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1
Kurt M. Dahlstrom
1
, Dianne K. Newman
1,2
2
3
1
Division of Biology and Biological Engineering and
2
Division of Geological and Planetary Sciences,
4
California Institute of Technology, Pasadena, CA, USA
5
6
7
8
Correspond
e
nce: Dianne K. Newman
9
Email: dkn@caltech.edu
10
11
ORCID: Kurt Dahlstrom,
0000
-
0001
-
6590
-
6020
; Dianne Newman, 0000
-
0003
-
1647
-
1918.
12
13
14
15
16
17
Keywords:
phenazines, bacteria, fungi,
protective
partnership
, microbial interactions
, inter
-
kingdom
18
19
Author Contributions:
K.M.D
. performed
the experiments
.
K.M.D and D.K.N.
designed
the
research
20
strategy, analyzed data, and wrote the paper.
21
22
This PDF file includes:
23
Main text
,
Figures 1 to
6
24
Supplemental Figures S1 to S
4
25
Supplemental Tables S1 & S2
26
.
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;
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Summary
27
Many environmentally and clinically important fungi are
sensitive to
toxic,
bacterially
-
produced,
28
redox
-
active molecules called phenazines. Despite being vulnerable to phenazine
-
assault, fungi
inhabit
29
microbial communities that contain phenazine p
roducers. Because many fungi cannot withstand
30
phenazine challenge, but some bacterial species can, we hypothesized that
bacterial partner
s
may
protect
31
fungi in
phenazine
-
replete
environment
s
. In the first soil sample we collected, we co
-
isolated several su
ch
32
physically associated pairings. We
discover
ed
the novel species
Paraburkholderia edwinii
and
33
demonstrate
d
it
can
protect
a co
-
isolated
Aspergillus
species from phenazine
-
1
-
carboxylic acid (PCA)
by
34
sequestering
it
, acting as a toxin sponge
; in turn, it a
lso gains protection
.
W
hen challenged with
PCA
,
P.
35
edwinii
changes its morphology
, forming
aggregates within the growing fungal colony. Further, the
36
fungal partner trigger
s
P. edwinii
to
sequester
PCA
and maintain
s
conditions that limit
PCA
toxicity by
37
promoting an
anoxic
and highly reducing environment.
A mutagenic screen revealed this program
38
depends
on the stress
-
inducible transcriptional repressor HrcA
.
We show that one relevant stressor in
39
response to
PCA
chall
enge
is fungal acidification
and that acid stress cause
s
P. edwinii
to behave as
40
though the fungus
were
present. Finally, we
reveal
this
phenomenon as
widespread among
41
Paraburkholderia
with moderate specificity among bacterial and fungal partners, includin
g plant and
42
human pathogens. Our
discovery suggests
a
common
mechanism by which fungi can gain access to
43
phenazine
-
replete environments, and provide
s
a tractable model system
for its study
. These results have
44
implications for how
rhizosphere
microbial comm
unities as well as plant and human infection sites are
45
policed for
fungal
membership.
46
47
48
49
50
51
52
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Introduction
53
The presence or absence of
particular
fungal species in host
-
associated microbial communities
54
plays a central role in human and plant health, crop yield, and climate change
1
–
3
. However,
we lack an
55
understanding
of
how key fungal species are integrated into these communities
in the face of rampant
56
chemical warfare
.
It has long been
known that the soil is home to diverse microbes that produce natural
57
products with antibiotic activity. Important amongst these are phenazines
, redox active compounds that
58
can
restrict
fungal growth and
have been shown to be responsible for excluding
fungi
from
agriculturally
59
important
microbial communities
4,5
.
A recent metagenomic study revealed that phenazine biosynthesis
60
capacity is widespread in agricultural soils and crop microbiomes
6
.
Given that
drier soils are also
61
associated with higher rates of phenazine producers colonizing wheat
, th
is
suggests
that soil fungi may
62
need to contend with higher con
centrations of phenazines as the climate shifts
7,8
.
Paradoxically, many
63
fungi that are
sensitive
to phenazine
s
are rout
inely found living in close proximity to phenazine
-
producing
64
bacteria
,
including pathogenic fungi in the lungs of cystic fibrosis patients, beneficial and
65
phytopathogenic fungi in the rhizosphere, and in oceanic environments including coral
9
–
12
.
This pattern
66
of co
-
habitation
indicates
there may be a general way fungi are screened for membership
in microbial
67
comm
unities
that produce phenazines
that holds broad relevance.
