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Prevalence and correlates of phenazine resistance in culturable bacteria from a dryland wheat field
Elena K. Perry
1
, Dianne K. Newman
1,2
*
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
91125, USA
2
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125,
USA
Running title:
Phenazine resistance in wheat rhizosphere community
Contact information:
E
-
mail:
dkn@caltech.edu
Tel: 626
-
395
-
3543
SUPPLEMENTAL MATERIAL
Figure S1:
Structures of protonated and deprotonated oxidized PCA.
The negative charge on deprotonated PCA is thought to make PCA less toxic at higher pH by
decreasing uptake due to the negative charge on cell membranes.
Figure S2: Examples of image processing limitations for growth quantification.
Accurate quantification of growth and PCA susceptibility was hindered in cases where the strains
were transparent (e.g.
Microbacterium sp.
W4I4), grew to very low levels (e.g.
Neobacillus
drentensis
B4I4 in the presence of PCA), produced pigments (e.g.
Sphingomonas faeni
W4I17),
or had a tendency to turn mucoid and spread across the agar (
Chitinophaga ginsengisegetis
NBRC
109750). Such characteristics generally led to underestimation of growth. The displayed examples
were grown at pH 7.3.
Figure S3:
PCA sensitivity at pH 7.3 of different strains belonging to the same putative
species.
Dots represent the mean of two to four biological replicates for each strain (except for
S.
phaeochromogenes
B
-
1248 and
S. bobili
B
-
1338, for which only one replicate w
as available). Dots
are colored according to the source of the strain; dark blue represents wheat field isolates while
light blue represents strains procured from public culture collections. The high ratio for the culture
collection strain of
C. ginsengise
getis
reflects its tendency to become mucoid and spread while
growing agar, which PCA appeared to restrict (see Fig. S2).
A. ag.
=
Arthrobacter agilis
;
A. alb.
=
Agromyces albus
;
A. glo.
=
Arthrobacter globiformus
;
A. pas.
=
Arthrobacter pascens
;
A. ram
.
=
Agromyces ramosus
;
B. atr.
=
Bacillus atrophaeus
;
B. pum.
=
B. pumilus
;
C. gin.
=
Chitinophaga
ginsengisegetis
;
M. nia
. =
Massilia niastensis
;
M. pli.
=
Massilia plicata
;
N. dre.
=
Neobacillus
drentensis
;
N. nia.
=
Neobacillus niacini
;
P. alg.
=
Paenibacillus alginolyticus
;
P. bre.
=
Pseudomonas brenneri
;
P. eq.
=
Pseudarthrobacter equi
;
P. kyo.
=
Pedobacter kyongii
;
P. meg.
=
Priestia megaterium
;
P. mob.
=
Paenibacillus mobilis
;
P. phe.
=
Pseudarthrobacter
phenanthrenivorans
;
P. rhi.
=
Paenibaci
llus rhizoryzae
;
P. sic.
=
Pseudarthrobacter siccitolerans
;
P. sim.
=
Peribacillus simplex
;
S. afr.
=
Streptomyces africanus
;
S. aur.
=
Streptomyces
aurantiacus
;
S. bob.
=
Streptomyces bobili
;
S. can.
=
Streptomyces canus
(synonymous with
S.
ciscaucasicu
s
);
S. cya.
=
Streptomyces cyaneofuscatus
;
S. fae.
=
Sphingomonas faeni
;
S. lut.
=
Streptomyces luteogriseus
;
S. nov.
=
S. novaecaesareae
;
S. peu.
=
Streptomyces peucetius
;
S. pha.
=
Streptomyces phaeochromogenes
;
S. ris.
=
Streptomyces rishiriensis
;
V.
bor.
=
Variovorax
boronicumulans
;
V. par.
=
Variovorax paradoxus
.
Figure S4:
Growth of Actinobacteria strains over time with and without PCA.
Growth was quantified as described in the Methods. Solid lines represent the growth of spotted
cultures on P
CA
-
free agar, and dashed lines represent the growth of spotted cultures on agar
containing 100
μ
M PCA. Blue represents growth at pH 7.3 and pink represents growth at pH 5.1.
