RESEARCH ARTICLE
Natural transformation in the Ralstonia solanacearum species
complex: number and size of DNA that can be transferred
Bénédicte Coupat1, Fanny Chaumeille-Dole1, Saliou Fall2, Philippe Prior3, Pascal Simonet2, Xavier
Nesme1 & Franck Bertolla1
1
Université de Lyon, Université Lyon 1, CNRS, INRA, Ecologie Microbienne, UMR 5557, USC 1193, Villeurbanne, France; 2Génomique Microbienne
Environnementale, UMR CNRS 5005, Laboratoire Ampère, Ecole Centrale de Lyon, Ecully, France; and 3CIRAD-INRA, Pôle de Protection des Plantes (3P),
Bios UMR C53 – Ligne Paradis, Saint Pierre Cedex, La Réunion, France
Correspondence: Bénédicte Coupat,
Université de Lyon, Université Lyon 1, CNRS,
INRA, Ecologie Microbienne, UMR 5557, USC
1193, F-69622 Villeurbanne, France. Tel.:
133 472 432 758; fax: 133 426 234 468;
e-mail: [email protected]
Received 30 October 2007; revised 23 April
2008; accepted 21 May 2008.
First published online 25 July 2008.
DOI:10.1111/j.1574-6941.2008.00552.x
Editor: Kornelia Smalla
Keywords
Ralstonia solanacearum ; horizontal gene
transfer; natural transformation.
Abstract
Ralstonia solanacearum is a widely distributed phytopathogenic bacterium that is
known to invade more than 200 host species, mainly in tropical areas. Reference
strain GMI1000 is naturally transformable at in vitro and also in planta conditions
and thus has the ability to acquire free exogenous DNA. We tested the ubiquity and
variability of natural transformation in the four phylotypes of this species complex
using 55 strains isolated from different hosts and geographical regions. Eighty per
cent of strains distributed in all the phylotypes were naturally transformable by
plasmids and/or genomic DNA. Transformability can be considered as a ubiquitous physiological trait in the R. solanacearum species complex. Transformation
performed with two independent DNA donors showed that multiple integration
events occurred simultaneously in two distant genomic regions. We also engineered a fourfold-resistant R. solanacearum GMI1000 mutant RS28 to evaluate the
size of DNA exchanged during natural transformation. The results demonstrated
that this bacterium was able to exchange large DNA fragments ranging from 30 to
90 kb by DNA replacement. The combination of these findings indicated that the
natural transformation mechanism could be the main driving force of genetic
diversification of the R. solanacearum species complex.
Introduction
Horizontal gene transfer (HGT) is recognized as one of the
main forces behind the adaptation and evolution of bacteria
(Nielsen et al., 1998; Ochman et al., 2000). Sex between
bacteria, being defined as any exchange of genetic material,
has allowed the acquisition of new functions such as
pathogenicity or new metabolic pathways (Davison, 1999).
Among the three mechanisms of HGT (natural transformation, conjugation and transduction), natural transformation
requires that bacteria have the ability to develop competence
and to take up free foreign DNA. This mechanism is widely
distributed among trophic bacterial groups. At least 40
bacterial species are naturally transformable and are able to
colonize various environments such as water, soil and
sediment (Lorenz & Wackernagel, 1994; de Vries & Wackernagel, 2002).
Among naturally transformable bacteria, some are pathogens of animals or plants, such as the phytopathogenic
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c
bacterium Ralstonia solanacearum. This betaproteobacterium is the causal agent of bacterial wilt affecting more than
50 families of plants worldwide (Hayward, 1991); the disease
was initially restricted to tropical areas but has recently been
detected in temperate regions (Europe) (Wullings et al.,
1998). In addition, recent reports indicate that the range of
plants infected by R. solanacearum is increasing (Wicker
et al., 2007). These pathogenicity-related evolutionary
trends indicate that this soil-living bacterium exhibits a
strong adaptive potential that could be related to its HGT
potential based on results demonstrating competence development and natural transformation-mediated gene transfer
during plant colonization (Bertolla et al., 1999).
The genome sequence of R. solanacearum strain GMI1000
revealed the presence of alternative codon-usage regions
(Acurs), suggesting that these sequences were probably
acquired by HGT (Salanoubat et al., 2002; Genin & Boucher,
2004). The mosaic structure of R. solanacearum was confirmed by Nakamura et al. (2004), who showed that 16% of
FEMS Microbiol Ecol 66 (2008) 14–24
15
Natural transformation in R. solanacearum
the genome was acquired by HGT. Recently, methods based
on phylogenetic reconstruction of prokaryote homologous
gene families and on two codon-usage indices detected 151
genes (13.3%) of foreign origin in the R. solanacearum
GMI1000 genome and tentatively identified their bacterial
origin (Fall et al., 2007).
This bacterial genomic diversity is also found in the
R. solanacearum species complex (Gillings & Fahy, 1994),
which are distributed in four lineages termed phylotypes, which
were defined as monophyletic clusters of strains revealed using
phylogenetic analysis of sequence data (Fegan & Prior, 2005).
