Download Characterization of arsenic-resistant bacteria

236
Characterization of arsenic-resistant bacteria
from the rhizosphere of arsenic hyperaccumulator
Pteris vittata
Anhui Huang, Max Teplitski, Bala Rathinasabapathi, and Lena Ma
Abstract: Arsenic hyperaccumulator fern Pteris vittata L. produces large amounts of root exudates that are hypothesized
to solubilize arsenic and maintain a unique rhizosphere microbial community. Total heterotrophic counts on rich or defined
media supplemented with up to 400 mmol/L of arsenate showed a diverse arsenate-resistant microbial community from
the rhizosphere of P. vittata growing in arsenic-contaminated sites. Twelve bacterial isolates tolerating 400 mmol/L of arsenate in liquid culture were identified. Selected bacterial isolates belonging to different genera were tested for their resistance to osmotic and oxidative stresses. Results showed that growth was generally better under osmotic stress generated by
arsenic than under that generated by NaCl or PEG 6000, demonstrating that arsenic detoxification metabolism also crossprotected bacterial isolates from arsenic-induced osmotic stress. After 32 h of growth, all arsenate at 1 mmol/L was reduced to arsenite by strains Naxibacter sp. AH4, Mesorhizobium sp. AH5, and Pseudomonas sp. AH21, but arsenite at
1 mmol/L remained unchanged. Sensitivity to hydrogen peroxide was similar to that in broad-host pathogen Salmonella
enterica sv. Typhimurium wild type, except strain AH4. The results suggested that these arsenic-resistant bacteria are metabolically adapted to arsenic-induced osmotic or oxidative stresses in addition to the specific bacterial system to exclude
cellular arsenic. Both these adaptations contribute to the high arsenic resistance in the bacterial isolates.
Key words: arsenic-resistant bacteria, Pteris vittata, osmotic stress, oxidative stress, arsenate reduction.
Résumé : La fougère Pteris vittata L. qui accumule de hauts niveaux d’arsenic, produit une grande quantité d’exsudat racinaire que l’on croit capable de solubilizer l’arsenic et de maintenir une communauté microbienne unique dans la rhizosphère. Les comptes totaux d’hétérotrophes sur du milieu riche ou défini supplémenté à l’arsenate à des concentrations
allant jusqu’à 400 mmol/L ont montré la présence d’une communauté microbienne résistante à l’arsenate dans la rhizosphère de P. vittata cultivée sur des sites contaminés à l’arsenic. Douze isolats bactériens tolérant 400 mmol/L d’arsenate
en culture liquide ont été identifiés. Des isolats bactériens sélectionnés appartenant à différents genres ont été testés quant
à leur résistance à des stress osmotiques et oxydatifs. Les résultats ont montré l’existence d’une meilleure croissance en
général sous un stress osmotique généré par l’arsenic comparativement au chlorure de sodium ou au PEG 6000, démontrant que le métabolisme de détoxication de l’arsenic protège les isolats bactériens du stress osmotique causé par l’arsenic
de façon croisée. Après 32 h de croissance, tout l’arsenate à 1 mmol/L était réduit en arsénite par les souches Naxibacter
sp. AH4, Mesorhizobium sp. AH5 et Pseudomonas sp. AH21, mais l’arsenite à 1 mmol/L demeurait inchangé. Leur sensibilité au peroxyde d’hydrogène était similaire à celle du pathogène à large spectre Salmonella enterica sv. Thypimurium
sauvage, à l’exception de la souche AH4. Les résultats ont suggéré que ces bactéries résistantes à l’arsenic sont métaboliquement adaptées au stress osmotique induit par l’arsenic ou au stress oxydatif, en plus de posséder un système bactérien
d’exclusion d’arsenic cellulaire spécifique. Ces deux adaptations contribuent à la haute résistance à l’arsenic des isolats
bactériens.
Mots-clés : bactéries résistantes à l’arsenic, Pteris vittata, stress osmotique, stress oxydatif, réduction de l’arsenate.
[Traduit par la Rédaction]
Introduction
Arsenic hyperaccumulator Pteris vittata L. (Chinese brake
fern) tolerates up to 1500 mg/kg of arsenic in soil and rapidly accumulates up to 2.3% arsenic in its aboveground biomass from contaminated soils (Ma et al. 2001). Pteris vittata
produces more root exudates than non-hyperaccumulator
fern Nephrolepis exaltata; the root exudates are hypothe-
sized to help P. vittata mobilize arsenic directly from soils
(Tu et al. 2004). Besides directly affecting arsenic mobilization from soils, root exudates also help sustain a rhizosphere
microbial community, which indirectly mobilizes arsenic
from soil.
In a symbiosis between plant roots and rhizosphere microbes, plants produce carbon compounds, while microbes
Received 27 April 2009. Revision received 30 November 2009. Accepted 11 January 2010. Published on the NRC Research Press Web
site at cjm.nrc.ca on 18 March 2010.
A. Huang, M. Teplitski, and L. Ma.1 Soil and Water Science Department, University of Florida, Gainesville, FL 32601, USA.
B. Rathinasabapathi. Horticultural Science Department, University of Florida, Gainesville, FL 32601, USA.
1Corresponding
author (e-mail: [email protected]).
Can. J. Microbiol. 56: 236–246 (2010)
doi:10.1139/W10-005
Published by NRC Research Press
Huang et al.
help the plant to maintain nutrient recycling, provide resistance to microbial diseases, and tolerate toxic compounds
(Morgan et al. 2005). Microbial activities in the rhizosphere
of P. vittata impact the fate of arsenic by changing soil pH,
root exudation, and redox potential, thus playing a key
role in controlling arsenic bioavailability in soils (Fitz and
Wenzel 2002).
Besides interacting with plant roots, bacteria also play an
important role in arsenic biogeochemistry in the environment. They affect arsenic reduction and oxidation, methylation and demethylation, and sorption and desorption in soils.
