ISSN 00036838, Applied Biochemistry and Microbiology, 2010, Vol. 46, No. 1, pp. 38–43. © Pleiades Publishing, Inc., 2010. Original Russian Text © O.I. Sizova, V.V. Kochetkov, A.M. Boronin, 2010, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2010, Vol. 46, No. 1, pp. 45–50.
Rhizosphere Bacteria Pseudomonas aureofaciens and Pseudomonas chlororaphis Oxidizing Naphthalene in the Presence of Arsenic O. I. Sizovaa, V. V. Kochetkova, b, and A. M. Boronina, b a
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5 Pushchino, Moscow oblast, 142290 Russia email:
[email protected] b Pushchino State University, pr. Nauki 3, Pushchino, Moscow oblast, 142290 Russia Received December 18, 2008
Abstract—Rhizosphere strains of P. aureofaciens BS1393(pBS216, pKS1) and P. chlororaphis PCL1391(pBS216, pKS1), exhibiting the ability to stimulate the growth of plants and protect them from phy topathogens, have been obtained. In these strains, plasmid pBS216 ensures naphthalene degradation and plasmid pKS1 confers resistance to arsenic. In the presence of arsenic and naphthalene, the number of living cells and the growth rate of the arsenicresistant strains were higher than those of the arsenicsensitive strains BS1393(pBS216) and PCL1391(pBS216). During the cultivation of the resistant strains, arsenic had no inhibitory effect on the activity of the key enzymes of naphthalene biodegradation, except for catechol2,3 dioxygenase. In a model system containing plant–microbial associations, strains BS1393(pBS216, pKS1) and PCL1391(pBS216, pKS1) degraded as much as 97% of added naphthalene in the presence of arsenic. DOI: 10.1134/S0003683810010060
arsenic and the ability to degrade PHAs, to stimulate plant growth, and to protect plants against soil patho gens, and studying their characteristics may be useful for developing the strategy for bioremediation of soils polluted with crude oil and oil products. Earlier, Siunova et al. [3] obtained a P. chlororaphis strain car rying the plasmids that confer resistance to cobalt and nickel and ensure naphthalene degradation. This mul tiplasmid strain, in contrast to the sensitive strain, effi ciently degraded naphthalene in the presence of nickel. Published data on the strains that are able to degrade xenobiotics in the presence of arsenic are, as of yet, missing. The goals of this work was to obtain and character ize arsenicresistant strains of rhizosphere bacteria of the Pseudomonas genus that are able to degrade PAHs, to study the effect of arsenic on the activity of the key enzymes of PAH degradation, and to assess the effi ciency of naphthalene degradation by these strains in a model plant–microbial association.
INTRODUCTION Currently, complex pollution of soils with organic pollutants and metals (metalloids) is a serious prob lem. Soils polluted with polycyclic aromatic hydrocar bons (PAHs) and arsenic occur in oil field develop ment areas as well as on agricultural lands, plants of which were treated with arseniccontaining pesticides, herbicides, and defoliants. On chemical weapon destruction polygons, soils are polluted with mustard gas degradation products; such areas are often con comitantly polluted with oil [1, 2]. Waste rock piles produced as a result of gold mining operations contain arsenic and are polluted with PAHs because fuels and lubricants get into soil. In the case of complex soil pol lution, the number of rhizosphere bacteria decrease, the content of microbial populations changes, and the number of phytopathogenic fungi increases. The pos sibility of using microbial–plant associations is con sidered as one of phytoremediation strategies aimed at fighting against complex soil pollution. We propose to use associations of plants with plant growthstimulat ing rhizosphere bacteria (PGSRB) of the Pseudomonas genus. Obtaining PGSRB strains combining several properties in one strain, such as the resistance to
MATERIALS AND METHODS Bacterial strains. PGSRB used in this study were the rhizosphere strain P. aureofaciens BS1393 from the laboratory collection and the rhizosphere strain P. chlororaphis PCL1391, which was kindly provided by Dr. Lugtenberg (Leiden, Holland). These strains produce various antibiotics and exhibit a high antago nistic activity with respect to a broad spectrum of phy
Abbreviations: PAHs is polycyclic aromatic hydrocarbons, PGSRB is plant growthstimulating rhizosphere bacteria, MIC is minimum inhibitory concentration, MTC is maximum tolerated concentration, OD is optical density, NDO is naphthalene dioxy genase, SH is salicylate hydroxylase, C1,2O is catechol 1,2dioxy genase, C2,3O is catechol 2,3dioxygenase.
