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In response to rising growth temperatures, all organisms synthesize heat shock proteins (HSPs). Many of these inducible polypeptides are members of larger families based on similar size and sequence homology, and most include proteins synthesized constitutively under nonstress conditions. Members of most HSP families function as molecular chaperones in assisting the folding, assembly, and translocation of other proteins during normal and adverse growth regimens. One relatively new family of chaperones is HSP100/Clp. It can be divided into two basic groups, with proteins in the first (ClpA to -E) having two distinct ATP-binding domains (ATP-1 and ATP-2) while proteins in the second (ClpX and -Y) possess only one such domain (21). The main HSP of this chaperone family is ClpB, and most organisms produce at least two different types. Separate nuclear clpB genes in eukaryotes encode mitochondrial (78-kDa [11]) and cytosolic (100- to 110-kDa [19]) proteins, while plants have another ClpB isomer localized in chloroplasts (10). In contrast, a single gene in eubacteria encodes two differently sized proteins (ca. 79 and 93 kDa) via dual translational initiation sites within the clpB transcript (5, 23).

ClpB is essential for resistance to high-temperature stress. The cytosolic ClpB in Saccharomyces cerevisiae and plants confers acquired thermotolerance (9, 18, 19), which is commonly defined as the resistance developed by an organism to withstand an otherwise lethal temperature treatment by being preexposed to a nonlethal high temperature. Similarly, the heat shock-inducible ClpBI protein from the cyanobacterium Synechococcus sp. strain PCC 7942 (Synechococcus) is also crucial for thermotolerance (5), a role that can be complemented by Escherichia coli ClpB (6). In the case of ClpBI, both the full-length and truncated forms of the protein contribute to the thermotolerance acquired by Synechococcus (3).

Mechanistically, ClpB functions to dissolve inactive protein aggregates that accumulate at high temperatures (16). In both bacteria and eukaryotes, ClpB cooperates with the DnaK-DnaJ-GrpE proteins in a bichaperone network to prevent and revert protein aggregates during heat shock (7, 13, 14). ClpB apparently dissolves large, stable heat-inactivated proteins by directly binding to aggregates and exposing hydrophobic surfaces within the polypeptides by ATP-induced structural changes in the ClpB protein (4, 8).

All ClpB proteins studied to date are strongly induced by high temperatures, and most are vital for heat tolerance. Under nonstressed conditions, however, ClpB is characteristically a nonessential protein, with little or no phenotypic changes resulting from clpB gene inactivations (5, 9, 19, 23). We now describe the identification of a second clpB gene in Synechococcus that encodes a protein closely related in terms of primary sequence to ClpBI and other known ClpB proteins. We show that the Synechococcus ClpBII has characteristics so far unique to ClpB proteins in eukaryotes and bacteria, revealing an extra dimension to the functional importance of ClpB molecular chaperones.

Cloning and sequencing of clpBII gene. Degenerate primers specific for ATP-1 and ATP-2 of HSP100/Clp proteins (2) were used to clone the clpBII gene from Synechococcus by PCR. A single 1.4-kb fragment was amplified and identified by DNA sequencing as an internal portion of a putative clpBII gene. The clpBII fragment was later used to isolate a full-length clone from a Synechococcus genomic DNA library.

Inducibility of ClpBII by high temperature. To investigate if Synechococcus ClpBII is an HSP like all other ClpB proteins, wild-type cultures were shifted from the standard 37°C growth conditions (6) to 48.5°C for 90 min (Fig. 1). Cellular proteins were isolated during the heat shift, and both ClpBI and ClpBII were detected by immunoblotting using specific polyclonal antibodies. Each antibody was made to the C-terminal region downstream of ATP-2 in Synechococcus ClpBI or ClpBII (17), and no cross-reaction to the alternative ClpB isomer was detected for both antibodies. As shown in Fig. 1, wild-type Synechococcus induced both the full-length 93-kDa (ClpBI-93) and truncated 79-kDa (ClpBI-79) forms of ClpBI during the shift to 48.5°C (Fig. 1), consistent with previous observations (5). Using the ClpBII antibody, a single 97-kDa polypeptide was detected that corresponded to the predicted size of ClpBII. The amount of ClpBII, however, remained relatively unchanged during the heat shock treatment, indicating that ClpBII is not an HSP in Synechococcus.

