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1 Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
2 Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
Correspondence
Kenji Sonomoto
sonomoto{at}agr.kyushu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Russell et al., 1998; Szabo et al., 1994). The ATP-bound state of DnaK exhibits a low affinity toward substrates; therefore, the rates of binding and release of substrates are rapid. In contrast, the ADP-bound state exhibits a high substrate affinity, and the rates of binding and release of substrates are low. In the nucleotide-dependent chaperone cycle, DnaJ, one of the co-chaperones, stimulates the ATPase activity of DnaK and the binding of the substrate protein (Gassler et al., 1998; Suh et al., 1998, 1999). Moreover, the other co-chaperone, GrpE, induces ADP dissociation from the nucleotide-binding domain of DnaK and substrate release from the substrate-binding domain (Brehmer et al., 2004; Liberek et al., 1991).
Previous biochemical and structural analyses have demonstrated that GrpE is a homodimer that consists of the following three domains: paired N-terminal 
-helices (residues 40–88), four-helix bundles (residues 89–137) and C-terminal β-domains (residues 139–197) (Harrison, 2003; Harrison et al., 1997; Schonfeld et al., 1995). In addition, it has been shown that residues 1–33, when unstructured and removed for crystallization, are responsible for the interaction with the substrate-binding domain of DnaK and the release of substrate proteins from DnaK (Brehmer et al., 2004).
Many molecular chaperones are heat-shock proteins (Hsps) and play an important role in the protection of the host cell against various stresses, including high-temperature stress (Lindquist & Craig, 1988). Moreover, they have housekeeping functions and are essential for cellular homeostasis, even at physiological temperatures. For example, DnaK plays a key role in cell division, chromosome segregation and maintenance of low-copy-number plasmids. Bukau & Walker (1989) reported that deletion of dnaK affected both cell division and cell growth, and the defects were suppressed by the overexpression of FtsZ, a cell-division-related protein, suggesting functional interaction of DnaK and FtsZ. Some evidence exists that for the proper functioning of the cell-division machinery, DnaK and DnaJ proteins have to be present at appropriate levels. Physiological consequences of DnaK and DnaJ overexpression in E. coli have been reported (Blum et al., 1992). DnaK overexpression results in a defect in cell division and growth, but DnaJ overexpression does not. The effect of DnaK overexpression was found to be partially suppressed by co-expression of DnaJ. However, the precise role of the DnaK chaperone system in cell division and the physiological consequences of GrpE overexpression remain unclear.
In this report, we provide evidence that the ratio of GrpE, but not DnaJ, to DnaK is crucial for functionality of the DnaK chaperone system and cell-division machinery in vivo and in vitro. Overexpression of GrpE resulted in filamentous morphologies. Excess amounts of GrpE inhibited the chaperone activity of DnaK, leading to accumulation of protein aggregates in cells and irregular localization of FtsZ.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) were grown as described previously (Sugimoto et al., 2003, 2006). MC4100 (DE3) was constructed by infection of 
DE3 into MC4100 in order to overexpress target genes under the control of the T7 promoter; this was performed using a DE3 lysogenization kit (Novagen).
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). The DNA sequences of the cloned genes were determined by Macrogen and confirmed.
Using a site-directed mutagenesis kit (Quick Change, Qiagen), the primers G122D-S1 and G122D-A1 (5'-GCG ATG GTT GAA GAC ATT GAG CTG ACG CTG-3' and 5'-CAG CGT CAG CTC AAT GTC TTC AAC CAT CGC-3', respectively) and pGRPE(T7) as a template, a plasmid was constructed. This plasmid carried the grpE gene with the G122D point mutation (grpE-G122D), and Gly at amino acid 122 was substituted by Asp. The resulting plasmid was termed pG122D (Table 1). Since pGRPE(T7) was used as a template, the grpE-G122D gene was encoded under the control of the T7 promoter.
