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CURRENTMICROBIOLOGY,Vol. 2 (1979), pp. 15-20
Current
Microbiology
An International Journal
Transient Appearance of Lectin Receptors
on Rhizobium trifolii
Frank B. Dazzo,-~-* Maria R. Urbano,:~ and Winston J. Brillw
t$ Department of Microbiology and Public Health and -~Department of Crop and Soil Science, Michigan State University, East Lansing,
Michigan 48824, USA
wDepartment of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA
Abstract. The appearance on the surface of Rhizobium trifolii 0403 of determinants important to
both clover lectin (trifoliin) binding and adherence of the bacteria to clover root epithelial surfaces
was studied by quantitative agglutination, immunofluorescence, and direct microscopic techniques. These unique determinants were found for only transient periods of t i m e - - a s cells left lag
phase and as they entered stationary phase of growth in broth. When present, these receptors were
associated with a fibrillar polyanionic capsule surrounding the cells when grown on solid medium.
These studies support earlier proposals that the architecture of the rhizobial cell surface is not constant in composition, but changes with the phase of growth.
The selectivity of bacterial attachment is a major underlying basis for the tissue, organ, and host tropisms
of a variety of microorganisms [8,19]. In the nitrogen-fixing Rhizobium-clover symbiosis, the bacteria
selectively adhere to root hairs of the host prior to
specific infection of these specialized epidermal cells
[14]. Immunochemical [1 1,13] and genetic studies [4]
(F. B. Dazzo and W. J. Brill, submitted for publication) suggest that this selective adherence may be
initiated by a specific cross-bridging of antigenically
related saccharide determinants on the surface of the
bacterium and the cell wall of the host by a multivalent, host-coded lectin called trifoliin [15]. Competition studies using Fab antigen-binding fragments
show that trifoliin and antibody to the clover root
cross-reactive antigen bind to the same R. trifolii saccharide determinants that bind these bacteria to clover root hairs. The sugar 2-deoxyglucose specifically
inhibits the binding of trifoliin or the cross-reactive
antibody to these bacteria [11,13] and their attachment to clover root hairs [14]. Transfer of the R. trifolii genes controlling the synthesis of this determinant to Azotobacter vineIandii has resulted in a
genetic hybrid that can adsorb selectively to clover
root hair tips, the site of trifoliin accumulation
(Dazzo and Brill, submitted for publication). These
studies establish the importance of this unique sac*To whom offprint requests should be addressed.
charide determinant in the selective adherence of the
bacteria to target epithelial cells of the host.
The ability of R. trifolii to adhere selectively to clover root hairs should be influenced by conditions that
affect the saccharide receptor on the bacterium and
the accumulation of trifoliin on the host root surface.
Evidence supporting this hypothesis has been obtained from studies that showed fixed nitrogen ions
(eg., NO3-, NH4 ~) in the rooting medium regulate the
levels of trifoliin on clover root hairs and the concurrent ability of R. trifolii to adhere to these surfaces
[12]. This report also supports that hypothesis by
showing that the transient appearance of trifoliin receptors on Rhizobium trifolii may influence the ability
of these bacteria to attach to clover root hairs.
Materials and Methods
Agglutination studies. Rhizobium trifolii strain 0403 (obtained
from P. S. Nutman) was grown on a chemically defined medium
(Bill) containing 1% mannitol [1 I] and solidified with 1.3% purified agar (Difco Laboratories, Detroit, Michigan). Cells grown at
30~ for 5 days served as inoculum. Lawns were prepared daily
on freshly poured plates and incubated at 30~ The methods for
harvesting and washing of cells, removal of nondispersible flocs,
purification of clover trifoliin, and quantitative agglutination assays of this lectin have been described [11,15].
Electron microscopy.Cells grown on solid medium for 3 or 5 days
were prepared for transmission electron microscopy using ruth0343-8651/79/0002-0015 $01.20
9 1979 Springer-Verlag New York Inc.
16
CURRENT MICROBIOLOGY, VOI. 2 (1979)
enium red staining procedures as described by Pate and Ordal
[24]. Ultrathin sections were post-stained with uranyl acetate and
lead citrate, and examined in a Philips 300 transmission electron
microscope operating at 80 kV. Sections not post-stained served as
positive controls for ruthenium red staining of cell surfaces.
