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Isolation and molecular characterization of arsenitetolerant Alishewanella sp. GIDC-5 originated fromindustrial effluentsRaina Jain a , Sanjay Jha a , Hemanta Adhikary b , Prasant Kumar b , Vipul Parekh a , AnamikaJha c , Mahesh K. Mahatma & G. Naresh Kumar ba Department of Plant Molecular Biology and Biotechnology, ASPEE College of Horticultureand Forestry , Navsari Agricultural University , Navsari , Gujarat , 396456 , Indiab Department of Biochemistry, Faculty of Science , The Maharaja Sayajirao University ofBaroda , Vadodara , Gujarat , 390002 , Indiac Department of Biotechnology, Ashok & Rita Patel Institute of Integrated Study & Researchin Biotechnology & Allied Sciences , New Vidyanagar , Gujarat , 388121 , IndiaAccepted author version posted online: 09 Jul 2013.

To cite this article: Geomicrobiology Journal (2013): Isolation and molecular characterization of arsenite tolerantAlishewanella sp. GIDC-5 originated from industrial effluents, Geomicrobiology Journal, DOI: 10.1080/01490451.2013.811317

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Original Article

Title: Isolation and molecular characterization of arsenite tolerant Alishewanella sp.

GIDC-5 originated from industrial effluents

Authors: Raina Jain1, Sanjay Jha1*, Hemanta Adhikary2, Prasant Kumar2, Vipul Parekh1,

Anamika Jha3, Mahesh K. Mahatma, G. Naresh Kumar2

Institution at which work was performed:

1Department of Plant Molecular Biology and Biotechnology, ASPEE College of Horticulture and

Forestry, Navsari Agricultural University, Navsari, Gujarat-396456, India

2Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda,

Vadodara, Gujarat-390002, India

3Department of Biotechnology, Ashok & Rita Patel Institute of Integrated Study & Research in

Biotechnology & Allied Sciences, New Vidyanagar, Gujarat-388121.India

* Author for correspondence E-mail: sanjayjha14@nau.in

Phone: +91-2637-282745, Ext-659

Fax: +91-2637-282145

Abstract

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An As-hypertolerant Alishewanella sp. GIDC-5 (Accession no. HQ659190) was isolated

from an effluent treatment plant of the industrial area near Sachin, Gujarat (India). In vitro

studies revealed that GIDC-5 can tolerate 18 mM of arsenite [As(III)] and 220 mM of arsenate

[As(V)]. PCR analysis confirmed the presence of arsenite transporters [arsB and ACR3(1)] and

arsenite oxidase gene [aioB]. Specific activities of arsenite oxidase and arsenate reductase,

located on membrane and cytoplasmic fractions respectively, increased in dose dependent

manner with arsenite concentration. Also, specific activities of antioxidant enzymes viz.,

catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase increased in

presence of arsenite. Increased exposure to arsenite changes enzyme activities of the glycolysis,

Krebs and glyoxylate branches dramatically. These results reveal that along with ars operon,

metabolic adaptation and antioxidant activities participate in As(III) tolerance in Alishewanella

sp. GIDC-5.

Key words: Alishewanella, arsenite, ars operon, central carbon metabolism, antioxidant

enzymes.

Abbreviations

As, arsenic; As(III), arsenite; As(V), arsenate; CS, citrate synthase; G6PDH, glucose-6-

phosphate dehydrogenase; GDH, gluconate deydrogenase; ICDH, isocitrate dehydrogenase;

PDH, pyruvate dehydrogenase; α-KGDH, α- ketoglutarate dehydrogenase; MDH, malate

dehydrogenase; ICL, isocitrate lyase; OAA, oxaloacetate; PPC, phosphoenolpyruvate

carboxylase; PYC, pyruvate carboxylase; CAT, catalase; APX, ascorbabate peroxidase; SOD,

superoxide dismutase; GST, glutathione S-transferase; GR, glutathione reductase; TCA,

Tricarboxylic acidIntroduction

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Arsenic, a toxic metalloid of global concern, usually originated geogenically but can be

intensified by human activities (Wang and Mulligan 2006). Consequentially, elevated levels of

arsenic have been reported in soils and groundwater worldwide. The maximum concentration

limit (MCL) recommended for drinking water by the World Health Organization (WHO) is 10

µg l-1. Elevated levels of arsenic in drinking water can seriously impact human health and have

been implicated in human diseases and mortality (Doop et al. 2004). Around 6 million people in

West Bengal and more than 46 million people in Bangladesh are estimated to be at risk from

drinking water with arsenic above 50 µg l-1 (Wang and Mulligan 2006; Kinniburgh and Smedley

2001).

