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,aDepartment of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ. Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ-independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins. |
Introduction |
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DnaA stably binds ATP or ADP, but the unwinding of oriC DNA requires the ATP-bound form. The nucleotide forms of DnaA vary in a replication cycle-coordinated manner (Kurokawa et al. 1999). Before the initiation of replication, the number of ATP-DnaA molecules increases. After initiation, ATP-DnaA decreases in abundance, with a corresponding increase in the ADP-DnaA population. ATP bound to DnaA is hydrolysed by a DNA replication-coupled mechanism (RIDA, ‘regulatory inactivation of DnaA’) to yield ADP-DnaA, which is inactive for initiation (Katayama et al. 1998; Katayama 2001; Nishida et al. 2002). In the RIDA process, DnaA-ATP is hydrolysed by interaction with the Hda protein and the clamp subunit of DNA polymerase III holoenzyme loaded on to DNA (Kato & Katayama 2001; Su’etsugu et al. 2004).
The initiator function of DnaA is also controlled by its subchromosomal localization. A single cell contains 500–2000 DnaA molecules, depending on strain background and growth conditions (Sekimizu et al. 1988; Chiaramello & Zyskind 1989; Hansen et al. 1991; Katayama & Kornberg 1994; Katayama et al. 1997). The datA locus, an approximately 1 kb chromosomal region containing five DnaA boxes (Kitagawa et al. 1996, 1998), can bind 200–300 DnaA molecules, thereby restricting the number of DnaA molecules accessible to oriC.
After initiation, oriC DNA is temporarily in a hemimethylated state as newly synthesized strands are not yet methylated by Dam methyltransferase (Lu et al. 1994). SeqA protein preferentially binds to hemimethylated oriC, thereby inactivating oriC function temporarily (Lu et al. 1994; Slater et al. 1995; Wold et al. 1998; Taghbalout et al. 2000).
Transcriptional readthrough of oriC affects the timing of initiation (Su’etsugu et al. 2003). This transcription starts from the promoter of the mioC gene, which flanks oriC, and fluctuates in a replication cycle-dependent manner, such that it is repressed before initiation and induced after initiation (Theisen et al. 1993; Ogawa & Okazaki 1994). oriC DNA contains recognition motifs for the IHF and FIS proteins (Kornberg & Baker 1992), which may also be involved in the cell cycle-coordinated control of initiation by binding in a timely manner to oriC (Ryan et al. 2004).
The replicational initiation of some plasmid and bacteriophage replicons requires heat shock proteins such as DnaK, DnaJ and GrpE (Kornberg & Baker 1992). For example, replication of 
phage DNA is initiated by a nucleoprotein complex containing ori DNA and the O protein, a functional DnaA homolog. The DnaB-P protein complex is dissociated by the activities of DnaK, DnaJ and GrpE, when DnaB is loaded on to oriDNA. The P protein is a functional homolog of DnaC.
To further cultivate our understanding in the regulation of initiation, we have searched for factors that genetically interact with DnaA by isolating and analysing suppressors of specific dnaA mutants. By carrying out transposon mutagenesis of the dnaAcos mutant, which exhibits overinitiation of replication and inhibition of growth at 30 °C (Katayama & Kornberg 1994; Katayama 2001), we previously confirmed that the dam gene, which encodes the Dam methyltransferase, is a stimulatory factor for initiation (Katayama et al. 1997). A histone-like protein, H-NS, also supports overinitiation in the dnaAcos mutant (Katayama et al. 1996). When transcription of the mioC gene is rendered constitutive by a Tn5 promoter insertion, initiation is prevented or delayed (Su’etsugu et al. 2003). In this study, we have focused on the temperature-sensitive dnaA508 allele, which contains base substitutions (creating the P28L and T80I alterations) in DnaA domain I, a region that mediates DnaA-DnaB and DnaA-DnaA interactions (Hansen et al. 1992; Messer 2002). We isolated suppressor mutants by random transposon mutagenesis of the dnaA508 mutant and found a novel heat shock gene, hspQ, which encodes a protein that stimulates the degradation of mutant DnaA proteins.
