The clpP multigene family for the ATP-dependent Clp protease in the cyanobacterium Synechococcusc

Jenny Schelin1, Fredrik Lindmarka,1 and Adrian K. Clarkeb,1

Ume Plant Science Centre, Department of Plant Physiology, Ume University, 901 87 Ume, Sweden1

Author for correspondence: Adrian K. Clarke. Tel: +46 31 7732502. Fax: +46 31 7732626. e-mail: Adrian.Clarke{at}botinst.gu.se


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the cyanobacterium Synechococcus sp. strain PCC 7942 a multigene family of three different isozymes encodes the proteolytic subunit ClpP of the ATP-dependent Clp protease. In contrast to the monocistronic clpPI gene, clpPII and clpPIII are part of two bicistronic operons with clpX and clpR, respectively. Unlike most bacterial Clp proteins, the Synechococcus ClpP2, ClpP3, ClpR and ClpX proteins were not highly inducible by high temperatures, or by other stresses such as cold, high light or oxidation, although slower gradual rises occurred for all four proteins during high light, and for ClpP3, ClpR and ClpX at low temperature. Attempts to inactivate the clpPII, clpIII, clpR or clpX genes were only successful for clpPII, suggesting the others are essential for Synechococcus cell viability. The {Delta}clpPII mutant exhibited no significant phenotypic changes from the wild-type, including no change in ClpX content. Despite the apparent bicistronic arrangement of both clpPII-clpX and clpR-clpPIII, all four genes primarily produce monocistronic transcripts, although polycistronic transcripts were detected. Mapping of 5' ends for the clpX and clpPIII monocistronic transcripts revealed promoters situated within the 3' region of clpPII and clpR, respectively. Transcriptional and translational studies further showed differences in the expression and regulation between the clpP-clpR-clpX genes. Inactivation of clpPI caused a significant decrease in ClpP2 protein concomitant to small increases in both ClpP3 and ClpR. Inactivation of clpPII resulted in a large rise in clpPI transcripts but to a lesser extent in ClpP1 protein. Similar small increases in ClpP3, ClpR and ClpX proteins also occurred in {Delta}clpPII. These results highlight the regulatory complexity of these multiple clp genes and their functional importance in cyanobacteria.

Keywords: cyanobacteria, gene expression, protein regulation, proteolysis, stress

Abbreviations: Chl, chlorophyll; MBP, maltose-binding protein

c The GenBank accession numbers for the sequences of clpPII-clpX and clpR-clpPIII reported in this paper are U92039 and AJ132005, respectively.

a Present address: Department of Molecular Biology, Ume University, 901 87 Ume, Sweden.

b Present address: Botanical Institute, Göteborg University, Box 461, 405 30 Göteborg, Sweden.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Molecular chaperones and energy-dependent proteases are two vital contributors to cell homeostasis. Without an effective system constantly controlling and monitoring protein quality, many cellular processes would eventually cease, leading ultimately to cell death. Chaperones perform many roles, facilitating processes such as protein folding, assembly and membrane transport, while also enabling protein stabilization, renaturation and resolubilization under various adverse growth conditions. Targeted degradation by energy-dependent proteases is equally important by regulating the availability of regulatory proteins and removing non-functional but potentially harmful polypeptides arising from misfolding, denaturation or aggregation (reviewed by Gottesman, 1996 ). Many of these proteases incorporate chaperone activity, whereby substrates targeted for degradation require unfolding prior to proteolysis. One of the best-studied ATP-dependent proteases in Escherichia coli are the Clp proteases, which consist of regulatory ATPase/chaperone (ClpA, ClpX, ClpY) and proteolytic (ClpP, ClpQ) subunits.

Clp proteases have an overall conserved architecture that resembles the cytosolic 26S proteasome in eukaryotes. Two apposed annuli of the proteolytic subunit, heptameric rings for ClpP and hexameric for ClpQ, form a central cavity housing the proteolytic active sites (Rohrwild et al., 1997 ; Wang et al., 1997 ). Narrow axial pores that only unfolded polypeptides can traverse restrict access to the inner cavity. Flanking the proteolytic complex are single hexameric rings of the ATPase/chaperone subunit, in E. coli being ClpA and/or ClpX with ClpP (Grimaud et al., 1998 ), and ClpY with ClpQ (Rohrwild et al., 1997 ). Substrate recognition is conferred by the chaperone subunit, which, after binding, unfolds the protein substrate and enables its transfer into the proteolytic chamber (Singh et al., 2000 ). Once inside, the protein substrate is efficiently degraded to small peptide fragments that later diffuse out.

Clp proteins are widely distributed in nature and are found in all eubacteria, plants and mammals. Although ClpP is common to all these organisms, the type and number of ATPase/chaperone subunits vary. ClpA and ClpX are now known as members of the Clp/Hsp100 family of chaperones. This family is divided into two basic groups, with members of the first (ClpA–E) being characterized by having two distinct ATP-binding domains, whilst those of the second (ClpX, ClpY) have only one. Although ClpX is ubiquitous, ClpA appears restricted to Gram-negative eubacteria like E. coli. Instead of ClpA, Gram-positive bacteria, cyanobacteria and plant chloroplasts commonly have ClpC, with additional types also occurring in most Gram-positive bacteria (ClpE) and higher plants (ClpD) (reviewed by Porankiewicz et al., 1999 ).