A plasmid carrying the N-terminal GFP–grpE fusion gene (gfp–N–grpE) was constructed by splice-overlap-extension (SOE) PCR (Horton et al., 1989). The gfp and grpE genes were amplified using the following primer sets: SOE-GFP-S1 and SOE-GFP-A1 (5'-GCG AAA AAA ACG CGG AGA AAT TCA TGA GTA AAG GAG AAG AAC-3' and 5'-GCG TTT TCT GTT CTT TAC TAC TCA TTT TGT ATA GTT CAT CCA TGC-3') and SOE-GRPE-S1 and SOE-GRPE-A1 (5'-GCA TGG ATG AAC TAT ACA AAA TGA GTA GTA AAG AAC AGA AAA CGC-3' and 5'-TTC CTG TGA AAC CGC TGC GCG AGA GTG TG-3'), respectively. The PCR template for the gfp gene was pGreen (Table 1) (Miller & Lindow, 1997). SOE-GFP-S1 contained a region upstream of the start codon for the grpE gene (underlined) in order that the ribosome-binding site of grpE could be used. SOE-GFP-A1 and SOE-GRPE-S1 contained a sequence corresponding to the 5' region of the grpE gene (underlined) and a sequence corresponding to the 3' region of the gfp gene (underlined), respectively. Finally, the gfp–N–grpE gene was amplified using the primer set SOE-GFP-S1/SOE-GRPE-A1. The amplified fragment was ligated into a pGEM-T vector under the control of the lac promoter. The resulting plasmid was termed pGFP-N-GRPE (Table 1).
Cell morphology.
E. coli MC4100 cells were transformed with the self-ligated pGEM-T vector (pGEM), pGRPE(T7), pG122D, pGreen and pGFP-N-GRPE, and the resulting transformants were named MC4100GEM, MC4100GRPE, MC4100G122D, MC4100GFP and MC4100GFP-N-GRPE, respectively (Table 1). These transformants were grown on Luria–Bertani (LB) agar plates containing 50 µg ampicillin ml–1 overnight. Single colonies of the respective transformants were cultured in LB medium containing 50 µg ampicillin ml–1 without IPTG, since leaky expression of the respective genes was observed. The exponential-phase cells (OD600 0.8–1.2) were examined under a microscope (H550L, Nikon). GFP fluorescence was monitored with a GFP-specific filter to visualize the localization of GFP–N–GrpE. The DNA of the E. coli cells was stained with 4,6-diamidino-2-phenylindole (DAPI), as described elsewhere (Hiraga et al., 1998). The DAPI-stained samples were observed under a fluorescence microscope with a DAPI-specific filter.
SDS–PAGE and Western blotting.
Cell extracts of the E. coli transformants were prepared as described previously (Sugimoto et al., 2007) and subjected to 12 % (w/v) SDS–PAGE. The gel was stained with Coomassie brilliant blue (CBB) R-250 or transferred onto a PVDF membrane (Atto) and subsequently immunoblotted using an anti-GrpE-antibody (StressGen Biotechnologies), an anti-DnaK antibody (laboratory collection) and an anti-FtsZ antibody (a gift from Dr M. Wachi, Tokyo Institute of Technology). Detection was performed using the ECL detection kit (GE Healthcare) according to the manufacturer's protocol.
Monitoring of E. coli cell growth.
The E. coli transformants MC4100GEM, MC4100GRPE and MC4100G122D were grown at 37 °C in LB liquid medium containing 50 µg ampicillin ml–1. The OD600 of the culture was measured at the indicated times. The growth of the transformants was also examined on LB agar plates. When the OD600 of the culture in LB liquid medium reached 1.0, the culture was serially diluted as indicated, and 5 µl aliquots were spotted on LB agar plates containing 50 µg ampicillin ml–1 with or without 0.5 mM IPTG. The plates were incubated at 37 °C for 16 h.
Complementation assay.
Thermosensitive E. coli DA259 (Table 1) harbouring the grpE mutation (provided by Dr C. Georgopoulos, Centre Médical Universitaire, Switzerland) was transformed with the following plasmids: pGEM, pGRPE(lac) and pGFP-N-GRPE. The resulting E. coli transformants, DA259GEM, DA259GRPE and DA259GFP–N–GRPE, respectively (Table 1), were grown in LB medium with 50 µg ampicillin ml–1 at 30 °C. When the OD600 reached 1.0, the cultures were serially diluted as indicated, and 5 µl aliquots were spotted on LB agar plates. The plates were incubated at 30 and 44 °C for 16 h.
Luciferase-refolding activity assay.
