Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120

The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35°C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 μM·min⁻¹, respectively. The apparent K_m and k_cat/K_m values for Reactive Blue 5 were 3.6 μM and 1.2 × 10⁷ M⁻¹ s⁻¹, respectively, while the apparent K_m and k_cat/K_m values for H₂O₂ were 5.8 μM and 6.6 × 10⁶ M⁻¹ s⁻¹, respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to ∼50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.In textile, food, and dyestuff industries, reactive dyes such as azo and anthraquinone (AQ) and pthalocyanine-based dyes constitute one of the extensively used classes of synthetic dyes. However, it has been estimated that approximately 50% of the applied reactive dye is wasted because of hydrolysis during the dyeing process (26, 35). This results in a great effluent problem for the industries because of the recalcitrant nature of these dyes. With increased public concern and ecological awareness, in addition to stricter legislative control of wastewater discharge in recent years, there is an increased interest in various methods of dye decolorization. Dye decolorization using physicochemical processes such as coagulation, adsorption, and oxidation with ozone has proved to be effective. However, these processes are usually expensive, generate large volumes of sludge, and require the addition of environmentally hazardous chemical additives (26). There are several reports of microorganisms capable of decolorizing synthetic dyes. This has been attributed to their growth and production of enzymes such as laccase (1, 9, 40), azoreductases (3), and peroxidases, for example, lignin peroxidase (12, 25, 36), manganese peroxidase (10, 38), and versatile peroxidase (16). However, most of the synthetic dyes are xenobiotic compounds that are poorly degraded using the typical biological aerobic treatments. Furthermore, microbial anaerobic reductions of synthetic dyes are known to generate compounds such as aromatic amines that are generally more toxic than the dyes themselves (3). Therefore, for environmental safety, the use of enzymes instead of enzyme-producing microorganisms presents several advantages such as increased enzyme production, enhanced stability and/or activity, and lower costs by using recombinant DNA technology.Peroxidases are heme-containing enzymes that use hydrogen peroxide (H₂O₂) as the electron acceptor to catalyze numerous oxidative reactions. They are found widely in nature, both in prokaryotes and eukaryotes, and are largely grouped into plant and animal superfamilies. They are one of the most studied enzymes because of their inherent spectroscopic properties and potential use in both diagnostic and bioindustrial applications. In particular, their ability to degrade a wide range of substrates has recently stimulated interest in their potential application in environmental bioremediation of recalcitrant and xenobiotic wastes (10, 25, 26).Recently, a novel family of heme peroxidases characterized by broad dye decolorization activity has been identified in various fungal species such as Thanatephorus cucumeris Dec1 (18), Termitomyces albuminosus (15), Polyporaceae sp. (15), Pleurotus ostreatus (13), and Marasmius scorodonius (27). Because of their broad substrate specificity, low pH optima, lack of a conserved active site distal histidine, and structural divergence from classical plant and animal peroxidases (32), these proteins have been proposed to belong to the novel DyP peroxidase family. Over 400 proteins of prokaryotic and eukaryotic origins have been grouped in the DyP peroxidase family, Pfam 04261 (http://pfam.sanger.ac.uk/), and it is apparent from genome databases that many species possess DyP. The ability of these proteins to effectively degrade hydroxyl-free AQ and azo dyes as well as the specificity for typical peroxidase substrates illustrates their potential use in the bioremediation of wastewater contaminated with synthetic dyes. However, with the exception of a DyP from the plant pathogenic fungus T. cucumeris Dec1 (an anamorph of Rhizoctonia solani, a very common fungal plant pathogen), which has been characterized extensively (18, 28, 30-32, 34), little information is available on other members of the DyP family. In particular, studies on bacterial DyPs have been limited to only the automatically translated sequence or structural data (41, 42). Within the context of further understanding the structure-function and potential applicability of these novel types of enzymes in general, we have taken an interest in DyP-type enzymes, particularly, the less known bacterial groups.Cyanobacteria (blue-green algae) represent the most primitive, oxygenic, plant-type photosynthetic organisms and are thought to be involved in greater than 20 to 30% of the global photosynthetic primary production of biomass, accompanied by the cycling of oxygen. Anabaena sp. strain PCC 7120 is a filamentous, heterocyst-forming cyanobacterium capable of nitrogen fixation and has long been used as a model organism to study the prokaryotic genetics and physiology of cellular differentiation, pattern formation, and nitrogen fixation (14). This strain''s genome sequence is complete and annotated (17). From bioinformatics analysis of the Anabaena sp. strain PCC 7120 genome, we identified an open reading frame (ORF), alr1585, encoding a putative heme-dependent peroxidase exhibiting homology to T. cucumeris Dec1, DyP. Here, we report on the characterization of this novel bacterial DyP, designated AnaPX (for Anabaena peroxidase), from the cyanobacterium Anabaena sp. strain PCC 7120, with broad specificity for both aromatic compounds and synthetic dyes such as AQ dyes.     

