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Laboratory of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Introduction |
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) and hydrogen peroxide (H2O2), damage cellular components, including DNA, RNA, nucleotides, proteins and lipids, and have been implicated in cancer, aging and numerous diseases. To avoid eventual oxidative damage, cells express anti-oxidant defense proteins under oxidative stress conditions, which, in Escherichia coli, are organized under the SoxRS or OxyR regulons, such as catalase, superoxide dismutase, and endonuclease IV (Storz & Imlay 1999). A Saccharomyces cerevisiae dst1 null mutant, in which the gene encodes a eukaryotic transcription elongation factor S-II that promotes the read-through of RNA polymerase II-dependent transcription through the arrest sites (Fish & Kane 2002), exhibits increased oxidative stress sensitivity (Koyama et al. 2003). Although the increased sensitivity to oxidative stress is apparently correlated with a decrease in the fidelity of gene expression, it is not known how S-II mutant alters the oxidative stress sensitivity.
Bacterial RNA polymerase consists of 
2, ß and ß' subunits and has a central role in gene expression regulation, and studies of its mutants have contributed to a better understanding of the molecular mechanisms of bacterial transcription regulation (Jin & Zhou 1996). Most of the mutations causing resistance to rifampicin (Rif), a bacterial RNA polymerase inhibitor, were mapped in the rpoB gene, which encodes the ß subunit of RNA polymerase. These resistance mutations are clustered into four regions on the ß subunit, where the amino acid residues directly interact with rifampicin (Campbell et al. 2001).
The process of transcription termination, revealed by detailed studies in E. coli, is divided into at least the two different systems. One is Rho-dependent and the other is Rho-independent termination (Nudler & Gottesman 2002; Richardson 2002). Some rifampicin-resistant rpoB alleles suppress the temperature-sensitive phenotypes of the rho mutation, whereas others are incompatible with the mutation. The in vivo phenomena are closely correlated with in vitro changes in the transcription elongation rate and termination efficiency of RNA polymerase containing each mutant ß subunit; that is, an increase or decrease of the transcription elongation rate decreases or increases the termination efficiency of each process, respectively (Jin et al. 1992; McDowell et al. 1994). The involvement of bacterial transcription termination control in physiologic processes, however, especially in stress responses, is not known.
We showed that either a multicopy supply of the rof gene or bicyclomycin, both of which inhibit the transcription termination Rho factor, suppressed the increased sensitivity to oxidative stress of the rifampicin-resistant rpoB mutation in Escherichia coli. Multi-copy supply of the rnk gene also suppressed oxidative stress sensitivity, coincident with the recovery of the reduced concentration of nucleoside triphosphates in the mutant cells, which is one of the factors that affects transcription termination efficiency in vitro. Thus, an appropriate, nonexcessive termination frequency at Rho-dependent transcription terminators might contribute to oxidative stress survival.
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Results |
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To clarify the molecular interrelation between oxidative stress survival and transcription elongation and/or termination in bacteria, we screened for an oxidative stress sensitive mutant among rifampicin-resistant E. coli strains, in which some of the mutant RNA polymerases exhibit aberrant transcription elongation and/or termination control (Heisler et al. 1996). Rifampicin-resistant mutants (50 µg/mL) were collected using a mutator strain MK602 (mutT mutant) as the parent strain, and tested for their sensitivity to paraquat (PQ), a redox-cycling drug that accelerates the formation of reactive oxygen species by catalyzing the transfer of electrons from redox enzymes (Hassan 1984). Of 80 rifampicin-resistant mutants, 6 strains exhibited sensitivity to paraquat at 12.5 µg/mL. One of the mutants, named HAM023, was tested further and exhibited cold-sensitive growth at 28 °C and manganese-sensitive growth on Luria Bertani (LB) agar plates containing 5 mM MnCl2. The paraquat-sensitivity and manganese-sensitivity of HAM023 was complemented by a plasmid, pLBC-R, harboring the wild-type rpoB gene (data not shown).
