Download Molecular diversity of thermophilic bacteria isolated from Pasinler

A. ADIGÜZEL, K. İNAN, F. ŞAHİN, T. ARASOĞLU, M. GÜLLÜCE, A. O. BELDÜZ, Ö. BARIŞ
Turk J Biol
35 (2011) 267-274
© TÜBİTAK
doi:10.3906/biy-0908-7
Molecular diversity of thermophilic bacteria isolated from
Pasinler hot spring (Erzurum, Turkey)
Ahmet ADIGÜZEL1, Kadriye İNAN2, Fikrettin ŞAHİN3, Tulin ARASOĞLU4, Medine GÜLLÜCE5,
Ali Osman BELDÜZ2, Özlem BARIŞ5
1
Department of Molecular Biology and Genetics, Faculty of Science, Atatürk University, 25240, Erzurum - TURKEY
2
Department of Biology, Faculty of Arts and Science, Karadeniz Technical University, 61080 Trabzon - TURKEY
3
Department of Genetic and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University,
4755 Kayışdağı, İstanbul - TURKEY
4
İstanbul Regional Hygiene Institute, Ministry of Health Turkey, 34020 Zeytinburnu, İstanbul - TURKEY
5
Department of Biology, Faculty of Science, Atatürk University, 25240 Erzurum - TURKEY
Received: 03.08.2009
Abstract: The present study was conducted to determine the phenotypic and genotypic characterization of thermophilic
bacteria isolated from Pasinler hot spring, Erzurum, Turkey. Fatty acid profiles, BOX PCR fingerprints, and 16S
rDNA sequence data were used for the phenotypic and genotypic characterization of thermophilic bacteria. Totally
9 different bacterial strains were selected based on morphological, physiological, and biochemical tests. These strains
were characterized by molecular tests including fatty acid and BOX profiles, and 16S rDNA sequence. The data of fatty
acid analysis showed the presence of 14 different fatty acids in the 9 bacterial strains examined. Additionally, 5 of these
fatty acids, 15:0 iso, 15:0 anteiso, 16:0 iso, 17:0 iso, and 17:0 anteiso fatty acids, were found in all isolates. Based on
fatty acid profiles, it was determined that the bacterial strains were classified into 2 phenotypic groups, and these data
were confirmed by BOX PCR genomic fingerprint profiles and 16S rDNA sequence analysis. The first group, identified
as Bacillus licheniformis, was represented by 4 strains, and the second group, identified as Aeribacillus pallidus, was
represented by 5 strains.
Key words: Thermophile bacteria, FAMEs, BOX-PCR, 16S rDNA sequencing
Pasinler kaplıcasından izole edilen termofilik bakterilerin moleküler farklılıkları
(Erzurum, Türkiye)
Özet: Bu çalışma Erzurum, Pasinler kaplıcasından izole edilen termofilik bakterilerin genotipik ve fenotipik
karakterizasyonunu belirlemek için yapılmıştır. Termofilik bakterilerin genotipik ve fenotipik karakterizasyonu için yağ
asidi, BOX PCR profilleme metodları ve 16S rDNA dizileme verileri kullanılmıştır. Morfolojik, fizyolojik ve biyokimyasal
testlere dayanarak, toplam 9 bakteri suşu seçilmiştir. Bu suşlar, yağ asidi, BOX PCR profilleme ve 16S rDNA dizi analiz
yöntemlerini içeren moleküler testlerle ileri seviyede karakterize edilmiştir. Yağ asidi analiz verileri, çalışılan 9 bakteri
suşunda 14 farklı yağ asidinin varlığını göstermiştir. 15:0 iso, 15:0 anteiso, 16:0 iso, 17:0 iso ve 17:0 anteiso yağ asitlerini
içeren beş yağ asidi ise bütün suşlarda bulunmuştur. Yağ asidi profilleri temel alınarak yapılan analizler sonucunda
267
Molecular diversity of thermophilic bacteria isolated from Pasinler hot spring (Erzurum, Turkey)
bakteri suşlarının iki fenotipik gruba ayrıldığı tespit edilmiş, bu veriler, BOX PCR parmakizi ve 16S rDNA dizi analiz
sonuçları ile de desteklenmiştir. Bacillus licheniformis olarak tanılanan ilk grup dört suş, Aeribacillus pallidus olarak
tanılanan ikinci grup ise beş suş ile temsil edilmiştir.