Fluorescence microscopy. One-tenth milliliter of early stationaryphase cells (adjusted to 5.8 • 107 cells/ml using the Klett-Summerson colorimeter with the red filter, 660 nm) was inoculated
into 20 ml of broth in 300-ml sidearm flasks and incubated with
shaking (180 rpm) at 30~ Cell densities were measured periodically with the Klett-Summerson colorimeter, followed by aseptic
transfer of 15 ~tl of culture to an etched ring (9-mm diameter) on
clean fluorescent antibody slides (Clay Adams, Parsippany, New
Jersey). Slides were dried at room temperature in a dust-free
chamber, heat-fixed, and rinsed gently with distilled water. The
smears were incubated with rabbit antiserum against clover root
antigens [13] for 4 h, rinsed, and then incubated for 4 h with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit g a m m a
globulin (Difco) diluted 1 : 5 with [0 mM phosphate-buffered
physiological saline, pH 7.2 [15]. The smears were rinsed, airdried, mounted in glycerol mounting fluid (pH 9, Difco), and examined for immunofluorescence with transmitted illumination using a Zeiss Photomicroscope l equipped with an HBO 200 Hg
lamp, K G I and BG 38 filters, a BG 12 exciter filter, and a no. 50
barrier filter. The percentage of cells reactive with the antibody
was calculated from the ratio of fluorescent cells to total cells in 510 randomly selected microscope fields (1,250x). Total cell counts
in the same microscope field were measured under dark field illumination. The results were averaged from two separate experiments. No indirect immunofluorescence was obtained when norma[ p r e i m m u n e serum was substituted as a control [13]. Binding
of trifoliin to cells grown for 3, 5, and 7 days on BIII agar was examined by indirect immunofluorescence [15].
Adherence of R. trifolii to clover root hairs, The ability of R. trifolii to attach to root hairs of clover seedlings was examined by a
previously described assay [1 I, 15]. Cells for this purpose were harvested by centrifugation from mid-exponential and early and late
stationary-phase broth cultures, washed in nitrogen-free Fahraeus
medium [18], and resuspended in this medium at 2 x 10 7 cells/ml
for root-binding assays. Cells were also harvested from BIll plates
after growth tbr 3, 5, and 7 days, then assayed. Five 24-h-old sterile ckwer seedlings (Trifolium repens vat. Louisiana Nolin) were
incubated with the bacteria for 20 rain and then rinsed in Fahraeus medium. The roots were scanned and representative fields
were recorded photographically.
Results
Culture age effects on agglutination by trifoliin. Rhi-
zobium trifolii 0403 began to produce visible colonies
on BIll agar after 2-3 days' incubation at 30~
The
ability of trifoliin to agglutinate cells was influenced
profoundly by the cell culture age (Fig. 1). The specific activity of trifoliin (in agglutinating units/mg
protein) was highest when the agglutination assay
was performed with cells grown for 5 days on BIII
agar.
Electron microscopy of cells grown on B i l l agar. Because the cells' surface changed with age, properties
of cells grown for 3 or 5 days were examined in detail
Fig. 1. The eft'cot of culture age on the agglutination of Rhizo&um
trifolii 0403 by trifoliin. Cells were grown on BIll agar medium,
harvested, and assayed for quantitative agglutination (agglutinating units/rag trifoliin protein) [I 1,15].
by ruthenium red staining followed by transmission
electron microscopy. The most obvious difference in
the topography of cells of these two culture ages was
the exclusive presence of a ruthenium red-staining
glycocalyx of fibrillar capsular material surrounding
cells of the 5-day-old culture (Fig. 2). Reserve polymer inclusions and capsular material accumulated at
the same cell poles when cell polarity was observed
(Fig. 3). The majority of the cells from the 7-day-old
culture were unencapsulated (not shown).
Fluorescence microscopy of cells grown on B i l l agar.
Binding of trifoliin to cells was examined by indirect
immunofluorescence. Trifoliin did not bind to cells
grown for 3 days. Trifoliin bound uniformly on heavily encapsulated cells grown for 5 days. Photomicrographs representative of this positive reaction have
been published elsewhere [15]. Most of the cells
grown for 7 days were unencapsulated and did not
bind trifoliin. Only some of the remaining encapsulated cells of this culture age bound trifoliin uniformly (Fig. 4). The remainder of the encapsulated
cells either bound trifoliin asymmetrically along one
cell pole or did not bind trifoliin at levels detected by
this fluorescence assay.
