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PCR Detection and 16S rDNA Sequence Analysis of

Different Acidithiobacillus Ferrooxidans Isolates
a

Ralitsa Ilieva , Veneta Groudeva & Mihail Iliev

a

Sofia University St. Kliment Ohridski, Faculty of Biology, Sofia, Bulgaria

Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, Atelier

Pasteur, Sofia, Bulgaria
Published online: 16 Apr 2014.

To cite this article: Ralitsa Ilieva, Veneta Groudeva & Mihail Iliev (2011) PCR Detection and 16S rDNA Sequence Analysis
of Different Acidithiobacillus Ferrooxidans Isolates, Biotechnology & Biotechnological Equipment, 25:sup1, 47-49, DOI:
10.5504/BBEQ.2011.0127
To link to this article: http://dx.doi.org/10.5504/BBEQ.2011.0127

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Article

DOI: 10.5504/bbeq.2011.0127

A&EB

PCR DETECTION AND 16S rDNA SEQUENCE ANALYSIS OF DIFFERENT

ACIDITHIOBACILLUS FERROOXIDANS ISOLATES
Ralitsa Ilieva1, Veneta Groudeva1, Mihail Iliev2
1
Sofia University St. Kliment Ohridski, Faculty of Biology, Sofia, Bulgaria
2
Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, Atelier Pasteur, Sofia, Bulgaria
Correspondence to: Ralitsa Ilieva
E-mail: foxi_vr@abv.bg

ABSTRACT

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

Introduction

Acidithiobacillus 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 physicochemical 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.
WHO WE ARE AND WHAT WE ACHIEVED

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 Methods

Microorganisms
DNA 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; 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.
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 extraction
Total 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
47

Sequence analyses, revealing identity to type strain

Isolate

Identity to ATCC 23270

(%)
96%
95%
96%
96%
95%
95%
98%
96%
96%
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).

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B-1
B-2
BA-4
F-3
G-12
m-1
R
TF-2
U1
V3

Number of pairs
(bp)
950
944
920
950
950
950
950
949
950
950

TABLE 1

Fig. 2. Partial alignment of 16S rDNA sequence of the tested isolates.

kit. The concentration of the obtained DNA was determined by

QubitFluorometer (Invitrogen). DNA yield was checked by
electrophoresis in 1% agarose gel (30 min at 120 V and 60
mA) stained with 5 l gel stain (GelRedTM Nucleic Acid Gel
Stain, 10 000, Biotium).
Primers
The 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-CA3(R1-Thio).
48

16S rDNA amplification by PCR

PCR was carried out by Ready-To-Go PCR kit (GE
Healthcare), final volume 25 L. 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 l 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
Biotechnol. & Biotechnol. Eq. 25/2011/4, Suppl.

analysis of the sequenced fragments was done by specific

software (BLAST, MultAlin (2), Sequence scanner v1.0,
Applied Biosystems, USA).

Results and Discussion

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16S rDNA PCR

Amplification 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.

Acknowledgements

This 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.

WHO WE ARE AND WHAT WE ACHIEVED

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