Refolding of chemically denatured firefly luciferase was carried out as described previously (Sugimoto et al., 2007), with some modifications. The protein concentrations used were 5 µM (DnaK), 1 µM (DnaJ), 1–50 µM (GrpE) and 10 nM (luciferase). The refolding reaction was carried out at 30 °C for 30 min.
Cell-free extracts from the E. coli transformants MC4100GEM, MC4100GRPE and MC4100G122D were used as folding enzymes instead of the purified chaperone proteins. The cell-free extract was prepared as follows. E. coli cells were grown at 37 °C in LB medium under shaken conditions. When the OD600 of the culture reached 1.0, the cells were harvested by centrifugation at 9000 g for 20 min and stored at–30 °C until further use. The cells were resuspended in phosphate buffer containing 10 mM sodium phosphate (pH 6.5), 1 mM EDTA, 20 % (w/v) sucrose and 1 mg lysozyme ml–1 (Seikagaku Corporation) and incubated on ice for 30 min. After cell disruption by sonication with a Sonifier cell disruptor 350 (Branson Ultrasonics) (output 3, 30 % duty, 10 s, 2 cycles), the cell debris was removed by centrifugation at 15 000 g for 20 min, and the supernatant was collected. Using the cell-free extract instead of purified chaperones, the luciferase-refolding reaction was carried out as described previously (Sugimoto et al., 2007). The protein concentration of the cell-free extract used was 10 mg ml–1. At the indicated times, aliquots of the sample were withdrawn and the luciferase activity was measured.
Isolation of protein aggregates.
Protein aggregates from E. coli cells grown at 37 °C in LB medium were isolated and analysed according to a previously described protocol (Sugimoto et al., 2008).
Indirect immunofluorescence microscopy.
Indirect immunofluorescence microscopy was carried out according to the procedure of Hiraga et al. (1998). To detect FtsZ proteins in MC4100GEM, MC4100GRPE and the dnaK deletion mutant BM271 (Table 1), anti-FtsZ antibody and Cy3-conjugated goat anti-rabbit IgG (GE Healthcare) were used as primary and secondary antibodies, respectively. The immunostained samples were observed under a fluorescence microscope.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Kang & Craig, 1990). In addition, DnaK overexpression results in a defect in cell division and growth, although DnaJ overexpression does not (Blum et al., 1992). Here, the effect of GrpE-overexpression on E. coli morphology was tested (Fig. 1). MC4100GEM (vector-control strain) presented typical rod-shaped cells (Fig. 1a), while MC4100GRPE cells (GrpE-overexpressing strain) were filamentous (Fig. 1b). Next, we examined whether the effect of GrpE on cell morphology was direct or indirect. Previously, Bukau's group reported that both overexpression and deletion of DnaK produce a similar filamentous morphology, indicating that the effect of GrpE-overexpression could be related to the function of DnaK. In order to confirm that DnaK is absolutely required for this morphological change, dnaK deletion mutants should be used as host cells. However, dnaK deletion mutants are, by nature, filamentous and therefore not suitable for this test. As an alternative, we examined the effect of the GrpE mutant GrpE-G122D that has been recently characterized as having an impaired DnaK cooperative function (Gelinas et al., 2002; Grimshaw et al., 2005). As expected, MC4100G122D (GrpE-G122D-overexpressing strain) cells were rod-shaped (Fig. 1c). SDS-PAGE along with CBB staining revealed that there was no apparent difference in expression profile of the total proteins, except for the GrpE proteins of MC4100GEM, MC4100GRPE and MC4100G122D (Fig. 1d, upper panel). Western blotting using the anti-GrpE antibody also showed that there was no significant difference in the expression levels of GrpE variants (Fig. 1d, lanes 3 and 4). These results implied that the effect of GrpE overexpression on cell morphology is related to cooperation with DnaK.
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; Dai & Lutkenhaus, 1992; Weart et al., 2007). To address the question of whether overexpression of GrpE affects the expression levels of DnaK and FtsZ, Western blotting analyses using anti-DnaK antibody and anti-FtsZ antibody were performed. As shown in Fig. 1(d) (lower panels), the amounts of DnaK and FtsZ proteins in MC4100GRPE were very similar to those in MC4100GEM and MC4100G122D, indicating that overexpression of GrpE did not affect the expression levels of DnaK and FtsZ.