The Sachin region in the south Gujarat, India being the part of “Golden Corridor” has

witnessed rapid industrial development. These industrial areas contain many individual industrial

units, including dye factories, textile, rubber, pesticide and paint manufacturers, pulp and paper

producers, engineering and chemical companies. Effluents of these industries are rich in

persistent organic compounds, heavy metals and metalloids (Bruno 1995, CPCB 1996), and one

which is arsenic with its level up to 150 µg l-1 i.e., about 15 times more than India’s permissible

limits (10 µg l-1). The groundwater arsenic contamination has been increasing day by day. It has

been established that the arsenic concentrations are of geogenic origin. In microcosm-

experiments with sediments from affected aquifers, microbes have been shown to trigger arsenic

release either indirectly by using solid-phase minerals such as iron hydroxides for energy gain or

after changing the aquifer redox conditions by direct reduction of arsenate to arsenite (Akai et al.

2004; Islam et al. 2004). Microorganisms play a major role in the biochemical cycle of arsenic

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and can convert it to different oxidation states with different solubility, mobility and toxicity

(Silver and Phung 2005).

Arsenic (‘As’) occurs in nature in four oxidation states (+5, +3, 0, –3), with pentavalent

arsenate [+5, As(V)], and trivalent arsenite [+3, As(III)] being the most common forms (Jackson

et al. 2003). Arsenate is chemically similar to inorganic phosphate and the potential to substitute

for phosphate in glycolytic and cellular respiration pathways makes it toxic for cells. Arsenate

cannot form the high-energy phosphate bonds that are needed and eventually oxidative

phosphorylation is uncoupled. Therefore, ATP with its high-energy phosphate bonds is not

formed (Ralph, 2008; Mandal and Suzuki, 2002). Adverse effects of arsenate are partially due to

its transformation to trivalent arsenite. Since, arsenite has greater mobility, it is considered more

toxic than arsenate (Ehrlich, 2005; Tamaki and Frankenberger, 1992). Arsenite inhibits

numerous cellular enzymes through sulfhydryl group binding. It inhibits cellular glucose uptake,

gluconeogenesis, fatty acid oxidation, and decreases production of cellular ATP and further

production of acetyl CoA. Exposure to arsenicals either in vitro or in vivo in model organisms

caused the induction of heat shock proteins (hsp), superoxide dismutase (SOD), catalase (CAT),

glutathione reductase (GR), glutathione peroxidase enzymes (Hughes, 2002).

A wide variety of microorganisms are found in water bodies receiving industrial

effluents. Many of the microorganisms show adaptation to the toxic materials constantly released

into their environment. They have developed strategies to defy, tolerate, metabolize, and to

detoxify these toxic substances (Parsek et al. 1995). Several heavy metal tolerance mechanisms

have been reported in different types of cells (Reed and Gadd 1990), viz., cell wall binding,

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decreased cell membrane permeability, active extrusion and complexation with chelating agents

(Gekeler et al. 1988; Rauser 1990). The most widespread resistance mechanisms found in

bacteria against arsenic are mediated through the ars operons (Mukhopadhyay and Rosen 2002).

The current study deals with the effects of arsenic exposure on an As-hypertolerant

Alishewanella sp. strain GIDC-5, isolated from industrial effluents of Gujarat Industrial

Development Corporation area of Sachin, Gujarat, India. Our results clearly demonstrate that in

addition to the ars operon, adjustments in antioxidant machinery and central carbon metabolic

pathway are the means of adaptations in Alishewanella sp. GIDC-5 to arsenite exposure.

Materials and Methods

Isolation of arsenite tolerant bacteria: - The water sample was collected from the effluents of the

Gujarat Industrial Development Corporation (G.I.D.C.) area, located at Sachin, Gujarat, India

(Latitude: 21° 29' 46" N; Longitude: 72° 76' 51" E). Arsenic-oxidizing bacteria were isolated by

enrichment culture method. Different dilutions of the water sample were inoculated in modified

M9 minimal medium (Sambrook and Russell, 2001) containing total P < 100 µM and

supplemented with 1 mM of Sodium arsenite (NaAsO2). The media composition included

Na2CO3.10H2O, 36g/L; KH2PO4, 0.015g/L; KCl, 8g/L; NH4Cl, 5g/L; NaCl, 2.5g/L; 2mM

MgSO4; 0.1mM CaCl2 and micronutrient cocktail. The micronutrient cocktail was constituted of

FeSO4.7H2O, 3.5 mg/L; ZnSO4.7H2O, 0.16 mg/L; CuSO4.5H2O, 0.08 mg/L; H3BO3, 0.5 mg/L;

CaCl2.2H2O, 0.03 mg/L and MnSO4.4H2O, 0.4 mg/L. Glucose was used as carbon source. pH of

the medium was adjusted to 7.4 and was sterilized by autoclaving for 20 min at 121°C. Flasks

were incubated at 28°C on a rotary shaker for 48-72 h. The grown culture was harvested and

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transferred to fresh arsenite-amended medium. After 5-6 sub-culturing with increasing

concentrations of sodium arsenite from 1 to 20 mM (As(III) : NaAsO2; 0, 5, 10, 15, 18, 20), an

enrichment culture was established. Sequential purification of culture was done by streaking on

As(III)-supplemented modified M9 agar plates. Morphologically distinct colonies tolerating

maximum arsenite concentration were further purified and stored at -20°C as 50% glycerol stock

for future use. The strain was grown to mid-exponential phase in modified M9 minimal medium

at 28°C with shaking at 180 rpm, prior to use.