Taken together, the transformability and genomic plasticity of this organism led us to envisage that natural
transformation could be a major driving force of the
evolution and the adaptability of the whole species complex
of R. solanacearum. Our objectives here were to determine
(1) if the natural transformable property is ubiquitiously
shared among strains belonging to the four R. solanacearum
phylotypes and (2) the number and size of DNA fragments
that can be transferred in R. solanacearum during natural
transformation. Answers to these questions could help us to
understand better the role of natural transformation in the
genomic plasticity in the R. solanacearum species complex
and its contribution to the adaptive evolution of plant
pathogenicity functions.
Materials and methods
Bacterial strains, culture media and growth
conditions
The bacterial strains and plasmids used in this study are
listed in Table 1. Wild-type and transformant strains of R.
solanacearum were grown at 28 1C on B agar plates or B
liquid medium (Boucher et al., 1985). Escherichia coli cells
were grown at 37 1C on liquid or solid Luria–Bertani (LB)
medium. Ralstonia solanacearum and E. coli transformants
were grown on their respective media supplemented with
the appropriate antibiotics to the following final concentrations: 50 mg mL1 for kanamycin (Km), 40 mg mL1 for
spectinomycin (Sp), 10 mg mL1 for gentamicin (Gm),
30 mg mL1 for chloramphenicol (Cm) and 100 mg mL1 for
ampicillin (Amp).
pGEM-T-cloning vector (Promega, Madison, WI) to constitute the plasmid pnagI. The kanamycin resistance cassette,
extracted from the plasmid pMKm (Murillo et al., 1994) via
restriction with AccI (Fermentas, Ontario, Canada), was
ligated into the NarI-linearized pnagI plasmid to give
pnagI<Km. Plasmids pRSc1152<Sp and precO<Cm were
constructed by a similar protocol. RSc1152 and recO genes
were amplified by PCR with F1573/F1574 and F2621/F2622
primers and cloned in the pCR2.1 TOPO TA cloning vector
(Invitrogen, Carlsbad, CA). Plasmids pRSc1152 and precO
were linearized with the enzyme SalI (Fermentas) to insert
streptomycin or chloramphenicol cassettes obtained, respectively, from pMSm (Murillo et al., 1994) and pMCm
(Murillo et al., 1994) via SalI digestion (Fermentas). The
plasmid pcomA<Gm (Fall et al., 2007) carried the comA
gene labelled with the gentamicin antibiotic cassette (Brau
et al., 1984).
For each plasmid construction, E. coli DH5a cells were
transformed with ligation products according to the manufacturer’s instructions (Invitrogen). Plasmids were extracted
and purified with a Wizard Plus SV minipreps DNA
purification system (Promega).
Construction of the fourfold R. solanacearum
mutant
The isogenic mutant RS28 was engineered by successive
gene replacements into the recO, RSc1152, nagI and comA
genes in R. solanacearum GMI1000. The four recombination
steps were obtained by successive natural transformation
with precO<Cm, pRSc1152<Sp, pnagI<Km and pcomA<Gm. Indeed, precO<Cm was used to transform the
wild-type R. solanacearum GMI1000 to give the mutant
RS25, which was then transformed by pRSc1152<Sm to
create the RS26 mutant. RS26 was then transformed by
pnagI<Km to select the RS27 mutant. Finally, the RS28
mutant was obtained after transformation of the RS27
mutant by pcomA<Gm. Sensitivity of the recombinant R.
solanacearum to ampicillin confirmed the absence of the
replicative plasmid vector and also the successive double
cross-over events, which replace the wild-type genes by the
chimeric constructions.
Plasmid constructions
Natural transformation of R. solanacearum
strains
In order to evaluate the size of transferred DNA, we chose
the recO gene and three other genes, namely nagI, comA and
RSc1152, which are 30, 60 and 90 kb, respectively, distant
from the first (Fig. 1a). Each target gene was amplified by
PCR from the R. solanacearum GMI1000 strain, cloned in
appropriate cloning vector and disrupted by a specific
antibiotic resistance cassette. The nagI gene was amplified
with the F2619/F2620 primers (Table 2) and cloned in the
Ralstonia solanacearum strains were transformed according
to the protocol of Bertolla et al. (1997) with some modifications. Fifty microliters of competent bacteria were transformed with 200 ng of plasmid DNA or 400 ng of genomic
DNA. GMI1000 was naturally transformed in vitro with
digested and nondigested DNA of the RS28 mutant. This
genomic DNA was restricted with SspI (Fermentas), which
digests DNA only between the target loci (Fig. 1a). These
FEMS Microbiol Ecol 66 (2008) 14–24
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Published by Blackwell Publishing Ltd. All rights reserved
