Mutagenesis Advance Access originally published online on June 15, 2006
Mutagenesis 2006 21(4):255-260; doi:10.1093/mutage/gel025
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ß-Glucosidase as a reporter for the gene expression studies in Thermus thermophilus and constitutive expression of DNA repair genes
School of Life Science, Tokyo University of Pharmacy and Life Science 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70–75°C. Because the frequency of DNA damage, such as deamination, depurination and single-strand breaks, increases as the temperature rises, the regulation of expression as well as the specificities and activities of T.thermophilus DNA repair systems are of particular interest. To study those systems, we developed a gene expression vector using the T.thermophilus ß-glucosidase gene (bgl) with host strain JOS9 (
bgl) derived from the T.thermophilus wild-type strain HB27. Since HB27 has two putative ß-galactosidase genes, the use of a single bgl gene as a reporter in combination with a bgl host strain permits the study of gene expression against a low background level. We assayed Bgl activity with 2-nitrophenyl-ß-D-glucopyranoside as the substrate at 80°C. We measured the expression of seven genes involved in DNA repair—three nucleotide excision repair genes (uvrA, uvrB and uvrC) and four recombinational repair genes (recA, ruvA, ruvB and ruvC). Expression levels of uvrA and uvrB were about three times those of uvrC, while those of ruvA, ruvB and ruvC were almost equal. Both ruvA and ruvC formed an operon with their adjacent 5'-upstream gene paaG and ftsQAZ, respectively. recA was transcribed as an operon of four genes, amt-cinA-ligT-recA. All seven DNA repair genes were expressed constitutively, and the DNA damaging agent mitomycin C did not increase their expression.
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Introduction |
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Thermus thermophilus HB27, isolated from a Japanese thermal spa, is an extremely thermophilic eubacterium that grows at 50–85°C and optimally at 70–75°C, pH 7.5 (1
). It is a nonsporulating, gram-negative, aerobic, obligate heterotroph. Its thermostable enzymes have been subjected to extensive research, both basic and applied, and its genome has been sequenced recently (2). The genome consists of 
2200 putative genes divided between a 1.89 Mb chromosome and a 0.23 Mb megaplasmid named pTT27 with high (69%) GC content. Although T.thermophilus can grow rapidly in nutrient medium at 70–75°C (<30 min/generation), it is assumed that DNA damage, such as deamination, depurination, depyrimidination and single-strand breaks, occurs at a much higher frequency in thermophilic compared with mesophilic cells. Therefore, it would be interesting to characterize the DNA repair systems that function in extremely high-temperature environments.
T. thermophilus shows natural transformation competence throughout its growth phase with an efficiency on the order of 104 transformants/µg DNA (3). A chemically defined medium for HB27 (4) and a host-vector system that uses the cryptic plasmid pTT8 carrying a trpB gene have been developed (5). Antibiotic resistance genes from mesophilic bacteria, however, do not function at >70°C, and no natural antibiotic resistance plasmids have been found in Thermus spp. The only available gene at present is the thermostable kanamycin-resistant gene (HTK) developed by Hoseki et al. (6), who increased the thermostability of kanamycin nucleotidyltransferase from Staphylococcus aureus to 80°C by directed evolution method. HTK provides an excellent tool for gene disruption in T.thermophilus (7) as well as in vector plasmid construction. It is still difficult, however, to disrupt multiple genes to make double or triple mutants without other positive selection markers like drug-resistance. For these reasons, genetic studies using mutant DNA repair strains have been limited.
On the other hand, a convenient reporter gene for gene expression study is indispensable, and an attempt has been made to use the ß-galactosidase gene from Thermus sp. T2 (8) or A4 (9) for that purpose. The HB27 shows high background levels of ß-galactosidase activity and a mutant strain in which the ß-galactosidase gene was disrupted with kanamycin-resistant gene was constructed (9). The specific activity of ß-galactosidase in the mutant, however, was still half the normal value, which prevented its use as a reporter gene for the analysis of low promoter activity. The high background activity of ß-galactosidase was probably due to the existence of two putative chromosomal genes (TTP0220 and TTP0222) for ß-galactosidase (2). In this study, we report the use of the T.thermophilus HB27 ß-glucosidase gene (bgl, TTP0042) as a reporter. We developed a convenient gene expression vector using bgl in combination with a bgl mutant as the host strain. With this system, we investigated the expression of the recA, ruvABC and uvrABC genes of T.thermophilus with and without mutagen treatment. We also report here that T.thermophilus recA is transcribed as part of a 4-gene operon.
