Current Biology, Volume
32
Supplemental Information
Soil bacteria protect fungi
from phenazines by acting as toxin sponges
Kurt M. Dahlstrom and Dianne K. Newman
Fungus (
-
) PCA
Fungus (+) PCA
Co
-
colony (+) PCA
Aspergillus
sp.
Coniochaeta
sp.
+
Paraburkholderia
sp.
+
Luteibacter
sp.
2 mm
P.
chlororaphis
P.
chlororaphis
::
phzB
P. fluorescens
P. fluorescens
::
phzD
Aspergillus sp.
A
B
Fig
ure
S1
.
Fungi from identified bacterial
-
fungal pairings are sensitive to PCA
, related to Figure
1
and STAR Methods
. A)
Further bacterial
-
fungal pairings isolated as visualized in Figure 1A (see STAR
Methods).
Coniochaeta
and
Aspergillus
species grown on PDA, PDA supplemented with 300 μM PCA,
and finally in the presence of PCA along with co
-
isolated bacterial partners, a
Pa
raburkholderia
species
and a
Luteibacter
species, respectively. Plates were grown for 48 hrs at 30 ºC.
B)
Phenazine production is
required to inhibit growth of the
Aspergillus
species as seen in Figure 1D.
Pseudomonas chlororaphis
and
Pseudomonas fluoresce
ns
inhibit fungal growth, but the
::phzD
and
::phzB
strains, respectively, fail to
inhibit fungal growth after growing on PDA for 48 h
rs
at 30 ºC.
Eubacteria HCR
DAPI
Merge
(
-
) PCA
(+) PCA
A
B
C
D
E
F
Fig
ure
S2
.
Bacterial aggregates accumulate along the ridge of co
-
colonies treated with PCA
, related
to Figure 2
.
A band of bacteria can be seen in both control (panels A, B, C) and control (panels D, E, F)
colonies between the inn
er and outer zones that were imaged in Figure 2. In the PCA treated colony, this
band is thicker with more well
-
formed aggregates, and is representative of the region larger aggregates
are visible to the eye during co
-
colony growth on PCA. Samples were cle
ared using the MiPACT
protocol, and images were captured on a Nikon Ti2 Eclipse. Red represents the signal produced by
eubacterial HCR probes coupled to Alexa fluor 647 nm shown in panels A and D. DAPI staining is
visible in panels B and E. Panels C and F
represent the merged images. Colonies were grown for 48 h
rs
at
30 ºC before clearing.
Fig
ure
S3
.
P. edwinii
does not degrade PCA in liquid culture
, related to Figure 3
.
P. edwinii
was
grown in potato dextrose broth shaking at 30 ºC spiked with and without 300 μM PCA, and with PCA and
its fungal partner. PCA concentration was measured every 24 hours for three days with no significant loss
of the molecule observed. This demonstrates t
hat the PCA sequestered by
P. edwinii
in Figures 3B and C
is unlikely to be degraded by the organism, and therefore degradation of the molecule is not part of the
mechanism of protection. A control lacking
P. edwinii
was included to confirm PCA did not deg
rade over
this time period. Quantification occurred by measuring absorbance at 365 nm. Error bars represent
standard deviation of four biological replicates.
2 mm
Aspergillus
Alone
Aspergillus
+
Δ
hrcA
Aspergillus
+ WT
Δ
hrcA
WT
(
-
) PCA
(+) PCA
(+) PCA +
Aspergillus
sp
Fungus
(μm)
A
B
C
Fig
ure
S4
.
Morphology of WT and Δ
hrcA
P. edwinii and properties of the
Aspergill
u
s isolate with
and without PCA challenge
, related to Figures 4 and 5
.
A)
Comparison of the
Aspergillus
isolate
grown alone (left, top), with WT
P. edwinii
(center), and with the Δ
hrcA
mutant (bottom). Note similar
large aggregates as seen in Figure 4B also from here in the Δ
hrcA
mut
ant despite the absence of PCA (red
arrows).
B)
WT (left) the the Δ
hrcA
mutant (right) grown in the absence (top) and presence (center) of
PCA, and next to its partner fungus when challenged with PCA (bottom). The WT strain shows a
thickening of the colony
in response to PCA, as well as an increased sheen. A similar thickening and
sheen is visible for the Δ
hrcA
mutant in all conditions with enhanced yellowing next to its fungal partner.
See Figures 4C and E for impact on fungal growth and PCA sequestration
ability, respectively. Red
arrows indicate bacterial colonies, white indicate fungus.
C)
The
Aspergillus
isolate produces an
oxidizing environment when growing on potato dextrose agar in the absence of PCA, but generates a
more reducing environment during
challenge with 300 μM PCA, potentially contributing to the enhanced
reduction seen in Figure 5B. Error bars represent the standard deviation of 3 measurements at each depth.
