Introduction

The heat shock response (HSR) entails rapid and increased expression of heat shock proteins (Hsps). The major heat-shock regulons in bacteria are (i) positively regulated by σ32 (RpoH) or (ii) negatively regulated by HrcA (Narberhaus 1999; Yura and Nakahigashi 1999). The RpoH protein in association with RNA Polymerase binds to σ32 promoters upstream of heat shock genes, thereby enhancing their expression during heat shock. Low RpoH level at ambient temperature is controlled at the transcriptional and translational level and by manipulation of its stability (Yura et al. 1993). The negative regulation by a repressor protein HrcA involves a consensus 9 bp inverted repeat sequence [TTAGCACTC-N9-GAGTGCTAA] termed Controlling Inverted Repeat of Chaperone Expression (CIRCE) in close proximity to the promoter (Zuber and Schumann 1994). The HrcA binds to the CIRCE element preventing transcription of the downstream genes. During heat stress, the inability of denatured HrcA to bind to the CIRCE element derepresses transcription of the downstream genes (Roberts et al. 1996; Mogk et al. 1997). X-ray crystallographic studies have revealed that the native HrcA protein is a dimer (Liu et al. 2005). Synthesis of GroEL during heat stress regenerates HrcA dimers and shuts off HSR (Schumann et al. 1998). The presence of CIRCE element and the corresponding hrcA gene has been observed in over 40 bacteria, including the HrcA-based negative regulation at CIRCE has been demonstrated only in few bacteria (Narberhaus 1999).

Cyanobacteria neither possess a rpoH-like gene nor the cis elements similar to σ32-like promoter and do not show the typical σ32-based positive regulation of HSR. Deletion of three alternate sigma factors SigB, SigC and SigD in Synechocystis PCC6803 hampered growth at higher temperatures. Of these, SigB has been shown to be essential for hspA expression, while the downstream target genes of SigC and SigD are unknown (Tuominen et al. 2006, 2008). Recent studies on heat-shock regulation in cyanobacteria have identified three putative negative regulatory mechanisms: (i) the hspA gene of Synechococcus vulcans is negatively regulated by an upstream AT-rich imperfect inverted repeat sequence (ACAAgcAAA-N x -TTTagTTGt), with the aid of a DNA-binding protein (Kojima and Nakamoto 2002), (ii) the mutation in hik34, a gene encoding a putative histidine kinase of Synechocystis PCC6803, constitutively expresses certain heat shock genes, suggesting their possible negative regulation by Hik34 protein (Suzuki et al. 2005, Slabas et al. 2006), and (iii) the groESL operon and the cpn60 (groEL2) gene of Synechocystis PCC6803 (Chitnis and Nelson 1991; Lehel et al. 1993) harbour the CIRCE element and are negatively regulated by HrcA (Nakamoto et al. 2003) in this unicellular cyanobacteria. Additionally, two cis-elements termed, the N-box upstream of the groESL1 gene of Synechocystis PCC6803 and the K-box found in the hsp60 promoters and dnaK2 of Synechocystis PCC6803 and Synechocuccus elongatus PCC7942, appear to positively regulate hsp60 expression in response to heat and light (Kojima and Nakamoto 2007; Sato et al. 2007). Occurrence of such elements upstream of the hsp60 genes seems to differ in different cyanobacteria.

All cyanobacteria possess at least two or rarely three hsp60 genes (Lund 2009). One gene, groEL, is found with its co-chaperonin-encoding gene, groES, in a bicistronic groESL operon; the other, cpn60 (groEL2), is present as a single gene. Occurrence of hrcA gene and CIRCE in hsp60 genes/operons of several cyanobacteria suggests that HrcA-based negative regulation of hsp60 genes may occur in these microbes. However, the following recent observations have seriously questioned the paradigm of CIRCE/HrcA interaction in the regulation of cyanobacterial hsp60 genes: (i) absence of CIRCE element upstream of some groE genes e.g. the groEL2 in Synechococcus vulcanus that possess hrcA (Furuki et al. 1996), (ii) presence of CIRCE-like elements but absence of hrcA gene in certain marine cyanobacteria, e.g. Prochlorococcus marinus (http://genome.kazusa.or.jp/cyanobase), (iii) up-regulation of genes which do not possess CIRCE element in ΔhrcA mutants of Synechocystis PCC6803, e.g.. the clpB2 gene (Nakamoto et al. 2003) or pilA1, pilA2 and dnaK2 genes (Singh et al. 2006), and (iv) the presence of additional cis-elements (N-box and K-box), which positively regulate hsp60 expression in response to light and heat, irrespective of CIRCE and HrcA (Kojima and Nakamoto 2007). The CIRCE/HrcA-negative interaction thus appears to be neither exclusive nor universal among cyanobacteria.