As a result, bacteria have developed different detoxification
strategies to withstand the growth restriction under arsenic
stress. On one hand, bacterial ability to tolerate osmotic and
oxidative stress contributes to their arsenic resistance. Metabolism under hyperosmotic conditions or in a low nutrient
environment could cross-protect cells from other stresses
such as oxidative burst, heavy metal stress, and sodium hypochlorite (Pichereau et al. 2000). Similar to other heavy
metals, arsenic causes oxidative stress by inducing reactive
oxygen species (ROS). Therefore, it is not surprising to see
the correlation between arsenic resistance and hydrogen peroxide (H2O2) resistance (Liu et al. 2001). On the other hand,
arsenic-resistant bacteria usually have a specific genetic system, which is directly involved in arsenic transformation and
sequestration. One example is the reduction–detoxification
mechanism, which has been found in bacteria isolated from
arsenic-contaminated soils and mine tailings (Jones et al.
2000; Macur et al. 2001). The functional genes are encoded
by the arr operon in dissimilatory arsenic-reducing prokaryotes (DARP) (Rosen 2002) or by the ars operon in
arsenite-specific expulsion prokaryotes (ASEP) (Kuai et al.
2001).
The fact that the soil where arsenic hyperaccumulation
was first found in P. vittata has been contaminated for approximately 60 years (Komar 1999), coupled with the ability
of P. vittata to solubilize arsenic in the rhizosphere, provides
a good opportunity to discover microbes highly tolerant
to arsenic. The objectives of this study were to isolate
and characterize arsenic-resistant bacteria from arseniccontaminated soils where P. vittata naturally grows. The
ability of the bacterial isolates to tolerate osmotic and oxidative
stresses induced by different sources was studied, and functional genes in the specific genetic system were investigated.
Materials and methods
Bacterial isolation and enumeration by total
heterotrophic counting
Soil (bulk and rhizosphere) and plant samples were collected from 2 arsenic-contaminated sites where P. vittata
grows naturally in north central Florida in April 2007. The
first site (CCA site) was contaminated by chromated copper
arsenate (CCA), which was used for pressure-treating lumber from 1951 to 1962 (Komar et al. 1998). The second site
(RES site) was a residential site where CCA-treated timber
was used for stairs and decks. Rhizosphere soil, defined as
the soil attached to the roots, was removed from the roots
by gentle shaking. Bulk soil without plant influence was collected from the soil near the sampling plant at the same site.
Soil samples were kept at 4 8C for 20 days before bacterial
237
isolation. For arsenic analysis, soil and plant samples were
air dried (22 8C), mixed thoroughly, and digested by
HNO3–H2O2 (US Environmental Protection Agency Method
3051) on a heating block in triplicates (Environmental Express, Ventura, Calif.). Arsenic concentrations in the solutions were analyzed by a graphite furnace atomic absorption
spectrophotometer (240Z, Varian, Walnut Creek, Calif.).
To select arsenic-resistant bacteria, 5 soil samples from
the CCA and RES sites with different arsenic concentrations
were used. The CCA soils included 2 bulk soils (92.8 and
167 mg/kg of arsenic) and 1 rhizosphere soil (80.0 mg/kg
of arsenic), and the RES soils included one bulk soil
(12.9 mg/kg of arsenic) and one rhizosphere soil (28.2 mg/kg
of arsenic). Soil samples (0.3 g) were suspended in 10 mL
of sterilized water and vortexed vigorously.
Soil suspensions (2, 20, and 200 mL) were plated onto 2
agarose media: modified tryptone – yeast extract – glucose
(TYEG, 1/10 strength) or the National Botanical Research
Institute’s phosphate growth medium (NBRIP). The 1/10
strength TYEG medium contained 1 g/L of tryptone, 0.3 g/L
of yeast extract, 0.5 g/L of glucose, and 1% agarose (TYEG
medium used in this study was all at 1/10 strength). The
NBRIP agarose medium contained 5 g/L of glucose, 5 g/L
of MgCl 26H 2O, 0.25 g/L of MgSO47H2O, 0.2 g/L of KCl,
0.1 g/L of (NH4)2SO4, 10 mg/L of FeSO4, 14 mg/L of
nitric acid, and 1% agarose, which was spiked with inorganic or organic phosphorus sources (5 g of phosphate
rock, Ca 10(OH)2(PO4)6(F, Cl), or sodium phytic acid,
Na 12C 6H 6O 24P6). After 2 days, the 3 media with the highest
number of single colonies among 3 volumes’ worth of inocula were inoculated to plates containing 6 different levels of
arsenate (10, 50, 100, 200, 300, and 400 mmol/L of sodium
arsenate). The number of surviving bacterial colonies was
counted after 2 days of growth.
Single colonies from plates containing 400 mmol/L of arsenate were subcultured 3 times to obtain a pure culture.
Since arsenic availability in solid media might be limited,
bacteria isolated from solid plates containing 400 mmol/L
of arsenic were inoculated into TYEG liquid medium containing 400 mmol/L of arsenic to screen the most arsenictolerant bacteria. All bacterial incubations were conducted
at room temperature (22 8C).
Identification of arsenic-resistant bacteria
The 12 most arsenic-resistant bacteria were identified by
sequencing and analyzing the 16S rRNA gene. Bacterial genomic DNA was extracted using the standard phenol–
chloroform method followed by ethanol precipitation. The
16S rDNA fragments were PCR amplified using Taq polymerase and primers 8F 5’-AGAGTTTGATCCTGGCTCAG-3’
and 1489R 5’-TACCTTGTTACGACTTCA-3’ (Bruneel et
al. 2006). The PCR products were cloned using the TOPO
TA cloning kit (Invitrogen Inc., Carlsbad, Calif.) and sequenced at the Interdisciplinary Center for Biotechnology
Research (ICBR) Sequencing Facility at the University of
Florida (Gainseville, Fla.). Bacteria were identified based
on 16S rDNA sequences. Two-sequence comparisons among
the 12 isolates were analyzed by BLAST (Altschul et al.
1990).
Published by NRC Research Press
238
Bacterial growth under osmotic stress
Eight bacterial isolates tolerating 400 mmol/L of sodium
arsenate in liquid culture and belonging to 8 different genera
according to 16S rRNA gene sequences (Naxibacter sp.
AH4, Rhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, Bacillus sp.
AH22, Acinetobacter sp. AH23, and Caryophanon sp.
