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Plant and Soil 254: 115123, 2003.

2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Genomic fingerprinting of Frankia strains by PCR-based techniques.


Assessment of a primer based on the sequence of 16S rRNA gene of
Escherichia coli
Jose M. Igual1 , Angel Valverde1 , Raul Rivas2, Pedro F. Mateos2 , C. Rodrguez-Barrueco1 ,
Eustoquio Martnez-Molina2, Emilio Cervantes1 & Encarna Velazquez2.3
1 Instituto

de Recursos Naturales y Agrobiologa-CSIC Apartado 257. 37080 Salamanca. Spain. 2 Departamento


de Microbiologa y Genetica. Edificio Departamental. 37007 Salamanca. Spain. 3 Corresponding author

Received 19 July 2002. Accepted in revised form 20 August 2002

Key words: biodiversity, Clavibacter, Frankia, genomic fingerprinting, PCR, taxonomy

Abstract
Polimerase Chain Reaction (PCR)-based genomic fingerprints of 16 Frankia isolates were obtained using two
different primers. rep-PCR DNA fingerprints were obtained by using DR1R primer, and Randomly Amplified
Polymorphic DNA (RAPD) fingerprints by using a large primer derived from the 16S rDNA sequence of Escherichia coli (879F primer). According to the results obtained, primer DR1R generates strain-specific patterns.
However, primer 879F yielded an identical band patterns in two Frankia strains, CcI3 and UGL 020603, isolated
from different Casuarina species and geographical origins, indicating that it could identify genomic fingerprints
at a higher taxonomic level (subspecies or species) than DR1R primer. To verify this hypothesis, we tested the
primer 879F with eight strains of Clavibacter michiganensis, another actinobacterium with high G+C content,
which includes several well-defined subspecies. Primer 879F identified a unique band pattern in all strains within
the same subspecies but different patterns for other subspecies, indicating that it generates subspecies-specific
genomic fingerprints. Consequently, Frankia Cc13 and Frankia UGL 020603 should be included in the same
subspecies and the remaining Frankia strains used in this study in different subspecies. An UPGMA dendrogram
of the 879F-PCR fingerprint patterns shows that the Frankia strains used in this study could be clustered into groups
that broadly reflects the host plants from which they were derived. These results are consistent with those obtained
by other authors using different techniques. Because primer 879F yields genomic fingerprints at taxonomic level
upper than strain level, it may be a valuable tool in the polyphasic approach to Frankia taxonomy, where other
classical taxonomic methods are barely applicable. Taking into account that several primers can be designed from
ribosomal sequences in order to obtain RAPD patterns, we propose to name these methods according to the position
of such primers in the 16S rRNA sequences of Escherichia coli. In this way, we name 879F-RAPD fingerprinting
the procedure used in this study.
Introduction
Bacteria included in genus Frankia are actinomycetes
able to induce nitrogen fixing nodules in the root of
25 plant genera of nonleguminous angiosperms, collectively designated as actinorhizal plants (Benson and
Silvester, 1993). The study of Frankia and the biology
of the actinorhizal symbiosis have been hampered by
FAX No: +34-923-224876. E-mail: evp@gugu.usal.es

the difficulty in obtaining these bacteria in pure culture


and by their slow growth rate in laboratory conditions.
In spite of such drawback, several hundred of isolates
have been obtained in diverse laboratories throughout
the world since Callahan et al. (1978) first reported the
isolation of an infective Frankia strain from nodules of
Comptonia peregrina.
Diverse studies have shown that the grouping of
Frankia isolates broadly reflects the host plants from
which they were derived, but there are controver-