Clp proteases in E. coli degrade a variety of substrates, including SsrA-tagged polypeptides as part of the protein quality control system for removal of unstable or misfolded proteins subsequent to translation (Gottesman et al., 1998 ). Despite such roles, however, loss of ClpP in E. coli produces no obvious phenotypic changes. In contrast, more diverse and crucial roles for Clp proteins occur in Gram-positive bacteria, cyanobacteria and plants. In Bacillus subtilis, for example, proteins like ClpC, ClpX and ClpP are vital for resistance to many stresses, and for many cellular and developmental processes such as cell division, motility, sporulation and genetic competence (reviewed by Porankiewicz et al., 1999 ). The ClpXP protease is also mainly responsible for degradation of SsrA-tagged proteins in B. subtilis like the homologous protease in E. coli (Wiegert & Schumann, 2001 ). ClpC and ClpP functions are equally essential in cyanobacteria and plants, as shown by various genetic studies (Shanklin et al., 1995 ; Clarke & Eriksson, 1996 ; Clarke et al., 1998 ; Shikanai et al., 2001 ).

Cyanobacteria are a diverse group of eubacteria that can be found in nearly all habitats. They constitute one of the largest and most ecologically important bacterial groups, being one of the major contributors to biomass accumulation on Earth. Cyanobacteria are also the only prokaryotes that perform oxygenic photosynthesis like algae and higher plants, and are generally considered the progenitors of plastid evolution, according to the endosymbiotic theory. With such attributes, cyanobacteria have served as valuable model organisms for investigating many cellular processes common to photosynthetic organisms. One feature common, but not exclusive to cyanobacteria and plants is the presence of multiple ClpP isomers. These photosynthetic organisms furthermore have the ClpR variant of ClpP that lacks the three active site amino acids representative of ClpP proteases and whose function remains unclear (Clarke, 1999 ). To date, little is known about the regulation and functional importance of the ClpP and ClpR proteins in cyanobacteria. Previously, we described the clpPI gene in the cyanobacterium Synechococcus sp. strain PCC 7942 (Synechococcus) and its importance for stress acclimation (Clarke et al., 1998 ). We now identify the remaining Clp proteins in this cyanobacterium and examine their complex regulatory characteristics under a range of physiological conditions.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Culture conditions.
All Synechococcus strains were grown on solid or in liquid BG-11 (Clarke et al., 1995 ). Liquid cultures were grown in 80 ml glass tubes in 37 °C water-baths with continuous light of 70 µmol photons m-2 s-1 and bubbled with 5% CO2 in air (standard growth conditions). Cells in exponential growth phase with a chlorophyll (Chl) concentration of 2·5–3·5 µg ml-1 were used for all experiments. Mutant strains were maintained on solid plates and in liquid cultures supplied with 5 µg kanamycin ({Delta}clpPI) or chloramphenicol ({Delta}clpPII) ml-1 to maintain selection. No antibiotic, however, was added to the experimental cultures to eliminate the possibility of antibiotic-induced phenotypic changes.

Cloning and sequencing of clp genes.
The clpPII-clpX and clpR-clpPIII operons from Synechococcus were identified using degenerate oligonucleotides specific for highly conserved domains within known ClpX and ClpP3 homologues, respectively. Each primer was 27–35 bases long and included EcoRI restriction sites at the 5' ends to facilitate cloning. Primers for clpX were 5'-GTIGAATTCGTIGCIGTITA(CT)AA(CT)CA(CT)TA(CT)AA and 5'-AT(CT)TTGAATTCIGC(CT)TG(CT)TGIACICC(CT)TCICC, and for clpPIII were 5'-CCIGAATTCCA(AG)TA(CT)GA(AG)(AC)GITGGATIGA(CT)ATITA and 5'-GCCGAATTCIGG(CT)TG(AG)TGIATCATIAT. The expected 200 (clpX) and 260 (clpPIII) bp fragments were PCR-amplified from Synechococcus genomic DNA, cloned into the plasmid pUC19 and verified by DNA sequencing. Fragments were used as specific DNA probes to isolate clones containing the complete clpPII-clpX and clpR-clpPIII operons from partial Synechococcus genomic libraries constructed from 8–10 kb SacI and 2·5–4 kb HindIII restriction fragments in pUC19, respectively. Clones were isolated by colony hybridization as described previously (Eriksson & Clarke, 1996 ). DNA sequencing was done using the ABI PRISM Dye Termination Cycle Sequencing Ready Reaction Kit (Perkin Elmer), analysed on an automated sequencer (ABI377; Perkin Elmer) and viewed with AutoAssembler computer software.

Construction of clpPII-inactivation plasmid and transformation.
The {Delta}clpPII plasmid was made by replacing a 218 bp fragment in the middle of the complete clpPII gene with a 1·8 kb chloramphenicol resistance cassette (Shapira et al., 1983 ). An EcoRI–XbaI fragment covering the 5' end of clpPII and a HindIII–XbaI fragment covering the 3' end were joined together with the XbaI-cut chloramphenicol resistance cassette. This construct was cloned into the EcoRI–HindIII site of pUC19 and the resultant plasmid was transformed into E. coli DH5{alpha}. Positive transformants were selected on media containing chloramphenicol and restriction endonuclease digests verified plasmids with the correct construct. Wild-type Synechococcus was transformed with the linearized inactivation plasmid (van der Plas et al., 1990 ). Putative transformants were selected on BG-11 plates supplemented with 5 µg chloramphenicol ml-1. Correct insertion of the {Delta}clpPII construct and its complete segregation in selected transformants were confirmed by Southern blot analysis (Eriksson & Clarke, 1996 ).

