Ultrastructure of Rhizobium japonicum in Relation to Its Attachment to Root Hairs

Transcription

1 JOURNAL OF BACrERIOLOGY, Mar. 1978, p /78/ $02.00/0 Copyright i 1978 American Society for Microbiology Vol. 133, No. 3 Ultrastructure of Rhizobium japonicum in Relation to Its Attachment to Root Hairs ARYA K. BAL,* S. SHANTHARAM, AND S. RATNAM Department of Biology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada AIB 3X9 Received for publication 7 December 1977 Printed in U.S.A. In Rhizobiumjaponicum strain Nitragin 61A76, morphologically distinct types of bacteria were found to occur in yeast extract-mannitol broth cultures, at both mid-log and stationary phases. Of these only the capsular form, characterized by a smooth cell envelope, storage granules (glycogen and poly-,8-hydroxybutyric acid), and an amorphous extracellular capsule, bound soybean lectin. The binding site was localized in the capsular material. Less than 1% of the bacterial population differentiated into these capsular forms, which were also able to attach to the soybean root hair surface. The nitrogen-fixing bacteria of the genus Rhizobium are known to associate symbiotically with roots of leguminous plants, forming nodules. Lectins commonly found in legume seeds have been implicated in the specific recognition process involving rhizobia and the root hair surface (1, 6, 15, 19, 21, 24, 30, 34). Varying degrees of lectin binding are exhibited by different strains of Rhizobium species (6). Although strains which do not bind lectin are incapable of nodulating soybeans, there is no quantitative relation between effective nodulation and rich or poor lectin-binding bacteria. Therefore, a few lectin-binding bacterial cells seem to be sufficient to cause infection and nodulation. Such a parallel exists also among pathogenic bacteria, where pleomorphism is common and only a small fraction of cells represent the virulent type (1, 8, 11, 25, 32, 35), which are capsulated forms. Such forms also occur in Rhizobium (17), and specific polysaccharides have been detected in their capsules (9). The present study was carried out with a poorly lectin-binding strain of R. japonicum, Nitragin 61A76, which produces effective nodulation in soybean roots. An ultrastructural analysis revealed distinct morphological types, of which only the capsular form was found to bind soybean lectin and attach to the root hair surface. MATERLALS AND METHODS R. japonicum Nitragin 61A76 was grown in yeast extract-mannitol nutrient medium (mannitol, 10 g; K2HPO4, 0.5 g; MgSO4, 0.2 g; NaCl, 0.1 g; yeast extract, 1.0 g; distilled water, 1 liter, ph 6.8 to 7.0) and was maintained as stationary cultures at 23 C. For experiments, cultures were taken at mid-log phase (8 h) and stationary phase (24 h) Electron microscopy. Bacterial cultures at midlog phase and stationary phase were centrifuged, and pellets were fixed (a) in a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde (23) in 0.1 M Sorensen's phosphate buffer, ph 7.2, for 1 h at 23 C and (b) in 2.5% glutaraldehyde in the same buffer under the same conditions. After thorough washing in buffer, the pellet clumps were treated with 1% osmium tetroxide in the same phosphate buffer, ph 7.2, for 1 h at 4 C. This was followed by dehydration in ethanol series and subsequent embedding in Spurr's medium (33). Ultrathin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate. Some sections were also stained with phosphotungstic acid (PTA) and chromic acid mixture (31), and others were stained with periodic acid-lead (29) specific stain for glycogen. For staining of extracellular polysaccharides, ruthenium red (1 mg/ml) was mixed with both aldehydes and osmium tetroxide during fixation (28). For whole-mount preparations bacterial suspensions were mounted on carbon-stabilized Formvar-coated grids and stained with PTA. Observations were made on a Zeiss 9S electron microscope. Soybean lectin binding. For in vivo binding, stationary-phase bacterial cultures were washed in 0.1 M phosphate buffer, ph 7, to remove the nutrient media and treated with soybean lectin (Miles Laboratories) conjugated with ferritin (3) for 1 h at 25 C. After five washes in buffer (repeated pelleting and suspending), the final pellet was fixed in aldehyde fixatives and processed for electron microscopy as above. Pellets prefixed in paraformaldehyde-glutaraldehyde mixture (a) at 0 C were thoroughly washed in cold buffer for 18 h and then treated with ferritin-lectin conjugate for 1 h at 25 C. This was followed by repeated washing in buffer for 18 h at 35 C with continuous shaking (4). After treatment with osmium tetroxide, the pellet clumps were processed for electron microscopy. Attachment of bacteria to soybean root hair. Roots of 5-day-old soybean seedlings grown in sterile vermiculite were placed in a bacterial suspension (10 ml of 24-h bacterial broth culture was added to 490 ml of sterile distilled water; 19 x 108 bacteria/ml, final

