COST Action 873 Training School Agrobacterium ... - Cost 873

COST Action 873 Training School
Agrobacterium classical and molecular
phytobacteriology
5 - 9 September 2011
University of Lyon 1, Villeurbanne, France
Focus:
Classical, molecular, genomic and phytosanitary aspects of Agrobacterium related to
stone fruit
and nut health.
Topics:
- Detection, isolation and identification
- Phenotypic macroarrays and biochemical tests
- Pathogenicity tests
- Plasmid profiles
COST 873 - Lyon 4-9 September 2011 1

Place:
Université Lyon 1, Microbial Ecology UMR 5557 / USC INRA 1193, Villeurbanne,
France
http://www.univ-lyon1.fr/ http://www.ecologiemicrobiennelyon.fr/
Training course will take place Building Darwin A - room M3
10, rue Rafael Dubois - 69100 Villeurbanne Cedex - France
Local organizer:
Dr. Céline Lavire, Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC
INRA 1193,
Bâtiment Forel, 43 boulevard du 11 Novembre 1918, 69100 Villeurbanne Cedex –
France
celine.lavire@univ-lyon.fr Tel: +33 (0)4 26 24 82 05 Fax: +33 (0)4 72 43 12 23
Scientific general organiser:
Dr. Xavier Nesme, Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC
INRA 1193,
Bâtiment Mendel, 43 boulevard du 11 Novembre 1918, 69100 Villeurbanne Cedex –
France
nesme@univ-lyon1.fr Tel: +33 (0)4 72 44 82 89 Fax: +33 (0)4 72 43 12 23
COST 873 chair:
Dr. Brion Duffy, Federal Department of Economic Affairs FDEA, Agroscope Changins-
Wädenswil
ACW, Schloss 1 Postfach, CH-8820 Wädenswil, Switzerland
duffy@acw.admin.ch Tel. +41 44 783 64 16 Fax +41 44 783 63 05
Dr. Joël F. Pothier, Federal Department of Economic Affairs FDEA, Agroscope
Changins-
Wädenswil ACW, Schloss 1 Postfach, CH-8820 Wädenswil, Switzerland
joel.pothier@acw.admin.ch Tel. +41 44 783 61 14 Fax +41 44 783 63 05
Trainers:
Dr. Xavier Nesme, Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC
INRA 1193,
Villeurbanne, France
Pr. María M. López, Instituto Valenciano de Investigaciones Agrarias, Moncada, Spain
Dr. Joanna Pulawska, Research Institute of Horticulture, Skierniewice, Poland
Dr. Daniel Muller, Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC INRA
1193,
Villeurbanne, France
Mrs. Laurence Loiseau, Université Lyon 1, PAR-MIC, Villeurbanne, France
Mr. Tony Campillo, Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC
INRA 1193,
Villeurbanne, France
Mr. Malek Shams Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC
COST 873 - Lyon 4-9 September 2011 2

INRA 1193,
Villeurbanne, France
Mr. Florent Lassale Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC
INRA 1193,
Villeurbanne, France
Mr. David Chapulliot Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC
INRA 1193,
Villeurbanne, France
Dr. Céline Lavire, Université Lyon 1, Microbial Ecology UMR CNRS 5557 / USC
INRA 1193,
Villeurbanne, France (local organiser)
Dr. Joël F. Pothier, Agroscope Changins-Wädenswil Research station, Wädenswil,
Switzerland
(COST 873 chair representative)
COST 873 - Lyon 4-9 September 2011 3

What is so uncommon with Agrobacterium that is
necessary you remember before you undertake crown gall studies.
1- Agrobacterium tumefaciens is not the causative agent of crown gall: Ti plasmids
are the true crown gall causative agents Agrobacterium are only Ti plasmid
vectors
2- Agrobacterium tumefaciens does not necessarily induce crown gall, nor Rhizobium
rhizogenes hairy root: because these are taxon names and no longer indicator of
pathogenicity traits.
3- More than one strain of Agrobacterium are generally involved in a crown gall
outbreak: more than one isolate is necessary to have an idea of pathogenic
population involved.
1.00
0.05
1.00
1.00
0.95
0.97
0.95
R. rhizogenes
R. tropici
0.98
B. japoponicum
Rh. palustris
Az. caulinodans
M. loti
E. medicae
E. meliloti
R. etli
R. leguminosarum bv. trifolii
R. phaseoli
1.00
G7
G3
A. undicola
A. larrymoorei
NCPPB 1650
A. rubi
G1
G5
G13
G4 = A. radiobacter
G2
G9
G6
G8 = A. fabrum
A. vitis
Fig.1 Revised nomenclature for Agrobacterium as proposed by the
subcommittee of taxonomy for Rhizobium and Agrobacterium
recA phylogeny constructed using Maximum-Likelyhood with PhyML ln(L) = -9626.2,
1501 sites, GTR, 4 rate classes. A., Agrobacterium; R., Rhizobium; E., Ensifer; M.,
Mesorhizobium; Az., Azorhizobium; Rh. Rhodopseudomonas; B., Bradyrhizobium.
COST 873 - Lyon 4-9 September 2011 4

