Characterization and 16S rDNA Identification of

Journal of Applied Sciences Research, 3(10): 994-1000, 2007
© 2007, INSInet Publication
Characterization and 16S rDNA Identification of
Thermo-tolerant Bacteria Isolated from Hot Springs
Reda A.I. Abou-Shanab
Department of Environmental Biotechnology, Mubarak City for Scientific
Research and Technology Applications, Borg El Arab, P.O. 21934, Alexandria, Egypt
Abstract: High water temperature exerts selection pressure on microbial species leading to specific flora
that survives and tolerates heat stress. A total of 229 bacterial cultures were isolated from ten different
hot springs at Siwa, Matrouh, Egypt. The number and percentage of heat tolerant bacteria were assessed.
Only 13 (5.6%) of bacterial isolates were able to tolerate and survive 65°C. These bacterial isolates were
genetically diverse according to RAPD and Box- PCR analyses using different primers. RAPD, Box-PCR
and 16S rRNA sequence analysis confirmed the abundance of bacterial genotypes and that they were
closely related to Bacillus licheniformis and Bacillus pumilus, based on 100% similarity in their 16S
rDNA gene sequences. Bacillus licheniformis responded to one hour of thermal stress at elevated
temperature from 30°C to 65°C by synthesizing different heat shock proteins (HSPs) with molecular
weights ranging between .30 – 120 kDa.
Key words: Bacteria, Hot spring; Thermotolerance, PCR, Heat Shock Protein
Over the past decade, studies of the microbiology
of high temperature terrestrial hot springs by both
molecular-ecological and culture-based approaches have
revealed phylogenetic and physiological diversity[2 3 ,1 2 ,1 4 ].
Bacterial communities are difficult to study because of
their immense complexity and because of potential
problems in culturability of many of the members.
H owever, D N A-based analyses can contribute
significantly in characterization of bacteria that have
been successfully isolated from these environments.
G e no m ic D N A f in g e r p r in ting using ra nd o m
amplification of polymorphic DNA (RAPD-PCR) and
BOX-PCR have been found to be useful in
differentiating between very closely related bacteria.
The RAPD and BOX-PCR techniques are a polymerase
chain reaction (PCR)-based assay that was developed
to detect polymorphisms in genomic DNA [2 9 ,2 6 ].
Besides being simpler and cheaper, these methods are
as effective as the more labor intensive RFLP for
establishing genetic relationships and identifying
bacterial genomes [2 1 ].
In this study, thermotolerant bacterial isolates from
different hot springs were characterized using genomic
patterns obtained through RAPD and BOX–PCR with
the objective of identifying the dominant thermotolerant
bacteria and to find out whether these thermophilic
bacteria also responded to abrupt temperature changes
with a heat shock-like response.
INTRODUCTION
Temperature is one of the most important factors
that govern species abundance and distribution. High
temperatures in soil and/or water exert pressure on
microbial species leading to the selection of specific
flora
capable
of tolerating and surviving heat
stress [1 0 ]. Some species can survive at the elevated
temperatures of hot springs, or in various other adverse
environments. The defense mechanism cells utilize
when confronted with high temperatures in their local
environment is known as the heat shock response. This
response has been described extensively in both
eukaryotes and prokaryotes [5 ]. W hen thermal stress is
applied, the most prominent physiological reactions
are the production of a set of novel proteins or an
increase in the quantity of certain types of existing
proteins. These proteins are known as heat shock
proteins (HSPs) [1 1 ]. HSPs have been shown to play
important roles in the protection of organisms
under heat stress [1 5 , 1 6]. Heat shock proteins can be
classified into three categories according to their
molecular
size:
1) high molecular size, with
molecular size between 39 and 68kDa,) mediummolecular
between 39 and 68 kDa and 3) lowmolecular size, with molecular weight below 38
kDa [6 ].
