This article was downloaded by: [5.2.189.6]On: 10 August 2015, At: 02:07Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 HowickPlace, London, SW1P 1WGBiotechnology & Biotechnological EquipmentPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tbeq20PCR Detection and 16S rDNA Sequence Analysis ofDifferent Acidithiobacillus Ferrooxidans IsolatesRalitsa Ilievaa, Veneta Groudevaa & Mihail Ilievba Sofia University St. Kliment Ohridski, Faculty of Biology, Sofia, Bulgariab Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, AtelierPasteur, Sofia, BulgariaPublished online: 16 Apr 2014.To cite this article: Ralitsa Ilieva, Veneta Groudeva & Mihail Iliev (2011) PCR Detection and 16S rDNA Sequence Analysisof Different Acidithiobacillus Ferrooxidans Isolates, Biotechnology & Biotechnological Equipment, 25:sup1, 47-49, DOI:10.5504/BBEQ.2011.0127To link to this article:http://dx.doi.org/10.5504/BBEQ.2011.0127PLEASE SCROLL DOWN FOR ARTICLETaylor & Francis makes every effort to ensure the accuracy of all the information (the Content) containedin the publications on our platform. 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Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions47ARTICLEDOI: 10.5504/BBEQ.2011.0127A&EBWHO WE ARE AND WHAT WE ACHIEVEDBiotechnol. & Biotechnol. Eq. 2011, 25(4), Suppl., 47-49Keywords:Acidithiobacillusferrooxidans,16SrDNA,PCR detectionIntroductionAcidithiobacillusferrooxidansisawellknownacidophilic, chemolitoautotrophicGramnegativebacteriumcapableof aerobic growth via the oxidation of Fe(II) to Fe(III) or reduced inorganicsulphurcompounds.Morerecentlythisspecious hasbeenwidelystudiedforitscapacityforbioleaching, biodegradationanddesulphurisationofcoalandnaturalgas (14,16).ThehabitatsofAcidithiobacillusferrooxidansare geographicallyextremelydiverseandvaryintheirphysico-chemical conditions (presence of particular sulphide minerals andtheirratio,pH,temperatureandthecontentoftoxic compounds).ThismightexplainthepolymorphismofA. ferrooxidans strains concerning their physiological properties (1,6)andgenotypiccharacteristics(9,12).Thestrains belongingtothesameA.ferrooxidansspeciesexhibitahigh degreeofinterstraingeneticvariabilitywithrespecttothe DNAG+Ccontent,levelofDNA-DNAhomologyoftotal genomes, the number and sizes of plasmid and chromosomal DNA structure (13, 15). Classicalmicrobiologyschemefordetectionandtyping of A. ferrooxidansisolates hasseriouslimitations concerning the need of complex nutritious medium, long time incubation and diverse morphological and biochemical characteristics of the strains. In the last few years, several molecular techniques for typing of A. ferrooxidans have been developed using PCR methodology such as 16S rDNA analysis by restriction enzymes (11), analysis of spacing regions from ribosomal operons (10), analysisofphilogeneticgroups(3),DGGE(Denaturating GradientGelElectrophoresis)(4)andetc.Howevermostof thesetechniquesareslowandincasesofcomplexsamples irrelevant microorganisms could also be detected.Due to its relatively high conservative 16S rDNA is a good tool for both identifcation and inferring inter and intrageneric relationships among the species. The 16S rDNA PCR protocol used in this study is a rapid and specifc tool for identifcation ofA.ferrooxidansstrainsisolatedfromdifferentecological niches.Materials and MethodsMicroorganismsDNAfromtenA.ferrooxidansstrainswasusedastemplate forPCR.Bacteriawereculturedin9K+Fe2+medium: (NH4)2SO4, 3.0 g; KCl, 0.1 g; K2HPO4, 0.5 g; MgSO47H2O, 0.5 g; Ca(NO3)2, 0.01 g; distilled water, 700 ml; containing 9 g/L Ferro iron, pH 2.3-2.5. Samples were cultivated at 28 C for one week with aeration.DNAwasextractedfromA.ferrooxidansisolates originatingfromdifferentlocations(B2 Isolatedfrom sulphide ore, Bulgaria; R from India; m-1 Shabla, Bulgaria; G12 Greece; TF-2 mine Vlaikov Vruh, Bulgaria; F-3 Italy; V3 and B-1 mine Radka, Bulgaria; BA4 Finland; U1 Simitly, Bulgaria). Genomic DNA extractionTotalDNAwasextractedfromliquidsamplesbyusing commercial DNA extraction kit (genomic Prep Mini Spin Kit) (GE Healthcare) with slight modifcation of the manufactures protocol concerning harvesting the bacterial cells. To increase thequantity andquality oftheobtained DNA fraction and to reducethenegativeeffectofpossiblePCRinhibitors,DNA isolationprocedurestartedwithcentrifugationofatotal volume of 50 ml bacterial suspension at 1000 rpm for 2 min to eliminate the inorganic pellet.The supernatant was further centrifuged at 5000 rpm for 15 min. The obtained cell depot was washed twice with 1x PBS, pH 1.2 and cells were