View larger version (51K): [in this window] [in a new window]   FIG. 1.   Levels of ClpBI and ClpBII in Synechococcus during heat shock. Wild-type Synechococcus cultures grown at 37°C were shifted to 48.5°C for 90 min, with all other growth factors kept constant. Cellular protein samples were taken at the indicated times and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the basis of equal Chl content (0.25 µg). ClpBI and ClpBII proteins were detected immunologically with specific polyclonal antibodies. Shown is a representative result from one of three replicates. Molecular mass standards (in kilodaltons) are indicated on the left.

The clpBII gene produces no truncated protein form. In addition to no ClpBII induction at high temperatures, no truncated version of ClpBII was detected throughout the 48.5°C shift (Fig. 1). Given that the ClpBII antibody was directed against the C terminus, such a truncated form should have been detected if it were present along with the full-length ClpBII, as both ClpBI-79 and -93 proteins were detected by the ClpBI antibody. This observation was consistent with the lack of a putative ribosome-binding site and start Val_GTG codon in the region of the clpBII sequence that corresponds to the second translational initiation site of Synechococcus ClpBI and E. coli ClpB. In fact, the sequence of this region of ClpBII is the most divergent within the entire protein relative to ClpBI and all other bacterial homologues.

ClpBII is essential for cell viability. To investigate the function of ClpBII, we attempted to inactivate the clpBII gene in wild-type Synechococcus. Being naturally competent, Synechococcus is an easily transformable cyanobacterial strain that has been frequently used to successfully study protein function via gene disruption. As for clpBI (5), a deletion-insertion strategy was used to inactivate the clpBII gene. Three different plasmid constructs were tested. In the first, 580 bp of the clpBII sequence was deleted and a Cm^r marker was inserted. The resulting construct was linearized to ensure double recombination and transformed into both wild-type and clpBI strains according to the method of Van der Plas et al. (24). For both strains, no viable transformants were recovered, even after several attempts. The second construct was as for the first but with the antibiotic resistance marker changed to one for erythromycin. Again, no variable transformants were obtained from both wild-type Synechococcus or clpBI cells. For the third construct, a larger internal portion of clpBII (1,960 bp) was replaced with the Cm^r cassette, but again after transformation with the linearized plasmid all transformed cultures lost viability. As a control for the transformation protocol, the third construct was kept intact and transformed into wild-type Synechococcus as the circular plasmid. Numerous viable transformants were obtained, but all derived from plasmid integration via a single crossover event, with no disruption to clpBII as verified by Southern blot analysis (data not shown). Overall, these results suggest that ClpBII is an essential protein for Synechococcus cell viability.

ClpBII is not induced by other stresses. Given the lack of heat inducibility of ClpBII and its importance for cell viability, we examined whether other stresses influenced the constitutive level of ClpBII in wild-type Synechococcus (Fig. 2). Conditions were selected based on the known stress sensitivity of Synechococcus and the degree to which the wild type could acclimate to them during the chosen time course. For all experiments, wild-type cultures were shifted from the standard growth condition (37°C, 50 µmol of photons m² s¹, 5% CO₂ in air) to either cold (25°C) or high light intensity (1,000 µmol of photons m² s¹) or were treated with high salt (150 mM NaCl) or H₂O₂ (0.5 mM) concentrations. Each condition produced a transient cessation in growth after the shift (1 to 3 h) followed by resumption at a lower rate. For each treatment, ClpBII content did not increase rapidly during the inhibitory period of the shift as would be expected for a stress-inducible protein. Instead, the level of ClpBII protein rose gradually over the duration of cold, high-light and high-salt treatments, being most abundant once cultures had acclimated to the new condition. In contrast, the level of ClpBII upon addition of H₂O₂ decreased gradually throughout the time course of oxidative stress, remaining low even after cultures had acclimated and resumed growth.

View larger version (43K): [in this window] [in a new window]   FIG. 2.   Levels of ClpBII protein during different stresses. Synechococcus wild type grown under standard conditions was either chilled at 25°C for 24 h, photoinhibited at 1,000 µmol of photons m2 s1 for 24 h, or treated with either 0.5 mM H2O2 for 24 h or 150 mM NaCl for 48 h. Cellular protein extracts were taken at the indicated times and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on equal Chl content (0.25 µg), and ClpBII was detected immunologically. For each stress, the figure shows a representative result from three replicates.