The heat regulation of hsp60 genes has not been investigated in filamentous heterocystous nitrogen-fixing Anabaena strains. The HrcA proteins from different Anabaena strains show 93–95% similarity of amino acid residues among themselves, but only 45–50% similarity with the HrcA of Synechocystis PCC6803 and about 18–25% with that of heterotrophic bacteria, wherein it is restricted to specific regions (http://blast.ncbi.nlm.nih.gov). Binding of a protein from cell-free extract of the thermophilic cyanobacterium Thermosynechococcus elongatus to CIRCE was revealed recently (Sato et al. 2008), though the identity of the binding protein was not established. Direct physical interaction of HrcA with CIRCE has never been demonstrated in cyanobacteria and deserves attention in view of the above facts. Additional cis-elements are also present upstream of the groESL operon in Anabaena strains. The K-box element is found upstream of the groESL operon, but not upstream of the cpn60 (groEL2) gene, in Anabaena sp. strain PCC7120, hereafter referred to as Anabaena 7120, and Anabaena sp. strain L-31, hereafter referred to as Anabaena L-31. The N-box which contributes to both light- and heat-induction in Synechocystis is not detected upstream of the groESL operon of both Anabaena 7120 and Anabaena L-31 but is present upstream of the cpn60 (groEL2) gene of Anabaena 7120 (Kojima and Nakamoto 2007). Anabaena L-31 harbours several unique additional direct/inverted repeats upstream of the groESL operon. The role(s) of these various elements in hsp60 regulation remains to be explored in Anabaena strains. In this study, we demonstrate negative regulation of hsp60 genes in Anabaena L-31, by direct binding of the HrcA dimer at the CIRCE element. A new negative heat-regulatory element designated as H-box has been identified along with light-specific positive regulatory K-box upstream of the groESL operon in Anabaena L-31.

Materials and methods

Organism and growth conditions

Escherichia coli cells were grown in Luria–Bertani (LB) medium at 37°C. Axenic cultures of Anabaena L-31 and Anabaena 7120 were grown in BG-11 liquid medium, pH 7.0 (Castenholz 1988) with (BG-11, N+) or without (BG-11, N−) combined nitrogen (17 mM NaNO3) under continuous illumination (30 Μe m−2 s−1) and aeration (3 L min−1) at 27°C. Heat-shock treatment involved exposure to 42°C. The different antibiotics used were 100 μg carbenicillin mL−1 (Cb100), 34 μg chloramphenicol mL−1 (Cm34) and 50 μg kanamycin mL−1 (Kan50) for E. coli and 25 μg neomycin mL−1 (Neo25) in BG-11 agar plates or 10 μg neomycin mL−1 (Neo10) in BG-11 liquid medium for recombinant Anabaena 7120 strains.

PCR amplification and DNA electrophoresis

PCR amplification of DNA fragments involved genomic DNA of Anabaena L-31 (100 ng) and 1 μmole each of specified forward and reverse primers (Table 1), 100 μM dNTPs, 1U Taq DNA Polymerase in the Taq Buffer provided (Roche Diagnostics, Germany). DNA fragments were electrophoretically resolved on 0.8–1.2% agarose gels in TBE at 80 V for 2 h.