Procedure for Agrobacterium isolation and identification
NB. Populations of Agrobacterium in soils, rhizospheres and even tumors are always
heterogenous. Thus, extreme cares must be taken during the isolation procedure
because a single colony may often consisted of different strains. Performing three
time the isolation stepsdescribed below. A phase of hydration of several hours in
water to allow efficient cell separations from mucous polysaccharides is imperative.
Experiment 1
Agrobacterium isolation from: soil, maize rhizosphere and crown gall.
Soil: weigh 0.5 g in sterile 1.5 ml microtube. Add 500 µl sterile water, vortex. Pipet
100 µl and isolate using bead on agar media (1A-Te,2E-Te and MG-Te).
Rhizosphere: gently wash plant in sterile distilled water, weigh 0.5 g of root in sterile
1.5 ml microtube. Add 500 µl sterile water, vortex. Crush roots. Streak 25 µl of
crushed root on 1A-Te, 2E-Te and MG-Te agar plates (2 plates per selective media).
Crown gall: gently wash plant in sterile distilled water, weigh 0.5 g of root in sterile
1.5 ml microtube. Add 500 µl sterile water, vortex. Crush roots. Streak 25 µl of
crushed tumor on 1A-Te,2E-Te and MG-Te agar plates (2 plates per selective
medium).
Incubate 4/5 days at 28°C.
Experiment 2
Day 1: Select four Agrobacterium like colonies and transfer them in microplate wells
containing 200 µl of sterile water. Incubate overnight at 28°C (for diet and efficient
bacterium cell separation).
Day 2: From microplates streak each selected bacteria onto 1A, 2E, MG and LPG
plates (4 bacteria per 1 plate).
COST 873 - Lyon 4-9 September 2011 5

Day 4: For colonies grown on LPG or MG agar plates, do colonies PCR experiments
(See colony PCR section).
Experiment 3
Source: Pure cultures grown in separate colonies on 1A-Te, 2E-Te, MG-Te, and MG,
1A, 2E and LPG.
Select 2 strains (1 per trainee) for further characterization and identification
Day 1
Observation of colony morphology of selected strains on each medium.
Transfer 6 isolated colonies from LPG (or MG if necessary) into sterile capped tube
containing 4 ml of NaCl 0.8% incubate overnight at 28°C (see phenotypic microarray
section).
Transfer 1 colony into 200 µl of sterile water (microplate wells or 1.5 ml microtube).
Use it to perform biochemical tests (see biochemical tests section).
Day 2
Streak each selected bacteria onto MG and LPG plates.
For phenotypic macroarrays experiments prepare PM1 and PM2 plates. See
phenotypic macroarray section.
Day 4
For colonies grown on LPG or MG agar plates, do colonies PCR experiments (See
colony PCR section).
COST 873 - Lyon 4-9 September 2011 6

Standard culture medium
LPG
Yeast extract 5 g
Peptone
10 g
D-Glucose 10 g
Agar
15 g
Adjust volume to 1 L with H2O
Adjust to pH 7.2
Selective media
Selective media of Brisbanne and Kerr were modified by Mougel et al. to improve
their selectivity in particularily difficult environments such as soils or necrosed
tumors.
A stock solution (100 mg ml
1 ) of K 2 TeO 3 was prepared in ultra-pure water and
sterilized by filtration (NB: potassium tellurite doses differ according to biovar)
- 1A and 1A-Te (for biovar 1 [i.e. A. tumefaciens complex))
L-Arabitol
3.04 g
NH 4 NO 3
0.16 g
KH 2 PO 4
0.54 g
K 2 HPO 4
1.04 g
MgSO 4 ,7H 2 0
0.25 g
Taurocholic acid sodium salt hydrate 0.29 g
Crystal violet (stock solution 1 %) 200 µl
Agar
15 g
Adjust volume to 1 L with H2O
Sterilize by autoclaving (20 minutes at 120° C)
For 1A-Te, after autoclaving add:
Tellurite of potassium (K 2 TeO 3 ) 80 µg. ml -1
- 2E and 2E-Te (for biovar 2 [i.e. Rhizobium rhizogenes]):
Erythritol
3.05 g
NH 4 NO 3
0.16 g
KH 2 PO 4
0.54 g
K 2 HPO 4
1.04 g
MgSO 4 ,7H 2 0
0.25 g
Taurocholic acid sodium salt hydrate 0.29 g
Yeast extract
0.01 g
Malachite green (stock solution 1 %) 200 µl
Agar
15 g
Adjust volume to 1 L with H2O
Sterilize by autoclaving (20 minutes at 120° C)
For 2E-Te, after autoclaving add:
Tellurite of potassium 320 µg. ml -1
-MG and MG-Te
D-Mannitol
L-glutamic acid
5 g
2 g
COST 873 - Lyon 4-9 September 2011 7

