Romanian Biotechnological Letters Copyright 2009 Bucharest University Romanian Society of Biological Sciences

Vol. 14, No. 6, 2009, pp. 4779-4785 Printed in Romania. All rights reserved ORIGINAL PAPER

Phylogenetic Analysis on 16S Ribosomal DNA of Pseudomonas Strains from Oil Polluted Soil
Received for publication, April 1, 2009 Accepted, October 20, 2009 ANA-MARIA TANASE1, CRISTINA TRASCA3, TATIANA VASSU1, ALEXANDRU OLTEANU4, DIANA PELINESCU1, ORTANSA CSUTAK1, IONESCU ROBERTINA2, ILEANA STOICA1*. 1Faculty of Biology, University of Bucharest, Bucharest, Romania 2MICROGEN, University of Bucharest, Bucharest, Romania 3Max Plank Institute for Marine Microbiology, Bremen, Germany 4Politechniq University, Bucharest, Romania *Author to whom correspondence should be addressed: E-Mail; ileana@botanic.unibuc.ro; Tel: + 40213118077

Abstract
In the current study we report molecular analysis of two bacterial isolates from an oil-polluted soil sample taken from the surroundings of an oil extraction field near Pitesti. This study focuses on two strains designated SQ2a and, respectively, SQ2b, because of their similar behave but very different colonies morphology. ARDRA patterns and sequencing results suggested that in fact we dialed with a single strain instead of two as we assumed at the beginning. Sequencing and phylogenetic analysis of 16S rDNA indicated a 99% affiliation at the Pseudomonas stutzeri species.

Keywords: ARDRA, phylogenetic tree, oil pollution.

Introduction
The genus Pseudomonas includes species with functions of ecological, economic, and health-related importance. Some species or strains are well recognized for their metabolic versatility, making them attractive candidates for use in bioremediation [3, 10, 11, 14]. Many studies have described the potential of Pseudomonas species to degrade a variety of compounds [7, 9, 15 ,20]. This bacterial genus is also a very heterogenic one, therefore many species initially classified by general microbiological characters being finally re-classified,, for example Burkholderia cepacia, is now Pseudomonas cepacia [1,2,3,10,11]. Isolation of a new strain is mostly performed after a fastidious initial isolation and may be achieved by various methods, the original tools used to identify the bacterial strains being mainly based on biochemical and serological differentiation, and other conventional microbiological tests [15]. These methods are being replaced lately by faster DNA-based tools, many of these methods being based on the 16S rDNA sequence for various reasons. First, the 16S rDNA has been sequenced for all recognized species and is required when describing a new one [1, 3, 10, 11]. Secondly, the 16S rDNA sequences have lower intraspecific variability than most protein encoding genes [1,3,9,11]. The objective of this study was to differentiate between two strains, isolated from oil contaminated soil near an extraction pipe, by enrichment cultures using quinoline as carbon source. These strains were formally classified as Pseudomonas sp. using conventional identification tests [8]. On this purpose we analyzed ARDRA patterns obtained with 9

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different endonucleases, and finally sequenced the 1500bp 16S rDNA amplicons for a better affiliation and for the reconstruction of the phylogenetic tree. Quinoline, due to its low-solubility and low-biodegradability, has become one of the most common contaminants in ground water and soil, especially near landfills, coal tar distillation, as well as creosote wood preservation and fossil fuel facilities. Many studies have shown that quinoline and its derivatives have toxic, carcinogenic and mutagenic activity to animals and humans [20]. It is of great significance to find more bacterial species with advantages such as wide availability, high environmental endurance and strong degradation capacity, and also fast molecular analysis to identify them [3, 7, 14, 15].