16S rRNA gene amplification and sequencing: - NaCl - CTAB method (Sambrook and Russell,

2001) was used for DNA isolation from grown cells. 16S rRNA gene fragment was amplified

with thermal cycler (Eppendorf, Germany) using degenerate universal primer pair 27f and 1492r

(Aksornchu et al. 2008). After an initial denaturation step at 94°C for 5 min, 30 cycles consisting

denaturation at 94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 1.5 min

were performed. Final extension step was kept at 72°C for 10 min. PCR products were examined

by agarose gel electrophoresis. The amplified 16S rRNA fragments were used as templates for

cycle sequencing reaction (Bigdye v3.1) followed by DNA sequencing (ABI Prism 3130 genetic

analyzer, Applied Biosystems, U.S.A) using 27f and 1492r primers. The derived sequences were

analyzed by performing online BLASTn sequence homology test

(http://www.ncbi.nlm.nih.gov/BLAST/). The phylogenetic tree was constructed by using the

neighbor-joining method in the program Phylogeny.fr (Dereeper et al. 2008).

Biochemical and Phenotypic characterization: - Growth curve of GIDC-5 was plotted by

growing the cells in modified M9 medium at 28°C in 150 ml Erlenmeyer flasks, with 0, 15, 18

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and 20 mM NaAsO2 with continuous shaking at 180 rpm in the orbital shaker. At different time

intervals, 2 ml culture broth was withdrawn till bacteria reached stationary phase. Growth was

monitored by measuring optical density (OD) of the cultures at 600 nm using spectrophotometer

(CARY 50 UV, Australia).

Phenotypic characters of bacterial cells were observed by performing Gram staining and

subsequently analyzing under compound microscope (100x). Biochemical characterization of

strain was done by performing various tests as described by Lanyi (1987) using cultures grown at

28°C in modified M9 broth.

Cross tolerance of bacterium isolate for other heavy metals was checked by determining

the minimal inhibitory concentrations (MIC) for the metals: As (V) 0.0–250 mM; Co, Cu, Ni,

Cd, Hg 0.0-0.5 mM, as described by Drewniak et al. (2008). Medium supplemented with the

respective metal compounds was inoculated with mid-log phase grown culture to a final density

of ~106 cells/ml and then incubated for 24 h.

Amplification and sequencing of arsenical resistance genes: - The amplification of genes for

arsenite oxidase (aioB), arsenite transporter (arsB), and arsenite membrane pump, (ACR3(1) and

ACR3(2)) were performed using four pairs of degenerate primers [#1F and #1R for aioB,

darsB1F and darsB1R for arsB, dacr1F and dacr1R for ACR3(1), dacr5F and dacr4R for

ACR3(2)] as described by Cai et al. (2009). The PCR products were purified, sequenced and

analyzed.

Transformation of arsenite and arsenate: - Conversion of supplemented arsenite to arsenate in

intracellular and extracellular fractions was determined by using Inductively Coupled Plasma

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Optical Emission Spectrometry (ICP-OES) equipped with hydride generator. To prepare

intracellular and extracellular fractions, bacterial cells were inoculated in modified M9 minimal

medium with 5 mM of arsenite, and allowed to grow till late log phase (OD 1.3, at 600 nm). 20

ml grown cultures were collected and centrifuged at 7,168xg for 3 min. Supernatant was

collected as extracellular fractions. Intracellular fractions were washed, resuspnded in 100 mM

Phosphate buffer (pH 7.0), subsequently disrupted and arsenic speciation analysed by the method

described by Cumming et al. (1999). To differentiate between intracellular and extracellular

fractions, assay for cytosolic marker (G6PDH enzyme) was performed. Standard solutions of

arsenic availed from Merck (India) was used in the experiment. HCl (for As(V)) or mixture of

KIO3 and HCl (for As(III)) served as blank, whereas, heat killed bacterial cell fractions (intra and

extracellular) were used as controls.

Analysis of organic acids: - Cells of bacterial isolate were grown in amended M9 minimal

medium (containing 100 mM glucose as a carbon source) with and without 5 mM of NaAsO2. 5

ml of late log phase cultures with pH below 5.5 were collected and centrifuged at 9,200 ×g for 5

min, supernatant was filtered through 0.22 µm filters. Pellets were washed twice with 50 mM

Phosphate buffer (pH 7.0) and resuspended in 2 ml of same buffer. These cell suspensions were

sonicated and centrifuged at 10,800 ×g for 30 min at 4°C to obtain intracellular fractions.

Intracellular fractions were filtered (0.22 µm membranes) and used for HPLC analysis.