KH 2 PO 4
NaCl
MgSO 4 ,7H 2 0
Yeast extract
Agar
Adjust volume to 1 L with H2O
Adjust to pH 7.2
0.5 g
0.2 g
0.2 g
0.5 g
15g
Sterilize by autoclaving (20 minutes at 120° C)
For MG-Te, after autoclaving add:
Tellurite of potassium 200 µg. ml -1
Reference:
For 1A-Te and 2A-Te:
- Brisbane, P. G., and A. Kerr (1983) Selective media for the three biovars of Agrobacterium. J. Appl.
Bacteriol. 54:425–431.
- C. Mougel, B. Cournoyer and X. Nesme (2001) Novel Tellurite Amended media and specific
chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium Biovars 1
and 2. Applied and environmental Microbiology, volume 67, issue 1, pages 65-74.
For MG media:
- Keane, P. J., A. Kerr, and P. B. New (1970) Crown gall of stone fruit. II. Identification and
nomenclature of Agrobacterium isolates.Aust. J. Biol. Sci. 23:585-595
Fig. 2. Plating of a 10 -1 dilution suspension of soil on 1A medium (a) or 1A
medium amended with 60 ppm of K 2 TeO 3 (b). (c) Enlarged (magnification, 30 x)
typical black colonies of A. tumefaciens on the amended medium. Some
agrobacterial colonies are indicated by arrows (Mougel et al. 2001).
COST 873 - Lyon 4-9 September 2011 8

Phenotypic macroarrays (Omnilog®) and biochemical
tests (presumptive identification of Agrobacterium
members)
PROCEDURE for PM 1 – 2 Inoculation
Cell Suspension Preparation and PM Inoculation
Monday
Preparation of Cells :
1. From pure strains isolated on agar plates and incubated 48h at 28°C.
2. Transfer isolated colonies into sterile capped tube containing 4 ml of NaCl
0.8%. Stir gently cell suspension with the swab and incubate overnight at
28°C.
Tuesday
3. Centrifuge , rinse and resuspend cell pellet in 2 ml of IF0a
Preparation of PM Inoculating Fluids
1. Pipet 10 ml of this IF-0 into a 20 x 150 mm sterile capped test tube.
2. Pipet 16 ml of 1.2x IF-0 into a 120 ml sterile plastic vial.
+ dye mixA 0.240ml
+ sterile water 3.76ml
Inoculation of PM Panels (see procedure in Fig A)
Step 1: Prepare Cell Suspensions
1. Transfer into the sterile capped tube containing 10 ml IFOa 1.2X
Check the turbidity of this IF0a to achieve 100% T (transmittance) in the Biolog
Turbidimeter.
2. Add the cell suspension to achieve 42% T to the tube containing 10 ml of IF0a
3. Transfer 4 ml of the 42%T cell suspension prepare in point 2 into the sterile
pre-filled vial containing 20 ml of IF-0+dye +water.
Mix completely but gently. DO NOT create air bubbles in the cell
suspension.
The final cell density is 85% T.
Step 2: Inoculate PM 1 – 2
4. Transfer this cell suspension into a sterile reservoir.
5. Inoculate PM 1 – 2 with this cell suspension, 100 µl / well.
COST 873 - Lyon 4-9 September 2011 9

