Plant and Soil 229: 3539, 2001.

2001 Kluwer Academic Publishers. Printed in the Netherlands.

35

Cellulase isoenzyme profiles in Frankia strains belonging to different

cross-inoculation groups
Jose M. Igual1 , Encarna Velazquez2 , Pedro F. Mateos2,3 , Claudino Rodrguez-Barrueco1 ,
Emilio Cervantes1 & Eustoquio Martnez-Molina2
1 IRNA-CSIC.

Apartado 257. 37080-Salamanca. Spain. 2 Departamento de Microbiologa y Genetica. Edificio

Departamental. Universidad Salamanca 37007-Salamanca. Spain. 3 Corresponding author
Received 4 May 2000. Accepted in revised form 5 September 2000

Key words: actinorhizal plants, cellulase, Frankia, isoenzyme profiles

Abstract
Carboxymethyl cellulase activities were evaluated in eight strains of Frankia from diverse host specificity groups
and geographical origins. Cellulase activity was detected in culture supernatants of all strains in the absence of
CMC (Carboxymethylcellulose) as an inducer using both double-layer plate and reducing sugar assays, indicating
a constitutive production of CM-cellulases by Frankia. CM-cellulase isoenzyme profiles were visualized using
activity gel electrophoresis of concentrated culture supernatants. Different electrophoretic profiles were observed
among the eight strains tested, which correlate with the host specificity and taxonomic grouping of Frankia.

Introduction
Bacteria that induce nodules in plants are currently included in two families, Rhizobiaceae and Frankiaceae,
which form symbiotic relationships with legumes and
actinorrhizal plants, respectively. Frankia is a genus
of actinomycetes characterized by the ability to induce
nitrogen fixing nodules in the roots of 25 plant genera
of nonleguminous angiosperms. The plants involved
in this process are named actinorhizal plants (Baker
and Schwintzer, 1990).
Although the actinorhizal nodules have their origin
in the pericycle, as lateral roots (Berry and Sunell,
1990), some of the steps involved in the infection
process are similar between rhizobia and Frankia (i.e.
root hair curling and infection thread formation). Because of that, the tools used by biologists working
with Rhizobium can be useful in molecular characterization of the infection process in actinorrhizal plants.
Electron microscopy studies of the infection process
in legumes have shown a localized degradation of the
root hair wall at the infection site, which suggests
FAX No: +923-224876. TEL No: +923-294532. E-mail:

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the involvement of cell wall hydrolytic enzymes in

the penetration process (Callaham and Torrey, 1981;
Higashi and Abe, 1980; Ridge and Rolfe, 1985; Turgeon and Bauer, 1985). In many of these reports, the
principal studied enzymes are cellulases using soluble carboxymethylcellulose (CMC) as substrate. It
remains unknown whether the degrading enzymes are
produced by the micro-organism, by the host plant or
simultaneously by the two partners.
Several authors have detected CMC-ases in different species of Rhizobium (Martnez-Molina et al.,
1979; Mateos et al., 1992; Morales et al., 1984; SalehRastin et al., 1991). Jimnez-Zurdo et al. (1996a)
have confirmed the presence of cellulolytic enzyme
activities in all the strains of rhizobia and bradyrhizobia studied. The same authors demonstrated that a
cellulase produced by Rhizobium leguminosarum biovar. trifolii ANU843 is encoded by the symbiotic
plasmid (pSym) (Jimnez-Zurdo et al., 1996b). The
results obtained in these studies showed that the production of CM-cellulases by rhizobia is constitutive.
Cellulase activity of these bacteria is very low, requiring sensitive methods of detection and quantification
(Jimnez-Zurdo et al., 1996a; Mateos et al., 1992). Using SDS-PAGE electrophoresis, Jimenez Zurdo et al.