Corresponding Author: Reda A.I. Abou-Shanab, Department of Environmental Biotechnology, Mubarak City for Scientific
Research and Technology Applications, Borg El Arab, P.O. 21934, Alexandria, Egypt
[email protected] Fax: 203 459 3407; Tel 20128963380
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J. Appl. Sci. Res., 3(10): 994-1000, 2007
proteins. The protein profile of bacterial strains was
assayed by (Sodium Dodecyl Sulfate-Polyacrylamide
Gel electrophoresis) SDS-PAGE Samples containing
10 mg protein/ml were prepared and electrophoresis
was carried out according to the methods described
in [2 0 ]. The stacking gel was 5% (W/V) acrylamide and
resolving SDS-containing gel was 10% (W /V) acrylamide.
M ATERIALS AND M ETHODS
Sampling and Isolation of Bacteria: W ater samples
for viable bacterial counts were taken in sterile 250-ml
screwcap bottles from ten different hot springs that are
located at Siwa, Matrouh, Egypt. Temperature was
measured in situ with a mercury bulb thermometer. The
bottles were filled completely and then closed tightly
to prevent the loss of dissolved gases. The water
samples were brought back to the laboratory and
analyzed within 24 h. Bacteria were isolated and
enumerated using the standard plate method. 0.1 ml of
the proper dilution was used to surface inoculate R2A
media [1 7 ]. From each dilution, five plates were
inoculated and incubated at 30°C. Colonies were
counted every 2 days until maximal plate counts were
obtained. Numbers were expressed as colony forming
units (c.f.u.) ml -1 . Colonies obtained on R2A were
isolated and further purified on R2A medium. The
isolates were stored at-80°C in R2A broth medium
containing 50% glycerol.
DNA Extraction, RAPD and BOX-PCR Analysis:
Bacterial D NA of thirteen thermotolerant bacterial
isolates was extracted from 10-ml bacterial cultures
grown overnight according to the method described
by [3 ]. DNA amplification reactions were conducted
in a Perkin-Elmer 9600 thermocycler. RAPD-PCR
amplification was performed as described by [2 1 ,2 2 ]
using the primers: A01, -5-CAGGCCCTTC-3 - ; A02,
5-TGCCGAGCTG-3 -; and A03 -5-AGTCAGCCAC-3 -.
The PCR protocol was a 35-cycle PCR (initial
denaturation, 95°C for 5 min; subsequent denaturation,
95 °C for 30 sec; annealing temperature, 45°C
for 2 min; extension temperature, 72 °C for 1 min
and final extension, 72 °C for 10 min). The
oligonucleotide primers BOXA1R (5 --CTACGGCA
AGGCGACGCTGACG-3 -) used to amplify bacterial
DNA [2 6 ]. The PCR was initiated by incubating the
reaction mixture at 95°C for 10 min, followed by 30
cycles of 30 sec at 94°C; 1 min at 52°C; and 6.5 min
at 65°C. The reaction was terminated with an extension
step consisting of 16 min incubation at 65°C. The PCR
products were analyzed on 1.5% agarose gel and
visualized by ultraviolet illumination after staining with
0.5µg mlG 1 ethidium bromide. The fingerprint patterns
resulting from RAPD and BOX-PCR analyses were
scored for each template DNA by recording the
presence or absence of bands to construct a
rectangular binary matrix. The matrix was used to
derive
simple
matching
coefficients used in
clustering analysis
and the construction of
dendrograms to illustrate the genetic relationships using
NTSYS-PC 2.0 [1 9 ].
Heat Tolerance: All isolates were examined for their
ability to tolerate heat stress. The number and
percentage of heat tolerant bacteria were assessed by
transferring each colony onto R2A agar medium. The
plates were incubated at different temperatures; 30, 37,
45, 50, 55, 60 and 65°C. Results of the inoculated
plates were read in time intervals of 2 days for at least
4 days of incubation. Numbers and the percentage of
bacteria grown at different temperatures were
calculated.
