RESEARCH LETTER
Indole toxicity involves the inhibition of adenosine
triphosphate production and protein folding in
Pseudomonas putida
Jisun Kim, Hyerim Hong, Aram Heo & Woojun Park
Department of Environmental Science and Ecological Engineering, Korea University, Seoul, Korea
Correspondence: Woojun Park, Department
of Environmental Science and Ecological
Engineering, Korea University, Seoul
136-713, Korea. Tel.: +82 82 3290 3067;
fax: +82 2 953 0737;
e-mail: [email protected]
Received 13 January 2013; revised 8 March
2013; accepted 15 March 2013. Final version
published online 22 April 2013.
DOI: 10.1111/1574-6968.12135
Editor: Peter Lund
MICROBIOLOGY LETTERS
Keywords
indole; microarray; NADH/NAD+ ratio; ATP;
protein folding; Pseudomonas.
Abstract
High concentrations of indole are known to be toxic to cells due to perturbations in membrane potential. Here, we report for the first time a transcriptome
analysis of a soil model bacterium, Pseudomonas putida KT2440, under indole
treatment. We demonstrated that 47 genes are differentially expressed, including 11 genes involved in the tricarboxylic acid cycle (TCA cycle) and 12 genes
involved in chaperone and protease functions (hslV, hslU, htpG, grpE, dnaK,
ibpA, groEL, groES, clpB, lon-1, lon-2, and hflk). Mutant analysis supported the
observation that protease genes including hslU are essential for the indole resistance of Pseudomonas strains. Subsequent biochemical analyses have shown that
indole increases the NADH/NAD+ ratio and decreases the adenosine triphosphate (ATP) concentration inside cells, due to membrane perturbation and
higher expression of TCA cycle genes in the presence of indole. This energy
reduction leads to a reduction in cell size and an enhancement of biofilm
formation in P. putida. The observed upregulation in many chaperones and
proteases led us to speculate that protein folding might be inhibited by indole
treatment. Interestingly, our in vitro protein-refolding assay using malate dehydrogenase with purified GroEL/GroES demonstrated that indole interferes with
protein folding. Taken together, our data provide new evidence that indole
causes toxicity to P. putida by inhibiting cellular energy production and
protein folding.
Introduction
Indole is widespread in nature, because various species of
bacteria produce considerable amounts of indole (Lee &
Lee, 2010). Indole is known to participate in various biological events such as biofilm formation (Lee et al., 2007),
pathogenicity (Chu et al., 2012), plasmid stabilization
(Field & Summers, 2012), spore formation (Stamm et al.,
2005), acid resistance (Hirakawa et al., 2010), and persister cell formation (Vega et al., 2012). Indole has
received great attention owing to the broad spectrum of
its effects on bacterial physiology. Recent interesting findings also suggest that indole plays an important role in
bacterial persister cell formation under antibiotic treatment (Vega et al., 2012). Interestingly, a tnaA mutation
(non-indole-producing) in Escherichia coli decreased persister cell formation (Vega et al., 2012). Cells of E. coli
FEMS Microbiol Lett 343 (2013) 89–99
that are lysed by antibiotics protect the majority of neighboring cells of the same kind by producing indole as a
defense signal molecule (Lee et al., 2010a). Stationaryphase cells of E. coli under nutrient-rich conditions could
also produce indole, which might be linked to stress
defense (Vega et al., 2012). It has been speculated that
the survival of bacterial population might be enhanced
for turning on antibiotic defense systems, such as multidrug efflux pumps and oxidative stress–protective
pathway by producing indole (Lee et al., 2010a).
