diversity of thermophilic bacteria isolated from hot...

. . . 40(2) 524-533 (2555) KKU Sci. J. 40(2) 524-533 (2012)

Diversity of Thermophilic Bacteria Isolated from Hot Springs

(

30, 3.65 x 105 1. x 103 CFU/ml

(a, b, c, d, e f

16S rRNA 1. kb Polymerase Chain Reaction-Restriction Fragment

Length Polymorphisms (PCR-RFLP) fI 16S

rRNA a ( YTM2)

(97%) b ( YTM4)

c ( YTM5) (98%) d (

KSII_1) (99%) e ( Kao_P2)

(99%) f ( Kao_P4)

(97%)

1 . . 34190 *Corresponding Author, E-Mail: [email protected], [email protected]

ABSTRACT This research aims to study the diversity of thermophilic bacteria of three hot springs in

southern Thailand. Total bacteria number in Tanoh Maroh, Kaochaison and Ban Natungpo hot

spring were 30, 3.6x105 and 1.45x103 CFU/ml, respectively. The 13 isolates were put into 6

groups according to their distinct restriction cutting profiles of 16S rRNA gene obtained from

employing PCR-RFLP technique using restriction enzyme I. The 6 groups were named group

a, b, c, d, e and f, and analysis of their partial 16S rRNA gene sequence showed that group a

(isolate YTM2) had 97% similarity with , group b (isolate YTM4) is related to

members of the genera and , group c (isolate YTM5) is related to

(98%), group d (isolate KSII_1) is related to (99%), group e (isolate

Kao_P2) is related to (99%) and group f (isolate Kao_P4) is related

to (97%).

: 16S rRNA gene PCR-RFLP

Keywords: Thermophile, Hot spring, 16s rRNA gene, PCR-RFLP

( ,

2553)

45

(Bae et al., 2008)

DNA Polymerase

(Brock and

Madigan, 1991

(Brock

and Madigan, 1991; Abdelnasser et al., 2007;

Adiguzel et al., 2009; Elnasser et al., 2007;

Research

Kanso 2003; Reda et al., 2007)

112

16S

rRNA

1.

1.1

( 2

)

( 3

) (

) (

)

1.2

Spread plate

10 100

0.5X Nutrient agar (NA)

24 48

0.5X NA CFU/ml.

0.5X NA

1.3

1.2

24

2. 16S rRNA

2 . 1

(Chromosomal DNA)

CTAB/NaCl

0.5X NB 30

ml. 24

5,000 15

TE buffer pH8

1 ml. TE 13,000

3

TE buffer pH8 400µl.

10 mg/ml Lysozyme 30 µl.

37 1

10%SDS (Sodium dodesyl sulfate) 30 µl.

20mg/ml Protienase K 6 µl.

50 oC 1

0.7 M NaCl 5 M NaCl

CTAB/NaCl 0.1

65 15 25:24:1 Phenol:

Chloroform: Isoamyl alcohol

13,000 4 15

1.5 ml.

(cell

debris) 24:1 Chloroform:

Isoamyl alcohol

13,

1. ml.

0.6

13, 4

(70%) 500 µl.

13,

1 TE

buffer pH8 80 µl. 10 mg/ml Dnase free

Rnase µl 37

20

2.2 16S rRNA

(Polymerase chain reaction,

PCR)

16S

rRNA Fd1 (5’AGAGTTTGATCCTGGCAG3’)

Rd1 (5’AAGGAGGTGATCCAGCC3’)

25 µl

12.5 µl 2X PCR Go Taq

Colorless Master Mix, 5µM Fd1 primer, 5µM

Rd1 primer, 5 µl Nuclease free water 2.

µl DNA template

Preheat 94

Denaturation 94 30

Annealing 55 30

Extension 72 2

(agarose gel elec-

trophoresis)

(Lambda III DNA size marker)

2.3

PCR-RFLP

(PCR product

fI 13.3 µl

Deionized water, 0.2 µl 10 µg/ µl Acetylated

BSA, 2 µl 10X buffer, 0. µl U/ µl fI

µl PCR product 37

2

(100 bp DNA ladder)

2.4

16S rRNA

PCR-RFLP

16S rRNA

GenBank BLASTn

(http://www.ncbi.hlm.gov/)

1.