To examine whether the rifampicin-resistance and paraquat-sensitivity were caused by the same mutation, P1 phage transduction analysis was performed. The thiA39::Tn10, which was co-transducible with the rpoB gene (Singer et al. 1989), was used for selection of the transductants. When tetracycline-resistant transductants were examined for their sensitivities to the drugs, rifampicin-resistance and paraquat-sensitivity were co-transducible (Table 1). The rifampicin-resistance was also coincident with cold-sensitive growth at 28 °C and manganese-sensitive growth. Quantitative analysis of the paraquat-sensitivity of the rifampicin-resistant transductant KW04 revealed that 160 µM paraquat decreased the mutant's viable cell number to less than 10–3, whereas wild-type KW03 cells were not decreased (Fig. 1A). In addition, KW04 lost the colony-forming ability when cells were pretreated with 8 mM H2O2 for 30 min, whereas KW03 cells required more than 32 mM of H2O2; therefore, there is at least a four-fold difference between them (Fig. 1B). These results suggest that the mutation causing rifampicin-resistance was responsible for the increased oxidative stress sensitivity.
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Sequencing analysis of the rpoB gene of HAM023 was performed to determine the mutation site, and revealed a single base transition of A1577C in the open reading frame (ORF), which results in an amino acid substitution, His526Pro. We named this mutation rpoB2070, as suggested by the E. coli Genetic Stock Center. The H526 residue is assigned to one of the amino acids that directly interacts with rifampicin and is substituted with high frequency in rifampicin-resistant strains of E. coli and Mycobacterium tuberculosis (Campbell et al. 2001). In E. coli, the rpoB2 (H526Y) mutation is well-studied. The rpoB2 mutation causes temperature-sensitive growth and has decreased termination efficiency at the terminator in vivo and in vitro (Jin & Gross 1989). In contrast to rpoB2, rpoB2070 induced cold-sensitive growth and increased oxidative stress sensitivity that have not been reported in rifampicin-resistant mutants. Although the H526P substitution mutation in the rpoB gene was reported previously as a polymerase chain reaction (PCR)-mutagenesis-generated mutation that conferred rifampicin-resistance when expressed on a multicopy plasmid, phenotypes other than rifampicin-resistance were not described (Severinov et al. 1993).
Isolation of an S. aureus rpoB mutant responsible for both rifampicin-resistance and oxidative stress sensitivity
Next, we asked whether such an rpoB mutation causing both rifampicin-resistance and oxidative stress sensitivity occurs in other bacterial species. The gram-positive S. aureus strain RN4220 was used as a parent strain, mutagenized with ethylmethanesulfate, and rifampicin-resistant strains at 100 µg/mL were collected. Of 84 rifampicin-resistant strains, 14 were more sensitive to menadione, an oxidative stress-inducing drug, than the parent strain on a gradient plate (data not shown), and had cold-sensitive growth at 25 °C like the E. coli rpoB2070 mutant (data not shown). Three mutants among them were used in the phage transduction experiment with phage 80alpha and a drug-resistant marker inserted near the rpoB gene. The menadione-sensitivity was linked with rifampicin-resistance and cold-sensitive growth (data not shown). The transductants of the three mutants were each sensitive to 32.5 µM menadione (Fig. 2). Sequencing analyses revealed that the three mutants each had a single base transition in the rpoB gene at the same position of C1441T in the ORF, which resulted in an amino acid substitution mutation His481Tyr where H481 corresponds to H526 in the E. coli rpoB gene. The results suggest that this finding might be general among bacterial species and that the H526 residue of the ß subunit of E. coli RNA polymerase has a crucial role for survival under oxidative stress conditions.