Indole has been reported to function as an intercellular
signal in bacterial quorum sensing. There might be cross
talk between indole-based and acyl-homoserine lactone
(AHL)-based signaling, because a recent study demonstrated that indole-producing E. coli could inhibit quorum sensing–regulated virulence factors of Pseudomonas
aeruginosa (Chu et al., 2012). The E. coli LuxR homolog,
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90
SdiA, is considered to be related to this indole signaling
(Lee et al., 2007). In our previous study, the addition of
exogenous indole increased the expression of the ppoR
gene, an sdiA homolog, in Pseudomonas putida (Lee et al.,
2010b). Furthermore, increased biofilm formation and
reduced swimming motility were also observed upon
treatment with indole, but these effects were inhibited by
the expression of the cviI gene (encoding N-hexanoyl
homoserine lactone synthase, HHL) in P. putida. Thus,
we speculated that the PpoR-HHL complex inhibits the
effects of indole, and that indole may act as a signal via
PpoR (Lee et al., 2010b). However, there is still no direct
evidence showing that indole can bind to any SdiA
homolog, and it is unclear how indole and SdiA act
together in controlling many cellular processes. In contrast to all these observations, it has been recently argued
that SdiA might not respond to indole in E. coli and in the
Salmonella enterica serovar Typhimurium (Sabag-Daigle
et al., 2012). Thus, the detailed linkage between indole
signaling and bacterial quorum sensing remains unclear.
It has been reported that millimolar concentrations of
indole are present in human intestines (Sabag-Daigle et al.,
2012). The Mtr transporter might be involved in indole
import and export in E. coli (Yanofsky et al., 1991). However, indole has been recently known to be freely diffusible
across bacterial membranes (Pi~
nero-Fernandez et al.,
2011). Indole has been reported to act as a proton ionophore at high concentrations and inhibit cell division
(Chimerel et al., 2012). The electrochemical potential is
reduced when indole passes through the cytoplasmic membrane. Thus, high indole concentrations are toxic to bacterial cell growth, apart from the fact that indole functions as
a signal. Here, we report the first transcriptome data of
P. putida upon indole treatment and directly show that
exogenous indole can cause a reduction in adenosine triphosphate (ATP) production by membrane perturbation.
Furthermore, we demonstrated that indole inhibits the
protein-folding process inside cells, which might cause the
higher expression of many proteases in P. putida.
Materials and methods
Bacterial strains, culture conditions, and DNA
manipulation
The bacterial strains and plasmids utilized in this study
are shown in Table S1. Pseudomonas putida KT2440 and
P. aeruginosa PAO1 were grown at 30 and 37 °C in LB
and modified M9 media [Na2HPO47H2O (6.8 g L 1),
KH2PO4 (3 g L 1), NaCl (0.5 g L 1), NH4Cl (1 g L 1),
MgSO4 (2 mM), and CaCl2 (0.1 mM)] containing
10 mM glucose with aeration by shaking. When required,
antibiotics were added at the following concentrations:
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J. Kim et al.
kanamycin (50 lg mL 1) and ampicillin (100 lg mL 1).
Indole was added to the growth medium at various concentrations (0.25–5 mM). Growth was monitored by
measuring the OD600 nm of the cultures using a BioPhotometer (Eppendorf, Hamburg, Germany).
HslU mutant construction
The primers utilized in this study are listed in Table 1. A
589-bp fragment of the internal region of the hslU gene
was amplified using the PP_5001 (hlsU-SC)-F and
PP_5001 (hlsU-SC)-R primers. The polymerase chain
reaction (PCR) product for the hslU mutant was digested
with the EcoRI and KpnI restriction enzymes. Each fragment was subsequently inserted into a pVIK112 vector
via ligation. The constructed plasmids were then transformed into the E. coli S17-1 k pair. Conjugation was
performed using the biparental filter mating method with
the KT2440R strain.
Indole sensitivity test
To conduct sensitivity tests, all cells were collected at the
exponential phase (OD600 nm = 0.4) and then washed three
times with PBS. Cells were inoculated into PBS at
approximately 107 CFU mL 1 and serially diluted. The
diluted samples were spotted on LB plates containing different concentrations of indole. Then, cells were incubated at
30 °C for 24 h.
Microarray analysis
The cells were grown to the exponential phase (OD600 nm
= 0.4) at 30 °C with aeration. The cells were then treated
with 1 mM indole for 10 min. Total RNA was isolated
using the RNeasy Mini kit (Qiagen, Valencia, CA)
according to the manufacturer’s instructions. Following
procedure was conducted, as previously described (Lee
et al., 2010b) and detailed methods were located in Supporting Information. Genes that showed changes of more
than 1.5-fold (upregulated genes) and < 0.67-fold (downregulated genes) in at least two replicates were selected.
