To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain. The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress. However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain. The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB. These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress.
It is well known that the synthesis of heat shock proteins (Hsps) is induced by heat in a variety of organisms . In general, Hsps have been known to function for the stabilization and the protection of intracellular proteins from heat stress [1–6]. Moreover, the induction of some Hsp(s) has been suggested to make cells tolerant to heat and some other stresses, such as ethanol and oxidative stresses . The Hsps are (1) chaperones, such as GroES/GroEL and DnaK/DnaJ/GrpE, (2) proteases, such as Lon, ClpP/ClpX, HtrA and HflB, (3) proteins having other functions and (4) proteins having unknown functions [1–6]. In Escherichia coli, the expression of hsp genes is regulated by either σ32 or σE[1,6].
Recently, the small heat shock protein (sHsp) family has been reported to be widely distributed in different organisms [8,9]. These proteins have molecular masses of 15–30 kDa per monomer and form a large oligomeric complex structure [8–10]. The sHsps contain a region similar to the ‘α-crystallin domain’ of an eye lens protein, α-crystallin [8,10,11]. Further, some sHsps have been found to suppress protein aggregation by heat and to stabilize proteins [8–10,12,13]. Unlike ATP-dependent chaperones, such as the Hsp70 and Hsp60 families, sHsps have a chaperone-like activity in vitro even in the absence of ATP [8–10,12]. Other investigators have reported that the sHsp oligomer binds to a variety of proteins in heat-treated cells and that its refolding activity arises in cooperation with Hsp70 [13,14]. Although there are a lot of in vitro studies on the function of sHsps, only a few reports on their intracellular functions have been published. However, it has also been found that the overproduction of sHsps makes mammalian cells resistant to heat and oxidative stresses [8,15,16].
We have previously reported that a subset of E. coli 15-kDa Hsps, C14.7 and G13.5, are synthesized during periods of heat treatment and recovery after heat stress and also that they were found not only in the soluble fraction of the cell extracts but also in the sedimentary fraction obtained by ultracentrifugation . We also confirmed that these proteins were IbpA and IbpB, respectively, designated by Allen et al. , based on N-terminal amino acid analysis (Kitagawa et al., unpublished).
Although IbpA and IbpB are homologous to sHsp family proteins, their functions in detail are not known. Thomas and Baneyx  have indicated that defects of both IbpA and IbpB make cells slightly sensitive to heat and also that this effect was enhanced by the introduction of dnaK756 mutation. This result suggests that the functions of IbpA and IbpB are not straightforward and they may cooperate with some other Hsps.
In this study, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also a mutant defective in both genes to elucidate their roles in the resistances to heat and oxidative stresses.
2Materials and methods
2.1Bacterial strains and growth conditions
E. coli BL21(DE3) was used as a host strain of pET8c . A wild-type strain, E. coli OW6 , and a temperature-sensitive rpoH amber mutant of E. coli, K165 , which cannot substantially induce heat shock proteins at nonpermissive temperatures, were used as host strains of pUC19 . E. coli OW6 was also used for the construction of an ibpAB-disrupted mutant and as its parent strain. All of these three strains were also used for the overexpression of ibpA and ibpB. E. coli JM109 was a host strain for gene manipulation . All strains used were grown in M9 medium containing 20 L-amino acids (AM9 medium) at 37 or 30°C . Ampicillin (35 μg ml−1) or kanamycin (25 μg ml−1), if necessary, was added to the medium. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to induce the overexpression of ibpA and ibpB.