AH28) were used in this study. TYEG medium was used
for this study. The experiment was conducted under –1.5
MPa of osmotic stress using 3 different sources: 176 mmol/
L of sodium arsenate, 400 mmol/L of NaCl, and 26% polyethylene glycol 6000 (PEG 6000) (Sosa et al. 2005), with
the TYEG medium as a control. Bacteria were grown at
room temperature (22 8C) with constant shaking (150 r/
min). Bacterial growth was measured at 0, 5, 16, 25, 38,
and 44 h after inoculation by using a spectrophotometer
(Shimadzu BioSpec-Mini; Shimadzu Biotechn Inc., Columbia, Md.) at 600 nm (1 cm light-path length).
Bacterial growth inhibition under oxidative stress
The same 8 bacterial isolates were tested for their resistance to oxidative stress by measuring inhibition area for
H2O2 on TYEG agar. Salmonella enterica sv. Typhimurium
GS014 oxyR::Tn10 and Salmonella enterica sv. Typhimurium 14028 wild type were used as controls. For the assays,
50 mL aliquots of bacterial culture grown overnight were
plated in TYEG agar medium, and bacterial isolates were allowed to grow for 4 h. A sterile paper disc (6 mm in diameter) was placed inside each plate, to which 25 mL of 3% (v/v)
H2O2 was applied. Plates were incubated at 22 8C, and the
diameters of the inhibition zones were measured after 24
and 48 h. The experiments were carried out in triplicate.
Can. J. Microbiol. Vol. 56, 2010
Sigma-Aldrich, St. Louis, Mo.). Total soluble proteins were
extracted by vortexing with glass beads and centrifuging at
4 8C. Protein concentration was determined using the BioRad protein assay (Peterson 1979) (Bio-Rad Laboratories,
Hercules, Calif.). The arsenate reductase assay solution contained 150 mL of protein extract, 1 mL of reaction buffer,
10 mmol/L of sodium arsenate, 0.5 mmol/L of NADPH,
1 mmol/L of glutathione, 2 U of yeast glutathione reductase,
and 0.02 mmol/L of Escherichia coli glutaredoxin 2. Enzyme
activity was determined by measuring the absorption at
340 nm (Gladysheva et al. 1994; Shi et al. 1999).
Bacterial arsenate reductase gene
The primers used for arsC gene amplification were
5’-GCATTCTTTCCGAAGCCATGTTCAA-3’ (forward)
and 5’-AGCTCACGCTTGAGCTGGTCGCGAT-3’ (reverse),
which were designed based on Pseudomonas aeruginosa
PAO1 arsC gene. Pseudomonas aeruginosa PAO1 was used
as a positive control, and the PCR product from PAO1 was
confirmed by sequencing before being used used as a probe
for Southern hybridization. The PCR conditions used were
94 8C for 7 min and 29 cycles at 94 8C for 1 min; 56.8 8C
for 1 min; 72 8C for 1 min; and a final extension at 72 8C
for 10 min, with 0.2 mmol/L of primers. The Southern blot
method of Sambrook and Russell (2001) and the user manual of the DIG DNA Labeling and Detection Kit (Roche Diagnostics, Indianapolis, Ind.) were followed.
Results
Arsenic transformation by arsenic-resistant bacteria
Arsenic transformation by 3 arsenic-resistant bacterial isolates (Naxibacter sp. AH4, Mesorhizobium sp. AH5, and
Pseudomonas sp. AH21) and a non-arsenic resistant bacterium, Sinorhizobium meliloti MG32, were analyzed. The experiment included a control without bacteria and 3 replicates
per treatment. The growth medium contained either 1 mmol/L
of arsenite (AsIII) or arsenate (AsV). All bacterial cultures
had the same initial cell density (OD600 = 0.1). Cell cultures
were grown at room temperature with constant shaking at
150 r/min. Shake cultures were sampled after 4, 8, 16, and
32 h. Total arsenic was analyzed by a graphite furnace
atomic absorption spectrophotometer. Arsenic speciation
was performed using an arsenic speciation cartridge (Metal
Soft Center, Highland Park, N.J.) (Meng et al. 2001).
Diversity of the arsenic-resistant microbial community
Arsenic-resistant bacteria were isolated from 2 arseniccontaminated soils by growth in both rich medium, TYEG,
and defined medium, NBRIP. For both sites, the rhizosphere
of P. vittata sustained a substantially higher number of
arsenic-resistant bacteria compared to that found in the bulk
soils with similar arsenic concentrations (Tables 1 and 2).
Taking the CCA site for example, 43% of the colonies from
the rhizosphere soil grew in TYEG containing 400 mmol/L
of arsenate, while the numbers in the 2 bulk soils from the
same site were 17% and 3.4%, respectively (Table 1). In
the RES site, the percentage of surviving isolates in TYEG
plates containing 400 mmol/L of arsenate was 13% and
3.1% for rhizosphere soil and bulk soil, respectively. In total, 53 isolates, designated AH1–AH53, were obtained from
the plates containing 400 mmol/L of arsenate. Isolates AH1–
AH4 were also phosphate-solubilizing bacteria isolated in
the defined NBRIP medium.
Arsenate reductase assay
Cell culture of arsenic-resistant Pseudomonas sp. AH45
was grown in 50 mL of TYEG medium overnight. To induce the synthesis of proteins regulated by arsenic,
0.1 mmol/L of sodium arsenate was added to the medium,
which was incubated for 3 h. The medium was then incubated in a shaker (200 r/min) for another 4 h before the cells
were harvested by centrifugation. Cell pellets were resuspended in reaction buffer (10 mmol/L of Tris (pH 7.5),
1 mmol/L of Na2EDTA, 1 mmol/L of MgCl2, and 1 mmol/L
of DTT) (Anderson and Cook 2004) with 0.1 mmol/L of
proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF,
Twelve most arsenic-resistant bacterial isolates
Chemoheterotrophic bacteria tolerating 400 mmol/L of arsenate were isolated from rhizosphere soils. Because TYEG
is a nutrient-rich medium and NBRIP is a defined medium,
bacteria recovered through growth in NBRIP were able to
grow in TYEG medium. Therefore, for better comparison in
the growth study, TYEG medium was used for all isolates.
Considering that arsenic availability in agarose plates is
lower than that in liquid culture, 53 isolates were further
screened in TYEG liquid culture containing 400 mmol/L of
arsenate. Those able to grow were further separated and
identified based on 16S rRNA gene sequence analyses using
Published by NRC Research Press
Huang et al.