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RHIZOSPHERE BACTERIA Pseudomonas aureofaciens
topathogenic fungi and bacteria. In this study, we used the previously obtained variants of these strains con taining the naphthalene degradation plasmid pBS216 [4]. Plasmid pBS216 contains the nah operon, which allows the strain to grow on naphthalene and salicylate as the only sources of carbon and energy. We also used the previously obtained recombinant plasmid pKS1, which confers resistance to arsenic. This plasmid con tains the arsenic resistance operon (arsRBC) cloned into the pUCP22 vector [5]. Culture media and cultivation conditions. Strains were cultivated in M11 mineral medium containing 1 g/l (NH4)2SO4, 2 g/l KH2PO4, 1 g/l K2HPO4, 0.1 g/l NaCl, and 0.5 g/l NaHCO3 or in Luria–Bertani (LB) nutrient medium [6] containing 10.0 g/l tryptone, 5.0 g/l yeast extract, and 10.0 g/l NaCl. Glucose (2 g/l) or naphthalene (1 g/l) was added to the medium as a carbon and energy source. Arsenic in the form of sodium arsenite (NaAsO2) was added at a con centration of 0.5 mM. Bacteria were cultured at 24°С in Petri dishes or in a liquid medium in Erlenmeyer shake flasks (150 rpm). Seed sterility was monitored on King B medium containing 20 g/l peptone, 10 g/l glycerol, 1.5 g/l K2HPO4, 1.5 g/l MgSO4 ⋅ 7H2O, and 15 g/l agar. Plants were cultivated in sterile model sys tems in Murashige–Skoog mineral medium (Sigma, United States) [7] containing no amino acids and sac charides. Cell transformation with plasmid DNA. Cells were transformed with plasmid DNA as described earlier [4]. The transformation frequency was determined as the number of plasmidcontaining clones per 1 μg of plasmid DNA. Isolation of plasmid DNA. Plasmid DNA was iso lated from the strains by alkaline lysis as modified by Birnboim and Doly [6]. Determination of physiological parameters of bac terial growth. The growth kinetics of a batch culture of microorganisms cultivated in flasks with the mineral medium (200 ml) in the presence of naphthalene (1 g/l) was studied as described in [8]. Plasmid stability. To determine plasmid stability, successive passages of the plasmidcarrying strains in the glucosecontaining liquid mineral medium were performed as described earlier [9]. The stability of plasmids in the rhizosphere of plants was determined on day 7. For this purpose, the root of one plant was taken and washed with 10 ml of saline. Thus, obtained liquid was used to prepare serial dilutions and subse quent plating on agar LB medium. Then, individual bacterial colonies were replated on selective media using a replicator. Plasmid stability was determined as the ratio of the number of grown bacterial cells to the total number of cells and expressed in percent. Determination of the minimum inhibitory concen tration (MIC) and the maximum tolerable concentra tion (MTC) of arsenic. MIC and MTC of arsenic for APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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the strains used in the study was determined in the mineral medium as described earlier [10]. Determination of enzyme activity. Enzyme activity was determined in cellfree extracts with a UV160A spectrophotometer (Shimadzu, Japan). Cellfree extracts for determination of enzyme activity were obtained by destroying a frozen biomass with a press constructed at the Institute of Biochemistry and Phys iology of Microorganisms (Russia). The undestroyed cells and cell debris were precipitated by centrifuga tion at 16000 g for 60 min at 0°С in a Beckman J221 centrifuge (Beckman, United States). The activity of naphthalene dioxygenase (NDO) and salicylate hydroxylase (SH) was determined by a decrease in the NADH absorption at 340 nm (ε = 6.22 μM cm–1) [11, 12]. The activity of catechol 2,3dioxygenase (C2,3O) was determined by the rate of αhydroxymuconic