Standard biochemical tests
Agrobacteria and especially A. tumefaciens / biovar 1 have esculinase and fast
acting urease. Positive response to esculine and urease tests add to a Gram
negative coloration (using rapid test with just prepared KOH solution insteed) and
colony morphology in agar plates tests are strongly indicating the presence of
agrobacteria. Together with the ability to produce 3-keto-lactose indicated by an
intense yellow precipitate, these tests sign the presence of A. tumefaciens / biovar 1.
Esculine
- peptone 10g
- Esculine 1 g
- ferric ammonium citrate 20 g
- distilled water (qsp) 1 L
Urease
- L-tryptophane 3 g
- Urée 20 g
- KH 2 PO 4 1 g
- KH 2 PO 4 1 g
- NaCl 5 g
- Ethanol 95 % 10 ml
- Phenol red 25 mg
- distilled water (qsp) 1 L
3-keto-lactose
Streak bacteria on plates containing lactose medium, grow for 48h then pour
Benedict's reagent onto plate.
Lactose medium
- Lactose 10 g
- Yest extract 1 g
- Agar 15 g
COST 873 - Lyon 4-9 September 2011 10

Distilled water (qsp) 1 L
Benedict's reagent
Solution A
- sodium citrate 173 g
- Na 2 CO 3 100 g
- Distilled water (qsp) 850 ml
Solution B
- CuSO 4 18 g
- Distilled water (qsp) 150 ml
Gently add solution B to solution A.
Fig. 3. Positive and negative reactions to the 3-keto-lactose test of Bernaert &
De Ley of various A. tumefaciens complex and R. rhizogenes members.
COST 873 - Lyon 4-9 September 2011 11

Agrobacterium and Ti plasmid direct detection from
complex environments : metagenomic DNA extraction
and molecular detection
1- Metagenomic DNA Extraction using Mobio Powersoil DNA
isolation kit.
NB. Tumor samples have to be dissected into small pieces with a sterile scalpel and
crushed into a 1.5 ml microtube with a sterile micropestle before extraction.
0.5 g tumor, 0.5 g soil, 0.5 g rhizosphere directly into Mobio tubes.
1. To the PowerBead Tubes provided, 0.5 grams of soil sample or crown gall or
rhizospheric soil (root plus adherent soil).
2. Gently vortex to mix.
3. Check Solution C1. If Solution C1 is precipitated, heat solution to 60°C until
dissolved before use.
4. Add 60 µl of Solution C1 and invert several times or vortex briefly.
5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube
holder for the vortex. Vortex at maximum speed for 10 minutes.
6. Centrifuge tubes at 10,000 x g for 30 seconds at room temperature.
7. Transfer the supernatant to a clean 2 ml Collection Tube (provided).
Note: Expect between 400 to 500 µl of supernatant. Supernatant may still contain
some soil particles.
8. Add 250 µl of Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5
minutes.
9. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
10. Avoiding the pellet, transfer up to, but no more than, 600 µl of supernatant to a
clean 2 ml Collection Tube (provided).
11. Add 200 µl of Solution C3 and vortex briefly. Incubate at 4°C for 5 minutes.
12. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
13. Avoiding the pellet, transfer up to, but no more than, 750 µl of supernatant into a
clean 2 ml Collection Tube (provided).
14. Shake to mix Solution C4 before use. Add 1200 µl of Solution C4 to the
supernatant and vortex for 5 seconds.
15. Load approximately 675 µl onto a Spin Filter and centrifuge at 10,000 x g for 1
minute at room temperature. Discard the flow through and add an additional 675 µl of
supernatant to the Spin Filter and centrifuge at 10,000 x g for 1 minute at room
COST 873 - Lyon 4-9 September 2011 12

temperature. Load the remaining supernatant onto the Spin Filter and centrifuge at
10,000 x g for 1 minute at room temperature.
NB: A total of three loads for each processed sample are required.
16. Add 500 µl of Solution C5 and centrifuge at room temperature for 30 seconds at
10,000 x g.
17. Discard the flow through.
18. Centrifuge again at room temperature for 1 minute at 10,000 x g.
19. Carefully place spin filter in a clean 2 ml Collection Tube (provided). Avoid
splashing any Solution C5 onto the Spin Filter.
20. Add 100 µl of sterile H 2 0 to the center of the white filter membrane.
21. Centrifuge at room temperature for 30 seconds at 10,000 x g.
22. Discard the Spin Filter. The DNA in the tube is now ready for any downstream
application. No further steps are required.
2- PCR detection of Agrobacterium and Ti plasmid in metagenomic
DNA
Primer Target size (bp) Annealing temp.
F809-PA 16s 1477 60
F810-PH
F8360 A. tumefaciens generalist (pyr) 453 52
F8361
F8533 A. radiobacter / A.tumefaciens G4 511 60
F8827
F6786 A. fabrum / A.tumefaciens G8 502 60
F6784
F7386 Rhizobiaceae generalist TRL 779 52
F7387
VCF3 (F8521) Ti plasmid 414 54
VCR3 (F8521)
FGP tmr530 FGP tmr701! FGP nos 1236!
tmr
ons
6b
nos
T-DNA
172bp
3600bp
256bp
FGP virA2275 FGP virB 2164!
Vir
vir A
vir B2
1673bp
FGPS6 FGPS1509! FGPL132!
rrs
IGS
rrl
1479bp
2500-2700bp
500bp
COST 873 - Lyon 4-9 September 2011 13