Detection and quantification of different organic acids was done on Knauer advanced

High Performance Liquid Chromatogram (HPLC) equipped with PDA detector (Merck,

Germany). The mobile phase was 0.02% ortho-phosphoric acid (Merck, Germany) in the

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gradient of flow rate as described by Vyas and Gulati (2009). Eluates were detected at λ 210 nm

and identified by retention time and co-chromatographed by spiking the sample with the

authentic organic acids. Organic acid standards prepared in double distilled water were filtered

(0.22 µm nylon membranes) and their retention times were determined under similar conditions.

Comparison of peak areas with external standards was used for quantification.

Enzymatic preparations and assays: - Overnight grown cells in amended M9 minimal media

supplemented with various concentrations of As(III) (0, 1, 5 and 10 mM) were harvested during

mid-log to late-log growth phase by centrifugation at 9,200 ×g for 2 min at 4°C. The preparation

of cell-free extracts for pyruvate carboxylase (PYC), phosphoenol pyruvate carboxylase (PPC)

and glucose-6-phosphate dehydrogenase (G6PDH) assays was carried out according to Kodaki et

al. (1985). The cell pellet was washed with 80 mM phosphate buffer (pH 7.5) followed by

resuspension in same buffer containing 20% glycerol and 1 mM DTT (Dithiothreitol). The cells

were lysed by sonication (1 – 1.5 min at 4°C) and centrifuged at 9,200 ×g at 4°C for 30 min to

remove the cell debris. The supernatant was used as cell-free extract for the enzyme assays and

pellets were used as membrane fractions for different enzyme assays. In case of arsenite oxidase

and arsenate reductase enzymes, 50 mM Tris-Cl (pH 7.0) buffer was used, and assay was

performed in cell-free extracts and membrane fractions both, to localize the activity of respective

enzymes. The whole cell preparation for glucose dehydrogenase (GDH) assay was carried by

washing the harvested cells thrice with normal saline and resuspending it in 0.01 M phosphate

buffer (pH 6.0) with 5 mM MgCl2.

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Total catalase (CAT) (EC 1.11.1.6) activity was determined in the homogenates by

measuring the decrease in absorption at 240 nm as the consumption of H2O2 (ε = 39.4 mM-1 cm-

1) according the method of Aebi (1984). Ascorbate peroxidase (APX) (EC 1.11.1.11) activity

was measured in fresh extract by following the decrease in absorbance, due to ascorbate

oxidation, at 290 nm (ε = 2.8 mM-1 cm-1) (Nakano and Asada 1981). Total superoxide dismutase

(SOD) (EC 1.15.1.1) activity was measured spectrophotometrically based on inhibition in the

photochemical reduction of nitroblue tetrazoilum (NBT) as described by Van Rossun et al.

(1997). Enzyme unit of SOD was calculated according to formula given by Constantine and

Stanley (1977). Glutathione reductase (GR) (EC 1.6.4.2) activity was measured by following

NADPH reduction at 340 nm following the method of Schaedle and Bassham (1977).

Gluatathione S-transferase (GST) (EC 2.5.1.18) activity was measured by observing the

conjugation of 1-chloro, 2, 4-dinitrobenzene (CDNB) with GSH (Boyland and Chasseaud 1969).

The molar extinction of CDNB was taken as 9.6 mM-1 cm-1 at 340 nm.

Citrate synthase, CS (EC 2.3.3.1) activity was estimated by measuring the absorbance of

5,5΄-dithiobis(2-nitrobenzoic acid) at 412 nm, which would change due to its reaction with the

thiol group of CoA (Serre, 1969). The assay mixture contained the following in 1.0 ml: Tris/HCl

(pH 8.0), 93 mM; acetyl-CoA, 0.16 mM; oxaloacetate, 0.2 mM; 5,5΄-dithiobis(2-nitrobenzoic

acid), 0.1 mM and cell lysate. The reaction was started by addition of oxaloacetate. The molar

absorption coefficient was taken as 13.6 mM-1cm-1 at 412 nm. Phosphoenol pyruvate

carboxylase, PPC (EC 4.1.1.31) and pyruvate carboxylase, PYC (EC 6.4.1.1) activities were

monitored by following NADH oxidation at 340 nm in an assay combined with malate

dehydrogenase (MDH); Glucose 6-phosphate dehydrogenase, G6PDH (EC 1.1.1.49) and

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isocitrate dehydrogenase, ICDH (1.1.1.42) activities were determined by following the reduction

of NADP at 340 nm; isocitrate lyase, ICL (4.1.3.1) activity was monitored by measuring

glyoxylate formation at 324 nm with the aid of phenylhydrazine-HCl; and glucose

dehydrogenase, GDH (EC 1.1.5.2) was assayed by following the coupled reduction of 2,6-

dichlorophenolindophenol at 600 nm, as described by Buch et al. (2008).