36
(1996a) observed several cellulase isoenzymes among
different strains tested.
The study of Frankia and the biology of the actinorhizal symbiosis has been hampered by the difficulty in obtaining these bacteria in pure culture and
by their slow growth rate in laboratory conditions.
Moreover, the precise delimitation of species within
the genus Frankia is not clearly established because
of difficulties in developing discriminatory identification systems. Only a few studies have been made
of enzyme production in this genus. Safo-Sampah
and Torrey (1988) studied polysacharide-hydrolyzing
activity in five strains of Frankia. Cellulase activity,
using carboxymethylcellulose (CMC) at 1% as substrate, was detected in the five strains studied, which
also showed the capacity to produce reducing sugar
as hydrolytic products. Optimum activity depended
upon pH and primary carbon source, with pH 5.0 and
pyruvate or propionate producing highest activities. In
fractionation studies, the highest activity was in the
culture supernatants with lesser activity in the cell-free
extract and cell wall fraction.
Therefore, with the above mentioned results in
mind, our objective in this work was to detect and
quantify endo- and exo-cellulase activities and to
compare by SDSPAGE electrophoresis the cellulase
isoenzyme profiles of eight strains of Frankia belonging to different cross-inoculation and taxonomic
groups.

Materials and methods

Bacterial strains and culture conditions
The Frankia strains used in this study are listed in
Table 1. Frankia ArI3 was included as reference because Safo-Sampah and Torrey (1988) reported this
strain as a cellulase producer. All strains were grown
for 10 weeks at 28 C in sterile, plastic centrifuge
tubes (50 mL) filled with 20 mL of BAP medium with
propionate as the sole C source (Murry et al., 1984).
A separate set of tubes containing BAP-propionate
medium was supplemented with 0.5% CMC to test
whether all strains produce cellulases constitutively.
Preparation of culture supernatant
Supernatants were obtained by centrifugation of
Frankia cultures at 9000 g in a microspin centrifuge
for 10 min at 4 C and the pH was adjusted to 5.0 by
addition of PCA buffer (K2 HPO4 17.4 g L1 , citric

acid 8.4 g L1 ). For quantitative enzyme assays and

SDSPAGE electrophoresis, supernatants were concentrated 30 fold by filtration through an Ultrafree
filter of polysulfone, 10 kDa cut off (Millipore Co.,
MA, USA), which does not bind cellulases.
Double-layer plate assay
The in vitro plate assay described by Mateos et al.
(1992) was used. An aliquot (20 L) of the supernatant was pipetted onto double layer agarose plates
containing 0.7% agarose in the bottom layer and
0.5% agarose with 0.2% CMC in 100 mM potassium
phosphate-citric acid buffer (PCA; pH 5.0) in the top
layer and incubated at 28 C for 14 h. Cellulolytic
activities were revealed by flooding the plates with an
aqueous solution of 1% Congo red for 30 min followed
by decoloration with 1 M NaCl.
Quantitative enzyme assays
A micromethod based on the 2,20 -bicinchoninate assay (BCA) described by Waffenschmidt and Jaenicke
(1987) was used. Cellulolytic activities were determined after 5 h of incubation of 40 L of each concentrated supernatant with 40 L of 1% CMC in PCA
buffer (pH 5.0) and 80 L of PCA buffer. The reagents
were mixed and an aliquot of 80 L was frozen at 20
C. The remainder of the sample was incubated at 30
C for 5 h. The whole procedure was done according
to Mateos et al. (1992).
Cellulolytic enzyme profiles
CM-cellulase isoenzymes were detected using activity
gel electrophoresis in a vertical slab unit (Miniprotean II, BioRad) (Mateos et al., 1992). The separating
gel contained 12% acrylamide and the stacking gel
4% acrylamide. Protein standards (Bio-Rad Laboratories, Richmond, CA) were included in SDSPAGE
electrophoresis as markers of molecular masses. Electrophoresis was run at 200 V. Proteins were renatured
by shaking the gel for 2 h in 10 mm PCA buffer
with periodic changes every 30 min. CMC-agarose
films were constructed using a 0.4-mm layer of 0.2%
CMC and 0.5% agarose (w/v) in 100 mM PCA buffer on electrophoresis film (Sigma). The running gel
was placed on the substrate film and kept in a moist
chamber at 30 C for 14 h after which the activities
were revealed by staining with aqueous 1% Congo red
solution for 30 min and rinsed in 1 M NaCl to detect
areas of enzymatic activity.