239
Table 1. Percentage of arsenate-resistant bacterial colonies recovered in
tryptone – yeast extract – glucose (TYEG) medium inoculated with bacteria
isolated from 2 arsenic-contaminated soils.a
Arsenate concentration in TYEG medium
(mmol/L)
Soilb
CCA B1
CCA B2
CCA R3
RES B4
RES R5
Soil arsenic
(mg/kg)
92.5
167
80.0
12.9
28.0
10
62.3
40.9
68.1
15.9
78.7
50
46.8
37.5
66.0
9.50
76.0
100
42.9
36.4
57.4
4.80
74.7
200
40.3
21.6
46.8
3.10
72.0
300
36.4
15.9
42.5
3.10
69.3
400
16.9
3.40
42.5
3.10
13.3
a
Percentage of surviving colonies as a proportion of c.a. 80 single colonies in each soil
tested.
b
CCA site, chromated copper arsenate contaminated site; RES site, residential
contaminated site; B, bulk soil; R, rhizosphere soil.
Table 2. Percentage of bacterial colonies surviving in the National Botanical
Research Institute’s phosphate growth medium (NBRIP, containing phosphate rock as phosphorus source) under different arsenate concentrations.a
Arsenate concentration in NBRIP medium
(mmol/L)
Soilb
CCA B1
CCA B2
CCA R3
RES B4
RES R5
Soil arsenic
(mg/kg)
92.5
167
80.0
12.9
28.0
10
87.2
98.9
54.3
0
53.7
50
38.5
73.3
0
0
0
100
25.6
14.4
20.0
0
24.1
200
15.4
4.40
20.0
0
0
300
5.1
0
0
0
0
400
0
0
0
0
0
a
Percentage of surviving colonies as a proportion of c.a. 80 single colonies in each soil
tested.
b
CCA site, chromated copper arsenate contaminated site; RES site, residential
contaminated site; B, bulk soil; R, rhizosphere soil.
Table 3. The 12 most arsenic-resistant bacteria identified from 2 arsenic-contaminated
soils.
Bacterial IDa
AH4
AH5
AH6
AH10
AH21
AH22
AH23
AH25
AH28
AH34
AH43
AH45
Soil arsenic
(mg/kg)
28.2
92.8
92.8
92.8
80.0
80.0
80.0
80.0
80.0
80.0
28.2
28.2
Soil typeb
R
B
B
B
R
R
R
R
R
R
R
R
Phylogenetic neighborc,
GenBank accession No.
Naxibacter sp., AM774589
Mesorhizobium sp., AM181745
Methylobacterium sp., AM910536
Enterobacter sp., Z96079
Pseudomonas sp., AF368755
Bacillus sp., FJ188297
Acinetobacter sp., AM945567
Pseudomonas sp., EF178450
Caryophanon sp., AF385535
Pseudomonas sp., EF178450
Pseudomonas sp., AF368755
Pseudomonas sp., EF178450
a
All bacteria were from the CCA site (chromated copper arsenate contaminated site) except for
AH4, AH43, and AH45, which were from the RES site (residential contaminated site).
b
R, rhizosphere soil; B, bulk soil.
c
16S rRNA gene; reference is selected from published data with similar cellular morphologies.
Published by NRC Research Press
240
BLAST (Table 3). The 12 most arsenic-resistant soil isolates
were tentatively identified as Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter
sp. AH10, Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, Pseudomonas sp. AH25, Caryophanon
sp. AH28, Pseudomonas sp. AH34, Pseudomonas sp. AH43,
and Pseudomonas sp. AH45. Their most probable phylogenetic neighbors, which were selected based on published
data and similar colony morphologies, showed that 2 groups
of isolates (AH21 and AH43; AH25, AH34, and AH45)
shared the same most probable neighbors. The results were
consistent with comparative analyses of 2 sequences at a
time via BLAST based on 600 informatic nucleotides of the
16S rRNA genes among the 12 isolates (data not shown).
The two-sequence comparisons via BLAST also showed
that, while 2 groups of isolates (AH21 and AH43; AH25,
AH34, and AH45) had 99% similarity; other isolates all had
similarity below 97%.
Bacterial growth under osmotic stress
To test whether the observed resistance to arsenate simply
resulted from a high resistance to osmotic stress, growth of
arsenic-resistant bacteria was tested under various osmotic
stresses. The growth characteristics of the 8 most arsenicresistant bacteria were studied under –1.5 MPa osmotic
stress induced by 400 mmol/L of NaCl, 26% (m/v) of PEG
6000 or 176 mmol/L of sodium arsenate. A laboratory
strain, P. fluorescens CHA0, which was pretested for its sensitivity to osmotic stress, was used as a control.
Except for the control CHA0, all bacteria tolerated –1.5 MPa
osmotic stress (Fig. 1). Figure 1 shows the data for bacterial
isolates AH4, AH10, and AH23. Bacterial isolates AH5,
AH6, AH21, AH22, and AH28 were comparable with
AH10 (data not shown). Compared to the growth curve in
TYEG, all 8 bacterial isolates tested had a longer lag phase
under osmotic stress generated by PEG 6000. All 8 isolates
grew better under NaCl-induced osmotic stress than under
that induced by PEG 6000. At the end of the experiment,
all bacterial isolates grew better under arsenic than under
NaCl or PEG 6000, except for AH23. The control P. fluorescens CHA0 tolerated –1.5 MPa of osmotic stress, but
was unable to grow in the presence of arsenic.
To compare bacterial ability to tolerate different stresses, a
resistance index (RI) was calculated. The RI was defined as
the ratio of the exponential growth rate in the medium with
stress to that in the control medium. The closer RI was to 1,
the smaller the arsenate toxicity was. The RI for arsenateinduced stress for the 8 isolates was the highest for AH28
(0.94) and the lowest for AH23 (0.20) (Fig. 2). The RI index
showed that, among the 3 sources of stress, bacterial growth
rate in arsenate was the highest, with a mean RI of 0.62 for
the 8 strains studied, followed by NaCl with mean of 0.37,
and PEG6000 was the lowest with mean of 0.29. AH23 was
unusual among the arsenic-resistant bacteria in that it exhibited greater tolerance to NaCl than to arsenate (Fig. 2).