resuspended in 1 ml of PBS. The further isolation of genomic DNA was carried out by a DNA extraction PCR DETECTION AND 16S rDNA SEQUENCE ANALYSIS OF DIFFERENT ACIDITHIOBACILLUS FERROOXIDANS ISOLATESRalitsa Ilieva1, Veneta Groudeva1, Mihail Iliev21Sofa University St. Kliment Ohridski, Faculty of Biology, Sofa, Bulgaria2Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, Atelier Pasteur, Sofa, BulgariaCorrespondence to: Ralitsa IlievaE-mail: foxi_vr@abv.bgABSTRACTAcidithiobacillus ferrooxidans is one of the most important microorganisms involved in bioleaching, acid mine drainage and biodesulphurization of coals. In this study A. ferrooxidans is detected in liquid samples using specifc primers for 16S rDNA PCR. The applied PCR technique is accomplished using template DNA from pure cultures of the microorganisms. PCR amplifcation products were subjected to bioinformatical analysis. The results obtained from these analyses are a good foundation for further molecular experiments with different A. ferrooxidans isolates.Downloaded by [5.2.189.6] at 02:07 10 August 2015 48 BIOTECHNOL. & BIOTECHNOL. EQ. 25/2011/4, SUPPL.Fig. 2. Partial alignment of 16S rDNA sequence of the tested isolates.TABLE 1Sequence analyses, revealing identity to type strainIsolateNumber of pairs (bp)Identity to ATCC 23270(%)B-1950 96%B-2944 95%BA-4920 96%F-3950 96%G-12950 95%m-1950 95%R 950 98%TF-2949 96%U1950 96%V3 950 95%Fig.1.16SrDNAfragmentsamplifedfromthegenomicDNAofA. ferrooxidans. 1 Ladder (100 bp), 2 G-12, 3 TF-2, 4 F-3, 5 V-3, 6 B-1, 7 BA4, 8 U-1, 9 B2, 10 R, 11 m-1 , 12 Ladder (100 bp).kit. The concentration of the obtained DNA was determined by QubitFluorometer (Invitrogen). DNA yield was checked by electrophoresisin1%agarosegel(30minat120Vand60 mA) stained with 5 l gel stain (GelRedTM Nucleic Acid Gel Stain, 10 000, Biotium).PrimersThespecifcprimersusedinthePCRreactionsforA. ferrooxidans, targeting a conservative 16S rDNA region, were describedbyEscobaretal.(5). Thenucleotidesequencesof theprimersare5-ATG-CGT-AGG-AAT-CTG-TCT-TT-3 (F1_Thio)and5-GGA-CTT-AAC-CCA-ACA-TCT-CA-3(R1-Thio).16S rDNA amplifcation by PCRPCRwascarriedoutbyReady-To-GoPCRkit(GE Healthcare), fnal volume 25 L. The amplifcation programme consistedofonestepofinitialdenaturation,35cyclesof95 C/1 min, 58.5 C/30 sec, 72 C/30 sec and a fnal extension stepat72C/8min.Thereactionswerecarriedoutinan EppendorfThermocycler.Theamplifcationproductswere separatedby3%agarosegelelectrophoresisstainedwith 5lgelstain(GelRedTMNucleicAcidGelStain,10000, Biotium). The amplifed fragments (980 bp) from all isolates were sequenced by ABI 3730 XL with BigDye Terminator v3.1CycleSequencingkit(AppliedBiosystems,USA).The Downloaded by [5.2.189.6] at 02:07 10 August 2015 49 WHO WE ARE AND WHAT WE ACHIEVEDanalysisofthesequencedfragmentswasdonebyspecifc software(BLAST,MultAlin(2),Sequencescannerv1.0, Applied Biosystems, USA).Results and Discussion16S rDNA PCRAmplifcation conditions were optimized using genomic DNA frompureculturesoftestedisolates.Theelectrophoretical analysis of the PCR products (Fig. 1) showed that the size of the fragments amplifed from all isolates matched the expected size of 980 bp. Also, no other amplifcation bands were observed, which demonstrated the specifcity of the chosen primer pair. Althoughthis16SrDNAPCRprotocolwasimplementedin thisworkonlyfordetectionofA.ferrooxidans,itcouldbe applied for other species like A. thiooxidans, A. criptum. Sequencing analysis of amplifed 16S rDNA fragments Theobtained16SrDNAsequencesweresubjectedto bioinformatical analysis aiming to reveal genetic homogeneity of the chosen target among the tested isolates and to estimate identitywiththetypestrainA.ferrooxidansATCC23270 (GenBank AF329205). The results are presented in Table 1.The results confrmed the high genetic homogeneity of 16S rDNAamongdifferentstrainsofA.ferrooxidansdescribed by other authors (7, 8). Based on these data a dendrogram of thetestedisolatesandthetypestrainwasconstructed.The alignmentofthe16SrDNAsequencesrevealedthepresence of highly homogeneous region consisting of 387 bp (Fig. 2).Thisregioncouldbeusedasatargetforconstructionof specifcprimersinfurthermolecularbasedexperimentswith the tested isolates.AcknowledgementsThisworkwassupportedbygrantBG051PO001-3.3.04/32 fnancedbyOperationalProgrammeHumanResources Development (2007 2013) and co-fnanced by the European Social Fund of the European Union.REFERENCES1.AgeevaS.,KondratevaT.,KaravaikoG.(2001) Mikrobiologia, 70, 226-234. (In Russian)2.Corpet F. (1988) Nucl. 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