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Materials and methods |
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Bacterial culture media and chemicals
T. thermophilus strains were cultured in PY medium (0.8% polypeptone, 0.4% Difco yeast extract, 0.2% NaCl, 0.35 mM CaCl2 and 0.4 mM MgCl2) at 70°C with shaking. MSG minimal medium was described previously (10
). Medium was solidified with 0.7% gellan gum (Wako Pure Chemical Industry, Tokyo). Kanamycin sulfate (Sigma–Aldrich Co., MO, USA) was added at 500 µg/ml. Escherichia coli strains were cultured in Luria–Bertani medium (1% tryptone, 0.5% yeast extract and 1% NaCl) with ampicillin (Sigma–Aldrich) added at 100 µg/ml. 2-Nitrophenyl-ß-D-glucopyranoside (2NPGlc) and 5-bromo-4-chloro-3-indolyl-ß-D-glucopyranoside (X-Glc) were obtained from Wako Pure Chemical and Funakoshi Co. (Tokyo), respectively. Mitomycin C (MMC) was purchased from Kyowa Hakko Kogyo Co. (Tokyo).
Isolation of bgl mutant
The strains of T.thermophilus and plasmids used in this study are listed in Table I. Strains HB27 and MT111 (pyrE) and plasmid p3TSDN1 carrying a T.thermophilus orotate phosphoribosyltransferase (pyrE) gene were gifts from Dr M. Tamakoshi (Tokyo University of Pharmacy and Life Science, Japan). The bgl gene (TTP0042, 1.3 kb) was amplified from HB27 chromosomal DNA by PCR to give an EcoRI site and cloned into the pUC18 vector. The middle part of the bgl gene (bases 270–928) was removed by digestion with EcoT14I and the pyrE cassette amplified from p3TSDN1 by PCR was inserted. The resulting plasmid with the correct orientation was designated pOSG153 (bgl::pyrE). A logarithmically growing cell culture (0.5 ml) of MT111 (pyrE) in PY medium at 70°C was mixed with 25 µl of pOSG153 linearized by ScaI digestion and cultured for 1 h at 70°C with gentle shaking to allow homologous recombination. Cells were washed with 100 mM Na-phosphate buffer (pH 7.4) by centrifugation and spread on MSG plate for the selection of Ura+ (pyrE+) transformants. Plates were wrapped in PVC wrapping film and incubated at 70°C for 3 days. Transformants showing the Bgl– phenotype were then selected on PY plates containing 50 µg/ml X-Glc.
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Construction of pGLS expression vector plasmid
The plasmid pUC18-pJHK3 carrying an HTK gene was provided by Prof. S. Kuramitsu (Osaka University, Japan). Plasmid pJHK3 was derived from pTT8, a cryptic plasmid in T.thermophilus HB8, and its copy number was reported to be eight (13
). E. coli XL1-Blue was used as the host. Plasmid pJIN43 was constructed by replacing the pyrE gene (NdeI–BamHI) on p3TSDN1 with a promoterless bgl gene fragment (NdeI–BglII) amplified from pUC18BGL by PCR (Figure 1a). A 1.1 kb fragment containing the promoter region of the uvrB gene (TTC1531) was amplified from HB27 chromosomal DNA using PCR primers having 5'-Aor13HI and 3'-HindIII sites and cloned into pGEM-T Easy vector (Promega Corp., WI, USA) to make pJIN41. Then the promoterless bgl gene cassette (HindIII–XbaI) from pJIN43 was inserted into the HindIII–SpeI site on pJIN41 resulting in pJIN54 (Figure 1a). A 2.4 kb fragment (Aor13HI–PstI) from pJIN54 was ligated with an E.coli–Thermus shuttle vector (pTAP60) that had been digested with Aor13HI and PstI. The resulting plasmid carrying the uvrB'-bgl gene was designated pGLS1531 (Figure 1b). Plasmids pGLS1075 (uvrA'-bgl), pGLS1182 (uvrC'-bgl), pGLS1696-1 (ruvA'-bgl), pGLS1696-2 (paaG-ruvA'-bgl), pGLS0038 (ruvB'-bgl), pGLS0725-1 (ruvC'-bgl), pGLS0725-2 (ftsQAZ-ruvC'-bgl), pGLS1466-1 (recA'-bgl), pGLS1466-2 (ligT-recA'-bgl), pGLS1466-3 (amt-cinA-ligT-recA'-bgl), pGLS1467 (ligT'-bgl), pGLS1468 (cinA'-bgl) and pGLS1469 (amt'-bgl) were constructed by replacing the Aor13HI–HindIII region (uvrB') of pGLS1531. Plasmid pBGL73 carries bgl with a 0.4 kb 5'-flanking region (bgl promoter), and pBGL85R carries bgl ORF without the promoter region.