Anahtar sözcükler: Termofilik bakteri, FAMEs, BOX-PCR, 16S rDNA dizileme
Introduction
Since they can propagate under the conditions
where other organisms either cannot grow or grow a
little, microorganisms living in extreme environments
always have been considered as a popular research
subject by scientists. In particular, the tolerance
of these microorganisms’ cell components to high
temperature has caused thermophilic bacteria to be
used extensively in different types of biotechnological
applications (1). Molecular characterization of
thermophilic bacteria has been done for many
geothermal areas such as Turkey, Bulgaria, Greece,
China, India, Yellowstone National Park (USA), and
Iceland (2).
Aerobic spores forming thermophilic bacteria
growing at 70 °C were characterized for the first time
by Miquel in 1888 (3). Then a number of strains of
the spore forming thermophilic bacteria particularly
those belonging to the genera Bacillus and Clostridium
have been studied (4,5).
Recent studies based on 16S rDNA sequencing
analyses revealed enough evidence supporting
reclassification of thermophilic members of the
genus Bacillus as Amphibacillus, Alicyclobacillus,
Paenibacillus, Aneurinibacillus and Brevibacillus,
Halobacillus, Virgibacillus, Gracilibacillus, Sulfobacillus
and Salibacillus, Anoxybacillus, Coprobacillus,
Thermobacillus, Filobacillus, Geobacillus, Ureibacillus,
Jeotgalibacillus, Sulfobacillus, and Marinibacillus (2).
Advances in molecular biology techniques
such as fatty acid methyl ester and genomic
fingerprinting, and 16S rDNA sequencing have
provided an excellent opportunity for identification
and characterization of microorganisms at species
and subspecies levels (2,6). In addition, rep PCR
[REP (repetitive extragenic palindromic), ERIC
(enterobacterial repetitive intergenic consensus)
and BOX elements] methods are well known
genomic profiling techniques commonly used for
the characterization of Actinomycetes, gram-positive
268
and -negative bacteria. These methods have also been
used for studying the diversity in the ecosystem,
presenting the phylogenetic relation between strains,
and discriminating between microorganisms that are
genetically close to each other (1,7).
The objective of the present study was to identify
and characterize thermophilic bacteria isolated from
Pasinler hot spring water, Erzurum, Turkey, by using
phenotypic and genotypic methods.
Materials and methods
All the methods used in the present study were
described in our previous work with some minor
modifications (2).
Isolation of strains
The water and sludge samples were obtained
from Pasinler hot spring, Erzurum, Turkey, and
transferred into laboratory under aseptic conditions.
The samples (G3B, P38, G1, Ah2, G19B, P45, Ah23,
P26, and M71) were streaked and inoculated onto
Nutrient Agar (NA) plates, and then incubated in an
aerobic incubator with an adjusted temperature of 5560 °C for 24-48 h. After incubation, different colonies
developed in the media were selected and purified
by subculturing. Isolated and purified bacterial
strains were stored in Nutrient Broth containing 15%
glycerol at –86 °C for further studies.
Morphological, physiological and biochemical
characterizations of isolates
The temperature range for growth was determined
by incubating the isolate from 30 to 80 °C with 5 °C
intervals. The effect of NaCl on the thermophilic
bacterial growth was studied in NB medium
containing 2.0, 3.0, 4.0, 5.0, 8.0, and 10.0% (w/v)
NaCl. The pH dependence of growth was tested in
the pH range of 4.0-11.0 in nutrient broth medium.