Heat Shock Treatment: The highest thermotolerant
bacterial isolate was grown overnight in 250-ml
Erlenmeyer flasks containing 100ml of sterilized R2A
broth on a shaker at 150 rpm. at 30°C until late log
phase. Subsamples (5ml) of bacterial suspensions were
transferred to test tubes. The bacterial cultures in each
tube were then placed in water baths set at 37°C,
45°C, 55°C, 60°C and 65°C for 15 min as pretreatment of heat shock [6 ]. The cultures were maintained
at the different temperatures for 60 minutes and then,
after the heat shock treatment, bacterial cells were then
harvested by centrifugation (14,000 xg, 20°C, 15 min)
and the pellets were washed twice with sterile distilled
water.
1 6 S R ib o s o m a l R N A (r R N A ) S eq u en c in g :
Oligonucleotide primers with specificity for eubacterial
16S rRNA genes, primers M16Sa and M16Sb were
used to amplify the 16S rRNA gene fragments with
template DNA originating from bacterial isolates and
using PCR protocols described by [1 8 ]. Subsamples
(10µl) of the reaction mixtures were analyzed by 1%
horizontal agarose gel electrophoresis to confirm the
presence of products [2 5 ]. PCR products were purified
using Q IA quick Spin columns (Qiagen Inc.,
Chatsworth, CA). Applied Biosystem 3100 Genetic
analyzer in combination with a Dye Deoxy Terminator
Electrophoresis Analysis of Proteins: Cell pellets was
washed three times with 10mM ice-cold tris-HCl
buffer, pH 7.6. Cells were suspended in 0.5ml 2x
treatment buffer and disrupted by repeated rapid
freezing at -80 0 C followed by thawing in a 45 0 C water
bath. Cell preparations were centrifuged (10,000 xg) to
obtain a clear supernatant containing the soluble
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J. Appl. Sci. Res., 3(10): 994-1000, 2007
Cycle Sequencing Kit (Perkin Elmer, Foster City, CA)
were used for sequencing the purified PCR products as
described previously [2 4 ]. A search of GenBank with
BLAST [1 ] was used to identify named bacterial species
with 16S rRNA gene sequences similar to those of the
isolates.
bacterial cultures isolated from Aen Zahra, Bar
El-Dakrour and Bar Quarash, respectively, were able to
grow at 65°C. A total of thirteen bacterial isolates with
the highest rates of heat tolerance were selected for
RAPD and BOX-PCR analysis.
The development and increased availability of
techniques in molecular biology have made it possible
to obtain information regarding the diversity of
bacterial cultures isolated from different habitats [2 ,9 ].
One such technique, a polymerase chain reaction
(PCR)-based assay to fingerprint genomes using
random amplification of polymorphic DNA (RAPD)
and BOX are useful for differentiating between
bacterial isolates [2 7 ,8 ].
RESULTS AND DISCUSSIONS
Bacterial Count and Response to Temperature: Total
bacterial population size in the ten hot springs ranged
from 2 to 40 x 10 2 CFU ml -1 water. This relatively low
bacterial count can be attributed to the relatively high
temperatures ranged from 50 to 55°C as shown in
Table 1. Elevated levels of temperatures affect the
qualitative as well as the quantitative structure of
microbial communities. Several studies found that
temperature influence microorganisms by adversely
affecting their growth, morphology and biochemical
activities, resulting in decrease biomass and
diversity [2 8 ,4 ,7 ,1 3 ]. A total of 229 morphologically distinct
isolates were randomly selected from the ten different
hot springs. All isolates were screened for their ability
to grow at different temperatures. The cumulative
percentages of bacterial strains resistance to various
temperatures are shown in Table 2. The results shows
that only 8 (20%), 4 (16.7%) and 1 (5.6%) of
RAPD Fingerprinting: RAPD analysis of the thirteen
isolates led to a minimum of 4 and a maximum of 7
discrete visible bands ranging in size from 0.1 to 3.5
kb (Figure 1). RAPD-PCR with primer A02 was the
most informative since the highest numbers of
polymorphisms were observed among the isolates.