Bacterial growth inhibition under oxidative stress
Arsenate also causes oxidative stress in bacteria. To evaluate bacterial oxidative stress tolerance, H2O2 inhibition of
the 8 isolates was compared to inhibition of Salmonella enterica sv. Typhimurium GS014 oxyR::Tn10 and Salmonella
Can. J. Microbiol. Vol. 56, 2010
enterica sv. Typhimurium 14028 wild type. These controls
were chosen because they were well characterized for their
responses to oxidative stress. The bacteria were tested for
antioxidative stress by measuring the H2O2 inhibition zone
relative to the controls. Under the experimental conditions,
the largest inhibition diameters occurred for AH4, with
47 mm after 24 h and 43 mm after 48 h (Fig. 3). The smallest inhibition was observed for AH28, with 22 mm after
24 h and 21 mm after 48 h. The results showed that, except
for AH4, the arsenic-resistant bacteria were as resistant as
Salmonella enterica sv. Typhimurium 14028 wild type to
H2O2. AH4 was as sensitive as the oxyR mutant (Fig. 3)
(p < 0.001), suggesting that the arsenic and NaCl tolerances
of AH4 are not related to its H2O2 tolerance.
In vitro arsenic transformation by arsenic-resistant
bacteria
Three arsenic-resistant bacteria, Naxibacter sp. AH4,
Mesorhizobium sp. AH5, and Pseudomonas sp. AH21, were
tested for their ability to transform arsenic during 32 h of
growth in TYEG medium, which was spiked with 1 mmol/L
of arsenate or arsenite (375 mg for 5 mL of cell culture). A
non-arsenic resistant strain, Sinorhizobium sp. MG32, which
was pretested for its resistance to different arsenic species,
was used as a control.
Speciation of arsenic in TYEG medium without bacterial
inoculation confirmed that both arsenate and arsenite were
stable in shaking TYEG medium. While there was neither
oxidation nor reduction in the medium with Sinorhizobium
sp. MG32, all 3 arsenic-resistant bacterial isolates reduced arsenate to arsenite efficiently in 32 h (Fig. 4a), with the halflife of arsenate being 22 h for AH4, 18 h for AH5, and 12 h
for AH21. Figure 4b shows the final OD at 600 nm after 32 h
of growth in the presence of arsenic. The growth of control
bacterium Sinorhizobium sp. MG32 was inhibited by
1 mmol/L of arsenite, whereas all 3 arsenic-resistant bacteria
tolerated both arsenate and arsenite.
Bacterial arsenate reductase and the functional gene
To test the presence of arsenate reductase in the bacterial
isolates, protein extract from AH45 was tested for activity in
vitro. The coupled assay experiment tested the activity of
glutaredoxin-dependent arsenate reductase (glutaredoxin 2
from E. coli), with NADPH/arsenate being the electron
donor/acceptor. Compared to the control without protein extracts or glutaredoxin, arsenate reduction by E. coli glutaredoxin 2 was observed, with absorption decreasing at a rate
of 0.0012 ABS/min (Fig. 5). Enzymatic reduction by soluble
protein from Pseudomonas sp. AH45 was detected from the
decrease of NADPH absorption at 340 nm, with absorption
decreasing at rate of 0.0045 ABS/min, translating to a specific activity of 3.58 nmolmin–1mg–1 (Gladysheva et al.
1994).
Although a portion of the arsC gene was successfully amplified using PCR, with the genomic DNA of the positive
control as the template, and the partial arsC gene confirmed
by sequencing, there was no positive PCR product amplified
from arsenic-resistant bacteria by the primers designed
based on the arsC gene of P. aeruginosa PAO1. Southern
hybridization with the arsC probe (partial gene sequence,
Published by NRC Research Press
Huang et al.
241
Fig. 1. Growth curve of Pseudomonas fluorescens CHA0 (A) and 3 arsenic-resistant bacteria, AH4, AH10, and AH23 (B, C, and D), in
tryptone – yeast extract – glucose (TYEG) medium under osmotic stress (–1.5 MPa) generated by 400 mmol/L of NaCl, 176 mmol/L of
sodium arsenate, or 26% (m/v) of PEG 6000. Data are means and standard errors of 3 replicates. Standard errors were not shown when they
were smaller than the depicted points.
A
0
-0.5
-1
-1.5
-2
-2.5
-3
0
-0.5
-1
-1.5
-2
TYEG
NaCl
PEG
As
-2.5
-3
0
5
10
15
20
25
30
35
40
0
0.5
C
Growth (log10 OD600 )
0.5
Growth (log10 OD600 )
B
0.5
Growth (log10 OD600 )
Growth (log10 OD600 )
0.5
0
-0.5
-1
-1.5
-2
-2.5
5
10
15
20
25
10
15
20
25
Time (h)
30
35
30
35
40
D
0
-0.5
-1
-1.5
-2
-2.5
-3
-3
0
5
10
15
20
25
Time (h)
30
35
0
40
5
40
Fig. 2. Resistance index (RI) of 8 arsenic-resistant bacteria (AH4–AH28) based on growth at –1.5 MPa of osmotic stress. RI was defined as
the ratio of the growth rate in medium with –1.5 MPa of osmotic stress to that in the control medium. Pseudomonas fluorescens CHA0 was
a laboratory control strain. Data are means and standard errors of 3 replicates.
1.2
CHA0
AH4
AH5
AH6
0.8
AH10
AH21
0.6
AH22
AH23
Resistance Index
1
0.4
AH28
0.2
0
PEG
NaCl
As
Stress
408 nt) from PAO1 also showed a negative result (data not
shown).
GenBank accession No. of the 16S rRNA gene sequences
of the 12 isolates are as follows: AH4: FJ621305; AH5:
FJ621306; AH6: FJ621307; AH10: FJ621308; AH21:
FJ621309; AH22: FJ621310; AH23: FJ621311; AH25:
FJ621311; AH28: FJ621313; AH34: FJ621314; AH43:
FJ621315; AH45: FJ621316.
Discussion
Previous studies have reported that microbial arsenic
resistance is associated with P. vittata. For example, the
bacterial isolate AsRB1 from the phyllosphere exhibited resistance to arsenate, arsenite, and antimony and reduced arsenate to arsenite (Rathinasabapathi et al. 2006). Plant
growth and arsenic uptake were increased by either inoculation with whole arbuscular mycorrhizal fungal community
(Al Agely et al. 2005) or with specific fungus Glomusmosseae or Gigasporamargarit (Trotta et al. 2006). The fungi
that were studied all tolerated the spiked arsenic (£100 mg/kg)
in the soil. However, so far there has been no study focused
on the rhizosphere bacterial community associated with P.
vittata.