Arsenate reductase (AR) and arsenite oxidase (AIO) assays were performed both in

membrane and cytosolic fractions. AR activity was measured using a coupled assay system that

measures the arsenate-dependent oxidation of NADPH (Mukhopadhyay et al. 2000). This

activity was measured as a change in absorbance at 340 nm. The quantity of NADPH oxidized

was calculated using an extinction coefficient of 6,200 M-1 cm-1. The assay of arsenite oxidase

enzyme was done by following the method of Anderson et al. (1992). The reduction of the

artificial electron acceptor 2,4-dichlorophenolindophenol was monitored at 600 nm in the

presence of 1, 5 and 10 mM As(III).

All enzyme activities were determined at 28 ± 2°C, against appropriate controls lacking

the substrate or the enzyme source in the reaction mixture. One unit of specific enzyme activity

was defined as the amount of protein required to convert 1 nM substrate min-1mg-1 total protein,

unless specified. Total protein concentration of crude extracts and whole-cell suspensions was

measured by a modified Lowry method (Peterson 1977) using BSA as standard. All enzyme

activities are expressed in nM min-1 mg-1 total protein except CAT, GST, APX, GR, MDH, AIO

and AR are depicted in µM min-1 mg-1 total protein and SOD is expressed in U mg-1 total protein.

The values are depicted as Mean ± S.E.M of 3 independent observations. Statistical significance

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level is indicated as p-values: ***, p<0.001, **, p<0.01, *, p<0.05, ns, p is non-significant. All

parameters are compared to the control i.e., culture without arsenite in medium.

Results

Isolation and characterization of arsenite tolerant bacterium: -

The isolation procedure described above yielded eight arsenite tolerant bacterial isolates

from industrial effluents of Sachin, Gujarat, India. Isolate GIDC-5 showed hypertolerance to

both the species of arsenic, As(III): 18 mM and As(V): 220 mM. Also, GIDC-5 showed a

tolerance level of 48 mM As(III) when grown in LB medium. Cross metal tolerance experiments

demonstrated that GIDC-5 could grow in presence of 0.5 mM copper, cobalt and cadmium each.

NCBI-BLAST analysis of the partial 16S rRNA sequence of GIDC-5, showed 99%

identity to 16S rRNA genes of Alishewanella genera (Fig. 1; Accession no.: HQ659190).

Microbiological tests indicated Alishewanella sp. GIDC-5 to be a Gram negative, bacilli shaped,

aerobic, motile bacterium. It can survive in high salt concentrations of up to 12% NaCl. Catalase

and oxidase tests were positive for GIDC-5. Results of growth kinetic study are shown in Fig. 2.

Stationary phase was obtained at 21 – 24 h in presence of As(III).

DNA fragments for arsenical resistance genes were amplified by PCR using genomic

DNA and gene specific primers. Positive amplification for arsenite oxidase (aioB, ~450bp),

arsenite transporter (arsB, ~700 bp) and arsenite membrane pump (ACR3(2), ~750 bp) genes was

obtained (Fig. 3). The nucleotide sequences of putative ACR3(2) gene are deposited to NCBI

Gene Bank database (Fig. 4; HQ659195 and HQ659196).

Transformation of arsenite and arsenate: -

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ICP-OES was performed to study the amount of accumulation, expulsion and

transformation of As(III) and As(V) in the Alishewanella sp. GIDC-5. G6PDH activity in

extracellular fractions was found to be 0.04 ± 0.001 µM min-1 mg-1 total protein, and in cytosolic

fractions was 33.18 ± 0.07 µM min-1 mg-1 total protein. Intracellular As(III) concentration was

0.072 ± 0.034 mM, much lower than extracellular concentration, 2.539 ± 0.53 mM. Similarly,

intracellular and extracellular As(V) concentrations were 0.12 ± 0.017 mM and 2.077 ± 0.019

mM, respectively. As shown in Fig. 5, Arsenite oxidase (AIO) was more prominent in membrane

fractions and a positive correlation between As(III) concentrations and activity of membrane

bound arsenite oxidase was observed. In presence of 5 mM As(III), the AIO activity in

membrane fractions was observed to be 140.05 ± 3.9 µM min-1 mg-1 total protein , which is

significantly higher than that in cytoplasmic fractions (2.31 ± 0.13 µM min-1 mg-1 total protein).

Also, it is 3.9 fold higher than activity of control in membrane fractions. Contrastingly, arsenate

reductase (AR) activity was observed mainly in the cytosolic fractions of the cell. At 5 mM

As(III) concentration, AR activity was observed to be 12.59 ± 0.07 µM min-1 mg-1 total protein

in cytosol, which was significantly higher than 2.55 ± 0.1 µM min-1 mg-1 total protein compared

to membrane fraction of cell. It is 16.7 fold higher than activity of control in cytosolic fractions.

At 10 mM As(III) concentrations, both AIO and AR activities were further increased in

respective fractions. At this concentration, AR activity (13.63 ± 0.12 µM min-1 mg-1 total

protein) was 18.1 fold and AIO activity (333.07 ± 3.11 µM min-1 mg-1 total protein) was 9.3 fold

increased compared to control. No AIO and AR activities were detected in any of the heat-killed

controls.