Preparation of Clp-specific antibodies.
Polyclonal antibodies specific for Synechococcus ClpP2 or ClpX were made against fusion proteins overexpressed in E. coli using the pMAL-c2 overexpression plasmid (New England Biolabs). The complete clpPII gene and the 3' end downstream of the clpX ATP-binding domain were PCR-amplified from their genomic clone with the high-fidelity pfu DNA polymerase (Stratagene). PCR products were separately ligated in-frame to the 3' end of the malE gene, encoding the maltose-binding protein (MBP), on the plasmid pMAL-c2, and then transformed in E. coli DH5{alpha}. Overexpression of pMAL/clpPII and pMAL/clpX under control of the tac promoter was induced by adding IPTG to actively growing cells, and the resulting MBP/ClpP2 or MBP/ClpX fusion proteins were purified as described previously (Riggs, 1990 ). For Synechococcus ClpP3 and ClpR, synthetic peptides were designed for regions specific for each isomer (ClpP3: MPIGVPSVPYRLPGS; ClpR: LESIQAVQAPYYGDV; Fig. 1), and conjugated at the C terminus to the carrier protein keyhole limpet haemocyanin. All purified proteins were injected into rabbits intramuscularly and subcutaneously to produce the specific antibodies (AgriSera AB).



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Fig. 1. Sequence alignment of Synechococcus ClpP and ClpR proteins. Amino acid sequences were aligned using the PILEUP program from the UWGCG package. Amino acids identical for all (black) or three (grey) of the four Clp proteins are shaded as indicated. Gaps are shown as dashes, while asterisks indicate the Ser, His and Asp active site residues. The underlined regions in ClpP3 and ClpR indicate the synthetic peptides used for specific antibody production. The boxed Ala residue in ClpP2 shows the putative amino-terminal processing site for the precursor protein.

 
Stress treatments.
All stress treatments were performed with cultures grown under standard conditions and taken in early exponential growth phase at a Chl concentration of 2·5–3·5 µg ml-1. For all stresses, standard conditions were maintained except for the specified stress factor. No changes in Clp protein content relative to Chl concentration occurs during standard, non-stressed growth of cultures over the time period used for the stress treatments. For heat shock, culture flasks were directly shifted from a 37 °C water-bath to one at 50 °C for 2 h, whereas for cold shock they were moved to a 25 °C water-bath for 24 h. For high light, cultures were directly shifted to 1000 µmol photons m-2 s-1 for 6 h, while the oxidative stress was done by adding 0·5 mM H2O2 (final concentration) directly to culture flasks. For each stress, cell samples were taken at the selected time points, pelleted by centrifugation and then frozen in liquid N2 to await protein isolation.

Sample preparation and immunodetection.
Total proteins were extracted from frozen cell pellets (Clarke et al., 1993 ). Samples containing equal Chl (0·4 µg) were separated on 4–12% polyacrylamide Bis-Tris NuPAGE gels (Novex). Proteins were transferred to PVDF (0·45 µm) membranes (Millipore) or supported nitrocellulose (0·2 µm) (Bio-Rad). Each Clp protein was detected using specific polyclonal antibodies, with the antibody for Synechococcus ClpP1 described elsewhere (Clarke et al., 1998 ). All primary antibodies were detected with a horseradish peroxidase-conjugated, anti-rabbit secondary antibody made in donkey and visualized by enhanced chemiluminescence (Amersham Pharmacia).

RNA isolation.
Synechococcus cultures grown to a Chl concentration of approximately 3·5 µg ml-1 were pelleted and resuspended in 1 ml DEPC-treated H2O per 50 ml culture. Cells were then frozen in liquid N2 and ground to a fine powder, to which 1 ml Trizol Reagent was added per 50 ml culture. Total RNA from Synechococcus was extracted using the Trizol Reagent method (Invitrogen). Isolated RNA samples were treated with DNase when used for RT-PCR analysis. RNA purity and concentration was determined spectrophotometrically.

Northern blotting, RT-PCR and 5'-RACE.
Northern blot analyses were made with total RNA using the Northern Max-Gly/Blotting Kit and method (Ambion). For Northern blots, 19 µg total RNA was denatured by glyoxylation and separated on a 1% agarose gel. Separated RNA was transferred to nylon membrane (BrightStar-Plus; Ambion) and cross-linked under UV light. Membranes were prehybridized with heated ULTRAhyb (Ambion) for at least 1 h at 42 °C, followed by addition of 32P-labelled DNA probes. Hybridizations were done overnight at 42 °C. DNA probes were specific for each of the different Synechococcus clp transcripts (as determined by Southern blotting) and prepared by PCR amplification from genomic clones. The gene probes corresponded to the following DNA regions relative to the start ATG: clpPI, -100 to +296; clpPII, +265 to +705; clpPIII, +80 to +545; clpR, +121 to +599. Following hybridization, membranes were washed at 42 °C once with low-stringency solution for 10 min, twice with high-stringency solution for 15 min (Northern Blotting Kit; Ambion) and then exposed to X-ray film.


RT-PCR reactions were performed with Synechococcus total RNA using the SuperScript One-step RT-PCR System kit (Life Technologies). Varying amounts of template RNA (10–100 ng) were used to determine the amount suitable for non-saturated amplifications. The following primers were used, along with their predicted product size: clpPII/X, 5'-TTGGTACCGGTAGTGGCTGGTATTA-3' and 5'-CCTTCGAGTTGCGCCGTAGTAGATG-3' (1098 bp); clpR/P3, 5'-TTGTTCTCGTCTGACGATGTGA-3' and 5'-TCAGCAGACATGAAGTAGTCGCGA-3' (1154 bp). For each set of RT-PCR reactions, an extra control reaction was included without reverse transcriptase, but with Taq DNA polymerase to detect potential DNA contamination. RT-PCR products were separated on 1% agarose gels and viewed with an AlphaImager and associated software (Alpha Innotech). The identity of each RT-PCR product was verified by DNA sequencing.