2.2Construction of ibpA-, ibpB- and ibpAB-overexpression plasmids
The DNA fragment of ibpAB region was amplified by PCR , using the chromosomal DNA of E. coli OW6  as a template and two sets of oligonucleotide primers, 5′-TTATAGGGATCCCTTGCTGAAAATAAC-3′ and 5′-GCTGCAGATTTGCCTTATAGCGCACT-3′, including restriction sites, BamHI and PstI, respectively, and also 5′-CAGGAGCTATTGATCATGAGAAACT-3′ and 5′-GATTATGGCTGGATCCGTGAAGCAG-3′, including restriction sites, BspHI and BamHI, respectively. These primer sets were designed based on the sequence around the ibpAB region . Amplified fragments were digested with BamHI and PstI or with NcoI and BamHI and then cloned into pUC18 or pET8c, respectively. The resultant plasmids were designated pUC-IbpAB and pET-IbpAB, respectively. pET-IbpA and pET-IbpB were constructed from pET-IbpAB by enzymatic deletion of the ibpB and the ibpA regions, respectively. The fragment obtained from pET-IbpAB by digestion with XbaI and BamHI was subcloned into pUC19 and the resultant plasmid was designated pUI IbpAB (Fig. 1).
2.3Construction of an ibpAB-disrupted strain
For the disruption of ibpA and ibpB genes, pUC-IbpAB was digested with AccI and BalI, sites of which were located in the ibpAB region. The kan cartridge, derived from pUC4K , was inserted to the pUI IbpAB at the AccI–BalI site. The ibpAB::kan fragment in the resultant plasmid was subcloned into pKF38s (a gift from Dr. Hashimoto-Gotoh, Kyoto Prefectural University), the replication of which was temperature sensitive. The plasmid obtained was designated pKFs-IbpABK. E. coli OW6 was transformed with pKFs-IbpABK and an ibpAB-disrupted mutant was constructed by the homologous recombination method described by Hamilton et al. .
2.4Treatments with heat, paraquat, hydrogen peroxide and ethanol
The bacterial culture at mid-exponential phase was transferred from 30 to 48, 50 or 52°C and then kept at the transferred temperature. In treatments with 10% ethanol, 2.4 mM hydrogen peroxide and 1.2 mM paraquat, these agents were added to the culture grown to mid-exponential phase at 37°C. The viability of cells was determined by the colony count method, using M9 plates containing 0.1% vitamin-free casamino acid (Difco), 0.2% glucose and 1.5% agar (EM9 agar).
2.5Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and protein analysis
2D PAGE was performed by isoelectric focusing described by O'Farrell , followed by SDS–polyacrylamide gel electrophoresis by the method of Laemmli . Gel was stained by using either Coomassie brilliant blue (R250) or Silver Stain II Kit Wako (Wako Pure Chemical Co.). Cell extracts were prepared by sonication. An aggregated protein (AP) fraction was prepared from the cell extracts by centrifugation at 13 000×g for 10 min at 4°C. The protein content was determined by the method of Lowry et al. , using bovine serum albumin as a standard.
3.1Stress resistances of the ibpAB-disrupted strain
To investigate the functions of sHsps, IbpA and IbpB, in E. coli, an ibpAB-disrupted strain was constructed by the homologous recombination method. Proteins produced in the ibpAB-disrupted strain and its parent strain were analyzed by 2D PAGE. After heat treatment at 50°C, IbpA and IbpB spots on the gel visualized by silver stain were detected in the cell extracts of the parent strain, but not in the mutant (data not shown). These proteins were undetectable in unstressed cells in both the mutant and its parent strain (data not shown).
The mutant grew normally in AM9 medium, suggesting that both IbpA and IbpB were dispensable for cell viability and growth. Furthermore, its resistance to heat was similar to that of its parent strain. Also, no substantial differences in resistances to hydrogen peroxide, paraquat, which is a superoxide generator, and ethanol, were observed between the mutant and its parent strain (Fig. 2 and data not shown).
It has already been reported that IbpA and IbpB are induced by non-lethal heat stress . Thus, we compared the heat resistance at 48, 50 and 52°C between the mutant and its parent strain after preheating at 44°C for 15 min upon the induction of IbpA and IbpB syntheses. Although these preheated cells of both strains increased their resistance to the subsequent heat challenge, no substantial difference in the resistance could be detected (data not shown).
