ISSN 0003-6838, Applied Biochemistry and Microbiology, 2008, Vol. 44, No. 3, pp. 276–281. © Pleiades Publishing, Inc., 2008. Original Russian Text © A.T. Adylova, T.N. Chernikova, A.A. Abdukarimov, 2008, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 3, pp. 308–313.
Phenol Biodegradation by a Pseudomonas sp. Strain Tagged with the gfp Gene A. T. Adylova, T. N. Chernikova, and A. A. Abdukarimov Institute of Plant Genetics and Experimental Biology, Academy of Sciences of Uzbekistan, Yukori-Yuz Village, Tashkent oblast', 702151 Uzbekistan email:
[email protected] Received December 28, 2006
Abstract—Conjugal transfer of the pAG408 suicide vector from E. coli S17-1 to Pseudomonas sp. cells able to consume phenol yielded transconjugates brightly luminescing under UV illumination. It was shown that tagging of the Pseudomonas sp. cells with the gfp gene did not affect their ability to consume phenol. The change of the population density of the tagged bacteria after their introduction to soil was studied. The potential of the resulting bacterial strain in remediation of phenol-polluted soils is discussed. DOI: 10.1134/S0003683808030083
Many environment protection problems caused by chemical pesticides and industrial sewage can be solved by employing the ability of microorganisms to degrade xenobiotics [1–3]. The potential of methods involving biological degradation of pollutants is broadly extended by application of microorganisms tagged with marker genes, which facilitate their monitoring in target ecosystems without affecting their degrading ability. Advantages of using a bioluminescent label for examination of the behavior of bacterial strains under conditions close to natural have been demonstrated for some bacterial plant pathogens and their antagonists [4, 5]. The gfp gene is finding increasing use for tagging microbial degraders [6]. The advantages of this marker in comparison with other genes (antibiotic-resistance genes, the lux gene, etc.) are its visualization without special substrates, safety of the gene product for the host cell, simple detection of microorganisms harboring the gene (luminescence under long-wave UV illumination), and the possibility of quantitative assessment of bacterial population density without isolating the bacteria [7, 8]. Formerly, we studied bacteria consuming phenol as the sole carbon and energy source [9, 10]. The reason for choosing phenol-degrading bacteria was that phenol belonged to the most common pollutants of surface waters, which are released to the environment in the course of anthropogenic industrial and agricultural activity. In addition, phenol and its metabolite, pyrocatechol, are among major intermediate products of processing of most aromatic pesticides by soil bacteria, which adds to phenol accumulation in soil. These facts make phenol pollution a challenge. Therefore, it is reasonable to introduce relevant bacteria to natural ecosystems.
The objectives of the present study are tagging of a phenol-degrading strain with the gfp gene, investigation of the viability and activity of these bacteria after their introduction to soil, and test of the possibility of their application to remediation of phenol-polluted areas. MATERIALS AND METHODS Bacterial strains and growth conditions. Experiments were performed with the following bacterial strains: Escherichia coli S17-1 λ-pir (pAG408), E. coli HB101 (RK600) [6], and Pseudomonas sp. (PhÂ+) [9, 10]. Escherichia coli cells were grown in LB medium [11] supplemented with antibiotics. The final antibiotic concentrations (mg/l) were the following: ampicillin, 100; kanamycin, 50; chloramphenicol, 30. The Pseudomonas sp. strain was grown in minimal M9 medium [11] supplemented with phenol as a carbon source or in the pseudomonad medium of the following composition (g/100 ml): K2HPO4, 0.1: KH2PO4, 0.1; NaCl, 0.02; MgSO4, 0.005; NH4NO3, 0.05; peptone, 0.2; glucose, 0.2; and FeSO4, traces; pH 