The 5' end for clpX and clpPIII monocistronic transcripts was determined using the 5'-RACE kit and method (Invitrogen). DNase-treated total RNA (3 µg) was used for the first strand cDNA synthesis together with the gene-specific primers clpX, 5'-TGGTCTAGATATCGCTTGATGTCGTG-3', and clpPIII, 5'-TCATAATCCGCGAATGAGGCAATG-3'. After cDNA purification, 10 µl was used in the TdT-tailing reaction from which 5 µl was used in the final nested PCR amplification using additional gene-specific primers: clpX, 5'-GGAATCTGCGACAGCGTTAGCGATC-3'; clpPIII, 5'-ATTGCATCGTGTCGTAGATCGCCAT-3'. PCR products were purified and then identified by DNA sequencing.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequencing of Synechococcus clp genes
Extra clp genes were cloned from Synechococcus using the PCR strategy of degenerate primers specific for conserved regions to amplify an internal portion of putative clpX and clpPIII genes from wild-type DNA. After sequence verification, these fragments were used as specific probes to isolate full-length clones from a Synechococcus genomic DNA library. Sequencing of the clpX clone revealed no extra ORFs over 500 bp downstream of the clpX termination, but upstream was the clpPII gene only 12 bp from the start ATG of clpX. The same scenario occurred for the clpPIII clone, with the clpR gene identified only 42 bp upstream of the clpPIII start codon, whilst no ORFs were found approximately 800 bp downstream of clpPIII. Additional sequencing upstream of clpPII and clpR revealed no further ORFs, suggesting clpPII/X and clpR/PIII are organized as bicistronic operons. The predicted ORFs for clpPII, clpPIII, clpR and clpX are 723, 600, 687 and 1353 bp, respectively. According to Southern blot analysis, only one copy of each clp gene occurs in the Synechococcus genome (data not shown).

Analyses of protein sequences
The predicted amino acid sequences for the new ClpP/R isozymes were compared in Fig. 1 with that of ClpP1 (Clarke et al., 1998 ). As for ClpP1, both ClpP2 and ClpP3 possess the three conserved amino acids that compose the catalytic triad (Ser-His-Asp) characteristic of serine-type proteases. Equally characteristic was the apparent absence of this catalytic triad in the ClpR protein (Clarke, 1999 ), the significance of which remains unknown. Of the three ClpP isomers, each is 75–80% identical to its homologous protein in the related strain Synechocystis sp. strain PCC 6803, and 57–70% to the other two. The three Synechococcus isozymes have a similar low level of conservation (42–50% identity) in comparison to ClpP forms in plants and non-photosynthetic eubacteria. Of the three, ClpP2 appears to be homologous to the single ClpP in Gram-negative eubacteria like E. coli, in terms of both sequence similarity and gene arrangement with clpX. Synechococcus ClpP2 also has an extended N terminus like the E. coli protein that is post-translationally removed to produce the active mature form (Maurizi et al., 1990a ). Comparison with E. coli ClpP suggests a processing site in ClpP2 at an Ala residue in position 36 (Fig. 1).

Like the ClpP isomers, the Synechococcus ClpR is also closely related to its counterpart in Synechocystis (78% identity), but much less to the ClpP proteins in both Synechococcus and Synechocystis (41–47%). ClpR has two short extensions in the first half of the protein, which is conserved for ClpR in other cyanobacteria and the plant Arabidopsis thaliana. When overlaid with the known structure of E. coli ClpP, these stretches are situated in the ‘head’ region, the first being between ß-sheet 2 and helix B, and the other inside helix C (Wang et al., 1997 ). Of the remaining protein, Synechococcus ClpX contained all the conserved amino acid domains characteristic of its class of Clp protein, in particular the signature single ATP-binding domain. Furthermore, the presence of the two Zn-finger, DNA-binding motifs in the N-terminal domain indicates the Synechococcus ClpX is homologous to other bacterial types rather than to eukaryotic ClpX proteins, which usually lack this domain (Halperin et al., 2001 ).

Preparation of specific antibodies
To characterize in more detail the new Clp proteins in Synechococcus, antibodies were made for each of them. Fusion proteins with MBP were used as antigens for the entire Synechococcus ClpP1 and ClpP2 proteins, and the C-terminal domain of ClpX, whereas specific synthetic peptides conjugated to keyhole limpet haemocyanin were used for ClpP3 and ClpR (Fig. 1). When tested against cell protein extracts from wild-type Synechococcus, each antibody detected a single polypeptide of the expected size (Fig. 2). The estimated sizes are: ClpP1, 22 kDa; ClpP2, 23·5 kDa; ClpP3, 21 kDa; ClpR, 28 kDa; ClpX, 52 kDa.



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Fig. 2. Specificity of antibodies raised to Synechococcus ClpP1–3, ClpR and ClpX proteins. Total cell proteins were isolated from wild-type Synechococcus grown under standard culture conditions. Protein samples (0·4–0·5 µg Chl) were separated electrophoretically by SDS-PAGE and then analysed by immunoblotting. Identity of Clp protein recognized by each polyclonal antibody is indicated above the lane. Molecular mass standards are indicated on the left.

 
Clp protein levels during stress
Since most Clp proteins in bacteria are induced by various stresses, especially heat, we examined if Synechococcus ClpP2, ClpP3, ClpR and ClpX were also similarly affected. Stresses known to severely impair Synechococcus phototrophic growth were chosen: heat, cold, high irradiance and oxidation (Fig. 3). Heat shock at 50 °C for 2 h produced no significant induction for any of the tested Clp proteins, similar to that observed for ClpP1 (Clarke et al., 1998 ). A similar lack of Clp protein induction was also observed after the addition of 0·5 M H2O2, a severe oxidative stress. Indeed, the oxidative stress caused a significant decrease in ClpP2 content (approx. 25% of control level) after 30 min, followed by a partial recovery (approx. 50% of control) after 2 h (Fig. 3). A drop in ClpX protein was also observed during the oxidative stress, but only after 4 h and decreasing to approximately 60% of control levels at 6 h. During a cold shift (25 °C for 6 h) in which ClpP1 is strongly induced (Porankiewicz et al., 1998 ), no change in ClpP2 or ClpX content occurred throughout, except for an increase in ClpX after 6 h. For ClpP3 and ClpR, a slight rise in protein content occurred after 2 h at 25 °C, but which then remained unchanged for the next 4 h. Like the cold treatment, high light exposure of 1000 µmol photons m-2 s-1 for 6 h caused strong induction of ClpP1 (Clarke et al., 1998 ) and this same treatment also produced increases in ClpP2, ClpP3, ClpR and ClpX protein content, but to a lesser extent (Fig. 3).