PCR are performed in a 30 µl reaction mixture containing (n+1 reactions):
Components
Volume
10 X PCR buffer 3 µl
DMSO 1.2 µl
2 mM dNTP mixture 2 µl
50 mM MgCl2 0.4 µl
10 µM Primer forward 1.2 µl
10 µM Primer reverse 1.2 µl
Taq DNA polymerase 0.1 µl or 0.2 µl
Distilled water (qsp) 28.5 µl for metagenomic extraction or 30 µl for PCR colony test.
For n reactions, prepare a mix for n+1 reactions in a 1.5 ml microtube.
Aliquot 28.5 µl for metagenomic extraction or 30 µl for PCR colony
Add template DNA (metagenomic extraction) 1.5 µl
Or for PCR colony: add a small amount of colony from agar plates (inoculated
Monday).
NB. Use a tooth pick: the amount of cells must be very small. Just a touch will do!!
A negative control and positive control are included for each PCR assay.
PCR cycling:
-95°C for 5 min
-Perform 30 cycles of PCR amplification:
Denature 95°C for 45 s
Anneal (see temperature table 1) for 45s
Extend 72°C for 1 min
-Final extend 72°C for 1 min
Amplification products are analyzed by agarose gel electrophoresis (1% in T.B.E
buffer).
Before loading the samples mix loading dye ( X 10 µl) with 10 µl of the PCR product
Don’t forget appropriate molecular weight standards (5 µl of 1 Kb Plus DNA ladder).
COST 873 - Lyon 4-9 September 2011 14

Pathogenicity
Virulence assays (pathogenicity test) are conducted by wounding stems of 3-weekold
tomato plant (or 4-week-old tomato) with a scalpel (incision between 4 cm and 6
cm in length) then inoculating a dense suspension of 48-h-old cultures in sterile
water.
Tumors are visible 3 weeks postinoculation.
Fig. 3. Symptoms of crown gall disease on
rose plants. Galls developed frequently on
rootstocks (A, B, and C) and roots (D and E)
but rarely on scions (F) (Pionnat et al., 1999).
Fig. 4. Tumor formation induced by pathogenic Agrobacterium on tomato.
COST 873 - Lyon 4-9 September 2011 15

Plasmid status of Agrobacterium isolates
The back trap method is modified by Wheatcroft et al (2) from the original protocol
Eckhardt (1) originally based on gentle lysis of bacteria in the wells of a gel.
Materials
Sterile tubes (5 ml) with 2 ml of medium, 1,5 ml eppendorf tubes
* 11 x 14 cm gel (BRL tank) or equivalent
* 20 well comb, 2 mm thick teeth
Solutions
Na N-lauroyl sarcosinate 0,3% (4°C)
Na N-lauroyl sarcosinate 30 mg
H2Oup up to 10 ml
Tris 10 mM, EDTA 10 mM
Tris base 10 mM
0,605 g
EDTA 10 mM
1,861 g
H2O up to 500 ml
Solution F: Tris 10 mM, EDTA 10 mM, 20% Ficoll (400,000) (store at -20°C)
Ficoll (400,000)
10 g
Tris 10 mM, EDTA 10 mM up to 50 ml
Solution S : SDS 10%, xylene cyanole (1 mg/ml) (store at -20°C)
Lauryl sulfate
10 g
Xylene cyanole FF 0,1 g
H2Oup up to 100 ml
Lysozyme (10 mg/ml)
lysozyme
H2Oup
10 mg
1 ml
Solution L : Tris 10 mM, EDTA 10 mM, RNase type A (0,4 mg/ml),
bromophenol red (1 mg/ml) (store at -20°C)
Tris 10 mM, EDTA 10 mM 10 ml
RNase A (0,4 mg/ml) 10 µl RNase A (800 mg/2ml)
bromophenol red (1 mg/ml) 0,01 g
Methods
Bacterial cell culture
- Bacterial cells are grown at 28°C in 2 ml liquid medium
Gel stacking
- Prepare a 0.75% agarose gel in 1 x TBE ( 0.60 g agarose /80 ml TBE1x). Prepare
the gel 1 h before deposits
- Put the electrophoresis tank in an ice bath.
- Place gel in 1 x TBE: buffer flush with the surface of the gel without covering it
(buffer should not enter in the wells)
COST 873 - Lyon 4-9 September 2011 16