37
Table 1. Strains of Frankia used in this study
Designation

Host source

Location

Reference or source

HrI1 (DDB 14010110)

55005 (DDB 02060510)
UGL 020603
CeI2 (DDB 02060210)
ArI3 (HFP 013003)
AvsI3 (DDB 01360610)
Ar 112.2
AcN14a

Hippophae rhamnoides
Casuarina equisetifolia
C. equisetifolia
C. equisetifolia
Alnus rubra
A. viridis ssp. sinuata
A. rubra
A. crispa

USA
USA
Egypt
USA
USA
USA
France
Canada

Baker (unpublished)
Baker (1987)
Velazquez et al. (1998)
Lechevalier (1986)
Lechevalier (1986)
Lechevalier (1986)
Moiroud (unpublished)
Normand and Lalonde (1982)

Results and discussion

The double layer plate assay detected CM-cellulase
activities in the supernatants of eight Frankia strains
grown in both 0.5% CMC-supplemented media and
non-supplemented media, indicative of the constitutive synthesis of extracellular CM-cellulases in
Frankia. Since the absence of CMC facilitates the concentration of supernatants using ultra-free filters, we
selected CMC-free media for further assays. Cellulolytic activities in concentrated supernatants determined using the 2,20 -bicinchoninate- reducing sugar assay (BCA) ranged from 7.7 for Frankia CeI2 to 1.3
units for Frankia ArI3 (one unit of enzyme activity is
the amount releasing 1nmol reducing sugar at 28 C
and pH5 mg1 h1 ).
Detection of the synthesis of constitutive CMCases by all Frankia is in agreement with the results of
Safo-Sampah and Torrey (1988) for different Frankia
strains. Although they have a low activity, cellulases can be easely detected in culture supernatants of
Frankia. In rhizobia, cellulase activities were only detected after sonication, indicating that, unlike Frankia,
cellulases are bound in the cells (Mateos et al., 1992).
Jimnez-Zurdo et al. (1996a) obtained the cellulase isoenzyme profiles of Rhizobium leguminosarum
bv phaseoli ATCC 14482 (nodulating common beans),
R. leguminosarum bv trifolii ANU843 (nodulating
clover) and R. loti ATCC 33669 (nodulating chickpea). Currently R. loti has been reclassified as
Mesorhizobium loti (Jarvis et al., 1997) and the strain
R. leguminosarum bv phaseoli ATCC 14482 as R.
etli (Segovia et al., 1993). The profiles of cellulase
isoenzymes were different among the three strains
suggesting that different rhizobial species have different cellulase isoenzyme profile. Thus, M. loti ATCC
33669 showed an isoenzyme with approximate mo-

lecular mass 44.3 kDa, R. leguminosarum bv phaseoli

ATCC 14482 showed an isoenzyme with approximate
molecular mass of 33.3 KDa and R. leguminosarum
bv trifolii ANU843 showed two different isoenzymes
with approximate molecular masses of 44.3 and 33.3
kDa.
These results prompted us to study the cellulase
isoenzyme profiles of several Frankia strains, the other
bacteria able to induce the formation of symbiotic
nodules in plants. In the current study, the eight
Frankia strains selected were isolated from diverse
host plant species (Table 1) belonging to different
taxonomic groups, according to previous results obtained by different molecular techniques: 16S rRNA
sequences (Nazaret et al., 1991), PCR-RFLP and DNA
homology (Lumini et al., 1996), LMW RNA profiles
(Velzquez et al., 1998) and polypeptides and isozyme
profiles (Gardes and Lalonde, 1987; Gardes et al.,
1987). Thus, Frankia CeI2 and UGL020603 grouped
together with other Casuarina isolates, whereas the
strain Frankia 55005, unable to reinfect Casuarina but
able to nodulate Eleagnus (Baker, 1987), was included
in a separate group along with strains isolated from
Eleagnus (Velzquez et al., 1998). Frankia strains isolated from Alnus grouped close together (Nazaret et al.,
1991, Velzquez et al., 1998); however, Frankia ArI3
and AcN14a can be differentiated by their polypeptide
and isozyme profiles (Gardes and Lalonde, 1987a;
Gardes et al., 1987b). Finally, Frankia HrI1, as well
as other isolates from Hippophae rhamnoides, belongs
to a divergent group (Lumini et al., 1996; Velzquez et
al., 1998).
Figure 1 shows the cellulase isoenzyme profiles of
the eight Frankia isolates. Two different profiles were
found in the concentrated supernatants of the Frankia
strains isolated from Alnus (ACN14a, Ar112.2, AvsI3
and ArI3) (Figure 1, lanes 58). All have in common