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Fig. 3. Stress effects on Synechococcus Clp protein levels. Wild-type cells grown under standard culture conditions (Chl concentration of 2·5–3·0 µg ml-1) were shifted to either 50 °C for 2 h (heat), 25 °C for 6 h (cold) or 1000 µmol photons m-2 s-1 for 6 h (high light), or were treated with 0·5 M H2O2 for 6 h (oxidative); all other growth parameters were kept constant. Cells were collected at the indicated times during each treatment. Protein samples (0·4 µg Chl) were separated electrophoretically and analysed by immunoblotting. The figure shows results representative of two to three replicate experiments.

 
Inactivation of clp genes
Earlier observations with a clpPI mutant showed that loss of ClpP1 produced pleiotropic changes in Synechococcus. Apart from a filamentous morphology (Clarke et al., 1998 ), the {Delta}clpPI strain could not acclimate to cold or moderate UV-B irradiation (Porankiewicz et al., 1998 ). To evaluate the roles of the new clp genes, we attempted to prepare gene-specific inactivation strains using the same deletion/insertion strategy that was successful for clpPI and other genes in Synechococcus (Clarke & Campbell, 1996 ; Eriksson & Clarke, 1996 ; Clarke et al., 1998 ). Of the four genes, however, viable transformants were only obtained for clpPII, suggesting the ClpP3, ClpR and ClpX proteins are all essential for Synechococcus under standard growth conditions. Characterization of the {Delta}clpPII strain revealed no growth impairments relative to wild-type Synechococcus under standard culture conditions despite the loss of ClpP2 (see Fig. 6b). Neither did {Delta}clpPII exhibit the same phenotypic changes as {Delta}clpPI. There was no significant change in cell morphology and ClpP2 was unnecessary for acclimation to or growth at either high light or cold (data not shown).



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Fig. 6. Levels of mRNA (a) and protein (b) for clpPI-III and clpR in wild-type Synechococcus, {Delta}clpPI and {Delta}clpPII grown under standard conditions. (a) Northern blot of total RNA (19 µg) from each strain as described in Fig. 4. Size standards are indicated on the left. (b) Cell protein extracts from each strain were separated on linear 18% Tris/glycine gels (NOVEX) on the basis of equal Chl content (0·4 µg). The ClpP and ClpR proteins were detected by immunoblotting using the specific polyclonal antibodies described in Fig. 2. Molecular mass markers are shown on the left.

 
Transcriptional regulation
Due to the short intervening distances between the clpPII/X and clpR/PIII genes (12 and 42 bases, respectively), we investigated their transcriptional regulation more closely. Genomic organization suggested that both gene pairs are arranged as bicistronic operons and are thus expressed as polycistronic messages. To test this, Northern blot analysis was performed using total RNA from wild-type Synechococcus in early exponential growth under standard conditions. As in Fig. 4(a), only monocistronic mRNAs were detected for both gene pairs, despite their apparent operon organization: the 0·9–1·0 kb transcripts for clpPII, clpIII and clpR and the less distinct transcript for clpX at 1·5 kb. Degradation of the main monocistronic mRNA was also evident for clpX and clpPIII by the shorter smeared signals, suggesting both transcripts are less stable than those for their upstream partners.



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Fig. 4. Expression of Synechococcus clpPII-III, clpR and clpX genes. (a) Northern blot of total RNA extracted from wild-type in early exponential growth under standard conditions. Total RNA (19 µg) was separated electrophoretically and hybridized with 32P-labelled DNA probes specific for each transcript (see Methods for probe details). Molecular size standards are indicated on the left. (b) Detection by RT-PCR of wild-type polycistronic mRNA for clpPII/clpX and clpR/clpPIII. Primers were selected that spanned the intervening gap between the clpPII/clpX or clpR/clpPIII genes, as depicted in the diagram. Template RNA (10 and 50 ng) was used for each clp gene pair in separate RT-PCR reactions, along with a control reaction (C) with 50 ng template RNA in which reverse transcriptase was replaced with Taq polymerase to detect possible DNA contamination. Molecular size markers are shown on the right. (c) ClpX content in wild-type (wt) Synechococcus, {Delta}clpPI and {Delta}clpPII strains grown under standard culture conditions. Protein samples were isolated from each strain at matching growth stages and separated by SDS-PAGE on the basis of equal Chl content (0·4 µg). ClpX protein was detected by immunoblotting using the specific polyclonal antibody described in Fig. 2.

 
Despite the main transcripts for clpPII, clpIII, clpR and clpX being monocistronic, the possibility remained of less abundant polycistronic mRNAs due to the weak detection of longer transcripts. For both gene pairs, faint smears were observed at the expected sizes for bicistronic transcripts: 2·5 kb for clpPII/X and 1·8 kb for clpR/PIII (Fig. 4a). Like for the monocistronic clpX and clpPIII messages, the degraded signal for the polycistronic transcripts suggests they are less stable than the monocistronic ones for clpPII and clpR. To confirm the existence of polycistronic transcripts, the more sensitive approach of RT-PCR was employed (Fig. 4b). By choosing a complementary primer annealing within the downstream gene of the operon and one annealing within the upstream gene, any resulting RT-PCR product would span the intervening gap and originate only from a polycistronic message. Primers were also selected whereby any resulting products would be longer than the monocistronic transcripts detected by Northern blotting. As shown in Fig. 4(b), products of the expected length were indeed obtained for clpPII/X and clpR/PIII (approx. 1·1 kb each), thereby verifying the existence of polycistronic messages.