We set out to
identify such a putative
68
screening mechanism
, a necessary step
towards the goal of
manipulat
ing these microbial communities
for
69
human benefit.
70
Our drive to
understan
d
how
particular
fungi are incorporated or rejected from a microbial
71
community is motivated by the large impact fungal composition can have on the outcome for human and
72
plant health. Fungi in complex polymicrobial infections act as marker
s
of disease severity, p
articularly in
73
the lungs of patients with cystic fibrosis
13
. In this environment,
Aspergillus fumigatus
and
Candida
74
albicans
are two opportunistic fungal pathogens that are susceptible to phenazines, yet are routinely
75
isolated from patients who are co
-
infected with the proli
fic phenazine producing bacterium
Pseudomonas
76
aeruginosa
9,10
. Likewise, fungi play prominent roles in the rhizosphere, where they can help the host
77
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plants acquire nutrients and water a
s well as withstand stress and pathogens
14,15
;
plant
-
growth promoting
78
fungi such as
Trichoderma
and
Penicillium
species are often found in rhizospheres containing phenazine
79
producing
bacteria
, yet
their growth is inhibited by
phenazines
11,16
. Conversely, the ability of
80
phytopathogenic fungi to e
nter
the rhizos
p
here
is of interest due to fungi being responsible for a third of
81
all lost crops annually
17
. This is despite phenazines being credited as a primary factor in stopping a
82
variety of
fungal phytopathogens from infecting food crops, including pseudomonads that can suppress
83
Gaeumannomyces graminis
var.
tritici
and
Fusarium oxysporum
f sp.
radicis
-
lycopersici
, two fungal
84
pathogens of tomato
and wheat
, respectively
4,5
. Finally, plant associated fungi known as mycorrhizae
85
play
an outsized role in carbon sequestration
:
mycorrhizae
-
associated vegetation sequester approximately
86
350 gigatons of carbon a year compared to 29 gigatons stored by nonmycorrhizae
-
associated veg
etation
3
.
87
Notably, phenazine pro
ducers are found in
diverse
environments beyond food crops, including in forests
88
and grasslands, thus pointing to another niche of consequence where fungi must navigate phenazine
89
assault
6
.
90
How do fungi maintain an active presence in microbial communities where they run the risk of
91
encountering phenazines
? Recognizing that some
soil
bacteria can tolerate phenazines well
18
–
20
,
we
92
hypothesize
d
that one mechanism by whi
ch fungi
might
gain
navigate such
hostile
environments
is
93
through
association with
a protective bacterial partne
r. P
recedent for such relationships exists
.
For
94
example,
members of the
Burkholderiaceae
family form associations with fungi.
Trichoderma aspere
llum
95
is a biocontrol fungus
that
suppres
ses
the wheat pathogen
Fusarium oxysporum
.
Paraburkholderia terrae
96
associate
s
with the mycelium of
T. asperellum
and can be induced to migrate in the direction of mycellial
97
growth, as well as promote fungal growth in the presence of crude supernatant derived from antagonistic
98
bacteria
21
. However, this family of bacteria can also empower pathogenic fungi.
Rhizopus microsporus
99
is a necrotic plant pathogen of rice. The primary toxin it s
ecretes that is required for infection is actually
100
produced by the intracellular bacterium
Paraburkholderia rhizoxinica
that resides inside the fungal cells
101
22
. Other
Paraburkholderia
with less clear roles associate extracellularly with fungal pathogens, such as
102
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P. fungorum
, found isolated with the
white
-
rot fun
gus
Phanerochaete chrysosporium
23
. While the
103
roles each of these bacteria play for their host fungus may differ, fungal association with bacteria of
104
this family is well established.
105
In addition to these isolated examples
,
data from
a recent metagenomic survey of
soil microbes
106
across many
climate conditions
support the
notion
that cooperation
with bacteria
might underpin
fungal
107
ecological success
24
. Specifically, this study
found that the presence of bacterially
-
derived genes
108
regulating antibiotic tolerance were correlated with fungal biomass in the community. Although this co
-
109
occurrence
was
suggested to indicate inter
-
domain antagonism
,
where bacterial groups use these genes
to
110
defend themselves against fungally
-
produced antibiotics, an alternative and non
-
mutually exclusive
111
explanation may be that these
bacterial
stress response genes
help
fungi
navigate
an otherwise
112
inhospitable
environment
.
Given that fungi can be excluded
from microbial communities by phenazine
-
113
producing bacteria, it stands to reason other phenazine resistant bacteria in the community that associate
114
with fungi may have the power to affirm their presence.