3.2The overexpression of IbpAB makes cells resistant not only to heat but also to superoxide stress
To examine further the functions of IbpA and IbpB in E. coli cells, we constructed ibpA, ibpB and ibpAB recombinant plasmids derived from pET8c, which contains T7 promoter, and then transformed E. coli strain BL21(DE3) with these plasmids. The cells of strain BL21(DE3) bearing the ibpA and/or ibpB recombinant plasmids, pET-IbpA, pET-IbpB and pET-IbpAB, were grown to the mid-exponential phase after the addition of IPTG in AM9 medium. IbpA and/or IbpB in these overexpressing strains were confirmed to be induced with the addition of IPTG by 2D PAGE followed by Coomassie brilliant blue (data not shown). The ibpA and/or ibpB overexpression was found to make cells resistant to heat treatment at 50°C (Fig. 3 and Table 1). Also, to paraquat, the cell survival was improved by the overexpression of ibpA and/or ibpB (Fig. 4 and Table 1), whereas to hydrogen peroxide, its resistance did not seem to be increased with the overexpression (Table 1). Furthermore, the ibpA- and/or ibpB-overexpressing strains were slightly more resistant to 10% ethanol, compared with strain BL21(DE3) bearing pET8c (Table 1).
Table 1. Survival of the ibpA- and/or ibpB-overexpressing strains exposed to various stresses
aValues are averages±S.D. obtained from three independent experiments. See the legends to Figs. 3 and 4 for experimental conditions.
bPlasmids with which strain BL21(De3) were transformed.
48°C, 30 min
50°C, 30 min
52°C, 30 min
1 mM, 30 min
2 mM, 30 min
2 mM, 30 min
4 mM, 30 min
10%, 30 min
We also examined resistances to heat and paraquat of strain OW6 carrying the ibpAB recombinant plasmid, pUI-IbpAB, and that carrying pUC19. To the mid-exponential phase cells grown in AM9 at 30°C, IPTG was added at a final concentration of 4 mM and the culture was then incubated for a further 2 h. The viabilities of those cells after heat treatment at 50°C for 20 min of strain OW6 carrying pUI-IbpAB and that carrying pUC19 were 17.5±2.0 (S.D.) and 5.9±4.1%, respectively. When cells grown at 37°C were incubated for 1.5 h after the addition of IPTG at a concentration of 1 mM, the viabilities of strain OW6 carrying pUI-IbpAB and that carrying pUC19 after the treatment with paraquat at 2 mM for 30 min were 79.6±7.7 and 57.2±6.3%, respectively. These results indicate that the overexpression of IbpA and IbpB make the cells of strain OW6 resistant to heat and paraquat, as in the case of the cells of strain BL21(DE3) carrying pET-IbpAB. It seems, therefore, that IbpA and IbpB contribute to the cellular resistances to heat, superoxide stress and ethanol.
3.3Heat sensitivity of a rpoH mutant is suppressed by the overexpression of ibpAB
To investigate whether IbpA and IbpB need the help of some other Hsps for their function, we constructed an ibpAB recombinant plasmid, pUI-IbpAB, which was derived from pUC19, and then transformed a temperature-sensitive rpoH amber mutant, K165, with pUI-IbpAB. IbpA and IbpB in this overexpressing strain were confirmed to be induced with the addition of 1 mM IPTG by 2D PAGE (data not shown). Strain K165 bearing the pUI-IbpAB was found to be more resistant to heat treatment at 44°C than that bearing pUC19 (Fig. 5). This result suggests that the overproduction of IbpA and IbpB partially suppress the heat sensitivity of cells of strain K165.