2 1394 BAL, SHANTHARAM, AND RATNAM concentration) for 1 h in the dark. The roots were then washed by repeated dipping in a series of five separate 500-ml beakers containing sterile distilled water over a period of 2 min. After washing, free-hand sections were stained with rose bengal. Root hairs showing attached bacteria were counted. Sections of roots were also fixed and processed for electron microscopy. RESULTS R. japonicum Nitragin 61A76 grown in yeast extract-mannitol medium exhibited a typical sigmoid pattern (Fig. 1). It appeared to be a fastgrowing strain, attaining stationary phase at 14 h. Heterogeneity in the bacterial population was evident even at mid-log phase. The typical gramnegative Rhizobium in the mid-log phase of growth had a double-layered envelope (Fig. 2), which represented a majority of the bacteria. The inner and outer cell membranes were separated by a periplasmic space. The outer membrane was differentiated into minute projections (Fig. 3), which could be extremely slender and filamentous (5.5 nm wide), as shown in Fig. 4. Peritrichous flagella (13.8 nm wide; Fig. 6) appeared in sections at random planes (Fig. 3). Whole mounts of bacteria (Fig. 6) showed extracellular filamentous structures and flagella. The outer envelope of the bacteria (Fig. 5) revealed undulations, which had become apparent as a result of the shrinkage of cytoplasm in the median. A variety of morphologically distinct forms of bacteria are shown in Fig Figure 7 represents a population which was characterized by the presence of glycogen granules (g) and the distinctive outer cell envelope projections, which were longer than the other forms described earlier. A different type of population is represented by Fig. 8. The bacterial cells had a smooth outer cell envelope structure, which was PTA negative. Storage granules such as glycogen and poly-,8-hydroxybutyric acid were present. These bacteria showed a rather amorphous extracellular substance, which condensed into a capsule surrounding the cells, as shown in Fig. 10. The capsule in fixed preparation showed somewhat patchy amorphous configuration and was not stainable with ruthenium red. Only less than 1% of the bacterial cells were capsular forms in this strain at 24 h (stationary phase). The bacterial population represented in Fig. 9 revealed an outer thick cell envelope with undulations on the surface comparable to the whole mount in Fig. 5. Only the capsule of the bacteria was found to bind soybean lectin when bacteria were treated with ferritin-labeled soybean lectin (Fig. 12 and 13). The capsular population being very low, F0 'a f 10 TIME IN HOURS FIG. 1. Growth pattern of R. japonicum Nitragin 61A76 in yeast extract-mannitol broth culture (0.1% yeast extract) grown at 23 C. repeated scanning of various parts of the bacterial pellet was necessary to locate such cells. There was no lectin binding in any other form of bacteria. The capsular material appeared filamentous in the in vivo lectin-binding experiment (Fig. 13). The number of root hairs which attached bacteria after exposure to a bacterial suspension was low. Of 1,798 root hairs counted, only about 5% revealed attached bacteria, and the number of bacteria attached to each root hair was highly variable (1 to 16). Bacteria adhered to the surface in a random fashion, and no special point of attachment could be located with a light microscope. Electron microscopy showed that the attached bacteria were all capsular forms (Fig. 11). It was difficult to fix the root hair as well as the bacteria with the same fixative. In our preparations the root hair cell plasmolyzed, and therefore the cytoplasm is not visible in Fig. 11. The bacterial morphology was quite distinct as a result of the presence of capsule, J. BACTERIOL. smooth cell envelope, glycogen, and poly-,f-hydroxybutyric acid granules. DISCUSSION Occurrence of different types of bacteria in pure culture is common (1, 8, 11, 35), but ultrastructural and functional characterization of such types has been made only in recent years (10, 12, 22, 27). In R. japonicum strains very little progress has been made along these lines. Recently, the bacteroidal population of the soybean nodule was analyzed, and four different