Table 1 Primers and PCR products used in this study
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Cloning, overexpression and purification of Anabaena L-31 HrcA protein from E. coli

Amplification of Anabaena L-31 chromosomal DNA with hrcAFwd and hrcARev primers (Table 1) yielded a 1.1-kb fragment. The ends of the PCR product were filled using dNTPs and Klenow enzyme and ligated to pUC19 vector at EcoRV restriction site. Restriction analysis ensured that the hrcA gene was cloned in the same direction as the lacZ promoter to achieve HrcA expression in E. coli DH5α (data not shown). The resulting construct was designated as pUChrcA (Table 2). The gene sequence of hrcA was submitted to Gen Bank (Accession No. AY897588).

Table 2 Plasmids used in this study
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The 1.1-kb NdeI-XhoI fragment from pUChrcA was ligated to pET29b at identical sites to obtain pEThrcA (Table 2) and transformed into E. coli BL-21(pLysS) cells. The cells were induced with 1 mM IPTG at 37°C for 1 h, then resuspended in lysis buffer containing 8 M urea and lysed using One Shot model Cell Disruptor (Constant Systems, UK). The HrcA protein was purified from the cell lysate using Ni2+-nitrilotriacetic acid (NiNTA) column as per the manufacturer’s protocol (Qiagen, Germany). HrcA protein was obtained in native form by gradual removal of urea by dialysis against decreasing concentration of urea at 4°C. The purified HrcA protein was used to raise specific anti-Anabaena HrcA antibodies in rabbit.

Cloning of the putative regulatory regions of the hsp60 genes

The upstream regulatory region of the groESL operon (Fig. 1a) was amplified from the cloned groESL operon of Anabaena L-31 (Rajaram et al. 2001) using P gro Rev1 (Table 1) as a reverse primer in combination with P gro CFwd, P gro DR12Fwd, P gro IR11Fwd, P gro DR10Fwd and P gro Fwd1 (Table 1) as the five forward primers. Positions of these primers are indicated in Fig. 1a, I. The different PCR products were designated P gro C, P gro DR12, P gro IR11, P gro DR10 and P gro , respectively (Fig. 1a, II; Table 1). The groESL promoter element lacking the CIRCE element was generated by amplification with P gro Fwd2 and P gro Rev2 primers (Table 1; Fig. 1a), which resulted in a 92 bp PCR product, designated as P gro ΔCIRCE. The upstream regulatory region of the cpn60 (groEL2) gene was obtained by amplification of the cloned cpn60 gene of Anabaena L-31 (Rajaram and Apte 2008) using P cpn Fwd and P cpn Rev primers (Table 1), and the resulting 125-bp PCR product was designated P cpn (Fig. 1b). All the PCR products were cloned in pBluescript SKII vector and sequenced to confirm their identity with the original sequence.

Fig. 1
figure 1

Upstream DNA sequence of (a) groESL operon and (b) cpn60 gene of Anabaena L-31. The CIRCE element [TTAGCACTC-N x -GAGTGCTAA] is marked with striped arrows. The −35 and −10 regions of the putative σ70-dependent promoter are underlined. The +1 indicates the putative transcription start sites. The first nucleotide of different forward and reverse primers is indicated by a plus sign. a (I) Nucleotide sequence of the upstream putative regulatory sequence of the groESL operon. The 12-bp direct repeats (DR12) [CAGTTATCAGTTATCAGTT] are marked with arrows pointing in the same direction (→). The 11-bp inverted repeat (IR11) [CAGTTATCAGT-N x -ACTGATAACTG] is marked by arrows facing each other (→ and ←). The 10-bp direct repeat (DR10) [TCCACTGTTC-N x -TCCACTGTTC] is marked by block arrows. The boldface nucleotides represent the 5-bp direct repeats (CAGTT and ACTGT). The K-box sequence [TGTTCGGAAACCCGTA] has been boxed. (II) Different PCR products with their corresponding sizes and relative positions upstream of the groESL operon. The left (L) and right (R) arms of IR11 constituting the proposed H-box are marked in I and II. b Nucleotide sequence of the upstream region of cpn60 gene