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In response to oxidative stress, E. coli cells induce genes regulated by SoxRS, OxyRS, and/or 
S (Storz & Imlay 1999). Therefore, we next examined whether oxidative stress genes were induced in the rpoB2070 mutant of E. coli after H2O2 treatment, because loss of the induction might explain the oxidative stress sensitivity of the rpoB2070 mutant. Using Northern hybridization experiments, mRNA expression was examined for the nfo gene, which is regulated under the SoxRS system, the katG gene (under OxyRS), and the xthA gene (under S). Each gene was transiently induced by H2O2 in both the wild-type and mutant cells (Fig. 3), which was consistent with the literature (Zheng et al. 1998). The results suggest that the mutant induces oxidative stress genes that are regulated by each transcription factor, and that the oxidative stress sensitivity of the E. coli rpoB2070 mutant is not due to defects in induction.
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To further elucidate molecular basis of the oxidative stress sensitivity of the rpoB2070 mutant, multicopy suppressor genes for the oxidative stress sensitivity of the mutant were determined. Genomic DNA from the E. coli W3110 strain was digested with HindIII and ligated in the HindIII site of pBR322, and the resultant plasmid library was electroporated into mutant cells. Of 3000 transformants, 2 were resistant to 160 µM paraquat. Comparison of the nucleotide sequences of both ends of each insert with genomic DNA sequence information revealed that one of the inserts contained a region from the ahpC gene to the citG gene, including the rnk gene, at 13.9 min of the E. coli genome. Suppression experiments using plasmids harboring a series of deletion constructs suggested that the rnk gene was responsible for the suppression (data not shown). Consistent with this result, a plasmid harboring the single rnk gene region amplified by the PCR method suppressed the oxidative stress sensitivity of the rpoB2070 mutant (Fig. 4A,B), although the colony size of the transformants was more heterozygous than that in the absence of paraquat (Fig. 4A). The suppression was partial; colony formation efficiency of the mutant cells with a plasmid harboring the rnk gene was 53 ± 2% on LB plates containing 160 µM paraquat (Fig. 4B). Under the same conditions, the colony formation efficiency of mutant cells with a vector plasmid was less than 0.3% (Fig. 4B). The rnk gene encodes the regulator of nucleoside diphosphate (NDP) kinase, and E. coli cells have at least two NDP kinases (Shankar et al. 1995). The specific activity of the total NDP kinase activity in a cell extract from the rpoB2070 mutant cells decreased to approximately 60% that of wild-type cells, and assays using either UDP, CDP, or GDP as a substrate provided similar results (Fig. 4C). The rpoB2070 mutant cells had reduced concentrations of UTP, CTP, GTP and ATP and this reduction was recovered by a multicopy supply of the rnk gene (Fig. 4D). Thus, the rpoB2070 mutation caused defects in the maintenance of nucleoside triphosphate (NTP) concentrations, and its recovery by the rnk gene is considered to be a factor involved in the suppression of the oxidative stress sensitivity.
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The other plasmid that suppressed paraquat-sensitivity of the rpoB2070 mutation contained 4.5 kbp fragment at 4.6 min of the E. coli genome harboring the rof gene and five other ORFs. Deletion analysis of the inserted DNA fragment indicated that the rof gene is a candidate for the suppression, and plasmids containing the single rof genes amplified by PCR suppressed the paraquat-sensitivity phenotype of the rpoB2070 mutation with a colony formation efficiency of 32 ± 7% (Fig. 4A,B). The rof gene product binds to Rho factor, a protein largely responsible for factor-dependent transcription termination, and inhibits the Rho-dependent transcription termination (Pichoff et al. 1998). Because the antibiotic bicyclomycin (Bcm) inhibits the functions of the Rho factor (Zwiefka et al. 1993; Park et al. 1995), we examined whether bicyclomycin suppresses paraquat-sensitivity of the rpoB2070 mutant. Low concentrations of bicyclomycin that did not inhibit the E. coli cell growth suppressed paraquat-sensitivity (Fig. 4E). These results suggest that the Rho-dependent transcription termination is provoked more efficiently than normal in the rpoB2070 mutant cells, which might cause the oxidative stress sensitive phenotype of the mutant.