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Transformation of T. thermophilus
Plasmids were introduced into JOS9 (
bgl) by selection of a Kmr transformant. A logarithmically growing cell culture (0.5 ml) in PY medium at 70°C (1 x 108 cells/ml) was mixed with 20 µl of the plasmid DNA solution and cultured for 1 h at 70°C with gentle shaking. A portion of the culture was spread on PY plates containing kanamycin. Plates were incubated at 70°C for 1 day.
ß-Glucosidase assay
Logarithmically growing cells in PY medium at 70°C were used for the ß-glucosidase (Bgl) assay. When MMC treatment was incorporated, cells were incubated with MMC at varying doses at 37°C for 10 min and then cultured at 70°C for 120 min. Cells were washed and resuspended in Z buffer (14) prior to measurement of cell density at OD600. To disrupt the cell membrane for the Bgl assay, 0.01 ml of toluene was added and the mixture was immediately vortexed for 30 s. Then 0.2 ml of the toluenized cells was added to 0.8 ml of Z buffer. Enzyme activity was assayed at 80°C unless otherwise described. The reaction was started by the addition of 0.05 ml of 10 mg/ml 2NPGlc solution. After incubation in a water bath at 80°C for 60–120 min, tubes were removed from the bath and 0.2 ml of 1 M Na2CO3 was added to stop the reaction. The mixture was centrifuged at 14 000 g for 5 min and the supernatant was collected for measurement of the OD420 and OD550. Since 2NPGlc gradually decomposed at 70–80°C and turned pale yellow, a concurrent blank control (2NPGlc alone) was used so that the background OD420 value could be subtracted. Bgl activity (units/OD600) was calculated by the formula of Miller (15). In some experiments, cells were disrupted by sonication (Ultra homogenizer VP-5s; Taitec, Tokyo) for 3 min. After centrifugation at 14 000 g for 5 min to remove cellular debris, Bgl activity was measured with X-Glc as a substrate at OD620.
Northern blotting
Total RNA from 40 ml of the HB27 culture was isolated with hot phenol (65°C) in the presence of 1% SDS. After being extracted twice with phenol-chloroform (1:1) and precipitated with ethanol, RNA samples were treated with DNase I (RNase-free, Promega Corp.), extracted with phenol-chloroform and precipitated with ethanol. The RNA samples (40 µg) were denatured at 50°C for 30 min in the presence of 1 M glyoxal and loaded on 1% agarose gel. RNA High AGN (BioDynamics Lab Inc., Tokyo) was used as an RNA size marker. After electrophoresis, the gel was stained with ethidium bromide to mark the position of molecular size marker RNA. The RNA on the gel was then transferred to Hybond-N+ filters (GE Healthcare Bio-Sciences Corp., NJ, USA) and hybridized with DIG-labeled RNA probes (0.5–0.6 kb) for recA, ligT, cinA and amt. Probes were labeled and detected using DIG RNA Labeling Kit (SP6/T7) with anti-DIG-AP and CDP-Star (Roche Diagnostics, Tokyo).
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Results |
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Bgl activity of JOS9 cells
We first isolated a
bgl mutant (JOS9) from the T. thermophilus wild-type strain HB27 to use as a host strain for a vector plasmid. The Bgl activity of HB27 and JOS9 cells at different temperatures are shown in Figure 2. Activity (units/OD600) increased in the wild-type HB27 cells as the temperature rose. JOS9 cells, on the other hand, lacked Bgl activity. The low background level of Bgl activity observed in JOS9 cells (Figure 2) may be due to the catalysis of 2NPGlc by intact ß-galactosidase. The absence of Bgl activity was also confirmed with JOS9 cells disrupted by sonication with X-Glc as a substrate (data not shown). In the present study, Bgl showed higher activity at 80°C than at 70°C. We, therefore, assayed Bgl activity at 80°C as follows. Bgl activity was restored by introducing pBGL73 carrying an intact bgl gene into JOS9 (Figure 3). On the contrary, when the bgl gene without the promoter region (pBGL85R) was introduced, no Bgl activity was observed. Thus Bgl activity was completely dependent on the presence of promoter and we considered JOS9 to be suitable for the assay of Bgl activity, even at low expression levels.
