1
Title:
Prevalence and correlates of phenazine resistance in culturable bacteria from a dryland wheat field
1
2
Running title:
Phenazine resistance in wheat rhizosphere community
3
4
Authors:
Elena K. Perry
1
, Dianne K. Newman
1,
2
*
5
6
Affiliations:
7
1
Division of
Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
8
91125, USA
9
2
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125,
10
USA
11
12
Contact information:
13
E
-
mail:
dkn@caltech.edu
14
Tel: 626
-
395
-
3543
15
Abstract
16
Phenazines are a class of bacterially
-
produced
redox
-
active
natural antibiotics that have
17
demonstrated potential as a sustainable alternative to traditional pesticides for the
bio
control of
18
fungal
crop
diseases
. However, the prevalence of
bacterial
resistance to agriculturally
-
relevant
19
phenazines is poorly understood, limiting
both
the understanding of how these molecules might
20
shape rhizosphere bacterial communities and the abilit
y to perform risk assessment for
off
-
target
21
effects.
Here, we describe
profiles of
susceptibility
to
the antifungal agent
phenazine
-
1
-
carboxylic
22
acid (PCA)
across
more than 100 bacterial strains isolated from a wheat field where
PCA
23
producers are indigenou
s
and abundant
.
We find that
Gram
-
positive bacteria are typically more
24
sensitive
to PCA than Gram
-
negative bacteria,
but that
there is
also
significant variability in
25
susceptibility
both within
and across
phyla.
Phenazine
-
resistant strains
are
more likely
to be
26
AEM Accepted Manuscript Posted Online 9 February 2022
Appl Environ Microbiol doi:10.1128/aem.02320-21
Copyright © 2022 American Society for Microbiology. All Rights Reserved.
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2
isolated from the wheat rhizosphere, where PCA producers
are
also more abundant, compared to
27
bulk soil.
Furthermore,
PCA toxicity is pH
-
dependent for most susceptible strains
and broadly
28
correlates with PCA reduction rates,
suggesting
that uptake and redox
-
cycling are important
29
determinants of
phenazine
toxicity
.
Our results shed light on which
classes
of bacteria are most
30
likely to be susceptible to phenazine toxicity
in acidic or neutral soils
. In addition,
the
taxonomic
31
and phenotyp
ic divers
ity of our
strain
collection
represents a valuable resource
for future
studies on
32
the role of natural antibiotics in shaping wheat rhizosphere communities
.
33
Importance
34
Microbial communities
contribute to
crop health
in important ways
.
For example,
p
henazine
35
metabolites are a class of redox
-
active molecules
made by diverse soil bacteria
that
underpin
the
36
biocontrol of wheat and other crops.
Their physiological functions are nuanced: in some contexts
37
they are toxic, in others, beneficial.
While much i
s known about phenazine production and the
38
effect of phenazines on producing strains,
our ability to predict how phenazines
might
shape the
39
composition of
environmental
microbial communities is poorly constrained; that phenazine
40
prevalence in
the
rhizosphere is predicted to increase in
arid
soils as the climate changes provides
41
an impetus for further study.
As a step towards
gaining a predictive understanding of phenazine
-
42
linked microbial ecology
,
we
document the effects of phenazines on diverse ba
cteria that were co
-
43
isolated from a wheat rhizosphere
and identify conditions and
phenotypes that correlate with
how a
44
strain will respond to phenazines
.
45
46
Introduction
47
Diverse microorganisms
produce
natural
antibiot
ics
that can kill or inhibit the growth
of
48
other microbes
(1, 2)
. Several
such
compounds have been commercialized
as antimicrobial drugs
49
for the treatment of infections
, beginning with penicillin in the 1940s
(3, 4)
.
Unfortunately, the
50
s
elective pressure
s
exerted by the
widespread
use of
antibiotics
in medicine and agricu
lture
have
51
led to
worrisome
increases in the prevalence of antimicrobial resistance
among human and animal
52
pathogens
over the past several decades
(5, 6)
.