Effect of As(III) on antioxidant enzymes: -

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A positive effect of As(III) was observed on the activities of various antioxidant enzymes

viz. catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and glutathione

S-transferase (GST), except glutathione reductase (GR) whose total activity was decreased at 10

mM As(III) concentration. At 5 mM As(III) a 7.5 fold increase in total CAT activity, 8.75 fold in

APX, 6.22 fold in SOD and 3.54 fold in GST activity was observed, in comparison to control.

GR activity was 1.8 times lower in presence of 10 mM As(III), respectively (Table 1).

Effect of As(III) on carbon metabolic pathway: -

Carbon metabolic pathways and organic acid concentrations were analyzed for

Alishewanella sp. GIDC-5 in response to arsenite exposure. GDH activity was 1.9 fold higher in

sample treated with 5 mM As(III) as compared to untreated control. But, G6PDH activity at

same As(III) concentration was 2.5 fold decreased. Similarly, a 1.6 and 1.2 fold inhibition in the

CS and ICDH activities was observed at 5 mM As(III). Major effect of increasing concentrations

of As(III) was observed on PYC, PDH and α-KGDH enzymes, which were 10.9, 8.6 and 7.5 fold

decreased compared to control, respectively. In contrast to all these enzyme activities which

were made vulnerable by As(III), PPC, MDH and ICL activities were increased 1.7, 3.1 and 1.9

fold in presence of 5 mM As(III) compared to respective controls (Table 2).

Intracellular organic acid levels of Alishewanella cells growing in presence of 5 mM

As(III) were quantified in HPLC (Table 3). The results demonstrate that the levels of citric (1.8

times), malic (2.2 times), fumaric (2.0 times), succinic (2.6 times) and acetic (2.8 times) acids

were significantly decreased; whereas, gluconic (2.6 times), α-ketoglutaric (2.2 times), pyruvic

(1.3 times) and oxaloacetic (5.4 times) acid levels were increased.

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Discussion

An As-hypertolerant Alishewanella sp. GIDC-5 was isolated from an effluent treatment

plant in the industrial area near Sachin, Gujarat (India). Groundwater contamination due to

arsenic mobilization is a problem of geomicrobiology (Oremland and Stolz 2005). Hence,

isolation and study of microbes involved in arsenic transformation will add to the knowledge of

how microbes interact with arsenic-bearing minerals and control arsenic pollution in

groundwater. Thus, present study was an attempt to analyze the arsenite tolerance of

Alishewanella sp. GIDC-5 by ascertaining a link between its multiple gene products (antioxidant

enzymes, arsenite oxidase and arsenate reductase) and central carbon metabolic pathways.

Phylogenetic analysis of 16S rRNA gene of GIDC-5 clearly indicates that it belongs to

the genera Alishewanella. Further, phylogenetic study of its arsenite efflux pump gene, ACR3(2),

reveals that this gene is similar to other arsenite tolerating microbes reported. In addition to this

it explain the divergence of this bacterium from other bacteria of Alishewanella genus. The most

studied mechanism of arsenic detoxification in bacteria is ars operon. Ars-mediated tolerance

involves the reduction of arsenate (AsV) to arsenite (AsIII) via cytoplasmic AsV reductase (ArsC),

followed by the extrusion of AsIII by a membrane-associated ArsB efflux pump that is efficient at

removing AsIII (Mobley and Rosen, 1982). Most arsenite tolerant bacteria possess both arsenate

reductase and arsenite oxidase activities (Silver and Phung 2005; Mateos et al. 2006). ICP-OES

analysis of As-treated Alishewanella sp. GIDC-5 revealed the presence of extracellular and

intracellular As(III) and As(V). AIO activity was localized to membrane fractions, and was ~25

folds higher than AR activity, which was more prominent in cytosolic fractions. This indicates

that biological oxidation of arsenite could be a detoxification mechanism (Tamaki and

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Frankenberger 1992) converting more toxic As(III) (present in the inhabiting environment of

GIDC-5) into less toxic As(V), before it could cross the cell membrane. Arsenate, As(V), a

structural analogue of phosphate, thus formed entered the cells of GIDC-5 via phosphate-specific

transporters present on membranes of bacterial cells (Rosen, 2002). Prominent AR activity was

found in cytosolic fractions of GIDC-5 cells, suggesting that As(V) thus formed was again

transformed back to As(III) in the cells. As(III) inside the cytoplasm could be extruded out via

arsenite efflux pump (arsB) and arsenite transporter (ACR3(1)). PCR analysis for marker genes

(aioB, arsB and acr3(1), further corroborated the presence of an ars-operon, a genetic

mechanism for arsenic tolerance and arsenic transformation, in GIDC-5.

Arsenic lead bacteria to increase the rate of H2O2 production which increase intracellular

ROS concentrations, by depleting GSH concentrations and membrane lipid peroxidation

reactions. This in turn induces major components of the oxidative stress response (Huang et al.

