1
Supplementary Material – Schwager et al.
2
3
Experimental Procedures
4
Organisms and culture conditions
5
All bacterial strains used in this study are shown in Table S1. Bacteria were routinely grown
6
in LB at 37°C with shaking. Antibiotics were added as required at the following
7
concentrations: ampicillin (100µg/ml), kanamycin (50 µg/ml), chloramphenicol (25 µg/ml),
8
and gentamycin (20 µg/ml). Growth of liquid cultures was monitored with an Ultraspec3100
9
Pro spectrophotometer (Biochron, Cambridge, England) by measurement of optical density at
10
600 nm. For plant experiments, seeds of Arabidopsis halleri were surface sterilized,
11
germinated and grown on half-strength MS medium according to (Blom et al., 2011). After
12
four weeks, they were transferred to hydroponic cultures (500 ml pots containing a solution
13
containing the following nutrients (final concentration): 5mM C6H13NO4 x H2O, 3mM
14
CaNO3, 2mM KNO3, 2mM Fe(III)EDTA, 0.8 mM NH4H2PO4, 98 µM MgSO4 x H2O, 4.6µM
15
H3BO3, 0.5 µM MnCl3 x 4H2O, 76nM ZnSO4 x 7 H2O, 32nM CuSO4 x 5 H2O, 11nM MoO3.
16
pH was adjusted to 6). The nutrient solution was aerated continuously and renewed every
17
second week. Plants were grown in controlled conditions (16h/8h day/night and 20°C/15°C).
18
Bacterial inoculation was performed as follows: 8 ml of an overnight culture of RFP-tagged
19
B. cenocepacia H111 harbouring a cadA::gfp reporter plasmid were harvested, the pellet was
20
resuspended in 2 ml 0.9% NaCl and inoculated into the plant pots.
21
22
DNA manipulations, plasmid conjugation and sequence analysis of Tn5 mutants
23
All plasmids used in this study are shown in Table S2. General molecular methods were as
24
described by Sambrook (Sambrook, 1989). Plasmid DNA was routinely isolated using the
25
Qiagen miniprep kit. Following PCR amplification or restriction digestion, DNA was purified
26
using the Qiagen PCR purification kit. Plasmids were introduced into B. cenocepacia H111
27
by conjugation. Bacterial conjugations were carried out using a filter mating technique
28
(Herrero et al., 1990) . Briefly, donor, recipient, and helper E. coli HB101 (pRK600) or
29
MM294 (pRK2013) were grown at 37°C overnight in 10 mL LB media supplied with the
30
appropriate antibiotics. 2 mL overnight culture of each was harvested using a microcentrifuge
31
(6000 rpm, 5 min), washed and resuspended in 500 μL LB. 100 μL donor cells and 100 μL
32
helper cells were mixed and incubated at RT for 10 min. 200 μL recipient cells were added.
33
The mixture was spot-inoculated onto the surface of prewarmed LB agar plates and incubated
34
overnight at 37 °C. The cells were scraped off and resuspended in 1 mL 0.9% NaCl and
35
plated on Pseudomonas Isolation Agar (DIFCO) medium containing antibiotics for
36
counterselection of donor, helper and untransformed cells. For identification of the DNA
37
sequences flanking transposon insertion sites in the transposon mutants, arbitrary PCR was
38
carried out essentially as described by (O'Toole and Kolter, 1998). Briefly, a first round of
39
PCR
40
GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCC-3´)
41
specific primer luxCext2 (5´-AGTCATTCAATATTGGCAGG-3´). The first round PCR
42
conditions consisted of (i) 5 min at 95 °C; (ii) 6 x [30 s at 95 °C, 30 s at 30 °C, 1 min at 72
43
°C]; (iii) 30 x [30 s at 95 °C, 30 s at 45 °C, 1 min at 72 °C]; (iv) 5 min at 72°C. The second
44
round of PCR amplification used 5 µl purified first round PCR product as template and the
45
following primers: ARB2 (5´-GGCCACGCGTCGACTAGTAC-3´) and luxCint2 (5´-
46
GGATTGCACTAAATCATCAC-3´), with the following PCR conditions: (i) 30 x [30 s at 95
47
°C, 30 s at 45 °C, 1 min at 72 °C]; (ii) 5 min at 72 °C. Amplicons were sequenced using an
48
ABI3730 automated sequencer (Applied Biosystems) and reads were mapped to the genome
49
of B. cenocepacia H111 (WGS Accession No. CAFQ00000000).