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Discussion |
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The H526 residue of the E. coliß subunit (H406 of Thermus aquaticusß) is one of the residues that directly interact with rifampicin, and the position is supposed to be near the active site for the transcription elongation reaction (Campbell et al. 2001). The rpoB2 (H526Y) mutation, which had a mutation at the same position as the rpoB2070 (H526P) isolated in this study, cannot stop efficiently at Rho-dependent and independent transcriptional terminators in vivo (Jin et al. 1988) and increases the transcription elongation rate and decreases pausing, a condition where elongation is slowed or halted temporarily, in vitro (McDowell et al. 1994). In addition, the rpoB2 mutation is incompatible with the rho15 mutation, which decreases Rho function in vivo (Jin & Gross 1989). These previous findings suggest that the H526 residue has a critical role in the transcription termination reaction. As described in detail below, the present study indicates that the oxidative stress sensitivity of the rpoB2070 mutation is suppressed by the inhibition of Rho function or by multicopy supply of the rnk gene and that NTP pools were decreased in the mutant. Based on these results, we hypothesized that the effects of the rpoB2070 mutation were opposite to those of the rpoB2 mutation with respect to changes in their transcription elongation rate and read-through frequency. That is, the mutation might decrease the transcription elongation rate and terminate with an increased efficiency at transcription terminators. Although direct evidence for this model has not been accumulated yet, this notion is consistent with their dissimilar phenotypes; the rpoB2070 mutation causes cold-sensitive growth and increased oxidative stress sensitivity, while the rpoB2 mutation causes temperature-sensitive growth (Jin & Gross 1989) and does not cause increased oxidative stress sensitivity (data not shown). The H526 residue seems to have a role in survival under oxidative stress conditions via its involvement in transcription elongation rate and termination reactions.
Oxidative stress sensitivity caused by the rpoB2070 mutation was suppressed by a multicopy supply of the rof gene or by bicyclomycin, both of which inhibit Rho function. Rho-dependent termination occurs in some operons and also in some ORFs (Richardson 2002), and thus such downstream genes in operons might have a role in survival under oxidative stress conditions and their expression might be reduced because the RpoB2070 RNA polymerase terminates more efficiently than normal at Rho-dependent transcription terminators. In contrast to our observations, Italiani et al. (2002) reported that a loss of function mutation of Rho in Caulobacter crescentus caused oxidative stress sensitivity. Although it is unknown whether similar phenomena take place in E. coli rho mutant, it is interesting to speculate and to examine a notion that controlled Rho function to a moderate level is required for the oxidative stress survival.
Oxidative stress sensitivity of the rpoB2070 mutant is also suppressed by a multicopy supply of the rnk gene, and the mutant has a decreased total NDP kinase activity and reduced NTP concentrations. Such a reduction in the NTP concentration decreases the transcription elongation rate and conversely increases the efficiency of transcription termination at transcription terminators (Heisler et al. 1996). Therefore, the reduction of the NTP concentration might further decrease the transcription elongation rate of the mutant RNA polymerase, and this change might affect the gene expression profile under oxidative stress conditions via the efficient termination of transcription. Recovery of NTP concentration by multicopy supply of the rnk gene in the mutant cells might underlie the suppression of the oxidative stress sensitivity.
E. coli cells have at least two NDP kinases; one is encoded by the ndk gene and the other is called the alternative kinase, the gene of which is not known (Shankar et al. 1995). Repression of one of the two kinases via a disruption of the rnk or sspA genes does not decrease the total NDP kinase activity of each mutant, which grows normally (Shankar et al. 1995). Therefore, the rpoB2070 mutation might reduce the expression of both the NDP kinases or repress their activities. To test and refine our model, it will be necessary to identify genes that are under Rho control and responsible for the oxidative stress sensitivity phenotype and to evaluate quantitatively their expression in rpoB2070 mutant with or without the multicopy suppressor genes.