semialdehyde formation at 375 nm (ε = 33.4 μM cm–1); the activity of catechol 1,2dioxygenase (C1,2O) was determined by the rate of ciscismuconate formation at 260 nm (ε = 16.9 μM cm–1) [13]. The specific activ ity of enzymes was expressed in nmoles of the cofactor utilized in 1 min or the reaction product formed in 1 min per 1 mg of total bacterial protein, which was determined spectrophotometrically [14]. Seed sterilization and seedling bacterization. Rape (Brassica napus ssp. Oleifera L.) seeds were sterilized with 10% sodium hypochlorite for 60 min and, then, washed four times with sterilized tap water for 2 h. The seeds were placed on agar King B medium and incu bated at 24°С for 48 h to control seed sterility. Sterile seeds (seedlings) were inoculated for 15 min in a sus pension of strains at the exponential growth phase at a density of approximately 108 CFU/ml. Microvegetation experiment in a model system under sterile conditions. The effect of polyfunctional strains on rape growth was studied in the sterile system described earlier by Simons et al. [15]. Seedlings were grown in closed plastic flasks (77 × 77 × 97 mm; Sigma, United States) in 150 g of sand at a 10% humidity. Naphthalene was added to a concentration of 200 mg/kg sand. Arsenic was added in the form of sodium arsenite solution to a concentration of 15 mg/kg sand. Mineral nutrition of plants was pro vided using Murashige–Skoog medium. The plants grown in the presence of naphthalene and without inoc ulation were used as a control. Twenty seedlings were planted in one pot. The plants were grown under 12 h light/12 h dark regime at 20 and 10°С, respectively. Determination of naphthalene content in samples. The residual naphthalene content in the model sys tems with plants was determined on day 7. For this purpose, the entire content of plastic flask was extracted with 100 ml of methanol for 1 day under periodic stirring. Then, 1ml aliquots of the extract were taken and the content of naphthalene in them was determined by HPLC in a C18 column (Nova Pak Waters, United Kingdom) in the methanol–water sys tem at a flow rate of 1 ml/min under a UV detector
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Table 1. Growth characteristics of strains cultured under different conditions Naphthalenecontaining medium
Naphthalene and arseniccontaining medium
Strain lag phase, h
µ, h–1
lag phase, h
µ, h–1
BS1393(pBS216)
—
0.30
24
0.2
PCL1393(pBS216)
—
0.28
24
0.18
BS1393(pBS216, pKS1)
—
0.28
4
0.25
PCL1393(pBS216, pKS1)
—
0.26
2
0.26
(λ = 254 nm). Naphthalene concentration was calcu lated by the peak area of the experimental sample referred to the peak area of the control sample. Statistical data processing. Data were processed using the Stadia statistical program package [16]. All experiments were performed in triplicate. RESULTS AND DISCUSSION Obtaining polyfunctional strains P. chlororaphis PCL1391 and P. aureofaciens BS1393. To obtain polyfunctional strains able to degrade naphthalene in the presence of arsenic, the previously obtained strains P. chlororaphis PCL1391(pBS216) and P. aureofaciens BS1393(pBS216) were transformed with the arsenic resistance plasmid pKS1. The transformants BS1393(pBS216, pKS1) and PCL1391(pBS216, pKS1) were able to grow in the presence of arsenic in a min eral medium containing naphthalene as the only source of carbon. Plasmid DNA contained in these strains was isolated and analyzed by electrophoresis. The electrophoregram confirmed the presence of two plasmids in the transformants (data not shown). MIC and MTC of arsenic for the obtained strains. The study of the resistance of the resultant strains to arsenic showed that strain P. chlororaphis PCL1391(pBS216, pKS1) was more resistant than strain P. aureofaciens BS1393(pBS216, pKS1). The MIC and MTC of arsenic for strain BS1393(pBS216, pKS1) were 15 and 2 