50
amplification
was
performed
using
the
arbitrary
primer
and
the
ARB6
(5´-
Tn5-transpon
51
Construction of a cosmid library of B. cenocepacia H111 and complementation of the
52
transposon mutant
53
Chromosomal DNA of B. cenocepacia H111 was partially digested with EcoRI and ligated
54
into the cosmid vector pLAFR3 cut with the same enzyme. In vitro packaging into E. coli
55
HB101 was done by the aid of the Gigapack III Gold Packaging Reaction Kit from Invitrogen
56
as described by the manufacturer. The library was transformed into mutant 35aA12 and
57
transformants were selected on PIA medium containing 2 mM CdSO4.
58
59
Construction of a defined cadA mutant
60
A 406 bp internal cadA fragment was amplified using the primer pair CadF417 (5’-
61
GTTCCGGATCATGCAGATGG-3’) and CadR825 (5’-GATGTTCAGGTTGCCGTTGC-
62
3’). The amplicon was digested with NotI and cloned into the suicide vector pSHAFT2 cut
63
with the same enzyme. The resulting plasmid was transferred to B. cenocepacia H111 by
64
triparental mating and integrants were selected on PIA medium containing 90 µg/ml
65
chloramphenicol. The correct genetic structure of the mutant was verified by PCR analysis.
66
67
Measurement of cadA promoter activity
68
A transcriptional fusion of the cadA promoter with gfp was constructed as follows: a 386 bp
69
BamHI fragment of the BCAE0587 promoter region was PCR amplified using primers
70
CadA_Pro_F
71
TGGATCCGTATGACGGTGTCGTGG-3’). The amplicon was digested with BamHI (the
72
introduced restrictions sites are underlined) and inserted into the promoter probe vector pGA-
73
G1 cut with the same enzyme. The resulting plasmid, pMS1, was transferred to B.
74
cenocepacia H111. An overnight culture of B. cenocepacia H111 (pMS1) was used to
75
inoculate mineral base medium (adjusted to pH 5 or pH 7; OD600 of 0.001; Van Nostrand et
(5’-
AGGATCCGTGATCGTCGAGCAGC-3’)
and
CadA_Pro_R
(5’-
76
al., 2005) supplemented with 1 mM citrate as carbon source, 20 μg/ml gentamycin and 1 mM
77
of each of the following heavy metals: ZnSO4, CdSO4, CoCl2 x 6H2O, CuSO4 x 5 H2O, NiCl2 x
78
6 H2O, Pb(NO3)2. Following 18 h of incubation at 37 °C, the OD600 was measured and
79
cultures were adjusted with 0.9 % NaCl to an OD600 of 2.0. Green fluorescence was measured
80
using a 300 µl sample in the microtitre plate reader SynergyTM HT (MWG Biotech,
81
Germany) with an excitation wavelength of 485 nm and emission detection at 528 nm. The
82
data were processed with KC4 software (BioTek Instruments). The fluorescence
83
measurements were corrected for autofluorescence. Specific fluorescence units, i.e. relative
84
fluorescence divided by the OD600, are shown.
85
86
Table S1. Strains used in this study.
87
Species
Description/ Characteristics
Source, Reference
H111
CF isolate
Romling et al. (1994)
35aA7
cadmium sensitive Tn5 insertion mutant
This study
35aA12
cadmium sensitive Tn5 insertion mutant
This study
40G3
cadmium sensitive Tn5 insertion mutant
This study
3G12
cadmium sensitive Tn5 insertion mutant
This study
7G10
cadmium sensitive Tn5 insertion mutant
This study
8D3
cadmium sensitive Tn5 insertion mutant
This study
8E11
cadmium sensitive Tn5 insertion mutant
This study
ΔcadA
BCAE0587 knock-out mutant, CmR
This study
dsRed
H111 tagged with pUT-Km-dsRed; KmR
Lumjiaktase et al., (2010)
HB101
recA thi pro leu hsdM+, SmR
Boyer and Roulland (1969)
Burkholderia cenocepacia
Escherichia coli
DH5α
CC118 λpir
88
89
supE44 ∆lacU169 (ΦlacZ∆M15) hsdR17 endA1
recA1 gyrA96 thi1 relA1
Δ(ara-leu) araD ΔlacX74 galE galK phoA20 thi-1
rpsE rpoB argE(Am) recA1 Δpir lysogen
Hanahan (1983)
Herrero et al. (1990)
MM294
hrs hrm+ thi endA supE0
Meselson and Yuan (1968)
XL1 blue
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac
Bullock et al. (1987)
Table S2. Plasmids used in this study.