Published by NRC Research Press
242
Can. J. Microbiol. Vol. 56, 2010
Fig. 3. Diameter of the growth inhibition zone of 8 arsenic-resistant
bacteria (AH4–AH28), Salmonella enterica sv. Typhimurium
oxyR::Tn10 (GS014), and S. enterica sv. Typhimurium wild type
(WT) in a glass disc containing 25 mL of 3% hydrogen peroxide.
The y-axis represents the diameter of the inhibition area on Petri
dishes. Data are means and standard errors of 3 replicates.
50
24 h
48 h
Inhibition(mm)
40
30
20
10
0
AH4
AH5
AH6 AH10 AH21 AH22 AH23 AH28 GS014 WT
Strain
Fig. 4. Arsenic transformation by arsenic-resistant bacteria AH4,
AH5, and AH21 and control strain Sinorhizobium sp. MG32 during
32 h of growth in tryptone – yeast extract – glucose (TYEG) medium spiked with 375 mg of arsenate (As(V)) or arsenite (As(III)).
(A) Arsenic speciation in the medium. The y-axis shows the
amount of the transformed arsenic (reduction on the left axis and
oxidation on the right axis). Solid and broken lines denote the
amount of arsenite and arsenate, respectively, that was transformed
by bacterial isolates. R, reduction test; O, oxidation test. (B)
Growth of AH4, AH5, AH21, and M32 after 32 h in TYEG. Medium spiked with 375 mg of arsenate (solid column) or arsenite
(open column). Points and columns are means and standard errors
of 3 replicates.
400
400
300
300
200
200
100
100
0
0
-100
-100
4
8
R AH4
R Control
O MG32
12
16
20
Time(h)
24
R AH5
O AH4
O Control
B
28
32
R AH21
O AH5
Reduction
R MG32
O AH21
Oxidation
1.2
OD600
AsV oxidation (µg)
AsIII reduction (µg)
A
0.8
0.4
0
AH4
AH5
Strain
AH21
MG32
Diversity of arsenic-resistant microbial community
Under the selective pressure that existed in the contaminated sites for several decades, the microbial community
was selected for its ability to overcome growth restrictions
through arsenic detoxification. The proportion of arsenicresistant bacteria was presented as a percentage of the
number of bacteria colonies tested (Table 1), which minimized the effects of nutrition and soil heterogeneity. The
fact that a higher percentage of arsenic-resistant bacteria
was from the rhizosphere soils than from the bulk soils indicated that, during plant arsenic accumulation, a greater
amount of bioavailable arsenic was mobilized from soil that
selected for the arsenic-resistant microbial community in the
rhizosphere.
Phosphorus plays a major role in arsenic detoxification by
P. vittata. Under arsenic stress, the tendency of P. vittata
to take up more phosphorus has been attributed to arsenicinduced phosphorus deficiency (Tu and Ma 2003).
Phosphorus availability in phytic acid or phosphate rock
was much lower than that provided in the TYEG medium.
The fact that the percentage of phosphorus-solubilizing bacteria surviving at a high arsenic concentration was much
lower than the percentage of isolates surviving in TYEG
(Table 2) implies that bacterial ability to solubilize phosphorus was not critical to its ability to tolerate arsenic. Alternatively, the bacteria failed to obtain sufficient phosphorus to
help detoxify the arsenic. Moreover, by supplying bacteria
with a high arsenic concentration in the NBRIP medium,
phosphorus-solubilizing bacteria might have taken up more
arsenic through the nonspecific phosphorus transporter and
thus suffered more arsenic toxicity.
A previous study described a similar level of arsenic resistance in Corynebacterium glutamicum, which tolerated
500 mmol/L of arsenate on tryptic soy agar plates (Ordóñez
et al. 2005). However, arsenic bioavailability in solid medium is reduced not only because of low arsenic activity,
but also because of the small bacterial surface area that directly contacts the arsenic. This study used 1/10 strength
TYEG medium to simulate the low nutrient environment in
soil, and more importantly, minimized the potential of a
nutrient-induced reduction in arsenic availability. Therefore,
bacterial arsenic resistance was tested in a liquid culture.
The difference in arsenic bioavailability between solid and
liquid cultures was obvious from our data. Among the 53
isolates surviving on solid 1% agarose plates containing
400 mmol/L of arsenate, only 12 (23%) grew in liquid culture with the same arsenic concentration. Therefore, those
isolates represent the most arsenic-resistant bacteria.
The 12 strains were identified by sequencing the 16S
rRNA gene. Two-sequence comparisons via BLAST showed
that 8 of the strains had a similarity <97% (data not shown),
which was the borderline similarity level to define a species
(Paster et al. 1991). The high similarity (99%) among isolates AH25, AH34, and AH45 suggested that the 3 isolates
might be from the same species or strain.
Bacterial growth under osmotic stress
In soils under –1.5 MPa of osmotic stress (the permanent
wilting point for many plants), the water film thickness surrounding soil matrices is estimated to be ~10 H2O molecules
(Halverson and Firestone 2000). Under the same osmotic
Published by NRC Research Press
Huang et al.
243
Fig. 5. Arsenate reductase assay of arsenic-resistant bacterium AH45, where enzyme activity was detected by measuring NADPH absorption
at 340 nm. The total protein concentration in the reaction was 101 mg/mL. Data are means and standard errors of 3 replicates.
1.5
ABS (340 nm)
1.45
1.4
1.35
1.3
1.25
Background
Grx no protein
AH45
1.2
0
1
2
3
4
stress in bacterial medium, bacteria are expected to desiccate
because of reduced water availability. This study showed
that arsenic-resistant bacteria grew in the presence of
400 mmol/L of arsenate in 1/10 strength TYEG medium, a
medium simulating the low nutrient conditions in soil. However, most of the isolates were unable to grow under similar
ionic strength induced by 2.4 mol/L of NaCl (data not
shown). Instead of using a lethal dose of NaCl, this growth
experiment used a lower concentration (400 mmol/L), which
the 8 arsenic-resistant isolates were able to tolerate.
