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115
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
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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
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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
France
USA
USA
USA
Argentina
Chile
Hr114.2
HrI1 (DDB14010110)
MpI1 (LLR162001)
PtI1 (DDB17020110)
DtI2
TtI1
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
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.
PCR amplification
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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-
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Figure 3. Ethidium bromide stained agarose gel pattern of 879F-PCR products from genomic DNA of Frankia strains as stated in Figure 1.
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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.
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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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