CURRENT MICROBIOLOGY Vol. 49 (2004), pp. 321326 DOI: 10.

1007/s00284-004-4351-2

Current Microbiology
An International Journal
Springer ScienceBusiness Media, Inc. 2004

Effect of Chromium(VI) Action on Arthrobacter oxydans

Nino V. Asatiani,1 Marina K. Abuladze,1 Tamar M. Kartvelishvili,2 Nugzar G. Bakradze,1 Nelly A. Sapojnikova,1 Nelly Ya. Tsibakhashvili,1 Leila V. Tabatadze,1 Lia V. Lejava,1 Lali L. Asanishvili,1 Hoi-Ying Holman3
1 2

Institute of Physics, Georgian Academy of Science, 6 Tamarashvili Street, 0177 Tbilisi, Georgia Institute of Molecular Biology and Biophysics, Georgian Academy of Sciences, 14 Gotua Street, 0160 Tbilisi, Georgia 3 Center for Environmental Biotechnology, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA Received: 16 March 2004 / Accepted: 23 April 2004

Abstract. Arthrobacter species is of interest because of its high potential for bioremediation. Bacteria can detoxify chromium, by either reduction or accumulation inside the bacteria and/or absorption of chromium(VI) (CrVI) on their surface, and efux pump. The possible pathway of Cr(VI) reduction by Arthrobacter oxydans isolated from Columbia basalt rocks at a US DOE highly contaminated site (USA) has been considered in the present study. FTIR absorption spectroscopy showed that these bacteria reduce Cr(VI). In the present study the threshold Cr(VI) nontoxic concentration (35 g/mL) for A. oxydans growing in liquid medium was estimated. Complete uptake of this concentration was achieved in about 10 days after chromium addition into the medium. At this concentration an increase in the protein isolated from the cell wall of A. oxydans was observed. This increased protein predominated independently of the growth phase at which Cr(VI) was added. Thermal analysis was used to identify any inuence of Cr(VI) on the DNP complex of A. oxydans. According to the data obtained it can be supposed that Cr(VI) reduction predominantly occurs on the bacterial surface and that cell wall represents a permeable barrier for these bacteria at the non-toxic chromium action.

Chromium (Cr) is a widespread environmental pollutant and a strong oxidant. It has been detected in groundwater due to industrial and military operation. The toxicity of chromium is dependent on its oxidation state. In the environment Cr occurs primarily in the valence forms Cr(VI) and Cr(III). The dominant form of Cr(VI) in aqueous solution (CrO42) is highly toxic and carcinogenic, unlike the less soluble Cr(III). The different biological effects of Cr(VI) and Cr(III) are believed to be caused by the cellular uptake process. The oxyanion (CrO42) can cross cellular membranes via surface anion transport systems (SO42 and HPO42 channels) and is biologically active [7, 31]. Bacteria can detoxify chromium. This is achieved basically by either reduction or accumulation inside the bacterium and/or absorption of Cr(VI) on its surface and efux pump [6, 22, 25]. Some chromium-resistant microorganisms have been described: Salmonella typhimurium [28], Escherichia coli [7, 30], Pseudomonas uorescens [16, 27],
Correspondence to: N.V. Asatiani; email: nina_asatiani@yahoo.com

Pseudomonas ambigua [15], Alcaligenes eutrophus [26], Enterobacter cloacae [35], Bacillus sp. [36], and Arthrobacter sp. [22]. Heavy-metal-resistant bacteria may be used for biotechnological processes, namely for bioremediation of metal-contaminated environments. In this respect bacteria capable of reducing Cr(VI) with subsequent efux of the reduced chromium products are of the most interest. Once inside a cell Cr(VI) as transition metal can be reduced to Cr(V/IV/III) by different nonspecic reductants such as glutathione (GSH), glutathione reductase (GR), cysteine, carbohydrates, NADH, NADPH, nucleotides, and ascorbic acid [8, 9, 32]. The by-products of the Cr(VI) reduction process (reactive oxygen species, ROS) can attack DNA and proteins and cause them damage. On the other hand, considering the composition of the bacterial cell wall (teichoic acids, polycarbohydrates, and other diol-containing substances [29] that possess reducing capability [8]) and the activity of different specic and nonspecic membrane- associated reductases [5, 6, 16, 20, 23], it is suggested that Cr(VI) can be at least partly reduced on the bacterial cell wall. In