B
ecause the number of environments w
h
ere
115
susceptible
fungi are found living in proximity to phenazine producers
is likely to be high
,
we reasoned
116
that
finding an example of
such a hypothetical
protective association
could be
of
great value in
117
understanding the recruitment versus repression of fungi in micro
bial communities
containing phenazine
118
producers
.
119
Accordingly, we set out to identify a model
bacterial/fungal partnership in the presence of
120
phenazines.
Using an accessible soil on the Caltech campus from which we had previously isolated
121
phenazine
degradi
ng bacteria
19
, we designed a procedure to select for such partnerships. Here, we report
122
the
isolation
and initial mechanistic characterization
of a genetically tractable fungal
-
bacterial
system
123
where the bacterial partner protects the fungus from PCA assault
. The ease with which we were able to
124
experimentally
validate the existence of
a hypothetical
bacterial/fungal
association suggests that
this
type
125
of partnership may be widespread in nature.
126
127
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Results
128
129
Isolation of
p
rotective
b
acterial
p
artner and
p
hysically
a
ssociated
f
ungus.
130
131
T
o identify fungi that resist phenazine assault with protective bacterial partners,
we sampled
132
top
soil
from the base of a
blood orange citrus
tree
outside of the Beckman
I
nstitute on the
Caltech
133
campus.
We chose this
site
because soil
represents an easily a
ccessible and broad niche containing many
134
microbial species and because we had isolated
a strain of
Mycobacterium
from this same plot that can
135
degrade
phenazines, suggesting the presence of bacteria capable of
producing and interacting with
these
136
molecules
19
.
137
We collected t
he top three centimeters of soil from this site, and
developed
a protocol to find
138
strong bacterial
-
fungal pairs
. We
washed and sonicated
100 mg to separate microbes that were not
139
strongly
associated with
one another
, thereby enriching
for strongly adherent partners (
Fig.
1A
). To
select
140
a first fungal culture, the washed samples were diluted to extinction and plated on potato
dextrose agar.
141
Fungal colonies that grew after approximately three days were screened for the presence of bacteria via
142
PCR amplification of the 16S
rDNA
region.
Fungal/bacterial
pairings were
then challenged with
300
μ
M
143
phenazine
-
1
-
carboxylic acid (
PCA
)
.
W
e used PCA because it is the biosynthetic starting product for
144
modification into more specialized phenazine types and is known to play important roles in excluding
145
fungal pathogens from wheat rhizosphere communities
5
.
Co
-
colonies that were able to grow when
146
challenged with PCA were repeatedly sub
-
cultured to isolate t
he
partner
bacterium, while the fungus was
147
re
-
plated
in the absence of PCA
with
bacteriocidal
antibiotics to cure it of the bacterium.
Isolated
fungi
148
and bacteria were retreated with PCA to
check
phenazine
-
sensitivity and tolerance, respectively, and
149
susce
ptible fungi were then supplemented with their co
-
isolated bacterium in the presence of PCA to
150
confirm
that the
partner bacterium conferred phenazine tolerance.
151
Using this process we uncovered t
hree fungal
-
bacterial
partnerships.
G
enus
-
level identification
152
was performed with ITS and 16S
rDNA
sequencing, respectively. Two
Paraburkholderia
isolates were
153
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found protecting an
Aspergillus
and a
Lecythophora
isolate. A
Luteibacter
species
also
provide
d
154
protection to a second
Aspergillus
isolate
(
Fig
.
S1
A
). In each case, the fungal growth
was
negatively
155
impacted when challenged with PCA alone, but
was
restored to varying degrees when supplemented with
156
its natively co
-
isolated bacterial partner. Of these three co
-
isolates, we selected the
Paraburkholderia
-
157
Aspergillus
pairing for further analysis due to the dramatic level of protection the bacterium
provi
ded
the
158
fungus, as well as the radical morphological change the bacterium
underwent
as it form
ed
spherical
159
aggregates within the fungal colony when
the two
were
challenged with PCA (
Fig
.
1B
).
Moreover,
while
160
the
Aspergillus
species is sensitive to PCA
and
P. edwinii
resists its toxic effects,
P. edwinii
is
still
161
vulnerable to engulfment by the PCA producer
P. fluorescens
, but not to a strain that cannot make PCA
,
162
suggesting a mutual benefit
(
Fig. 1B
).
Finally
, this pairing was attractive
because
previous reports of
163
Burkholderiaceae
family members being isolated with fungi
21,25
suggest
ed
that
such associations may be
164
common
in the soil
.
