Chapter 16

Introduction of Genetic Material in Ralstonia solanacearum

Through Natural Transformation and Conjugation
Anthony Perrier, Patrick Barberis, and Stéphane Genin

Abstract
Ralstonia solanacearum is a soil-borne plant pathogen, responsible of the bacterial wilt disease. Its unusual
wide host range (more than 250 plant species), aggressiveness, and broad geographic distribution have
made of this bacterium the main plant pathogenic model in the beta-Proteobacteria class. Many
R. solanacearum strains have the ability to internalize exogenous DNA through natural transformation.
This property is widely used in reverse genetics studies to create mutants or reporter gene constructs, in the
aim to study the molecular bases of pathogenesis of this bacterium. In this chapter, we describe three in vitro
methods (natural transformation, electrotransformation, and conjugation) commonly used to produce
recombinant R. solanacearum cells after introduction of exogenous DNA.

Key words Natural competence, Bacterial conjugation, Electroporation, Plant pathogen, Reverse
genetics

1 Introduction

The R. solanacearum species complex is divided in four monophy-

letic groups, designated as phylotypes I to IV, which are generally
associated with the geographic origin of the strains [1, 2]. In the
present chapter we present protocols primarily used and optimized
for the phylotype I strain GMI1000, but can be extended to some
other strains from the species complex with variable efficiency
[3, 4]. The transfer of exogenous DNA inside the bacterial cells is
a prerequisite to all the basic molecular techniques used to study
this pathogen including the generation of mutants or gene reporter
fusions and the completion of genetic complementation assays
[5–7].
Many R. solanacearum strains, but not all, are naturally com-
petent and can be transformed in vitro [3]. Although both the
comA gene product and type IV pili were shown to be required
for natural transformation [8, 9], the precise mechanism of DNA
uptake and transport remains unknown. Natural transformation is

Carlos Medina and Francisco Javier López-Baena (eds.), Host-Pathogen Interactions: Methods and Protocols, Methods
in Molecular Biology, vol. 1734, https://doi.org/10.1007/978-1-4939-7604-1_16, © Springer Science+Business Media, LLC 2018

201
202 Anthony Perrier et al.

related to the development of a competent state, which depends on

the physiological state of the cells. Only cells in exponential growth
achieve the competent state, which declines rapidly during the log
phase [7]. The competent state is induced by culturing the bacterial
cells in a limiting growth medium [4, 10]. To do so, bacteria are
grown in a minimal growth medium supplemented with 2% of
glycerol as sole carbon source, as it is poorly metabolized by
R. solanacearum. The limit in size of the exogenous DNA frag-
ments incorporated is rather large since DNA blocks up to 30 kb
were reported to be integrated after natural transformation
[11]. Different types of DNAs can be used for natural transforma-
tion. Genomic DNA fragments carrying a selectable antibiotic
resistance gene can be used for quick generation of deletion or
disruption mutants (see for example ref. 12). Linearized plasmids
containing two regions of homology are used for stable chromo-
somal insertion allowing trans-complementation or reporter gene
fusion analysis [7]. Circular plasmids containing chromosomal
homology region can also be incorporated during the competent
state, such as the nonreplicative plasmid pK18mobsacB [13] used to
generate chromosomal deletion or site-directed mutagenesis.
We detail in the present chapter the three main methods com-
monly used to introduce genetic material in R. solanacearum and
generate modified strains.

2 Materials

1. Escherichia coli strains pRK2013, pRK2073. R solanacearum

strain. . .
2. BG medium: 10 g/L bacto peptone, 1 g/L casamino acids,
1 g/L yeast extract. For agar plates, BG medium is supplemen-
ted with agar (15 g/L), glucose (5 g/L), and optionally with
triphenyl-tetrazolium chloride (0.05 g/L) to ease phenotypic
characterization.
3. Minimal medium: 0.125 mg/L FeSO4·7H2O, 0.5 g
(NH4)2SO4, 0.05 g/L MgSO4·7H2O, 3.4 g/L KH2PO4.
The pH is adjusted to 6.5 with KOH. This medium corre-
sponds to one-quarter strength M63 [14]. Supplement with
2% glycerol when necessary.
4. LB medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L
NaCl. For agar plates, LB medium is supplemented with agar
(15 g/L).
5. Antibiotics used at the following final concentrations: 25 mg/
L kanamycin, 10 mg/L gentamycin, 10 mg/L tetracycline,
50 mg/L ampicillin, 25 mg/L chloramphenicol for E. coli.
50 mg/L kanamycin, 40 mg/L spectinomycin, 10 mg/L gen-
tamycin, 10 mg/L tetracycline for R. solanacearum.
 In vitro Methods to Transform R. solanacearum 203

6. 10% glycerol in ultrapure water.