3.4The overexpression of ibpA and ibpB suppresses the aggregation of intracellular proteins
Since some investigators have reported that sHsps bind and stabilize unfolded proteins to protect them from aggregation [13,14], we compared the level of aggregated proteins in cells between strain BL21(DE3) bearing pET-IbpAB and that bearing pET8c after exposure to stresses. After heat treatment at 50°C for 30 min, the degree of protein aggregation in the ibpAB-overproducing strain was 5.8±3.1%, while 10.6±2.7% in the strain carrying pET8c. This substantial reduction in the degree of protein aggregation in the ibpAB-overexpressing strain suggests that IbpA and IbpB may rescue proteins apt to unfold from aggregation. When cells were treated with 2 mM paraquat or 4 mM hydrogen peroxide for 30 min, however, the level of aggregated proteins was rather low in both the control strain and the ibpAB-overexpressing strain, being less than 1% of the total. This result demonstrates that IbpA and IbpB can suppress the protein aggregation caused by heat, although such an effect of both proteins can not be elucidated for oxidative stress.
In this study, we report that the overexpression of ibpA and ibpB in E. coli cells increases the resistances to heat, ethanol and superoxide stress, but not the resistance to hydrogen peroxide. The suppression of heat sensitization was also partially obtained with a rpoH amber mutant bearing an ibpAB recombinant plasmid. These findings suggest that IbpA and IbpB may contribute to cellular resistances to various stresses, as have also been indicated for the overexpression of sHsps in other organisms [8,15,16].
Unexpectedly, however, the ibpAB-disrupted strain was not more sensitive to the above stresses than its parent strain. Similar results on the heat sensitization have been obtained with an E. coli strain deficient in Hsp90  and a hsp26 deletion mutant of Saccharomyces cerevisiae[33,34]. Another ibpAB-disrupted strain of E. coli constructed by Thomas and Baneyx  has been reported to grow at temperatures below 45°C but not above 46°C, indicating its slight sensitivity to heat. This result is different from ours, possibly due to different genetic background of parent strains or the kind of medium used.
Our results on the ibpAB mutant suggest that both IbpA and IbpB are dispensable for cell growth and survival at high temperature, being apparently incompatible with the results of ibpAB overexpression described above. Although this contradiction is difficult to be explained at present, some other functional Hsps, probably as chaperones, might substitute for IbpA and IbpB. Thomas and Baneyx  indicated that the introduction of dnaK756 mutation sensitizes the ibpAB-disrupted strain to heat. Veinger et al.  have also indicated that IbpB displays a refolding activity for unfolded proteins only in cooperation with DnaK. These results suggest that DnaK may help the functions of IbpA and IbpB.
sHsps including IbpA and IbpB have been suggested to function as molecular chaperones [8–10,12]. Some investigators have reported that sHsps bind to and stabilize unfolded proteins to protect them from aggregation [8–10,12,13]. In fact, IbpA and IbpB were able to suppress, to some extent, the protein aggregation caused by heat. Considering the relationship demonstrated between the cellular resistance to heat and protein aggregation, increased levels of IbpA and IbpB due to their overexpression may protect cells from thermal death through their chaperoning function, similar to mammalian sHsps [8–10,12,13].
In the case of oxidative stress, however, such a relationship between the resistance and aggregation is unclear at present. Oxidative stress in general causes no drastic conformational changes in proteins, even though substantial inactivation of those is observed. IbpA and IbpB might play some role for reducing intracellular levels of reactive oxygen species. In mammalian cells, it has been reported that sHsps work as regulators responding to the intracellular redox state to reduce the level of reactive oxygen species and also to maintain the intracellular level of glutathione . Recently, Jakob et al.  and Zavialov et al.  have reported that Hsp33 in E. coli and Hsp25 in murine play important roles in the defense against oxidative stress. Heat stress in the presence of oxygen has been reported to be accompanied with oxidative stress . Further, some Hsps, such as DnaK and GroEL, are known to be induced not only by heat stress but also by oxidative stress . It is likely that IbpA and IbpB may function for the defense against heat and oxidative stresses by themselves or in cooperation with some other Hsps, such as DnaK and Hsp33.
We are grateful to Professor H. Obata, Kansai University, for his encouragement and to Dr. T. Hashimoto-Gotoh, Kyoto Prefectural University, for providing us with pKF38s. We also thank N. Noro, K. Matsushita and T. Marutani for their technical assistance. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 07650966).