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16
B. Coupat et al.
Table 1. Bacterial strains, mutants and plasmids used in this study
Strains and plasmids
Characteristics
R. solanacearum
Environmental strains
GMI1000
NCPPB4011
T41
CFBP765
R292
ACH092
T3
NCPPB3190
M41
JT523
MAFF211266
CIP65
R288
PSS194
CFBP2968
CFBP2964
CIP365
ACH1023
M71
PSS241
ICMP7963
CFBP712
JT510
CIP120
UW009
UW469
UW20
CFBP2957
CIP301
NCPPB3987
CFBP1419
UW11
ANT307
A3909
CFBP6783
Molk2
CMR34
IPO1609
B34
JT516
UW134
NCPPB3059
NCPPB332
CFBP734
CIP358
NCPPB1029
NCPPB283
NCPPB342
NCPPB505
JT525
ACH732
MAFF301558
R24
PSI7
JT663
Other designation
RUN54
RUN80
RUN95
RUN33
RUN91
RUN44
RUN94
RUN78
RUN577
RUN333
RUN69
RUN578
RUN90
RUN274
RUN38
RUN37
RUN47
RUN10
RUN68
RUN87
RUN55
RUN463
RUN59
RUN42
RUN450
RUN109
RUN96
RUN36
RUN45
RUN81
RUN465
RUN579
RUN16
RUN9
RUN17
RUN74
RUN147
RUN35
RUN22
RUN160
RUN55
RUN39
RUN75
RUN477
RUN46
RUN77
RUN478
RUN76
RUN480
RUN60
RUN14
RUN71
RUN88
RUN83
RUN64
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c
Origin
Host
Phylotype
Biovar
French Guyana
China
Indonesia
Japan
China
Australia
Indonesia
Malaysia
Malaysia
Reunion Island
Japan
Costa Rica
China
Taı̈wan
Guadeloupe
Martinique
Philippine
Australia
Malaysia
Taı̈wan
Kenya
Burkina Faso
Reunion Island
Peru
Costa Rica
Brazil
Venezuela
Martinique
Peru
Brazil
Costa Rica
Costa Rica
Martinique
Hawaii
French West Indies
Philippines
Cameroon
The Netherlands
Brazil
Reunion Island
Kenya
Burkina Faso
Zimbabwe
Madagascar
Cameroon
Reunion Island
Zimbabwe
Zimbabwe
Zimbabwe
Reunion Island
Australia
Japan
Indonesia
Indonesia
Indonesia
Tomato
Morus alba
Tomato
Tobacco
Morus alba
Zingiber officinale
Tomato
Tomato
Peanuts
Tomato
Tomato
Chilli pepper
Morus alba
Tomato
Eggplant
Eggplant
Potato
Strelizia reginae
Soil
Tomato
Potato
Eggplant
Potato
Potato
Heliconia sp.
Potato
Banana
Tomato
Potato
Potato
Musa sp.
Heliconia
Anthurium
Heliconia sp.
Heliconia caribea
Musa sp.
Tomato
Potato
Banana
Potato
Patato
Eggplant
Potato
Potato
Potato
Pelargonium capitatum
Solanum panduraforme
Tobacco
Symphytum sp.
Pelargonium asperum
Tomato
Potato
Clove
Tomato
Clove
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
III
III
III
III
III
III
III
III
III
IV
IV
IV
IV
IV
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
5
5
5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2T
1
1
1
1
1
1
1
1
1
2T
2
2T
2T
RSY
RSY
FEMS Microbiol Ecol 66 (2008) 14–24
17
Natural transformation in R. solanacearum
Table 1. Continued.
Strains and plasmids
Characteristics
R. solanacearum
Mutant strains
RS3
RS13
RS25
RS26
Origin
Source
Fall et al. (2007)
Fall et al. (2007)
This study
This study
Host
RS27
This study
RS28
This study
E. coli
DH5a
Invitrogen
F-B 80lac ZDM15 D(lac ZYA-arg F) U169
rec A1 end A1 hsd R17(rk-, mk1) pho A
sup E44 thi-1 gyr A96 relA1 tonA
Plasmids
pBBR1-MCS5
pCR2.1-TOPO
pGEM-T
pMCm
pMKm
pMSm
pMGm
precO
Kovach, 1995
Invitrogen
Promega
Ubben & Schmitt (1987)
Keen et al. (1988)
Ubben & Schmitt (1987)
Ubben & Schmitt (1987)
This study
Broad host range cloning vector, GmR
PCR cloning vector, Apr, Kmr, lac POZ
PCR cloning vector, Apr, lac POZ
Cmr
Kmr
Spr
Gmr
recO PCR fragment from GMI1000
inserted in pGEM-T
RSc1152 PCR fragment from
GMI1000 inserted in pCR2.1-TOPO
nagI PCR fragment from GMI1000
inserted in pGEM-T
pGEM-T, recO<Cm
pCR2.1-TOPO, RSc1152<Sm
pGEM-T, nagI<Km
pUC18, comA<Gm
pCR2.1-TOPO, ftsK<Gm
pCR2.1-TOPO, recA<Gm
pRSc1152
This study
pnagI
This study
precO<Cm
pRSc1152<Sm
pnagI<Km
pcomA<Gm
pftsK<Gm
precA<Gm
This study
This study
This study
Fall et al. (2007)
Fall et al. (2007)
Fall et al. (2007)
Phylotype
Biovar
Description/relevant genotype
GMI1000 mutant, recA<Gm
GMI1000 mutant, ftsK<Gm
GMI1000 mutant, recO<Cm
GMI1000 mutant, recO<Cm;
RSc1152<Sp
GMI1000 mutant, recO<Cm;
RSc1152<Sp; nagI<Km
GMI1000 mutant, recO<Cm;
RSc1152<Sp; nagI<Km; comA<Gm
ACH, C. Hayward, University of Queensland, Australia; CIP, International Potato Center, Peru; CFBP, Collection Française des Bactéries Phytopatho-
gènes, France; ICMP, International Collection of Microorganisms from plants, Auckland, New Zealand; MAFF, Collection of the Ministery of Agriculture,
Forestry and Fisheries, Tsukuba, Ibaraki, Japan; NCPPB, National Collection of Plant Pathogenic Bacteria, York, UK; UW, University of Wisconsin; R,
Rothamsted Experimental Station, UK; RUN, CIRAD collection, Reunion Island, France.