Due to this bacteri
um
’s ability to help its fungal partner prosper despite the presence
165
of a
toxin
, and
its phylogenetic placement
, we named
it
Paraburkholderia edwinii
, derived from th
e Old
166
English “Edwin”, meaning prosperous friend.
167
168
P. edwinii
p
rotects its
f
ungal
p
artner from
p
henazine
a
ssault
169
170
We next
sought
to characterize the
range of the
bacterium’s
ability to protect
its partner fungus
171
from phena
z
ines
. The minimal inhibitory concentration of PCA toward fungal targets has been reported to
172
be in the 1
-
50
μ
M range
26
. The 300
μ
M PCA used for our isolation assay there
fore
represents a strong
173
phenazine challenge intended to identify bacterial partners with a robust protection ph
enotype.
The
174
advantage of
using
a high concentration of PCA
in our laboratory experiments
is that it may better
mimic
175
local gradients of PCA that exist within
rhizosphere
microbial communities
that
likely exceed bulk
176
measurement
s.
177
T
o
determine whether
P.
edwinii
can protect
Aspergillus
against
actual
phenazine
-
producer
s
in
178
addition to purified PCA
, we
tested
the response
in the presence of different
phenazine
-
producing
179
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Pseudomonads
.
Aspergillus
was plated adjacent to
Pseudomonas fluorescens
,
Pseudomonas
180
chlororaphis
,
Pseudomonas chlororaphis
sub
-
species
aureofaciens
, and
Paraburkholderia phenazinium
.
181
T
he primary phenazine product
made by these
species
under are growth conditions
is PCA, phenazine
-
1
-
182
carboxamide (PCN), 2
-
hydroxy
-
phenazine, and i
odinin, respectively. When grown
close
to one another,
183
each phenazine producing bacteri
um
impe
ded
Aspergillus
growth (
Fig.
1C
). However, when the fungus
184
was supplemented with
P. edwinii
, growth was partially restored. Intriguingly, bacterial aggregates again
185
formed within the co
-
colonies
proximal
to phenazine producers, suggesting this
morphological phenotype
186
reflect
ed
a general
protective
response (
Fig.
1C
). Finally, to verify that the
s
e responses
were
specifically
187
due to phenazine assault, mutants of
Pseudomonas fluorescens
and
Pseudomonas chlororaphis
were
188
obtained
that could not make phenazines
27
. While the
WT
strains were capable of suppressing fungal
189
growth, the fungus grew unimpeded in proximity of the non
-
phenazine producing mutants (
Fig.
S
1B
),
190
confirming that
the protection provided by
P. edwinii
is specific to phenazine assault and can occur in a
191
mixed microbial system
.
192
193
P. edwinii
undergoes a morphological shift in response to phenazine
-
induced fungal stress.
194
195
To
understand
how
P. edwinii
respond
s
to its pa
rtner fungus during phenazine assault
,
we
imag
ed
196
the bacterium inside the co
-
colony. While a ring of what appear
ed
to be one or two dozen bacterial
197
aggregates form
ed
on the co
-
colony surface, it remained possible these were fungal structure
s
.
To
198
distinguis
h between these possibilities, we adapted a
tissue clearing technique developed in our lab
199
termed Microbial identification after PASSIVE Clarity Technique (MiPACT)
to
render the fungal tissue
200
transparent
(see Materials and Methods)
. This allowed us to visualize bacteria
within
the fungal structure
201
using
in situ
fluorescence detection of
16S rRNA
with the hybridization chain reaction (HCR)
28
.
202
Because the exterior of the colonies show
ed
putative bacterial aggregates within the center, we
203
hypothesized that may be where the bacteria
were
concentrated. We first
imaged
the outer 2/3
of the
204
colonies. In the control
,
whole
-
colony samples untreated with PCA,
P. edwinii
was
found
concentrating
205
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near the tip
s
of the outward
ly
-
growing mycelium, with relatively low amounts of bacteria found further
206
inward among older mycelial growth.
The prop
ensity of
P. edwinii
to track with the mycelial edge in the
207
untreated samples is in agreement with reports suggesting other
Paraburkholderia
are capable of
208
identifying the growing edge of expanding fungi
21
.
Conversely, in the PCA treated sample, this
209
population of bacteria w
as largely absent (
Fig.
2
).
210
To
gage whether
a lack of bacteria among the outer mycelium in the PCA treated samples was
211
due to bacterial aggregation, we imaged the center of each
co
-
colony
. In untreated sample
s
, bacteria were
212
mixed homogenously throughout
the fungal mass without identifiable structure or patterning
(
Fig.