7.5. Phenol was added either by direct inoculation into the medium or soil at the ratio 300 mg/l or 1 g/kg, respectively, or by incubation of bacteria in phenol vapor, applying phenol crystals onto the inner surfaces of Petri dish lids. Soil was inoculated with suspended cells sampled at the log phase of growth (density 107 cfu/g soil). Experiments with soil were carried out at 20–25°ë with regular loosening and watering. Conjugal mating of the recipient and donor cells was carried out as in [12]. Escherichia coli S17-1 λ-pir (pAG408) Ampr, Kmr, and E. coli HB101 (RK600) Cmr kindly provided by Dr. Timmis (Germany) [6] were used as donors in the experiments on pAG408 transfer to pseudomonads. Pseudomonas sp. (PhÂ+) strain from
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the collection of the Institute of Plant Genetics and Experimental Biology, AS Uzbekistan [9, 10] was used as a recipient. The donor, recipient, and helper (harboring a mobilizing plasmid) strains were grown in 5 ml of liquid LB medium for 16 h. For biparental mating, the donor cell suspension was diluted tenfold and mixed with the equal volume of the recipient cell suspension. For triparental mating, suspensions of the donor, recipient, and helper strains were mixed at the ratio 4 : 1 : 1. The conjugal mixture (0.2 ml) was inoculated to 0.22 µm Millipore filters (United States) placed on the surface of LB agar medium. After 16 h of incubation, bacteria were washed from the filters with 0.85% NaCl. The resulting suspension was appropriately diluted and inoculated to dishes with minimal M9 medium supplemented with antibiotics. Luminescent conjugates were selected by their resistance to ampicillin, kanamycin, chloramphenicol, and phenol. The following antibiotic concentrations (mg/l) were used in conjugal transfer experiments: ampicillin, 100; kanamycin, 50; and chloramphenicol, 30. Luminescence was induced by UV illumination with an M20 transilluminator (Appligene, United States). Conjugal transfer rate was determined as the ratio between the number of transconjugates and the number of donor cells. Phenol assay in culture liquid and soil. Phenol concentration in the culture liquid was determined by HPLC in a 0.46 × 25-cm C18 Ultrasphere reverse-phase column (Beckman, United States) with a Beckman Gold System 110 chromatograph equipped with software for result processing. Chromatography conditions: mobile phase 70% methanol with 0.1% trifluoroacetic acid; flow rate 1 ml/min; detection at 278 nm. Samples were injected under formerly developed standard conditions. One arbitrary unit in the chromatogram corresponds to 440 mg/l phenol. To determine bacterial population densities, 1-g soil samples were mixed with 3 ml of sterile 0.85% aqueous NaCl and extracted for 1 h. End-point dilutions were inoculated to M9 supplemented with phenol and antibiotics depending on experiment conditions. Phenol was added in one portion. For phenol analysis, a 1-g soil sample was extracted with 10 ml of methanol at vigorous agitation for 1 h. The extract was cleared by centrifugation, and phenol was assayed in the supernatant by HPLC. The change in phenol content was determined by comparing peak areas in the experiment and control. All experiments were done in triplicate. RESULTS AND DISCUSSION Conjugal mating of Pseudomonas sp. with donor cells. Formerly, we reported isolation of 16 strains resistant to high (up to 1 g/l) phenol concentrations from phenol industry sewage [9]. These strains consumed 30–40% of phenol depending on starting concentrations. Six of these strains were assigned to the genus Pseudomonas according to their morphological, cultural, and physiobiochemical traits. Study of genetic APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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Fig. 1. Colonies of Pseudomonas sp.1 cells tagged with the gfp gene. The photograph was taken under UV illumination in darkness.