transformation mixtures were plated on BG medium supplemented with different antibiotic combinations (Table 4)
for enumeration of all single, double, triple or quadruple
antibiotic-resistant clones.
To test the occurrence of multiple DNA integrations, R.
solanacearum GMI1000 was simultaneously transformed
with plasmids and or genomic DNAs containing the ftsK or
recO genes disrupted by the Gm or Cm antibiotic resistance
cassettes, respectively. According to the genome sequence,
the ftsK and recO loci are separated by 1.5 Mb on the
GMI1000 chromosome (accession no. AL646052).
The transformation frequency was defined as the number
of colonies growing on the appropriate selective media and
exhibiting the expected DNA fragments after PCR amplifiFEMS Microbiol Ecol 66 (2008) 14–24
cation, divided by the total number of viable bacteria. For
the experiments detailed in Tables 4 and 5, the transformation frequencies are the results of at least three independent
experiments and the spontaneous mutation rate was determined by plating on selective media control samples on
which transformations were carried out without DNA. For
determination of transformability in R. solanacearum, 55
strains were transformed once by each of three donor DNAs.
Genomic DNA extraction
Genomic DNA was extracted following the technical protocol of the ‘DNeasy tissue kit’ (Qiagen, Hilden, Germany).
However, some modifications were performed to improve
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18
B. Coupat et al.
22 ORFs
0 SspI
(a)
Donor DNA :
RS28 mutant
5’
Recipient DNA:
GMI1000 strain
3’
33 ORFs
30
33 ORFs
SspI
SspI
90 kb
3’
Cm
Km
Gm
Sp
recO
nagI
comA
RSc1152
recO
nagI
comA
5’
RSc1152
(b) 1E-05
Transformation frequency
1E-06
1E-07
1E-08
1E-09
1E-10
0
10
20
30
40
50
60
70
Length of integrated DNA (kb)
80
90
100
Fig. 1. Relationship between natural
transformation frequencies and length of
exogenous DNA integrated in the Ralstonia
solanacearum genome. (a) Locations of possible
recombination events between the RS28 mutant
and the GMI1000 strain. Crosses represent the
possible sites of crossover between the GMI1000
and the RS28 DNA. (b) Correlation between the
length of exchanged DNA between the RS28
DNA and the GMI1000 strain and transformation
frequencies. Solid and dotted lines represent
transformation frequencies obtained with
genomic DNA or SspI-digested genomic DNA
from the RS28 mutant, respectively. Closed
and open squares or triangles indicate the
combination of selective antibiotics used to
select the transformant, 5 0 –3 0 or 3 0 –5 0 direction,
respectively. Horizontal dotted line represents
the limit of detection.
Table 2. List of primers used in this study
Primer pair
Gene
Annealing temperature ( 1C)
PCR fragment length (bp)
Sequence
F1024/F1025
F1573/F1574
F1784/F1785
F2251/F2252
F2619/F2620
F2621/F2622
F2838/F2839
F2901/F2902
F3044/F3045
F3775/F3776
F3777/F3778
F3779/F3780
comA
RSc1152
RSc1152
ftsK
nagI
recO
recO
nagI
Km
RSc1076
soxA2
RSc1132
58
57
58
50
55
55
50
55
50
60
58
60
2036
2266
496
2376
2093
2048
242
225
356
4212
4396
4424
CGAGCTGCCCGAAGTCAC/CCTGCGGATGCGGATGAC
GCAGATGGCGACCGATTC/ACGTGAAGGCGAACAGCA
ATACTGCGCGCGGAAGTC/ CGTGAGCGCAAGCAAAGC
TTCCAGGGATGCGGTAAC/CCCGAGCTTCCACCACT
GTCGGAACTGAAGATGGA/GTAGCGCAGCTTGACAC
GCGGCGCGTGTTCGTC/CAAAGCGCGTGGCAAGAA
CACGGCGAGCCTGTTGC/GTGGCCGGACGGAATAA
CAGACACTGCCGCCTGAG/TCCCGGGTTCTCCAGCAC
GCGCAACGGAACATTCATCA/GAGACGCAACGTGGCTTTGT
CGCGCTGCTCTGGCTGACA/GGCGATGTTGCGGGTGAAGA
AGGGCCTGTACTTCAACTGC/CGGCTTGCCAGAGAGAAAC
GCCGCCGAGATCCTTGAACT/CGCTGGCTCGACGAACTGAA
A standard PCR programme was performed and consisted of a classical amplification over 35 cycles except for ftsK, recO and Km () for which a
touchdown PCR programme was used: the annealing temperature was programmed to decrease by 1 1C at each cycle over the first 10 cycles. This
programme amplified DNA during 30 cycles.