Based on our model, other efficient termination mutants of the rpoB gene (i.e., rpoB7 and rpoB8) (Jin et al. 1988) or the rho gene (i.e., rhos-81 and rhos-82) (Tsurushita et al. 1989) might exhibit oxidative stress sensitivity. However, our initial investigations on rpoB7 and rpoB8 mutation revealed that they did not exhibit oxidative stress sensitivity (data not shown), implying a possibility that the phenotype might be allele specific for the H526 residue. To investigate how much the Rho-dependent termination would be modulated by rpoB mutations is also important to this matter.
S. aureus is a major pathogen for human infectious diseases and the spread of multidrug-resistant strains of S. aureus (MRSA) has caused severe problems in clinical fields. Noticeably, over 50% of these MRSA strains are resistant to rifampicin (Voss et al. 1994). Because the S. aureus rpoBH481Y mutation, which is sensitive to oxidative stress, is identified with high frequency among rifampicin-resistant S. aureus mutants (Aubry-Damon et al. 1998), our results suggest that an anti-bacterial drug that induces oxidative stress might kill some of these rifampicin-resistant MRSA strains. This might also be effective for tuberculosis, for where rifampicin is used as a critical drug and the emergence of drug-resistant strain causes clinical problems. Therefore, the finding that a rifampicin-resistant bacteria is sensitive to oxidative stress is important for selecting a therapeutic course for infectious disease caused by these multidrug-resistant strains and to develop drugs for that purpose. Some anti-bacterial substances produced by insects kill bacteria via oxidative stress (Akiyama et al. 2000; Akiyama & Natori 2003). Clinical application of such anti-bacterial substances should be studied.
The present study provides another view regarding our prior work (Koyama et al. 2003), which described oxidative stress sensitivity of the yeast S-II null mutant; the transcription elongation complex are more susceptible to terminate in the absence of S-II, and thus the mutant exhibited the oxidative stress sensitivity as is the case in E. coli rpoB2070 mutant. In agreement with the idea, the yeast S-II deletion mutant actually decreases the expression of a specific subset of genes (Shaw & Reines 2000; Shimoaraiso et al. 2000). Future studies are needed to determine which gene expression, that regulated by either Rho factor in bacteria or S-II in yeast, is responsible for the organism survival under oxidative stress conditions.
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Experimental procedures |
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E. coli AB1157(thr-1 leuB6 thi-1 lacY1 galK2 ara-14 xyl-5 mtl-1 proA2 his-argE3 rpsL31 tsx-33 supE44 sup-37) was obtained from National Institute of Genetics, Mishima, Japan. MK602 (leu+mutT) is derived from AB1157 (Tajiri et al. 1995), and was a kind gift from Dr H. Hayakawa (Kyushu University, Fukuoka, Japan). HAM023 is a rpoB2070 mutant derived from MK602 in this study. KW01 is the thiA39::Tn10 derivative of HAM023 constructed by phage transduction using CAG8160 as a donor (Singer et al. 1989). KW03 or KW04 is the AB1157 derivative harboring a rifampicin-sensitive or resistant allele, respectively, constructed by phage transduction using KW01 as a donor. The oxidative stress sensitivity of the rpoB2070 mutation was examined in the AB1157 background, but not in the W3110 background, because the findings were not consistent in the W3110 background. E. coli W3110 [IN(rrnD-rrnE)] and S. aureus RN4220 (Novick et al. 1993) were from our laboratory stock. S. aureus CK1004 is an RN4220-derivative harboring a rifampicin-resistant allele constructed by phage transduction using CK1002 as a donor. CK1002 is one of the S. aureus mutants isolated from mutagen-treated RN4220, which represented the rifampicin-resistant and menadione-sensitive phenotypes and was inserted in the chloramphenicol-resistant gene near the rpoB gene described below. S. aureus CK1003 had the wild-type rpoB allele and insertion of the chloramphenicol-resistant marker, which had been backcrossed into RN4220 by phage transduction.