Preparation of bacterial lysates
- Measure the OD 600 nm of each culture tube.
- Make dilutions with 0.9% NaCl so as to have 1 ml at OD 600 nm of 0.2.
- Centrifuge (13,000 g, 3 min, 4 ° C).
From this stage, work on ice
- Resuspend the pellet in 500µl H 2 O (4°C), Vortex
- gently place the suspension in 1 ml Na N-lauroyl sarcosinate
0.3% (4 ° C).
The bacterial suspension migrates in the sarcosyl. quickly go to
the next step to avoid lysis insarcosyl.
- Centrifuge (13,000 g, 3 min, 4 °C).
- Promptly remove the supernatant.
- Dry traces of supernatant.
- Resuspend the pellet in 40 µl solution F (stored at -20 °C).Vortex 10 sec, put tubes
15 min on ice (after 5 to 10 minutes, vortex again a few seconds to fully resuspend).
It is possible to leave tubes on ice over15 minutes.
- During those 15 minutes, deposited 25 µl solution S in the wells of the gel. Run 10
to 15 minutes at 100 V (reverse polarity) until the migration line is one cm in front of
the wells.
- Cover the gel with about 150 ml 1 x TBE (1 mm above the gel), wells must be
covered.
- Prepare the lysis solution: 25 µl lysozyme (10 mg / ml) to be added to an Eppendorf
tube containing 230 µl solution L.
- Add 10 µl of lysis solution to the bacterial suspension, mix gently (3 laps with the
pipette) and deposit immediately 25 µl in a well.
This is the most difficult step to get a beautiful profile: everything is done in the ice
next to the electrophoresis tank thus prepare a pipetteman set to 10 µl, and another
pipette set to 25 µl.
- Migrate (40 V, 30 min) do not forget to change polarity, this slow migration allows
SDS to return to the wells and then 100 V, 3 h or more if necessary.
- Reveal by a ethidium bromide bath.
Plasmid content. Plasmid profiles are determined by a modified Eckhardt agarose
gel electrophoresis technique as described. Plasmid sizes are estimated by
comparison with those of A. fabrum C58 (fig baktrap profil)
Reference:
- Eckhardt T. 1978. A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria.
Plasmid 1: 584-588.
- Wheatcroft R., McRae D.G., and Miller R. W. 1990. Changes in the Rhizobium meliloti genome and
the ability to detect supercoiled plasmids during bacteroid development. Mol. Plant Microbe
Interactions. 3: 9-17.
COST 873 - Lyon 4-9 September 2011 17

Sequence and phylogenetic analyses
This module intend to present the utilization of bioinformatic tools in a standard
procedure for phylogenetic typing of isolated strains, from the elaboration of PCR
primers for amplification of marker genes to the construction of phylogenetic trees
replacing the unknown strain's allele into a well-defined taxonomic context.
A set of diverse bioinformatic softwares and web-based resources are used in
this training session. Web links for resources or downloading of installation files are
listed in annex.
I. Definition of oligonucleotidic primers for PCR
For amplification of a marker gene from an unknown Agrobacterium strain, we need
to define generalist primers able to hybridize with any allele of the gene. For this
purpose, the primers must be nested in region highly conserved in all targeted recA
alleles among agrobacteria strains.
1. Determination of conserved regions from sequence alignments
The marker gene used for routine typing of agrobacteria is the gene recA. This gene
has been sequenced in all known representatives of the taxon and its allele diversity
is well known. This diversity is summarized in the sequence bank file in FASTA
format recA_allAgro.fas. Open this file with the software Seaview (see annex). The
sequences are named according to the species and the allele type of the strain (for
example, « recA-G9-2 » stands for allele type 2 of genomovar G9 of A. tumefaciens).
Also, sequences are already aligned, allowing us to search for putative conserved
regions. The recA gene has been chosen as a marker because it codes the
recombinase A, a housekeeping protein under strong purifying selection that should
display good conservation among the taxon. Search the alignment for such
conserved region.
2. Definition of primers candidate regions
To have a specific hybridization, a PCR primer must be at least 18 nt-long. One must
then identify regions of that size where each site is invariant across all known allele
types. However, such long conserved regions are quite rare, and regions with low
variation might be good for primers, since they are defined taking into account the
constrains due to the molecular mechanisms of PCR. Indeed, efficient amplification
of the product rely mostly on the good annealing of the 3'-end of the primer to the
template DNA for the Taq polymerase to elongate the DNA strand. Then, a few
mismatches can be tolerated between the primer and template sequences, since
they are far from the 3'-end of the primer (at least 5 or 6 nt). Pay attention that many
mismatches impair the specific hybridization between the primer and its target.
Consecutive mismatches must be avoided in primer design because they create a
non-annealed bulge in the primer-target DNA duplex and impair their affinity
properties.
3. Fine-tuning of primer design
When good primer regions are defined, it is recomended to optimize the primer
design to make the pair of oligonucleotides compatible, especially for their melting
COST 873 - Lyon 4-9 September 2011 18