bacterial lysis. Cells were washed with TE buffer, pH 8, and
then lysed via the addition of 25 mL of proteinase K
(50 mg mL1), 25 mL of sarkosyl at 10% and 4 mL of RNAse
A (100 mg mL1) in a final volume of 500 mL, over 4 h at
room temperature. The size and quality of extracted DNA
were verified by pulsed-field electrophoresis with a 1%
PFGE agarose gel (Biorad, Hercules, CA) in TBE (0.5 ),
at 6 V cm1 over 22 h with pulse times ranging from 60 to
120 s, in a CHEF-DR III pulse field electrophoresis system
(Biorad).
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c
Transformant verification
Integration of antibiotic resistance cassettes into the target
genes in R. solanacearum mutants and transformants were
checked using PCR amplification. In the RS25, RS26, RS27
and RS28 mutants and in transformants, these stable integrations were checked with primers F1024/F1025 (comA),
F1784/F1785 (RSc1152), F2838/F2839 (recO), F2901/F2902
(nagI) and F3044/F3045 (Km). Additional PCR verifications
were performed in the fourfold-resistant clones with primers
FEMS Microbiol Ecol 66 (2008) 14–24
19
Natural transformation in R. solanacearum
F3775/F3776, F3777/F3778 and F3779/F3780, in order to
check for the presence of DNA regions between target genes.
Verification of transformants resulting from multiple
transformation events was performed with primers F2251/
F2252 (ftsK) and F2838/F2839 (recO). PCR reactions were
carried out with 100 ng of DNA and Taq Platinum DNA
polymerase (Invitrogen).
Results and discussion
Competence and transformability within the
R. solanacearum species complex
Fifty-five isolates were selected among the four phylotypes
defined in the R. solanacearum species complex (Fegan &
Prior, 2005) to be tested for their ability to be naturally
transformed by naked DNA. These strains were systematically subjected to the competence development treatment
that yields the best transformation efficiency in strain
GMI1000. In agreement with data acquired with other
naturally competent bacteria (Lorenz & Wackernagel,
1994), we transformed R. solanacearum in limiting growth
conditions that permitted higher transformation efficiency
(Bertolla et al., 1997). Detection of even a very low number
of transformants was sufficient to demonstrate that the
strain considered was actually equipped with the molecular
machinery to develop a competence state. In order to
increase the range of conditions that enabled detection of
transformation events, tests were also carried out with both
plasmid and chromosomal DNA. Two plasmids were used,
plasmid pBBR1MCS-5 (Kovach et al., 1995), which is able to
replicate autonomously in R. solanacearum, and plasmid
precA<Gm, which only yields detectable transformants after
homologous recombination-based genome integration inside the recA gene. Genomic DNA from the recA mutant
RS3, in which the gentamicin resistance gene was inserted in
the recA gene, was used as the ‘chromosomal’ donor DNA.
Transformation-mediated acquisition of the gentamicin
resistance phenotype was detected in 80% (43/55) of the R.
solanacearum strains tested. In these naturally transformable
strains, transformation frequencies always diverged significantly from the spontaneous mutation rates, which are
below 1 1010 (Table 3 and supplementary Table S1). The
presence of the gentamicin resistance gene was demonstrated
by PCR in several strains, confirming that their new phenotype resulted from transformation events with donor DNA.
Comparison of transfer frequencies in R. solanacearum
between the three DNA sources suggest that recombinationmediated DNA integration is more efficient than plasmid
replication, confirming results observed with other models
(Lorenz & Wackernagel, 1994). Transformability was detected in 33 of the 55 strains tested when the isolates were
subjected to the suicide plasmid precA<Gm or the genomic
FEMS Microbiol Ecol 66 (2008) 14–24
DNA RS3, whereas only 24 strains yielded detectable colonies
by using the broad-host-range plasmid pBBR1MCS-5. However, among these 24 strains, two were not transformable with
the integrative DNAs (Table 3 and supplementary Table S1),
indicating that tests with the replicative plasmids were
necessary to complete the list of strains developing competence. It is known that a high sequence divergence between
the donor DNA and the recipient strain can limit the
recombination step and affect transformation efficiency
(Mercier et al., 2007). Indeed, whatever the donor DNA used,
transformation tests permitted the determination of transformable strains in each phylotype, providing strong evidence
that competence is a ubiquitous physiological trait in the R.
solanacearum species complex. The ability to transform
naturally is a major factor contributing to genetic variability
and could also preserve and restore genetic integrity, if
necessary (Fall et al., 2007). It is, however, still difficult to
relate the experimentally acknowledged transformability of
biovars with their actual genetic variability or stability.