Although polycistronic transcripts were detected for clpPII/X and clpR/PIII by RT-PCR, their low abundance as detected by Northern blot analysis suggests that the monocistronic messages for the four genes are the most abundant under standard growth conditions. In the case of clpPII/X, we were able to examine this further by examining the relative ClpX content in the {Delta}clpPII strain. If the polycistronic mRNA was the main source of ClpX protein, then its disruption by the inactivation of clpPII should significantly decrease the amount of ClpX in the {Delta}clpPII strain. However, as seen in Fig. 4(c), no such polar decrease in ClpX content was observed in {Delta}clpPII compared to the wild-type, consistent with ClpX protein originating primarily from a monocistronic mRNA independent of clpPII gene expression. The absence of any ClpX loss in the {Delta}clpPII strain also precludes the unlikely possibility that the monocistronic transcripts for clpPII and clpX derive from rapidly processed polycistronic mRNAs, although this possibility cannot be excluded as yet for clpR/PIII.

Promoters for monocistronic clpX and clpPIII mRNA
The existence of monocistronic clpX and clpPIII messages infers that dedicated promoters exist for both genes. The short gap between clpPII/X (12 bp) and clpR/PIII (42 bp) equally implies that these independent promoters for clpX and clpPIII must be located close to or inside the 3' region of their upstream partner. To locate these promoter regions, we first identified the 5' end of the monocistronic clpX and clpPIII transcripts. By 5'-RACE analysis on total wild-type RNA, the 5' end of the clpX mRNA was located inside the clpPII gene, 9 bases upstream from the clpPII stop codon (Fig. 5a). Putative constitutive -10 and -35 promoters for clpX gene expression were also identified, consistent with the lack of stress induction for ClpX. For clpPIII, the transcript 5' end was situated 1 bp downstream of the clpR stop codon (Fig. 5b). Although no obvious promoter motifs were found for clpPIII gene expression, they must be located inside the clpR gene like those for clpX in the clpPII gene.



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Fig. 5. Mapping the 5' ends of the monocistronic transcripts for clpX (a) and clpPIII (b) genes. The 5' end of each transcript was identified by 5'-RACE from total RNA isolated from wild-type Synechococcus. The nucleotide and deduced amino acid sequences are shown for the relevant region between clpPII-clpX and clpR-clpPIII. An open arrow indicates the 5' end of each transcript, whereas putative RBS and -10/-35 promoter regions are boxed.

 
Differential regulation of clp genes
Although multiple clpP forms, including the variant clpR, are now recognized in cyanobacteria and plants, little is known about their transcriptional and translational regulation. In particular, do changes in the amount of one Clp protein affect the level of one or more of the others? To analyse this, we compared the levels of mRNA and protein for the clpP/R genes in wild-type Synechococcus and the two viable mutants {Delta}clpPI and {Delta}clpPII. Levels of mRNA for each gene were detected by Northern blotting (Fig. 6a) using the gene-specific probes described in Fig. 4(a), while Clp protein contents were examined by immunoblotting (Fig. 6b) using the antibodies in Fig. 2. It should be noted that in the absence or low level of ClpP1 or ClpP2, as in the two mutants, the corresponding antibodies cross-react to the other ClpP isozymes since they were made to the entire ClpP1 or ClpP2 proteins.

Inactivation of the clpPI gene resulted in an increase of both transcript and protein levels for ClpP3 and ClpR (Fig. 6). In the case of ClpP3, it was the level of polycistronic transcript that rose (Fig. 6a). Although no change could be detected in the amount of clpPII mRNA, there was a significant decrease in ClpP2 protein content (Fig. 6). Concomitant to this loss of ClpP2 in {Delta}clpPI was a marked rise in ClpX protein content (approx. 75%, Fig. 4c), further indicating the regulatory separation between the clpPII and clpX genes. Contrary to the {Delta}clpPI strain, mutation of the clpPII gene caused much less dramatic alterations. Despite a significant increase in the amount of clpPI mRNA, the level of the ClpP1 protein increased to a much lesser extent (Fig. 6). Larger increases in clpPIII mRNA were detected compared to a smaller increase in the amount of clpR mRNA (Fig. 6a). However, the protein levels for ClpP3 and ClpR remained relatively unchanged (Fig. 6b).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study we have characterized four new clp genes in Synechococcus: clpPII, clpPIII, clpR and clpX. Together with clpPI, the discovery of the clpPII and clpPIII genes now confirms the presence of a multigene clpP family in this cyanobacterium. The arrangement of these genes in Synechococcus (i.e. monocistronic clpPI and bicistronic clpPII-clpX and clpR-clpPIII) is conserved in the genomes of Synechocystis sp. strain PCC 6803 (Kaneko et al., 1996 ) and other strains (Synechococcus WH8102, Nostoc punctiforme and Prochlorococcus marinus). Including the monocistronic clpC, clpBI and clpBII genes, cyanobacteria appear to have a common clp gene complement (eight in total), types and organization. Furthermore, multiple ClpP isomers and the ClpR variant also seem characteristic of all oxygenic photosynthetic organisms. Higher plants in particular have a great complexity of Clp proteins, with six distinct clpP and four clpR genes in the Arabidopsis genome (Adam et al., 2001 ). In contrast, archaea and lower eukaryotes like yeast (Saccharomyces cerevisiae) and Drosophila lack clpP, whilst mammals and most eubacteria have only one clpP gene and no clpR (reviewed by Porankiewicz et al., 1999 ). However, multiple clpP genes are not a feature exclusive to cyanobacteria and plants, since up to five clpP genes exist in different Streptomyces strains (Viala et al., 2000 ).