3

(

1

2

Research

3 4

1

(oC) (oC) (CFU/ml)

80 70a 30

55 55 3.65 x 105

( ) 50 50 1.45 x 103 a 70 NA

2

(µm) (mm)

1 YTM1 1 3 Positive Bacilli 0.5x1-3



4 YTM4 1 Positive Bacilli 0.5x2-6


3

(µm) (mm)

1 Kao_P1 2 3 Positive Bacilli 0.5x1-3

2 Kao_P2 1-2 Positive Bacilli 0.5x1-3

3 Kao_P3 1-2 Positive Bacilli 0.5x1-3

4 Kao_P4 1 Positive Bacilli 0.5x1-2

4 ( )

(µm) (mm)

1 KSII_1 2 4 Positive Bacilli 0.5x1-4




2. 16S rRNA PCR-RFLP

1 16S rRNA

Fd1 Rd1 ( 2

16S rRNA

1, bp

Non-specific

Product 700-800 bp

YTM1, YTM2 YTM3

1-3

PCR-RFLP

13 a, b, c, d, e

f

fI ( 3 a

YTM1, YTM2 YTM3 b

YTM4 c YTM5 d

KSII_1, KSII_2, KSII_3 KSII_ e

Kao_P1, Kao_P2 Kao_P3 f

Kao_P4

1 1 = YTM1, =YTM2 =

YTM3 4 =YTM4, 5 = YTM5, 6 = KSII_1, = KSII_2 8 = KSII_3, = KSII_4, = Kao_P1,

= Kao_P2, = Kao_P3, = Kao_P4, 14 = Lambda III DNA marker

2 M = Lambda III DNA marker,

1 = YTM1, =YTM2 = YTM3 4 =YTM4, 5 = YTM5, 6 = KSII_1, = KSII_2 8 = KSII_3, =

KSII_4, = Kao_P1, = Kao_P2, = Kao_P3, = Kao_P4

Research

3 16S rRNA fI 1 = YTM1, 2

=YTM2, 3 = YTM3, 4 =YTM4, 5 = YTM5, 6 = KSII_1, 7 = KSII_2, 8 = KSII_3, 9 = KSII_4, 10 =

Kao_P1, 11 = Kao_P2, 12 = Kao_P3, 13 = Kao_P4, M = 100 bp DNA marker, a, b,

c, d, e f

5 16S rRNA GenBank

(% Similarity)

YTM2

97

94

93

491/503

451/447

468/477

a YTM4

80

80

80

395/488

395/448

395/488

YTM5

sp. C56-T3,

sp. A27

98

98

98

502/511

502/511

502/511

KSII_1

strain 1245

IS13

99

99

99

497/500

497/500

495/501

Kao_P2

sp.

99

99

98

497/501

497/501

497/501

Kao_P4

97

94

92

440/451

441/456

441/453 a 16S rRNA YTM4 Similarity YTM4

531

3. 16S

rRNA BLASTn

fI

5 a YTM2

BLASTn

(97%) b

YTM4

80% Similarity

YTM4

YTM4

Sequence

trace Peak (

( 3 4

16S rRNA (1.5 kb)

c YTM5

(98%) d KSII_1

(99%)

e Kao_P2

(99%)

f Kao_P4

(97%)

YTM1, YTM2, YTM3,

YTM4, YTM5, KSII_1, KSII_2, KSII_3, KSII_4,

Kao_P1, Kao_P2, Kao_P3 Kao_P4

PCR-RFLP

16S rRNA

fI

YTM1, YTM2 YTM3

YTM2

(97%) b

YTM4 c

YTM5

(98%) d

KSII_1, KSII_2 KSII_3

KSII_1

(99%) e

Kao_P1, Kao_P2 Kao_P3

Kao_P2

(99%) f

Kao_P4

(97%)

CGS2 (Wen-

Ming et al., 2005)

T4

Research

(Shang-Kai et al., 2004)

(Yasser et al.,

2008)

(Vicki

et al., 2003)

pH

2553).

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