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The different promoter fragments were ligated to the vector pAM1956, at SacI-KpnI restriction sites. The vector pAM1956 has a promoterless gfpmut2 gene coding for Green Fluorescent Protein (GFP) (Yoon and Golden 1998). The corresponding promoter constructs were designated P gro C:gfp, P gro DR12:gfp, P gro IR11:gfp, P gro DR10:gfp, P gro :gfp and P cpn :gfp (Table 2) and were maintained in E. coli strain DH5α, hereafter referred to as ‘Ec’. For some experiments, E. coli clones carrying different promoter:reporter constructs were co-transformed with plasmids pUChrcA or pUC19 and selected on LBCb100Kan50 plates. Such co-transformants were confirmed by cell-based PCR using appropriate corresponding primers (data not shown).

Mutagenesis of hrcA gene in Anabaena 7120

The pUChrcA plasmid DNA was linearised with XbaI, ends filled and ligated with the 1.1-kb HincII-SmaI fragment of pBSnptII comprising the nptII gene (Fig. 2a). The resulting construct pUChrcA − (Table 2) was electroporated into Anabaena 7120 as described earlier (Chaurasia et al. 2008), and the electrotransformants were selected on BG-11, N+, Neo25 plates for several generations to allow complete segregation. The hrcA mutant of Anabaena 7120 thus obtained was designated AnhrcA − (Table 3).

Fig. 2
figure 2

Hsp60 expression in the hrcA mutant of Anabaena 7120. a Schematic representation of the construction of the plasmid pUChrcA −. The different restriction sites indicated are B: BamHI, S: SmaI and X: XbaI b PCR amplification of Anabaena 7120 and AnhrcA − cells with hrcAFwd and hrcARev primers. The PCR products obtained are indicated by arrows. c Immunodetection of HrcA. Protein extracts from three-day-old nitrogen-fixing cultures of Anabaena 7120 and AnhrcA − were resolved by 10% SDS–PAGE and electroblotted. HrcA protein was immunodetected with an anti-AnHrcA antibody. d and e Immunodetection of Hsp60 proteins. Three-day-old nitrogen-fixing Anabaena 7120 (An7120) and AnhrcA − cells were incubated either at 27°C or at 42°C for 16 h. Immunodetection was carried out with (d) anti-AnGroEL or (e) anti-AnCpn60 antibodies. Values below the lanes depict protein levels relative to wild-type Anabaena 7120 under control (27°C) conditions (c) and during heat stress (d, e)

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Table 3 Strains used in this study
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Transfer and expression of the promoter constructs in Anabaena 7120

The different promoter constructs and the empty vector, pAM1956, were conjugated into Anabaena 7120 using a conjugal E. coli donor [HB101(pRL623 + pRL443)] (Table 3) as described earlier (Elhai and Wolk 1988) and exconjugants selected on BG-11, N+, Neo25 plates. The different transgenic Anabaena 7120 strains obtained were designated as AnP gro C:gfp, AnP gro DR12:gfp, AnP gro IR11:gfp, AnP gro DR10:gfp, AnP gro :gfp, AnP cpn :gfp and AnpAM (Table 3).

Fluorimetric analysis of GFP expression

Three-day-old Anabaena 7120 cultures were directly subjected to heat stress (42°C). The stressed and control cells were centrifuged, the pellet washed three times and resuspended in BG-11, N− medium. Fluorimetric analysis was carried out using a Perkin Elmer Fluorimeter (Model LS50B) with excitation at 480 nm and emission measured at 510 nm. Turbidity (OD) of the cells was estimated at 750 nm. Promoter activity was measured as ‘GFP fluorescence (arbitrary units) (OD)−1.

Protein electrophoresis, western blotting and immunodetection

Proteins were resolved by 10% SDS–PAGE and electroblotted on positively charged nylon membranes (Roche Diagnostics, Germany), as described previously (Alahari and Apte 1998). Immunodetection was carried out with anti-AnGroEL, anti-AnCpn60 (Rajaram and Apte 2008) or anti-AnHrcA antibodies raised against the corresponding purified proteins of Anabaena L-31, hence the prefix ‘An’.