322 this respect, Arthrobacter sp. is of interest because of its potential for bioremediation [22]. In the present study we investigated bacterial cell culture of Arthrobacter oxydans isolated from the Columbia basalt rocks of a US DOE contaminated site (USA). FTIR absorption spectroscopy showed that these bacteria reduce Cr(VI) [14]. The purpose of the present study was to identify the possible pathway of Cr(VI) reduction by A. oxydans. As physiological changes in cell metabolism at the different growth phases can cause various alterations (e.g., changes in ROS level) which can switch on supplementary detoxifying mechanisms during oxidative stress [11], we studied the response of A. oxydans to Cr(VI) at different periods of the life cycle. Materials and Methods
Bacterial culture and growth conditions. Bacterial culture of A. oxydans was isolated from the Columbia basalt rocks of a US DOE contaminated site (USA) and cultivated in the Lawrence Berkeley National Laboratory. In our laboratory the cells were maintained as a batch culture in a standard medium recommended for Arthrobacter species [10] at a temperature of 21C with constant shaking. Culture growth was monitored by measuring optical density at 490 and 590 nm. The viability was detected by cell growth on agar plates with a cell suspension dilution. In all cases bacterial culture growth without or with Cr(VI) proceeded without medium replenishment. Cell lysate preparation. Cells were harvested by centrifugation at 10,000 rpm for 10 min (4C) and washed twice in 0.15 M NaCl. The cell pellets were treated with 500 L of lysis buffer B-PER, pH 7.5 (Pierce) per 2109 cells overnight at 4C. The lysate was centrifuged at 15,000 rpm for 20 min (4C). Cell wall protein extraction method. The experimental procedure for preparation of cell wall from A. oxydans was a modication of the method of Strashinskaya et al. [33]. The cells (2109) were harvested by centrifugation at 10,000 rpm for 10 min (4C) and washed twice in 0.15 M NaCl and once in ice-cold deionized water. The cells suspended in ice- cold deionized water were sonicated by linear probe (44 kHz) for 51 min bursts (1 min interval between each burst) on ice using a sonicator (Sredmash, Russia). Cells debris was removed by centrifugation at 3,000 rpm for 20 min (4C), and cell wall was recovered by centrifugation at 15,000 rpm for 20 min (4C). The upper white friable layer of the pellets was the cell wall. This was additionally washed several times with ice-cold water in order to remove cytoplasm and membrane debris [24]. Cell wall extract was prepared by adding 0.25 N HCl, 6 M urea for 1 h followed by centrifugation at 15,000 rpm for 20 min (4C). Six volumes of acetone were added to precipitate the acid-soluble protein fraction. Microcalorimetry. The melting process of biological structures in A. oxydans was studied by using differential scanning calorimetry (DSC) designed at the Institute of Physics, Georgian Academy of Sciences [3]. A. oxydans cells, growing as a batch culture, were rinsed with buffer (50 mM Tris-HCl, pH 7.0, 0.1 M NaCl, 0.1 mM PMSF (phenylmethylsulfonyl uoride), 0.1 mM benzamidine). The measurement was performed in the same buffer within the temperature range 40 110C. Thermal spectra were normalized relative to the dry weight of the sample.

CURRENT MICROBIOLOGY Vol. 49 (2004) Analytical methods. Protein concentration was determined by using the BCA (bicinchoninic acid) protein assay reagent from Pierce (Rockford, IL). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli [19], using 10% gels. Molecular weight markers used were as follows: fructose6-phosphate kinase (84.0 kDa), bovine serum albumin (66.3 kDa), glutamate dehydrogenase (55.4 kDa), ovalbumin (45.0 kDa). Twentyve micrograms of the total protein was run per lane. The gels were stained with Coomassie blue R-250. Cr(VI) was determined spectrophotometrically by the diphenylcarbazide method. Cells were harvested by centrifugation at appropriate times, and the chromium content in the supernatant was estimated by adding 0.2 N H2SO4 and an acetone solution of 1,5-diphenylcarbazide. The absorbance was measured at 540 nm [12].

Results and Discussion

Growth phase effect on Cr(VI) reduction in Arthrobacter oxydans. The effective nontoxic concentration of Cr(VI) as potassium chromate for A. oxydans grown as batch culture was determined by the loss of Cr(VI) in the bacterial medium over a time course of up to 10 days and by the estimation of cell viability (%) on agar plates at the end of the cell incubation with chromium. The effects of Cr(VI) at the concentration range 10 200 g/mL on A. oxydans bacterial culture after chromate addition to mid-logarithmic phase cells are presented in Table 1. The data show that as the concentration increased up to 50 g/mL the Cr(VI) loss from the medium slowed down, but in all cases it reached 100% in about 10 days. High Cr(VI) concentration (70 200 g/mL) causes the diminution of Cr(VI) loss from the medium depending on Cr(VI) concentration, accompanied by a signicant reduction in bacterial survival. It follows from our data that 50 g/mL is already toxic for A. oxydans (bacterial survival essentially decreased), which coincides well with data for another Arthrobacter sp. reported by Megharaj et al. [22]. From our data the threshold concentration after which chromate displays a toxic effect on A. oxydans growth in liquid medium is 35 g/mL of Cr(VI). A typical bacterial growth curve is presented in Fig. 1A. The Cr(VI) loss in the culture medium depending on the growth phase was investigated. For this purpose chromate (35 g/mL of Cr(VI)) was introduced into the bacterial medium at different growth phases: along with the bacterium cell inoculation (0 h) in the liquid medium, at the middle of the logarithmic phase (16 h), and at the early stationary phase (24 h). The times of chromium introduction are indicated by arrows in Fig. 1A. The lowest rate of chromium loss took place in the bacterial medium when 35 g/mL of Cr(VI) was added along with the bacterium inoculation in the liquid medium (Fig. 1B). If chromium was added at the early stationary growth phase (24 h) or in the middle of the log phase (16 h), the