temperature (Tm). This can be done using Primer3 software or its web resource (see
annex).
NB: when ready to order the primers, do not forget that the reverse primer
must have been converted to its reverse complement relatively to the sequence
alignment. This can be done on diverse web resources like Sequence Editor.
II. Curation of raw sequence data
Once the PCR performed and the products sequenced, the sequencing company or
platform is likely to return you a pair of files : the sequence file and the raw
sequencing data consisting of an electrophoregram. The latter is usually in .ab1 file
format, which can be opened with softwares like Sequence Scanner (Applied
Biosystems, freeware). We will use this program on the file electrophoregram.ab1.
1. Reading and editing the electrophoregram
The 5'-end of the electrophoregram is otfen of low quality, leading to undetermined
bases (N) in the deduced sequence. It is however easy to infer visually some of the
actual bases from the electrophoregram. In the Sequence Scanner interface, you
can simply edit the nucleotidic sequence, and then export it to a new FASTA file.
NB: the file can be accompagnied by a Quality Control file. This file contain
expert information from the company/platform that may be useful, notably
concerning the detection of unpure PCR products.
III. Identification of the sequence with BLAST
BLAST is an algorithm used to align rapidly a sequence against a (large) database of
sequence, and rank all the possible alignments given their length and quality. It is an
useful tool to get a raw idea of the taxonomic provenance of a sequence by finding its
most similar known homologs. However, there is none elaborated evolutionary model
underlying a BLAST search, and it is therefore not recommended to use its results for
taxonomic assignation.
BLAST is available as a web service on the websites of the NCBI and EBI (see
annex). We will use this program to identify an unknown agrobacterial recA sequence
stored in the file unknown_sequence.fas.
1. blastn search against recA “gold standard” database
On the NCBI BLAST website, choose the blastn algorithm (for nucleotides). On the
blastn page, check the “Align two or more sequences” option. In the Query (upper)
box, paste or upload the sequence to identify. In the Subject (lower) box, paste or
upload the recA_allAgro.fas file. Run BLAST, and browse results.
2. blastn search against universal nucleotidic databases
The large and constantly maintained databases that contain all known sequences (nr
at NCBI (non-redundant part of GenBank); EMBL at EBI) can be searched as well to
see if new sequences closer to our unknown sequence may have been added
COST 873 - Lyon 4-9 September 2011 19