All colonies were grown for 48 hrs at 30 ºC.
Mutant Phenotype
Transposon Hit
(in nearest relative)
Homologue of Encoded Protein
Actively inhibits
fungal growth
BTO02_09010, Chr 1
Pyruvate dehydrogenase
Decreased Protection
BTO02_10735, Chr 1
photosystem reaction center subunit H
Decreased Protection
BTO02_08765, Chr 1
bacterioferritin
Decreased Protection
BTO02_27095, Chr 2
FAD-binding oxidoreductase
Decreased Protection
AciPR4_3613
short-chain dehydrogenase/reductase SDR
Decreased Protection
BJG93_11340
Glutamate-Cysteine Ligase
Decreased Protection
BTO02_09675, Chr 1
lysophospholipid transporter LplT
Decreased Protection
BTO02_00075, Chr 1
basal rod FlgB
Decreased Protection
Burkholderia sp.
JP2-270 plasmid p1
cobyrinic acid ac-diamide synthase
Decreased Protection
(2x) BTO02_18750, Chr 1
phosphoribosyl-AMP cyclohydrolase HisI
Decreased Protection
BTO02_30205, Chr 2
alpha,alpha-trehalase
Decreased Protection
BTO02_00530, Chr 1
tRNA uridine-5-carboxymethylaminomethyl
Decreased Protection
(2x) BTO02_15090, Chr 1
ABC Transporter
Decreased Protection
(2x) BTO02_32630, Chr 2
D-xylose ABC transporter ATP-binding protein
Decreased Protection
BTO02_09325, Chr 1
potassium transporter Kup
Decreased Protection
BTO02_01560, Chr 1
cation-efflux pump
Decreased Protection
BTO02_11025, Chr 1
HipA Toxin
Decreased Protection
BTO02_12180, Chr 1
Secretion Factor VirJ
Decreased Protection
C2L64_00505
lysine transporter LysE
Decreased Protection
BTO02_13845, Chr 1
TetR transcriptional regulator
Decreased Protection
BTO02_01100, Chr 1
D-threo-aldose 1-dehydrogenase
Decreased Protection
BTO02_07430, Chr 1
LysR family transcriptional regulator
Decreased Protection
BTO02_23235, Chr 2
GlxA family AraC-like transcriptional factor
Decreased Protection,
altered aggregates
BTO02_10435, Chr 1
LysR family transcriptional regulator
Increased Protection
(2x) BTO02_00065, Chr 1
flagellar hook protein FlgE
Increased Protection
(2x) BTO02_00055, Chr 1
flagellar basal body FlgF
Increased Protection
BTO02_00745, Chr 1
gspN, type II secretion
Increased Protection
BTO02_14620, Chr1
Toxin BrnT
Increased Protection
BTO02_02275, Chr 1
Peptidase
Increased Protection
BTO02_17485, Chr 1
heat-inducible transcriptional repressor HrcA
Increased Protection
BTO02_04810, Chr 1
serine protease
Increased Protection,
Mucoid
BTO02_12310, Chr 1
Domain of Unknown Function
Increased Protection,
Mucoid
BTO02_15085, Chr 1
glycosyl transferase
Table S1.
T
ransposon mutants found in
P. edwinii
, related to
Figure
4 and STAR M
ethods
.
Listed
are mutants
that alter its ability to protect its partner fungus, including phenotype (left), closest
homologue of
P. edwinii
gene each transposon hit was found in based on NCBI BLAST using arbitrary
PCR on the mutant strains (center), and any apparent homologue the g
ene encodes (right). See STAR
Methods for genome annotation method which may differ from the automated annotation provided by
NCBI upon genome submission. Original author
-
annotated chromosomes available upon request.
Strains
Genotype
Source or Reference
Paraburkholderia
Paraburkholderia edwinii
; DKN_2541
WT
This Study
DKN_2542
Δ
hrcA
This Study
DKN_2543
::
pdhA
This Study
DKN_2544
::
hrcA
This Study
DKN_2545
::
flgE
This Study
DKN_2546
Transposon insertion into
putative potassium transporter
This Study
DKN_2547
Transposon insertion into
putative LysR transcriptional regulator
This Study
DKN_2548
Transposon insertion into gene
of unknown function
This Study
Paraburkholderia
isolate KMD_2017
WT
This Study
Paraburkholderia SOS3
WT
Provided by
Sustainable
Organic Solutions
Pty. Ltd.