features of the selected pseudomonad strains showed that four strains contained plasmids with molecular weights from 20 to 85 kb [10]. As the location of the character under study in plasmids threatens its loss under nonselective conditions, we chose for manipulations a plasmidless Pseudomonas sp. strain, which retained its phenol-degrading potential throughout multiple passages in rich medium. This strain was tagged with the pAG408 plasmid [6]. This vector is a suicide plasmid derived from a mini-Tn5 transposon. It is maintained in a special E. coli strain, S17-1 λ-pir. These cells do not luminesce by themselves, but they serve as donors for transferring the gfp gene to recipient strains by bacterial cell mating. In our experiments on biparental mating, the rate of gfp insertion into Pseudomonas sp. cells was about 10–8 per recipient cell. The presence of the HB101 helper, harboring the pRK600 plasmid with the mob gene, increased this index by nearly two orders of magnitude. A wide range of phenol- and antibiotic-resistant colonies was obtained. They varied in luminescence intensity, and there were colonies producing a bright green glow (Fig. 1). Further studies were performed just with these strains. Phenol consumption by tagged Pseudomonas sp. cells in liquid culture and in soil. It was necessary to find out whether the introduction of the gfp gene into the Pseudomonas sp. genome preserved the ability of the bacteria to degrade phenol. Figure 2 presents the results of chromatographic assay of phenol in the culture medium after 1 day of the experiment (Fig. 2a) and 4 days after growth of the tagged (Fig. 2b) and the initial pseudomonad strains. The corresponding peak areas calculated with appropriate software were 6.74711, 4.27286, and 4.19175, respectively. We deduced from these results that the selected Pseudomonas sp. transconjugate grown in minimal medium with phenol as the sole carbon source could consume phenol (Figs. 2a and 2b) approximately within the same range
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–0.0010 0.05 1.08 2.11 3.13 4.16 5.19 6.22 7.24 8.27 0.57 1.59 2.62 3.46 4.67 5.70 6.73 7.76 8.79 min Fig. 2. Chromatographic assay of phenol in the culture liquid; a, 1 day of cell growth; b, 4 days of transconjugate growth; c, 4 days of original strain growth. APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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Fig. 3. Kinetics of phenol degradation (%) in soil after Pseudomonas sp.1 introduction; 1, control (without bacterial inoculation); 2, experiment (with inoculation).
as the initial strain (Figs. 2b and 2c). In both experiments, after 4 days of growth, phenol concentration decreased approximately by 30–40% in comparison with the concentration on the first day of the experiment. Phenol loss in the control medium without bacteria within the same time was 3–5%, probably, owing to evaporation. Assessment of the effect of introduction of tagged bacteria on the degree of soil purification from phenol showed that the difference in phenol content between the control (no inoculation) and experimental (with bacteria) samples appeared after 5 days of the experiment (Fig. 3). After 10 days, phenol content in inoculated soil was 24% in comparison with 58% in the control. After 20 days, only traces of phenol were detected in soil with bacteria. In the control soil sample, phenol loss was less than 60%. Notable phenol concentrations were present in the control soil even after 25–30 days of the experiment. Owing to adsorption of part of the pollutant by soil and the resulting decrease in its accessibility for bacteria, biodegradation rate in soil was expected to be lower than in liquid medium. Although highly toxic, phenol does not belong to highly resistant chemicals. Such physical factors as evaporation, soil moistening, and temperature add to self-remediation of soil from phenol. Also, soil contains enzymes (dehydrogenases, peroxidases, and polyphenol oxidases) performing degradation and condensation of aromatic compounds and converting them to humus components. Finally, aboriginal soil microflora can participate in aromatic compound degradation [13]. Apparently, the combination of these factors may be responsible for phenol loss in the control soil sample (not inoculated with bacteria). APPLIED BIOCHEMISTRY AND MICROBIOLOGY
In this context, the loss of phenol in the experimental sample is considered to be a combined effect of the introduced bacteria and the listed biotic and abiotic factors. The population density of tagged bacteria inoculated to soil. Three experimental variants of transconjugate introduction to soil were performed: (1) inoculation of tagged cells to sterile (autoclaved) soil, (2) inoculation to nonsterile soil, and (3) inoculation to nonsterile soil with simultaneous addition of phenol. Numbers of colony-forming units were determined in soil after 5, 10, and 25 days of the experiment. Examination of the microflora of soil extracts showed that in the first experimental variant the density Kinetics of the population density of phenol-degrading bacteria tagged with the gfp gene in soil cfu × 107/g soil Experimental variant 5 days
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Fig. 4. Soil microflora 30 days after the inoculation of the transconjugate tagged with the gfp gene: a, in daylight; b, in darkness under UV illumination.