Transformation frequencies varied over more than four
orders of magnitude between strains. Some strains exhibited
a very low transformation frequency, such as ICMP7963
(phylotype II) (3.8 1010), only slightly higher than the
detection threshold (1010), while strain GMI1000 (phylotype I) exhibited the highest frequency of up to 3.8 106,
even though both strains were transformed by the same
replicative plasmid precA<Gm. These wide differences
might indicate that the level of physiological competence is
highly variable among strains, resulting from markedly
different genetic potentials (Wilson et al., 2003). However,
experimental biases cannot be excluded, including those
related to the transformation protocol used, which was
optimized for strain GMI1000 but which could be far from
optimal for other strains, mainly those of phylotypes II, III
and IV. In addition, transfer efficiency in strains classified in
these phylotypes can also be affected by activity of the
methyl mismatch repair (MMR) system, which would
decrease efficiency of the recombination process in integrating donor DNA sequences diverging significantly from those
of the recipient genome. The combination of the two
parameters could have led to some phylotype III and IV
strains being classified as lacking the competence machinery
when in fact just transformation protocols and donor DNA
were not adapted to yield transformant production.
Comparison of transformation efficiencies also indicated
that these were usually higher for chromosomal DNA than for
integrative plasmids in spite of the same sequences in both
constructions. These results could be due to the longer flanking
regions in the chromosomal donor compared with the plasmid, which provided a greater probability of short sequences
termed minimum efficiently processed segment (MEPS) (Majewski & Cohan, 1999; Majewski et al., 2000) or microhomologies and GC-rich regions (de Vries & Wackernagel, 2002;
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20
B. Coupat et al.
Table 3. Natural transformation ability of Ralstonia solanacearum
Number of transformable strains
Phylotype
Number of strains
pBBR1-MCS5
precA<Gm
recA<Gm
Transformation frequencies
I
II
III
IV
Total
20
20
10
5
55
12
9
1
2
24
15
13
3
2
33
15
10
6
3
34
7.7 1010–3.8 106
3.8 1010–4.6 107
1.2 108–5.9 108
3.2 109–5.1 107
Transformation frequency is expressed as the number of transformants per recipient cells.
Meier & Wackernagel, 2003), favouring recombination around
the target gene. This phenomenon is notably observed for
strains belonging to phylotype III, for which four strains were
transformable only with genomic DNA (Table 3 and supplementary Table S1).
(a)
1
2
3
4
5
6
7
8
4000 bp
2376 bp
Multiple integration into the R. solanacearum
genome
Experimental methods for detecting HGT in bacterial genomes are based on acquisition of selective markers such as
genes encoding the resistance to antibiotics or heavy metals
or the utilization of rare carbon sources (Davison, 1999; de
Vries & Wackernagel, 2002). However, such methods detect
the recombination-based transfer events involving DNA
sequences flanking the marker gene but are unable to
determine if other positions in the genome were simultaneously modified by independent transfer events involving
the same or different DNA molecules. This methodological
limitation can lead to misinterpretations of the actual
impact of HGT on the genome structure in the case of
additional HGT events. We addressed this question by
subjecting the recipient strain to a mixture of two DNA
types each labelled by a different marker gene. Two integrative plasmids were constructed based on the recO and ftsK
genes distantly separated by 1.5 Mb on the GMI1000 chromosome. These genes were labelled with a chloramphenicol
and a gentamicin resistance gene to yield the plasmids
precO<Cm and pftsK<Gm, respectively. In a second step,
chromosomal DNAs from the recO and ftsK recombinant
strains (RS25 and RS13) were also used as donor DNA.
Transformation tests with plasmid and chromosomal DNA
mixtures allowed the recovery of transformants exhibiting
resistance to both chloramphenicol and gentamicin. The
presence of these antibiotic markers in the recombinant
clones was checked using PCR with primers complementary
to part of the target genes (Fig. 2). The size of PCR products
was around 4000 and 3000 bp as expected. These results
indicate that two independent recombination events occurred simultaneously to integrate two different DNA fragments in separate regions in the R. solanacearum genome, in
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(b)
1
2
3
4
5
6
7
8
3200 bp
242 bp
Fig. 2. PCR-based control of the multiple integration of DNA in
Ralstonia solanacearum chromosome. PCR verification of gentamicin
and chloramphenicol cassettes in (a) the ftsK and (b) recO gene: 1,
1-kb1 ladder; 2, control; 3, GMI1000; 4A, pftsK<Gm; 4B, precO<Cm;
5, 6 and 7, multiple plasmid DNA transformants; 8, multiple genomic
DNA transformant.
accordance with results obtained with Bacillus subtilis
(Zawadzki et al., 1995). However, these transformants were
detected at frequencies of 5.4 ( 1.5) 109and 6.1
( 4.4) 109, i.e., two and three orders of magnitude
lower than those of transformants resulting from one single
transfer event by one DNA source [7.4 ( 1.9) 107 and
8.3 ( 0.8) 106] (Table 4). In addition, no triple transformant was detected when a mixture containing three
different plasmid or chromosomal DNAs was used.