7. Nitrocellulose filter (pore size 0.45 μm).

3 Methods

3.1 Induction of the 1. Cultivate bacteria from
80  C stock on BG agar plates with
Competent State the appropriate antibiotics during 2 days at 28  C.
2. Inoculate a single colony from the BG agar plate into 10 mL of
minimal medium supplemented with 2% glycerol. Incubate
2 days under shaking at 180 rpm at 28  C.

3.1.1 Natural 1. Transfer 50 μL of competent cells into a sterile 1.5 mL micro-

Transformation centrifuge tube.
2. Add 5 μL of plasmidic DNA (300–500 ng) or 10 μL of geno-
mic DNA (2–4 μg) to the competent cells. Mix gently.
3. Spot the entire volume into the center of a BG agar plate (see
Notes 1 and 2).
4. Incubate at 28  C for 2 days.

3.1.2 Selection of the 1. Transfer the bacteria with a sterile 10 μL inoculation loop into a
Transformants sterile 1.5 mL micro-centrifuge tube containing 1 mL of liquid
BG medium. Gently resuspend the cells by pipetting up
and down.
2. Plate 100 μL of the resuspended cells on BG agar plate contain-
ing the appropriate antibiotics (see Note 3).
3. Centrifuge the 900 remaining microliters at 16,000  g for
2 min. Discard the supernatant and resuspend the pellet with
200 μL of liquid BG medium.
4. Plate 100 μL of the resuspended cells on two agar plates con-
taining the appropriate antibiotics (see Note 4).
5. Incubate the plates at 28  C for 2 days. Identify growing
colonies and streak on a fresh agar plate containing the appro-
priate antibiotics. Further steps such as the confirmation of a
correct DNA recombination event in the genome start here.

3.2 Electro- While most of the DNA types can be integrated in the cells with
Transformation of high efficiency by natural transformation, electrotransformation
R. Solanacearum Cells could be useful for the integration of high molecular weight plas-
mid such as pLAFR-derived replicative plasmids [15]. Electrocom-
petent cells are made by concentrating and washing cells cultivated
overnight in rich BG medium (see Note 5). The application of an
electrical field to the electro-competent chilled cells will increase
their permeability and facilitate the incorporation of the charged
DNA [16].
204 Anthony Perrier et al.

3.2.1 Preparation of the
Electrocompetent Cells
1. Cultivate bacteria from
80  C stock on BG agar plates with
the appropriate antibiotics during 2 days at 28  C.
2. Inoculate one colony from the BG agar plate into a desired
volume of liquid BG medium (see Note 6). Incubate overnight
under shaking at 180 rpm at 28  C.
3. Centrifuge 1 volume of culture at 16,000  g for 2 min.
4. Discard the supernatant. Resuspend the pellet with 1/2 vol-
ume of ultrapure water (see Note 7) and centrifuge at
16,000  g for 2 min at 4  C.
5. Discard the supernatant. Resuspend the pellet with 1/4 volume
of ultrapure water and centrifuge at 16,000  g for 2 min at
4  C.
6. Discard the supernatant. Gently resuspend the pellet with 1/8
volume of ultrapure water supplemented with 10% glycerol and
centrifuge at 16,000  g for 2 min at 4  C.
7. Discard the supernatant. Gently resuspend the pellet with
1/80 volume of ultrapure water supplemented with 10%
glycerol.
8. Prepare 50 μL aliquots in 1.5 mL microcentrifuge tubes.
9. Add 5 μL (300–500 ng/μL) of dialyzed plasmid DNA to a
50 μL aliquot and mix gently.

3.2.2 Electro- 1. Transfer the mixture to an electroporation cuvette (1 mm).

transfomation 2. Electroporate at 1.8 kV for 5 ms.
3. Resuspend the cells into 1 mL of BG medium.
4. Incubate 2 h under shaking at 180 rpm at 28  C.