The three Synechococcus ClpP isozymes are typical of serine-type proteases, with the characteristic catalytic triad of Ser, His and Asp residues (Maurizi et al., 1990b ). While the ClpP1 type appears specific for cyanobacteria, ClpP2 is homologous to the single ClpP in E. coli, in terms of both sequence similarity and gene arrangement with clpX. In contrast, ClpP3 has higher sequence similarity to the plastomic ClpP (ClpP1) in plants. Moreover, Synechococcus ClpP3 is essential for photosynthetic growth like the plant ClpP1 (Huang et al., 1994 ; Shikanai et al., 2001 ). Existence of a cyanobacterial homologue to ClpP1 in plants is consistent with the endosymbiotic theory for plastid evolution.

Clp proteins in most eubacteria are induced by one or more stresses and some are crucial for cellular acclimation to these less favourable conditions. ClpX and ClpP are typically induced by high temperatures (Kroh & Simon, 1990 ; Östers et al., 1999 ) and other stresses like high salt, oxidation and glucose deprivation (Völker et al., 1994 ). In B. subtilis, ClpP also plays a vital role in stationary-phase adaptive responses like competence development, motility, degradative enzyme synthesis and sporulation (Msadek et al., 1998 ). In contrast, none of the studied Synechococcus Clp proteins responded to heat shock, similar to our previous observation for ClpC and ClpP1 (Clarke & Eriksson, 1996 ; Clarke et al., 1998 ). Cold stress also failed to elicit significant increases in ClpX, ClpP2, ClpP3 and ClpR protein, unlike that observed for ClpP1 (Porankiewicz et al., 1998 ), whereas all four Clp proteins were induced by high light intensities to different degrees, but again to a lesser extent than ClpP1 (Clarke et al., 1998 ). Instead, ClpX, ClpP3 and ClpR appear to be primarily constitutive proteins that perform roles essential for cell viability, as shown by the inability to inactivate each gene. Indispensable ClpX and ClpP proteins have also been observed in another eubacterium, Caulobacter crescentus, in which loss of these proteins blocks cell division (Jenal & Fuchs, 1998 ). In contrast, the functional importance of ClpP2 in Synechococcus remains unclear. Indeed, the lack of obvious phenotypic changes resulting from clpPII inactivation implies ClpP2 is redundant under the conditions tested in this study. Interestingly, a similar lack of significant functional importance was observed for the homologous ClpP in E. coli (Maurizi et al., 1990b ).

In terms of transcriptional regulation, the single clpP gene in E. coli is one of the best studied. It is situated upstream of clpX like the Synechococcus clpPII gene, but with a longer intervening distance (125 bases; Gottesman et al., 1993 ). In contrast to clpPII, however, the E. coli clpP gene is mainly co-transcribed with clpX as a single polycistronic mRNA. In E. coli, the clpX gene can also be expressed independently of clpP from a promoter proximal to the 3' end of clpP, but the amount of ClpX produced from this monocistronic transcript is relatively low (Yoo et al., 1994 ). More transcriptional variation exists for clpX and clpP in Gram-positive bacteria. Both genes in Salmonella enterica are arranged like those in E. coli and are expressed only as a single polycistronic transcript (Yamamoto et al., 2001 ), whereas clpP and clpX in C. crescentus are separated by the cicA gene and transcribed monocistronically (Östers et al., 1999 ). In B. subtilis, clpP and clpX are located at different chromosomal loci and are also transcribed as monocistronic genes (Msadek et al., 1998 ). Yet another variation occurs in Synechococcus with both genes transcribed monocistronically despite being separated by only 12 bases. Indeed, the 5' end of the monocistronic clpX mRNA is situated inside the 3' region of the clpP gene, along with the putative promoters. To our knowledge, this is the first example of such a regulatory organization for these two clp genes, or indeed for any monocistronic genes in eubacteria.

As for clpPII/X, monocistronic transcripts predominate for the Synechococcus clpR/PIII genes, despite the bicistronic gene arrangement. However, the polycistronic message for clpR/PIII is relatively more abundant than that for clpPII/X, particularly in the {Delta}clpPI strain. Although the exact function of ClpR is unknown, results so far suggest that it interacts in some way with ClpP3. Besides the conserved gene arrangement in cyanobacteria, both clpR and clpPIII are essential for Synechococcus cell viability. Transcript and protein levels for ClpR and ClpP3 match closely in wild-type Synechococcus under various stress regimes, and increase equally in {Delta}clpPI in response to the loss of ClpP1. Moreover, ClpR homologues are localized in the stroma of plant chloroplasts, as is the ClpP isomer homologous to the cyanobacterial ClpP3. Although ClpR proteins are characterized by lacking the ClpP catalytic triad, whether it functions as a regulatory subunit or as a novel proteolytic one remains to be resolved.

The regulatory variations in clpX gene expression suggest an evolutionary selective pressure to uncouple ClpX protein synthesis from that of ClpP in certain eubacteria. In strains like E. coli, expression of clpP and clpX remains tightly co-ordinated and regulated from common promoters proximal to the upstream clpP gene, suggesting ClpX in these eubacteria functions primarily in a ClpXP protease. In cyanobacteria and many Gram-positive strains, however, the uncoupled expression of both clp genes infers that ClpX chaperone activity, independent of ClpP, may be increasingly crucial. ClpX can function as a chaperone apart from ClpP (Levchenko et al., 1995 ) and it is the importance of this function under different growth conditions that may underlie the development of independent clpX gene expression in certain eubacteria. The unchanged level of ClpX in the Synechococcus {Delta}clpPII strain is consistent with this proposal. Moreover, in {Delta}clpPI where ClpP2 content also drops significantly concomitant to ClpP1 loss, the amount of ClpX protein instead increased, again supporting a role for ClpX independent of ClpP2. Alternatively, in strains that have many ClpP forms like Synechococcus, ClpX may function as the regulatory partner of Clp proteolytic complexes with other ClpP isomers, thereby requiring clpX expression independent of the upstream clpP gene.