Effect of GrpE overexpression on the growth rate of E. coli
The E. coli transformants were precultured in LB medium at 37 °C overnight and then inoculated into fresh LB medium. Their OD600 was monitored at the indicated times (Fig. 2a). MC4100G122D showed a similar growth profile to that of MC4100GEM. In contrast, the lag phase of MC410GRPE was longer than that of the other transformants (Fig. 2a); this indicated that overexpression of wild-type GrpE delayed the growth of the host cells. After 5 h, MC4100GRPE grew rapidly with a similar growth rate to that of other transformants. At this stage of growth, the expression level of GrpE was much lower than that at the start of culture, and this led to wild-type growth rates and a decrease in the number of elongated cells (data not shown). Bukau and colleagues have reported that strong overexpression of GrpE in grpE mutant cells leads to decreased colony formation (Brehmer et al., 2004). We also tested the colony-formation ability of these transformants. As in the results from Bukau's group, MC4100GRPE showed a much lower colony-formation activity in the presence of 0.5 mM IPTG than MC4100GEM (Fig. 2b). In contrast, MC4100G122D did not display defective colony-formation activity; this is consistent with the results described above (Fig. 2a). Notably, in the absence of IPTG, there was no difference in colony formation among these three transformants (Fig. 2c), even though a significant delay in growth was observed in the case of MC4100GRPE cultured in LB liquid medium in the absence of IPTG (Fig. 2a). This discrepancy may result from the fact that the expression level of GrpE in E. coli cells cultured on LB agar without IPTG was much less than that in LB liquid medium without IPTG (data not shown). Furthermore, an excess of GrpE appears to be deleterious to the chaperone function of the DnaK system (Grimshaw et al., 2005); this may be responsible for the defects in cell division, cell growth and colony formation.
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). GFP–N–GrpE was expressed using the lac promoter (Table 1), which is functional in the grpE mutant strain DA259 that was used in the complementation test. A GFP moiety was connected to the N terminus of GrpE, since the C-terminal region interacts directly with DnaK (Harrison et al., 1997) and the GFP moiety could inhibit an interaction between GrpE and DnaK by steric hindrance. The construct was transformed into both the wild-type E. coli strain MC4100 (DE3) and the grpE mutant strain DA259 (Table 1).
First, we confirmed that GFP–N–GrpE can complement the function of GrpE in vivo, because we realized that the GFP moiety might affect the activity and conformation of GrpE. As shown in Fig. 3(a), DA259GEM (vector-control strain) did not grow at 44 °C, whereas DA259GRPE (intact GrpE-expressing strain) and DA259GFP–N–GRPE (GFP–N–GrpE-expressing strain) did. These results indicated that GFP–N–GrpE expression rescued the growth of the grpE mutant DA259 at 44 °C, as observed in the case of intact GrpE expression, and that GFP–N–GrpE functioned as a co-chaperone in a similar manner to intact GrpE.
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). The morphology of MC4100GFP was the same as that of MC4100GEM, indicating that the GFP moiety did not directly affect cell morphology. In contrast, filamentous cells were observed in MC4100GFP–N–GRPE (Fig. 3c, d). However, the number of elongated cells versus rod-shaped cells when GFP–N–GrpE was overexpressed was less than that when intact GrpE was overexpressed. A possible reason for the decreased number of filamentous cells is the lower expression level of GFP–N–GrpE in MC4100GFP–N–GRPE compared with that of GrpE in MC4100GRPE (data not shown). The fluorescence of GFP was diffused throughout the cytoplasm in MC4100GFP (Fig. 3f). On the other hand, the GFP fluorescence of GFP–N–GrpE was specifically localized and appeared as several large deposits in the cell (Fig. 3g, h). The localization patterns were irregular among these cells, and GFP–N–GrpE was localized at the centre, polar (Fig. 3g) and non-specific positions (Fig. 3h). To confirm the subcellular localization of GFP–N–GrpE, fluorescence images (Fig. 3g, h) were compared with phase-contrast images (Fig. 3c, d). GFP fluorescence in MC4100GFP–N–GRPE overlapped with the dark deposits in the phase-contrast images, which should be inclusion bodies (Carrio & Villaverde, 2005). However, it was improbable that GFP–N–GrpE aggregated, since GFP–N–GrpE complemented a thermosensitive grpE mutation (Fig. 3a) and it was not present in the inclusion bodies observed after centrifugation of the MC4100GFP–N–GRPE cell-free extract (data not shown). In addition, dots of GFP fluorescence were not observed when GFP–N–GrpE was overexpressed in the dnaK deletion mutant BM271 (Fig. 3e, i), suggesting that the localization of GFP–N–GrpE was dependent on DnaK.