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sial results concerning the relative taxonomic position
in dendrograms of strains isolated from diverse host
plants. Molecular methods based on PCR can characterize cultured as well as unculturable Frankia strains,
now make it possible to study Frankia populations
directly in soil or inside nodules (Hahn et al., 1999).
The analysis of 16S or 23S rDNA genes by DNA sequence analysis have become more useful approaches
to assess the phylogenetic and taxonomic diversity of
bacterial isolates (Woese, 1987). Using complete 16S
rDNA sequences, Normand et al. (1996) proposed an
emendation of the family Frankiaceae to contain only
the genus Frankia, excluding the other two earlier included genera: Geodermatophilus and Blastococcus
(Hann el al., 1989). From the analysis of such sequences, Frankia was clustered in four groups: Cluster
1 included strains that infect Alnus and Casuarina species; Cluster 2 included the uncultured endophytes of
Dryas, Coriaria and Datisca species; Cluster 3 included strains of the Elaeagnus host infection group;
and Cluster 4 included atypical non-nitrogen-fixing
strains (Normand et al., 1996). Other authors have
generally accepted this grouping (Benson et al., 1996;
Clawson et al., 1998; Hnerlage et al., 1994; Maunuksela et al., 1999; Mirza et al., 1994; Ramirez-Saad et
al., 1998; Ritchie and Myrold, 1999).
However, because of the conserved nature of rRNA
sequences, the power of rDNA based protocols resides
at a low level of phylogenetic or taxonomic resolution, ranging from the genus to even the kingdom
level, but it is insufficient to classify bacteria at the
(sub)species level (Fox et al., 1992; Stackebrandt and
Goebel, 1994; Woese, 1987). Thus, DNADNA hybridization studies are necessary to define species and
subspecies within a genus. However, in the case of
Frankia, such studies are hampered by the difficulty to
isolate and grow some strains in laboratory media as
well as by the low efficiency of DNA extraction from
Frankia cultures.
Polyphasic taxonomy is increasingly being accepted as a comprehensive approach to microbial systematics (Vandamme et al. 1996). In this regard, PCRbased genomic fingerprinting methods can function as
core techniques in polyphasic taxonomy. During the
past decade, PCR-based DNA fingerprinting of microorganisms has been developed using a wide variety
of techniques and primers designs. In the so-called
rep-PCR (Versalovic et al. 1994), primers targeting
at consensus sequence motifs of repetitive elements
common to prokaryotic genomes such as REP (repetitive extragenic palindromic), ERIC (enterobacterial

repetitive intergenic consensus) and BOX elements,


have been used to amplify intervening sequences of
Frankia genomic DNA (Jeong and Myrold, 1999;
Murry et al., 1995; Prez et al., 1999). In a similar way, Jeong and Myrold (1999) designed a primer
corresponding with conserved DNA sequences of directs repeats in Mycobacterium bovis (DR1R primer)
and demonstrated its utility for genomic fingerprinting
of Ceanothus-microsymbiotic Frankia. On the other
hand, Random Amplified Polymorphic DNA assay
(RAPD), also referred to as arbitrarily primed PCR
(AP-PCR), employs a single short (typically 10 bp)
primer that is not targeted to any specific bacterial
DNA sequence to obtain genomic fingerprints (Williams et al., 1990). In the case of Gram positive bacteria
with high G+C content, primers rich in G+C should
be used in order to obtain adequate RAPD patterns, as
was demonstrated in Clavibacter (Pastrik and Rainey,
1999). Both rep-PCR and AP-PCR produce genomic
fingerprints with a resolution at strain level and, thus,
are inappropriate for taxonomic analysis at species
level. However, it has been recently shown that the
PCR reaction products generated by using oligonucleotide primers complimentary to partial 16S rDNA
sequences result, once separated electrophoretically,
in species or subspecies-specific fingerprints (Dong et
al., 2000; Rivas et al., 2001).
Taking into account the aforementioned results and
that the taxonomy of Frankia is far from being wellestablished, the objective of this study was to test the
usefulness of a novel PCR-based procedure, which
uses a large primer targeting at a partial sequence of
the 16S rRNA gene of Escherichia coli (879F primer),
as tool for polyphasic taxonomy of this actinomycete.
879F-RAPD profiles of 16 Frankia isolates were contrasted with their rep-PCR (DR1R-PCR) fingerprints
to determine the resolution level of this new technique
for strain differentiation. In order to have a reference
of the taxonomic usefulness of this technique, we also
obtained the 879F-RAPD profiles of eight strains belonging to five well-defined subspecies of Clavibacter
michiganensis, another Gram positive bacterium with
high G+C content.