Genetic studies revealed interesting regulatory features for the multiple Synechococcus clpP genes at both the transcriptional and translational level (Fig. 6). While clpPI inactivation did not produce a significant change in the level of clpPII transcript, the ClpP2 protein was rendered highly unstable. Concomitant increases in both clpPIII and clpR mRNA and protein in the {Delta}clpPI strain suggested a possible compensatory response, although this would clearly be insufficient to substitute for ClpP1 function as shown by the pleiotrophic phenotype of {Delta}clpPI. Instead, the increased levels of ClpP3 and ClpR may be directly related to the degradation of ClpP2 in {Delta}clpPI. Absence of ClpP1 may somehow target ClpP2 for degradation by a Clp protease containing ClpP3 and/or ClpR. One possibility is that ClpP2 activity requires post-translational processing at the N terminus as does the homologous ClpP in E. coli, in which the first 14 aa are removed (Maurizi et al., 1990a ). For Synechococcus ClpP2, this processing event may be facilitated by ClpP1 rather than being completely autoproteolytic as for E. coli ClpP. Accumulation of the immature, non-functional ClpP2 precursor would explain its instability and low constitutive level in the {Delta}clpPI strain. It would also explain why no reciprocal response occurred in {Delta}clpPII, in which the level of ClpP1 instead rose in response to the loss of ClpP2. Despite a large increase in the amount of clpPI transcript, there was only a slight increase in ClpP1 protein content in the clpPII mutant. Given that {Delta}clpPII showed no phenotypic changes, it is likely that the small increases in ClpP1 and ClpP3 are sufficient to compensate for the absence of ClpP2. Such compensatory responses by either ClpP1 or ClpP2, however, are clearly unable to substitute for ClpP3 and ClpR function given that both are apparently essential for Synechococcus viability.

Such differential regulation and functional importance of clp genes as shown in Synechococcus has also recently been observed in the high G+C Gram-positive bacterium Streptomyces lividans (de Crécy-Lagard et al., 1999 ; Viala et al., 2000 ). Interestingly, high G+C Gram-positive bacteria are now believed to be more closely related to Gram-negative bacteria, and in particular cyanobacteria, than to other Gram-positive bacteria (Gupta, 1998 ). S. lividans has five clpP genes, of which only the first four have so far been studied. Like the clpP homologues in Synechococcus, none of the four genes in S. lividans is induced by heat stress (de Crécy-Lagard et al., 1999 ; Viala et al., 2000 ). The S. lividans clpPI and clpPII genes are located in tandem upstream of clpX, and both genes produce active ClpP proteins that require post-translational processing like the E. coli ClpP. In these regards, S. lividans ClpP1 and ClpP2 proteins are homologous to Synechococcus ClpP2. In contrast, however, inactivation of clpPI/II in S. lividans causes several phenotypic changes, including thermosensitivity at 37 °C and no aerial mycelium formation (de Crécy-Lagard et al., 1999 ), whereas the Synechococcus {Delta}clpPII strain exhibits no such obvious changes from the wild-type.

Similarities also exist between the clpPIII-IV genes in S. lividans and clpR-PIII in Synechococcus as shown in this study. In S. lividans, expression of the clpPIII-IV bicistronic operon is induced upon inactivation of the clpPI-II operon (Viala et al., 2000 ). Both transcript and protein levels for Synechococcus ClpP3 and ClpR are also elevated in the {Delta}clpPII strain, although more so in {Delta}clpP1. This up-regulation of S. lividans clpPIII-IV, however, is unable to fully compensate for the loss of ClpP1 and ClpP2, since the lack of aerial mycelium formation in the {Delta}clpPI strain persists (Viala et al., 2000 ), a finding reminiscent of the Synechococcus {Delta}clpPI strain with increased ClpP3 and ClpR proteins. This suggests that the ClpP isomers in both S. lividans and Synechococcus are not fully isofunctional. Despite such similarities, the S. lividans clpPIII-IV genes also differ from Synechococcus clpR-PIII in several regards. Whereas the clpPIII-IV genes in S. lividans are expressed polycistronically (Viala et al., 2000 ), the clpR-PIII genes in Synechococcus are expressed primarily as monocistronic transcripts. Inactivation of the two Synechococcus genes is also lethal, while a viable mutant of clpPIII-IV was obtained in S. lividans without obvious phenotypic changes (Viala et al., 2000 ). Furthermore, although the ClpP4 protein in S. lividans lacks the active site His residue in the expected position, another His residue lies just 6 aa upstream and may well function within the catalytic triad (Viala et al., 2000 ). In contrast, the Synechococcus ClpR lacks all three conserved amino acids of the catalytic triad and cannot be considered a true ClpP protein. The position of clpR upstream of clpPIII gene in Synechococcus also differs from the clpPIV position downstream of clpPIII in S. lividans. Expression of the S. lividans clpPIII-IV genes is also controlled by the activator protein PopR (Viala et al., 2000 ), whereas no homologue of this regulatory protein has yet been identified in cyanobacteria.


   ACKNOWLEDGEMENTS
 
We thank Mats-Jerry Eriksson for experimental samples and expert technical advice. This research was funded by a Swedish Natural Science Research Council project grant.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 4 January 2002; revised 8 March 2002; accepted 13 March 2002.