mm, respectively, and 17 and 8 mM, respectively, for strain PCL1391(pBS216, pKS1). This difference is determined by the higher resistance of the initial strain PCL1391 to arsenic compared to strain BS1393. Assessment of plasmid stability in strains. The sta bility of the naphthalene biodegradation plasmid pBS216 and the arsenic resistance plasmid pKS1 depended on the host strain. The plasmids were more stable in the strain PCL1391 variants. Apparently, strain PCL1391 is a more preferred host for both plas mids. When strains were cultured in the mineral medium, plasmid pBS216 was stably maintained in strains PCL1391(pBS216) and PCL1391(pBS216, pKS1) for seven passages. In strain BS1393, approximately 50%
of the cell population retained plasmid pBS216 after 7 days of culturing. The presence of the second plasmid, pKS1, in strain BS1393(pBS216, pKS1) decreased the stability of plasmid pBS216 in this strain. The stability of plasmid pKS1 in strains BS1393(pBS216, pKS1) and PCL1391(pBS216, pKS1) after seven passages accounted for 5 and 25%, respectively. The higher sta bility of plasmid pBS216 compared to plasmid pKS1 is due to the fact that pBS216 is a natural plasmid found in the P. putida strain. The stability of plasmids (espe cially pKS1) in the rhizosphere of plants was consider ably higher, which was associated with the presence in the medium of selection pressure (arsenic and naph thalene). As much as 90% of strain cells retained plas mid pBS216. The stability of plasmid pKS1 in the rhizosphere of plants was 70% after seven days of cul turing. Strain growth in the mineral medium. When cul tured on naphthalene, all strains grew at approxi mately the same rate (μ = 26–30 h–1) (Table 1); how ever, the variants of strains carrying two plasmids grew somewhat more slowly. The experiments on selection of arsenic inhibitory concentrations in the mineral medium showed that, even at a low concentration (0.5 mM), arsenic had a strong inhibitory effect on the strain growth. Arsenic significantly inhibited the growth of sensitive strains. The latter had a long lag phase (24 h), during which the number of bacteria drastically decreased (see figure), and grew more slowly than the resistant strains. Further increase in the growth rate and in the CFU number was appar ently due to the fact that, in the course of culturing, arsenic bound to certain components in the medium and dead cells and, hence, had no toxic effect of the bacterial cells. The arsenicresistant strains had a short lag phase (2–4 h) followed by the exponential growth phase, which was apparently caused by the ars operon induction. Activity of the key enzymes of naphthalene catabo lism. The activities of the enzymes of naphthalene catabolism in the plasmidcarrying Pseudomonas strains were measured at two points—in the exponen tial growth phase (OD 0.3) and in the stationary phase
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RHIZOSPHERE BACTERIA Pseudomonas aureofaciens
(OD 0.6). The results of experiments showed that all strains cultured in the naphthalenecontaining medium had similar levels of salicylate hydroxylase (SH), catechol 1,2dioxygenase (C1,2O), and cate chol 2,3dioxygenase (C2,3O) activities irrespective of the growth phase (Table 2). An exception to this ten dency was naphthalene dioxygenase (NDO), whose activity was higher in the twoplasmid strains in the exponential growth phase. However, this pattern changed in the presence of arsenic in the culture medium. Arsenic had an inhibitory effect on the activ ity of the key enzymes of naphthalene degradation in the arsenicsensitive strains. The activity of NDO, the first enzyme in the naphthalene mineralization path way, was not detected in the stationary growth phase. The activity of SH in this growth phase was also absent. The activity of C1,2O was recorded in both phases but was very