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We carried out minitransposon (mini-kan) random insertion mutagenesis of the dnaA508 (Ts) mutant NKN1 and isolated 57 independent clones that could form colonies at 40 °C on LB plates containing kanamycin. P1 transduction was then performed to identify clones in which the mini-transposon insertion site was genetically linked with the suppression of temperature sensitivity. For 19 of the independent suppressor mutants, the co-transduction frequency of elements conferring both kanamycin and temperature (40 °C) resistance was 100%, when NKN1 was infected with P1 phage lysates isolated from these mutants. One of the re-constructed suppressor mutants was named NKN79. In contrast, co-transduction of the two phenotypes was not seen for the other 38 strains, which means that these contained spontaneous suppressor mutations or were true revertants with an additional insertion of mini-kan at another site. By shotgun-cloning into pUC19, we isolated a mini-kan-containing EcoRI-restriction fragment derived from each of the 19 strains. Restriction enzyme analysis revealed that these independent plasmid clones all contained a 7.7 kb DNA fragment with the same structure.
To identify the location of this fragment, a 2 kb chromosome-derived fragment isolated from it was used as a probe for hybridization with the minimal set of Kohara phage DNA aligned on a nitrocellulose membrane (Kohara et al. 1987). Only clone #223, covering 22.1–22.2 min of the genomic map, gave a specific hybridization signal (data not shown). The restriction map of this region was consistent with the cloned fragment. To confirm the mapping and to determine the insertion site, we subcloned a fragment that contains the border region between the chromosome-derived DNA and mini-kan. Sequencing of this region and a homology search revealed that mini-kan is inserted in an ORF (o223#11, b0966 or yccV) located at 22.2 min (Fig. 1A,B) (Oshima et al. 1996; Blattner et al. 1997). We named this novel gene hspQ (heat shock protein Q), based on the following findings.
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To confirm that this ORF is a functional gene, we performed a plasmid complementation test. Introduction of a low-copy mini-R vector pKP1673 (Miki et al. 1992) and its derivative containing the wild-type hspQ (pNKN269) into the suppressor mutant NKN79 (dnaA508 hspQ79: :mini-kan) revealed that this ORF represses, and thus complements, the temperature-resistant phenotype of this strain at 40 °C (Fig. 1A, Table 1). These results suggest that this region contains a functional gene that is related to suppression of the dnaA508 mutation. Using an in vitro coupled transcription-translation system, we obtained evidence that hspQ encodes a protein. pNKN255, a pBR322 derivative bearing hspQ, expressed a protein of about 14 kDa, the size expected for this gene, in the so-called Zubay system (Zubay 1973), whereas pBR322 alone did not (data not shown).
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DE3), and transcription by T7 RNA polymerase was induced by the addition of IPTG (Tabor & Richardson 1985; Studier et al. 1990). SDS-polyacrylamide gel electrophoresis revealed that a protein of about 14 kDa was expressed in IPTG-treated cells containing the Sau3AI fragment (Fig. 1D). This protein was transferred on to a PVDF membrane, and the first six amino acid residues were determined by the Edman degradation method to be M-I-A-S-K-F, which indicates that the second ATG triplet is used as the initiation codon in vivo (Fig. 1C). The theoretical molecular mass and pI of the HspQ protein were calculated to be 11.8 kDa and 4.53, respectively. hspQ is a heat shock gene
The expression of heat shock proteins is regulated at the transcriptional level in E. coli (Yura et al. 1993; Gross 1996). When the heat shock response is induced, the alternative RNA polymerase sigma factor 32 replaces 70, the usual sigma factor. We found that the putative promoter region of the hspQ gene shares significant nucleotide homology with 32-dependent promoters (Fig. 2A). Overall, this region shares 67% homology with the heat shock promoter consensus sequence, comparable to the hslV promoter (Fig. 2B).