Electrophoreticmobility shift assays

The 84-bp P gro and 92-bp P gro ΔCIRCE DNA fragments used for electrophoreticmobility shift assays (EMSA) (Fig. 1a) were 3′-end labelled using DIG-11-ddUTP and terminal transferase (Roche Diagnostics, Germany). Labelled DNA was incubated with purified HrcA protein from Anabaena L-31 for 20 min in binding buffer [25 mM HEPES, pH 7.9; 5 mM MgCl2, 25 mM NaCl, 5 μg BSA and 0.5 μg poly (dI-dC)] (Koksharova and Wolk 2002). For supershift assay, anti-AnHrcA antibody was added to the mixture after 20 min of binding at 1:100 dilution and incubated for another 30 min. The DNA/HrcA or DNA/HrcA/anti-AnHrcA antibody mixtures were electrophoretically separated on 12% polyacrylamide gel in Tris–Acetate buffer, pH 7.4 at 4°C, blotted onto nylon membrane and detected by chemiluminescence as per the manufacturer’s protocol (Roche diagnostics, Germany).

Statistical analyses

All experiments were repeated at least three times, and typical results from one experiment are shown. Variations between experiments were less than 10%. Each treatment comprised of three replicates, and mean ± SE values are shown.

Results and discussion

De-repression of Hsp60 proteins in Anabaena 7120 hrcA mutant

The conserved CIRCE element upstream of both the groESL operon (Fig. 1a) and cpn60 (groEL2) gene (Fig. 1b) and the presence of hrcA gene are common occurrence in the genomes of different Anabaena strains including Anabaena L-31. In order to assess the involvement of hrcA in regulating hsp60 expression, a hrcA mutant was constructed in Anabaena 7120 as shown in Fig. 2a. The mutant, AnhrcA −, yielded a single DNA fragment of 2.2 kb upon PCR amplification with hrcAFwd and hrcARev primers and lacked the 1.1-kb fragment corresponding to the native hrcA gene (Fig. 2b), indicating gene replacement. Immunodetection studies revealed that while the 40-kDa HrcA protein could be detected in wild-type Anabaena 7120, the corresponding protein was absent in the mutant, AnhrcA − strain (Fig. 2c). The mutant cells expressed the 59-kDa GroEL (Fig. 2d) and the 61-kDa Cpn60 (GroEL2) (Fig. 2e) proteins at elevated levels under control (27°C) growth conditions, unlike Anabaena 7120 (Fig. 2d, e), suggesting that negative regulation of hsp60 expression by HrcA occurs in Anabaena strains. An unexpected additional increase in the levels of the GroEL protein, but not of Cpn60 (GroEL2) protein, was observed upon heat stress in the AnhrcA − cells (Fig. 2d, e). This suggested that, even the HrcA protein, may not be the sole regulator of the groESL operon and a second level of thermal regulation may operate. This was ascertained by: (a) assessment of negative regulation by HrcA at CIRCE and (b) deletion analysis of DNA elements from groESL promoter region.

Constitutive expression of Anabaena L-31 hsp60 promoters in E. coli and their repression by Anabaena L-31 HrcA

To ascertain if HrcA of Anabaena L-31 can per se regulate Anabaena L-31 hsp60 promoters, GFP expression driven from the Anabaena L-31 groESL (P gro ) and cpn60 (P cpn ) promoters (Fig. 1b) was monitored in E. coli. E. coli does not have a hrcA-like gene, while the Anabaena L-31 hsp60 genes have typical E. coli σ70-like promoters. Thus, it provides a clean background, wherein the possible interaction between the cis-elements and trans-acting proteins of Anabaena L-31 can be unambiguously assessed. E. coli cells harbouring P cpn :gfp and P gro :gfp plasmids strongly expressed GFP both at 30°C (Fig. 3a, I) and during heat shock (Fig. 3a, II). Co-transformation of such cells with pUChrcA resulted in (a) strong expression of Anabaena L-31 HrcA in E. coli as confirmed by immunodetection with anti-AnHrcA antisera (Fig. 3b) and (b) loss of GFP expression from both P gro :gfp and P cpn :gfp plasmids at ambient temperature (Fig. 3a, V). Co-transformation with empty pUC19 vector did not affect GFP expression (Fig. 3a, III and IV), indicating that the observed result is not due to low copy number of the pUC clones at 30°C. The HrcA repression of P gro and P cpn promoters was alleviated upon shift to higher growth temperatures (42°C) (Fig. 3a, VI).