Expression of an Adenylate Cyclase Gene of Anabaena cylindrica in the Cyanobacterium Anabaena sp. Strain PCC 7120

A transformant of Anabaena 7120 was made by introducing a plasmidthat includes an adenylate cyclase gene of Anabaena cylindrica.Expression of this gene was driven by the bacterial tac promoter.Transformants accumulate cAMP 170 fold higher than the concentrationin the parental strain. The transformation resulted in the fragmentationof filaments in both nitrogen-replete and nitrogen-free media.It was suggested that this fragmentation caused the inhibitionof growth under nitrogen-fixing conditions. (Received December 26, 1997; Accepted April 30, 1998)     

Catabolic Function of Compartmentalized Alanine Dehydrogenase in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

In the diazotrophic filaments of heterocyst-forming cyanobacteria, an exchange of metabolites takes place between vegetative cells and heterocysts that results in a net transfer of reduced carbon to the heterocysts and of fixed nitrogen to the vegetative cells. Open reading frame alr2355 of the genome of Anabaena sp. strain PCC 7120 is the ald gene encoding alanine dehydrogenase. A strain carrying a green fluorescent protein (GFP) fusion to the N terminus of Ald (Ald-N-GFP) showed that the ald gene is expressed in differentiating and mature heterocysts. Inactivation of ald resulted in a lack of alanine dehydrogenase activity, a substantially decreased nitrogenase activity, and a 50% reduction in the rate of diazotrophic growth. Whereas production of alanine was not affected in the ald mutant, in vivo labeling with [¹⁴C]alanine (in whole filaments and isolated heterocysts) or [¹⁴C]pyruvate (in whole filaments) showed that alanine catabolism was hampered. Thus, alanine catabolism in the heterocysts is needed for normal diazotrophic growth. Our results extend the significance of a previous work that suggested that alanine is transported from vegetative cells into heterocysts in the diazotrophic Anabaena filament.Cyanobacteria such as those of the genera Anabaena and Nostoc grow as filaments of cells (trichomes) that, when incubated in the absence of a source of combined nitrogen, present two cell types: vegetative cells that perform oxygenic photosynthesis and heterocysts that perform N₂ fixation. Heterocysts carry the oxygen-labile enzyme nitrogenase, and, thus, compartmentalization is the way these organisms separate the incompatible activities of N₂ fixation and O₂-evolving photosynthesis (9). In Anabaena and Nostoc, heterocysts are spaced along the filament so that approximately 1 in 10 to 15 cells is a heterocyst. Heterocysts differentiate from vegetative cells in a process that involves execution of a specific program of gene expression (12, 15, 39). In the N₂-fixing filament, the heterocysts provide the vegetative cells with fixed nitrogen, and the vegetative cells provide the heterocysts with photosynthate (38). Two important aspects of the diazotrophic physiology of heterocyst-forming cyanobacteria that are still under investigation include the actual metabolites that are transferred intercellularly and the mechanism(s) of transfer (10).Because the ammonium produced by nitrogenase is incorporated into glutamate to produce glutamine in the heterocyst and because the heterocyst lacks the main glutamate-synthesizing enzyme, glutamine(amide):2-oxoglutarate amino transferase (GOGAT; also known as glutamate synthase), a physiological exchange of glutamine and glutamate resulting in a net transfer of nitrogen from the heterocysts to the vegetative cells has been suggested (21, 36, 37). On the other hand, a sugar is supposed to be transferred from vegetative cells to heterocysts. Because high invertase activity levels are found in the heterocysts (34) and because overexpression of sucrose-degrading sucrose synthase in Anabaena sp. impairs diazotrophic growth (4), it is possible that sucrose is a transferred carbon source. Indeed, determination of ¹⁴C-labeled metabolites in heterocysts isolated from filaments incubated for short periods of time with [¹⁴C]bicarbonate identified sugars and glutamate as possible compounds transferred from vegetative cells to heterocysts (13). However, this study also identified alanine as a metabolite possibly transported from vegetative cells to heterocysts.The cyanobacteria bear a Gram-negative type of cell envelope, carrying an outer membrane (OM) outside the cytoplasmic membrane (CM) and the peptidoglycan layer (9, 15). In filamentous cyanobacteria, whereas the CM and peptidoglycan layer surround each cell, the OM is continuous along the filament, defining a continuous periplasmic space (10, 19). In Anabaena sp. strain PCC 7120, the OM is a permeability barrier for metabolites such as glutamate and sucrose (27). Two possible pathways for intercellular molecular exchange in heterocyst-forming cyanobacteria have been discussed: the periplasm (10, 19) and cell-to-cell-joining proteinaceous structures (11, 22, 25). Whereas the latter would mediate direct transfer of metabolites between the cytoplasm of adjacent cells, the former would require specific CM permeases to mediate metabolite transfer between the periplasm and the cytoplasm of each cell type (10).In Anabaena sp. strain PCC 7120, two ABC-type amino acid transporters have been identified that are specifically required for diazotrophic growth (29, 30). The N-I transporter (NatABCDE), which shows preference for neutral hydrophobic amino acids, is present exclusively in vegetative cells (30). The N-II transporter (NatFGH-BgtA), which shows preference for acidic and neutral polar amino acids, is present in both vegetative cells and heterocysts (29). A general phenotype of mutants of neutral amino acid transporters in cyanobacteria is release into the culture medium of some hydrophobic amino acids, especially alanine (16, 23, 24), which is accumulated at higher levels in the extracellular medium of cultures incubated in the absence than in the presence of a source of combined nitrogen (30).Thus, alanine is a conspicuous metabolite in the diazotrophic physiology of heterocyst-forming cyanobacteria, and the possibility that it moves in either direction between heterocysts and vegetative cells has been discussed (13, 29, 30). Alanine dehydrogenase, which catalyzes the reversible reductive amination of pyruvate, has been detected in several cyanobacteria (8). In Anabaena spp., alanine dehydrogenase has been found at higher levels or exclusively in diazotrophic cultures (26), and in the diazotrophic filaments of Anabaena cylindrica it is present at higher levels in heterocysts than in vegetative cells (33). Open reading frame (ORF) alr2355 of the Anabaena sp. strain PCC 7120 genome is predicted to encode an alanine dehydrogenase (14). In this work we addressed the expression and inactivation of alr2355, identifying it as the Anabaena ald gene and defining an important catabolic role for alanine dehydrogenase in diazotrophy.     