Building 1015, Gate
4, 80-120
Meiers Road,
Indooroopilly, 4068
QLD, Australia
Paraburkholderia unamae
isolate
WT
S1
Paraburkholderia phenazinium
WT
S2
Lutibacter
Lutibacter
isolate KMD_2017; DKN_2549
WT
This Study
Pseudomonas
P. fluorescens
2-79
WT
S3
P. fluorescens
phz(-)
::
phzD
S4
P.chlororaphis
PCL 1391
WT
S5
P.chlororaphis
phz(-)
::
phzB
S5
P. chlororaphis
subsp.
Aureofaciens
30-84
WT
S6
E. coli
S17
λ(pir)
thi pro hsdR-hsdM+ "recA RP4-
2::TcMu-Km::Tn7
S7
Beta2155
thrB1004 pro thi strA hsdsS
lacZD M15 (F`lacZD M15
lacIq traD36 proA+ proB+)
D dapA::erm (Ermr) pir::RP4
[::kan (Kmr)]
S8
Fungi
Aspergillus
ADI1; DKN_2550
WT - coisolate of
P. edwinii
This Study
Aspergillus
isoalte_02 KMD_2017; DKN_2551
WT
This Study
Lecythophora
isolate KMD_2017; DKN_2552
WT
This Study
Fusarium
isolate KMD_2018; DKN_2553
WT
This Study
Aspergillus fumigatus
isolate KMD_2018; DKN_2554
WT
This Study
Penicillium
isolate_01 KMD_2018; DKN_2555
WT
This Study
Penicillium
isolate_02 KMD_2018; DKN_2556
WT
This Study
Plasmids
Originally Reported
pMQ30
S9
pMQ30
Δ
hrcA
This Study
pBBR1 MSC-2
S10
pBBR1 MSC-2 hrcA
This Study
mini-mariner plasmid
S11
Table S2.
List of strains and plasmids used in this study, related to STAR Methods.
Primer
Purpose
Sequence
8bF
Bacterial 16S Identification
GRG TTT GAT CCT GGC TCA G
1512uR
Bacterial 16S Identification
ACG GHT ACC TTG TTA CGA CTT
ITS-1
Fungal ITS Identification
TCC GTA GGT GAA CCT GCG G
ITS-4
Fungal ITS Identification
TCC TCC GCT TAT TGA TAT G
Del-hrcA upstream Fwd
Construction of pMQ30-∆
hrcA
AGC TAT GAC CAT GAT TAC GAA TTC
GAG CTC GGT ACC CCC CGA CAC GAT
CTG GAT GCT GAC
Del-hrcA upstream Rev
Construction of pMQ30-∆
hrcA
TGC TGG CTC ACT GCC GAT GGC GAA
AAA TTT TAG CTC AAT GAC GAC GCT G
Del-hrcA downstream Fwd
Construction of pMQ30-∆
hrcA
GAG CTA AAA TTT TTC GCC ATC GGC
AGT GAG CCA GCA GTG AAT C
Del-hrcA downstream Rev
Construction of pMQ30-∆
hrcA
AGC TTG CAT GCC TGC AGG TCG ACT
CTA GAG GAT CCC CCA CTC GAC CGG
TGT CAG TTC CAG
pBBR1-hrcA
Construction of pBBR1-
hrcA
ATA AGC TTG ATA TCG AAT TCC TGC
AGC CCG TCA TTG AGC TAA AAT TTT
TCG CCA TGT TAG
pBBR1-hrcA
Construction of pBBR1-
hrcA
CGG TGG CGG CCG CTC TAG AAC TAG
TGG ATC CCC CTC ACT GCT GAC TCA
GCG TCA ACG
SS9arb1 Round 1
Arbitrary PCR to identify
transposon insertion site
GAC CAC GAG ACG CCA CAC TNN NNN
NNN NNC ATG C
SS9arb2 Round 1
Arbitrary PCR to identify
transposon insertion site
GAC CAC GAG ACG CCA CAC TNN NNN
NNN NNA CTA G
SS9arb8 Round 1
Arbitrary PCR to identify
transposon insertion site
GAC CAC GAG ACG CCA CAC TNN NNN
NNN NNG ATA T
Mariner_3 Round 1
Arbitrary PCR to identify
transposon insertion site
CTT CTT GAC GAG TTC TTC TGA GC
Mariner_4 Round 1
Arbitrary PCR to identify
transposon insertion site
TAG GGT TGA GTG TTG TTC CAG TT
arb3 Round 2
Arbitrary PCR to identify
transposon insertion site
GAC CAC GAG ACG CCA CAC T
Mar4 Round 2
Arbitrary PCR to identify
transposon insertion site
TCA CCG TCA TGG TCT TTG TAG TC
Table S3.
List of primers used in this study, related to STAR Methods
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