The microarray data were deposited in the National
Center for Biotechnology Information (NCBI) GEO site
(under accession number GSE 41254). Microarray data
were confirmed by quantitative reverse transcriptase PCR
for the 12 heat-shock and protease genes. Detailed experimental procedure described in Supporting Information.
Measurement of the NADH/NAD+ ratio
Nicotinamide adenine dinucleotide (NAD+) and NADH
concentrations were measured using the EnzyChromTM
FEMS Microbiol Lett 343 (2013) 89–99
91
Indole toxicity in Pseudomonas species
NAD+/NADH Assay kit (BioAssay Systems, Hayward, CA)
according to the manufacturer’s instructions. Exponentially growing cultures were treated with 1 mM indole,
1 mM tryptophan, and 1 mM indole acetic acid (IAA) for
30 min, and cells were collected for NADH and NAD+
extraction. Approximately 108 cells for each condition
were harvested after treatment and washed with cold PBS.
The samples were homogenized in either 100 lL NAD
extraction buffer for NAD determination, or 100 lL
NADH extraction buffer for NADH determination. Then,
extracts were heated at 60 °C for 5 min, and 20 lL assay
buffer and 100 lL of the corresponding extraction buffer
were added to neutralize the extracts. The samples were
centrifuged at 14 000 g for 5 min, and the supernatants
used for NAD+/NADH assays. The assay is based on a lactate
dehydrogenase cycling reaction, in which the formed
NADH reduces a formazan (3-(4,5-Dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide; MTT) reagent. The intensity of the reduced product color, measured at 565 nm, and
the change in absorbance during the reaction between
enzyme and substrates were calculated from the standard
curve. A microplate spectrophotometer (PowerWaveXS;
Bio-Tek, Winooski, VT) was used for measuring optical
density. NAD+ and NADH concentrations were normalized
by the amount of protein.
Determination of ATP concentrations
To measure intracellular ATP concentrations, the ENLITEN ATP assay system bioluminescence detection kit
(Promega, Madison, WI) was used in accordance with
the manufacturer’s instructions. To confirm ATP concentrations, exponentially growing cultures were treated with
0.5, 1, 2, and 3 mM indole for 30 min and cells were harvested for ATP extraction using 1% trichloroacetic acid
(TCA) buffer. Before carrying out the assay, samples in
TCA buffer were diluted fivefold with Tris-acetate buffer
to neutralize the extracts. The luminescence was measured
using a microtiter plate reader (VICTOR3; Bio-Rad). The
ATP concentration was expressed as molar concentration
per mg of protein.
Malate dehydrogenase denaturation, refolding,
and enzymatic assays
To demonstrate that indole affects protein folding, we
used mitochondrial malate dehydrogenase (mMDH) as a
substrate in the GroE-assisted protein-folding reaction.
The protein-folding assay was performed as described by
Diamant et al. (1995, 2001). mMDH (35 lM) was denatured at 25 °C for 2 h in 6 M guanidium HCl containing
40 mM MOPS buffer and 20 mM dithiothreitol and
incubated at 47 °C for 30 min to ensure complete
FEMS Microbiol Lett 343 (2013) 89–99
denaturation. The refolding reaction was initiated by 100fold dilution of the guanidium/heat-treated mMDH into
40 mM MOPS buffer, pH 7.5, 200 mM KCl, 4 mM
MgCl2 1 mM ATP, and 5 lL of a GroEL/GroES 1 : 1
mixture (25 mg mL 1; Sigma, St. Louis, MO) at 37 °C.
During the refolding process, 1 mM indole and ethanol
were added. The activity of mMDH was assayed at 25 °C
in 40 mM MOPS buffer, pH 7.5, 10 mM dithiothreitol,
0.5 mM oxaloacetate, and 0.28 mM NADH. Native
malate dehydrogenase (MDH) was used as an enzyme
activity control. The time-dependent oxidation of NADH
by MDH was monitored at 340 nm. The refolding activity was expressed as a relative percentage, taking the
activity of native MDH after 60 min of refolding to be
100%.