Materials and methods


Bacterial strains and culture conditions
Sixteen Frankia and eight Clavibacter michiganensis
strains used in this study are listed in Tables 1 and

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Table 1. Frankia strains used in this study
Designation

Host source

Location

Reference or source

ArI3 (HFP013003)
S14 (ENS010714)
UGL011301
AcN14a

Alnus rubra
A. glutinosa
A. inokumai
A. cordata

USA
Morocco
S. Korea
Canada

Ar 112.2
UGL020603
CSI020602
CeI2 (DDB0206210)
CcI3 (HFP020203)
Cj (DDB000320)

A. rubra
Casuarina equisetifolia
C. equisetifolia
C. equisetifolia
C. cunninghamiana
Soil beneath
Ceanothus jepsonii
Hippophae rhamnoides
H. rhamnoides
Myrica pennsylvanica
Purshia tridentata
Discaria trinervis
Trevoa trinervis

France
Egypt
Spain
USA
USA
USA

Murry et al. (1984)


Velazquez et al. (1998)
Sayed et al. (1997)
Normand and Lalonde
(1982)
Velazquez et al. (1998)
Velazquez et al. (1998)
Velazquez et al. (1998)
Lechevalier (1986)
Zhang et al. (1984)
D D Baker, unpublished

France
USA
USA
USA
Argentina
Chile

Velazquez et al. (1998)


D D Baker, unpublished
Lechevalier (1986)
Baker (1987)
Clawson et al. (1998)
Clawson et al. (1998)

Hr114.2
HrI1 (DDB14010110)
MpI1 (LLR162001)
PtI1 (DDB17020110)
DtI2
TtI1

Table 2. Clavibacter michiganensis strains used in this study


Designation
Clavibacter michiganensis
1154
1792
Clavibacter michiganensis
121.1
093.3F
Clavibacter michiganensis
CECT 4209
Clavibacter michiganensis
CECT 4263
Clavibacter michiganensis
CFBP 5042T

subsp. sepedonicus ATCC 33113T

Host source

Location

Solanum tuberosum
S. tuberosum
S. tuberosum

Canada
USA
USA

Lycopersicon lycopersicon
L. lycopersicon

Spain
Spain

Zea mays

USA

Triticum aestivum

USA

Medicago sativa

USA

subsp. michiganensis

subsp. nebraskensis
subsp. tessellarius
subsp. insidiosus

2, respectively. Frankia strains were grown at 28 C


in sterile, plastic centrifuge tubes (50 mL) filled with
20 mL of BAP medium with propionate as C source
(Murry et al., 1984). After 8 weeks of growth, and
before manipulations for DNA extractions, 1 mL of
each culture was plated in blood agar medium and
incubated for 10 days at 28 C in order to check the
absence of contaminants. Clavibacter michiganensis
strains were grown in TY medium (triptone 0.5%,
yeast extract 0.3%, CaCl2 0.0 1%) at 28 C for 24 h.

DNA extraction
Total genomic DNA from Frankia isolates was extracted according to the methods employed by Rivas et
al. (2001) with minor modifications. Cells were harvested by centrifugation at 9000 g in a microspin
centrifugue for 10 min. at room temperature and lyophilized during 5 h. DNA was extracted with 100
L of 0.05 M NaOH (DNA-free) heating at 100 C
for 5 min. Samples were then placed in an ice bath

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Figure 1. Ethidium bromide stained agarose gel pattern of DR1R-PCR products from genomic DNA of the following Frankia strains: S14 (lane
1), UGL011301 (lane 2), Ar112.2 (lane 3), ArI3 (lane. 4), AcN14a (lane 5), CeI2. (lane 6), CcI3 (lane 7), CSI020602 (lane 8), UGL020603
(lane 9), HrI1 (lane 10), Hr112.2 (lane 11), PtI1 (lane 12), DtI2 (lane 13), DDB000320 (lane 14), MpI1 (lane 15), TtI1 (lane 16). The DNA
molecular weight marker (lane MW) is Standard VI (Boehringer-Roche, Indianapolis, IN, USA) with 2176, 1766, 1230, 1033, 653, 517, 453,
394, 298, 234, and 154 bp.

and 900 L of water was added to each microtube and


mixed thoroughly. After an additional centrifugation at
9000 g, 700 L of the supernatants were harvested
and frozen at 20 C.