3 VOL. 133, 1978 RHIZOBIUM ROOT SURFACE ATTACHMENT I IAb 0 Z-.. Li.-.. 'm Downloaded from FIG Ultrastructure of R. japonicwn Nitragin 61A76 showing typical cell envelope 8tructure with minute projections of the outer cell membrane, which appear very slender and filamentous in Fig. 4. The two cell membranes of the envelope (arrow) are clearly shown in Fig. Z, andprofiles offlagella (arrow) are shown in Fig. 3 (stain, uranyl acetate and lad citrate). functional and developmental types were found to be present (T. M. Ching, S. Hedtke, and W. Newcomb, Plant Physiol. 59(Suppl.):50, 1977). In R. japonicum Nitragin 61A76 broth culture, we have been able to distinguish the following four different morphological types of bacteria: (i) the typical gram-negative form (13) with minute outer cell envelope projections, (ii) a form with glycogen granules and prominent outer cell envelope projections, (iii) a form with smooth cell envelope, glycogen and poly-,8-hydroxybutyric acid granules, and extracellular capsular material, and (iv) cells with a thick cell envelope and undulated outer membrane. Of all these cell types only the capsular bacteria were found to bind soybean lectin. Ferritinconjugated lectin was localized in the capsular material, which is known to be mucopolysaccharide in nature (2, 5, 9, 15-18, 20, 37). The specificity of soybean lectin lies in the presence of galactose residues of the mucopolysaccharides. Although we have not analyzed the capsular on November 14, 2018 by guest

4 1396 BAL, SHANTHARAM, AND RATNAM J. BACTERIOL. I..%. 'o a.i I,] -Agwl v 0.5pm lpm 0 FIG. 5 and 6. Whole mounts of bacteria stained with phosphotungstic acid. Figure 5 shows the undulations ofthe cell envelope. The electron-dense part represents the cytoplasm after shrinkage. Figure 6 shows bacteria with pertrichous flagella and some extracellular filamentous projections. material chemically in this particular strain, the staining property (lack of ruthenium red staining) suggests that the mucopolysaccharide is not acidic in nature (26), confirming earlier reports (20). Rhizobium lipopolysaccharides show specific binding with plant lectins (1, 24, 30, 36), and the lipopolysaccharide fraction of the cell envelope has been emphasized in the specificity of the recognition phenomenon. In our experiments, no lectin binding could be detected in the cell envelope; lectin was bound solely to the capsular material outside the cell envelope. Although it is not known whether this capsule also contains lipopolysaccharide in addition to the mucopolysaccharides, the polysaccharides of the capsule material appear to be the primary binding site of lectin. It should be pointed out that in fixed preparations the capsular material is not seen to be directly in contact with the cell surface. The rather irregular gap between the bacteria and ; the capsule is most likely an artifact of fixation (Fig. 10 and 12). When the lectin-binding experiment was done on unfixed bacteria, the capsular material appeared morphologically different (Fig. 13). A fibrous component was apparent in these preparations. In the unfixed bacteria lectin could bind freely to the available sites, and after thorough washing in buffer the bound lectin would be cross-linked by aldehyde fixation and would withstand extraction during processing for electron microscopy. However, in both experiments, pre- or postfixation did not alter the lectin-binding site. Therefore, the capsular polysaccharides of Rhizobium are the primary binding sites of soybean lectin, and specificity possibly lies in the capsule. Root hair binding experiments also support the above observations. Only the capsular bacteria were found to remain attached to the root hair surface after thorough washing. Very few bacteria were found attached to the root hair, as 0

5 i o-5pm SW tl,, -.-92:... s25i X; it \'w, t_ye -,.n: at.. ^ ffi _ is#^ os >:>mwe *t 7, SE o _s. *\41 S ;....* c _.a c.. +..iip,.f ':,. 15 h:i 6 _J; E e o.5pm o.5pm p;b k \ - F.-N ". 1 m o.5 lam o.5pjm FIG. 7. Ultrastructure of a morphologically distinct type of bacteria with glycogen granules (g) and very prominent outer cell envelope projections (arrow). (Stain, uranyl acetate and lead citrate). FIG. 8. Ultrastructure of a morphologically distinct bacterial type showing smooth cell envelope, glycogen (g), and poly-,8-hydroxybutyric acid (pb) granules. Note also the diffused amorphous extracellular component (arrow). (Stain, uranyl acetate and lead citrate). FIG. 9. Ultrastructure of a morphologically distinct type of bacteria showing thick cell envelopes and undulations on the outer membrane (cf Fig. 5). (Stain, uranyl acetate and lead citrate). FIG. 10. Ultrastructure ofmorphologically distinct bacteria showing smooth cell envelope, storagegranules (glycogen [g] and poly-,8-hydroxybutyric acid [pb]), and amorphous capsular material (c) (cf. Fig. 8) surrounding the bacteria. Note lack of stain in the cell envelope. (Stain, periodic acid-phosphotungstic acid). FIG. 11. Section of root hair surface showing attachment of capsular bacteria (cf. Fig. 10) to the root hair surface (RH). The root hair cytoplasm is plasmolyzed in this fixative and therefore not seen in the figure. Note the capsular material (c) and its attachment to the root hair surface (RH). (Stain, uranyl acetate and lead citrate) ¾% TC