Biofilm formation
Biofilm formation of the KT2440 strain was analyzed as
described by Jackson et al. (2002). Bacterial cells were
inoculated into LB or modified M9 media with glucose
(10 mM) as a carbon source and incubated for 24 h at
30 °C. The cultures were then washed with PBS, and
106 CFU mL 1 of cells were inoculated into LB or M9
with 10 mM glucose. Bacterial cultures were grown in
96-well polystyrene microtiter plates (Costar, Washington,
DC) for 24 h (1 day), 48 h (2 days), and 72 h (3 days)
at 30 °C in static conditions. Biofilm formation was measured by staining the attached cells with crystal violet.
After staining, the attached cells were resuspended in
ethanol, and the absorbance was measured at 595 nm.
A microplate spectrophotometer (PowerWaveXS; BioTek) was used for measuring the optical density.
Microscopic observation
Indole was added to the cells at the exponential phase
(OD600 nm = 0.4), and the cells were then incubated for
2 h at 30 °C. One milliliter of cells was collected and
washed twice with PBS, and 2 lg mL 1 of 4′,6-diamidino2-phenylindole (DAPI) was added to the cell suspension,
followed by staining for 10 min by rocking at room
temperature. Excess DAPI was removed by washing, and
pellet was resuspended in PBS. Then, 5 lL of cells was
placed on a glass slide and observed.
Results
The effect of indole toxicity on the growth of
P. putida
Indole is produced by bacteria that possess tryptophanase
(TnaA), which catalyzes the synthesis of indole from
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92
J. Kim et al.
tryptophan. Pseudomonas putida KT2440 does not have
the tnaA gene in its genome. To confirm that KT2440
cells cannot produce indole, Kovacs reagent was used
(Gabriel & Gadebusch, 1956). In the presence of indoleproducing bacteria (Vibrio harveyi and E. coli O157:H7
Sakai), a cherry-red layer is formed on top of the medium following the addition of Kovacs reagent. We
observed that KT2440 cells cannot produce indole (data
not shown). The growth of KT2440 cells in LB media
with various indole concentrations (0–5 mM) was measured to determine the minimal concentration of indole
toxicity. Millimolar concentrations of indole have been
used in many previous studies (Lee & Lee, 2010), and
they reflect the actual concentrations in animal intestines
(Sabag-Daigle et al., 2012). No apparent toxicity of indole
was observed at 0.25, 0.5, and 1 mM indole, but the
growth rate of KT2440 cells sharply decreased at 2 mM
indole (Fig. 1a). The KT2440 cells did not grow in indole
concentrations of 3 mM or greater during 24-h incubation. The effect of ethanol as a solvent for indole was
evaluated; no difference was observed at 3 mM indole
(data not shown). We observed some degradation of
indole following long-term (5 days) incubation of
KT2440 cells (Fig. S1). However, the cells could not grow
well in LB containing 3 mM indole (OD600 nm, < 0.1),
which suggested indole inhibition cannot be overcome by
longer-term growth. KT2440 cells grown on agar plates
with increasing concentrations of indole were also sensitive to indole (Fig. 1b), and surviving colonies on indolecontaining agar plates were smaller than in the control.
Thus, high concentration of indole exhibits toxic effects
on the growth of KT2440 cells in both liquid and solid
media.
Transcriptome analysis of Pseudomonas putida
under indole treatment
Microarray analysis of KT2440 cells was conducted to
investigate their response to indole. Our duplicate microarray data demonstrated that a total of 47 genes in
(a)
(b)
Fig. 1. The effect of indole toxicity on the
growth of Pseudomonas putida KT2440.
(a) Growth curve of KT2440 in LB media
containing various concentrations of indole
(0–5 mM). Cells were grown at 30 °C, and
the growth at each condition was monitored
by measuring the OD600 nm of cultures. EtOH
control indicates treatment with ethanol
instead of indole, corresponding to 5 mM
indole. (b) Indole sensitivity test. When the
cells reached the exponential phase (OD600 nm
= 0.5), they were diluted serially, spotted onto
individual plates.