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Expression of recA genes and operon structure
The genes TTC1469–TTC1466 in T. thermophilus HB27, namely amt (aminomethyltransferase), cinA (competence-damage inducible protein), ligT (2'–5' RNA ligase) and recA are linked on the chromosome (2
). We constructed six plasmids, pGLS1466-1, pGLS1466-2, pGLS1466-3, pGLS1467, pGLS1468 and pGLS1469 (Figure 3), and investigated Bgl expression. Bgl activity was not observed in cells carrying pGLS1466-1 (recA'-bgl), indicating there is no promoter region within the ligT ORF in the adjacent 5'-upstream region. The highest Bgl activity was detected in cells carrying pGLS1466-3 (amt-cinA-ligT-recA'-bgl). Bgl activity was also detected in cells carrying pGLS1466-2 (ligT-recA'-bgl) or pGLS1467 (ligT'-bgl), but the level was about half that of cells carrying pGLS1466-3. The expression level of amt'-bgl (pGLS1469) was almost equal to that of pBGL1466-3. Only weak Bgl activity was detected with pGLS1468 (cinA'-bgl). These results strongly suggested that a recA is transcribed as a 4-gene operon consisting amt-cinA-ligT-recA. At the least, transcription of recA gene was not monocistronic. We performed northern blotting using mRNA isolated from HB27 to confirm the operon structure. As shown in Figure 4, the labeled probe for amt, cinA, ligT and recA was detected as a band of 3.7 kb. Shorter bands corresponding ligT-recA (1.6 kb) and recA (1.0 kb) were not observed, but a faint 2.8 kb band corresponding to cinA-ligT-recA was revealed with a recA probe. Since amt-recA is 3.5 kb, we considered the four genes to be transcribed in the same mRNA. On the other hand, when cells were treated with the DNA damaging agent MMC at 1–40 µg/ml for 10 min at 37°C and then incubated at 70°C for 2 h, recA expression did not change (Figure 5). At the highest dose, cell growth was not observed. We also conducted time-course experiments for gene expression. Bgl activity was measured at 15, 30, 60, 90 and 120 min after MMC treatment at 10 µg/ml. No induction was observed at any incubation time (data not shown).
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Expression of ruvABC and uvrABC genes
The expression of DNA nucleotide excision repair genes and recombinational repair genes are shown in Table II. It is interesting that the expression levels among uvrA, uvrB and uvrC are not same. Expression levels of uvrA'-bgl (pGLS1075) and uvrB'-bgl (pGLS1531) were about three times that of uvrC'-bgl (pGLS1182). Since the 5'-upstream gene of uvrC is an opposite direction in the genome map (2
), it is not likely that the uvrC gene is organized in an operon-like structure with adjacent upstream genes. Similar Bgl activity was detected for pGLS1696-2 (paaG-ruvA'-bgl), pGLS0038 (ruvB'-bgl) and pGLS0725-1 (ruvC'-bgl). We believe that the ruvA gene forms an operon with its 5'-upstream gene, paaG, because pGLS1696-1 (ruvA'-bgl) revealed no Bgl activity. Cells carrying pGLS0725-2 (ftsQ-ftsA-ftsZ-ruvC'-bgl) had higher Bgl activity than cells carrying pGLS0725-1. The ruvC gene may form an operon with ftsQAZ genes. All the six DNA repair genes were expressed constitutively, and they did not increase expression in response to MMC treatment (Figure 5).
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Discussion |
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Purified T. thermophilus Bgl is a thermostable enzyme with an optimum temperature of 88°C and broad substrate specificity. It catalyzes the hydrolysis of ß-D-galactoside, ß-D-glucoside and ß-D-fucoside derivatives, although the catalytic efficiency is much higher for ß-D-glucoside than for ß-D-galactoside (16
). In the present study, Bgl showed higher activity at 80°C than at 70°C. Disruption of the HB27 bgl gene (TTP0042) by insertion of a pyrE gene cassette caused loss of Bgl activity in JOS9 cells. The low background level of Bgl activity in JOS9 cells (Figure 2) may be due to the catalysis of 2NPGlc by intact ß-galactosidase. The T. thermophilus chromosome contains two putative ß-galactosidase genes (TTP0220 and TTP0222) (2). When one is deleted, expression is half that of the wild-type cells (9). The bgl strain developed in the present study is suitable as a host cell for the Bgl assay and has the advantage that kanamycin-resistant HTK gene can be used as a positive selection marker of an introduced plasmid.