Cell morphology of isolates was investigated by light
microscopy. Cell morphology, Gram reactions, the
A. ADIGÜZEL, K. İNAN, F. ŞAHİN, T. ARASOĞLU, M. GÜLLÜCE, A. O. BELDÜZ, Ö. BARIŞ
presence of catalase, oxidase and amylase reduction
were investigated according to the methods described
by Harley and Prescott (8).
Extraction and analysis of fatty acid methyl
ester (FAME) profiles
Preparation and analysis of FAME from
whole cell fatty acids of bacterial strains were
performed according to the method described by
the manufacturer`s manual (Sherlock Microbial
Identification System version 4.0, MIDI, Inc.,
Newark, DE, USA) (9,10). FAMEs were separated by
gas chromatography (HP6890, Hewlett Packard, Palo
Alto, CA, USA) with a fused-silica capillary column
(25 m × 0.2 mm) with cross-linked 5% phenyl methyl
silicone. FAME profiles of each bacterial strain were
identified by comparing the commercial databases
(TSBA 60) with the MIS software package. The
identity of bacterial strains was revealed by computer
comparison of FAME profiles of the unknown test
strains with those in the library.
DNA extraction from pure cultures
Total genomic DNA was extracted from bacteria
samples using a modified method previously
described by Adıgüzel (1).
BOX-PCR analysis
DNA (50 ng) was subjected to PCR utilizing the
primer BOX A1R (CTA CGG CAA GGC GAC GCT
GAC G) as described by Versalovic et al. (11); 27 μL
of reaction cocktail was prepared as follows: Gitschier
Buffer 5 μL, Dimethyl sulfoxide 2.5 μL (100%, 20×),
dNTPs (10 mM) 1.25 μL, bovine serum albumin
1.25 μL (20 mg/mL), primer/primers (5 μM) 3.0 μL,
Taq polymerase (250 U) 0.3 μL, and water 13.7 μL.
A negative control (no DNA) was included in each
PCR assay.
PCR amplifications were performed in a DNA
Corbett Research Palm Cycler (Corbett CG1-96 AG,
Australia) with an initial denaturation step (95 °C, 7
min), followed by 30 cycles of denaturation (94 °C,
1 min), annealing (53 °C, 1 min) and extension (65
°C, 8 min), and a single final extension step (65 °C,
16 min).
The amplification products were resolved by
horizontal electrophoresis on a 1.5% agarose gel
(w/v) in 0.5 × Tris acetate-EDTA (TAE) buffer
with a row of 12 wells of 1.5 mm thickness. Fifteen
microliters of the amplification product were mixed
with 3 μL of 6 × gel-loading solution, and loaded
into the wells. Eight microliters of 10-kb DNA ladder
DirectLoadTM was loaded into one terminal well.
Electrophoresis was performed at 4 °C and 80 V (4 V
cm-1) in 0.5 × TAE buffer until the bromophenol blue
tracking dye reached the bottom of the gel (about 4
h). The gel was stained in 1 μg mL-1 ethidium bromide
solution prepared in 0.5 × TAE buffer for 45 min and
destained in distilled water for 10 min. The banding
patterns of the ethidium bromide-stained gel were
captured under UV light in a DNR-Imaging System
with a UV-soft analysis package (Israel).
The resulting fingerprints were transformed into
a binary character matrix (‘1’ for the presence and
‘0’ for the absence of a band at a particular position)
and analyzed by using SPSS (SPSS, version 11.0 for
Windows). Data were used to calculate a Jaccard
(1908) similarity (12).
All of the experiments in this study were repeated
at least twice.
PCR amplification and cloning of 16S rDNA
sequence
The 16S rDNA genes were selectively amplified
from purified genomic DNA by using oligonucleotide
primers designed to anneal to conserved
positions in the 3’ and 5’ regions of bacterial 16S
rDNA genes. The forward primer, UNI16S-L
( 5 ’ - AT T C TAG AG T T T G AT C AT G G C T C A ) ,
corresponded to positions 11 to 26 of Escherichia
coli 16S rDNA, and the reverse primer, UNI16S-R
(5’-ATGGTACCGTGTGACGGGCGGTGTGTA),
corresponded to the complement of positions 1411 to
1393 of Escherichia coli 16S rDNA (2). PCR reaction
conditions were carried out according to Beffa et al.