L of ethidium bromide mL1 and photographed on


a TUV transilluminator. Standard VI (BoehringerRoche, Indianapolis, IN, USA) was used as a size
marker.

PCR amplification

Data analysis and construction of dendrograms

The oligonucleotide PCR primers used in this study


were DR1R (5 -GCGGCAACGGCAAGGCGACGCT
GACG-3 ; Jeong and Myrold, 1999) and 879F (5 GCCTGGGGAGTACGGCCGCA-3 ). Crude DNA (2
L) was used as template for the PCR amplifications. PCR was performed using AmpliTaq Gold reagent kit (Perkin-Elmer Biosystems, California, USA)
following the manufacturers intructions: 2.5 L of
GeneAmp 10 buffer, 1 L of BSA 0.1%, 1.5 mM
MgCl2 , 200 M of each dNTP, 2U of AmpliTaq
Gold DNA polymerase, and 2 M of primer for 25
L of final reaction volume. PCR conditions were as
follow: Preheating at 95 C for 9 min; 35 cycles of
denaturing at 95 C for 1 min; annealing at 50 C for
1 min and extension at 75 C for 2 min, and a final
extension at 72 C for 7 min. Eight microliters of amplified PCR product was separated by electrophoresis
on 1.5% agarosa gels, in TBE buffer (100 mM Tris.,
83 mM boric acid, 1 mM EDTA; pH 8.5) for 2 h
at 6 V cm1 , stained in a solution containing 0.5

The bands present in each pattern were coded for input


into a data base that included all the Frankia strains
studied and Jaccards similarity coefficient was calculated to construct the distance matrix. Dendrograms
were constructed from the distance matrix using the
Unweighted Pair Group Arithmetic Mean (UPGMA).

Results and discussion


rep-PCR based fingerprinting
We used DR1R primer to obtain rep-PCR DNA fingerprints of 16 Frankia strains isolated from diverse
actinorhizal plants growing in different geographical
areas because this primer is based on conserved DNA
sequences of direct, repeats in an actino-bacterium,
Mycobacterium bovis (Jeong and Myrold, 1999), that
has a close phylogenetic relationship with Frankia.
DR-PCR patterns generated unique DNA fingerprints

119
for each of the 16 Frankia strains (Figure 1). Previous studies also revealed that the DNA fingerprints
obtained on Frankia by using primers based on repetitive sequences are strain-specific (Jeong and Myrold,
1999; Murry et al., 1995; Perez et al., 1999). Therefore, since Frankia DR1R-PCR fingerprints are strainspecific, they are more suitable for biodiversity studies
than for taxonomic studies.

879F-RAPD fingerprinting
According to the results obtained in other microbial
groups, primers targeting 16S rDNA sequences, when
used at low annealing temperatures (typically 50 or
55 C), yield RAPD patterns that allow to discriminate at species or subspecies levels (Dong et al., 2000;
Rivas et al., 2001). Also, primers with high G+C
contents yields the best fingerprint patterns in bacterial genomes rich in G+C, such as the genome of
Clavibacter (Pastrik and Rainey, 1999). These results prompted us to assess the usefulness of a primer
designated from 16S rDNA sequence of Escherichia
coli (879F primer, 75% G+C) as tool in studying
the taxonomy of the genus Frankia, an actinomycete
with high G+C content (66 to 75 mol%; Benson and
Silvester, 1993).
Because taxonomic levels below genus are not yet
well established in Frankia, we first tested the taxonomic level of resolution of this primer with eight
well-characterized strains of Clavibacter michiganensis belonging to five different subspecies (Table 2).
879F-RAPD band patterns of these strains are shown
in Figure 2. Strains ATCC 33113T 1154 and 1792
of C. michiganensis subsp. sepedonicus showed a
unique 879F-RAPD pattern (Figure 2; lanes 1, 2 and
3, respectively). In a similar way, strains 121.1 (Figure 2, lane 4) and 093.3F (Figure 2, lane 5) of C.
michiganensis subsp. michiganensis displayed a single
band pattern. However, 879F-RAPD patterns of C.
michiganensis subsp. nebraskensis CECT 4209 (Figure 2, lane 6), C. michiganensis subsp. tessellarius
CECT 4263 (Figure 2, lane 7) and C. michiganensis subsp. insidiosus CFBP 5042T (Figure 2, lane
8) were different from each other. Based on these
results, we concluded that 879F-RAPD fingerprinting allows to differentiate Gram positive bacteria at
the subspecies level. Moreover, other results obtained
in our laboratory working with several Gram negative bacteria, such as Ralstonia, Pectobacterium and
Sinorhizobium, also confirm the validity of this meth-