Third, we focused on the distribution of inclusion bodies, GFP–N–GrpE and nucleoids, since it was speculated that inclusion bodies might be relocalized by nucleoids. The GFP–N–GrpE fluorescence overlapped again with a dark dot corresponding to inclusion body (Fig. 4a, b). Furthermore, GFP–N–GrpE fluorescence was observed in the region where the nucleoid was absent (Fig. 4b–d). It has been reported that several DnaK molecules are localized on the surface of an inclusion body (Carrio & Villaverde, 2005) and that GrpE interacts strongly with the nucleotide-free or ADP-bound state of DnaK (Harrison, 2003; Harrison et al., 1997; Schonfeld et al., 1995). These earlier reports raise the possibility that GrpE colocalizes with DnaK on the surface of the inclusion body in MC4100GRPE (Fig. 4e). Additionally, a pull-down assay using the cell-free extract of His-tag-fused GrpE-overexpressing cells demonstrated that DnaK was bound to His-tag-fused GrpE (data not shown). Since the results indicated that most of the overexpressed GrpE proteins were detected in the soluble fraction (data not shown), it was concluded that interaction among GrpE, DnaK and inclusion bodies was transient.
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). Inclusion bodies are relocated to nucleoid-free regions (deduced cell-division sites or poles of the cell) by the nucleoid. GrpE proteins then transiently colocalize with DnaK proteins on the surface of the inclusion bodies.
Effect of a high GrpE concentration on the chaperone activity of the DnaK system
In vitro experiments using purified DnaK, DnaJ and GrpE proteins have demonstrated that a concentration of GrpE that is fivefold that of DnaK inhibits the chaperone activity of DnaK (Packschies et al., 1997), suggesting that a balance between ATPase stimulation and acceleration of nucleotide exchange by DnaJ and GrpE is crucial for DnaK-assisted protein folding. Here, we examined the effect of a high concentration of GrpE on the chaperone activity of DnaK in vitro.
A dose–response curve demonstrated that the maximum activity yield of refolded luciferase was obtained at a GrpE concentration of 
1 µM (Fig. 5a). Higher GrpE concentrations (>5 µM) resulted in lower amounts of reactivated luciferase; this is consistent with an earlier report (Packschies et al., 1997). Grimshaw et al. (2005) reported that GrpE-G122D, even at a high concentration (4 µM), only marginally assisted DnaK in the refolding of denatured proteins (luciferase and glucose-6-phosphate dehydrogenase), and in the presence of ADP, GrpE-G122D, in contrast to wild-type GrpE, did not apparently form a complex with DnaK.
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, the addition of the MC4100GEM cell-free extract stimulated the reactivation of luciferase to 23 % of the native activity after incubation for 2 h. The reason for the lower refolding yield than with purified proteins (Fig. 5a) may be the presence of various other molecular chaperones and proteases that might trap the substrate and delay refolding or degrade the substrate. The other reason appeared to be the low concentrations of DnaK, DnaJ and GrpE in the cell-free extract compared with the intracellular concentrations of these proteins. In contrast, the MC4100GRPE cell-free extract only subtly stimulated the refolding of luciferase (Fig. 5b). Interestingly, the cell-free extract of MC4100G122D assisted in the refolding of luciferase, but the yield and rate were lower than those obtained with the cell-free extract of MC4100GEM (Fig. 5b). An earlier report has stated that the affinity of the GrpE-G122D dimer for the DnaK monomer is much lower than that of the wild-type GrpE dimer (Grimshaw et al., 2005); this suggested that the chaperone activity of the DnaK system was not directly inhibited by the binding of the GrpE-G122D dimer, instead of wild-type GrpE dimer, to DnaK. We suspect that the GrpE-G122D monomer binds to the wild-type GrpE monomer and forms a heterodimer whose affinity may be lower than that of the wild-type GrpE homodimer, which might lead to a decrease in the chaperone activity of the DnaK system.