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32 by using reconstituted RNA polymerase holoenzyme containing the purified RNA polymerase core (E) and 32 or 70 (Fujita et al. 1987; Kurokawa et al. 1996). As controls for E32- and E70-dependent transcription, we used the promoters of the groE operon and the bla gene, respectively. The groE operon encodes the major heat shock proteins GroES and GroEL (Yura et al. 1993; Gross 1996). The groE and bla promoters were both carried on a plasmid, pBlue-groE (Kurokawa et al. 1996), and DdeI digestion fragments of this plasmid were used for in vitro run-off transcription assays (Fig. 2C). Similarly, pNKN255, the hspQ-bearing derivative of pBR322, was digested with NcoI and included in transcription assays. NcoI digestion of this plasmid yields a C-terminally truncated form (0.26 kb) of hspQ (Fig. 1). If the proposed 32-promoter shown in Fig. 2 A is indeed used for hspQ transcription, a transcript of about 280 bases should be produced. Under conditions where the E32- and E70-complexes specifically transcribed the groE and bla genes, respectively, an E32-dependent hspQ transcript was detected (Fig. 2C). The size of this transcript is consistent with the proposal that a 32-dependent promoter is used for transcriptional initiation of hspQ. We thus conclude that hspQ is a heat shock gene. Features of hspQ-dependent suppression of dnaA508 and other dnaA alleles
Suppression of dnaA508 temperature-sensitive colony formation by hspQ::mini-kan was observed on LB, LB containing glucose and supplemented M9 solid media (Fig. 3). Cells grown overnight at 30 °C were plated on each medium and incubated at 30, 37, 40, 41 and 42 °C. Suppression was greatest at 40 °C, and moderate levels of suppression were seen at 41 and 42 °C on each medium.
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Given that the disruption of hspQ permits viability of the dnaA508 mutant at 40 °C, we speculated that an excess of HspQ may inhibit the growth of this mutant at lower temperatures, such as 30 °C. Indeed, the transformation efficiency of the dnaA508 mutant with a pBR322 derivative bearing the hspQ gene was at least 103-fold lower than that with pBR322 (Fig. 5). Similar transformation experiments revealed that inhibition was specific for the dnaA508 and dnaA167 alleles (Fig. 5), consistent with the data concerning the suppression of temperature sensitivity by the hspQ disruption (Fig. 4). These results support the idea that HspQ directly acts on specific mutant DnaA proteins.
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We speculated that HspQ may enhance the degradation of mutant DnaA proteins, because proteases such as Lon, ClpXP, and HslVU are heat shock proteins (Gross 1996; Kanemori et al. 1997; Wickner et al. 1999; Dougan et al. 2002). If so, the suppression of the dnaA mutants by disruption of hspQ and the growth inhibition conferred by the over-expression of hspQ can be readily explained mechanistically.
To test this idea, we first quantified cellular levels of DnaA protein in wild-type and dnaA508 strains with or without hspQ::mini-kan by immunoblot analysis as previously described (Katayama & Kornberg 1994; Katayama et al. 1997). The introduction of hspQ::mini-kan did not affect the level of DnaA in the wild-type dnaA strain (Table 2). DnaA levels in the dnaA508 mutant cell were significantly decreased compared to that in the wild-type dnaA strain. This decrease was partially reversed by the presence of hspQ::mini-kan in NKN79.
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HspQ forms homomultimers in solution
We asked if HspQ forms homomultimers, as proteases such as HslVU form heteromultimers that include homomultimers of each subunit (Gross 1996; Dougan et al. 2002). HspQ protein was overproduced and was concentrated by preparing cleared lysates (fraction I), and ammonium sulphate precipitation (fraction II) (Fig. 7A). When proteins in fraction II were separated using a HiTrap-Q column and a linear gradient of KCl, HspQ eluted as two peaks (Fig. 7B). The first and major peak contained about 65% of HspQ present in the loaded fraction II. A part of the peak fractions was pooled, resulting in fraction III-1 (Fig. 7B). Purity of HspQ in fraction III-1 was about 90%. When fraction III-1 was applied to gel filtration of a Superose 12 column, HspQ was eluted around molecular mass 25–30 kDa (fraction IV-1; Fig. 7C). Given that the HspQ monomer has a calculated mass of about 12 kDa, this observation suggests that HspQ forms a homodimer. The total amount of eluted HspQ was about 90% of this protein present in the loaded fraction III-1. Purity of HspQ in fraction IV-1 was about 95%.