Figure 2. Ethidium bromide stained agarose gel pattern of


879F-PCR products from genomic DNA of Clavibacter michiganensis strains: C. michiganensis subsp. sepedonicus ATCC 33113T
(lane 1), 1154 (lane 2) and 1792 (lane 3); C. michiganensis subsp.
michiganensis 121.1 (lane 4) and 093.3F (lane 5); C. michiganensis
subsp. nebraskensis CECT 4209 (lane 6); C. michiganensis subsp.
tessellarius CECT 4263 (lane 7); C. michiganensis subsp. insidiosus
CFBP 5042T (lane 8); Molecular weight marker (lane MW) as in
Figure 1.

ods for the differentiation of bacterial subspecies (data


not shown).
The results obtained on Frankia using this new
procedure are shown in Figure 3. The reproducibility of the PCR reaction was examined by comparing
the banding patterns of the products in reactions repeated at least twice using the same DNA preparation
of the different strains. Fingerprints from individual
reactions amplified using primer 879F and the same
template DNA were virtually identical. Primer 879F
generated 15 different banding patterns among the
16 Frankia strains characterized. All Frankia strains
showed a distinctive 879F-RAPD profile except two
Casuarina isolates, Frankia CcI3 (Figure 3, lane 7)
and UGL 020603 (Figure 3; lane 9), which displayed
the same unique profile. Thus, taking into account
the results obtained on Clavibacter michiganensis,
Frankia CcI3 and UGL 020603 should be included,
in the same subspecies.
There is apparently genetic homogeneity among
Frankia strains nodulating Casuarina spp (Maggia
et al., 1992; Nazaret et al., 1989; Rouvier et al.,

120

Figure 3. Ethidium bromide stained agarose gel pattern of 879F-PCR products from genomic DNA of Frankia strains as stated in Figure 1.

1992). Fernandez et al. (1989) found a high degree of


DNA-DNA homology among Casuarinaceae-infective
Frankia strains isolated from geographically distant
areas, which grouped into a single genomic species. Moreover, all Frankia successfully isolated from
Casuarina spp., and most Frankia identified in nodules of Casuarina spp. growing outside their native
range belong to one group (type 1) (Prez et al., 1999;
Rouvier et al., 1996; Zimpfer et al., 1999) of the
seven phylogenetic groups found in nodules of Casuarinaceae from Australia (Simonet et al., 1998). In
this regard, the identical 879F-RAPD profile shared
between Frankia CcI3 (Figure 3, lane 7) and Frankia
UGL 020603 (Figure 3, lane 9), both isolated from
root nodules of Casuarina spp. growing at very distant geographic locations (Table 1), is also indicative
of the genetic homogeneity found previously among
all Frankia strains isolated from Casuarina spp. However, the other two Casuarina isolates included in this
study, Frankia CeI2 (Figure 3, lane 6) and Frankia
CSI 020603 (Figure 3, lane 8), showed different 879FRAPD profiles. This result indicates that this new
PCR-based technique has a resolution level in differentiating Casuatinaceae-Frankia strains higher than
other methods earlier used, such as sequence analysis
of hypervariable regions (Maggia et al, 1992; Rouvier
et al., 1992), symbiotic genes as hybridization probes
(Nazaret et al., 1989) or restriction analysis of PCR
products (PCR-RFLP) (Prez et al., 1999; Rouvier et
al., 1996).