38

Figure 1. Detection of CM-cellulase isoenzymes in concentrated supernatants of different Frankia strains. Lanes: (1) Frankia HrI1; (2) Frankia
55005; (3) Frankia CeI2; (4) Frankia UGL 020603; (5) Frankia AvsI3; (6) Frankia ArI3; (7) Frankia AcN14a; and (8) Frankia Ar112.2. The
molecular masses (in kilodaltons) of protein standards are indicated at the left of the panel. Arrowheads indicate the position of the different
isoenzymes in each lane.

an isoenzyme with an approximate molecular masse

of 50 kDa (Figure 1, lanes 58). Strains AvsI3 and
ArI3 produces another isoenzyme with a molecular
mass of approximately 100 kDa (Figure 1, lanes 5 and
6). Finally, strains ACN14a and Ar112.2 produce an
isoenzyme with an approximate molecular mass of 40
kDa (Figure 1, lanes 7 and 8).
The two strains isolated from Casuarina (CeI2
and UGL020603) which are able to reinfect their host
(named typical strains), shared the same isoenzyme
profile consisting of two bands with molecular masses
of approximately 45 and 34 kDa (Figure 1, lanes 3 and
4). However, the cellulase isoenzyme profile of strain
55005 isolated from Casuarina, but unable to reinfect
this host (named atypical strain), (Figure 1, lane 2),
was different to that shown by the two previous strains
from Casuarina, with a single band of approximately
60 kDa.
Frankia HrI1 (Figure 1, lane 1), isolated from Hippophae rhamnoides, produces a single CM-cellulase
isoenzyme, with an approximate molecular mass of 50
kDa, different from the isoenzyme profile of any other
strains studied.
In summary, our study shows that Frankia produces cellulases constitutively and their activity may
be detected after SDSPAGE electrophoresis. This
method has allowed the identification of different cellulase isoenzymes in several Frankia strains. Similar
to the results previously reported on rhizobial cellulase isoenzyme profiles (Jimnez-Zurdo et al., 1996a),

we have obtained different cellulase profiles among

eight Frankia strains belonging to different crossinoculation groups, which can be related to taxonomic
groupings obtained for this genus by the molecular
techniques mentioned above. Therefore, our results
suggest that the analysis of cellulase isoenzyme profiles could be an additional and useful tool for the
differentiation among strains of Frankia.

Acknowledgements
This work was supported by Grants SA35/99 and
CSI01-98 of La Consejeria de Educacin y Cultura
(Junta de Castilla y Len), and PB98-0266 of the
Ministerio de Educacin y Cultura.

References
Baker D D 1987 Relationships among pure-cultured strains of
Frankia based on host specificity. Physiol. Plant. 70, 245248.
Baker D D and Schwintzer C R 1990 Introduction. In The Biology
of Frankia and Actinorhizal Plants. Eds. CR Schwintzer and D
Tjepkema. pp 113. Academic Press, Inc., San Diego, California.
Berry A M and Sunell L A 1990 The infection process and nodule
development. In The Biology of Frankia and Actinorhizal Plants.
Eds. CR Schwintzer and JD Tjepkema. pp 6181. Academic
Press, Inc., San Diego, California.
Callaham D A and Torrey J G 1981 The structural basis for infection
of root hairs of Trifolium repens by Rhizobium. Can. J. Bot. 59,
16471664.