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The hspQhslU double mutant
We previously found that the hslU gene is required for inhibition of colony formation of the dnaA46 mutant at 40 °C (Katayama et al. 1996). The hslU heat shock gene encodes the ATPase subunit of bacterial proteasome HslVU that is nonessential for cell growth (Katayama et al. 1996; Kanemori et al. 1997; Wickner et al. 1999). To ask if the hslU gene is related to the dnaA508 temperature sensitivity, we constructed mutants bearing a disrupted hslU gene (hslU::cat) at 30 °C and incubated these mutants at higher temperatures (Fig. 9). At 40 °C and 41 °C cell growth of a wild-type dnaA strain was not significantly affected by introduction of both hslU::cat and hspQ::mini-kan (Fig. 9). Similar results were obtained when a wild-type dnaA strain bearing either hslU::cat or hspQ::mini-kan was used (data not shown). The doubling time of cells was similar at these temperatures among all these strains (data not shown). When hslU::cat was introduced to a dnaA508 mutant, suppression for the growth inhibition was not observed at 40 °C and 41 °C (Fig. 9). Suppression of dnaA508 by hspQ::mini-kan was not affected by introduction of hslU::cat (Fig. 9). These results suggest a certain specificity for targets of these heat shock proteins.
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Discussion |
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32, the rpoH gene product. The rpoH mRNA is transcribed constitutively, but its 5'-region adopts a stable secondary structure that inhibits translation (Morita et al. 1999). Elevated temperature destabilizes this secondary structure, thus permitting translation of 32.
In this study, we found a new heat shock gene that is transcribed in a 32-dependent manner (Figs 1 and 2). This gene, hspQ, stimulates the in vivo degradation of a mutant form of DnaA, DnaA508 (Fig. 6). HspQ might be a novel protease, but so far we have not detected protease activity in purified HspQ although we have incubated several proteins such as casein, purified DnaA and DnaA508, and denatured lysozyme in the presence of HspQ under various temperature, pH and buffer conditions. We speculate that HspQ requires a cofactor to form a functional hetero-oligomeric complex, as is the case for the ClpXP, ClpAP, and HslVU proteases. These proteins form multimeric complexes that adopt a ring-like configuration consisting of homo-oligomers of each subunit (Gross 1996; Wickner et al. 1999; Dougan et al. 2002). In solution, HspQ protein formed dimers and multimeric complexes containing about four monomers (Fig. 7). Structural analysis of these complexes and experiments to identify HspQ-binding proteins are important future steps. A search for sequences homologous to the hspQ gene and HspQ protein was carried out against all genes registered, but factors with significant similarity were not detected.
We determined the N-terminal sequence of HspQ and found that translation of the protein is initiated from the second ATG of the putative ORF (o223#11 b0699, or yccV) (Figs 1 and 2). During the preparation of this manuscript, a short note was published that described the overproduction and affinity column purification of hexahistidine-tagged YccV protein (d’Alençon et al. 2003). This protein includes the extra 17 amino acids that are seen in the putative ORF, because it was overproduced using a strong ribosome binding site located on the vector. In addition, d’Alençon et al. (2003) reported that hspQ(yccV)::mini-Tn10 can suppress the dnaA46 thermosensitivity at 40 °C, although experimental results indicating this are not shown. We observed very slight growth of the dnaA46 hspQ::mini-kan cells at 40 °C (Fig. 4). Some differences in experimental conditions, e.g. incubation time at 40 °C, might enhance this slight suppression by indirect effects, although d’Alençon et al. (2003) do not describe detailed experimental conditions.
Expression of dnaA gene is mainly regulated at the transcription level (Kornberg & Baker 1992; Messer 2002). In our results, the cellular DnaA content was not substantially affected by introduction of hspQ::mini-Tn10 (Table 2). Thus, we speculate that dnaA transcription is not affected by hspQ. Although d’Alençon et al. (2003) described that only 1.5-fold increase in dnaA transcription was detected by in vivo lacZ transcription assay, they do not describe the experimental data or detailed experimental conditions. There might be a possibility that some differences in experimental conditions such as those in cell culture can cause the slight effect on transcription indirectly.