Plasmids pBR322 and the R-plasmid pKP1673 (Miki et al. 1992) were from our laboratory stock. pLBC-R is a pKP1673 derivative carrying the rpoB and rpoC genes on the 11.3 kbp HindIII fragment of pLBC (Ishihama & Fukuda 1980) at the HindIII site, which was a kind gift from Dr A. Ishihama (NIG, Mishima, Japan). To construct pGEM-T-rnk and pGEM-T-rof, the rnk and rof genes were amplified from the W3110 genomic DNA using Ex Taq DNA polymerase (Takara Bio, Japan) and the following primers: rnk2, 5'-cgggatcctcaggagtggttttcgaggt-3'; rnk3, 5'-cgggatccggaattccctgaatgtgacg-3'; rof1, 5'-cgggatccaaagccggataagacgcatc-3'; rof2, 5'-cgggatcccagggaaaaagcagcatacg-3'. Amplified DNA fragments of 0.8 kbp and 0.4 kbp, respectively, were ligated into the pGEM-T vector using the pGEM-T vector systems (Promega). Cloning of the genes was confirmed by DNA sequencing. pCK20 (Ichihashi et al. 2003), a suicide vector in S. aureus, was used for recombinational integration of the chloramphenicol-resistant marker near the rpoB gene.
Culture of bacteria and chemicals
E. coli and S. aureus strains were cultured in LB medium [1% bactotryptone (Becton Dickinson), 0.5% yeast extract (Becton Dickinson), and 1% NaCl]. Transformants of E. coli harboring pBR322, pGEM-T, or their derivatives were cultured in LB medium supplemented with 100 µg/mL ampicillin. Those harboring pCK20, pKP1673, or their derivative were cultured in 12.5 µg/mL chloramphenicol. The rifampicin-resistant S. aureus strains were isolated by mutagenesis of RN4220 with ethylmethanesulfate (Sigma Chemicals Co.) as previously described (Inoue et al. 2001). The restriction enzymes, ligation kit, and random primer DNA labeling kit were purchased from Takara Bio (Otsu, Japan). [-32P]dCTP (6000 Ci/mmol) and [
-32P]ATP (3000 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Pscataway, NJ, USA) and Perkin Elmer, respectively. Oligonucleotides for PCR were purchased from Proligo Japan K.K. Paraquat and Menadione were purchased from Sigma Chemicals Co. Bicyclomycin was purchased from Fujisawa Pharmaceutical Co., Ltd (Osaka, Japan).
Northern blot analysis
RNA was extracted from E. coli cells by the SV Total RNA Isolation System (Promega). Probes were prepared from the W3110 genomic DNA using Ex Taq DNA polymerase and the following oligonucleotides: nfo, 5'-tcagaagaggattgccttgc-3' and 5'-attgccttcaccgaggctat-3'; katG, 5'-ctcgtcgtttcctcaacgat-3' and 5'-cagcactatgatatccgcca-3'; xthA, 5'-agctcggctacaacgtgttt-3' and 5'-atcagcctgtccatccattc-3', and were labeled with [-32P]dCTP using the random priming method. The blot was washed with 2x saline sodium citrate containing 0.1% SDS and with 0.1x saline sodium citrate containing 0.1% SDS with agitation at room temperature, and then visualized by autoradiography. Quantification of the signal was performed using a BAS-1800II PhosphorImager (FujiFilm, Japan).
Screening of multicopy suppressors for the rpoB2070 mutation
Chromosomal DNA extracted from E. coli W3110 cells was digested with HindIII and cloned into the HindIII site of pBR322, and the resultant HindIII plasmid library was transformed into the rpoB2070 mutant. Colonies grown on LB plate containing 200 µM of paraquat and 100 µg/mL of ampicillin at 37 °C after 2 days were re-streaked on LB plates containing 160 µM paraquat and 100 µg/mL of ampicillin to confirm suppression of the paraquat-sensitivity.
