5696 Nucleic Acids Research, 2010, Vol. 38, No. 17 Table 1. Sequences of oligonucleotides used in this study Name 10f 21f 21r 21rna 21sr 28sr 40sr 50sf O6m O4m 8OG rAP21 AP21 Ura21 Hyp30 Xan30 Length 10-mer 21-mer 21-mer 21-mer 21-mer 28-mer 40-mer 50-mer 18-mer 18-mer 21-mer 21-mer 21-mer 21-mer 30-mer 30-mer Sequence GGCCAGGTGG ATGACAACTAAAGCAACACCC GGGTGTTGCTTTAGTTGTCAT ATGACAACTAAAGCAACACCC CCTAGCGGCTGCCACCTGGCC GGGCACCATGCGGGCGGCCAAAATGCCC CGGGCGGCCTCCCCTCCACCCTAGCGGCTGCCACCTGGCC GGCCAGGTGGCAGCCGCTAGGGTGGAGGGGAGGCCGCCCGCATGGTGCCC GCCCGGCCAmGCTGCAGTT GCCCGGCCAmTCTGCAGTT TACTGTTGAoxoGTTGGTTGTGGG TACTGTTGArAPTTGGTTGTGGG TACTGTTGAAPGTTGGTTGTGGG TACTGTTGAUraTTGGTTGTGGG GCTCGTAGAGCGGTCHypTAGTCAAGATACCG GCTCGTAGAGCGGTCXanTAGTCAAGATACCG mG, O6-methylguanine; mT, O4-methylthymine; oxoG, 8-oxoguanine; rAP, reduced abasic site; AP, abasic site; Ura, uracil; Hyp, hypoxanthine; Xan, xanthine. procedure (21,35,36). The kcat and KM values were determined by ﬁtting the data to the Michaelis–Menten equation using Igor 4.03 (WaveMetrics, Lake Oswego, OR, USA). RESULTS Sequence comparison between TTHB178 and DnaQ superfamily exonucleases The tthb178 gene encoded a protein that comprised 296 amino acid residues and whose N-terminal region showed signiﬁcant sequence similarity to DnaQ superfamily exo- nucleases such as E. coli ExoI and ExoX (37,38) and the proofreading domains of the DNA polymerases. These regions contained exonuclease motifs I–III that included conserved Asp, Glu and His residues (Figure 1A). The DnaQ superfamily exonucleases had a wide variety of protein lengths and showed sequence diversities except for the exonuclease motifs (Figure 1B). Among them, the length of TTHB178 was comparable to that of E. coli ExoX; however, their C-terminal regions showed no detectable sequence similarity. Expression of tthb178 in T. thermophilus HB8 cells Unlike recJ, tthb178 has been annotated as a hypothetical protein, and there was no evidence for the in vivo expres- sion of tthb178 before the recent report on the CRP-dependent expression of tthb178 in cells (27). We ﬁrst performed time course transcription analysis of the tthb178 gene in T. thermophilus HB8 cells by using reverse transcription–PCR. Transcription of tthb178 was detected from the mid-log phase to the late stationary phase (Supplementary Figure S4). The gene was more actively transcribed in the late stationary phase (no. 6 in the ﬁgure) than in the mid-log phase (no. 3) or stationary phase (nos. 4 and 5). The result strongly suggested that tthb178 is not a pseudo-gene and is required for a cellular function in T. thermophilus HB8. Figure 2. Eﬀects of the disruptions of the tthb178 and recJ genes on the growth of T. thermophilus HB8. (A) Growth curves of the WT (circles), recJ (triangles), tthb178 (inverted triangles) and the double disruptant (squares). Growth was monitored by measuring the absorbance at 660 nm. (B) Spontaneous mutation rates of each strain to the streptomycin-resistant strain. (C) Sensitivity to 254-nm UV-C irradiation. The survival ratios are shown as a bar graph. (D) Sensitivity to H2O2. The survival ratios were plotted against the H2O2 concentration. The symbols are the same as in (A). In all panels, the data represent the averages of at least three independent experi- ments and each bar indicates the SD. Phenotypes of tthb178 and recJ disruptants To investigate the cellular functions of ssExos in vivo, we generated a tthb178 disruptant ( tthb178), recJ disruptant ( recJ) and tthb178–recJ double disruptant ( tthb178– recJ). All the disruptants grew in rich medium, indicating that these two genes were not essential under the condition examined. However, all of them exhibited a relatively long lag time prior to the exponential growth compared with the WT (Figure 2A). These disruptants also showed lower maximum cell density than the WT during the stationary phase (Figure 2A). Furthermore, the disruptants aggregated in the log phase whereas the WT did not (data not shown). Elongated cells of the disruptants were observed in the late log, stationary and