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FEMS Microbiol Lett 343 (2013) 89–99
93
Indole toxicity in Pseudomonas species
indole-treated KT2440 cells were either upregulated more
than 1.5-fold or downregulated < 0.67-fold, compared
with control (Table S2). The highest gene expression
among all upregulated genes was found in trpB (PP_0083,
tryptophan synthase beta subunit; 3.52-fold upregulated),
which is involved the synthesis of tryptophan from
indole. Interestingly, 11 genes involved in the TCA cycle
(kgdAB, PP_4012, more than twofold; sdhABD, sucCD,
gltA, lpdG, and PP_0897, more than 1.5-fold and less
than twofold) and 12 heat-shock or protease genes (hslV,
hslU, htpG, grpE, dnaK, ibpA, groEL, groES, clpB, lon-1,
lon-2, and hflk) were upregulated upon indole treatment.
Our microarray data were confirmed by qRT-PCR for the
12 heat-shock and protease genes (Fig. 2a). To uncover
the functions of these upregulated genes during indole
treatment, P. aeruginosa PAO1 mutants from the
Washington University Genome Center were obtained
and their sensitivities to indole were checked. The sensitivity test was conducted on the PAO1 mutants on plates
containing different concentrations of indole (0, 1, 3, and
5 mM; 0 and 3 mM indole-containing plates are shown
in Fig. 2b). All the mutants were unable to grow in the
presence of 5 mM indole. Among these mutants, the
PA0036 (trpB, tryptophan synthase subunit beta), PA0796
(prpB, carboxyphosphonoenolpyruvate phosphonomutase), PA5053 (hslV, ATP-dependent protease peptidase
subunit), and PA5054 (hslU, ATP-dependent protease
ATP-binding subunit) strains were very sensitive to
3 mM indole, compared with wild type. PrpB catalyzes
the synthesis of a phosphonate (C-P) bond (Hidaka et al.,
1990) and has been shown to be required for the catabolism of propionate in Salmonella typhimurium along with
prpBCDE operon (Horswill & Escalante-Semerena, 1997).
However, very little is known about regulation and
expression of the PrpB gene. Our data suggest that these
indole-sensitive gene products play an important role in
defense against indole toxicity, although other gene
functions are unknown.
The absence of HslU promotes indole
sensitivity and cell filamentation
To ascertain the effects of the proteases identified from
the indole microarray and our sensitivity tests, the hslU
(a)
(b)
Fig. 2. (a) Quantitative reverse transcriptase
PCR analysis of KT2440 cells treated with
1 mM indole or no indole. Relative expression
via qRT-PCR (black bar) of 12 genes involving
heat-shock proteins or proteases in KT2440 is
consistent with microarray data (gray bar).
(b) The sensitivity test was conducted on
plates with various indole concentrations (1, 3,
and 5 mM) in Pseudomonas aeruginosa PAO1
mutants. All mutant cells were selected based
on the Pseudomonas putida microarray results.
Serial dilutions of all mutants were spotted
and grown at 37 °C.
FEMS Microbiol Lett 343 (2013) 89–99
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94
J. Kim et al.
mutant of KT2440 was constructed. In E. coli, the HslVU
protease is an ATP-dependent protease (Missiakas et al.,
1996). HslV is known to have peptidase activity and HslU
has ATPase activity. Both HslV and HslU participate
together in the degradation of abnormal polypeptides
(Missiakas et al., 1996). Disruption of HslU decreased the
growth of KT2440 cells in LB media (Fig. 3a). The
growth rates (h 1) of wild-type and hslU mutant KT2440
cells were 1.47 0.05 and 0.98 0.03, respectively.
Unlike wild-type KT2440 cells, the hslU mutant showed
growth retardation upon the addition of 1 mM indole (at
time points between 8 and 10 h; Fig. 3a). Furthermore,
the growth of hslU mutant was severely inhibited by high
concentration of indole (> 2 mM; Fig. S2). When both
the wild-type and the hslU mutant cells were incubated
on indole-containing agar plates, the hslU mutant was
sensitive to indole, compared with the wild type (Fig. 3b).
Our data showed that the hslU mutant of KT2440 cells is
more sensitive to indole in both liquid and solid media.