Yet
while th
is
repercussion
of
human antibiotic use
53
has been well documented
,
comparatively
little is known about the
ecological
effects
of
54
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3
microbially
-
produced antibiotics in
natural environments
(7, 8)
.
R
ecent
studies have sug
gested that
55
natural antibiotics
may
serve a variety of
functions
for their producers
beyond
the
suppression of
56
competi
ng microbes
(9, 10)
,
including
nutrient acquisition
(11, 12)
,
conserv
ation of
energy
in the
57
absence of oxygen
(13, 14)
,
and
cel
l
-
cell signaling
(15, 16)
.
At the same time
,
toxicity
to
one or
58
more
microorganisms
is
by definition
a feature
of
these molecule
s
,
but
t
he
extent to which this
trait
59
shapes their influence on microbial communities
is
unclear
(2, 17, 18)
.
In addition, for
many if not
60
most natural antib
iotics,
the
determinants
and prevalence
of susceptibility or resistance
to their
61
toxicity
remain
unknown
or poorly characterized
(19)
.
T
hese gaps in knowledge
hinder our ability
62
to understand and predict the impacts of
these
metabolites
on microbial communities of interest
.
63
One
environmental context in which
natural
antibiotics are thought to be particularly
64
a
bundant
and ecologically relevant
is the rhizosphere
—
the narrow
plane of soil immediately
65
adjacent to plant roots
(20
–
22)
.
N
atural antibiotics
such as phenazines and 2,4
-
66
diacetylphloroglucinol
have been directly detected in
the rhizospheres of multiple crops
, including
67
wheat
, potato, and sugar beet
(22
–
24)
, and p
henazines have been shown to increase the fitness of
68
their producers
when competing
with other
microbes in
the rhizosphere
(25, 26)
.
P
roduction
of
69
these
molecules
has
also
been demonstrated to underpin
the ability of certain bacteria to control
70
fungal
crop diseases
(23, 26
–
29)
,
further
indicating that
natural antibiotics
can act as agents of
71
microbe
-
microbe antagonism in
the rhizosphere
.
As a result of
this
activi
ty, phenazine
-
producing
72
Pseudomonas
strains have received attention as potential biocontrol agents that
could serve as a
73
more sustainable alternative to traditional synthetic pesticides in agriculture
(19, 28)
.
However,
74
several challenges remain with respect to the
practical
application of
these strains,
incl
uding
75
inconsistent efficacy under field conditions
(20, 28)
,
limited understanding of the
mechanisms
and
76
evolutionary dynamics
of resistance to phenazines
(19, 30)
,
and
the possibility of
off
-
target effects
77
(28)
.
78
Im
portantly, with regard to the latter concern
,
p
henazines are toxic not only to fungi, but
79
also to some bacteria
(31
–
33)
.
Yet
while
the
utility of
phenazine
-
prod
ucing pseudomonads
for
80
biocontrol of fungal crop diseases
has been extensively investigated
,
the
potential
impact
of these
81
strains
on
non
-
target
bacterial residents of the rhizosphere
is less well understood
.
O
ne study
found
82
that
inoculation of
Pseudomonas
biocontrol strains shifted the rhizosphere community of maize
83
seedlings
,
pushing the ratio of
Gram
-
positive
to Gram
-
negative
bacteria
in favor of the latter
;
84
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4
however, this
analysis was
based on
profiling
colony growth rates and
whole
-
cell fatty
acids from
85
pooled cultured isolates
,
greatly
limiting the taxonomic resolution
and making it difficult to rule
86
out whether the Gram
-
negative biocontrol strains might themselves have directly contributed to the
87
shift
(34)
.
On the other hand, at least three studies found no
not
able
or consistent
effect
s
of
88
introduced
Pseudomonas
species
on the
rhizosphere
bacterial communities of wheat or potato
(35
–
89
37)
, albeit
the studies in wheat employed methods with limited discriminatory power
(
namely,
90
carbon
source
utilization profiling and terminal restriction fragment length polymorphism
)
. Given
91
these mixed results and the lack of fine
-
grained
spatial or taxonomic resolution in most studies on
92
this topic, whether phenazine
-
producing bacteria
actively
shape the surrounding rhizosphere
93
bacterial
community through antibiosis
, potentially
at the micro
n or millimeter
scale
,
remains an
94
open question.