Genome-Wide and Heterocyst-Specific Circadian Gene Expression in the Filamentous Cyanobacterium Anabaena sp. Strain PCC 7120

The filamentous, heterocystous cyanobacterium Anabaena sp. strain PCC 7120 is one of the simplest multicellular organisms that show both morphological pattern formation with cell differentiation (heterocyst formation) and circadian rhythms. Therefore, it potentially provides an excellent model in which to analyze the relationship between circadian functions and multicellularity. However, detailed cyanobacterial circadian regulation has been intensively analyzed only in the unicellular species Synechococcus elongatus. In contrast to the highest-amplitude cycle in Synechococcus, we found that none of the kai genes in Anabaena showed high-amplitude expression rhythms. Nevertheless, ∼80 clock-controlled genes were identified. We constructed luciferase reporter strains to monitor the expression of some high-amplitude genes. The bioluminescence rhythms satisfied the three criteria for circadian oscillations and were nullified by genetic disruption of the kai gene cluster. In heterocysts, in which photosystem II is turned off, the metabolic and redox states are different from those in vegetative cells, although these conditions are thought to be important for circadian entrainment and timekeeping processes. Here, we demonstrate that circadian regulation is active in heterocysts, as shown by the finding that heterocyst-specific genes, such as all1427 and hesAB, are expressed in a robust circadian fashion exclusively without combined nitrogen.     

ppGpp Metabolism Is Involved in Heterocyst Development in the Cyanobacterium Anabaena sp. Strain PCC 7120