To confirm that HslU functions as a heat-shock protein,
the indole sensitivity test was performed at temperatures
higher (35 °C, 37 °C) than the optimum temperature
(30 °C). However, no dramatic difference was observed
(data not shown). Indole has recently been shown to
inhibit E. coli cell division by acting as a proton ionophore (Chant & Summers, 2007; Chimerel et al., 2012;
(a)
(b)
(c)
Fig. 3. Influence of indole on growth and cell size in wild-type and hslU mutant KT2440 cells. (a) Growth curve of wild-type KT2440 and the
hlsU mutant. To compare the change in growth patterns of wild-type KT2440 and the hslU mutant, 1 mM indole was added at the exponential
phase. (b) Indole sensitivity test comparison between wild-type and hslU mutant cells. (c) Microscopic observation (scale bar, 10 lm) of indoletreated wild-type and hslU mutant cells. The average cell size was measured from 30 cells. The error bar indicates standard deviation; scale bar
indicates 10 lm.
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FEMS Microbiol Lett 343 (2013) 89–99
95
Indole toxicity in Pseudomonas species
Field & Summers, 2012). We investigated the effect of
indole on the cell division of KT2440 cells. DAPI staining
for microscopic observation showed that indole results in
smaller cell sizes in the wild type (Fig. 3c). The HslVU
protease degrades SulA (Wu et al., 1999), which is known
to inhibit FtsZ ring formation. FtsZ has GTPase activity
that is important for Z-ring formation during cell division (Bi & Lutkenhaus, 1993). Thus, we speculated that
the absence of HslU might increase cell filamentation due
to the lack of SulA degradation. The hslU mutant cells
were elongated approximately twofold compared with
wild-type cells (Fig. 3c), although cell size reduction by
indole was not changed in the mutant, suggesting that
HslU alone does not directly control cell size reduction
by indole.
(a)
(b)
Indole perturbs energy pools in bacterial cells
The expression of genes involved in the TCA cycle led us to
examine the NADH/NAD+ ratio and ATP levels under
indole treatment. The NADH/NAD+ ratio plays a critical
role in the regulation of cell metabolism (Ebert et al.,
2011). We observed higher NADH/NAD+ ratio by increasing the concentration of indole (Fig. 4a). The NADH/
NAD+ ratio was 3, 50-fold higher with 2 and 3 mM indole,
respectively. However, there was no change upon treatment
with the structurally similar compounds tryptophan and
indole-3-acetic acid. Furthermore, the NADH/NAD+ ratio
also showed no alteration in the presence of other amino
acids (1 mM) such as glycine, cysteine, and phenylalanine
(data not shown). Indole can interact with lipid membranes (Gaede et al., 2005; Mitchell, 2009). The increase in
the NADH/NAD+ ratio might be due to the inhibition of
electron transport in the membrane by indole, which lowers ATP production inside the cells.
Internal levels of ATP were reduced in the presence of
indole (Fig. 4b). We speculate that increased ATP consumption by many ATP-dependent proteases (Table S2,
Fig. 2a) and inhibition of ATP synthesis by reduction in
membrane potentials (Fig. 4) occurred in the presence of
a high concentration of indole. There was no significant
change in ATP level in the presence of lower concentrations of indole (< 2 mM, data not shown).
In our previous study, the addition of indole promoted biofilm formation in KT2440 cells (Lee et al.,
2010b). Furthermore, others have shown that indole
increases biofilm formation in other pseudomonads
including P. aeruginosa and P. fluorescens (Lee et al.,
2007). In contrast, biofilm formation and cell adhesion
decreased in E. coli upon treatment with indole (Domka
et al., 2006; Bansal et al., 2007; Lee & Lee, 2010; Lee
et al., 2010b), suggesting that indole acts differently in
different bacterial species. Biofilm formation in KT2440
FEMS Microbiol Lett 343 (2013) 89–99
Fig. 4. Indole affects the energy complexes of Pseudomonas putida
KT2440. (a) The effect of indole on the NADH/NAD+ ratio. The
NADH/NAD+ ratio was determined by measuring the absorbance at
565 nm, and each concentration was calculated from the standard
curve. NAD+ and NADH concentrations were normalized to the
amount of protein (mg). (b) Influence of indole on ATP generation. To
determine ATP concentrations, exponentially growing cultures were
treated with 0 and 3 mM indole for 30 min. The ATP concentration
was expressed as molar concentration per mg of protein. All data
show the average of four replicates, and one standard deviation is
shown.