Fluorescence microscopy of cells grown in Bill
broth. Cells were withdrawn from broth cultures at
various stages of growth and examined for reactivity
to anti clover root antiserum (Fig. 5). The percentage of cells that bound this cross-reactive antibody
peaked at two discrete phases of culture growth:
when cells left lag phase and as they entered stationary phase. In both cases, this appearance of surface
F. B. Dazzo, M. R. Urbano, and W. J. Brill: Lectin Receptors
17
Fig. 2. Ultrastructure of Rhizobium triJblii 0403 stained with ruthenium red. Cells were grown on Bill agar plates for 3 days (a) or 5 days
(b). (a) Bar = 1.24 #m; x24,218. (b) Bar = I /~nl; •
antigens cross-reactive with the host was transient, as
only a low percentage of the cells reacted while in
mid-exponential phase or late stationary phase.
Adherence of Rhizobium trifolii to clover root hairs.
The ability of R. trifolii 0403 to adhere to clover root
hairs was influenced significantly by the culture age.
When R. trifolii cells were harvested from BIlI broth
at various phases of growth, cells that were entering
stationary phase attached to clover root hairs in the
highest numbers (Fig. 6). Cells grown for 5 days on
BIII agar plates attached to clover root hairs in
higher numbers than cells grown for 3 or 7 days in
this medium.
Discussion
Our previous studies have indicated that selective adherence of Rhizobium trifolii to clover root hairs may
be mediated by specific residues on the bacterial
polysaccharide, which are cross-linked by a clover
lectin to similar determinants on the cell wall of the
plant [11-15]. This study shows that these important
determinants on the bacterial cell surfaces are accessible to binding by trifoliin and the antibody for only
a short period when the cells are grown in broth or
on agar surfaces. It is during this time that the greatest number of R. trifolii cells can adhere to the epithelial surface of clover roots. Comparable growth
rates in broth and on agar are not implied in this
study.
The appearance of the trifoliin receptors is concurrent with the accumulation of the fibrillar polyanionic capsule on R. trifolii 0403. These results support our earlier observation that the bacterial cells
that are most reactive with trifoliin are surrounded
by a definite capsule [11,13,15]. However, the inability of trifoliin to bind to most encapsulated cells
grown for 7 days on BIll agar suggests that either
these receptors eventually become masked, modified
to produce a lower affinity, or are shed from the bacterial capsule.
Similarly, an ultrastructural analysis has revealed distinct morphological types of a nodulating
strain of R. japonicum, of which only the capsular
form was found to bind the N-acetylgalactosaminespecific soybean lectin (SBL) [1,6] and attach to soybean root hair surfaces [1]. Bhuvaneswari, Pueppke,
and Bauer [3] have shown that most strains of R. ja-
18
Fig. 3. Transmission electron micrograph of Rhizobium trifolii
0403 grown for 5 days on Bill agar. Note the polarity of the capsular polysaccharide. Bar = 0.6 p.m; x49,890.
ponicum had their highest percentage of cells reactive
with SBL in the mid-exponential phase of growth.
The proportion of galactose residues in the capsular
polysaccharide, which p r e s u m a b l y binds SBL, is
greatest during this culture age, and diminishes as
the culture enters stationary phase [22]. Concurrent
Fig. 4. Asymmetric and nonuniform binding of trifoliin to Rhizobium trifolii 0403 grown for 7 days on Bill agar. Cells were examined by indirect immunofluorescence. Bar scale: 1 #m.
CURRENT MICROBIOLOGY, Vol. 2 (1979)
Fig. 5. The effect of culture age on the percentage of Rhizobium
trifolii 0403 cells reactive with anti-clover root antiserum. Cells
were grown in Bill broth at 30~ with shaking. Culture growth
(O) was measured with a Klett-Summerson colorimeter at 660 nm
and the cross-reactive antigen (O) by indirect immunofluorescence.
with a decline in galactose content is a rise in percentage of 4-O-methyl galactose residues.