6 .$~ sq_- ; *~~~ -ir's ~ I * &.A±CWtr,4.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. '8~~~~~~~~~~~~~~4 Downloaded from on November 14, 2018 by guest FIG. 12. Capsular bacteria showing ferritin-conjugated lectin (arrow) in the amorphous capsule material. Lectin binding was carried out in fixed bacteria. Note the bacterial cell envelope (phosphotungstic acid negative) and compare with the noncapsulated bacteria (NCB) (stain, periodic acid-phosphotungstic acid). FIG. 13. Capsular bacteria showing ferritin-lectin binding (arrow). In this in vivo experiment,lectin binding was carried out in live bacteria, which were fixed after thorough washing. Note fibrous structure in the capsular material (Stain, uranyl acetate and lead citrate). 1398

7 VOL. 133, 1978 would be expected from a low lectin-binding strain, where only less than 1% of the population possessed capsule. Polarity of attachment as described in some strains (7, 15) was not observed, as the lectin-binding capsule circumscribed the bacteria in this particular strain. Polarized attachment seems to be correlated with a polarized lectin-binding capsule. Involvement of lectin in the binding process implies either that the root hair surface secretes lectins or that there are specific sites in the root hair that have bound lectins. Soybean lectin, being divalent, can bind capsular polysaccharides on one site and some unknown root hair binding site on the other, resulting in attachment. Although glycoproteins have been shown to be secreted by legume roots (14), which have a possible chemotactic function, it is yet to be clarified whether soybean lectin is secreted or is attached to the root hair surface, mediating legume-rhizobium interaction. ACKNOWLEDGMENTS We are thankful to D. P. S. Verma for the preparation of ferritin-lectin conjugate and to J. Gow for helpful suggestions during the course of study. We are also grateful to J. C. Burton, Nitragin Co., for supplying us with the Rhizobium strain. The work was supported by the National Research Council of Canada (operating grant 6207 to A.K.B.). LMRATURE CIMD 1. Albersheim, P., and A. J. Anderson-Prouty Carbohydrates, proteins, cell surfaces and the biochemistry of pathogenesis. Annu. Rev. Plant Physiol. 26: Amarger, N., M. Obaton, and H. Blachere Polysaccharides extracellulaires de Rhizobium meliloti. Can. J. Microbiol. 13: Andres, G. A., K. G. Hsu, and B. C. Seegal Immunologic techniques for the identification of antigens or antibodies by electron microscopy, p In D. M. Weir (ed.), Handbook of experimental immunology. Blackwell Scientific Publications Ltd., Oxford. 4. Bal, A. K., D. P. S. Verma, H. Byrne, and G. A. Maclachlan Subcellular localization of cellulases in auxin-treated pea. J. Cell Biol. 69: Bjorndal, H., C. Erbing, B. Lindberg, G. Fahraeus, and H. Ljunggren Studies on extracellular polysaccharides from Rhizobium meliloti. Acta Chem. Scand. 25: Bohlool, B. B., and E. L. Schmidt Lectins: a possible basis for specificity in the Rhizobium-legume symbiosis. Science 185: Bohlool, B. B., and E. L. Schmidt Immunofluorescent polar tips of Rhizobium japonicum: possible site of attachment for lectin binding. J. Bacteriol. 125: Brinkenhoff, L. A Variation inxanthomonas malvacearum and its relation to control. Annu. Rev. Phytopathol. 8: Chaudhari, A. S., C. T. Bishop, and D. W. Dudman Structural studies on the specific capsular polysaccharide from Rhizobium trifolii TA-1. Carbohydr. Res. 28: Cheng, K.-J., and J. W. Costerton Ultrastructure RHIZOBIUM ROOT SURFACE ATTACHMENT 1399 of cell envelopes of bacteria of the bovine rumen. Appl. Microbiol. 