cells increased in the presence of indole in both rich and
minimal media (Fig. S3A,B). The changes in NADH/
NAD+ levels and ATP concentrations along with the
small cell and colony size imply that cells experienced
metabolic burdens or stresses. Thus, high indole concentration stresses bacterial cells and affects their adherence
to some surfaces.
Indole inhibits protein folding
Indole is a structurally simple and hydrophobic molecule.
It has a bicyclic structure, consisting of a benzene ring
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96
fused to a nitrogen-containing pyrrole ring. It is generally
accepted that proteins are organized with a hydrophobic
core and a hydrophilic exterior (Meirovitch & Scheraga,
1981). Our microarray data, which revealed that many
protease genes are highly upregulated, led us to speculate
that indole can intercalate into proteins during the process of folding, which could interfere with hydrophobic
interactions that are needed for folding. Unfolded proteins are known to induce the bacterial heat-shock
response (Neidhardt et al., 1984; Goff & Goldberg, 1985;
VanBogelen et al., 1987).
To test whether indole had any effect on chaperonemediated protein folding, a MDH refolding assay was
performed with the GroEL/GroES chaperones. The data
showed an increase in MDH refolding with time
(Fig. 5a). After 60 min of MDH refolding in the presence
of the GroEL/GroES chaperones, up to 70% of native
MDH activity was regained (Fig. 5b). Although GroEL
and GroES supported MDH refolding in the presence of
indole, refolding rates were lower than those seen with
only chaperones (Fig. 5a). The same volume of ethanol
corresponding to that of indole treatment did not affect
mMDH refolding (data not shown). In our microarray
data, 12 heat-shock or protease genes including GroEL
and GroES were upregulated by indole. Our transcriptome and protein-refolding assay supported our hypothesis that indole might interfere with protein folding. It is
worth noting that another hypothesis, fully consistent
with the array data and with the experiments on MDH
refolding, would be that indole directly inhibits the action
of GroEL and GroES.
Discussion
Studies of the effect of indole on bacterial cells have been
focused on E. coli, which produces indole. High temperature (50 °C), low pH, and the presence of the antibiotics
affect indole production in E. coli (Han et al., 2011). Thus,
indole production may be affected by the surrounding
environment, and this will in turn affect bacterial communities and their life. The impact of exogenous indole on the
physiological characteristics of non-indole-producing bacteria has been poorly explored. Indole production during
mixed-culture growth between P. aeruginosa and E. coli
prevented pyocyanin production and quorum sensing–regulated virulence factors in P. aeruginosa (Chu et al., 2012).
Indole could be degraded by various oxygenases from bacteria, fungi, and plants (Lee & Lee, 2010) and quickly
reduced in P. aeruginosa (Lee et al., 2009). Degradation
and incorporation of indole into tryptophan biosynthesis
may be the first mechanism to prevent stress caused by
indole. Our microarray data also showed the highest
expression of trpB genes in the presence of indole
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J. Kim et al.
Fig. 5. Indole inhibits protein folding. Detailed procedures for the
protein-folding assay were described in ‘Materials and methods’.
Denatured mMDH was refolded in ATP and GroEL/GroES 1 : 1
mixture at 37 °C. One millimolar indole was added during this
refolding process. The activity of mMDH was assayed at 25 °C, and
the oxidation of NADH by mMDH was monitored at 340 nm. (a)
Time-dependent protein folding. (b) The activity of refolded mMDH
after 60 min of refolding, expressed as a percentage of native MDH
control activity.