We constructed expression vectors with bgl gene as a reporter gene. The bgl gene of HB27 is organized with its upstream genes for sugar permease (TTP0041), sugar transport protein (TTP0040) and sugar-binding protein (TTP0039) (2). Although there are no typical promoter sequences reported in T. thermophilus (17) upstream of bgl ORF, introduction of pBGL73 carrying a cloned bgl gene and its 0.4 kb upstream region complemented Bgl activity in JOS9 cells (Figure 3). Therefore, bgl transcription was independent, at least in part, of upstream genes. On the other hand, absence of Bgl activity in JOS9 cells carrying pBGL85R with bgl ORF and no promoter region (Figure 3) indicated that possible homologous recombination between bgl ORF in pBGL85R and the residual part of the bgl' fragment (400 bp) in the chromosome had no affect on the Bgl assay. The pGLS series of expression vectors with the bgl host strain JOS9 made it possible to measure low levels of Thermus gene expression.
We demonstrated that in T. thermophilus, the expression of DNA repair genes such as recA, ruvABC and uvrABC was in low levels (1–3 units/OD600). We also showed that recA and upstream genes ligT, cinA and amt seemed to belong to the same transcription unit. In contrast, Castan et al. (18) reported that the HB27 recA gene was transcribed as monocistronic mRNA, but the size of their recA mRNA was not indicated. In the present study, our northern blot data using four kinds of probes (recA, ligT, cinA and amt) clearly showed a 3.7 kb band, which is consistent with the combined length of the four genes (3.5 kb). The longer bands shown in Figure 4 may include an adjacent gene (TTC1465, 1.7 kb) downstream from recA or part of TTC1465 since there is a terminator-like sequence in the middle of TTC1465. On the other hand, recA forms a polycistronic operon with genes cinA and ligT in the phylogenetically close genus, Deinococcus radiodurans (19). We concluded that the recA gene of T.thermophilus was also transcribed polycistronically.
In contrast to the report by Castan et al. (18), under our test conditions, MMC did not induce expression of seven DNA repair genes including recA. MMC treatment was conducted at 37°C for 10 min prior to incubation at 70°C for 120 min. Therefore, we considered that MMC-provoked DNA damages were formed before the chemical was inactivated by high temperature. We also tried with UV irradiation at doses of 40, 60 and 80 J/m2, but no induction was observed (unpublished data). T. thermophilus has uvdE gene for UV endonuclease which does not exist in E.coli. Neither a uvdE mutant nor a uvrA mutant showed UV sensitivity. Increased UV sensitivity was observed only in a uvdE, uvrA double mutant (10). It may be necessary to investigate the expression in a uvdE, uvrA background. T. thermophilus HB27 does not seem to have an inducible SOS response system, which has been found in E.coli and many other bacteria, because no homologous gene to lexA was found (2). LexA repressor regulates recA as well as several other DNA repair genes including uvrA, uvrB, ruvA and ruvB in E.coli, and those genes are strongly induced by MMC and DNA damaging agents (20,21). On the other hand, LexA protein from D.radiodurans is not required for recA induction, although the recA is inducible by 
-irradiation (22). D. radiodurans PprI protein is thought to be involved as a regulatory protein for radiation response (23). The lack of induction by MMC treatment in the present study does not contradict to the absence of lexA gene in T. thermophilus. However, this does not exclude the possibility of the existence of recA regulation. Further genetic studies would provide insight into the unique DNA repair systems of extreme thermophilic microorganisms. The convenient bgl expression vector developed here can be used for studies of transcriptional regulation.
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Acknowledgments |
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We thank Miriam Bloom for her critical reading of manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (18602003).
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Notes |
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*To whom correspondence should be addressed. Tel: +81 426 76 7093; Email: ohta{at}ls.toyaku.ac.jp
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References |
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Received on March 28, 2006; revised on May 4, 2006; accepted on May 11, 2006.
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