low. In the arsenicresistant strains, arsenic had no significant inhibitory effect on the activity of these enzymes. The activity of NDO and SH remained at the same level that was observed during culturing in the presence of naphthalene regardless of the growth phase. In the arseniccontain ing medium, C2,3O activity was absent in all strains irrespective of the growth phase. However, when arsenic was added to the medium containing the cell free extract of the strain grown in the presence of naphthalene immediately before the measurement of activity, no inhibition of C2,3O activity was observed. Salicylate degradation genes are under the control of one promoter and are transcribed sequentially. There fore, if the activity of SH, the product of the first gene nahG in the nah operon, is detected, the other genes are also transcribed, including the nahH gene encod ing C2,3O. Apparently, arsenic has a toxic effect on C2,3O at the stage of formation of the active protein form. However, arsenic had no inhibitory effect on C1,2O. Earlier, it was shown in our laboratory that heavy metals (nickel and cobalt) have an inhibitory effect on the activity of the key enzymes of naphthalene degra dation [4]. When nickel was added to the culture medium, the activity of NDO, SH, C1,2O, and C2,3O in the nickelsensitive P. chlororaphis strain was recorded in the exponential growth phase but was absent in the stationary phase. In the resistant strain, the activity of NDO and C1,2O was absent in the sta tionary phase but was detected in the exponential growth phase. In our experiments, the resistant strains retained NDO and SH activity when cultured in the presence of arsenic, and the absence of activity of C2,3O, the product of a gene located on plasmid pBS216, was compensated by the activity of C1,2O, the product of a chromosomal gene. Assessment of naphthalene degradation by bacteria in the model system containing plant–microbial associ ations. The analysis of the content of naphthalene in the model system after the end of the experiment showed that up to 50% of added naphthalene was vol APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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CFU/ml 1.0E+10
1
1.0E+09
2 1.0E+08 3 1.0E+07
4
1.0E+06
1.0E+05
0
12
24
30
48
72
95 h
Strain growth in the naphthalenecontaining medium in the presence of arsenic (CFU/ml): (1) P. chlororaphis PCL1391(pBS216, pKS1), (2) P. aureofaciens BS1393 (pBS216, pKS1), (3) P. aureofaciens BS1393(pBS216), and (4) P. chlororaphis PCL1391(pBS216).
atilized naturally during the experiment (Table 3). There are published data demonstrating that plants are able to consume and metabolize a number of organic compounds including pesticides and herbicides as well as aliphatic, monocyclic, and polycyclic hydrocar bons. The ability of plants to degrade compounds con taining aromatic rings was demonstrated in experi ments with plants grown under field and sterile condi tions [1]. However, the contribution of plants to naphthalene mineralization in soils experiencing complex pollution with PAHs and heavy metals (met alloids) is insignificant, which was confirmed in our experiments in the naphthalenecontaining medium. When plants were grown without inoculation with the microorganisms, the content of naphthalene in sand after the end of experiment was comparable to that in the control. When rape plants were inoculated with the plas midfree strains, the residual naphthalene content after the end of the experiment was also comparable to that in the control. Apparently, the presence of these strains in the substrate somehow prevents naphthalene volatilization owing to its binding by the bacteria and their metabolites. The inoculation of plants with the strains carrying the pBS216 plasmid significantly reduced the residual naphthalene content in sand. When the experiment was finished, the content of naphthalene in these samples accounted for 8–10% of the control. In the experiments with sand containing both naphthalene and arsenic, the residual content of