3.2.3 Selection of the
Transformants
1. Plate 100 μL of the resuspended cells on BG agar plate contain-
ing the appropriate antibiotics.
2. Centrifuge the 900 remaining microliters at 16,000  g for
2 min. Discard the supernatant and resuspend the pellet with
200 μL of liquid BG medium.
3. Plate the resuspended cells on two BG agar plates containing
the appropriate antibiotics.
4. Incubate the plates at 28  C for 2 days.

3.3 Triparental Triparental mating is a natural way to introduce circular plasmids

Mating into R. solanacearum cells, especially for those of high molecular
size. For example, a 560 Kb plasmid was successfully delivered in a
recipient strain using this technique [17]. In case of the absence of
tra genes in the mobilizable plasmid (which is generally the case for
all small replicative plasmids in E. coli), the conjugative transfer will
require a helper plasmid providing the tra genes [18]. Thus, three
different strains are required in this so-called “triparental mating”: a
 In vitro Methods to Transform R. solanacearum 205

donor E. coli strain carrying a mobilizable plasmid of interest, a

helper E. coli strain containing a conjugative plasmid (pRK2013
carrying kanamycin resistance or its derivative pRK2073 carrying
spectinomycin resistance [18]) and a recipient R. solanacearum
strain.
1. Cultivate the E. coli donor and helper strain from
80  C stock
on LB agar plates with the appropriate antibiotics during 1 day
at 37  C.
2. Cultivate the R. solanacearum recipient strain from
80  C
stock on BG agar plate with the appropriate antibiotics during
2 days at 28  C.
3. Inoculate a single colony of the E. coli donor and helper strain
from the LB agar plate into 5 mL liquid LB medium with the
appropriate antibiotics. Incubate overnight under shaking at
180 rpm at 28  C.
4. Inoculate one colony of the R. solanacearum recipient strain
from the BG agar plate into 5 mL liquid BG medium with the
appropriate antibiotics. Incubate overnight under shaking at
180 rpm at 28  C.
5. Add 500 μL of the overnight E. coli cultures into 4.5 mL of LB
medium. Incubate under shaking at 180 rpm at 37  C until mid
log phase (see Note 8).
6. Add 5  108 of both donor & helper E. coli cells for 108
recipient cells (with a volume of 500 μL per strain) in a sterile
2 mL micro-centrifuge tube.
7. Centrifuge at 16,000  g for 1 min.
8. Discard the supernatant. Gently resuspend the pellet with
100 μL of sterile water.
9. Transfer the bacteria into the center of a nitrocellulose filter on
a BG agar plate (see Note 1).
10. Incubate during 6–12 h at 28  C (see Note 9).
11. Transfer the filter into a sterile 1.5 mL micro-centrifuge tube
containing 1 mL of liquid BG medium.
12. Vortex during 30 s to resuspend the cells (see Note 10).
13. Plate 100 μL of the resuspended cells on BG agar plate contain-
ing the appropriate antibiotics.
14. Centrifuge the 900 remaining microliters at 16,000  g for
2 min. Discard the supernatant and resuspend the pellet with
200 μL of liquid BG medium.
15. Plate 100 μL of the resuspended cells on two BG agar plates
containing the appropriate antibiotics (see Note 4).
16. Incubate the plates at 28  C for 2 days.
206 Anthony Perrier et al.

4 Notes

1. At this step the BG agar plate should not contain glucose and
triphenyl-tetrazolium chloride.
2. Alternatively, the BG agar plate can be replaced by a minimal
medium agar plate supplemented with 2% glycerol to increase
the transformation efficiency.
3. When transforming with genomic DNA, the efficiency may
vary a lot depending on the DNA purity and integrity. It is
therefore recommended to also plate at ten times dilution of
the resuspended cells to avoid a bacterial lawn in case of high-
transformation efficiency.
4. As indicated in Note 3, this can result in a bacterial lawn, but in
case of low-transformation efficiency it is preferable to do it as a
backup.
5. Electro-competent cells of R. solanacearum cannot be stored
reliably at
80  C. In order to keep high transformation effi-
ciency, we recommend preparing the cells extemporaneously.
6. 4 mL of overnight culture is necessary to make 1 aliquot of
50 μL of electrocompetent cells.
7. The ultrapure water and the glycerol should be prechilled at
4  C and all the following steps should be done on ice.
8. It is important to use E. coli strains from exponential growth
phase (OD600 ¼ 0.5).
9. To keep high-transformation efficiency, this step should not be
longer than 12 h as R. solanacearum can have a deleterious
effect on the viability of E. coli cells.
10. At this step, if the recipient strain does not possess a selective
marker to counter-select E. coli cells, we recommend to add
some bacteriophage T4, which is specific for E. coli, to get rid
of the donor strain.

