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Article DOi: 10.5504/bbeq.2011.0127 A&eb

WHO WE ARE AND WHAT WE ACHIEVED

Biotechnol. & Biotechnol. Eq. 2011, 25(4), Suppl., 47-49Keywords: Acidithiobacillus ferrooxidans, 16S rDNA, PCR detection

IntroductionAcidithiobacillus ferrooxidans is a well known acidophilic, chemolitoautotrophic Gram negative bacterium capable of aerobic growth via the oxidation of Fe(II) to Fe(III) or reduced inorganic sulphur compounds. More recently this specious has been widely studied for its capacity for bioleaching, biodegradation and desulphurisation of coal and natural gas (14, 16). The habitats of Acidithiobacillus ferrooxidans are geographically extremely diverse and vary in their physico-chemical conditions (presence of particular sulphide minerals and their ratio, pH, temperature and the content of toxic compounds). This might explain the polymorphism of A. ferrooxidans strains concerning their physiological properties (1, 6) and genotypic characteristics (9, 12). The strains belonging to the same A. ferrooxidans species exhibit a high degree of interstrain genetic variability with respect to the DNA G + C content, level of DNA-DNA homology of total genomes, the number and sizes of plasmid and chromosomal DNA structure (13, 15).

Classical microbiology scheme for detection and typing of A. ferrooxidans isolates has serious limitations 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), analysis of philogenetic groups (3), DGGE (Denaturating Gradient Gel Electrophoresis) (4) and etc. However most of these techniques are slow and in cases of complex samples irrelevant microorganisms could also be detected.

Due to its relatively high conservative 16S rDNA is a good tool for both identification and inferring inter and intrageneric relationships among the species. The 16S rDNA PCR protocol used in this study is a rapid and specific tool for identification of A. ferrooxidans strains isolated from different ecological niches.

Materials and MethodsMicroorganismsDNA from ten A. ferrooxidans strains was used as template for PCR. Bacteria were cultured in 9K + Fe2+ medium: (NH4)2SO4, 3.0 g; KCl, 0.1 g; K2HPO4, 0.5 g; MgSO4×7H2O, 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.

DNA was extracted from A. ferrooxidans isolates originating from different locations (B2 – Isolated from 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 extractionTotal DNA was extracted from liquid samples by using commercial DNA extraction kit (genomic Prep Mini Spin Kit) (GE Healthcare) with slight modification of the manufactures’ protocol concerning harvesting the bacterial cells. To increase the quantity and quality of the obtained DNA fraction and to reduce the negative effect of possible PCR inhibitors, DNA isolation procedure started with centrifugation of a total 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 ISOLATES

Ralitsa Ilieva1, Veneta Groudeva1, Mihail Iliev2

1Sofia University “St. Kliment Ohridski”, Faculty of Biology, Sofia, Bulgaria2Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, Atelier Pasteur, Sofia, BulgariaCorrespondence to: Ralitsa IlievaE-mail: foxi_vr@abv.bg

ABSTRACTAcidithiobacillus 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 specific primers for 16S rDNA PCR. The applied PCR technique is accomplished using template DNA from pure cultures of the microorganisms. PCR amplification 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.

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 strain

Isolate Number of pairs (bp)

Identity to ATCC 23270 (%)

B-1 950 96%B-2 944 95%

BA-4 920 96%F-3 950 96%G-12 950 95%m-1 950 95%R 950 98%

TF-2 949 96%U1 950 96%V3 950 95%

Fig. 1. 16S rDNA fragments amplified from the genomic DNA of A. 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 qubit®Fluorometer (Invitrogen). DNA yield was checked by electrophoresis in 1% agarose gel (30 min at 120 V and 60 mA) stained with 5 µl gel stain (GelRedTM Nucleic Acid Gel Stain, 10 000×, Biotium).

PrimersThe specific primers used in the PCR reactions for A. ferrooxidans, targeting a conservative 16S rDNA region, were described by Escobar et al. (5). The nucleotide sequences of the primers are 5’- ATG-CGT-AGG-AAT-CTG-TCT-TT-3’ (F1_Thio) and 5’-GGA-CTT-AAC-CCA-ACA-TCT-CA-3’(R1-Thio).

16S rDNA amplification by PCRPCR was carried out by Ready-To-Go PCR kit (GE

Healthcare), final volume 25 µL. The amplification programme consisted of one step of initial denaturation, 35 cycles of 95 °C/1 min, 58.5 °C/30 sec, 72 °C/30 sec and a final extension step at 72 °C/8 min. The reactions were carried out in an Eppendorf Thermocycler. The amplification products were separated by 3% agarose gel electrophoresis stained with 5 µl gel stain (GelRedTM Nucleic Acid Gel Stain, 10 000×, Biotium). The amplified fragments (980 bp) from all isolates were sequenced by ABI 3730 XL with BigDye ® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA). The

49WHO WE ARE AND WHAT WE ACHIEVED

analysis of the sequenced fragments was done by specific software (BLAST, MultAlin (2), Sequence scanner v1.0, Applied Biosystems, USA).

Results and Discussion16S rDNA PCRAmplification conditions were optimized using genomic DNA from pure cultures of tested isolates. The electrophoretical analysis of the PCR products (Fig. 1) showed that the size of the fragments amplified from all isolates matched the expected size of 980 bp. Also, no other amplification bands were observed, which demonstrated the specificity of the chosen primer pair. Although this 16S rDNA PCR protocol was implemented in this work only for detection of A. ferrooxidans, it could be applied for other species like A. thiooxidans, A. criptum.

Sequencing analysis of amplified 16S rDNA fragments The obtained 16S rDNA sequences were subjected to bioinformatical analysis aiming to reveal genetic homogeneity of the chosen target among the tested isolates and to estimate identity with the type strain A. ferrooxidans ATCC 23270 (GenBank № AF329205). The results are presented in Table 1.

The results confirmed the high genetic homogeneity of 16S rDNA among different strains of A. ferrooxidans described by other authors (7, 8). Based on these data a dendrogram of the tested isolates and the type strain was constructed. The alignment of the 16S rDNA sequences revealed the presence of highly homogeneous region consisting of 387 bp (Fig. 2).

This region could be used as a target for construction of specific primers in further molecular based experiments with the tested isolates.

AcknowledgementsThis work was supported by grant BG051PO001-3.3.04/32 financed by Operational Programme Human Resources Development (2007 – 2013) and co-financed by the European Social Fund of the European Union.

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