Lucr 2 Ana Maria T

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Romanian Biotechnological Letters Vol. 14, No. 6, 2009, pp. 4779-4785

Copyright 2009 Bucharest University Printed in Romania. All rights reservedRomanian Society of Biological Sciences

ORIGINAL PAPER

4779

Phylogenetic Analysis on 16S Ribosomal DNA ofPseudomonas Strains fromOil Polluted Soil

Received for publication, April 1, 2009Accepted, October 20, 2009

ANA-MARIA TANASE1, CRISTINA TRASCA

3, TATIANA VASSU

1, ALEXANDRU

OLTEANU4, DIANA PELINESCU

1, ORTANSA CSUTAK

1, IONESCU ROBERTINA

2,

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; [email protected];Tel: + 40213118077

AbstractIn 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 genusPseudomonas includes species with functions of ecological, economic, andhealth-related importance. Some species or strains are well recognized for their metabolicversatility, making them attractive candidates for use in bioremediation [3, 10, 11, 14]. Manystudies have described the potential of Pseudomonas species to degrade a variety ofcompounds [7, 9, 15 ,20]. This bacterial genus is also a very heterogenic one, therefore manyspecies 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 andmay be achieved by various methods, the original tools used to identify the bacterial strainsbeing mainly based on biochemical and serological differentiation, and other conventionalmicrobiological tests [15]. These methods are being replaced lately by faster DNA-basedtools, 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 whendescribing a new one [1, 3, 10, 11]. Secondly, the 16S rDNA sequences have lowerintraspecific variability than most protein encoding genes [1,3,9,11].

The objective of this study was to differentiate between two strains, isolated from oilcontaminated soil near an extraction pipe, by enrichment cultures using quinoline as carbonsource. 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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ANA-MARIA TANASE, CRISTINA TRASCA, TATIANA VASSU, ALEXANDRU OLTEANU, DIANAPELINESCU, ORTANSA CSUTAK, IONESCU ROBERTINA, ILEANA STOICA

Rom. Biotechnol. Lett., Vol. 14, No. 6, 4779-4785 (2009)4780

different endonucleases, and finally sequenced the 1500bp 16S rDNA amplicons for a betteraffiliation 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 tardistillation, as well as creosote wood preservation and fossil fuel facilities. Many studies haveshown that quinoline and its derivatives have toxic, carcinogenic and mutagenic activity toanimals and humans [20]. It is of great significance to find more bacterial species withadvantages such as wide availability, high environmental endurance and strong degradationcapacity, 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. Enrichmentculture was started using 1g oil polluted soil, by incubation on liquid MSM (Mineral Salts

Medium: potassium hydrogen phosphate 1g, potassium dihydrogenphosphate 0.5g, magnesiumsulphate 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, afterincubation during 3 weeks at 280C and orbital agitation at 250rpm [14]. From the enrichmentculture there were isolated 13 strains on solid LB (peptone 10g, yeast extract 5g, sodium chloride10g, agar-agar 20g, pH 7-7,5) medium distributed in Petri dishes. All the isolated strains and theenrichments cultures were preserved in liquid LB medium supplemented with 20% glycerol at -70oC in the Microbial Collection of the Laboratory of Microbial Genetics and Biotechnologyfrom the Faculty of Biology, University of Bucharest [15]. The two strains that particulary drovedour attention were SQ2a and SQ2b. Reference strain used in this work was Pseudomonasaeruginosa 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 wasperformed 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 DNAstained with ethidium bromide 0.5 g/ml [4, 14, 15, 18].

PCR amplification of bacterial 16S rRNA genes: GM3f and 8 primers (5AGA GTT TGATC(A/C) TGG C3) and GM4r 1503 (5TAC CTT GTT ACG ACT T3) [8], which arecomplementary to conserved regions of 16S rDNA, were used in a 50 l reaction mixturecontaining: 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 negativecontrol. An initial denaturating step of 94oC for 10 minutes was followed by 25 cycles ofamplification (1 min 94oC, 2min 57oC, 3min 72oC), and a final extension step at 72oC for 10min. 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, untildigested and cloning [4].


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

Rom. Biotechnol. Lett., Vol. 14, No. 6, 4779-4785 (2009) 4781

ARDRA: Restriction was performed separately with each enzyme by incubation with 5Uendonuclease 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 referencestrain. 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. Therestriction 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 109using TOPO TA Cloning Kit Invitrogen. Clones were selected by cultivation on LP-IPTG-X-Gal-Amp100 and the white colonies checked for correct insert size, randomly, and by vector-targeted primers PCR and gel electrophoresis.

Sequencing and phylogenetic analysis: Sequencing was performed with the Big DyeTerminator 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 BiosystemsDNA sequencer 3100 (ABI prism). In order to obtained full length sequences, was usedSequencer 4.0 program, and then analyzed using Basic Local Alignment Search Tool(BLAST) program at the National Center for Biotechnology Information and the SequenceMatch and the Classifier programs of the Ribosomal Database Project II. For construction ofa phylogenetic tree the sequences were aligned with known bacterial 16S RNAs obtainedfrom 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 thispaper will appear in the NCBI nucleotide sequence databases under the accession no.DQ388084.