(13) and the PCR product was cloned to a pGEM-T
vector system (Promega, UK).
Sequencing analysis
Following PCR amplification and cloning of
the 16S rDNA genes of our isolates, the 16S rDNA
gene sequences were determined with an Applied
Biosystems model 373A DNA sequencer by using
the ABI PRISM cycle sequencing kit (Macrogen,
Korea). The sequences consisting of about 13971414 nucleotides (nt) of the 16S rDNA gene were
269
Molecular diversity of thermophilic bacteria isolated from Pasinler hot spring (Erzurum, Turkey)
determined. These sequences were compared with
those contained within GenBank (14) by using a
BLAST search (15). The 16S rDNA gene sequences
of the species most closely related to our strains were
retrieved from the database. Retrieved sequences
were aligned by using the Clustal X program (16) and
manually edited. A phylogenetic tree was constructed
by the neighbor-joining method using the software
package MEGA 4.0 (17).
analysis (FAMEs)] and genotypic [BOX PCR and 16S
rDNA sequence analysis] data.
FAMEs analyses
In total 14 different FAMEs were detected in the
9 bacterial strains tested in the present study (Table).
Five of these, 15:0 iso, 15:0 anteiso, 16:0 iso, 17:0 iso,
and 17:0 anteiso fatty acids, appeared in all strains.
However, 17:1 iso w10c fatty acid was only present
in the P38 strain. Bacterial strains of G3B, P38, G1,
and Ah2 have similar fatty acid profiles, containing
15:0 iso, 15:0 anteiso, 17:0 iso, and 17:0 anteiso at the
concentration ranges of 10.08%-29.42%. However,
fatty acid 19:0 iso, which is present in all other
remaining strains, is not found. These strains were
identified as Bacillus spp. according to FAME data. The
remaining 5 bacterial strains (G19B, P45, Ah23, P26,
and M71) also showed similar FAME composition,
including 15:0, 16:0 iso, 17:0 iso, and 19:0 iso fatty
acids. These strains were identified as members of
the Geobacillus. These results showed that FAMEs
analysis is an appropriate phenotypic method for the
discrimination of Bacillus and Geobacillus strains
at genus level, but not at species level. As a result, it
can be concluded that FAME profiling may be useful
for the characterization of Bacillus and Geobacillus
strains at genus level. Similar findings related to the
FAME profiles of Bacillus and Geobacillus have been
reported in the literature (2,19-22).
Results and discussion
The bacterial strains isolated in this study were
subjected to various biochemical and physiological
tests. The results showed that all strains (G19B, P45,
Ah23, P26, M71, G3B, P38, G1 and Ah2) were grampositive, catalase and oxidase positive, endospore
forming, and mobile rods. Four strains (P26, P45,
G1, and G3B) were amylase positive. The optimum
pH and temperature for all strains was found as 7.58.5 and 55 ± 1 °C, respectively. All strains were able
to grow in a salt concentration range of 2%-5%. These
met the criteria of thermophilic bacteria, which grew
at temperatures above 50 °C (18).
Identification and characterization of bacterial
strains isolated in this study were performed by
using phenotypic [morphological, biochemical and
physiological characteristics, cellular fatty acids
Table. Cellular fatty acid composition (% w/w) of strains.