95
In addition
to
the lack of clarity regarding
the
effects of phenazines on
rhizosphere
bacterial
96
communities, t
he
taxonomic
and physiological
correlates of
phenazine resistance
in bacteria
97
remain incompletely understood.
The toxicity of phenazines
is
generally
ascribed
to the generation
98
of reactive oxygen species
(ROS)
and interference with respiration
(33, 38
–
40)
. Previous studies
99
have suggested that efflux pump expression, cell permeability, oxidative stress responses, and the
100
composition of the respiratory electron transport chain can affect bacterial susceptibility to
101
phenaz
ines
(39
–
44)
.
In addition, a comparison of 14 bacterial strains
indicated
that Gram
-
negative
102
bacteria as a group may be more resistant to phenazine toxicity than Gram
-
positive bacteri
a
(33)
.
103
However, all of these studies
focused on
a specific phenazine, pyocyanin, that is
particularly toxic
104
(45)
and best known for its role as a virulence factor during infections
of humans and animals
(46,
105
47)
. W
hether the same observations hold true for more agriculturally
-
relevant phenazines such as
106
phenazine
-
1
-
carboxylic acid (PCA)
(22, 23, 27, 48)
is unknown
.
107
In this study, we set out to lay a foundation for addr
essing
unresolved
question
s
about the
108
ecological
impact
of phenazine toxicity
in the rhizosphere
by determining
the prevalence of
109
phenazine resistance among
bacteria
isolated from
a wheat field in the Inland Pacific Northwest
,
a
110
region
where phenazine production and the biocontrol potential of indigenous
Pseudomonas
111
species have been studied for decades
(22, 23, 25, 27)
.
W
e
designed
a culture
-
based
assay to
112
measure sensitivity to
PCA
,
which is
the best
-
studied and
one of the
most abundant phenazine
s
in
113
this environment
(22, 23, 48)
. We
also
performed full
-
length 16S rRNA gene sequencing of
our
114
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5
isolates in order to
assess the relationship between taxonomy and
PCA
resistance
. Finally, to assess
115
potential physiological
correlates of PCA resistance, we measured PCA reduction rates for a subset
116
of phenotypically
-
diverse strains and investigated the effects of broad
-
spectrum efflux pump
117
inhibitors on growth in the presence of PCA
.
118
Results
119
Taxonom
ic diversity
of culturable
bacteria from dryland wheat rhizosphere
s
and bulk soil
120
A total of
166
strains of bacteria were isolated from 12 soil samples collected from a wheat
121
field at Washington State University’s Lind Dryland Research Station
in early August 2019,
122
shortly after th
e wheat harvest
. The samples comprised 4 replicates each of wheat rhizosphere
123
(“
Wheat
”)
, bulk soil collected between planted rows
(“Between”)
, and bulk soil from a virgin
124
patch of uncultivated soil adjacent to the field
(“Virgin”
)
.
Full
-
length 16S rRNA gene sequencing
125
revealed that the isolates represented
2
4
genera across 4 phyla: Actinobacteria, Bacteroidetes,
126
Firmicutes, and Proteobacteria.
The vast majority of isolates from the bulk soil samples (both
127
Between and Virgin) were Act
inobacteria or Firmicutes
. I
n
Wheat
samples,
on the other hand,
the
128
combined
proportions of these
two
phyla were lower,
accounting
for 18
-
50%
and
10
-
25% of
129
isolates
respectively
,
while
Proteobacteria accounted for approximately 25
-
60% of isolates
130
depending
on the replicate, and
1
-
3 strains of
Bacteroidetes were also detected
in each
replicate
(5
-
131
12% of isolates)
(
Fig. 1
)
.