When deprived of a combined-nitrogen source in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) can form heterocysts capable of nitrogen fixation. The process of heterocyst differentiation takes about 20 to 24 h, during which extensive metabolic and morphological changes take place. Guanosine tetraphosphate (ppGpp) is the signal of the stringent response that ensures cell survival by adjusting major cellular activities in response to nutrient starvation in bacteria, and ppGpp accumulates at the early stage of heterocyst differentiation (J. Akinyanju, R. J. Smith, FEBS Lett. 107:173–176, 1979; J Akinyanju, R. J. Smith, New Phytol. 105:117–122, 1987). Here we show that all1549 (here designated rel_ana) in Anabaena, homologous to relA/spoT, is upregulated in response to nitrogen deprivation and predominantly localized in vegetative cells. The disruption of rel_ana strongly affects the synthesis of ppGpp, and the resulting mutant, all1549Ωsp/sm, fails to form heterocysts and to grow in the absence of a combined-nitrogen source. This phenotype can be complemented by a wild-type copy of rel_ana. Although the upregulation of hetR is affected in the mutant, ectopic overexpression of hetR cannot rescue the phenotype. However, we found that the mutant rapidly loses its viability, within a time window of 3 to 6 h, following the deprivation of combined nitrogen. We propose that ppGpp plays a major role in rebalancing the metabolic activities of the cells in the absence of the nitrogen source supply and that this regulation is necessary for filament survival and consequently for the success of heterocyst differentiation.     

在鱼腥藻7120中建立反义glnA系统

应用反义技术对鱼腥藻7120切的内源glnA基因的表达进行调控,首次获得了人工反义系统的蓝藻品系。先从编码谷酰胺合成酶（GS）的基因glnA中取得部分结构基因片段,与表达质粒载体pRL-439及穿梭质粒载体pDC－8相连接。通过酶切鉴定筛选出反向克隆的穿梭表达质粒pDC－AM,然后应用三亲接合转移法把它转入鱼腥藻对7120.通过新霉素筛选,酶谱鉴定,斑点杂交,质粒的交叉转化以及内源glnA基因表达的GS活性分析,GS相关的胞外泌氨分析及所获藻株的形态学变化,证明已在鱼腥藻7120中建立了人工反义glnA基因的品系。     

Control of Nitrogenase mRNA Levels by Products of Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120

Nitrate inhibited nitrogenase synthesis and heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Inhibition of dinitrogen fixation by nitrate did not take place, however, in nitrate reductase-deficient derivatives of this strain. Hybridization of total RNA isolated from cells grown on different nitrogen sources with an internal fragment of the nifD gene showed that regulation of nitrogenase activity by nitrate is exerted through a negative control of the nitrogenase mRNA levels.     

Biochemical Characterization of an Adenylate Cyclase, CyaB1, in the Cyanobacterium Anabaena sp. Strain PCC 7120

cyaB1 gene encodes a novel type of adenylate cyclase. The catalytic domain is located in the carboxyl-terminal half, while the GAF and PAS domains are conserved in the amino-terminal half. Recombinant CyaB1 and a truncated CyaB1 lacking the amino-terminal domain (ΔN–CyaB1) were purified and characterized. The purified CyaB1 is activated by divalent cations, such as Mg²⁺ and Mn²⁺, like other types of adenylate cyclase. The activity of CyaB1 was slightly elevated by forskolin, but was not affected by cGMP, irrespective of the presence of the cGMP binding motif in the GAF domain. The specific activity of ΔN–CyaB1 is one-eighteenth that of CyaB1, whereas the Km values of both proteins are almost the same. The results suggest that the amino-terminal half has a positive regulatory effect on the catalytic activity. Received 27 April 2001/ Accepted in revised form 6 July 2001     

A TRAP Transporter for Pyruvate and Other Monocarboxylate 2-Oxoacids in the Cyanobacterium Anabaena sp. Strain PCC 7120