Materials and methods

Sampling and strains isolation: Soil sample was taken from nearby of an oil pomp. Enrichment culture was started using 1g oil polluted soil, by incubation on liquid MSM (Mineral Salts Medium: potassium hydrogen phosphate 1g, potassium dihydrogenphosphate 0.5g, magnesium sulphate 0.2g, sodium chloride 1g, ammonium sulphate 1g, distillated water 1000ml) supplemented with 0,03%(v/v) of 98% pure quinoline (SIGMA) as unique carbon source, after incubation during 3 weeks at 280C and orbital agitation at 250rpm [14]. From the enrichment culture there were isolated 13 strains on solid LB (peptone 10g, yeast extract 5g, sodium chloride 10g, agar-agar 20g, pH 7-7,5) medium distributed in Petri dishes. All the isolated strains and the enrichments cultures were preserved in liquid LB medium supplemented with 20% glycerol at 70oC in the Microbial Collection of the Laboratory of Microbial Genetics and Biotechnology from the Faculty of Biology, University of Bucharest [15]. The two strains that particulary droved our attention were SQ2a and SQ2b. Reference strain used in this work was Pseudomonas aeruginosa ATCC 27853, as previously described [8, 14]. Isolation and purification of chromosomal DNA: Was performed after a CTAB protocol [4] with some modifications [8, 14, 18]. Electrophoretic analysis of DNA extracts: Electrophoretic analysis of the DNA extract was performed using horizontal submerse agarose gel 1% (wt/vol) in TBE buffer (Tris 0.089M, boric acid 0.089M EDTA 0.002M, pH=8.5). Electrophoresis was run at 2.5V/cm and DNA stained with ethidium bromide 0.5 g/ml [4, 14, 15, 18]. PCR amplification of bacterial 16S rRNA genes: GM3f and 8 primers (5AGA GTT TGA TC(A/C) TGG C3) and GM4r 1503 (5TAC CTT GTT ACG ACT T3) [8], which are complementary to conserved regions of 16S rDNA, were used in a 50 l reaction mixture containing: 1X buffer, BSA 3mg/ml, 200M of each deoxynucleotide triphoshpate, 50M of both primers and 1U Red-Taq DNA-Polymerase, 1l template DNA or water for the negative control. An initial denaturating step of 94oC for 10 minutes was followed by 25 cycles of amplification (1 min 94oC, 2min 57oC, 3min 72oC), and a final extension step at 72oC for 10 min. DNA amplification was checked by electrophoresis of 5 l of PCR product in a 1% agarose gel TBE (Tris 0.089M, boric acid 0.089M, EDTA 0.002M, pH=8,5), at 2.5V/cm and by staining with ethidium bromide. Amplification products were stored at -20oC, until digested and cloning [4].

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Phylogenetic Analysis on 16S Ribosomal DNA of Pseudomonas Strains from Oil Polluted Soil

ARDRA: Restriction was performed separately with each enzyme by incubation with 5U endonuclease in a final volume of 25 l Amplified rDNA restriction analysis (ARDRA) was performed in order to differentiate between the strains and in comparison to ATCC reference strain. PCR products were digested for 2,5h at 370C with nine different restriction endonuclease (PROMEGA) HaeIII, RsaI, AluI, DdeI, MpsI, HinfI, NotI, Sau3AI, CfoI, according to the manufactorys instructions, in separate reactions, using 0.5U/reaction. The restriction patterns were obtained by electrophoresis running on 3% (wt/vol) agarose gel (SIGMA) at 2.5V/cm for ~ 4h [4, 5, 12, 13, 15]. PCR amplicon cloning: PCR products were purified using a Sephadex G-50 Superfine, Amersham Millipore column and then were cloned in the Escherichia coli strain JM 109 using TOPO TA Cloning Kit Invitrogen. Clones were selected by cultivation on LP-IPTG-XGal-Amp100 and the white colonies checked for correct insert size, randomly, and by vectortargeted primers PCR and gel electrophoresis. Sequencing and phylogenetic analysis: Sequencing was performed with the Big Dye Terminator Cycle Sequencing Reaction Kit (APPLIED BIOSYSTEMS) and primers M13F (5GTA AAA CGA CGG CCA G3) [19], M13R (5CAG GAA ACA GCT ATG AC 3) [19], GM1 (5 CCA GCA GCC GCG GTA AT 3) [8], on automated Applied Biosystems DNA sequencer 3100 (ABI prism). In order to obtained full length sequences, was used Sequencer 4.0 program, and then analyzed using Basic Local Alignment Search Tool (BLAST) program at the National Center for Biotechnology Information and the Sequence Match and the Classifier programs of the Ribosomal Database Project II. For construction of a phylogenetic tree the sequences were aligned with known bacterial 16S RNAs obtained from the GenBank database by using ARB software package. Phylogenetic trees parsimony, neighbour-joining, and maximum-likelihood analysis with different sets of filters were calculated [6, 16]. Nucleotide sequence accession numbers: The nucleotide sequence data reported in this paper will appear in the NCBI nucleotide sequence databases under the accession no. DQ388084.