132
Taxonomic and spatial distribution of
PCA resistance phenotypes
133
We screened our isolates for resistance to PCA at both circumneutral
and acidic pH (7.3
134
and 5.1, respectively), because
the
toxicity of PCA to diverse organisms is known to vary
135
depending on pH
(49, 50)
, and because the bulk soil and rhizosp
here pH of wheat fields in the
136
Inland Pacific Northwest can vary by >3 units (pH 4.3
-
8.0) depending on geographic location and
137
fertilizer treatment status
(51)
. Th
e pH
-
dependency of PCA toxicity
has been attributed
to the
fact
138
that the
deprotonated form
of PCA
is negatively charged
(
Fig.
S1
)
;
the negative
charge on bacterial
139
cell walls and the outer membrane of Gram
-
negative bacteria (or the
negative membrane potential
140
of eukaryotic cells)
likely
hinder
s
uptake of this species
. T
he protonated form
of PCA
, on the other
141
hand,
is neutral and
presumably can
passively diffuse across cell membranes given the small size
142
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6
and hydrophobic nature of the
molecule
(50, 52)
.
The pKa of PCA is 4.24 at 25 °C; thus, at
pH 7,
143
only 0.
2
% of PCA in solution
is
protonated compared to 14.8
% at pH 5
,
typically
leading to
144
greater toxicity at the lower pH
(49)
.
In designing the screen, we additionally decided to focus on
145
comparing growth at a single concentration of PCA versus a PCA
-
free control condition, rather
146
than performing a minimum inhibitory concentration assay based on a dilution series, to enable
147
resour
ce
-
efficient comparison of a large number of strains.
We chose 100 μM as the
working
148
concentration of PCA as this is likely to be in a physiologically relevant range
based on
149
concentrations
measured
both in pure cultures and in the field
. In broth cultures
of biocontrol
150
strains of
Pseudomonas
, PCA accumulates to concentrations ranging from dozens to hundreds of
151
micromolar
(53, 54)
.
In
natural
wheat
rhizospheres
, PCA
has
been detected at
nanomolar
152
concentrations
(
22)
, but these bulk measurements almost certainly underestimate local
153
concentrations at the micron scale
given that
bacteria colonize the rhizosphere in a patchy manner
154
(23)
.
Notably,
PCA can accumulate in biofilms to concentrations 360
-
fold greater on a per volume
155
basis compared to broth cultures
(53)
, suggesting that local concentrations of PCA in the
156
rhizosphere, where biocontrol strains form
robust
biofilms
(55)
, may be orders of magnitude higher
157
than the
reported
bulk
values
.
158
The scree
n was performed
using 24
-
well plates and 0.1x
tryptic soy agar (TSA)
that was
159
either left unadjusted (pH 7.3
-
7.5
) or adjusted to pH 5.1.
Each isolate was spotted onto agar in
160
separate wells
to prevent crosstalk and antagonism between the strains
, and
image analysis
was
161
used
to derive quantitative information about the growth of each strain
over the course of 7 days
162
(with one timepoint per day)
.
Because several
strains among our
166
isolates appeared to be
163
duplicates
of each other based on 16S sequence
and colony morphology
, w
e restricted
the screen
to
164
1
08
strains
that
we judged likely to be unique (
Table
1
); wh
e
re
multiple strains appeared identical,
165
we chose
a
representative strain
.
We also included
30 strains obtained from public culture
166
collections
that
represent
ed species found among our
isolates (
Table
1
)
, in order to investigate the
167
extent to which PCA resistance phenotypes are consistent
within species
across different strains
168
isolated from different geographical locations
.
A
ccurate quantificatio
n of growth
was
not possible
169
in this screen
for
certain
strains that formed transparent colonies
,
produced
dark pigments
, or had a
170
tendency to turn mucoid and spread
(
Fig. S2
)
.
Nevertheless
,
this
assay
enabled us to derive
detailed
171
profiles of PCA
sensitivity and resistance
for the vast majority of our strains
.
Growth assays based
172
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