2010). Interestingly, specific activities of catalase (CAT), ascorbate peroxidase (APX),

superoxide dismutase (SOD) and glutathione S-transferase (GST) were found to increase

significantly, suggesting that the overall ROS-scavenging pathway is active in GIDC-5 in

response to arsenite exposure. Earlier studies have reported that cytoplasmic arsenate reductase

(arsC) uses glutathione (GSH) as a reactant, thus, the generated arsenite bound to GSH to form

As(III)-GS3 complex by the action of GST enzyme, followed by extrusion of the complex

through ABC transporter (Ghosh et al. 1999). This complex is a potent inhibitor of Glutathione

reductase (GR) activity in cells (Kala et al. 2000; Styblo et al. 1997). Notably, As(III) treated

GIDC-5 showed dose dependent increase in GST and decrease in GR specific activity,

suggesting the formation of arsenite-glutathione complex (As(III)-GS3) for extrusion by ABC

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transporters. However, these transporters are not capable of removing the entire amount of

As(III) as revealed by intracellular ICP-OES studies. We further investigated the effect of

leftover intracellular As(III) on central carbon metabolism (Glycolysis, TCA and Glyoxylate

cycles) in GIDC-5 cells. Different studies have shown that G6PDH (the enzyme mediating the

intracellular glucose oxidative phosphorylation) supplies the reducing power for antioxidant

regeneration (Lundberg et al. 1999), and plays a role in the mechanism of protection against

oxidative stress (Angelova et al. 2005). Therefore, we investigated the importance of G6PDH

during arsenite exposure. G6PDH showed a dose dependent decrease in its specific activity,

implying that As(III) affect the normal Embden Meyerhoff pathway (EMP) of pyruvate

production. Whereas increased glucose dehydrogenase (GDH) activity, followed by increased

gluconic acid concentration after arsenite exposure suggests that Alishewanella sp. GIDC-5

diverts glucose towards direct oxidative pathway.

Pyruvate enters the tricarboxylic acid cycle (TCA) via the reactions catalyzed by PDH

and pyruvate carboxylase (PYC), which are regulated to coordinate the utilization of pyruvate for

anabolism (via the anaplerotic production of oxaloacetate by PYC) and catabolism (via acetyl-

CoA production by PDH) (Zubay 1988). As(III) treated GIDC-5 showed pyruvate

dehydrogenase (PDH) and pyruvate carboxylase (PYC) enzymes inhibition. Whereas, two fold

increased PPC activity was observed, suggesting that As(III) treated GIDC-5 could overcome the

inhibitory effect of metalloid on PDH enzyme by adopting an alternative pathway for

Oxaloacetic acid (OAA) production via PPC. The activities of CS, ICDH and α-KGDH enzymes

were significantly reduced followed by reduced levels of citric acid. Since, Acetyl CoA

concentration is also limiting in the cells, which could be the consequence of PDH inhibition and

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pyruvate accumulation in the cells. Therefore, it is possible that the major control of citrate

synthase activity is by changes in the concentration of its substrates acetyl CoA. ICDH and α-

KGDH decarboxylate their respective substrates (isocitrate and α-KG) with the concomitant

formation of respective products (α-ketoglutarate and succinyl coA) and the reducing factors

NADPH or NADH. A reduction in the activity of ICDH would target isocitrate to isocitrate lyase

and lead to the production of glyoxylate (Hamel and Appanna 2001). Analysis of TCA bypass

pathway revealed that the activity of ICL enzyme, responsible for production of malic acid via

glyoxylate was up-regulated, simultaneously with reduced concentrations of succinic, fumaric

and malic acids, which suggests the metabolic shift from citric acid pathway to glyoxylate

pathway in arsenite exposed cells. Arsenite also significantly induced the activity of MDH

enzyme and resulted in intracellular OAA accumulation. The changes in the enzymatic activity

of the TCA cycles evidenced that this crucial metabolic pathway plays an important role in

Alishewanella sp. GIDC-5 to maintain an appropriate energy balance in order to adapt arsenite

stress.

In conclusion, arsenite oxidation, arsenite efflux mechanism, antioxidant enzymes and

central carbon metabolic pathway participates in Alishewanella sp. GIDC-5 to tolerate arsenite

stress in industrial effluent.

Acknowledgments

The work was supported by the Department of Biotechnology, Ministry of Science and

Technology, Government of India and Gujarat State Biotechnology Mission (GSBTM), Gujarat.

No Author has any conflict of interest.

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Legends to figures

Fig. 1. Phylogram of arsenic-resistant bacteria derived from 16S rRNA sequence data of

Alishewanella sp. GIDC-5 and closely related species. The scale bar represents 0.8

substitutions per nucleotide.

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Fig. 2. Growth curve of Alishewanella GIDC-5 on amended M9 minimal medium. The

values are plotted as Mean ± S.D. of 3 independent observations.