N.V. Asatiani et al.: Effect of CrVI on Arthrobacter oxydans Table 1. Survival of A. oxydans, and the Cr(VI) decrease in the culture medium under the different concentrations of Cr(VI) Cr(VI) concentration (g/mL) 10 20 35 50 70 100 200 Cr(VI) decrease (%) during an incubation time of: 1 day 100.0 70.0 42.8 26.0 31.4 10.0 25.0 6 days 100.0 97.5 74.3 74.0 62.8 30.0 23.0 10 days 100.0 100.0 100.0 99.0 64.2 40.0 20.0

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Survival (%) 82.90 81.50 89.20 9.10 0.85 2.15 1.52

Potassium chromate was added in the mid-log phase (16 h). Cells were grown at 21C. Survival of cells was determined by the colony forming ability of cells grown in Cr(VI)-containing medium and is represented as a percent of control.

Fig. 1. (A) Typical growth curve of A. oxydans grown without Cr(VI) as a batch culture. (B) Cr(VI) decrease in the growth medium after its addition at different growth phases of A. oxydans. Cr(VI) was added: () along with bacterial inoculation (0 h); () at the mid-log phase (16 h); () at the early stationary phase (24 h). 0-points to the Cr(VI) addition in the bacterial cell culture. The cells were grown at 21C. Data are the average of two sets of experiments.

Cr(VI) loss process was characterized by a higher rate. At earlier stages of Cr(VI) action (about 72 h) uptake corresponds to the cell number decrease in the stationary phase, namely the lowest cell density when Cr(VI) was added at the time of inoculation (0 h) and the highest cell density when Cr(VI) was added at the early stationary phase (24 h) (data not shown). The time required for complete loss (about 10 days) was almost independent of the growth phase at which chromium was added. Inuence of chromium on the protein composition of Arthrobacter oxydans. The inuence of cell growth time without medium replenishment on the protein composi-

tion was investigated. During long-term bacterial growth (168 h without chromium) it was observed that the protein composition was impoverished to some extent, which was accompanied by a decrease in the total protein concentration in crude cell extracts (data not shown). A similar result was observed for another species [20]. To estimate the inuence of chromium on protein composition 35 g/mL of Cr(VI) as potassium chromate was added at the same time points as above. The maximal duration of chromium action without medium replenishment was 144 h. The protein composition in response to chromium action was studied at different time

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CURRENT MICROBIOLOGY Vol. 49 (2004)

Fig. 2. The protein composition of A. oxydans in response to 35 g/mL of Cr(VI). (A) The total protein composition after 72 h of chromium action. Lane 1, cells grown without chromium for 72 h; lane 2, chromium was introduced along with the bacterial inoculation (0 h); lane 3, chromium was introduced at the mid-log phase (16 h); lane 4, chromium was introduced at the early stationary phase (24 h); lane 5, cells were grown without chromium for 96 h. (B). The total protein composition after 144 h of chromium action. Lane 1, chromium was introduced at the mid-log phase (16 h); lane 2, chromium was introduced at the early stationary phase (24 h); lane 3, cells were grown without chromium for 168 h. (C) The cell wall protein content in response to 35 g/mL of chromium for 72 h. Lane 1, cells grown without chromium for 96 h; lane 2, chromium was introduced along with the bacterial inoculation (0 h); lane 3, chromium was introduced at the mid-log phase (16 h); lane 4, chromium was introduced at the early stationary phase (24 h). Separation was performed on 10% SDS PAGE. Arrows indicate the major proteins increased in response to chromium action. Molecular weights were identied on a molecular size basis.

