... 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 10 5 1. x 10 3 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: scsungka@mail2.ubu.ac.th, samghamit@hotmail.com
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, 3.6x10 5 and 1.45x10 3 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 45 (Bae et al., 2008) DNA Polymerase 2553) (, (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 0.5X NA 1.3 1.2 24 rrna 1. 1.1 ( 2 ) ( 3 ) ( ) ( ) 1.2 16S Spread plate 10 100 0.5X Nutrient agar (NA) 24 48 0.5X NA CFU/ml. 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 o C 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, 13, 1. ml. 0.6 13, 4 (70%) 500 µl. 1 TE buffer ph8 80 µl. 10 mg/ml Dnase free Rnase µl 37 20 trophoresis) (Lambda 2.3 PCR-RFLP III DNA size marker) (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 2.4 16S rrna (100 bp DNA ladder) 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 PCR-RFLP 16S rrna GenBank (http://www.ncbi.hlm.gov/) 1. 3 1 BLASTn ( (agarose gel elec- 2
Research 3 4 a 1 ( o C) ( o C) (CFU/ml) 80 70 a 30 55 55 3.65 x 10 5 ( ) 50 50 1.45 x 10 3 70 NA 2 (mm) (µm) 1 YTM1 1 3 Positive Bacilli 0.5x1-3 2 YTM2 2 4 Positive Bacilli 0.5x3-5 3 YTM3 2 3 Positive Bacilli 0.5x3-6 4 YTM4 1 Positive Bacilli 0.5x2-6 5 YTM5 3 5 Positive Bacilli 0.5x2-6 3 (mm) (µm) 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 ( ) (mm) (µm) 1 KSII_1 2 4 Positive Bacilli 0.5x1-4 2 KSII_2 2 4 Positive Bacilli 0.5x1-4 3 KSII_3 2 3 Positive Bacilli 0.5x1-3 4 KSII_4 2 3 Positive Bacilli 0.5x1-3
2. 16S rrna PCR-RFLP Product 1, bp 16S rrna Fd1 Rd1 ( 2 16S rrna 1 Non-specific 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 a YTM2 a YTM4 YTM5 KSII_1 Kao_P2 Kao_P4 (% Similarity) 97 94 93 80 80 80 sp. C56-T3, 98 98 sp. A27 98 strain 1245 99 99 IS13 99 99 99 sp. 98 97 94 92 16S rrna YTM4 Similarity YTM4 491/503 451/447 468/477 395/488 395/448 395/488 502/511 502/511 502/511 497/500 497/500 495/501 497/501 497/501 497/501 440/451 441/456 441/453
531 3. 16S rrna BLASTn BLASTn YTM4 YTM4 fi 5 a YTM2 (97%) b 80% Similarity YTM4 Sequence trace Peak ( ( 3 4 16S rrna (1.5 kb) c YTM5 (98%) d KSII_1 (99%) e Kao_P2 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%) f Kao_P4 (99%) (97%) CGS2 Ming et al., 2005) T4 (Wen-
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