Fig. 3. PCR amplification results of arsenic related and 16S rRNA genes of Alishewanella

GIDC-5. Lane 1: aioB, Lane 2: arsB, Lane 3: ACR3(2), Lane 4: ACR3(1) (not present in

Alishewanella sp. GIDC-5), Lane 5: 16S rRNA gene segments, Lane L: marker.

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Fig. 4. Phylogram of arsenic-resistant bacteria derived from arsenic efflux pump gene

ACR3(2) gene sequence data of Alishewanella sp. GIDC-5 and closely related species. The

scale bar represents 0.6 substitutions per nucleotide. Bootstrap values (expressed as percentages

of 1000 replications) greater than 50% are given at the nodes.

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Fig. 5. Arsenite oxidase and arsenate reductase activity in Alishewanella GIDC-5. CF:

Cytoplasmic fraction; and MF: Membrane fraction. The activities have been estimated in late log

phase, without adding arsenite and after adding 1, 5 and 10 mM arsenite in the amended M9

minimal medium.

Legends to tables

Table 1. Activity of antioxidant enzymes in Alishewanella GIDC-5. All enzyme activities

were estimated in late log phase. All enzyme activities are expressed in µM min-1 mg-1 total

protein except SOD, which is depicted in U mg-1 total protein.

Table 2. Activities of PDH, α-KGDH, MDH, ICL, G6PDH, CS, GDH, ICDH, PPC and PYC

in Alishewanella GIDC-5. All the enzyme activities were estimated from mid log phase to late

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log phase cultures except CS and ICDH which were estimated in stationary phase. All enzyme

activities are expressed in nM min-1 mg-1 total protein except MDH which is depicted in µM min-

1 mg-1 total protein.

Table 3. Intracellular organic acid production from arsenite treated (5 mM) and untreated

samples of Alishewanella GIDC-5 (expressed in mM). Organic acid yields were estimated by

comparing the retention time of sample with standards. Stationary phase cultures grown on

amended M9 medium were collected.

Table 1

Antioxidant

Enzymes

(µM min-1

mg-1 total

protein)

As(III): 0 mM As(III): 1 mM As(III): 5 mM As(III): 10 mM

CAT 1.48 ± 0.06 4.95 ± 0.71** 11.07 ± 0.28*** 13.50 ± 0.47***

GST 3.40 ± 0.30 5.33 ± 1.07* 12.05 ± 0.36*** 18.33 ± 1.32***

SODa 62.90 ± 3.76 118.95 ± 3.85** 391.36 ± 6.15*** 525.61 ± 12.95***

APX 34.96 ± 3.44 223.09 ± 6.23*** 305.95 ± 3.91*** 627.59 ± 10.06***

GR 19.55 ± 0.94 21.37 ± 0.18* 18.00 ± 0.37ns 11.02 ± 0.65** aSOD is expressed in U mg-1 total protein.

Table 2

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Metabolic

enzymes (nM

min-1 mg-1

total protein)

As(III): 0 mM As(III): 1 mM As(III): 5 mM As(III): 10 mM

PDH 19.04 ± 0.04 5.82 ± 0.44*** 2.21 ± 0.21*** 1.20 ± 0.01***

α-KGDH 18.62 ± 0.29 11.72 ± 0.55*** 2.49 ± 0.11*** 1.05 ± 0.12***

MDHb 2.06 ± 0.07 5.12 ± 0.25*** 6.39 ± 0.18*** 10.00 ± 0.22***

ICL 4.67 ± 0.11 6.24 ± 0.80ns 8.72 ± 0.52** 8.55 ± 0.65**

G6PDH 84.14 ± 1.58 40.25 ± 8.41*** 33.18 ± 0.07*** 13.56 ± 2.92***

CS 19.51 ± 0.15 14.27 ± 0.62*** 12.27 ± 0.76*** 9.84 ± 0.70***

GDH 58.57 ± 1.46 56.37 ± 1.46ns 114.03 ± 6.04** 166.98 ± 7.16**

ICDH 109.74 ± 11.20 95.61 ± 0.14** 89.39 ± 0.07** 87.96 ± 0.39**

PPC 13.75 ± 0.35 18.70 ± 0.4** 23.40 ± 0.70*** 28.18 ± 0.88***

PYC 88.45 ± 2.57 23.20 ± 0.75*** 8.15 ± 0.90*** 2.31 ± 0.02***

b MDH is expressed in µM min-1 mg-1 total protein

Table 3

Intracellular

Organic acids

(mM)

As(III): 0 mM As(III): 5 mM

Gluconic acid 3.20 ± 0.00 8.40 ± 0.31***

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Pyruvic acid 2.53 ± 0.25 3.31 ± 0.19*

Succinic acid 2.68 ± 0.01 ND

Citric acid 4.50 ± 0.08 2.50 ± 0.00***

α-KG 1.37 ± 0.09 3.04 ± 0.03***

Fumaric acid 2.09 ± 0.08 1.03 ± 0.02***

Malic acid 5.87 ± 0.44 2.66 ± 0.06***

Oxaloacetic acid 0.65 ± 0.05 3.50 ± 0.10*** ND: not detected

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