FEMS Microbiol Ecol 66 (2008) 14–24
21
Natural transformation in R. solanacearum
Determination of the size of transferred DNA in
R. solanacearum GMI1000
Evaluation of the extent of gene flow and the size of DNA
that can be transferred by natural transformation during a
short time of exposure to exogenous DNA is an important
element in assessing the role of transformation in bacterial
adaptation and evolution. In order to address this question,
the fourfold mutant RS28 was elaborated by successive
integrations of four antibiotic resistance cassettes, namely
chloramphenicol, streptomycin, kanamycin and gentamicin,
in the four genes recO, RSc1152, nagI and comA, respectively. The correct integration of each antibiotic resistance
gene in its respective target gene was checked by PCR in the
successive mutants RS25, RS26, RS27 and RS28 (data not
shown). After cloning, marker genes were located in the
recipient genome and separated by a distance of about 30 kb.
The final strain RS28 thus had a 90-kb DNA fragment evenly
labelled by four markers (Fig. 1a). To limit transformation
bias due to DNA shearing and to ensure that the genomic
construction of 90 kb was included in a single DNA fragment, transformation tests were carried out with genomic
DNA carefully extracted from strain RS28. A pulsed-field
experiment confirmed the large size of this genomic DNA,
which was up to 100 kb (data not shown).
Thus, detection and enumeration of transformants expressing an increasing number of the marker genes can be
seen as a means to calculate the frequency to which large
DNA fragments are transferred. Interestingly, transformation of R. solanacearum GMI1000 plated on media supplemented with the four antibiotics led to the selection of
quadruply resistant transformants. Transformations occurred, however, at a low frequency, 3.4 109, which is a
drop of more than three orders of magnitude in comparison
with the frequencies of transformants selected on single
antibiotics, which reached 5 106–1.2 106 irrespective
of the antibiotic used (Table 5). Moreover, when the
corresponding antibiotic markers were located contiguously
in the donor DNA, double or triple resistant transformants,
frequencies reached 107 and 108, respectively (Fig. 1b). For
the quadruply resistant clones, the DNA region replacement
was confirmed by PCR through checking for the presence of
the four antibiotic resistance genes and also the neighbouring DNA genes between these selective markers (data not
shown). Consequently, we assumed that at least 90 kb of
transforming DNA would be exchanged between the transforming DNA and the GMI1000 genome. Ralstonia solanacearum was also transformed with donor DNA RS28
digested by SspI, whose restriction site was presented only
once between each allelic construction (Fig. 1a). No transformants were recovered when triple and quadruple-resistant
transformants were selected. The decrease of the transformation frequency below the detection threshold confirmed
that only the replacement of the whole fragment containing
the four genes could explain the simultaneous presence of
the four genes in the recombinant strains. This result was
also confirmed by the use of multiple-distant integrations in
which only two distinct DNA fragments could be integrated
simultaneously into the R. solanacearum genome.
In conclusion, we have clearly shown that the decrease in
transformation frequency is directly related to the size of the
DNA acquired by the cell and integrated by homologous
Table 4. Transformation frequencies depending on antibiotic selection and length of transferred DNA
Transformation frequency
Target gene
Antibiotic selection
Distance between target genes (kb)
Nondigested DNA
Digested DNA
recO
nagI
comA
rsc1152
recO. nagI
recO. comA
recO. rsc1152
nagI. comA
nagI. rsc1152
comA. rsc1152
recO. nagI. comA
nagI. comA. rsc1152
recO. comA. rsc1152
recO. nagI. rsc1152
recO. nagI. comA. rsc1152
Cm
Km
Gm
Sp
Cm, Km
Cm Gm
Cm, Sp
Km, Gm
Km, Sp
Gm, Sp
Cm, Km, Gm
Km, Gm, Sp
Cm, Gm, Sp
Cm Km Sp
Cm, Km, Gm, Sp
–
–
–
–
30
60
90
30
60
30
60
60
90
90
90
2.4 ( 1.1) 106
1.2 ( 0.8) 106
5 ( 1.6) 106
2.8 ( 1.1) 106
5.7 ( 1.7) 107
3.4 ( 1.1) 107
6.5 ( 2.9) 108
2.6 ( 0.9) 106
3.9 ( 1.9) 107
2.9 ( 1.3) 107
1.4 ( 0.8) 107
1.9 ( 1.1) 107
2.8 ( 1.9) 108
1.2 ( 0.4) 108
w
1.3 109
3.8 ( 2.3) 107
–
–
2.6 ( 1.8) 107
2.2 ( 1.5) 109
–
–
–
–
1.7 ( 0.9) 109
ND
ND
–
ND
ND
Transformation frequency is expressed as the number of transformants per recipient cells (mean SD).
w
No SD could be calculated because transformants were obtained only in one transformation experiment.
ND, not detected.