It is possible that the.effect of culture age on expression of lectin receptors may influence the competitiveness and ecological behavior of different rhizobial strains in soil and on the roots of their legume
hosts. The delay in appearance of the capsule surrounding cells of R. trifolii TA 1 [16,21] may improve
their competitiveness for nodulation of clover roots
in field soils [16]. Inoculum prepared from early stationary-phase cells of R. trifolii NA 30 gives rise to
more clover root hair infections than equivalent inocula prepared from exponentially growing or late
stationary-phase cells (C. A. Napoli, Ph.D. thesis,
University of Florida, Gainesville, Florida, 1976).
The one strain of R. japonicum (311b123) found to
accumulate the SBL-binding capsule during stationary phase [3] has been recognized as the most frequently found serogroup in soybean nodules from
m a n y soils of the central United States [9,20].
These studies support earlier proposals that the
architecture of the rhizobial cell surface is not constant in composition, but is a dynamic structure that
changes with culture age [2]. Several studies have
shown that polysaccharide antigens on microbial surfaces are particularly susceptible to alteration during
shifts in growth stages. The somatic O antigen of Salmonella lipopolysaccharide has a stronger expression
19
F. B. Dazzo, M. R. Urbano, and W. J. Brill: Lectin Receptors
such as we have observed occurs in both animal
[8,17,23,26] and plant hosts (M. Umali-Garcia et al.,
submitted for publication). This growth phase effect
in the Rhizobium-clover symbiosis can be explained
as a transient appearance of lectin receptors on the
rhizobial cell surface.
ACKNOWLEDGMENTS
Fig. 6. Attachment of Rhizobium trifolii 0403 in mid-exponential
phase (a), early stationary phase (b), and late stationary phase (c)
to clover root hairs. Cells in early exponential phase were not examined. Bar = 50 #m.
in stationary phase than in exponential phase as a result of an increase in the length of the polysaccharide
chains [25]. In continuous culture, the length of the
Salmonella 0 antigen is inversely related to the dilution rate of the culture [7]. The relative amount of
glucose in cell wall polysaccharides of cariogenic
Streptococcus mutans increases as the culture advances to stationary phase [5]. Nutrients that may become limiting as the culture enters stationary phase
could influence the composition of bacterial polysaccharides. For example, Ca +2 limitation modifies or
o b s c u r e s an a n t i g e n i c g r o u p in the l i p o p o l y saccharide of R. trifolii [27], and both Mg +2 and
PO4-2 limitations reduce the percentage of O-pyruval
linkages in the xanthan gum of Xanthomonas campestris [ 10].
The influence of the microbial growth phase
on the adsorption of bacteria to epithelial surfaces
This research was supported by the College of Agricultural and
Life Sciences, University of Wisconsin, Madison, by National Science Foundation grant AER 77-00879, by Michigan Agricultural
Experiment Station project no. 1314 H, and by the College of Natural Science, Michigan State University. We thank W. E. Yanke
and S. Pankratz for their skilled technical assistance. This paper is
Journal Series no. 8774 of the Michigan Agricultural Experiment
Station.
Literature Cited
1. Bal, A. K., Shantharam, S., Ratnam, S. 1978. Ultrastructure o f
Rhizobium japonicum in relation to its attachment to root
hairs. Journal of Bacteriology 133:1393-1400.
2. Bauer, W. D. 1977. Lectins as determinants of specificity in legume-Rhizobium symbiosis. Basic Life Sciences 9:283-297.
3. Bhuvaneswari, T. V., Pueppke, S. G., Bauer, W. D. 1977. Role
of lectins in plant-microorganism interactions. 1. Binding of
soybean lectin to rhizobia. Plant Physiology 60:486-491.
4. Bishop, P. E., Dazzo, F. B., Appelbaum, E. R., Maier, R. J.,
Brill, W. J. 1977. Intergeneric transfer o f genes involved in
the Rhizobium-legume symbiosis. Science 198:938-940.
5. Bleiweis, A. S., Taylor, M. C., Deepak, J., Brown, T. A.,
Wetherell, J. 1976. Comparative chemical compositions of
cell walls of Streptococcus mutans. Journal of Dental Research 55:A 103-A 108.
6. Calvert, H. E., Lalonde, M., Bhuvaneswari, T. V., Bauer,
W. D. 1978. Role o f lectins in plant-microorganism interactions. IV. Ultrastructural localization of soybean lectin
binding sites on Rhizobiumjaponicum. Canadian Journal o f
Microbiology 24:785-793.