Primers A01, A03 and A04 were uninformative
because no polymorphisms could be detected among
the isolates (data not shown). The dendrogram derived
from the RAPD dataset showed that bacterial isolates
2, 3, 4 and 5 from Bar El Dakrour hot springs shared
100% similarity and 8, 9, 10, 11 and 12 isolated
from Ain
Zahra
hot springs also shared 100%
similarity. The isolate numbers 7 and 13 shared 80%
similarity with isolates 8, 9, 10, 11 and 12. From the
RAPD patterns of the two remaining isolates it was
concluded that these were more diverse, with
similarities below 80% (Figure 2).
Total
colony form ing units (C FU ) of culturable
bacteria isolated from ten different hot springs at Siwa,
M atrouh, Egypt
Site Code
N am e of hot springs
Tem perature (°C) CFU x 10 2
A
Bar Q urash
50
30
B
Bar El-D akrour
52
40
C
Bar Kadous
50
20
D
Bar Aen Zahra
53
30
E
Bar El-Zaetoun
52
8
F
Bar Aen Shrouk
50
30
G
Aen Zahra
55
30
H
Aen Shrouk
51
4
I
Aen El-N akab
50
2
J
Aen El-D raat
50
2
Table 1:
BOX Fingerprinting: Genetic variation within thirteen
thermotolerant bacterial isolates was also assessed using
BOX-PCR and was shown to a useful method for
differentiating closely related bacteria [2 6 ,8 ]. BOX-PCR
analysis led to a minimum 9 and a maximum 15
characteristic bands ranging in size from 5.1 to 0.2 kb
as shown in figure 3. The dendogram derived from the
BOX-PCR dataset also showed that bacterial strains 2,
3, 4 and 5 shared 100% similarity and bacterial
cultures 7, 8, 9, 12 and 13 shared 100% similarity and
isolates number 7 and 13 shared 86% similarity with
isolates 7, 8, 9, 12 and 13 (Figure 4). Isolate 6 shared
82% similarity with isolates 7, 8, 9, 12 and 13. Isolate
number 1 shared 86% similarity with bacterial isolates
2, 3, 4 and 5.
The electrophoretic (RAPD and BOX-PCR)
patterns of bacterial isolates showed the bacterial
isolates 12 and 3 were predominant and represented
38.5% and 30.7% , re spe ctive ly, o f th e 13
thermotolerant bacterial isolates.
Table 2: Screening of 229 hot spring bacterial isolates to grow at
different tem peratures
30
37
45
50
55
60
65
70
---------------------------------------------------------------------Isolate Cum ulative % of bacterial isolates resistant at
Site No.
the following tem perature (°C)
A
18
100
72.2
72.2 33.3
11.2 5.6
5.6
0.0
B
24
100
91.7
91.7 58.3
25
16.7 16.7 0.0
C
31
100
90.3
77.4 51.6
9.7
0.0
0.0
0.0
D
27
100
100
81.5 18.5
11.1 0.0
0.0
0.0
E
13
100
92.3
76.9 0.0
0.0
0.0
0.0
0.0
F
27
100
88.9
77.8 11.1
7.4
0.0
0.0
0.0
G
40
100
72.5
52.5 35
32.5 22.5 20.0 0.0
H
15
100
80
60
26.6
20
0.0
0.0
0.0
I
14
100
78.6
57.1 0.0
0.0
0.0
0.0
0.0
J
20
100
80
50
0.0
0.0
0.0
0.0
0.0
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J. Appl. Sci. Res., 3(10): 994-1000, 2007
Fig. 1: RAPD-PCR products from thirteen thermotolerant bacterial isolates (1, 6, 7, 8, 9, 10, 11 and 12 were
isolated from Aen Zahra; 2, 3, 4 and 5 were isolated from Bar El-Dakrour and 13 was isolated from Bar
Qurash hot springs) generated by random primers A02. Lane M1, molecular size marker of Lambda DNA
EcoRI digested with HinDIII and M 2, 100 bP DNA ladder.