FEMS Microbiol Ecol 66 (2008) 14–24
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22
B. Coupat et al.
Table 5. Transformation frequencies with plasmid and genomic donor DNA
Donor DNA
Single DNA
precO<Cm
RS25
Control
pftsK<Gm
RS13
Control
DNA mix
precO<Cm1pftsK<Gm
RS251RS13
pftsK<Gm1RS25
Control
Type of DNA
Antibiotic selection
Transformation frequencies
Plasmid
Genomic
Cm
Cm
Cm
Gm
Gm
Gm
7.9 ( 2.3) 107
7.4 ( 1.9) 107
o 7.8 1010
8.3 ( 0.8) 106
6.55 ( 0.2) 106
o 1.1 1010
Cm1Gm
Cm1Gm
Cm1Gm
Cm1Gm
5.4 ( 1.5) 109
6.1 ( 4.4) 109
5.6 108w
o 4.5 1010
Plasmid
Genomic
Plasmid
Genomic
Plasmid1genomic
Control without DNA (threshold of detection).
w
No SD could be calculated because transformants were obtained only in one transformation experiment.
recombination (Fig. 1b; Table 5). This gene replacement is
important as it may involve up to 88 ORFs or 2% of the
recipient genome. Substitutive recombination might, however, have little effect on the genome plasticity of the
R. solanacearum, as this gene replacement is more probably
gene conversion than acquisition of new properties. Nevertheless, whether single nucleotide polymorphisms in converted genes could significantly modify the bacterial
phenotype of the recombinant remains an open question.
Concerning these genes, 33 coded for proteins involved in
the metabolism of small molecules (biosynthesis of cofactors, fatty acid and phosphatidic acid biosynthesis, carbon
compounds, degradation, amino acid biosynthesis, folic
acid, nucleotide biosynthesis, electron transport, glycolysis),
11 for proteins involved in the metabolism of large molecules (macromolecule degradation, macromolecule synthesis and modification, DNA repair), three for structural
elements, nine for cell processes (cell division, chaperoning)
and 12 for transcriptional regulators. Twenty of these
transferred genes coded for proteins with unknown functions. Moreover, among these 88 ORFs, six and eight genes
are known to be essential for B. subtilis and E. coli growth,
respectively, and are implied in metabolism (Gerdes et al.,
2003; Kobayashi et al., 2003).
In the present case, the substitution of large DNA
fragments by homologous recombination was also able to
facilitate the repair of damaged DNA by the replacement of
nonfunctional genes in closely related strains. The mutS
gene, located in the 90-kb transferred DNA region, was
considered as a transformation hot spot (Fall et al., 2007).
The replacement of a nonfunctional mutS copy by natural
transformation is in agreement with the hypothesis involving HGT as a mechanism for mutS-negative mutators to
reacquire a functional mutS copy to return to a more stable
wild-type phenotype (Denamur et al., 2000).
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As regards the gene transfers between phylogenetically
distant strains, these could allow the acquisition of new
metabolic functions. Recently, DNA microarray technology
has demonstrated that DNA blocks of up to 29 kb, containing type III effectors PopA, PopB and PopC, pathogenicityrelated functions or signal peptide belonging to the hrpB
pathogenicity regulons, can be transferred between phylotypes of the R. solanacearum species complex by natural
transformation around the target gene (A. Guidot, pers.
commun.). Transfer of large genomic sequences has also
been reported, such as the transfer of a 36-kb pathogenic
island between different Yersinia strains through the conjugation mechanism (Schubert et al., 2004) or the acquisition of a novel catabolic pathway implied in the degradation
of an organic xenobiotic (Springael & Top, 2004).
We have, thus, demonstrated that the amount of genetic
information exchanged between strains of the R. solanacearum species complex could be important. Indeed, 80% of a
collection of 55 strains developed a competence state, multiple recombination events can occur in the same genome and
the size of the exchanged DNA by homologous recombination can reach up to 90 kb. Natural transformation could be
considered as one of the major forces that drive adaptation
and evolution in this species and the cause of its mosaic
structure (Salanoubat et al., 2002). The gain of genomic
islands putatively involved in pathogenicity could potentially threaten to enlarge the current host range of
R. solanacearum strains. A new pathogenic variant of
R. solanacearum has recently been detected in the French
West Indies, which is affecting new host plants such as water
melon (Citrullus lunaus), pumpkin (Cucurbita moschata L.)
and cantaloupe (Cucumis melo L.) (Wicker et al., 2007).
Does natural transformation explain this genetic diversity
and this adaptation to new hosts? Further studies will be
needed to allow us to answer this question.
FEMS Microbiol Ecol 66 (2008) 14–24
23
Natural transformation in R. solanacearum
Acknowledgements
We are grateful to Sonia Assouaki for technical assistance.
B.C. received a grant from the French Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche. This work was partly supported by the biodiversity
programme of the Bureau des Ressources Génétiques
(BRG).
Authors’contribution
B.C. and F.C.-D. contributed equally to this study.
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Supplementary material
The following supplementary material for this article is
available online:
Table S1. Transformability in Ralstonia solanacearum environmental strains.
This material is available as part of the online article
from: http://www.blackwell-synergy.com/doi/abs/10.1111/
j.1574-6941.2008.00552.x (this link will take you to the
article abstract).
Please note: Blackwell Publishing is not responsible for
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