The osmotic stresses induced by NaCl and PEG 6000
were both hyperosmotic, representing low water stress with
and without electrolytes. The reason for using 2 different osmotic stresses was that, in some cases, a decrease in water
potential was due primarily to reduction in water activity
(e.g., PEG 6000) rather than to an increased concentration
of permeating salts (e.g., NaCl). High molecular weight
PEG 6000 was too large to penetrate cell walls, lowering
medium water potential in the way that a dry soil would.
Bacteria tolerate osmotic stress through accumulation of cytoplasmic solutes (Csonka 1989). However, ionic and nonionic stresses such as NaCl and PEG 6000 had different
effects on compatible solute uptake in bacteria, which can
affect the maintenance of the electrolyte balance in the cytoplasm, thus affecting bacterial growth (Molenaar et al.
1993). Therefore, NaCl stress might be expected to have effects more similar to those of arsenate stress than those of
PEG 6000 stress.
Heavy metal detoxification by bacteria can result from
sorption to biomass, sequestration, and intracellular precipitation. A previous study showed that arsenate interfered with
the regulation of Rhizobium sp. VMA301 cell wall biosynthesis, which might result from arsenic binding to cell walls
(Mandal et al. 2008). Moreover, phytochelatins, heavy metal
binding peptides, are present in many prokaryotes, including
cyanobacterium Nostoc (Anabaena) sp. PCC 7120, Prochlorococcus marinus strain MIT9313 (BX572098), and
Anabaena variabilis ATCC29413 (Hirata et al. 2005). Similar to plants hyperaccumulating heavy metals, phytochelatins
in bacteria would confer a high degree of heavy metal tolerance. Transformation–immobilization is not only affected
by bacterial functional enzymes, but also by the reduction–
oxidation potential in the environment, two processes that
are greatly influenced by microbial communities (Gadd
2004). Therefore, arsenic detoxification can be the byproduct
5
6
Time (min)
7
8
9
10
of normal metabolism, making it difficult to search for the
functional genes. From this angle, cellular physiological
studies are important in understanding mechanisms of arsenic resistance.
Compared to the bacterial growth curve observed for
TYEG, all bacteria had a longer lag phase under osmotic
stress (Fig. 1). Arsenic-resistant bacteria generally grew better under NaCl-induced osmotic stress than under osmotic
stress induced by PEG 6000 (Fig. 1). The presence of ions
in the media might help to maintain membrane potential
during the accumulation of osmoprotectants. Under osmotic
stress induced by PEG 6000, a low availability of cations
and anions might have dissipated the cell membrane. With
the same amount of inoculation, the initial absorption at
600 nm in bacterial culture with PEG 6000 was smaller
than in TYEG medium with either NaCl or arsenate for all
strains. This suggested that bacterial cells underwent plasmolysis because of the osmotic pressure (low water activity
outside cellular membrane).
Most bacteria except AH23 grew better with arsenic than
with NaCl or PEG6000, indicating the existence of crossprotection between arsenic toxicity and arsenate-generated
osmotic stress. The control P. fluorescens CHA0 tolerated
–1.5 MPa of osmotic stress, but was unable to grow in the
presence of arsenic (Fig. 1). Therefore, cellular growth
might be under the influence of bacterial ability to detoxify
arsenic and bacterial resistance to low water potential induced by permeating solute.
In fact, multiple tolerances frequently occur in bacteria
living under osmotic stress or starvation conditions as a result of global reprogramming of gene expression. For example, Hartke et al. (1998) demonstrated that under complete
starvation conditions induced in tap water, Enterococcus
faecalis cells became more tolerant to heat, acid, and sodium hypochlorite stresses and were significantly more resistant to UV (245 nm) irradiation. A recent study of gene
profile changes responding to pH change in Shigella flexneri
by whole-genome microarrays differentiated the expression
of 307 genes. The genes included global regulators such as
the s factors and specific genes that increased acid production and energy generation (Cheng et al. 2007). Northern
blot comparison of Listeria monocytogenes mRNA between
growth in neutral and alkaline environments revealed that
expression of ~60 sB-regulated genes was significantly
increased, and the bacterial strain became more resistant to
Published by NRC Research Press
244
subsequent exposure to ethanol, alkaline, or osmotic stress
(Giotis et al. 2008). Those studies discerned that global regulons conferred cross-protection against multiple stresses such
as oxidative burst, osmotic stress, as well as heavy metal
toxicity.
The higher RI induced by NaCl than that induced by PEG
6000 (Fig. 2) confirmed the different effects of ionic and
nonionic osmotic stresses on bacterial cell growth. Figure 2
also shows that most bacteria had a higher tolerance to arsenic than to NaCl, supporting the idea that arsenic detoxification metabolism likely cross-protected bacteria from
arsenic-induced ionic osmotic stress. The higher resistance
of AH23 to NaCl than to arsenic suggested that it might
have a unique arsenic detoxification mechanism, and its tolerance to osmotic stress could contribute more to the arsenic
resistance of AH23.
The closer the arsenic RI is to 1, the lower the arsenic
toxicity. Though RI is affected by the medium and growth
conditions, it can still provide a relative comparison of bacterial ability to detoxify arsenic among isolates in different
studies. For example, compared to the control, the growth
of isolate AsRB1 from the phyllosphere of P. vittata was
about 20% in Luria–Bertani medium containing 10 mmol/L
of arsenate (Rathinasabapathi et al. 2006). Both the arsenic
concentration and the growth percentage were much lower
than in our study, in which the lowest RI for AH23 was
even higher than 0.20 (Fig. 3).
Bacterial growth inhibition under oxidative stress
The oxyR regulon controls oxidative burst by increasing
ROS scavenging activities and limiting H2O2 generation in
the respiratory chain (González-Flecha and Demple 2000).
Under exposure to heavy metals, glutathione and sulfhydryl
groups in proteins are depleted after complexing with metals, which leads to production of ROS such as superoxide
ion, H2O2, as well as hydroxyl radicals (Stohs and Bagchi
1995). With Tn10 inserted in the oxyR gene, the mutant
GS014 is very sensitive to oxidative stress.