Whereas the degradation of DnaA508 was impeded by inactivation of the hspQ gene, the half-life of DnaA46 protein was not significantly affected, at least at 41 °C (Fig. 6). These findings indicate that the HspQ function has a certain specificity with respect to degradation substrates. In a previous study of the dnaA46 mutant, we isolated a suppressor at 40 °C by transposon mutagenesis and identified a transposon insertion in the hslU gene, which encodes the HslU subunit of the HslVU protease (Katayama et al. 1996). The temperature-sensitivity of colony formation was suppressed by the introduction of hslU1::mini-tet but not clpP1::CmR or lon::Tn10, indicating that the degradation of DnaA46 might be mediated primarily by HslVU. The HslVU protease is the bacterial proteasome (Wickner et al. 1999).
The half-life of DnaA508 was extended from about 20 min to 60 min, a three-fold increase, by the inactivation of the hspQ gene (Fig. 6). Although this result shows that HspQ plays a significant role in degradation of the mutant protein, the half-life of the wild-type DnaA is clearly longer than 60 min, indicating that HspQ is not the only factor that promotes degradation of DnaA508 and that other proteases must be cooperatively involved. Degradation of DnaA204 can be stimulated by at least three proteases ClpP, ClpQ (HslV) and Lon (Slominska et al. 2003).
In an hslVU mutant, induction of heat shock proteins is enhanced compared to the wild-type strain because of excessive accumulation of abnormal proteins (Kanemori et al. 1997). We analysed induction levels of heat shock proteins using the hspQ mutant and the 35S-Met pulse lavelling method as described (Yano et al. 1990; Kanemori et al. 1997). In our experiments, no significant difference in regulation of expression of heat shock proteins (GroEL and DnaK) was detected between the hspQ mutant and parental wild-type hspQ strain.
To determine if HspQ is involved in the regulation of chromosomal replication in wild-type dnaA cells, we analysed cells bearing a chromosomally borne hspQ::mini-kan mutation (NKN232) or a pBR322 derivative containing hspQ, as well as parental wild-type cells, by flow cytometry. Although cells were grown at various temperatures and in various media, no significant differences in cell cycle parameters (cell size distribution, timing of replicational initiation, oriC copy number and cellular doubling time) were observed between the mutant and wild-type cells (data not shown). Replicational initiation at multiple origins occurred synchronously in NKN232 cells under various conditions (data not shown). These observations suggest that the role of HspQ is specific to a subclass of certain denatured proteins, whereas some heat shock proteins such as DnaK, DnaJ and GrpE are required for replicational initiation of phage DNA (Kornberg & Baker 1992).
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Experimental procedures |
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All strains used are listed in Table 3. LB medium included 50 µg/mL thymine unless otherwise indicated. M9 medium was supplemented with 25 µg/mL thymine, 0.2% casamino acids, 25 µg/mL tryptophan, 0.0005% thiamin and 0.2% glucose. The Kohara phage #223 and #539 (Kohara et al. 1987) were gifts from Dr Y. Kohara and Dr H. Mori. 1105, which bears a Tn10-derivative minitransposon, mini-kan, was a gift from Dr N. Kleckner (Way et al. 1984). ME9018 was a gift from National Institute of Genetics.
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A previously described method was used (Katayama et al. 1996). Briefly, growing cells (1 x 109) of NKN1 at 30 °C were collected, infected with 1105 at a multiplicity of infection of 0.1 PFU/cell, and incubated at 37 °C for 1 h. Cells were washed in 0.9% NaCl, plated on LB agar containing 25 µg/mL kanamycin and 0.2% glucose, and incubated for 2 h at 37 °C and overnight at 40 °C. Colonies appearing at 40 °C were purified at the same temperature.
Mapping of cloned DNA using the Kohara phage DNA
Gene Mapping Membrane (Takara Biochemicals, Japan), on which a minimal set of the Kohara phage DNA was aligned, was used according to the manufacturer's instruction. For probe preparation, pNKN168 DNA was digested with EcoRI, BamHI and XhoI, and 2 kb-DNA fragments derived from the chromosomal region were isolated for labelling using a random primer kit (Amersham Biosciences) (Fig. 1). pNKN168 is a pUC19 derivative carrying an NKN79 chromosomally derived 7.7 kb EcoRI-fragment that contains mini-kan.