Fig. 3
figure 3

Analysis of Anabaena L-31 hsp60 promoters in E. coli. a Fluoromicrographs (×1,000 magnification) of E. coli DH5α (Ec) cells transformed with pAM1956, P gro :gfp or P cpn :gfp, and of cells co-transformed with pUC19 or pUChrcA, at 30°C and 42°C. b Proteins extracted from Ec (pUC19) (lane 1) and Ec (pUChrcA) (lane 2) were separated by 10% linear SDS–PAGE, followed by western blotting and immunodetection using anti-AnHrcA antibody. The 40-kDa HrcA protein is indicated by an arrow. c and d Fluorimetric analysis of GFP expression in E. coli DH5α (Ec) cultures transformed with Anabaena L-31 (c) groESL and (d) cpn60 promoters and exposed to heat stress

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The above results were also confirmed quantitatively by fluorimetric analysis (Fig. 3c, d), which clearly showed: (i) constitutive expression of GFP from both hsp60 promoters in E. coli at 30°C (control), (ii) loss of hsp60 promoter driven GFP expression at 30°C in E. coli co-expressing Anabaena L-31 HrcA, and (iii) restoration of hsp60 promoter-driven GFP expression in E. coli cells expressing Anabaena L-31 HrcA, at 42°C (Fig. 3c, d). It may be argued that such results obtained in an unnatural host (E. coli) may not truly reflect the physiological regulation native to Anabaena strains. But the data shown in Fig. 3 strongly suggested that Anabaena HrcA can directly represses Anabaena L-31 hsp60 genes at ambient conditions, and its inactivation during heat shock can result in expression from both the hsp60 promoters. Interestingly, in the absence of Anabaena L-31 HrcA, even recombinant E. coli showed over threefold induction of Anabaena L-31 P groESL promoter by heat stress (Fig. 3c), confirming the data shown in Fig. 2d and suggesting the possibility of a second level of regulation independent of HrcA.

Binding of HrcA dimer to CIRCE element

The physical binding of HrcA to the Anabaena L-31 CIRCE element was also ascertained. A recombinant His6-tagged Anabaena L-31 HrcA protein was purified from E. coli, its purity and identity ascertained by MALDI-ToF analysis–based peptide mass fingerprinting (UniProtKB/TrEMBL Acc. No. Q5EF74) and used in electrophoretic mobility shift assays (EMSA) (Fig. 4a, b). An 84-bp DNA fragment (P gro , Table 1; Fig. 1a) from Anabaena L-31 containing the CIRCE element showed a concentration-dependent mobility shift with purified HrcA protein (Fig. 4a), but not with heat-denatured (65°C, 15 min) HrcA protein. Anabaena L-31 HrcA did not bind to a 92-bp (P gro ΔCIRCE, Table 1) DNA fragment (Fig. 4a), which lacked the CIRCE element. A supershift with several slow migrating products was observed when anti-AnHrcA antibody was used along with HrcA protein in such assays (Fig. 4b), confirming that the observed mobility shift was indeed due to HrcA and not because of any co-eluting E. coli protein. The native HrcA protein displayed molecular mass of 81.2 kDa on Sephacryl S-400 gel filtration column suggesting that it was a dimer (Fig. 4c), unlike the heat-denatured HrcA (Fig. 4a), which corresponded to the monomeric molecular mass of 40.3 kDa (Fig. 4c), clearly indicating that HrcA dimer, but not its monomer, binds to the CICRE element.