Plasmid
Characteristic
Source or reference
pGA-G1
Broad host-range gfp-based promoter probe vector
Laboratory collection
pMS1
pGA-G1 containing a PcadA-gfp transcriptional fusion, Gmr
This study
pLAFR3
Broad host-range cosmid vector, TcR
Staskawicz et al. (1987)
pUT mini-Tn5Km2luxCDABE
delivery vector for mini Tn5Km2-luxCDABE, KmR
Winson et al. (1998)
pGEM-T easy
multicopy cloning vector, ApR
Promega
pRK600
ColE1oriV, RK-2Mob+ RK2-Tra+, helper plasmid, CmR
Kessler et al. (1992)
pRK2013
ColE1 RK2-Mob+ RK2Tra+ derivative, KmR
Ditta et al. (1980)
GmR
pBBR1 MSC-5
broad host-range cloning vector,
pSHAFT2
suicide plasmid, oriT+, oriR6K, CmR
Kovach et al. (1995)
M. S. Thomas, unpublished
90
Figure S1
91
92
93
94
95
Figure S1. Identification of genes involved in cadmium resistance in B. cenocepacia H111.
96
Screening of a Tn5-insertion mutant library resulted in seven mutants that had lost the ability
97
to grow on LB medium supplemented with 2mM CdSO4. The DNA sequences flanking the
98
transposon were determined using arbitrary PCR, essentially as described by (O'Toole and
99
Kolter, 1998). Amplicons were sequenced and reads were mapped to the genome of B.
100
101
cenocepacia H111.
102
References
103
104
105
106
Blom, D., Fabbri, C., Eberl, L., and Weisskopf, L. (2011) Volatile-mediated killing of
Arabidopsis thaliana by bacteria is mainly due to hydrogen cyanide. Appl Environ Microbiol
77: 1000-1008.
107
108
Boyer, H.W., and Roulland-Dussoix, D. (1969) A complementation analysis of restriction and
modification of DNA in Escherichia coli. J Mol Biol 41: 459-72.
109
110
111
Bullock, W.O., Fernandez, J.M., and Short, J.M. (1987) Xl1-Blue: A high-efficiency plasmid
transforming recA Escherichia coli strain with beta-galactosidase selection. BioTechniques 5:
376-379.
112
113
114
Ditta, G., Stanfield, S., Corbin, D., and Helinski, D.R. (1980) Broad host range DNA cloning
system for Gram-negative bacteria - construction of a gene bank of Rhizobium meliloti. Proc
Natl Acad Sci U S A 77: 7347-7351.
115
116
Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol
166: 557-580.
117
118
119
Herrero, M., Delorenzo, V., and Timmis, K.N. (1990) Transposon vectors containing nonantibiotic resistance selection markers for cloning and stable chromosomal insertion of
foreign genes in Gram-negative bacteria. J Bacteriol 172: 6557-6567.
120
121
122
123
Kessler, B., Delorenzo, V., and Timmis, K.N. (1992) A general System to Integrate Lacz
Fusions into the Chromosomes of Gram-Negative Eubacteria - Regulation of the Pm
Promoter of the Tol Plasmid Studied with All Controlling Elements in Monocopy. Mol Gen
Genet 233: 293-301.
124
125
126
Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M., 2nd and
Peterson, K. M. (1995) Four new derivatives of the broad-host-range cloning vector
pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175-176.
127
128
129
130
Lumjiaktase, P., Aguilar, C., Battin, T., Riedel, K., and Eberl, L. (2010) Construction of selftransmissible green fluorescent protein-based biosensor plasmids and their use for
identification of N-acyl homoserine-producing bacteria in lake sediments. Appl Environ
Microbiol 76: 6119-6127.
131
132
Meselson, M., and Yuan, R. (1968) DNA restriction enzyme from E. coli. Nature 217: 11101114.
133
134
135
O'Toole, G.A., and Kolter, R. (1998) Initiation of biofilm formation in Pseudomonas
fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic
analysis. Mol Microbiol 28: 449-461.
136
137
138
Romling, U., Fiedler, B., Bosshammer, J., Grothues, D., Greipel, J., Vonderhardt, H., and
Tummler, B. (1994) Epidemiology of chronic Pseudomonas aeruginosa infections in cystic
fibrosis. J Infect Dis 170: 1616-1621.
139
140
Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989) Molecular Cloning: A laboratory manual.
Cold Spring Harbor Laboratory Press, NY.
141
142
143
Staskawicz, B., Dahlbeck, D., Keen, N., and Napoli, C. (1987) Molecular characterization of
cloned avirulence genes from race-0 and race-1 of Pseudomonas syringae pv. glycinea. J
Bacteriol 169: 5789-5794.
144
145
146
Van Nostrand, J.D., Sowder, A.G., Bertsch, P.M., and Morris, P.J. (2005) Effect of pH on the
toxicity of nickel and other divalent metals to Burkholderia cepacia Pr1(301). Environ Toxicol
Chem 24: 2742-2750.
147
148
149
150
Winson, M.K., Swift, S., Hill, P.J., Sims, C.M., Griesmayr, G., Bycroft, B.W. et al. (1998)
Engineering the luxCDABE genes from Photorhabdus luminescens to provide a
bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5
constructs. FEMS Microbiol Lett 163: 193-202.
151