In the cyanobacterium Anabaena sp. strain PCC 7120, open reading frames (ORFs) alr3026, alr3027, and all3028 encode a tripartite ATP-independent periplasmic transporter (TRAP-T). Wild-type filaments showed significant uptake of [¹⁴C]pyruvate, which was impaired in the alr3027 and all3028 mutants and was inhibited by several monocarboxylate 2-oxoacids, identifying this TRAP-T system as a pyruvate/monocarboxylate 2-oxoacid transporter.The tripartite ATP-independent periplasmic transporter (TRAP-T) family of proteins (family 2.A.56 in the transporter classification database [19]) comprises transporters that consist of three components: a small membrane protein usually bearing 4 transmembrane segments (TMSs), a large membrane protein usually bearing 12 TMSs that is the membrane translocator, and a periplasmic substrate binding protein (10). The TRAP transporters use the energy of an electrochemical ion gradient to drive uphill substrate transport (7, 14). TRAP-T family members are widely present in bacteria and archaea, but only a few substrates, including different types of carboxylates, have been identified for them (20). In vitro binding analyses with the periplasmic solute binding proteins RRC01191 from Rhodobacter capsulatus (20) and TakP from Rhodobacter sphaeroides (8) have shown that they bind monocarboxylate 2-oxoacids, including pyruvate. Additionally, pyruvate induces the TRAP-T periplasmic solute binding protein {type:entrez-protein,attrs:{text:SMb21353,term_id:1320617611,term_text:SMB21353}}SMb21353 in Sinorhizobium meliloti strain 1021 (13). We are not aware, however, of any study showing a direct role of any of these proteins in pyruvate transport in vivo.Cyanobacteria are a morphologically diverse group of photoautotrophic bacteria that includes unicellular and multicellular (filamentous) organisms (18). Most cyanobacteria can use ammonium or nitrate ions as nitrogen sources, and some can also assimilate urea or fix atmospheric N₂ (5). Some filamentous cyanobacteria fix N₂ in differentiated cells called heterocysts that are formed under combined nitrogen deprivation (6). A TRAP transporter is involved in sodium-dependent glutamate uptake in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (17). It is composed of proteins GtrA and GtrB (small and large membrane subunits, respectively) and GtrC (periplasmic substrate binding protein). A cluster of open reading frames (ORFs), alr3026, alr3027, and all3028, encoding proteins similar to TRAP-T proteins, is found in the genome of the filamentous, heterocyst-forming Anabaena sp. strain PCC 7120 (9). The proteins are Alr3026, with 4 predicted TMSs; Alr3027, with 13 predicted TMSs (however, the N-terminal TMS is a predicted signal peptide that could be removed, producing a mature protein of 12 TMSs); and All3028, a predicted periplasmic solute binding protein. Whereas the two membrane proteins are most similar to proteins of the Synechocystis Gtr glutamate transporter (Alr3026 shares 63% identity with GtrA, and Alr3027, 77% identity with GtrB), the periplasmic solute binding protein, All3028, is more similar to Rhodobacter capsulatus RRC01191 (47% identity) and Rhodobacter sphaeroides TakP (49% identity) than to Synechocystis GtrC (about 18% identity in a 300-amino-acid overlap). It was of interest, therefore, to determine the substrate(s) for this Anabaena transporter, which we approached by mutation and transport analysis.     

Mutants of the Cyanobacterium Anabaena sp. PCC 7120 Altered in Nitrate Transport and Reduction

Nitrate assimilation-defective mutants SP7, SP9, and SP17 of the cyanobacterium Anabaena sp. PCC 7120 were isolated by use of transposon mutagenesis and screened on medium containing chlorate. SP7 and SP17 represented nitrate reductase-defective nature, while mutant SP9 appeared to be a regulatory mutant exhibiting pleiotropic behavior. Kinetics of nitrate uptake system exhibited K _s values of 31–38 μM for parent, SP7, and SP17 strains; however, mutant SP9 exhibited a high K _s value of 109.5 μM. Defective nitrate reductase was apparent in mutant SP7 and SP9, while mutant SP17 exhibited partial defective nature. Methyl viologen-dependent NR activity in parent strain presented a biphasic nature with K _m values of 0.13 and 2.47 mM, whereas a single K _m value (2.96 mM) was observed for mutant SP17. Mutant SP9 was also defective in nitrite uptake and reduction. Mutant strains exhibited derepressed nitrogenase activity in the presence of nitrate, while glutamine synthetase activity remained unaltered. Received: 20 April 1999 / Accepted: 22 May 1999     

Sucrose Synthesis in the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC 7120 Is Controlled by the Two-Component Response Regulator OrrA