Construction of plasmids bearing the hspQ gene
DNA was prepared from amplified Kohora phage #223, and a 3.75 kb EcoRI-BamHI fragment was isolated and ligated to EcoRI-BamHI-digested pBR322 to yield pNKN237. A 2.45 kb Eco47III-BamHI fragment of pNKN237, which includes the hspQ gene, was ligated to EcoRV-BamHI-digested pBR322 to yield pNKN252. A 1.2 kb hspQ-bearing ScaI-EcoRI fragment of pNKN252 was ligated to EcoRV-EcoRI-digested pBR322 to yield pNKN255. An hspQ-bearing 0.5 kb Sau3AI fragment of pNKN255 was cloned into the BamHI site of pUC19 and pT7-6, resulting in pNKN258 and pNKN261, respectively. An hspQ-bearing EcoRI-HindIII fragment of pNKN258 was ligated to EcoRI-HindIII-digested pKP1673, a low-copy mini-R vector, resulting in pNKN269.
In vitro run-off transcription assay using E32 and E70
Run-off single-round transcription experiments using purified RNA polymerase core and 32 or 70 were done according to a previously described method (Fujita et al. 1987; Kurokawa et al. 1996). Briefly, pBlue-groE and pNKN255 were digested with DdeI and NcoI, respectively, and deproteinized. The indicated amounts of the resulting DNAs were incubated for 10 min at 37 °C in 35 µL buffer A (50 mM Tris-HCl [pH 7.5], 3 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM dithiothreitol, 25 µg/mL bovine serum albumin, and 25 mM NaCl) containing the indicated amounts of RNA polymerase holoenzyme with 32 or 70. RNA synthesis was then promoted by adding buffer A (15 µL) containing 160 µM ATP, GTP and CTP, 50 µM[
-32P]UTP (100–150 cpm/pmol) and 200 µg/mL heparin (Sigma), and incubation was continued for 5 min at 37 °C. Reactions were terminated by the addition of a solution (50 µL) containing 50 mM EDTA and 300 µg/mL yeast tRNA. RNA products in each 90 µL sample were ethanol precipitated, dissolved in 95% formamide-containing standard sample buffer, denatured, and analysed by 8 M urea-polyacrylamide (5%) gel electrophoresis. RNA markers (Gibco BRL) were used as a size standard.
Immunoblot analysis
Immunoblot analysis of DnaA was performed as previously described (Katayama & Kornberg 1994; Katayama et al. 1997). Briefly, cells were grown exponentially at the indicated temperature in LB medium. At the indicated times, 5 mL aliquots were withdrawn, and 5% trichloroacetic acid was immediately added. Precipitates formed in the cold were collected by brief centrifugation and subjected to SDS-polyacrylamide (12%) gel electrophoresis. Separated proteins were blotted on to a PVDF membrane (Millipore) and detected using a polyclonal rabbit anti-DnaA antiserum and an alkaline phosphate-conjugated anti-rabbit antiserum (Bio-Rad). The intensities of the stained bands were quantified by scanning densitometry. The amounts of DnaA protein were determined using a purified DnaA standard.
Purification of HspQ
BL21(DE3) cells bearing pNKN276 were grown at 37 °C in 3.6 litre LB medium containing 50 µg/mL ampicillin. When the optical density (A660) of the culture reached 0.5, 1 mM IPTG was included and incubation was continued for 3 h. Cells were harvested, suspended in 33 mL ice-chilled HED buffer (25 mM HEPES-KOH [pH 7.6], 0.1 mM EDTA, 4 mM DTT and 15%[v/v] glycerol) containing 250 mM KCl, incubated for 30 min on ice in the presence of 20 mM spermidine-HCl, 20 mM EDTA and 0.2 mg/mL lysozyme, and frozen in liquid nitrogen. The following operations were carried out at 4 °C unless otherwise indicated. Frozen cells (35 mL) were thawed and soluble lysates were isolated by centrifugation at 44 000 r.p.m. for 20 min in a Beckman 70Ti rotor (fraction I, 24 mL, 19 mg/mL). Ammonium sulphate (0.28 g/mL of fraction I) was added slowly to fraction I with stirring. After additional stirring for 20 min, the suspensions were centrifuged at 18 000 r.p.m. for 20 min in a Beckman JA-20 rotor. Precipitants were rapidly dissolved in HED buffer, and the resulting solution was dialysed until the