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Table 2. Activity (nmol/min per mg protein) of the key en zymes of naphthalene biodegradation in strains cultured un der different conditions Strain Enzyme
OD
BS1393 PCL1391 BS1393 PCL1391 (pBS216, (pBS216, (pBS216) (pBS216) pKS1) pKS1)
Naphthalenecontaining medium NDO 0.3* 2.2 2.1 13.6 15.6 0.6** 2.2 5.6 6.1 2.8 SH 0.3 8.9 8.2 10.2 9 0.6 5.6 1.9 5.7 1.2 C1,2O 0.3 34.5 36.4 28.8 41 0.6 29 20.6 27.5 18 C2,3O 0.3 1.7 1.5 3.8 4.6 0.6 2.2 5.6 4.3 2 Naphthalene and arseniccontaining medium NDO 0.3 8.5 12.1 9.2 11.2 0.6 0 0 3.1 6.7 SH 0.3 8.5 4.1 26.4 25 0.6 0 0 1.2 1.5 C1,2O 0.3 1.6 5.6 28.4 23.8 0.6 1.1 3.1 1.15 3.50 C2,3O 0.3 0 0 0 0 0.6 0 0 0 0 Notes: * An optical density of 0.3 corresponds to the midexponen tial growth phase. ** An optical density of 0.6 corresponds to the stationary growth phase.
naphthalene in the variants with the arsenicsensitive strains was higher than in the experiments with sand containing only naphthalene, which was due to the toxic effect of arsenic on these bacteria. However, when plants were inoculated with the arsenicresistant
polyfunctional strains, the residual content of naph thalene in sand accounted for only 3–3.5% of the ini tial naphthalene content, which testifies to the effi ciency of the function of these strains under the com plex pollution conditions. Earlier, Springael et al. [17] showed that the Alcali genes eutrophus strain carrying a metal resistance plas mid and a plasmid that provides biodegradation of polychlorobiphenyls and 2,4dichloroacetic acid also degraded these antibiotics in the presence of nickel or zinc more efficiently than the sensitive strain. Our results demonstrate the possibility of combin ing the PAH degradation plasmid and the arsenic resistance plasmid in P. chlororaphis and P. aureofaciens strains. Although pKS1 plasmid showed low stability under nonselective conditions, it was sufficiently sta bly maintained by the bacterial population growing in plant rhizosphere in the presence of arsenic. During cultivation in the arseniccontaining medium, the activities of the key enzymes of naphthalene degrada tion in strains BS1393(pBS216, pKS1) and PCL1391(pBS216, pKS1) remained at a high level. The arsenicresistant polyfunctional strains capable of degrading naphthalene have a selective advantage in plant rhizosphere under conditions of complex sub strate pollution and degrade up to 97% of added naph thalene in the model system containing plant–micro bial associations. ACKNOWLEDGMENTS This study was supported by the Development of Scientific Potential of Higher School departmental scientific program (project 2.1.1/4341): Coupling of Microbial Processes of Degradation of Oil Hydrocar bons and Mineralization of Soil Organic Matter as a Factor of Disturbance of Natural Soil Formation under Conditions of Oil Pollution of Soils, and the Innovation and Development Support specialpur pose program of the Presidium of the Russian Acad emy of Sciences and also by FCP state contract no. 02.512.11.23.37.
Table 3. Naphthalene content in samples after the end of experiment Naphthalene, mg/kg Variant Control (start point) Control (end point) Plants without bacterization BS1393 BS1393(pBS216) BS1393(pBS216, pKS1) PCL1391 PCL1391(pBS216) PCL1391(pBS216, pKS1)
Naphthalenecontaining substrate
Naphthalene and arseniccontaining substrate 196.4
91.0 95.66 97.15 9.85 6.78 103.3 8.56 7.13 APPLIED BIOCHEMISTRY AND MICROBIOLOGY
91.15 97.0 119.0 27.9 2.77 106.2 14.1 3.21 Vol. 46
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RHIZOSPHERE BACTERIA Pseudomonas aureofaciens
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