29: Clasener, H Pathogenicity of L-phase of bacteria. Annu. Rev. Microbiol. 26: Costerton, J. W., H. N. Damgaard, and K.-J. Cheng Cell envelope morphology of rumen bacteria. J. Bacteriol. 118: Costerton, J. W., J. M. Ingram, and K.-J. Cheng Structure and function of the cell envelope of gram-negative bacteria. Bacteriol. Rev. 38: Currier, W. M., and G. A. Strobel Chemotaxis of Rhizobium spp. to a glycoprotein produced by Birdsfoot-Trefoil roots. Science 196: Dazzo, F. B., and W. J. Brill Receptor site on clover and alfalfa roots for Rhizobium. Appl. Environ. Microbiol. 33: Dudman, W. F Growth and extracellular polysaccharide production by Rhizobium meliloti in defined medium. J. Bacteriol. 88: Dudman, W. F Capsulation in Rhizobium sp. J. Bacteriol. 95: Dudman, W. F Detection of acidic polysaccharides in gels by DEAE-Dextran. Anal. Biochem. 46: Gleeson, P. A., and M. A. Jermyn Leguminous seed glycoproteins that interact with concanavalin A. Aust. J. Plant Physiol. 27: Hepper, C. M Extracellular polysaccharides of soil bacteria, p In N. Walker (ed.), Soil microbiology. Butterworths, London. 21. Hubbell, D. H., and G. H. Elkan Correlation of physiological characteristics with nodulating ability in Rhizobium japonicum. Can. J. Microbiol. 13: Irvin, R. T., A. K. Chatterjee, K. E. Sanderson, and J. W. Costerton Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium. J. Bacteriol. 124: Karnovsky, M. J A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27:137A-138A. 24. Leiner, I. E Phytohaemagglutinins (phytolectins) Annu. Rev. Plant Physiol. 27: Lippincot, J. A., and B. B. Lippincot The genus Agrobacterium and plant tumorigenesis. Annu. Rev. Microbiol. 26: Luft, J. H Selective staining of acid mucopolysaccharides by ruthenium red, p In C. J. Arcencaux (ed.) 28th Ann. Proc. Electron Microsc. Soc. Am., Los Angeles, Calif. 27. Murray, R. G. E., P. Steed, and H. E. Elson The location of mucopeptide in sections of the cell wall of Escherichia coli and other gram-negative bacteria. Can. J. Microbiol. 11: Pate, J. L., and E. J. Ordal The fine structure of Chondrococcus columnaris. III. The surface layers of Chondrococcus columnaris. J. Cell Biol. 35: Perry, M. M Identification of glycogen in thin sections of amphibian embryos. J. Cell Sci. 2: Planque, K., and J. W. Kijne Binding of pea lectins to a glucan type of polysaccharide in the cell walls of Rhizobium leguminosarum. FEBS Lett. 73: Roland, J. C., C. A. Lembi, and D. J. Morre Phosphotungstic acid-chromic acid as a selective electron-dense stain for plasma-membranes of plant cells. Stain Technol. 47: Schuster, M. L., and D. P. Coyne Survival mechanisms of phytopathogenic bacteria. Annu. Rev. Phytopathol. 12: Spurr, A. R A low viscosity epoxy-resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:31-43.

8 1400 BAL, SHANTHARAM, AND RATNAM 34. Sundberg, L, J. Parath, and K. Aspberg Simultaneous isolation of trypsin inhibitor and anti-a phytohaemagglutinin from Vicia cracea by means of biospecific adsorption. Biochim. Biophys. Acta 221: Wilson, G. S., and A. Ashley Miles Topley and Wilson's principles of bacteriology and immunity, p Edward Arnold (Publishers) Ltd., London. J. BACTERIOL. 36. Wolpert, J. S., and P. Albersheim Host symbiont interactions I. The lectins of legumes interact with the 0-antigen containing lipopolysaccharides of their symbiont Rhizobia. Biochem. Biophys. Res. Commun. 70: Zevenhuizen, L. P. T. M Chemical composition of exopolysaccharides of Rhizobium and Agrobacterium. J. Gen. Microbiol. 68: