ClpB in a Cyanobacterium: Predicted Structure, Phylogenetic Relationships, and Regulation by Light and Temperature

JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

Oct. 1998, p. 5173–5182 Vol. 180, No. 19

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

ClpB in a Cyanobacterium: Predicted Structure, PhylogeneticRelationships, and Regulation by Light and Temperature

MARTINA CELERIN,†* ANDREA A. GILPIN,‡ NICHOLAS J. SCHISLER,† ALEXANDER G. IVANOV,§EWA MISKIEWICZ, MARIANNA KROL, AND DAVID E. LAUDENBACH

Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7

Received 6 February 1998/Accepted 30 April 1998

The sequence of a genomic clone encoding a 100-kDa stress protein of Plectonema boryanum (p-ClpB) wasdetermined. The predicted polypeptide contains two putative ATPase regions located within two highly con-served domains (N1 and N2), a spacer region that likely forms a coiled-coil domain, and a highly conservedconsensus CK2 phosphorylation domain. The coiled-coil region and the putative site of phosphorylation arenot unique to p-ClpB; they are present in all ClpB sequences examined and are absent from the ClpB paralogsClpA, ClpC, ClpX, and ClpY. Small quantities of a 4.5-kb p-clpB transcript and 110-kDa cytosolic p-ClpBprotein were detected in cells grown under optimal conditions; however, increases in the quantities of thetranscript and protein were observed in cells grown under excess light and low temperature conditions. Finally,we analyzed ClpA, ClpB, and ClpC sequences from 27 organisms in order to predict phylogenetic relationshipsamong the homologs. We have used this information, along with an identity alignment, to redefine the Clpsubfamilies.

The Clp (caseinolytic protease) system was first identified asa heat shock-inducible, multicomponent, ATP-dependent pro-tease complex able to hydrolyze casein (24, 28; for a review, seereference 35). Subsequent studies showed that the Clp systemcan hydrolyze numerous other proteins and peptides in bothaggregated and nonaggregated forms (31). The Clp systemmembers include three nonhomologous gene families: clpAB-CXY, clpP, and clpQ. The ClpACX members (but not ClpB)facilitate the activities of ClpP, and some, such as ClpA (68)and ClpX (67), can function as independent chaperones inroles analogous to those of DnaK and DnaJ. In contrast, theClpP proteolytic subunit exhibits low levels of peptidolytic ac-tivity. However, when ClpP is complexed with either ClpA(28), ClpC (58), or ClpX (69), active holoenzymes (29) whichare able to cleave denatured proteins (70) are formed. Addi-tionally, ClpY interacts with ClpQ to form a proteolyticallyactive holoenzyme (40).

Distinctively, ClpB does not interact with ClpP (62).Whereas several molecular sequence analyses have predictedthat ClpB may or at least should be able to interact with ClpP(3, 30, 61), based on sequence similarity to ClpA and ClpC,empirical data to support this idea remain elusive (71). ClpB isunable to activate or facilitate ClpP in ATP-dependent proteinhydrolysis, and clpB null mutants of Saccharomyces cerevisiaefail to show any defect in proteolytic degradation (47; also seereference 61). Thus, unlike ClpA, ClpC, and ClpX, ClpB ap-pears invariably to function independently of ClpP.

Metabolic and cytological responses to adverse photosyn-thetic conditions have been documented for plants (17, 23, 46),

algae (38, 56, 60), and cyanobacteria (32, 36), including Plec-tonema boryanum UTEX 485 (4, 8, 39). Photosynthetic organ-isms typically undergo compensatory morphological and phys-iological changes on a variety of levels in response tophotosynthetic adjustments. These may include modulation ofpigment content and composition, changes in chlorophyll aand/or b light-harvesting complex II or phycobilisome abun-dance, increases in O2 evolution, upregulation of specific en-zymes (peroxidases, catalases, and superoxide dismutases[SOD]), increases in photorespiration, and increases in pig-ments (carotenoids) that are involved in both the dissipation oflight energy as heat (20, 51) and the scavenging of oxygenradicals by a mechanism of epoxidation (59). Oxygen radical-metabolizing enzymes (Fe-SOD, Mn-SOD, and catalase) in P.boryanum (8) and Synechoccocus sp. strain 7942 (1, 44) havebeen studied, and the induction of stress proteins, in particularthe Clp system, during growth under low (50) and high (14)temperatures in Synechoccocus sp. 7942 has been examinedrecently. In this study, we describe the isolation of the clpBgene of the cyanobacterium P. boryanum (p-clpB) and reportthe results of the investigation of its response to excess lightstress as enhanced by low temperature (ELLT). The results ofthe analysis of the predicted amino acid (aa) sequence ofp-ClpB show that it contains both of the highly conserved N1and N2 domains and the spacer region (61). We predict thatthe spacer region of p-ClpB, as well as those of other ClpBs,although divergent in predicted primary sequence among spe-cies, forms four coiled-coil structures. Further, we use phylo-genetic analyses and identity alignments to predict evolution-ary relationships among the ClpABC homologs and redefinethe families of the Clp proteins.

MATERIALS AND METHODS

Culture conditions and growth rates. Axenic cultures of P. boryanum UTEX485 were maintained on solidified (1% Difco-Bacto Agar) BG-11 medium (2)buffered to pH 8.0 with 20 mM HEPES. Standard liquid cultures were grown at29°C and irradiated with cool white bulbs to either 50 or 150 mmol of photonsm22 s21 (GaAs-photodiode connected to a Li-Cor light meter; measured in thecenter of the growth tube). The 50-ml culture tubes were placed in water-filledaquaria and sparged with 3% CO2. Cultures grown for protein analysis were notbubbled with CO2. Growth in ELLT was as described above, except that the

* Corresponding author. Mailing address: Department of Biology,Indiana University, Jordan Hall, Bloomington, IN 47405. Phone:(812) 855-3692. Fax: (812) 855-6705. E-mail: [email protected].

† Present address: Department of Biology, Indiana University,Bloomington, IN 47405.

‡ Present address: Department of Botany, University of Toronto,Toronto, Ontario, Canada M5S 1A1.

§ Present address: Institute of Biophysics, Bulgarian Academy ofSciences, 1113 Sofia, Bulgaria.

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liquid cultures were maintained at 15°C with either 150 or 300 mmol of photonsm22 s21. Periodic dilution with fresh growth medium ensured that cells wereharvested in their exponential growth phase.

Growth was measured as an increase in chlorophyll a concentration. Aliquotsof cells were filtered through glass microfiber filters (934-AH; Whatman, Maid-stone, United Kingdom) and transferred to 2-ml screw-cap tubes containing0.1-mm zirconium oxide beads and 1.5 ml of 90% acetone. Cells were brokenwith a Mini-Beadbeater (Biospec Products, Bartlesville, Okla.) for 60 s andsubsequently centrifuged at 12,000 3 g for 5 min. The amount of chlorophyll ain the supernatant was determined by the equations of Jeffrey and Humphrey(26). The specific constant rate (m), which represents the increase in biomass perunit time, was calculated by the equation dX/dt 5 mt, where X is the biomassconcentration (chlorophyll a concentration) and t is time. The doubling time, t2,was determined by the equation t2 5 0.693/m.

Analysis of pigment composition. Pigments were extracted with 100% acetoneat 4°C under dim light. The supernatant was filtered through a 0.22-mm-pore-sizesyringe filter, and samples were stored at 280°C. Pigments were separated andquantified by high-performance liquid chromatography (HPLC) as described byIvanov et al. (25), with some modifications. The system consisted of a BeckmanSystem Gold programmable solvent module 126, diode array detector module168 (Beckman Instruments, San Ramon, Calif.), and CSC-Spherisorb ODS-1reverse-phase column (5-mm particle size; inside diameter, 25 by 0.46 cm) withan Upchurch Perisorb A guard column (both columns from ChromatographicSpecialties, Inc., Concord, Ontario, Canada). Samples were injected with a Beck-man 210A sample injection valve with a 20-ml sample loop. Pigments were elutedisocratically for 6 min with a solvent system (acetonitrile–methanol–0.1 M Tris-HCl [pH 8.0] [72:8:3.5; vol/vol/vol]), followed by a 2-min linear gradient to 100%methanol-hexane (75:25 [vol/vol]), which continued isocratically for 4 min. Totalrun time was 12 min. The flow rate was 2 cm3 min21. Absorbance at 440 nm wasdetected, and peak areas were integrated by Beckman System Gold software.Retention times and response factors of chlorophyll a and b-carotene weredetermined by injection of known amounts of pure standards purchased fromSigma (St. Louis, Mo.). The retention times of zeaxanthin and of a carotenoid,which was tentatively identified as myxoxanthophyll, were determined by usingpigments purified by thin-layer chromatography (13). To determine the concen-tration of the putative myxoxanthophyll, the response factor was calculated by aspecific extinction coefficient of 2,160 at 478 nm (10).

PCR amplification and Southern blots. Two degenerate 20-mer oligonucleo-tides (oligo-I and oligo-II) were used to amplify DNA from the genome of P.boryanum, as described elsewhere (9). After the ends of the PCR product weremade blunt (T4 polymerase; Pharmacia, Mississauga, Ontario, Canada) and werephosphorylated (T4 polynucleotide kinase; BRL, Burlington, Ontario, Canada),the product was digested with Sau3AI (1 U; Pharmacia). The Sau3AI DNAfragments were purified by phenol-chloroform extraction and ethanol precipita-tion and then ligated (T4DNA ligase; Pharmacia) into M13mp19 plasmids thatwere linearized with SmaI. Single-stranded constructs were isolated for sequenceanalysis, and double-stranded inserts were purified (QIAprep-8 Plasmid kits;Qiagen, Santa Clarita, Calif.) for use as probes.

The cloned PCR product was radiolabelled by the random primer method withT7 polymerase (QuickPrime kit; Pharmacia) and [32P]dCTP (NEN; .3,000 Cimmol21). Southern blots were performed as described elsewhere (9).

Cloning of the clpB gene. Genomic DNA fragments ranging from 8.7 to 8.9 kb(generated by digestion with HindIII) were purified from an agarose gel. Thefragments were eluted and ligated into pUC18 plasmids that had been linearizedwith HindIII and dephosphorylated. The heterogeneous plasmid constructs wereelectroporated into Escherichia coli DH10B competent cells. Cells harboring aplasmid construct were selected by plating on 2% (wt/vol) agar–Luria-Bertaniplates supplemented with ampicillin (LBA; 50 mg ml21), 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal; 30 mg ml21), and isopropyl-b-D-thioga-lactopyranoside (IPTG; 60 mg ml21) (53). Colonies were picked in sets of 200and amplified (1 ml of Terrific Broth medium [53] and 50 mg of ampicillin ml21).Plasmids were isolated by the boiling miniprep method (53) and screened onSouthern blots for their ability to hybridize with a radiolabelled probe, the clonedSau3AI fragment of the PCR product. Clones were verified by performing theconverse experiments; inserts of positive clones were radiolabelled and used toscreen blots of P. boryanum genomic DNA, which was digested with each of therestriction enzymes BglII, HindIII, and EcoRI. A restriction map of one of thepositive clones, pMC8.8, was created by single- and double-enzyme digests. ASouthern blot of the digests was probed with the cloned Sau3AI fragment of thePCR product, and the smallest positive band, a 1.7-kb ClaI-AccI fragment, wassubcloned into pUC18. In addition, the 1.4-kb ClaI-ClaI DNA fragment, proxi-mal to the 1.7-kb ClaI-AccI DNA fragment in a restriction map of the 8.8-kbHindIII clone, was subcloned into pUC18. Inserts were released from vectorswith EcoRI and HindIII, radiolabelled by random priming, and used as probes inSouthern blots of total P. boryanum genomic DNA.

Sequencing. DNA sequencing with both single-stranded M13- and plasmid-based templates was performed by the dideoxynucleotide method as modified byU.S. Biochemicals Sequenase 2.0. Internal primers were made by Procyon Inc.,London, Ontario, Canada.

Structural and phylogenetic analysis of clpB. The DNA sequence obtainedwas translated in six reading frames and compared to all nonredundant polypep-tides in the translated NCBI database (GenBank, Bethesda, Md.) by using the

program BLAST (12) to identify similar sequences. Cellular localization waspredicted with the program PSORT, version 6.3 (42).

Alignments of Clp amino acid sequences were carried out with the ClustalWprogram, version 1.7 (27, 65). The program Sequence Similarity Presenter (ftp://ftp.bio.indiana.edu/) was used to visualize alignments (16, 18, 43, 64).

Maximum likelihood analyses were performed with the program Puzzle, ver-sion 3.1 (63), with the quartet puzzling tree search algorithm, the neighbor-joining (52) tree parameter estimation, the Dayhoff model of substitution (11),and a uniform rate model of rate heterogeneity. Maximum likelihood analysesused frequencies of puzzling steps in the quartet puzzling tree search as ameasure of confidence which is comparable to the number of bootstrap replicates(63). The program NJPlot (Manolo Gouy, University of Lyon, Lyon, France) wasused to visualize phylogenetic trees. The plastid ClpA sequence from Plasmo-dium falciparum was used as the outgroup in all cases.

Percent identities (pairwise comparisons) of amino acids between ClpB ho-mologs and ClpB spacer regions were calculated with Genetics Computer Group(GCG) Gap (12). In addition, the predicted amino acid sequences of ClpA,ClpB, and ClpC were analyzed by using Coils2 (37) and paircoils (5) programsdesigned to augur secondary structures. The Coils2 program was used with a21-residue scan window with all four combinations of weighted and unweightedMTK and MTIDK matrices.

Northern blot analysis of p-clpB. Total RNA from P. boryanum was isolated asdescribed elsewhere (32) from cells grown under optimal and ELLT conditions.RNA (5 mg per lane) was fractionated by electrophoresis in 1.2% agarose gelsunder denaturing conditions, transferred to nitrocellulose membranes (32), andhybridized with the radiolabelled 1.7-kb ClaI-AccI DNA fragment. The sizes ofthe RNA transcripts detected were estimated based on published rRNA sizes.

Preparation of total soluble protein fraction and thylakoid membranes. Cellswere harvested by centrifugation (4,500 3 g for 8 min at 10°C), washed in 60 mlof 5 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-NaOH buffer (pH 7.0), and treated with a saturated solution of NaI for 30 minat 37°C. After the treatment, the cells were washed twice in double-distilled H2Oand resuspended in 10 ml of 5 mM TES-NaOH (pH 7.0)–0.6 M sucrose–2 mMEDTA–3 mg of lysozyme, and the suspension was incubated for 2 h at 28°C withcontinuous shaking. Cells were collected by centrifugation as described above,washed with 60 ml of 20 mM TES-NaOH (pH 7.0)–0.6 M sucrose, resuspendedin 1 ml of the same medium plus 100 ml of 100 mM benzamidine, and passedthree times through a French pressure cell press (SLM, Inc., Rochester, N.Y.)operating at a pressure of 80 MPa. One hundred microliters of 100 mM phenyl-methylsulfonyl fluoride and 10 ml of 0.1 mg of DNase ml21 in 1 mM MgCl2 and10 mM Na acetate buffer (pH 5.6) were added to the homogenate. After incu-bation for 15 min on ice, the homogenate was centrifuged at 4,500 3 g for 10 minto remove the unbroken cells. For preparation of the total soluble proteinfraction, the supernatant was adjusted to 100 mM with NaCl and centrifuged for1 h at 100,000 3 g in a 50 Ti fixed-angle rotor. Soluble proteins were precipitatedby adjusting the supernatant to 10% trichloroacetic acid (vol/vol) and incubatingthe solution for 30 min on ice. Precipitated proteins were pelleted by centrifu-gation at 12,000 3 g at 4°C, washed once with ice-cold 90% (vol/vol) acetone,dried, and prepared for sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE). For preparation of thylakoid membranes, the supernatant,after DNase treatment (see above), was adjusted to 50% (wt/vol) sucrose, andthe thylakoids were isolated by centrifugation in a sucrose gradient at 130,000 3g for 16 h at 4°C as described elsewhere (41). Protein concentrations weremeasured by the standard bicinchoninic acid protein assay (Pierce, Rockford,Ill.).

SDS-PAGE and Western blot analysis of p-ClpB. Samples for electrophoresiswere solubilized in 0.1 M dithiothreitol–0.1 M Na2CO3–2.5% (wt/vol) SDS–30%(wt/vol) sucrose, boiled for 1 min, and separated by SDS-PAGE with a Mini-Protean II apparatus (Bio-Rad, Mississauga, Ontario, Canada). Electrophoresiswas performed with a 5% (wt/vol) stacking gel and a 15% (wt/vol) resolving gel.All samples were loaded on an equal protein basis (7 mg per lane), and a constantcurrent of 15 mA was applied for approximately 2 h at 4°C. Polypeptides wereeither stained with 0.2% (wt/vol) Coomassie brilliant blue R-250 or transferredby electrophoresis (Mini-Trans Blot; Bio-Rad) to nitrocellulose membranes(0.2-mm pore size; Bio-Rad [9]). Nitrocellulose filters were washed in blockingbuffer for at least 1 h at room temperature with continuous shaking. Afterblocking, the membranes were incubated in a 1:5,000 dilution of anti-Hsp78 (34),a monoclonal antibody that recognizes the 78-kDa mitochondrial heat shockprotein (a ClpB homolog) in S. cerevisiae. After the blots were washed with theblocking solution, the bound polypeptide-primary antibody complexes were in-cubated with goat anti-mouse immunoglobulin G conjugated with horseradishperoxidase (Sigma) as the secondary antibody at a 1:20,000 dilution. Complexeswere visualized with a chemiluminescence detection system (Amersham,Oakville, Ontario, Canada).

Nucleotide sequence accession number. The genomic DNA sequence of p-clpBis available in the GenBank data bank under accession no. AF061279.

RESULTS

Growth rates and culture morphology. Under optimalgrowth conditions (29°C/150 mmol of photons m22 s21), cells

5174 CELERIN ET AL. J. BACTERIOL.

of P. boryanum have a doubling time of 8.4 6 0.06 h. Con-versely, cells of P. boryanum grown under ELLT conditions(15°C/150 mmol of photons m22 s21) have a doubling time of55.74 6 0.73 h.

Macroscopically, cultures of P. boryanum grown under op-timal light and temperature conditions exhibited the blue-green hue typical of cyanobacteria (Fig. 1A). In contrast, cul-tures of P. boryanum grown under ELLT conditions showed amarked change in color (Fig. 1B); distinctively, the culture wastinged with an orange-red appearance. Analysis of the HPLC-separated cell pigments (Table 1) showed that cells grownunder ELLT conditions (Fig. 1B) had altered pigment compo-sitions compared to cells grown under optimal conditions (Fig.1A). The quantity of the carotenoids (Table 1) increased dif-ferentially; myxoxanthophyll and zeaxanthin showed approxi-mately five- and twofold increases, respectively, whereas theamount of b-carotene showed no appreciable increase. Carot-enoids have the ability to dissipate excess energy generatedunder excess light conditions and thereby provide protection tothe photosensitive cell components. This phenomenon hasbeen well-studied in plants (59), and, as expected, we havefound that the cellular quantities of these pigments also in-crease in P. boryanum. Additional environmental stresses, suchas low temperature, exacerbate the decreased photoutilization.Britten (7) and others have shown that, in plants, the amountof each carotenoid present varies but is dependent on growthconditions. Thus, it is expected that cells of P. boryanum grown

under ELLT conditions should exhibit (i) an overall decreasein growth rate, (ii) an increase in the quantity of carotenoids,and (iii) differential increases in specific carotenoids. Clearly,the changes in the quality and quantity of the carotenoidsestablish that cells of P. boryanum grown under ELLT condi-tions have undergone photoinhibition and, consequently, cel-lular stress.

Cloning of the p-clpB gene. Because we were interested incloning a catalase gene from a cyanobacterium, PCR primersbased on conserved amino acid sequences in both plants andbacteria were designed. Although catalase genes were not de-tected, serendipitously, the Sau3AI fragment of the PCR prod-uct proved to be a useful probe for detecting the clpB gene inP. boryanum (p-clpB). High-stringency Southern blot analysisof P. boryanum genomic DNA with the radiolabelled clonedSau3AI fragment of the PCR product as a probe showed onestrong (4.5-kb) and two weak (7.0- and 8.8-kb) signals in DNAdigested with HindIII (Fig. 2A, lane H). Both the 4.5- and8.8-kb HindIII DNA fragments were cloned, although re-peated attempts to clone the 7.0-kb HindIII fragment wereunsuccessful. Further analysis of the 4.5-kb HindIII DNA frag-ment indicated that it encodes the gene for kinesin light chain(9). When the 8.8-kb HindIII DNA fragment was used as theprobe for a genomic Southern blot (Fig. 2B), the fainter pat-tern observed in Fig. 2A (indicated by asterisks) was detected.Moreover, both the 1.7-kb ClaI-AccI DNA fragment (Fig. 2C)and the 1.4-kb ClaI-ClaI DNA fragment (Fig. 2D) subclonedfrom the 8.8-kb HindIII clone displayed this same patternwhen used as probes.

Sequence analysis revealed that the 1.4-kb ClaI-ClaI DNAsubclone harbored the 59 end of an open reading frame (ORF;p-clpB) and that the 1.7-kb ClaI-AccI DNA subclone harboredthe bulk of the same ORF. The DNA sequence of the 39 endof the ORF was obtained by sequencing from the plasmid thatcontained the 8.8-kb HindIII DNA fragment.

DNA and deduced amino acid sequences of p-ClpB. In total,of the 2,863 bases of nucleic acid sequence obtained, 2,649nucleotides are predicted to comprise an ORF of 883 aa (Gen-Bank no. 190195). The predicted polypeptide has a calculatedmolecular mass of 100,513 Da and a calculated isoelectricpoint of 5.37. In addition, the predicted polypeptide has twowell-conserved ATP-binding site motifs (66) at aa 219 to 226

FIG. 1. Cultures of P. boryanum grown under optimal (A) and ELLT (B)conditions.

FIG. 2. Southern blot analysis of the clpB gene in P. boryanum. In all panels,genomic DNA was digested with BglI (B), EcoRI (E), and HindIII (H). The blotwas probed with the Sau3A fragment of the PCR product (A), the 8.8-kb HindIIIgenomic DNA fragment (B), and the 1.7-kb ClaI-AccI and 1.4-kb ClaI-ClaIsubfragments (C and D, respectively). The hybridization pattern of clpB is indi-cated by the size markers 4.9 (BglII), 4.1 (EcoRI), and 8.8 (HindIII) kb. (Thestronger bands, i.e., 8.1 [BglII], 5.6 [EcoRI], and 4.5 [HindIII] kb, are the hy-bridization pattern for p-KLC [9].)

TABLE 1. Carotenoid composition of P. boryanum cells grownat 29 and 15°C

Carotenoid

Result under the following growth conditionsa

Optimal (29°C/150mmol m22 s21)

ELLT (15°C/150mmol m22 s21)

Myxoxanthophyll 0.101 6 0.015 0.484 6 0.051Zeaxanthin 0.035 6 0.007 0.074 6 0.012b-Carotene 0.249 6 0.009 0.329 6 0.059

a Results are micromolar concentrations per micromolar chlorophyll. Meanvalues 6 standard errors of the means were calculated from three to fourindependent experiments with three separate measurements in each experiment.

VOL. 180, 1998 ClpB IN A CYANOBACTERIUM 5175

and 622 to 629, as well as two chaperonin ClpAB signaturemotifs at aa 307 to 319 and 648 to 666.

The DNA sequence of the ORF was compared to all se-quences in the GenBank database (NCBI, Bethesda, Md.) withthe BLAST program (12). Overwhelming identity to the ClpBfamily of ATP-dependent proteins was observed, although sim-ilarities to the ClpC and ClpA families were also noted. Figure3 shows a representation of percent sequence identity acrossthe multiple sequence alignment profile (alignment not shown)of the predicted P. boryanum ClpB to the other members of theClpABC family. Several regions showed high sequence simi-larity, especially near and including the nucleotide bindingsites. The N1 and N2 regions, the regions of highest similarity,

are indicated. ClpB from P. boryanum was typical of othercyanobacterial and bacterial ClpB sequences.

Another feature of note in p-ClpB is the presence of aputative phosphorylation site for casein kinase II (CK2 [49][S-X-X-E-E-X-X-E]) adjacent to Serine-420. The putativephosphorylation site, which is adjacent to the N1 domain, is ahighly conserved feature of all ClpB sequences (Fig. 4) but isabsent from all ClpA, ClpC, ClpX, and ClpY sequences exam-ined. Surprisingly, in other unrelated proteins that have asite for phosphorylation adjacent to a predicted coiled-coildomain, the phosphorylation status is involved in regulatingthe three-dimensional (3D) conformation of the domain (21);we suspect that stabilization or regulation of the predicted

FIG. 3. Amino acid sequence identities of the ClpABC family of proteins. Sequences were aligned to the profile which was generated from all ClpABC sequencesused in this study. Species’ amino acid similarities to the profile are indicated by white (gaps), shades of gray (similarity with the profile), and black (complete identitywith the profile). Major structural regions including the N1 and N2 regions that contain the ATP-binding sites are identified above the alignment. The accessionnumbers for the sequences used in the alignment are as follows: Swiss-PROT P03815, P05444, P15716, P31541, P17422, P31539, P31542, P31543, P33416, P35100,P37571, P42730, P44403, P49574, P53532, P53533, P53533, and P77686; and GenBank 511145, 755163, 1001555, 1171612, 1314297, 1563721, 1653543, 1653648, 1705923,1705925, 1946209, 2058336, 2113980, 2146076, L35272, M67479, and U46549.


coiled-coil structures in ClpB (discussed below) may occur viaphosphorylation of the conserved serines.

Additional differences among the Clp sequences that arenoteworthy include the following (i): the clpB sequences fromMycoplasma are unusual in that they lack a leader region; (ii)ClpB from S. cerevisiae mitochondria has a truncated leaderregion, the amino acid sequence preceding the N1 domain; (iii)plants appear to have an extended trailer region; and (iv)Plasmodium berghei contains a longer spacer region that uponfurther analysis is predicted to form three coiled coils sepa-rated by 20 instead of 3 aa (data not shown), as is the case inall other ClpB spacers (described below).

Coiled-coil region predicted in the spacer of other ClpBgenes. The spacer region, a stretch of amino acids that sepa-rates the N1 and N2 regions, has been used to delineate rela-tionships among the Clp homologs (19, 61). Squires andSquires (61) assign Clp proteins into subfamilies based on thesize of the spacer region. ClpA family members have spacerregions composed of 5 aa, ClpC members have 62 to 69 aa, andClpB members have the longest spacers (123 to 131 aa). Thespacer region comprises 13.5% by the number of amino acids(14.6% by weight) of the total predicted polypeptide in p-ClpB.The programs Coils2 (37) and paircoils (5) predicted that the

spacer region of p-ClpB is composed of coiled coils (Fig. 5 andhttp://mold.bio.indiana.edu/mcelerin/index.htm). Also, Coils2was able to resolve the domain into four discrete helical re-gions. Each predicted helical region contains four sets of hep-tad repeats, a-b-c-d-e-f-g, where a and d are often hydrophobicresidues, and b, c, e, f, and g are hydrophilic and form thesolvent-exposed part of the helical surface (37). The sets offour are separated by three residues that are not part of aheptad repeat and thus are not part of the helical structure.Presumably, the three interhelical residues cause local insta-bility of the coiled coil that could result in bends or hinges inthe 3D structure, similar to those for the skip residues de-scribed elsewhere (45) for myosin. Of the nine residues thatcomprise the three sets of 3-aa interhelical hinges, seven aredefined by Bhaskaran and Ponnuswamy (6) as highly or excep-tionally flexible amino acids based on their computed flexibilityindices. Additionally, the calculated total bond length of the 3aa (10.1 Å) would permit a 90° turn in the direction of the helixin a manner similar to that of the 4 aa involved in the turn ofhelix-turn-helix motifs.

Because of the novel prediction that coiled coils are presentin the spacer region of p-ClpB, we pursued an analysis ofavailable ClpB sequences from the NCBI database and, usingCoils2 (37), we have found that all ClpB spacer regions arepredicted to form coiled-coil domains (http://mold.bio.indi-ana.edu/mcelerin/index.htm). Table 2 shows that although theoverall identity among pairwise comparisons of the ClpB se-quences from diverse organisms is high (mean, 51.1%; stan-dard deviation [SD], 7.9; n 5 78; shaded areas), the percentidentity of pairwise comparisons within the predicted coiled-coil region is surprisingly low (mean, 36.7%; SD 5 11.3; n 578; white areas), given the amount of conservation at the sec-ondary structural level. In all but two pairwise comparisons(Glycine max with Arabidopsis thaliana and Leishmania majorwith Trypanosoma brucei), the spacer region shows less percentidentity between sequences than the overall sequence percentidentity. This indicates that the predicted 3D structure of thedomain, but not the sequence per se, is conserved.

Phylogenetic analysis of ClpABC. Members of the ClpABCXY family in eubacteria and in eukaryotes, including plants,fungi, and protists, have been identified. The putative relation-ship of p-ClpB to other ClpA, ClpB, and ClpC sequences thatcontain both the N1 and N2 domains is shown in Fig. 3. SinceClpX and ClpY lack the leader region, spacer, and first nucle-otide-binding domain characteristic of ClpABC sequences,they were excluded from phylogenetic analysis.

Figure 6 shows the deduced phylogenies of ClpABC as cal-culated from maximum likelihood analyses of amino acid se-quences. Distance and maximum parsimony trees are availableat http://mold.bio.indiana.edu/mcelerin/index.htm. With theexception of the Plasmodium sequence, ClpB sequences arewell-resolved into bacterial and eukaryotic clades, with P. bo-ryanum ClpB appearing most closely related to Synechococcussp. and other cyanobacterial ClpB polypeptides.

The plant ClpA polypeptides appear to be more closelyrelated to ClpC than to ClpA sequences (Fig. 3). ClpA, ingeneral, is not particularly well-resolved, possibly due to thesmall number of taxa available for analysis (although it appearsdistinct from ClpB and ClpC by distance-based methods, albeitwith low bootstrap support). Since (i) the ClpC sequences inthe phylogenetic tree are predicted to be monophyletic and (ii)analysis of the amino acid alignment (described above) sug-gests that the spacer region, which is used to delineate Clphomologs, is absent from the ClpA plant sequences, we suggestthat ClpA plant polypeptides are, in fact, members of the ClpCsubfamily.

FIG. 4. Aligned amino acids proximal to the predicted coiled-coil domain.The conserved serine residue (aa 420), within a consensus CK2 phosphorylationdomain, is highlighted.

FIG. 5. Predictions of coiled-coil structures in ClpB from P. boryanum. Prob-abilities in excess of 90% were obtained with weighted and unweighted scoringmatrices, predicting strongly that the region does form a coiled coil in p-ClpB.The greatest percent difference between matrices was observed when compari-son was made of unweighted MTIDK and MTK (5.5%), a value that is still farbelow the critical maximum (20 to 30%) to confidently assign the region as beingpredicted to form a coiled coil (37).


Expression of p-clpB. A p-clpB transcript of approximately4.5 kb was identified by Northern blot analysis of total RNAfrom P. boryanum cells grown under optimal (29°C/50 mmol ofphotons m22 s21) conditions (Fig. 7A, lane 1b, large arrow-head). Total RNA collected from cells grown under ELLT(15°C/300 mmol of photons m22 s21) conditions also containedthe 4.5-kb p-clpB transcript (Fig. 7A, lane 2b); however, thequantity of transcript was fivefold more abundant in the ELLT-grown cells. In addition to the predominant 4.5-kb transcript,two larger transcripts were detected from cells grown underELLT conditions (Fig. 7A, small arrowheads). These may rep-resent stages of processing of a polycistronic message thatcontains the p-clpB transcript. Additionally, a large quantity ofRNA is detected through the length of the lane (Fig. 7A, lane2b). Based on the integrity of the rRNA bands (Fig. 7A, lane2a), we presume that the total RNA is not degraded; the smearof RNA may be reflective of the instability of the p-clpB tran-script.

We addressed the question of localization of p-ClpB usingimmunodetection. Differential sedimentation was used to sep-arate total soluble proteins from cell wall components, plasma,and thylakoid membranes. Figure 7BII (lane 2) shows that asingle protein band of approximately 110 kDa was detected inthe total soluble protein fraction from cells grown under opti-mal conditions. The apparent molecular mass of the protein isslightly higher than the predicted molecular mass of the p-ClpB polypeptide (100,513 Da), possibly a result of poor res-olution of large proteins by SDS-PAGE. The 110-kDa bandwas not detected in any other cell fraction (data not shown), afinding in agreement with the results of the analysis by PSORT(42), which predicted, based on sequence features related toprotein sorting signals, that p-ClpB would localize to the cyto-plasmic fraction of the cell components. Furthermore, a singleband of the same molecular mass but of greater intensity,which indicated that an increased quantity of the protein was

present, was detected only in the cytosolic fraction of cellsgrown under ELLT conditions (Fig. 7BII, lane 1). This isconsistent with results of the Northern blot analysis and sup-ports the idea that p-clpB encodes a single-copy gene that istranscribed and translated to make a single protein product.The results also indicate that a certain quantity of transcriptand protein is present in cells growing under optimal condi-tions but that the quantities of both increase during growthunder stressful conditions such as ELLT. It is interesting tonote that although the results of the Northern blot suggest thatthere is a fivefold induction of the p-clpB transcript after ELLTtreatment, there is not the same increase in the amount ofprotein present after ELLT treatment. Thus, the data suggestthat ELLT growth conditions may induce more transcriptionof clpB but not more synthesis of the protein.

DISCUSSION

We have cloned and sequenced the p-clpB gene from P.boryanum. Based on available sequence data, p-ClpB has77.3% identity with its most closely related subfamily member,ClpB from Synechococcus sp.; it has 44.2% identity with themost distantly related ClpB sequence (hsp104; S. cerevisiae,cytosolic). The predicted protein has a number of featureswhich enable us to suggest that ClpB from P. boryanum is aparalog of ClpA, ClpC, ClpX, and ClpY and an ortholog ofClpB. All ClpABCXY family members, which can show vari-ations in size (61), possess at least one ATP-binding motif (66)and one or both conserved N1 and N2 domains. Like otherClpB subfamily members, p-ClpB contains (i) both the N1 andthe N2 domains, each of which contain an ATP-binding motif(66); (ii) leader and trailer sequence; (iii) a central spacerregion, which we have predicted forms a coiled-coil domain;and (iv) a newly identified, highly conserved putative site forphosphorylation adjacent to the predicted coiled-coil domain.

TABLE 2. Pairwise comparison of the percent identities of the spacer region (white areas) and the entire amino acid sequences of ClpBs(shaded areas)

Species % Identity


The phylogenetic analysis in the present study supports pre-vious suggestions that the ClpA, ClpB, and ClpC subfamiliesare three separate yet evolutionarily related groups of polypep-tides. However, our analysis of the sequence data shows thatpreviously used nomenclature may not express accurately theevolutionary history of the proteins. We have redefined thesubfamily groupings in what seems to be a more credible re-flection of their amino acid sequence identities.

We suspect that the conserved recognition region for phos-phorylation may be involved in regulating conformation ofClpB. In some coiled-coil proteins, such as lamins, the phos-

phorylation states of the serine residues that are immediatelyadjacent to the coiled-coil domains are pivotal in regulating theconformation (21). Although ClpB is known to form homotet-ramers, we suspect that the coiled-coil domain is involved inheteropolymerization. ClpA and ClpB are similar over manyregions (reviewed in references 57 and 61), with the notableexception of the predicted coiled-coil domain. Moreover, ho-mopolymerization occurs in both ClpA and ClpB; by inference,the coiled-coil domain is unlikely to be involved in this process.

Based on the difference between ClpB and either the ClpAor the ClpC protein, namely, the presence or absence of the

FIG. 6. Deduced phylogeny of ClpABC amino acid sequences. The numbers at the nodes are percentages of puzzling steps which support the branch in thismaximum likelihood analysis.


predicted coiled-coil region, it is tantalizing to speculate thatthe lack of this domain may permit protein-protein interac-tions with ClpP. It would be interesting to determine if, byremoving the coiled-coil domain in ClpB, one could constructa truncated ClpB that may be able to interact with ClpP.

It is well-established that heat shock proteins (hsps) are infact stress proteins. In vivo, the occurrence of heat shock israre; heat stress is usually a gradual shift to an extreme con-dition. Therefore, other stresses such as exposure to heavymetals, excess salt, low temperature, and, in photosensitiveorganisms, excess light are more prevalent. Differential expres-sion of stress proteins is contingent on the type of stress (seereference 35) and the organism (48), but the hsps that tend tobe induced most consistently include hsp70-DnaK, hsp60-GroEL, and the hsp100-Clp complex members (3). ClpBs havecentral roles in ameliorating environmental stresses in additionto heat shock. Sanchez et al. (55) showed that the ClpB ho-molog of S. cerevisiae (hsp104) is responsible for tolerance notonly of heat but also of ethanol, arsenite, and long-term expo-sure to cold. Clarke and coworkers (14, 50) have demonstratedthat in Synechococcus, ClpB is responsible for sustained ther-motolerance at high temperatures and contributes to acclima-tion at moderately low (25°C) temperatures. However, Porank-iewicz and Clarke (50) also report that the level of ClpB foundin cells decreases with immoderately cold (15°C) conditions. Itis important to note that in the latter study, all cells were grownat 50 mmol of photons m22 s21; thus, the effect of increasedlight intensity in combination with low temperatures was notexamined.

Although all aerobic organisms must contend with reactiveoxygen species, the by-products of respiration that result fromthe incomplete reduction of molecular oxygen, photosyntheticorganisms have the added vicissitude of addressing a second

potential source of oxidative damage. The excitation of chlo-rophyll results in the singlet excited state, which eventually canlead to the production of singlet oxygen (51). Prolonged expo-sure of photosynthetic organisms to a combination of lightintensities and temperature such that the excitation energyexceeds the capacity for dissipation can result in the produc-tion of additional singlet oxygen. Oxidative damage can bemanifested as lipid peroxidation, protein oxidation, and DNAdamage, all of which potentially hamper normal cellular activ-ities. Numerous proteins, including stress proteins, must func-tion to compensate for these severe environmental conditionsin order to permit normal cellular metabolism and growth.

Under optimal growth conditions, either very low levels ofthe clpB transcript are detected (P. boryanum [present study]and Leishmania major [22]) or no clpB is observed (S. cerevisiae[54], Synecoccocus sp. [14], and Glycine max [33]). Remarkably,the quantity of clpB transcript present in P. boryanum increasesin cells exposed to excessive light (as exacerbated by low tem-peratures [present study]) and sulfur limitation (8a). At least inP. boryanum, both of these two stresses can induce SODs (8)and, thus, presumably cause oxidative damage (directly or in-directly). Porankiewicz and Clarke (50) showed that Synecoc-cocus sp. cells grown at 15°C (50 mmol of photons m22 s21)showed no induction of ClpB. They suggested that the lack ofinduction may be due to the retardation of protein synthesisduring growth under immoderately cold conditions. In thislight, the comparatively small (fivefold) induction observed inP. boryanum cells grown at 15°C with 300 mmol of photons m22

s21 is even more striking. Clearly, under these adverse condi-tions, cells must be expending a considerable amount of energyto produce this protein, and thus the end product, ClpB, musthave a critical role in cell survival. Alternatively, the differencesin induction of ClpB may simply be due to the fact that al-though both are cyanobacteria, P. boryanum and Synecoccocussp. are two different species, and thus each may employ dis-parate strategies for coping with stresses.

ClpB is a stress-induced protein, but it is not a protease.Parsell et al. (47) suggested that ClpB may function by con-trolling the aggregation or denaturation of vital cellular struc-tures rather than by operating as a proteolytic regulator. Oth-ers (48, 61, 71) have suggested that ClpB may function as amolecular chaperone, like hsp70-DnaK or hsp60-GroEL. Con-sequently, ClpB may either prevent the formation of proteinaggregates, catalyze ATP-dependent refolding, or reassembleunfolded proteins in stressed cells. Squires and Squires (61)also suggested that members of the ClpB family might beinvolved in controlling some enzymatic activities other thanproteolysis. Nonetheless, a clear answer to the question “Whatdoes ClpB do?” remains elusive.

ACKNOWLEDGMENTS

We thank Norm Huner and Mimi Zolan for their generosity duringthe final stages of manuscript preparation. Also, we thank ErinGerecke for extensive, helpful suggestions during the preparation ofthe manuscript; and John Coleman, Sean Turner, Peter Kuhlman, JoseBonner, and Sven Beushausen for critical reading. Anti-hsp78 was agenerous gift from T. L. Mason. Also, we thank an anonymous re-viewer for an insightful observation regarding our data.

This work was supported by a grant to D.E.L. from the NaturalSciences and Engineering Research Council of Canada (NSERC).Finally, the paper encompasses the last of the work that M.C. did inassociation with David Edgar Laudenbach, her cherished mentor andan outstanding scientist. It is dedicated to his memory.

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FIG. 7. (A) Northern blot analysis of clpB from P. boryanum UTEX 485.Total RNA was isolated from P. boryanum cells grown under optimal (lanes 1)and ELLT (lanes 2) conditions. The gels were stained with ethidium bromide,and RNA samples were loaded equally, based on the quantity of rRNA present(a). The p-clpB transcript was detected on Northern blots by using radiolabelled1.7-kb ClaI-AccI genomic DNA fragment of p-clpB (b). Large arrowhead indi-cates the 4.5-kb transcript. Smaller arrowheads indicate two additional tran-scripts of approximately 8 and 10 kb detected in the RNA of cells grown underhigh light and low temperature. (B) Characterization and immunolocalization ofthe p-ClpB protein. (I) Analysis of total proteins from P. boryanum by SDS-PAGE and stained with Coomassie G-250. Lanes 1 and 2, cells grown underELLT and optimal conditions, respectively. ST, protein standards. (II) Westernblot of gel (corresponding to gel in panel BI) probed with the polyclonal antibodyanti-hsp78 (34).


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ClpB in a Cyanobacterium: Predicted Structure, Phylogenetic Relationships, and Regulation by Light and Temperature

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