Inducible ClpBII fails to confer thermotolerance. The lack of heat shock induction of ClpBII indicated its regulation in Synechococcus differs from that of ClpBI. Given the primary sequence similarity between ClpBI and -II, however, we examined whether the two isomers were functionally equivalent despite their differential regulation. To test this, a plasmid construct was prepared to substitute for the native clpBII promoter with the heat shock-inducible clpBI promoter. A 200-bp fragment containing the clpBI promoter was ligated to a 5' fragment of clpBII (500 bp) at the site of the start Met_ATG codon (Fig. 3A). Upstream of the promoter was added a Cm^r cassette (not shown) as a selectable maker. The circular plasmid was then transformed into the Synechococcus clpBI strain to replace the native clpBII promoter with that of clpBI via a single homologous recombination event. Correct integration into the clpBII gene and its complete segregation in the clpBI chromosome were verified by Southern blotting, with the resulting strain termed HSB2.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Ectopic heat induction of ClpBII in HSB2. (A) Structural representation of the modified clpBII gene in clpBI whereby the native clpBII promoter was replaced with the heat-inducible clpBI promoter, resulting in the strain HSB2. (B) Heat shock induction of ClpBII in the HSB2 strain. Cultures of clpBI and HSB2 were shifted from 37 to 48.5°C for 90 min. Cellular protein extracts were taken at the indicated times and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on equal Chl content (0.25 µg), and ClpBII was detected immunologically. Shown is a representative result from three replicates. (C) Thermotolerance developed in wild-type Synechococcus, clpBI, and HSB2. Cultures grown at 37°C were preconditioned at 48.5°C for 90 min and then shifted to 54°C for 15 min. Viable cell numbers at each time point are expressed as percentages of the 37°C control values (100%). All values are averages ± standard errors (error bars) (n = 3).

Addition of a truncated form of ClpBII. The absence of a truncated form for Synechococcus ClpBII is so far unique among bacterial ClpB counterparts. For ClpBI, the truncated ClpBI-79 protein complements ClpBI-93 and contributes to thermotolerance in wild-type Synechococcus (3). To investigate this characteristic of ClpBII, a construct was first prepared to test whether a truncated form of ClpBII could functionally substitute for the native full-length ClpBII in wild-type Synechococcus. In this construct, a 500-bp fragment from the clpBII gene was PCR amplified, starting from the position 18 nucleotides downstream of where the second translation start codon (Val_GTG) occurs in the clpBI gene. Ligated to this was the clpBI promoter followed by Met_ATG and the 15 bases after the Val_GTG start codon from clpBI. Inserted upstream of the clpBI promoter fragment was the chloramphenicol resistance gene for selection purposes. Transformation of the circular construct into clpBI should modify the native clpBII gene via single recombination to produce a truncated 82-kDa protein (ClpBII-82) expressed from the clpBI promoter. However, repeated transformations with this construct failed to recover viable transformants, suggesting that the truncated ClpBII-82 cannot complement the native ClpBII protein.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Ectopic synthesis of truncated ClpBII-82 protein in HSB2-82. (A) Structural representation of the truncated clpBII gene with the clpBI promoter (2,229 bp) transformed into Synechococcus clpBI to create the HSB2-82 strain, which also retains the native clpBII gene (2,685 bp). (B) Induction of ClpBII-82 in HSB2-82. HSB2-82 and clpBI cultures were shifted from 37 to 48.5°C for 3 h, with cellular proteins isolated at 30-min intervals and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on equal Chl content (0.25 µg). The differently sized ClpBII proteins were detected immunologically using the antibody specific for the C terminus of ClpBII. (C) Decreased heat resistance in HSB2-82. Wild-type, clpBI, and HSB2-82 cultures grown at 37°C were shifted to 48.5°C for 3 h. For each strain, the numbers of viable cells at the indicated times are expressed as percentages of the 37°C values (100%). All values are averages ± standard errors (error bars) (n = 3).

Novel form of ClpB protein in Synechococcus. We have identified a second Synechococcus ClpB protein whose primary sequence is closely related to the first ClpB isomer and most other bacterial ClpB forms. The occurrence of genes for two distinct ClpB isomers is a characteristic of cyanobacteria apparently not shared by other eubacteria. Despite its structural similarity, ClpBII has features so far unique to both its bacterial and eukaryotic counterparts. Synechococcus ClpBII is not an HSP in contrast to all previously identified ClpB proteins. Inducible ClpB is crucial for thermotolerance and other forms of heat resistance in most organisms (5, 9, 19, 22, 23). In contrast, Synechococcus ClpBII is a constitutive protein that is unable to complement ClpBI function and confer thermotolerance, indicating its regulation and functional importance differs significantly from that of other ClpB homologues.