Results
Electrophoretical analysis of PCR products obtained from the amplification of 16S ARN genes confirmed that full length (1500pb) genes were amplified for both strains SQ2a and SQ2b, and the reference strain. In order to differentiate between SQ2a and SQ2b, we analyzed their ARDRA patterns obtained with 9 endonucleases comparatively with those of the reference strain P. aeruginosa ATCC 27853 (Fig. 1-3) as recommended by previous studies [1, 12, 14, 15, 17]. Since we couldnt determine any differences at 16S rDNA level of the tested strains using ARDRA analysis, we sequenced the two amplicons. Complete sequences were obtained after assembling the partial sequences corresponding to the M13f/r, and GM1 primer. Computer analysis and NCBI Data Base search resulted in taxonomical identification of the two strains as Pseudomonas stutzeri, with 99% similarity. Using complet sequences retreaved from SQ2a and SQ2b strains and also from different Pseudomonas species found in ARB Data Base, we reconstructed a phylogenetic tree

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using Maximum- Likelihood method and 50% filter variability for Gammaproteobacteria group (Fig. 4).
1.
3000bp 1500bp 900bp 500bp 300bp 100bp

2. 3. 4. 5. 6. 7. 8. 9. 10. Figure 1. HaeIII restriction patterns (type and reference strain). Lanes: 1. Molecular DNA Marker (GeneRuler 100bp DNA Ladder Plus, FERMENTAS); 2. SQ2a; 3.SQ2b; 4. P. aeruginosa ATCC 27583 ; RsaI restriction patterns (type and reference strain). Lanes: 5. SQ2a; 6. SQ2b; 7. P. aeruginosa ATCC 27583; AluI restriction patterns (type and reference strain). Lanes: 8. SQ2a; 9. SQ2b; 10. P. aeruginosa ATCC 27583;

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
3000bp 1500bp 900bp 500bp 300bp 100bp

Figure 2. DdeI restriction patterns (type and reference strain). Lanes: 1. Molecular DNA Marker (GeneRuler 100bp DNA Ladder Plus, FERMENTAS); 2. SQ2a; 3.SQ2b; 4. P. aeruginosa ATCC 27583; MspI restriction patterns (type and reference strain). Lanes: 5. SQ2a; 6. SQ2b; 7. P. aeruginosa ATCC 27583; HinfI restriction patterns (type and reference strain). Lanes: 8. SQ2a; 9. SQ2b; 10. P. aeruginosa ATCC 27583;

1. 2. 3. 4.
3000bp 1500bp 900bp 500bp 300bp 100bp

5. 6. 7. 8. 9. 10. Figure 3. NotI restriction patterns (type and reference strain). Lanes: 1. Molecular DNA Marker (GeneRuler 100bp DNA Ladder Plus, FERMENTAS); 2. SQ2a; 3.SQ2b; 4. P. aeruginosa ATCC 27583; Sau3AI restriction patterns (type and reference strain). Lanes: 5. SQ2a; 6. SQ2b; 7. P. aeruginosa ATCC 27583; CfoI restriction patterns (type and reference strain). Lanes: 8. SQ2a; 9. SQ2b; 10. P. aeruginosa ATCC 27583;