In addition, the amount of aggregated protein in the MC4100GRPE cell-free extract was compared with those of the MC4100GEM and MC4100G122D extracts (Fig. 5c). As expected, a large amount of protein aggregate accumulated for MC4100GRPE compared with the other extracts. In all cases, a small amount of GrpE was detected in the insoluble fraction by Western blotting (data not shown). The two major bands seemed to be OmpF and OmpA, both of which are usually identified as major insoluble proteins (Fig. 5c) (Mogk et al., 1999).
These results clearly indicated that overexpression of GrpE suppressed the activities of the folding enzymes in a cell. One explanation is that the activity of the DnaK chaperone system might be inhibited by the change in the balance between the ATP-bound and the ADP-bound state of DnaK (Packschies et al., 1997). Under physiological conditions, the synergistic action of the two co-chaperones DnaJ and GrpE controls the steady-state distribution between the high-affinity state (ADP-bound state) and low-affinity state (ATP-bound state) of DnaK and provides the substrate proteins with an opportunity to be efficiently folded into their correct structure (Fig. 6a). Under GrpE-overexpressing conditions, the balanced distribution of these states shifts towards the low-affinity state (ATP-bound state or nucleotide-free state), which decreases the fraction of substrate bound to DnaK in the high-affinity state; this drastically decreases the folding yield (Fig. 6b) (Grimshaw et al., 2005).
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). One of the proteins possibly affected is FtsZ, which plays a crucial role in the early stages of septum apparatus construction. It has been suggested that an abnormality in either the activity or the concentration of FtsZ probably results in the filamentous morphology of the dnaK deletion mutant and DnaK-overexpressing cells (Bukau & Walker, 1989). However, the precise roles of the DnaK chaperone system in the functioning or expression of FtsZ have been unclear. Our recent research has shown that the concentration of FtsZ in the dnaK deletion mutant is similar to that in wild-type cells (unpublished data). The activities and/or assembly of FtsZ or other cell-division proteins are probably deficient due to impairment of the DnaK chaperone system in GrpE-overexpressing cells. To address this, the effect of GrpE overexpression on the dynamics of FtsZ-ring formation was examined by indirect immunofluorescence microscopy with anti-FtsZ antibody and Cy3-labelled goat anti-rabbit IgG.
An FtsZ ring was observed as a red band at the centre of the MC4100GEM cell (Fig. 7). In contrast, the FtsZ ring was barely observed at potential division sites of the MC4100GRPE cell. Instead, several dots of FtsZ were observed at the poles and at non-specific positions in the filaments. The abnormal localization of FtsZ was also observed in the dnaK deletion mutant (BM271). Uehara et al. (2001) have reported that HscA, an Hsp70 family protein, is involved in the localization of FtsZ in E. coli K-12. In addition, the abnormal cell-division phenotype of the hscA knockout mutant was partially complemented by the plasmid-borne dnaK gene, suggesting that the roles of DnaK in cell division partially overlap with those of HscA (Uehara et al., 2001). Their report supports our conclusion that the loss in functionality of the DnaK chaperone system by GrpE overexpression leads to defective cell division via an abnormality in the cell-division machinery, including the FtsZ ring. To our knowledge, this is the first report that demonstrates the direct visualization of the dependence of FtsZ-ring formation upon DnaK. In contrast to GrpE, DnaJ overexpression did not affect cell division and growth in E. coli (Blum et al., 1992). The difference in contribution between GrpE and DnaJ should be further examined. The design of chemical compounds that regulate the function of the DnaK chaperone cycles will be one possible way to inhibit the growth of pathogens or enhance the viability of bacteria used in the food industry or in fermentation.
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ACKNOWLEDGEMENTS |
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Edited by: H. Ingmer
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REFERENCES |
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Received 6 February 2008;
revised 13 April 2008;
accepted 21 April 2008.
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), MC4100GRPE (
) and MC4100G122D (
) were grown in LB medium containing 50 µg ampicillin ml–1 at 37 °C. At the indicated times, 1 ml of culture was taken and the OD600 was measured. The OD600 of the cultures at 0 h was 
) or presence of the cell-free extract (10 mg ml–1) from MC4100GEM (