2
). One
213
exception to this observation was an apparent transition zone between the bacterially rich inner
co
-
colony
214
and more sparely populat
ed
outer
co
-
colony. This transition zone, or ring,
comprised
more densely
215
packed bacteria, but the region lacked further organization (
Fig.
S
2
). In the PCA treated sample, the
216
center of the
co
-
colony contained clear spherical structures that
lit up
with the eubacterial HCR probes
217
(
Fig.
2
). These
P. edwinii
aggregates
ranged from 50 to 100
μ
m in diameter and were ubiquitous
218
throughout the colony center, indicating that the
visible
bacterial aggregates
on
the surface
of the co
-
219
colony
were only a fract
ion of those being formed. The PCA treated
co
-
colony also had a transition ring
220
structure between the bacterially populated center and unpopulated outer colony. In this case, however,
221
the ring contained more
clearly defined
aggregates of bacteria, correspo
nding to
the region that produced
222
aggregates
visible to the naked eye
(
Fig.
S
2
). These results
reveal
that
P. edwinii
form
s
bacterial
223
aggregates inside
and in the center of
the fungal colony when challenged with PCA.
224
225
P. edwinii
a
cts as a
t
oxin
s
ponge
226
227
How does
P. edwinii
offer resistance from phenazine assault to its partner fungus?
Possible
228
mechanisms
include
d
phenazine
degradation, sequestration, and/or detoxification.
We first tested
229
d
egradation
.
To accurately measure the
PCA
concentration o
ver time,
we grew
P. edwinii
in shaking
230
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doi:
bioRxiv preprint
liquid
cultures spiked with 300
μ
M PCA
either
alone
or
in the presence of the
Aspergillus
species
,
in case
231
a fungal signal was necessary to trigger degradation of PCA. In no condition was PCA degraded (
Fig.
232
S
3
).
The lack of degradation suggested that
bacterial aggregate
formation
might
instead reflect a
PCA
233
sequestration and detoxification
response, wh
ich we proceeded to test
.
234
Because the bacterial aggregates are too small to probe or manipulate individually, we modified
235
our experimental set up to grow
P. edwinii
directly next to its
Aspergillus
partner in the presence of PCA
236
to generate bacterial auto
-
aggregation in the form of a colony
. To verify the protection phenotype is still
237
responsive in this assay, we grew the two organisms next to each other in the presence and absence of
238
PCA.
G
rowing
P
. edwinii
and the
Aspergillus
species at a distance in the presence of PCA resulted in
239
severely stunted fungal growth,
however
the fungus was able to grow toward
P. edwinii
when plated
240
adjacently (
Fig.
3A
). Intriguingly, the bacterial colony developed a de
ep yellow hue in the PCA treated
241
condition, but only did so in the presence of the fungus. PCA is a largely colorless molecule when
242
exposed to oxygen, but in the reduced state turns yellow.
We used LC
-
MS to
determine
whether
this
243
yellow
pigment was
PCA
and
its
presence was confirmed in the
bacterial sample grown next to the
244
fungus (
Fig.
3B
).
We
detected a
smaller amount of PCA in the colonies of
P. edwinii
grown alone in the
245
presence of PCA
than in the presence of PCA and the fungus
, suggesting that while
PCA
sequestration
246
may
be an intrinsic trait of the bacterium,
sequestration
is
stimulated by the
fungal partner (
Fig.
3C
).
247
Having confirmed the presence of PCA within the
bacterial colonies, we next wanted to assess its
248
redox state.
Previously, we had been measuring PCA sequestered from an agar plate where most of the
249
molecule would be expected to be oxidized due to atmospheric oxygen. A better comparator for the
250
fraction o
f reduced PCA found within a
P. edwinii
colony would therefore be another bacterial colony
251
containing PCA, but one that was not employing a protection response involving PCA detoxification.
To
252
this end
,
we grew
P. edwinii
and the
Aspergillus
species adjacent to the phenazine producer
P.
253
fluorescens
.
PCA is the sole phenazine produced
by P. fluorescens
.
Before the bacterial colonies were
254
harvested
,
the plates were transferred to an
anaerobic
chamber to
minimize the
atmospheric
oxidation of
255
any
reduced PCA. Reduced
,
but not oxidized
,
PCA has a peak excitation of
364 nm and emission of 520
256
.
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https://doi.org/10.1101/2021.03.28.437412
doi:
bioRxiv preprint