Fatty acid concentration (%)
Fatty acids
G3B
P38
G1
Ah2
G19B
P45
Ah23
P26
M71
14:0
15:0 iso
15:0 anteiso
15:0
16:0 iso
16:0
17:0 iso
17:0 anteiso
16:1 w7c
16:1 w11c
17:1 w10c
17:1 iso 1/Antei B
17:1 iso w10c
19:0 iso
5.36
25.79
19.27
8.05
6.23
10.08
18.12
1.56
2.79
1.54
1.22
-
4.45
20.08
16.11
0.62
9.25
7.78
18.42
14.69
3.06
2.12
1.45
1.98
-
6.22
23.36
22.91
5.89
5.37
12.69
19.08
1.58
2.05
0.86
-
4.46
29.42
20.01
7.02
8.04
12.54
13.60
1.98
1.75
1.19
-
3.16
7.85
5.31
24.55
21.23
6.15
19.45
6.87
2.25
2.01
1.16
9.01
6.20
26.0
20.59
2.95
23.26
8.25
1.58
1.05
1.07
0.25
9.68
7.92
25.52
21.85
3.58
21.63
8.12
1.71
2.65
7.54
7.23
24.09
22.15
21.37
5.95
2.13
2.94
2.05
1.03
0.85
6.12
5.01
26.77
23.21
2.82
21.19
9.64
1.68
1.95
0.68
0.94
270
A. ADIGÜZEL, K. İNAN, F. ŞAHİN, T. ARASOĞLU, M. GÜLLÜCE, A. O. BELDÜZ, Ö. BARIŞ
BOX-PCR
Genomic fingerprints involve analyzing the
whole genome of the targeted organisms. Rep-PCR
is one of the well-established genomic fingerprint
methods applied for bacterial identification and
characterization (2). This technique can simply
differentiate closely related strains of bacteria, and
it can assign bacteria potentially up to the strain
level based on the presence of repeated elements
within the genome examined (23). Therefore, repPCR was also used for the preliminary screening of
thermophilic strains as a rapid and reliable DNAbased fingerprinting method for identification
and characterization purposes, compared to other
traditional or conventional methods including
morphological, physiological, biochemical and
pathological test, FAMEs, SDS-PAGE and carbon
utilization profiles (2).
The BOX-PCR genomic fingerprints of the
bacterial strains revealed 2 distinct patterns with 18
different fragments ranging from 250 bp to 4500 bp
in size (Figure 1). Five bands of size 650 bp and 1550
bp were present in both genomic groups. However,
the remaining 13 fragments showed polymorphic
characteristics and discriminated 2 genomic groups.
Analysis of BOX-PCR results suggested that 9 strains
could be placed in 2 main groups (Figure 2), which
corresponded well to the groups formed by FAME
profiles.
M
1
2
3
4
CASE
0
Label Num
5
10
15
20
25
8
6
9
7
1
3
4
2
5
Figure 2. BOX-PCR cluster analyses; 1) G19B; 2) P45; 3) Ah23;
4) P26; 5) M71; 6) G3B; 7) P38; 8) G1; 9) Ah2.
For the closely related Bacillus and Geobacillus
genera, showing high sensitivity in the discrimination
of mesophilic and thermophillic species at the strain
level (24-26), rep-PCR genomic fingerprint protocols
and random amplified polymorphic DNA (RAPDPCR) technique were used (24-26). Additionally,
it was determined that, for the identification of
bifidobacteria, thermophilic lactic acid bacteria (LAB)
associated with dairy products, the most suitable rep
primer is BOXA1R primer (27). In the present study,
it was seen that BOX-PCR is a powerful technique to
differentiate Bacillus and Geobacillus strains.
Von der Weid et al. (28) reported the genetic
differences between the isolates belonging to
Paenibacillus polymyxa species by BOX PCR method.
Their findings show that this method clearly indicates
discrimination between the strains.
5
6
7
8
9
N
5000bp
4000bp
3000bp
2000bp
1500bp
1400bp
1000bp
750bp
500bp
400bp
300bp
200bp
Figure 1. BOX-PCR profile generated with the BOX A1R primer. Lanes: 1) G19B; 2) P45;
3) Ah23; 4) P26; 5) M71; 6) G3B; 7) P38; 8) G1; 9) Ah2; N; Negative Control;
M) Molecular Marker (10 kb).