Discussion
The major direction for the bioremediation technology consists in studyng bacteria from oil contaminated microbial community and especially those microbial strains that are capable to degrade oil and oil compounds. Therefore, it is very important to understand the faith of oil in natural contaminated ecosystems. Based on preliminary morpho-physiological tests, the microorganisms isolated in this presented in the current study, seemed to belong to the same species. At molecular level, ARDRA patterns revealed that for all restriction endonucleases used in this study, the
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Phylogenetic Analysis on 16S Ribosomal DNA of Pseudomonas Strains from Oil Polluted Soil

fragments number and length for the two strains SQ2a and SQ2b, presumed to be different [9], were extremely similar (Tab. 1), but different from the reference strain. In this matter, endonuclease NotI did not exhibited any restriction site for the two strains, as well as for the reference strain Ps. aeruginosa ATCC 27853 (Fig.3). Another similarity was observed between the patterns of endonucleases HinfI and HaeIII (Fig.1,2; Tab.1). For the rest of the restriction endonucleases, the fragments obtained for SQ2a and SQ2b, after 16S ribosomal DNA amplicon digestion, had different lengths than those obtained for the reference strain, but even in this case some of the fragments had similar length for all the analyzed strains as are indicate in Tab. 1 in bold characters. These results sustained the affiliation to Pseudomonas group of the strain SQ2a and SQ2b, but not to Ps. aeruginosa species.

Figure 4. Phylogenetic tree showing the position of SQ2a/b 16S rRNA gene sequence affiliated within the Pseudomonas group, constructed with maximum-likelihood method and a filter (50%) from the sequence dataset. In the present Pseudomonas group tree, E.coli K12 represents the outgroup. Scale bar represents 10% sequence difference. Table 1 Length of ARDRA fragments obtained with the 9 endonucleases for tested strains SQ2a and SQ2b, and the reference strain Ps. aeruginosa ATCC 27853.

Strain/ Endonuclease
HaeIII RsaI AluI DdeI MspI HinfI NotI Sau3AI CfoI

SQ2a
700; 220;180; 130 900; 380; 180;100 420; 220; 200 550;500;140;120;80 400; 350; 280; 100 1000; 200; 100; 80 1600 500; 250; 200; 100 400; 350; 300; 380; 220

SQ2b
700; 220;180; 130 900; 380; 180;100 420; 220; 200 550;500;140;120;80 400;350;280;100 80;100;200;1000 1600 500;250;200; 100 400; 350; 300; 380; 220

Ps. aeruginosa ATCC 27853

650; 220;180; 130 650;380; 280 420; 220; 550;320;150;140;120;80 300;340; 180; 120; 100; 80 80;100;200;1000 1600 500; 200; 100 450; 300; 380; 180

Since we could not determine any differences at 16S rDNA level of the tested strains using ARDRA, we proceeded to the sequencing of the two amplicons. Computer analysis of Rom. Biotechnol. Lett., Vol. 14, No. 6, 4779-4785 (2009)
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the complete sequences and NCBI Data Base search, indicated that the two strains are in fact the same one, having 99% similarity with Pseudomonas stutzeri. It is true that the two strains presented a colony dimorphism, SQ2a forming a very adherent rankle colonies, and SQ2b round smooth colonies (see [9]). It is possible that during biodegradative processes, the different aromatic compounds from oil induce a stress on fatty acids level as previously studies point out, but also on the growth and survival of the isotales [7]. We supposed that this potential stress factor could be involved in modification of the composition and structure of cellular wall fatty acids, and this modification could became definitive for SQ2b. On the other hand, 16S rDNA complet sequence, computer analysis and NCBI Data Base search resulted in taxonomical identification of the two strains as Pseudomonas stutzeri (99% similarity). The phylogenetic tree, shown in Fig. 4 indicates also that strain SQ2a/b is clustered with Pseudomonas stutzeri (bootstrap value 98%) and Pseudomonas putida and distinctively different from related genera, and also is not so closely related with Pseudomonas aeruginosa ATCC27853. In conclusion, phylogenetic analysis of the two strains SQ2a and SQ2b revealed that they are the same strain and are affiliated to Pseudomonas stutzeri species. Nevertheless, most probably the cellular response to oil and oil compounds, could induce an unreversable change of cellular wall determining a different morphology of the colonies for SQ2b. Finally, our results, presented here and also the preliminary ones [9], underline once again the importance of the polyphazic approach in the study of microbial strains, in general, and those isolated from natural ecosystems, in particular [11, 17].

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