Bacterial defenses against oxidative burst include antioxidant enzymes such as superoxide dismutase and catalase,
DNA repair systems following damage by ROS, scavenging
substrates, and competition with phagocytes for molecular
oxygen. Some of these defenses are regulated by global regulon oxyR (Hassett and Cohen 1989). The fact that the
arsenic-resistant bacteria were as resistant as Salmonella enterica sv. Typhimurium wild type was to H2O2 was consistent with a previous report that oxyR protected bacteria from
arsenic toxicity (Sukchawalit et al. 2005). Bacterial ability
to scavenge ROS during an oxidative burst might contribute
to their high arsenic resistance. The result was supported by
a previous study on P. aeruginosa, which showed that mutants lacking the arsenite membrane pump ArsB, superoxide
dismutase, catabolite repression control protein, or glutathione reductase were all more sensitive than the wild type
to arsenite (Parvatiyar et al. 2005).
Bacterium Naxibacter sp. AH4 was as sensitive as Salmonella enterica sv. Typhimurium GS014 to oxidative stress in
the inhibition test (Fig. 3). As a chemical analog of phosphate, arsenate is taken up by phosphate transporters in bacteria (Rosen 2002). With such high sensitivity to H2O2, AH4
needed to sequester arsenic by rapid efflux–immobilization
Can. J. Microbiol. Vol. 56, 2010
before arsenic-induced oxidative burst occurred inside cells
and prevented arsenic uptake. Therefore, lack of downstream oxidative detoxification ability in AH4 suggested a
different arsenic detoxification mechanism compared to that
in other isolates, probably a mechanism that involved suppressing the nonselective uptake system. Bacterium AH4
was also the only one able to grow in the presence of
2.4 mol/L of NaCl (data not shown). Further study is needed
to unveil the specific genes that confer the unique detoxification ability in this strain.
Bacterial arsenate reductase and functional gene
DARP and ASEP are 2 groups of known arsenate-reducing
bacteria (Plant et al. 2003). The transformation experiment
showed a high efficiency of arsenate reduction by the bacterial isolates, while no oxidation was observed (Fig. 4). The
experiment was carried out in rich medium under aerobic
conditions; therefore, those isolates tested might belong to
ASEP, and the functional genes were not constitutively
expressed according to the growth curve (Fig. 1). Under the
ASEP model, bacteria may take up arsenate through a
phosphate transporter, reduce it to arsenite using a specific
enzyme with the help of glutaredoxin, thioredoxin, or ferredoxin, and extrude the arsenite by membrane pumps (Rosen
2002). After being normalized on the basis of cell growth,
the RI indicates the comprehensive effects of the bacterial
ability to reduce and extrude arsenic in this model. The
high RI of bacterial isolates Mesorhizobium sp. AH5, Enterobacter sp. AH10, and Bacillus sp. AH28 reflects their enhanced ability to detoxify arsenic.
The chemical transformation results and enzymatic assay
proved the existence of a specific genetic system for transforming and detoxifying arsenic by arsenic-resistant bacteria
that belonged to DARP or ASEP. Although a positive control was sequenced and confirmed in the experiment cloning
the arsenate reductase gene in ASEP, there was no positive
PCR product amplified from 12 arsenic-resistant bacteria by
the primers designed based on the arsC gene sequence of P.
aeruginosa PAO1. The negative results were confirmed by
several degenerate primers designed based on reported ars
sequences that were phylogenetically close to our isolates.
A genomic DNA Southern blot using the arsC gene from P.
aeruginosa PAO1 as a probe also showed negative results
(data not shown), suggesting the probable sequence divergence from arsC known in P. aeruginosa PAO1.
Phylogenetic analysis of bacterial and archaeal arsC gene
sequences showed that arsC phylogeny is complex and is
likely the result of a number of evolutionary mechanisms;
in addition, the corresponding arsC gene sequences had little
or no sequence similarity and led to various homologies of
the corresponding arsC protein (60%–90%) (Jackson and
Dugas 2003; Branco et al. 2008). Therefore, our results suggested that either novel functional arsenate reductase genes
or low similarity between the arsenic-resistant bacteria and
the positive control resulted in difficulties in identifying the
target gene.
Previous studies showed that certain DARP species were
more sensitive to arsenic than others. For example, haloalkaliphile Bacillus selenitireducens grew well at 10 mmol/L of
arsenate, while Sulfurospirillum species was only able to
grow at 5 mmol/L of arsenate. This could be explained by a
Published by NRC Research Press
Huang et al.
generally weak arsenic-resistant system in Sulfurospirillum
species, in which an increase in the pH of the medium impeded the exit of arsenite from cells (Ahmann et al. 1994;
Switzer Blum et al. 1998). In our current study, while AH4
was sensitive to oxidative stress (Fig. 3), it tolerated high
levels of arsenic. AH23 had the highest NaCl resistance but
the lowest arsenic resistance (Fig. 2).
In summary, we isolated and characterized a group of
arsenic-resistant and (or) phosphorus-solubilizing bacteria
from the rhizosphere of the arsenic hyperaccumulator P. vittata. Twelve isolates tolerated 400 mmol/L of arsenate in
liquid culture, the highest arsenic resistance reported. Most
studies on arsenic-resistant bacteria have focused on specific
arsenic-resistant mechanisms, such as functional genes involved in detoxification mechanisms through transformation
and sequestration of arsenic species; however, little information is available on global metabolic adaptations such as the
osmotic stress between membranes when arsenic is stored
and (or) accumulated, oxidative stress generated during exposure to arsenic, and the impacts of those stresses on cell
growth. In this study, for the first time, we examined crosstolerances between the arsenic resistance of bacterial isolates
and their tolerance to stress induced by NaCl, PEG 6000,
and H2O2. While specific functional enzymes controlled the
uptake, reduction, and extrudation of arsenic, global metabolism counteracted arsenic-induced stresses, such as oxidative
burst or osmotic stress. Experimental results suggested that
bacterial ability to resist arsenic was due to bacterial efficiency in transforming and sequestering arsenic, scavenging
oxidative burst, and counteracting different osmotic stresses.
In addition, the bacterial isolates can potentially be used for
bioleaching to remove arsenic and phosphorus from soils.
These bacteria can also be used to increase the phosphorus
availability in agricultural soils as well as in arseniccontaminated soils to improve plants’ phosphorus nutrition.
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