points. It should be noted that the critical differences were found for 72 and 144 h of Cr(VI) action (Fig. 2A, B). Figure 2A shows the protein composition of A. oxydans in response to chromium action for 72 h. The major protein fractions of A. oxydans cells that increased in response to chromium action are approximately 80 kDa and 60 kDa, regardless of the growth phase at which chromium was added. As was shown in our previous study, A. oxydans cell wall contains an acid-soluble protein with approximately 60 kDa molecular weight, identied on a molecular-size basis by SDS-PAGE. The charge of the protein was positive, as identied by urea polyacrylamide gel electrophoresis and capillary electrophoresis [1]. To elucidate the difference in 60 kDa protein quantity in relation to the growth-phase effect, acid extracts from the same quantity of the cells grown without and with chromium added at the different phases of the growth cycle, were compared by electrophoresis. The data presented in Fig. 2C show that the quantity of this protein increases in the cells grown with chromium in all cases. At the earlier

stages of Cr(VI) action (72 h), while the Cr(VI) uptake process probably depends on cell number (maximal uptake at Cr(VI) addition at the early stationary phase), the greatest increase in 60 kDa protein was achieved with Cr(VI) addition in the middle log phase, which is the phase characterized by the most metabolic activity. Figure 2B shows the protein composition during long-term exposure to Cr(VI) (144 h) after the addition of chromium in the middle log phase, and at the early stationary phase. Long-term Cr(VI) exposure leads to the formation of insoluble Cr(III) colloid chromium hydroxide on the bacterial surface, accompanied by changes in total protein composition. In spite of an impoverished protein composition, the cell wall protein with approximately 60 kDa molecular weight was quantitatively increased. It should be noted that the increase of this protein under long-term Cr(VI) action was greater (especially after Cr(VI) addition in the middle log phase, where it was the dominant protein) than under Cr(VI) action for 72 h. The cell wall protein increase is universal in all cases of A. oxydans. Besides cell wall protein, the

N.V. Asatiani et al.: Effect of CrVI on Arthrobacter oxydans

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protein fractions with approximately 55-40 kDa molecular weight are predominant (Fig. 2B). Given the Cr(III) colloid hydroxide production after long-term Cr(VI) exposure, these proteins may be connected with the efux system of reduced chromium forms [21]. According to an atomic absorption analysis published earlier [34], even long-term Cr(VI) action (10 days) did not lead to signicant elevation of the total chromium in A. oxydans cells washed from Cr(VI)containing medium. These data conrm previous results about the inuence of chromate on the biomolecules of the cell surface of A. oxydans obtained by synchrotron FTIR absorption spectroscopy [13, 14]. In addition, ESR signal intensity characterizing the Cr(V) complex form was previously found on the A. oxydans surface, which points to the cell surface as a major site of Cr(VI) reduction [1, 34], though it does not, exclude negligible penetration of Cr(V) into the cells and an anion-transport mechanism for (CrO4)2 [7,8,31]. Thermal analysis of the inuence of Cr(VI) on Arthrobacter oxydans. As is generally known, the reduction of Cr(VI) to Cr(III) is not a one-step process and in the case of Cr(VI) intracellular penetration the intermediate forms of Cr(VI) reduction, such as Cr(V /IV /III) including ROS, can produce CrDNA adducts, DNA DNA, and DNAprotein crosslinks, as usually occurs in eukaryotic cells [8,18, 37]. On the other hand, there is a complex multistep defense system to prevent and repair feasible damage in bacterial cells. Thermal analysis was used to identify any Cr(VI) inuence on the DNA protein (DNP) complex of A. oxydans. The thermal spectrum of A. oxydans presented in Fig. 3 shows that Cr(VI) causes changes in the form and intensity of the part of the thermal spectrum within the temperature range 40 90C. In this temperature range the intracellular structural cell elements (except the DNP complex) and structural cell wall elements melt [4]. As regards the melting of the DNP complex, taking place in the temperature range 90 110C, Cr(VI) does not inuence the thermal stability of the DNP complex up to 144 h of chromium action. The calorimetric data show that intermediate forms of reduced chromium including Cr(III) do not generate adducts with DNA components and carboxylic and thiol groups of proteins contributing to the DNP complex inside cells. Considering bacterial cell design, there are two basic systems of detoxication that are connected with processes proceeding intracellularly or on the cell surface including the cell wall, although in some conditions synergism of these processes is quite possible. According to our data, Arthrobacter oxydans can be numbered among the chromium-reducing bacteria which basically

Fig. 3. Melting spectra of A. oxydans in 50 mM Tris-HCl, pH 7.0, 100 mM NaCl, 0.1 mM PMSF, 0.1 mM benzamidine calculated per gram of dry weight. Scanning rate 0.60C/min. 1, cells without chromium; 2, cells grown under 35 g/mL of Cr(VI) in the culture medium for 144 h. Chromium was added at the mid-log phase (16 h).

achieve this process on the cell surface irrespective of the growth phase at which Cr(VI) is introduced.
ACKNOWLEDGMENT

This work was supported by a grant (G-348) from the International Science and Technology Center (ISTC).

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