Data analysis and construction of dendrograms


An UPGMA dendrogram of the 879F-PCR fingerprint
patterns shows that the 16 Frankia strains used in this
study could be clustered into groups that broadly reflects the host plants from which they were derived
(Figure 4, Table 1). Despite having low similarity
coefficients among them, the five Alnus isolates, the
four Casuarina isolates and the two Hippophae isolates form three separated and well-defined groups.
These results are consistent with those obtained by
other authors using Low-Frequency Restriction Fragment Analysis (Beyazova and Lechevalier, 1992),
DNA-DNA hybridization (Fernandez et al., 1991),
16S rDNA sequencing (Nazaret et al., 1991; Normand
et al., 1996) and LMW RNA profiles (Velzquez et al.,
1998). Moreover, Frankia strains Cj (DDB000320),
isolated from soil beneath Ceanothus jepsonii, and
Frankia PtI1, isolated from nodules of Purshia tridentata (Table 1), also grouped together (Figure 4).
This grouping agrees with the analysis of partial 16S
rDNA sequences obtained from symbionts residing
in nodules from Ceanothus griseus and Purshia tridentata, which were found to be identical to each other
(Benson et al., 1996), and with the analysis of nearly
full-length 16S rDNA of the Ceanothus caeruleus
isolates Cea5.1 and Ceal.3, and the Frankia strain
PTI1, isolated from Purshia tridentata (Clawson et al.,
1998). Therefore, our results indicate that there is a
comparably good correlation between degrees of relatedness determined by 879F-PCR fingerprinting and

121

Figure 4. UPGMA dendrogram based on Jaccards coefficient derived from 879F-PCR DNA patterns of the Frankia strains analysed.

Figure 5. UPGMA dendrogram based on Jaccards coefficient derived from DR1R-PCR DNA patterns of the Frankia strains analysed.

122
comparative 16S rRNA sequence analysis of Frankia
strains.
On the other side, the topology of an UPGMA
dendrogram of the DR1R-PCR fingerprint patterns
(Figure 5) is, in general, similar to that of the 879FPCR fingerprint patterns (Figure 4). Consistent features of such trees include the grouping of strains
from Alnus sp., from Casuarina sp., and from Hippophae rhamnoides. By contrast, each Casuarina
isolate is here located in a different branch within
the same cluster because DR1R-PCR fingerprints are
strain-specific. Moreover, the Frankia strains PtIl and
DDB000320 group separately in the UPGMA dendrogram of the DR1R-PCR fingerprint patterns, indicating a lesser congruence with comparative 16S rRNA
sequence analysis of Frankia strains than the UPGMA
dendrogram of the 879-RAPD fingerprint patterns.
Therefore, for taxonomic purposes, 879F-RAPD fingerprint patterns seem to be better than DR1R-PCR
fingerprint patterns.
In conclusion, 879F-RAPD fingerprinting method
is easy to perform, require minimal technical skill, and
results can be obtained in two working days. Other
than a thermocycler and electrophoresis equipment,
no sophisticated or expensive apparatus are needed
to set up this method. Moreover, 879F-RAPD fingerprints were generated by using whole cell suspensions, which eliminated the need for DNA purification.
Therefore, this method is amenable to analyze large
collections of bacterial isolates. Since 879F-RAPD
fingerprinting allows to differentiate Frankia isolates
at levels higher than strain in a rapid, easy and reliable way, it can be included in polyphasic approach
to Frankia taxonomy, where other classical taxonomic
methods are less applicable.

Acknowledgements
This work was supported by Grants SA35/99 and
CSI01-98 of the Consejera de Educacin y Cultura
(Junta de Castilla y Len). We thank to J. L. Palomo
and P. Garcia Benavides for providing Clavibactcr
strains.

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