ecently. On the NCBI BLAST website, proceed as previously, but uncheck the “Align
two or more sequences” option. Chose instead the nr database and run BLAST.
NB : from the result page, you can download the homologous sequence hits in
FASTA format. This can be useful to build a sequence database for other
markers and/or species, or to refresh a routinely used dataset. To do so, on the
NCBI BLAST result page, go to the “Alignments” section and select the desired
sequences (you shall avoid the sequences corresponding to complete genomes
because the files are large and cannot be used for the further steps). Click on
“Get selected sequences” to reach the corresponding GenBank page. Then, use
the “Send” link (upper right corner of the page) to download the sequences in a file.
IV. Alignment of homologous sequences
Open the file recA_allAgro.fas with Seaview. In the “Edit” menu, choose “load
sequence”, paste the unknown sequence in the box and click on “add to alignment”.
Then, in the “Align” menu, select “align all” (performs alignment with MUSCLE
algorithm by default, can be changed in “alignment options”). Check visually the
alignment for artefacts.
V. Phylogenetic tree building
Seaview provides tools for phylogenetic tree construction from the alignment in its
“Tree” menu.
1. Distance methods
The BioNJ algorithm shall be prefered to the basic NJ as it is adapted to biological
purposes. Different distance models are proposed, from the simplest (Jukes &
Cantor, 1969, JC) to the most prameterized (Hazegawa et al., 1985, HKY). On
short alignments like the present one, calculation of BioNJ trees is very fast and allow
calculation of numerous bootstrap replicates. Build the tree and interpret it for
assignation of a sequence type or species to the unknown sequence.
2. Model-based methods
Unlees more computational time-consuming, model-based methods like PhyML
perform more accurate phylogenetic history reconstruction. Many options can be
specified, most of them concerning the estimation of the various parameters of the
evolutionary model. As estimation of all parameters from the data is recommended
rather than fixing arbitrary values, this make the calculation time grow, to a point that
is not bearable by the user (or by the computer). Similarly, bootstrap replicates are
very time-consuming and branch support estimation by aLRT (approximated
likelihood ratio test) may be prefered. Build the tree and compare it to the previous
one(s).
VI. Taxonomic assignation according to results
In the tree, the unknown sequence appears nested in the taxonomic group to wich it
belongs. This result must be taken carefully, as its accuracy depends on the supports
of the branch separating the sequence (or the group containing it) from other groups.
Low support of a branch (usually under 90%) indicate the bi-partition of the tree at
this point is unsure.
Also, a long branch without bifurcation in the tree must be interpreted cautiously, as a
common artefact in phylogeny will systematically locate a long branch nearer to the
COST 873 - Lyon 4-9 September 2011 20

tree basis than it should. In both cases, enhancing the species sampling is likely to
resolve an uncertain tree position.
This emphasize the need of good taxonomic coverage and regular sampling of the
biological diversity when constituting a dataset for sequence assignation. We are
proud to provide the present dataset for Agrobacterium typing, as it is the most
complete and evenly sampled yet built.
Fig. 5. Analysis of chromosome (ribotype) and Ti plasmid (vir) characters
shows the occurrence of genetic diversity for both cell and Ti plasmid of
agrobacteria involved in related crown gall outbreaks. Ponsonnet et al.
(unpublished)
COST 873 - Lyon 4-9 September 2011 21

Monday 5th Tuesday 6th Wednesday 7th Thursday 8th Friday 9th
Agrobacterium isolation and
Plasmid status of
phenotypic macroarrays and
9h00-9h45
CONFERENCE Daniel Muller
identification : results of
Agrobacterium isolates : gel
biochemical tests : results
isolation and PCR analyses
preparation
analyses
(vir/pyr/spe sp)
9h45-10h00
Coffee break
10h00-10h15 Coffee break Coffee break Coffee break
Plasmid status of
Pathogenicity tests :
agrobacterium isolates :
10h30-11h15
infection assay in
compilation of data and
preculture
metagenomic DNA
greenhouse and symptom
Results analyses
extraction from bulk soil,
11h15-11h45
visualisation.
standardized procedure for
rhizosphere and tumor and
Agrobacterium isolation.
visualisation on gel 1/2 concluding session : Xavier
11h45-12h30
CONF. Maria Lopez
Step 3
Nesme
lunch lunch lunch
13h30-14h30
Welcome and introductive CONFERENCE Joanna
metagenomic DNA
CONFERENCE Xavier Nesme
CONFERENCE. Xavier Nesme
Pulowska
extraction from bulk soil,
14h30-15h15
rhizosphere and tumor -
phenotypic macroarrays at
preparation and sterilisation
Plasmid visualisation : PCR analyses ( 16S, vir and
PARMIC facility and
of culture media
analyses of results
TRL) 2/2
biochemical tests. Step 2.
Coffee break Coffee break Coffee break Coffee break Coffee break
15h30-16h30
standardized procedure for
phenotypic macroarrays at
Agrobacterium isolation and
Agrobacterium isolation
PARMIC facility and
identification : analysis of
from bulk soil, rhizosphere
biochemical tests. Step 2.
biochemical tests
and tumor. Steps 1 and 2
sequence manipulation and
16h30-17h30 CONF. Maria Lopez phylogenetic analyses CONFERENCE Joel Pothier
phenotypic macroarrays and
17h30-18h00
biochemical tests. Step 1.
gel migration and results
analyses