39
Gardes M and Lalonde M 1987 Identification and subgrouping of
Frankia strains using sodium dodecyl sulphate-polyacrylamide
gel electrophoresis. Physiol. Plant. 70, 237244.
Gardes M, Bousquet J and Lalonde M 1987 Isozyme variation
among 40 Frankia strains. Appl. Environ. Microbiol. 53, 1596
1603.
Higashi S and Abe M 1980 Scanning electron microscopy of
Rhizobium trifolii infection sites on root hairs of white clover.
Appl. Environ. Microbiol. 40, 10941099.
Jarvis B D W, Van Berkum P, Chen W X, Nour S M, Fernndez
M P, Cleyet-Marel J C and Gillis M 1997 Transfer of Rhizobium
loti, Rhizobium ciceri, Rhizobium mediterraneum and Rhizobium
tianshanense to Mesorhizobium gen. nov. Int. J. Syst. Bacteriol.
47, 895898.
Jimnez-Zurdo J I, Mateos P F, Dazzo F B and Martnez-Molina
E 1996a Cell-bound cellulase and polygaracturonase production
by Rhizobium and Bradyrhizobium species. Soil Biol. Biochem.
28, 917921.
Jimnez-Zurdo J I, Mateos P F, Dazzo F B and Martnez-Molina.
E 1996b Influence of the symbiotic plasmid (pSym) on cellulase
production by Rhizobium leguminosarum bv. trifolii ANU843.
Soil Biol. Biochem. 28, 131133.
Lechevalier M P 1986 Catalog of Frankia strains. Actinomycetes
19, 131162.
Lumini E, Bosco M and Fernandez M P 1996 PCR-RFLP and total
DNA homology revealed three related genomic species among
broad-host-range Frankia strains. FEMS Microbiol. Ecol. 21,
303311.
Martnez-Molina E, Morales V M and Hubbell D H 1979 Hydrolytic
enzyme production by Rhizobium. Appl. Environ. Microbiol. 38,
11861188.
Mateos P F, Jimnez-Zurdo J I, Chen J, Squartini A, Haack S K,
Martnez-Molina E, Hubbell D H and Dazzo F B 1992 Cellassociated pectinolytic and cellulolytic enzymes in Rhizobium
leguminosarum biovar. trifolii. Appl. Environ. Microbiol. 58,
18161822.

Morales V M, Martnez-Molina E and Hubbell D H 1984 Cellulase

production by Rhizobium. Plant Soil 80, 407415.
Murry M A, Fontaine M S and Torrey J G 1984 Growth kinetics and
nitrogenase induction in Frankia sp. HFP Ar I3 grown in batch
culture. Plant Soil 78, 6178.
Nazaret S, Cournoyer B, Normand P and Simonet P 1991 Phylogenetic relationships among Frankia genomic species determined
by use of amplified 16S rDNA sequences. J. Bacteriol. 173,
40724078.
Normand P and Lalonde M 1982 Evaluation of Frankia strains isolated from provenances of two Alnus species. Can. J. Microbiol.
28, 11331142.
Ridge R W and Rolfe B G 1985 Rhizobium sp. degradation of
legume root hair cell wall at the site of infection thread origin.
Appl. Environ. Microbiol. 50, 717720.
Saleh-Rastin N, Petersen M, Coleman S and Hubbell D 1991 Rapid
plate assay for hydrolytic enzymes of Rhizobium. In The Rhizosphere and Plant Growth. Eds. D Keister and P Gregon. pp. 188.
Kluwer Academic Press, Dordrecht, The Netherlands.
Safo-Sampah S and Torrey J T 1988 Polysaccharide-hydrolizing
enzymes of Frankia (Actinomycetales). Plant Soil 112, 8997.
Segovia L, Young J P W and Martnez-Romero E 1993 Reclassification of American Rhizobium leguminosarum Biovar phaseoli
type I strains as Rhizobium etli sp. nov. Int. J. Syst. Bacteriol. 43,
374377.
Turgeon B C and Bauer W D 1985 Ultrastructure of infection-thread
development during the infection of soybean by Rhizobium
japonicum. Planta 163, 328349.
Velzquez E, Cervantes E, Igual J M, Peix A, Mateos P F, Benamar
S, Moiroud A, Wheeler C T, Dawson J O, Labeda D, RodrguezBarrueco C and Martnez-Molina E 1998 Analysis of LMW
RNA profiles of Frankia strains by staircase Electrophoresis.
System. Appl. Microbiol. 21, 539545.
Waffenschmidt S and Jaenicke L 1987 Assay of reducing sugars
in the nanomole range with 2,20 -bicinchoninate. Anal. Biochem.
165, 337340.