Fig. 2: Dendrogram derived from the analysis of RAPD fingerprint patterns generated using A02 primer showing
the diversity among thermotolerant bacterial isolates.
Fig. 3: Box polymerase chain reaction (PCR) fingerprint patterns of thermotolerant bacterial isolates. M 1,
molecular size marker of Lambda DNA EcoRI digested with HinDIII and M2, molecular size marker of
pBR322 digested with MspI.
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J. Appl. Sci. Res., 3(10): 994-1000, 2007
Fig. 4: Dendrogram derived from the analysis of BOX fingerprint patterns showing the diversity among thirteen
thermotolerance bacterial isolates.
Fig. 5: Effect of heat shock treatment on the pattern of protein synthesis in Bacillus licheniformis isolated from
Siwa hot spring. Bacterial cultures were placed in 30°C (control), 37°C 45°C, 55°C, 60°C and 65°C (heat
shock) water baths for 1 hour. The position of standard protein markers and the size of the heat shock
proteins are marked.
for the sickle fungus Fusarium oxysporum 40°C or
43°C. In general, a rise of 5°C above the normal
physiological temperature will induce the synthesis of
HSPs [1 1 ].
Thermophilic microorganisms normally grow at
high temperatures and therefore should have a higher
thermotolerance. Thus it was of interest to find out
whether these thermophilic bacteria (B. licheniformis)
as a dominant bacterial isolates in hot spring responded
to abrupt temperature changes with a heat shock-like
response. For this, a comparison was made of the
proteins which were synthesized in response to
different temperature. The response of B. licheniformis
to one hour of thermal stress at 65°C by synthesizing
11
heat
shock proteins (HSPs) with molecular
weights ranging between . 30 – 120 kDa as shown in
Figure 5.
16S rRNA Sequence Analysis: More than 1400 bp
of the 16S rRNA genes of strains 12 and 3 were
sequenced. Analysis of the 16S rRNA sequences
confirmed the strain 12 and 3 were closely related to
B a c illu s lic h en ifo r m is a n d B a cillu s pum ilus,
respectively, based on 100% similarity in their 16S
rDNA gene sequences.
Effects of elevated temperature on proteins
synthesis: A short exposure of cells to elevated
temperatures reduces the synthesis of normal cellular
proteins and at the same time induces a transient
overproduction of a specific group of proteins, the
so-called heat shock proteins (HSPs) [5 ]. The optimum
temperature for the production of HSPs varies from
organism to organism. The heat shock temperature
range for E. coli is 43- 47°C, for the yeast 36°C and
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J. Appl. Sci. Res., 3(10): 994-1000, 2007
Conclusions: In the present study, a high proportion of
heat tolerant bacteria were observed to be present in
water samples that were collected from Aen Zahra, Bar
El-Dakrour and Bar Qurash hot springs compared
with other hot springs. A selected thirteen bacterial
isolates could tolerate high temperature up to 65°C.
The genetic methods used in this study differentiated
these isolates. Similarity analysis of RAPD and
BOX-PCR screening revealed 6 unique patterns among
13 bacterial isolates. The most abundant bacterial
isolates were related to B. licheniformis (38%) and
B. pumilus (31%). B. licheniformis responded to heat
stress by synthesized different heat shock proteins. This
bacterial strain can be used as a candidate for many
industrial purposes like antibiotic and enzymes
productions.
9.
10.
11.
12.
ACKNOW LEDGM ENT
This work was supported by grant #71 Academy
of Scientific Research and Technology, Egypt, Program
of the National Strategy for Biotechnology and Genetic
Engineering.
13.
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