Fig. 4
figure 4

Binding of Anabaena L-31 HrcA to the CIRCE element. a and b DIG-labelled P gro or P gro ΔCIRCE PCR products (8 ng per lane) were incubated with specified amounts of purified native or heat-denatured HrcA protein (a) or native HrcA protein with or without anti-AnHrcA antibody (b). The observed shifts (arrowheads) and supershifts (arrows) are shown. c Molecular sieve Sephacryl S-400 chromatography of native or heat-denatured HrcA with standard proteins. Elution volumes of eluted native HrcA (circle) and heat-denatured HrcA (triangle) are indicated by arrows

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Additional cis-elements regulate groESL expression of Anabaena L-31

In silico analysis of the nucleotide sequence upstream of Anabaena L-31 groESL operon revealed the presence of several direct and inverted repeats (Fig. 1a, I) including the heat/light-regulated K-box reported in Synehcocystis PCC6803 (Kojima and Nakamoto 2007). Some of these were unique to Anabaena L-31 and were not found in Anabaena 7120. Their possible role in regulation of the groESL operon was analysed by sequentially deleting one or more of the elements from the 5′ end (Fig. 1a, II) and evaluating their effect on GFP expression in Anabaena 7120 (Fig. 5).

Fig. 5
figure 5

Functional analysis of groESL promoter by GFP expression from different regulatory elements. Recombinant Anabaena 7120 (An) strains grown at 27°C carrying different promoter constructs were subjected to different duration of heat stress at 42°C, either in light (a) or in dark (b). GFP expression in various strains was estimated by fluorimetric analysis and expressed as arbitrary units per unit turbidity

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Deletion of the first 7 bases of the left arm of DR12 as in P gro DR12 had no significant effect on GFP expression in Anabaena 7120 at 27°C or 42°C (data not shown). Deletion of the left arm of IR11, as in P gro IR11 (Fig. 1a), resulted in twofold increase in GFP expression under control conditions and a further five- to sixfold in heat due to HrcA denaturation (Fig. 5a, b). No further increase in GFP expression, either under control or during heat stress, was observed in light upon deletion of the left arm of DR10, as in P gro DR10 (data not shown), or partial deletion of the K-box as in P gro (Fig. 5a). These data suggested that the IR11 element acts as a negative regulatory element at ambient conditions, while the observed heat induction from all the constructs (Fig. 5a, b) could be attributed to alleviation of negative regulation by HrcA at CIRCE.

The K-box (described as MARS “multi-stress associated regulatory sequence” in Synechococcus elongatus PCC7942) has been implicated in heat/light regulation of groESL operon in Synechocystis 6803 (Kojima and Nakamoto 2007) and of dnaK gene in Synechococcus elongatus PCC7942 (Sato et al. 2007). K-box is found in groESL promoters of all Anabaena strains sequenced so far. The groESL promoters having the K-box (as in P gro C and P gro IR11) were differentially regulated in light and dark in Anabaena 7120, the expression being always higher in the presence of light (Fig. 5a, b). However, their expression became insensitive to light upon partial deletion of the K-box (as in P gro ) (Fig. 5a, b). This clearly showed that in Anabaena, the K-box acts solely as a positive light-responsive element. Repeated attempts to detect an H-box-binding protein from cell-free extracts or from a heparin-Sepharose purified preparation of DNA-binding proteins of Anabaena L-31 were not successful (data not shown). Based on the fact that in the absence of HrcA, the Anabaena L-31 groESL promoter showed heat-induction both in AnhrcA − (Fig. 2d) and in E. coli (Fig. 3c), it is tempting to speculate that H-box may possibly function as a cis-element per se without the aid of a regulatory protein.

The present work has revealed complex regulation of the groESL operon in Anabaena involving: (a) negative regulation by (i) binding of dimeric HrcA repressor to CIRCE and by (ii) the novel H-box element which forms an inverted repeat, and (b) positive regulation by a light-responsive K-box element. In Synechocystis PCC6803, which lacks H-box, the K-box is implicated in both light and heat regulation of groESL operon (Kojima and Nakamoto 2007). In contrast in Anabaena, the responsibility appears to be shared between exclusively light-regulated K-box and heat-regulated H-box and CIRCE/HrcA system.