Results

Electrophoretical analysis of PCR products obtained from the amplification of 16SARN genes confirmed that full length (1500pb) genes were amplified for both strains SQ2aand SQ2b, and the reference strain.

In order to differentiate between SQ2a and SQ2b, we analyzed their ARDRA patternsobtained with 9 endonucleases comparatively with those of the reference strainP. aeruginosaATCC 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 strainsusing ARDRA analysis, we sequenced the two amplicons. Complete sequences were obtainedafter assembling the partial sequences corresponding to the M13f/r, and GM1 primer.Computer analysis and NCBI Data Base search resulted in taxonomical identification of thetwo strains asPseudomonas stutzeri, with 99% similarity.

Using complet sequences retreaved from SQ2a and SQ2b strains and also fromdifferentPseudomonas species found in ARB Data Base, we reconstructed a phylogenetic tree


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using Maximum- Likelihood method and 50% filter variability for Gammaproteobacteriagroup(Fig. 4).

3000bp

1500bp

900bp

500bp

300bp

100bp

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.Figure 1.HaeIII restriction patterns (type

and reference strain). Lanes: 1. MolecularDNA Marker (GeneRuler 100bp DNALadder Plus, FERMENTAS); 2. SQ2a;3.SQ2b; 4. P. aeruginosa ATCC 27583 ;RsaIrestriction patterns (type and referencestrain). 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 ATCC27583;

3000bp1500bp

900bp500bp

300bp

100bp

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Figure 2.DdeI restriction patterns (type andreference strain). Lanes: 1. Molecular DNAMarker (GeneRuler 100bp DNA LadderPlus, 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 ATCC27583; HinfI restriction patterns (type andreference strain). Lanes: 8. SQ2a; 9. SQ2b;10.P. aeruginosa ATCC 27583;

3000bp

1500bp

900bp

500bp

300bp

100bp

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.Figure 3.NotI restriction patterns (type andreference strain). Lanes: 1. Molecular DNAMarker (GeneRuler 100bp DNA LadderPlus, 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 ATCC27583; CfoI restriction patterns (type andreference strain). Lanes: 8. SQ2a; 9. SQ2b;10.P. aeruginosa ATCC 27583;

Discussion

The major direction for the bioremediation technology consists in studyng bacteriafrom oil contaminated microbial community and especially those microbial strains that arecapable to degrade oil and oil compounds. Therefore, it is very important to understand thefaith of oil in natural contaminated ecosystems.

Based on preliminary morpho-physiological tests, the microorganisms isolated in thispresented 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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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 NotIdid not exhibited any restriction site for the two strains, as well as for thereference strain Ps. aeruginosa ATCC 27853 (Fig.3). Another similarity was observed

between the patterns of endonucleasesHinfI andHaeIII (Fig.1,2; Tab.1).For the rest of therestriction endonucleases, the fragments obtained for SQ2a and SQ2b, after 16S ribosomalDNA 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 asare indicate in Tab. 1 in bold characters. These results sustained the affiliation to

Pseudomonas group of the strain SQ2a and SQ2b, but not toPs.aeruginosa species.

Figure 4. Phylogenetic tree showing the position of SQ2a/b 16S rRNA gene sequenceaffiliated within thePseudomonas group, constructed with maximum-likelihood method

and a filter (50%) from the sequence dataset. In the presentPseudomonas group tree,E.coli K12represents 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, andthe reference strainPs. aeruginosa ATCC 27853.

Strain/

EndonucleaseSQ2a SQ2b Ps. aeruginosa ATCC 27853

HaeIII 700; 220;180; 130 700; 220;180; 130 650; 220;180; 130RsaI 900; 380; 180;100 900; 380; 180;100 650;380; 280AluI 420; 220; 200 420; 220; 200 420; 220;DdeI 550;500;140;120;80 550;500;140;120;80 550;320;150;140;120;80MspI 400; 350; 280; 100 400;350;280;100 300;340; 180; 120; 100; 80HinfI 1000; 200; 100; 80 80;100;200;1000 80;100;200;1000NotI 1600 1600 1600

Sau3AI 500; 250; 200; 100 500;250;200; 100 500;200; 100CfoI 400; 350; 300; 380; 220 400; 350; 300; 380; 220 450; 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


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the complete sequences and NCBI Data Base search, indicated that the two strains are in factthe same one, having 99% similarity withPseudomonas 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, thedifferent aromatic compounds from oil induce a stress on fatty acids level as previouslystudies 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 ofcellular wall fatty acids, and this modification could became definitive for SQ2b. On the otherhand, 16S rDNA complet sequence, computer analysis and NCBI Data Base search resulted intaxonomical 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 withPseudomonas stutzeri (bootstrap value 98%) and Pseudomonas putida and distinctivelydifferent from related genera, and also is not so closely related withPseudomonas aeruginosaATCC27853.

In conclusion, phylogenetic analysis of the two strains SQ2a and SQ2b revealed thatthey are the same strain and are affiliated toPseudomonas stutzeri species. Nevertheless, mostprobably the cellular response to oil and oil compounds, could induce an unreversable changeof cellular wall determining a different morphology of the colonies for SQ2b. Finally, ourresults, presented here and also the preliminary ones [9], underline once again the importanceof the polyphazic approach in the study of microbial strains, in general, and those isolatedfrom natural ecosystems, in particular [11, 17].

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