7. Collins, F. M. 1964. The effect o f the growth rate on the composition of S. enteritidis cell walls. Australian Journal o f
Experimental Biology and Medical Sciences 42:255-262.
20
8. Costerton, J. W., Gessey, G. G., Cheng, K. J. 1978. How bacteria stick. Scientific American 238:86-95.
9. l)amirgi, S. M., Frederick, L. R., Anderson, 1. C. 1967. Serogroups of Rhizobium japonicurn in soybean nodules as affected by soil types. Agronomy Journal 59:10-[2.
10. Davidson, 1. W. 1978. Production of polysaccharide by Xanthornonas carnpestris in continuous culture. FEMS Letters
3:347-349.
I I. Dazzo, F. B., Brill, W. d. 1977. Receptor sites on clover and alfalfa roots for Rhizobiurn. Applied and Environmental Microbiology 33:132-136.
12. Dazzo, F. B., Brill, W. J. 1978. Regulation by fixed nitrogen of
host-symbiont recognition in the Rhizobiurn-clover symbiosis. Plant Physiology 62:18-21.
13. Dazzo, F. B., llabbell, I). H. 1975. Cross-reactive antigens and
lectin as determinants of symbiotic specificity in the Rhizobium-clover association. Applied Microbiology 30:10171033.
14. Dazzo, F. B., Napoli, C. A., Hubbell, D. H. 1976. Adsorption
of bacteria to roots as related to host specificity in the Rhizobiurn-clover symbiosis. Applied and Environmental Microbiology 32:168- 171.
15. Dazzn, F. B., Yankc, W. E., Brill, W. J. 1978. Triloliin: A Rhizobiurn recognition protein from white clover. Biochimica
et Biophysica Acta 539:276 286.
16. Dudman, W. I-. 1968. Capsulation in Rhizobium species. Journal of Bacteriology 95:1200 - 120 I.
17. Ellen, R. P., Gibbons, R. J. 1974. Parameters affecting the adherence and tissue tropisms of Streptococcus p.yogenes. Infection and Immunity 9:85 91.
18. Fahraeus, G. 1957. The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique Journal of General Microbiology 16:374 381.
CURRENT MICROBIOLOGY, Vol. 2 (1979)
19. Gibbons, R. J. 1977. Adherence of bacteria to host tissue, pp.
395-406. In: Schlessinger, D. (ed.), Microbiology-1977.
Washington, D.C.: American Society for Microbiology.
20. Ham, G. E., Frederick, L. R., Anderson, 1. C. 1971. Serogroups of Rhizobiurn japonicum in soybean nodules sampled in Iowa. Agronomy Journal 63:69-72
21. Humphrey, B., Vincent, J. M. 1969. The somatic antigens o f
two strains of Rhizobiurn trifolii. Journal of General Microbiology 59:41 l 425.
22. Mort, A., Bauer, W. D. 1978. The chemical basis of lectin
binding to Rhizobiurn japonieurn. Abstracts of the Annual
Meeting of the American Society o f Plant Physiology
1978:325.
23. Ofek, 1., Mireman, D., Sharon, N. 1977. Adherence of Escherichia call to human mucosal cells mediated by mannose receptors. Nature 265:623-625.
24. Pate, J. L., Ordal, E. J. 1967. The fine structure of Chondrococcus colurnnaris. Ill. The surface layers of Chondrococcus
colurnnaris. Journal o f Cell Biology 35:37-51.
25. Schlecht, S., Fromme, I. 1975. Chemical and serological characterization of Salmonella lipopolysaccharides from different phases of growth. [In German, with English st, mmary.]
Z e n t r a l b l a t t far Bakteriologie, P a r a s i t e n k u n d e , lnfektionskrankheiten und Hygiene, Abt. 1 Orig. 233:199222.
26. Suegara, N., Morotomi, M., Watanabe, "I'., Kawai, Y., Mutai,
M. 1975. Behavior o f microflora in the rat stomach: Adhesion of lactobacilli to the keratinized epithelial cells of the
rat stomach in vitro. Infection and Immunity 12:173-179.
27. Vincent, J. M., Humphrey, B. A. 1968. Modification of the antigenic surface of Rhizobhtrn trifolii by a deficiency of calcium. Journal of General Microbiology 54:397-405.