. After the reagents were were used to demonstrate the production of both mixed, an 0.8 ml aliquot (considered T = 0 h) was enzyme activities by different wild-type strains of the frozen at - 80°C and the remaining sample was kept clover root-nodule symbiont, R. leguminosarum at 40°C for 5 h before quantitation of reducing sugar bv. trifolii. These enzymes were cell-bound rather products. Substrates (CMC medium viscosity and than extracellular, and were commonly produced NaPp; both obtained from Sigma Chemical Co., St by several wild-type strains isolated from different Louis, MO) were dialyzed for 2 days against distilled geographical regions. In strain ANU843, at least water before use. two carboxymethyl (CM)-cellulase isozymes and one Cellulolytic and pectinolytic activities were also polygalacturonase isozyme were found. The total and evaluated in cell extracts of the strain R. meliloti specific activities of these cell-bound enzymes in ATCC 9930 produced by treatment with lysozyme- sonicated cell extracts were unaffected by growth EDTA (Mateos et al., 1992). The presence of in defined B-INOS medium supplemented with cytoplasmic constituents in the intracellular and CM-cellulose or polygalacturonic acid substrates, or extracellular fractions of this strain was examined by by flavone 7,4’-dihydroxyflavone which activates measuring NADP-dependent glucose-6-phosphate expression of its pSym nodulation genes (Mateos dehydrogenase (E.C. 1.1.1.49) (Worthington, 1988) a et al., 1992). cytoplasmic enzyme marker. All enzyme samples These findings have prompted us to investigate were stored at 4C. whether production of these polysaccharide-degrad- CM-cellulase activity was also evaluated by ing enzymes by rhizobia is restricted to the clover measuring the decrease in viscosity of a solution of symbiont R. leguminosarum biovar trifolii, or are CMC (Martinez-Molina et al., 1979) using Cannon- commonly made by various species and biovars of the Fenske viscometers. Reaction mixtures for this assay root-nodule bacteria supporting the hypothesis of an contained 5 ml of a 1% solution of CMC in 100 mM involvement of these rhizobial hydrolytic enzymes in PCA buffer and 1 ml of the enzyme extract. the symbiosis. Sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) was performed (Mateos et al., 1992) in a vertical slab unit (Mini-Protean II; MATERIALS AND METHODS Bio-Rad). The separating gel contained 12% The strains of rhizobia used in this study were R. acrylamide and 0.33% bis. The proteins in SDS gels leguminosarum bv. trifolii ANU843 (B. Rolfe, were renatured by keeping for 2 h with shaking in 10 Australian National University), R. melifoti ATCC mM PCA buffer with periodic changes every 30 min. 9930, R. loti ATCC 33669, R. leguminosarum CMC-agarose overlays were constructed using a bv. phaseoli ATCC 14482, R. Ieguminosarum bv. 0.4-mm layer of 0.2% CMC and 0.5% agarose (w/v) oiceae ATCC 10004 and Bradyrhizobium japonicum in 100 ItIM PCA buffer on electrophoresis film ATCC 10324 (ATCC, American Type Culture (Sigma). The running gel was placed on the substrate Collection). overlay and kept in a moist chamber at 37 C. The Broth cultures were grown in 75 ml of B-INOS CMC-agarose overlay was immersed in aqueous defined medium (Mateos et al., 1992) in 250 ml flasks 0.1% Congo red solution for 20 min and rinsed in IM shaken at 125 rev min - ’ at 28°C. Inocula were NaCl to detect areas of enzymatic activity. prepared by suspending cells from 5-day-old plate cultures into sterile B-INOS medium, centrifuging aseptically at 4000 x g for 15 min and resuspending RESULTS AND DISCUSSION in B-INOS medium to an initial population density of 10’ cells ml - ‘. The results of tests to detect CM-cellulase and After 24 h, at 28’C, cells were pelleted by polygalacturonase activities in sonicated cell extracts centrifugation, resuspended in 100 mM potassium from each of these different rhizobia are summarized phosphate-citric acid buffer (PCA; pH 5.0) sonicated in Table I. All of the methods detected CM-cellulase in five cycles of I-min bursts, and recentrifuged. This and polygalacturonase activities from R. legumi- sonicated, cell-free extract and the original super- nosarum biovar trijblii ANU843 as the positive natant of extracellular culture fluid concentrated control. The double layer plate assay detected 15-fold by ultrafiltration using PM 10 membranes CM-cellulase activity in extracts from each test strain (Amicon Co., Danvers, MA, USA) were assayed for except R. meliloti ATCC 9930, but detected CM-cellulase and polygalacturonase activities by polygalacturonase activity with only the positive the double-layer plate assay, and quantitation of control R. leguminosarum biovar trifblii ANU843. reducing sugar products using methods described by Also, no pectate lyase activity was detected by the Cellulase and polygalacturonases in Rbizobium 919 Table 1. CM-ccllulase and polyplacturonase activities of Rhizobiu* CM-cellulasc Polygalacturonasc Rhizobiwntest strain Double layer plate Viscometric’ Reducing sugarsb Double layer plate Reducing sugar@ R.