Phage transductions
Generalized phage transduction in E. coli was achieved using phage P1 as previously described (Miller 1972). The transductants were selected by tetracycline (Tc)-resistance (20 µg/mL) by the thiA39::Tn10 marker derived from CAG8160. Phage transduction in S. aureus was achieved using phage 80alpha as previously described (Novick 1991). The chloramphenicol-resistant gene in a suicide vector pCK20 (Ichihashi et al. 2003) inserted near the rpoB gene was used for the selection of the phage transductants. The insertion was performed via single crossover recombination at the DNA region cloned into pCK20, which was amplified by PCR with RN4220 genomic DNA as a template and primers of 5'-tgaacaacgtattcaatatcaa-3' and 5'-acggcaacactaatgatcacac-3'. The insertion was confirmed by Southern blot analysis.
Nucleoside diphosphate kinase assay
NDP kinase assay was performed as previously described (Munoz-Dorado et al. 1990). Briefly, a log-phase culture was centrifuged, suspended in 3 volumes of 50 mM Tris-HCl (pH 7.5), and sonicated through 15 cycles of a 10-s pulse with a 10-s gap between pulses. The extract was centrifuged at 10 000 g for 10 min and the supernatant was collected. Protein was determined using the Lowry method with bovine serum albumin as a standard. For the assay, the reaction mixture consisted of 5 µL of buffer (100 mM Na/HEPES (pH 8.0), 200 mM NaCl, and 20 mM MgCl2), 1 µL of 20 mM ATP, 1 µL (10 µCi) of [-32P]ATP, 2 µL of 10 mM NDP, and 1 µL of the enzyme solution. After incubating at 30 °C for 20 min, 1 µL of the reaction mixture was taken to 10 µL of 5 mM EDTA, and 1 µL of this final mixture was loaded on a PEI-cellulose plate. Thin layer chromatography was performed with 0.75 M KH2PO4.
Nucleotide pool size analysis
The nucleotide pool extraction was performed as described (Olempska-Beer & Freese 1984). Briefly, exponentially growing cells in LB medium containing 10 µCi/mL [32P]orthophosphate at 37 °C were passed through nitrocellulose membranes, when the optical density at 600 nm reached 0.5. The filters were subjected to extraction with 1 M formic acid saturated with 1-butanol. After 10 min on ice, the extracts were centrifuged for 5 min at 15000 r.p.m. The supernatant were vacuum dried and spotted on polyethyleneimine-cellulose plates (20 cm x 20 cm, Merck). Thin layer chromatography was performed in two dimensions as described (Neuhard & Thomassen 1971); first dimension, 2 M LiCl and 2 M acetic acid (until start line) followed by 2.5 M LiCl and 2 M acetic acid (until 15 cm); second dimension, 0.75 M LiCl and 7.5% boric acid (until 2 cm) followed by 0.75 M LiCl and 10% boric acid (until 15 cm). The plates were autoradiographed, and each spot of the separated compounds was quantified.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: kurokawa@mol.f.u-tokyo.ac.jp
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Received: 25 November 2004
Accepted: 7 February 2005
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) or the rpoB2070 mutant KW04 (•) was diluted adequately, streaked on LB agar plates containing various concentrations of paraquat, and then incubated at 37 °C for 16 h. Colony numbers were normalized against one another in the absence of paraquat. (B) Hydrogen peroxide-sensitivity. Overnight culture of each strain was treated with the indicated concentration of H2O2 for 30 min, streaked on LB agar plates, and incubated at 37 °C for 16 h.
) and the mutant cells (
) were determined using UDP, CDP, or GDP as a substrate. (D) Nucleotide pools of exponentially growing wild-type cells (
) in LB medium containing [32P]orthophosphate at 37 °C were determined, and relative amounts to wild-type cells are shown for indicated nucleotides. (E) Suppression of the paraquat sensitivity of the rpoB2070 mutant by bicyclomycin, a Rho inhibitor. Overnight culture of the wild-type or rpoB2070 mutant cells that were streaked on LB agar plates containing both 160 µM paraquat and 10 µg/mL bicyclomycin, or with paraquat but without bicyclomycin, or without either drug. Plates were incubated at 37 °C for 16 h.