Data points are the mean of three technical replicates from a representative biolo
gical replicate for
each strain, and the shaded ribbon represents the standard deviation. Different lines of the same
color and line type (dashed vs. solid) represent separate biological replicates. For some biological
replicates, data were collected only
for a subset of days.
Figure S5:
Growth of Firmicutes strains over time with and without PCA.
Growth was quantified as described in the Methods. Solid lines represent the growth of spotted
cultures on PCA
-
free agar, and dashed lines represent the gro
wth of spotted cultures on agar
containing 100
μ
M PCA. Blue represents growth at pH 7.3 and pink represents growth at pH 5.1.
Data points are the mean of three technical replicates from a representative biological replicate for
each strain, and the shaded
ribbon represents the standard deviation. Different lines of the same
color and line type (dashed vs. solid) represent separate biological replicates. For some biological
replicates, data were collected only for a subset of days.
Figure S6:
Growth of
Bacteroidetes strains over time with and without PCA.
Growth was quantified as described in the Methods. Solid lines represent the growth of spotted
cultures on PCA
-
free agar, and dashed lines represent the growth of spotted cultures on agar
containing 10
0
μ
M PCA. Blue represents growth at pH 7.3 and pink represents growth at pH 5.1.
Data points are the mean of three technical replicates from a representative biological replicate for
each strain, and the shaded ribbon represents the standard deviation. Dif
ferent lines of the same
color and line type (dashed vs. solid) represent separate biological replicates. For some biological
replicates, data were collected only for a subset of days.
Figure S7:
Growth of Proteobacteria strains over time with and
without PCA.
Growth was quantified as described in the Methods. Solid lines represent the growth of spotted
cultures on PCA
-
free agar, and dashed lines represent the growth of spotted cultures on agar
containing 100
μ
M PCA. Blue represents growth at pH 7.3
and pink represents growth at pH 5.1.
Data points are the mean of three technical replicates from a representative biological replicate for
each strain, and the shaded ribbon represents the standard deviation. Different lines of the same
color and line ty
pe (dashed vs. solid) represent separate biological replicates. For some biological
replicates, data were collected only for a subset of days.
Figure S8: Derivation of PCA reduction rate from fluorescence readings over time.
This figure shows an exa
mple plot of fluorescence readings for reduced PCA over time for a single
strain. Linear regression was performed to find the line of best fit to the range of the data that was
judged by eye to be most linear, as depicted by the red line.
Figure S9:
Strain
-
specific synergistic toxicity of efflux pump inhibitors.
Growth curves of strains for which treatment with a combination of the efflux pump inhibitors
(EPI) reserpine and PA
β
N was toxic independently of PCA treatment. In all panels, data points
are
the mean of three biological replicates and the shaded ribbon represents the standard deviation.
The concentrations of reserpine and PA
β
N used for each strain can be found in Table S2.
Table S1:
16S rRNA gene sequences and BLAST hits for s
trains used in
this study.
This table
(see separate Excel file)
contains
16S rRNA gene sequences, closest BLAST hits, and
duplicate strain IDs for s
trains isolated in this study
.
A
n asterisk next to the percent identity to
the closest BLAST hit indicates that the match was on the basis of only t
he reverse sequencing
read of the 16S rRNA gene, due to multiple products in the forward read. Strains that are not
identified to species level had multiple equal top hits to different species in the NCBI 16S rRNA
gene sequence database.
Table S2:
Conc
entrations
of efflux pump inhibitors
used for different strains.
This table lists the concentrations of reserpine and PA
β
N used for each strain in the experiments
testing how efflux pump inhibitors (EPIs) affect susceptibility to PCA. The concentrations were
optimized
by determining the highest concentration of each EPI that, when administered
separately, did not affect growth in the absence of PCA.
Strain ID
Reserpine (
μ
g/mL)
PA
β
N (
μ
g/mL)
W1I9
10
10
W1I13
20
50
W1I16
20
25
W2I1
20
50
W2I8
20
50
W2I14
20
50
W3I1
10
25
W3I7
20
50
W3I9
20
50
W4I9
-
1
20
10
W4I11
20
50
W4I17
10
10
W4I20
5
5