(Table S2). If non-indole-producing bacteria cannot
degrade or use excess indole, defense mechanisms such as
molecular chaperones and proteases are turned on. Our
data demonstrate that indole increases biofilm formation
and reduces cell size in P. putida. These results are inconsistent with previously well-known studies using E. coli
strains. We speculate that the roles of indole may differ
between Pseudomonas species and indole-producing bacteria. Among indole derivatives, indole-3-acetic acid (IAA)
has been extensively studied in terms of a symbiotic relationship with soil bacteria and plants. When E. coli K-12
was treated with IAA, the majority of genes encoding cell
envelope components and adaptation-related proteins were
FEMS Microbiol Lett 343 (2013) 89–99
97
Indole toxicity in Pseudomonas species
differentially expressed (Bianco et al., 2006a, b). IAAtreated cells had enhanced biofilm formation due to an
increased production of lipopolysaccharide (LPS) and
exopolysaccharide. In addition, IAA induced higher levels
of the heat-shock protein DnaK, as also seen in our data.
In E. coli, sigma factor, r32, encoded by the rpoH gene,
positively regulates the induction of heat-shock proteins
such as DnaK, DnaJ and GrpE, and GroEL/ES (Kobayashi
et al., 2011). These heat-shock proteins have been extensively studied in other bacterial cells. Many heat-shock
proteins, which were highly expressed at high temperatures, include hslU, hslV, htpG, grpE, dnaK, ibpA, clpB, lon,
and hflK in E. coli (Richmond et al., 1999); htpG, grpE,
dnaK, groEL, and groES in Bacillus subtilis (Helmann et al.,
2001); dnaK, groEL, groES, clpB, and lon in Mycoplasma
pneumonia (Weiner et al., 2003); and ibpA, groEL, and
groES in Yersinia pestis (Motin et al., 2004). Many of
these heat-shock proteins were highly expressed in our
microarray analysis with indole (Table. S2) and are
reported to be expressed under various environmental
stresses, such as nutrient starvation, exposure to pollutants,
and changes in pH or osmolarity (Koide et al., 2006). In
case of P. putida KT2440, toluene or o-xylene treatment
induced those genes, such as groES, groEL, lon-1, lon-2,
ibpA, htpG, danK, grpE, hslV, and hslU (DomınguezCuevas et al., 2006).
Although indole has been reported to cause oxidative
stress (Vega et al., 2012), our microarray data did not
show any change in oxidative stress–related genes. To
investigate the degree of cellular oxidative stress in the
presence of indole, superoxide production was measured
using the nitroblue tetrazolium (NBT) assay. Exponentially growing cells were treated with different concentrations of indole and ethanol for 30 min. Concentrations of
1–3 mM indole did not cause oxidative stress (data not
shown). Therefore, indole probably stresses bacterial cells
by disrupting the electron transport system or energy
generation and protein folding, rather than by producing
oxidative stress, as seen in our study.
In this study, we have shown that indole, at concentrations above a certain level that appears to be toxic to
non-indole-producing Pseudomonas strains, altered the
expression of many genes in three functional categories:
(1) proteases; (2) molecular chaperones; and (3) TCA
cycle enzymes. These gene products play important roles
in conditions of indole-induced stress. The expression of
these genes might cause phenotypic and physiological
changes such as in cell morphology, biofilm formation,
the NADH/NAD+ ratio, and ATP concentrations. Our
data provide evidence that indole, which has been
recently spotlighted as a beneficial signal molecule for
E. coli, also exerts deleterious effects on non-indoleproducing Pseudomonas strains.
FEMS Microbiol Lett 343 (2013) 89–99
Acknowledgement
This work was supported by a grant (2012-0005277) from
the MEST/NRF program.
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Indole toxicity in Pseudomonas species
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Data S1. Materials and methods.
Fig. S1. Measurement of indole degradation via HPLC
analysis.
FEMS Microbiol Lett 343 (2013) 89–99
99
Fig. S2. Influence of indole on growth of wild-type and
the hslU mutant cells.
Fig. S3. Effects of indole on biofilm formation.
Table S1. Bacterial stains, plasmids and primers used in
this study.
Table S2. Genes differentially regulated upon treatment
with 1 mM indole in P. putida KT2440 cells compared to
untreated cells at the exponential phase.
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Published by Blackwell Publishing Ltd. All rights reserved