5694 Nucleic Acids Research, 2010, Vol. 38, No. 17 The plasmids for gene disruption were derivatives of the pGEM-T Easy vector (Promega Co., Madison, WI, USA), constructed by inserting the thermostable kanamycin- resistance gene, HTK (30), ﬂanked by 500-bp upstream and downstream sequences of the tthb178 and recJ genes (Supplementary Figure S1A). The 500-bp DNA fragments from upstream and downstream of the tthb178 gene were ampliﬁed by PCR using primer sets 50-ACTCGGGCGGC ACGATC-30 and 50-ATATGGTACCCGCCGTCAACG GGTACCG-30, and 50-ATATCTGCAGCATGTTGGTT ACGCTGCA-30 and 50-GCGCCGCCTCCACCACCT-30, respectively (the underlining indicates KpnI and PstI sites, respectively). The 500-bp DNA fragments from upstream and downstream of the recJ gene were also ampliﬁed by PCR using primer sets 50-CGGGACCCTCTTGGGCC T-30 and 50-ATATGGTACCCGCCGTCAACGGGTAC CA-30, and 50-ATATCTGCAGCATGTTGGTTACGCT GCA-30 and 50-CGGGGGCCTCCACCACCC-30, re- spectively (the underlining indicates KpnI and PstI sites, respectively). The ampliﬁed fragments were digested with KpnI and PstI, respectively, to obtain fragments I and II. The HTK gene was also ampliﬁed by PCR from plasmid pUC18/HTK (30) by using 50-ATATGGTACCCGTTGA CGGCGGGATATG-30 and 50-ATATCTGCAGCGTAA CCAACATGATTAA-30 as primers (the underlining indi- cates KpnI and PstI sites, respectively). The ampliﬁed fragment was then treated with KpnI and PstI to obtain fragment III. Fragments I, II and III were ligated into the pGEM-T Easy vector. For double gene knockout, the thermostable hygromycin-B-resistance gene was used for disrupting the recJ gene (Supplementary Figure S1B). The hygromycin-B-resistance gene was ampliﬁed by PCR from plasmid pHG206 (31) by using 50-GAATTCGAGCTCG GTACCCG-30 and 50-ATATCTGCAGGAATTCGAGG TCGCTACCCG-30 as primers (the underlining indicates KpnI and PstI sites, respectively). The ampliﬁed fragment was then digested with KpnI and PstI to obtain fragment IV. Fragments I and II from the recJ gene and fragment IV were ligated into pGEM-T Easy vector. The plasmid was transformed into T. thermophilus HB8 cells as previously described (29). Disruptions of the tthb178 and recJ genes were conﬁrmed by PCR ampliﬁcation using the isolated genomic DNAs as templates (Supplementary Figure S2). The absence of the mRNA transcribed from tthb178 and recJ was also conﬁrmed by reverse transcription–PCR (Supplementary Figure S3). Estimation of spontaneous mutation rates The spontaneous mutation rate of T. thermophilus HB8 was estimated based on the frequency of streptomycin-resistant strains measured by means of the modiﬁed Luria–Delbru¨ ck ﬂuctuation test (32). Cultured T. thermophilus HB8 wild-type (WT) and disruptants in the mid-exponential growth phase (A660 = 1.0–1.5) were appropriately diluted in TT broth and spread on TT agar plates with or without 50 mg/ml streptomycin. The numbers of colonies formed, colony-forming units (CFUs), were counted after incubation at 70 C for 24 h. The surviving fractions were expressed as the average obtained from at least three independent experiments. The spontaneous mutation rates (%) were calculated ac- cording to the formula mutation rate (%) = M/N 100, where M is the counted CFUs on the TT plates containing 50 mg/ml streptomycin and N is the mean of the CFUs on the TT plates without streptomycin. Examination of the sensitivities to UV irradiation and H2O2 addition Thermus thermophilus HB8 cells in the mid-exponential growth phase were spread on TT agar plates and irradiated with 254-nm UV light at the dose rate of 1.9 J m 2 s 1 for 40 s. The CFUs were counted after incu- bation at 70 C for 24 h and the surviving fractions were expressed as the average obtained from at least three in- dependent experiments. The sensitivity to H2O2 addition was measured as follows. Thermus thermophilus HB8 cells in the mid- exponential growth phase were mixed with equal volumes of 0-, 10-, 20- and 100-mM H2O2. The cells were further incubated at 70 C for 20 min and spread on TT agar plates. The CFUs were counted after incubation at 70 C for 24 h and the surviving fractions were expressed as the average obtained from at least three independent experiments. Overexpression and puriﬁcation of TTHB178 Escherichia coli Rosetta(DE3) (Novagen, Madison, WI, USA) was transformed with pET-11a/tthb178 (RIKEN BioResource Center, Tsukuba, Japan) and the trans- formed cells were cultured in L-broth containing 50 mg/ml ampicillin. When the cell density reached 1 108 cells/ml, isopropyl-1-thio-b-D-galactopyranoside was added to the culture to induce tthb178 gene expres- sion. The cells were further cultured for 6 h and harvested by centrifugation at 9000g under 4 C and stored at 20 C until use. All the steps for TTHB178 puriﬁcation except the heat treatment were performed at 4 C. The frozen cells were suspended in 50-mM Tris–HCl and 5-mM EDTA (pH 8.0; buﬀer A) and disrupted by sonication. The cell lysate was treated at 60 C for 10 min and the supernatant was re- covered after centrifugation at 34 000g for 1 h. (NH4)2SO4 was gradually added to the solution to a ﬁnal concentration of 1.5 M. The solution was applied to a Toyopearl Ether-650M column (bed volume of 20 ml; Tosoh Corp., Tokyo, Japan) equilibrated with 50-mM Tris–HCl, 1.5-M (NH4)2SO4, 100-mM KCl and 5-mM EDTA (pH 8.0; buﬀer B). The column was washed with buﬀer B and then eluted with a linear gradient of 1.5–0-M (NH4)2SO4 in 50-mM Tris–HCl, 100-mM KCl and 5-mM EDTA (pH 8.0). SDS–PAGE revealed that the target protein was eluted at 0.7–0.6-M (NH4)2SO4. The fractions containing TTHB178 were dialysed twice against 5 l of buﬀer A. The dialysed solution was diluted to 50 ml with buﬀer A and loaded onto a Toyopearl SuperQ-650M column (bed volume of 20 ml; Tosoh Corp.) equilibrated with buﬀer A. The column was substantially washed with buﬀer A and the proteins were eluted with a linear gradient of 0–1 M KCl in buﬀer A. The target protein was eluted at 100–150-mM KCl. The fractions containing