of tagged cell population in soil increased from the starting value 107 cfu/g soil to 7.8 × 107 cfu/g (table). With phenol, population density increased to 1.4 × 108 cfu/g, that is, by a factor of 14. However, bacterial population density in the control soil sample without phenol was lower than with phenol by one or two orders of magnitude throughout the experiment. The correlation of the kinetics of the population density of tagged bacteria with phenol presence brought us to some assumptions. Apparently, phenol produces selective conditions for growth and reproduction of phenol-degrading bacteria. This can explain the elevated density of luminescent cells in the first 5–10 days of growth of tagged bacteria in soil (third variant of the experiment). It is likely that the decrease in phenol concentration starting from the 10th day of the experiment activates the aboriginal microflora of soil, formerly suppressed by high phenol concentrations. The decrease in the density of introduced cells in the subsequent 15 days may have resulted from their decreasing competitive ability with regard to the aboriginal microflora. The inability of the tagged bacteria to increase their biomass in nonsterile soil without phenol (second variant) also seems to be related to its relatively low viability with the presence of aboriginal soil microflora. However, cells tagged with the gfp gene were detected in soil extract as late as 30 days after inoculation (Figs. 4a and 4b). Thus, in spite of the tendency for decreasing density of tagged cell population in soil, comparison of phenol contents in the experimental and control (without inoculation) soil samples points to a beneficial effect of the inoculation on soil remediation, manifesting itself as the shorter time of phenol removal.
Another result of our study is that the presence of the reporter gfp gene allowed monitoring of tagged bacteria without their isolation from the medium and provided information on their viability under conditions close to natural. It should be noted that the pAG408 vector used in our work had been designed for inserting the gfp gene mainly to chromosomes of Gram-negative bacteria [6]. We did not perform special studies to localize gfp in the transconjugate genomes. However, some experimental observations, in particular, the presence of a wide range of phenol- and antibiotic-resistant colonies differing in luminescence rate and the ability of cells to luminesce both under selective conditions and in complete LB medium, point to chromosomal location of gfp in the genomes of our transconjugates. Our results will allow development of practical recommendations on the application of bacterial strains tagged with the gfp gene to bioremediation of phenolpolluted soils. REFERENCES 1. Vel’kov, V.V., Biotekhnologiya, 1995, nos. 3−4, pp. 20−27. 2. Khabibulina, F.M, Shubakov, A.A., Archegova, I.B., and Romanov, G.G., Biotekhnologiya, 2002, no. 6, pp. 57–67. 3. Anokhina, T.O., Kochetkov, V.V., Zelenkova, N.F., Balakshina, V.V., and Boronin, A.M., Prikl. Biokhim. Mikrobiol., 2004, vol. 40, no. 6, pp. 654–658. 4. Shaw, J.J. and Kado, C.C.I., Biotechnology, 1986, vol. 4, no. 6, pp. 560–564. 5. Burmakina, S.I., Avdienko, I.D., Ismailov, Z.F., Miroshnikova, L.K., Chernin, L.S., and Stepanov, A.I., Biotekhnologiya, 1993, vol. 9, no. 1, pp. 35–38.
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10. Chernikova, T.N., Adylova, A.T., and Abdukarimov, A.A., Uzb. Biol. Zh., 1999, no. 1, pp. 7–10. 11. Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York: Cold Spring Harbor Lab., 1989. 12. Dunn, N.W. and Gunsalus, I.C., J. Bacteriol., 1973, vol. 114, no. 3, pp. 974–982. 13. Rabinovich, M.L., Bolobova, A.V., and Vasil’chenko, L.G., Prikl. Biokhim. Mikrobiol., 2004, vol. 4, no. 1, pp. 5–23.
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