References
1. Prior P, Ailloud F, Dalsing BL et al (2016)
Genomic and proteomic evidence supporting
the division of the plant pathogen Ralstonia
solanacearum into three species. BMC Geno-
mics 17:90
2. Genin S, Denny TP (2012) Pathogenomics of
the Ralstonia solanacearum species complex.
Annu Rev Phytopathol 50:67–89
3. Coupat B, Chaumeille-Dole F, Fall S et al
(2008) Natural transformation in the Ralsto-
nia solanacearum species complex: number
and size of DNA that can be transferred.
FEMS Microbiol Ecol 66:14–24
4. Le T, D Leccas D, Boucher C (1978) Transfor-
mation of Pseudomonas solanacearum strain
K60. In: proceedings of the 4th international
conference on plant pathogenic bacteria.
Angers (INRA ed). pp 819–822
5. Liu H, Zhang S, M a S, Denny TP (2005)
Pyramiding unmarked deletions in Ralstonia
solanacearum shows that secreted proteins in
addition to plant cell-wall-degrading enzymes
contribute to virulence. Mol Plant-Microbe
Interact 18:1296–1305
6. Cunnac S, Occhialini A, Barberis P et al (2004)
Inventory and functional analysis of the large
 In vitro Methods to Transform R. solanacearum
Hrp regulon in Ralstonia solanacearum: iden-
tification of novel effector proteins translocated
to plant host cells through the type III secre-
tion system. Mol Microbiol 53:115–128
7. Monteiro F, Solé M, van Dijk I, Valls M (2012)
A chromosomal insertion toolbox for pro-
moter probing, mutant complementation, and
pathogenicity studies in Ralstonia solana-
cearum. Mol Plant-Microbe Interact
25:557–568
8. Kang Y, Liu H, Genin S et al (2002) Ralstonia
solanacearum requires type 4 pili to adhere to
multiple surfaces and for natural transforma-
tion and virulence. Mol Microbiol 46:427–437
9. Barman A, Buragohain C, Ray SK (2017) Dis-
ruption of comA homolog in Ralstonia solana-
cearum does not impair its twitching motility. J
Basic Microbiol 57:218–227
10. Bertolla F, Van Gijsegem F, Nesme X, Simonet
P (1997) Conditions for natural transforma-
tion of Ralstonia solanacearum. Appl Environ
Microbiol 63:4965–4968
11. Guidot A, Coupat B, Fall S et al (2009) Hori-
zontal gene transfer between Ralstonia solana-
cearum strains detected by comparative
genomic hybridization on microarrays. ISME
J 3:549–562
12. González A, Plener L, Restrepo S et al (2011)
Detection and functional characterization of a
large genomic deletion resulting in decreased
pathogenicity in Ralstonia solanacearum race
207
3 biovar 2 strains. Environ Microbiol
13:3172–3185
13. Sch€afer A, Tauch A, J€ager W et al (1994) Small
mobilizable multi-purpose cloning vectors
derived from the Escherichia coli plasmids
pK18 and pK19: selection of defined deletions
in the chromosome of Corynebacterium gluta-
micum. Gene 145:69–73
14. Boucher CA, Barberis PA, Trigalet AP, Demery
DA (1985) Transposon mutagenesis of pseudo-
monas solanacearum : isolation of Tn5-
induced avirulent mutants. J Gen Microbiol
131:2449–2457
15. Friedman AM, Long SR, Brown SE et al
(1982) Construction of a broad host range
cosmid cloning vector and its use in the genetic
analysis of Rhizobium mutants. Gene
18:289–296
16. Neumann E, Schaefer-Ridder M, Wang Y,
Hofschneider PH (1982) Gene transfer into
mouse lyoma cells by electroporation in high
electric fields. EMBO J 1:841–845
17. Marchetti M, Capela D, Glew M et al (2010)
Experimental evolution of a plant pathogen
into a legume symbiont. PLoS Biol 8:
e1000280
18. Figurski DH, Helinski DR (1979) Replication
of an origin-containing derivative of plasmid
RK2 dependent on a plasmid function
provided in trans. Proc Natl Acad Sci U S A
76:1648–1652