conductivity reached that of 20 mM KCl (fraction II, 3.8 mL, 75 mg/mL). A portion (1 mL) of fraction II was subjected to a HiTrap Q column (1 mL bed volume; Amersham Biosciences) equilibrated with HED buffer containing 20 mM KCl, using a flow rate of 1 mL/min. This column was washed with 5 mL of the same buffer and proteins were eluted using a linear gradient (21 mL) of KCl from 20 mM to 400 mM in the same buffer. Aliquots of 1 mL were collected for fractions 1–21. The column was then washed with 1 M KCl in the same buffer (3 mL), and aliquots of 1 mL were collected for fractions 22–24. Two peaks of HspQ were eluted. Fractions of the first, major peak (fractions 10–12; fraction III-1) and the second, minor peak (fractions 14–15; fraction III-2) were separately collected. A portion (10 µL) of fraction III-2 (3 mL, 3.5 mg/mL) was diluted to 30 µL using HED buffer containing 100 mM KCl, and subjected to a Superose 12PC3.2/30 column (Amersham Biosciences) equilibrated with the same buffer using a flow rate of 40 µL/min. 40-µL fractions were collected, and fractions containing HspQ were pooled (fraction IV-1; 120 µL, 0.2 mg/mL). A portion (12 µL) of fraction III-2 (2 mL, 3.0 mg/mL) was similarly gel-filtrated, and fractions containing HspQ were pooled (fraction IV-2; 80 µL, 0.07 mg/mL).
Protein cross-linking experiments
HspQ fraction IV-1 (500 ng) was incubated for 1 h at 30 °C in HED buffer (25 µL) containing 100 mM KCl and the indicated concentrations of glutaraldehyde. Proteins were then precipitated in the presence of 10% trichloroacetic acid and analysed by SDS-polyacrylamide (15%) gel electrophoresis and silver-staining.
Construction of the hslU::cat mutants
A 1.3 kb-fragment bearing chloramphenicol-resistant gene (cat) was amplified from pACYC184 by PCR using primers, 5'-CCCAAGCTTGAGAGCCTGAGCAAACTG and 5'-CCGCTCGAGGTATACACTCCGCTAGCG. A 6.0 kb-fragment containing the hslU gene was amplified from Kohara phage #539 by PCR using primers, 5'-CGGAATTCGCTTCATACAATCGGAGC and 5'-CGGGATCCGAATCCCGATAAAGTCTCC. The resultant fragment was digested using BamHI and EcoRI, and ligated to pBR322 using the same restriction sites, resulting in pHSL01. To disrupt the hslU gene, a 10 kb-fragment was amplified from pHSL01 using primers, 5'-CCCAAGCTTGGCGAAACTGGTGAAC and 5'-CCGCTCGAGGCTTCTTCTTCAATCAGC. To insert the cat gene in the disrupted hslU gene, the resultant fragment and the cat fragment were digested with HindIII and XhoI, and ligated, resulting in pHSL02. Given that hslU is a nonessential gene under normal growth conditions (Katayama et al. 1996; Kanemori et al. 1997), this plasmid was digested with BamHI and NdeI, and transformed to ME9018 (recD::mini-tet) to replace the hslU gene with hslU::cat on the chromosome. After confirmed by PCR, the hslU::cat mutation was introduced to KH5402-1 and its derivatives using P1 transduction.
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Acknowledgements |
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Footnotes |
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Present addresses: aChugai Pharmaceutical Co., Tokyo, Japan;
bKaketsuken Institute, Kumamoto, Japan;
cDepartment of Pharmacy, Saga University Hospital, Saga, Japan
These authors contributed equally to this work.
* Correspondence: E-mail: katayama{at}phar.kyushu-u.ac.jp |
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Received: 31 May 2004
Accepted: 6 September 2004
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109 cells/mL), 5 µL serial dilutions were spotted on to LB agar medium containing 0.2% glucose, and the plates were incubated for 24 h at 30, 40, 41 or 42 °C. The following mutations are present in each dnaA allele: P28L and T80I for dnaA508, V157E for dnaA167, A184V and H252Y for dnaA46, A184V and G426S for dnaA5, A184V and P296Q for dnaA601, A184V and A347V for dnaA604, V383M for dnaA205, and I389N for dnaA204 (