271
Molecular diversity of thermophilic bacteria isolated from Pasinler hot spring (Erzurum, Turkey)
Geobacillus pallidus Ah23 (EU935598)
65
Geobacillus pallidus P45 (EU935593)
Geobacillus pallidus P 26 (EU935591)
100 Geobacillus pallidus M71 (EU935594)
Geobacillus pallidus (Z26930)
92
Geobacillus pallidus G19B (EU935595)
G.thermodenitrificans (Z26928)
G.stearohermophilus (X60640)
100
97
G.thermocatenulatus (Z26926)
G.kaustophilus (X60618)
99 G.thermoleoverans (M77488)
70 Bacillus licheniformis G3B (EU935596)
Bacillus licheniformis G1 (EU935597)
100
Bacillus licheniformis (EF427891)
Bacillus licheniformis P38 (EU935592)
54
Bacillus licheniformis Ah2 (EU935599)
Brevibacillus brevis (AB101593)
0.01
Figure 3. Dendrogram estimated phylogenetic relationships on the basis of 16S rDNA
gene sequence data of the thermophilic bacteria isolated from Pasinler hot
spring, Erzurum, Turkey, and some reference strains, using the neighborjoining method. The accession numbers are given in parentheses. Only
bootstrap values > 50% are shown at nodes (based on 1000 bootstrap
resamplings). The scale bar represents 1% divergence.
Meintanis et al. (26) concluded that REP- and
BOX-PCR methods are successful in showing the
discrimination between Geobacillus and Bacillus
strains. In this study, the difference between the
strains of the genera Geobacillus and Bacillus was
clearly determined by BOX PCR as reported similar
to the literature (26,29). The BOX-PCR data in our
study revealed the genomic relationship between the
strains in the first group (G3B, P38, G1, and Ah2)
as ≥ 94% similarity, and between the strains in the
second group (G19B, P45, Ah23, P26, and M71) as ≥
86% similarity (Figure 2).
16S rDNA sequence analysis
16S rDNA sequence analysis showed that the
strains in the first and second groups, which have
been classified according to FAME and BOX-PCR
profiles, have higher similarity with B. licheniformis
(G3B, P38, G1, and Ah2) and G. pallidus (G19B, P45,
Ah23, P26, and M71), respectively. In terms of the
16S rDNA gene sequences of Geobacillus and Bacillus
test isolates, a similarity value (99%) was retrieved
272
in agreement with Zeigler (30) and Meintanis et
al. (26), who confirmed that the 16S rDNA gene
sequences similarity of Geobacillus and Bacillus type
strains is higher than 98.5%. Although the 16S rDNA
gene is used as a framework for modern bacterial
classification, it has often been seen that its usage
shows limited variation for the discrimination of
closely related taxa and strains (26,31).
Existence of B. licheniformis and Aeribacillus
pallidus in thermal area was also reported in many
other studies (3,32-34). However, this is the first study
that demonstrated that B. licheniformis and G. pallidus
population are 2 common bacterial species present in
Pasinler hot spring water, Erzurum, Turkey.
Evolutionary relationships of thermophilic
bacterial strains
The neighbor-joining method was used for
identification of the evolutionary relationship of
the 9 bacterial strains and 8 closely related species.
The p-distance of nucleotide difference was used to
construct the tree (Figure 3) (35). The percentage of
A. ADIGÜZEL, K. İNAN, F. ŞAHİN, T. ARASOĞLU, M. GÜLLÜCE, A. O. BELDÜZ, Ö. BARIŞ
Corresponding author:
Medine GÜLLÜCE
Atatürk University,
Faculty of Arts and Science,
Department of Biology,
25240 Erzurum - TURKEY
E-mail: [email protected]
replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) is
shown next to the branches (36). The tree is drawn
to scale with branch lengths in the same units since
those evolutionary distances were used to infer the
phylogenetic tree. A total of 1371 positions were
obtained in the final dataset by eliminating the ones
containing gaps and missing data.
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