The filamentous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 accumulates sucrose as a compatible solute against salt stress. Sucrose-phosphate synthase activity, which is responsible for the sucrose synthesis, is increased by salt stress, but the mechanism underlying the regulation of sucrose synthesis remains unknown. In the present study, a response regulator, OrrA, was shown to control sucrose synthesis. Expression of spsA, which encodes a sucrose-phosphate synthase, and susA and susB, which encode sucrose synthases, was induced by salt stress. In the orrA disruptant, salt induction of these genes was completely abolished. The cellular sucrose level of the orrA disruptant was reduced to 40% of that in the wild type under salt stress conditions. Moreover, overexpression of orrA resulted in enhanced expression of spsA, susA, and susB, followed by accumulation of sucrose, without the addition of NaCl. We also found that SigB2, a group 2 sigma factor of RNA polymerase, regulated the early response to salt stress under the control of OrrA. It is concluded that OrrA controls sucrose synthesis in collaboration with SigB2.     

Overexpression of the groESL Operon Enhances the Heat and Salinity Stress Tolerance of the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC7120

The bicistronic groESL operon, encoding the Hsp60 and Hsp10 chaperonins, was cloned into an integrative expression vector, pFPN, and incorporated at an innocuous site in the Anabaena sp. strain PCC7120 genome. In the recombinant Anabaena strain, the additional groESL operon was expressed from a strong cyanobacterial P_psbA1 promoter without hampering the stress-responsive expression of the native groESL operon. The net expression of the two groESL operons promoted better growth, supported the vital activities of nitrogen fixation and photosynthesis at ambient conditions, and enhanced the tolerance of the recombinant Anabaena strain to heat and salinity stresses.Nitrogen-fixing cyanobacteria, especially strains of Nostoc and Anabaena, are native to tropical agroclimatic conditions, such as those of Indian paddy fields, and contribute to the carbon (C) and nitrogen (N) economy of these soils (22, 30). However, their biofertilizer potential decreases during exposure to high temperature, salinity, and other such stressful environments (1). A common target for these stresses is cellular proteins, which are denatured and inactivated during stress, resulting in metabolic arrest, cessation of growth, and eventually loss of viability. Molecular chaperones play a major role in the conformational homeostasis of cellular proteins (13, 16, 24, 26) by (i) proper folding of nascent polypeptide chains; (ii) facilitating protein translocation and maturation to functional conformation, including multiprotein complex assembly; (iii) refolding of misfolded proteins; (iv) sequestering damaged proteins to aggregates; and (v) solubilizing protein aggregates for refolding or degradation. Present at basal levels under optimum growth conditions in bacteria, the expression of chaperonins is significantly enhanced during heat shock and other stresses (2, 25, 32).The most common and abundant cyanobacterial chaperones are Hsp60 proteins, and nitrogen-fixing cyanobacteria possess two or more copies of the hsp60 or groEL gene (http://genome.kazusa.or.jp/cyanobase). One occurs as a solitary gene, cpn60 (17, 21), while the other is juxtaposed to its cochaperonin encoding genes groES and constitutes a bicistronic operon groESL (7, 19, 31). The two hsp60 genes encode a 59-kDa GroEL and a 61-kDa Cpn60 protein in Anabaena (2, 20). Both the Hsp60 chaperonins are strongly expressed during heat stress, resulting in the superior thermotolerance of Anabaena, compared to the transient expression of the Hsp60 chaperonins in Escherichia coli (20). GroEL and Cpn60 stably associate with thylakoid membranes in Anabaena strain PCC7120 (14) and in Synechocystis sp. strain PCC6803 (15). In Synechocystis sp. strain PCC6803, photosynthetic inhibitors downregulate, while light and redox perturbation induce cpn60 expression (10, 25, 31), and a cpn60 mutant exhibits a light-sensitive phenotype (http://genome.kazusa.or.jp/cyanobase), indicating a possible role for Cpn60 in photosynthesis. GroEL, a lipochaperonin (12, 28), requires a cochaperonin, GroES, for its folding activity and has wider substrate selectivity. In heterotrophic nitrogen-fixing bacteria, such as Klebsiella pneumoniae and Bradyrhizobium japonicum, the GroEL protein has been implicated in nif gene expression and the assembly, stability, and activity of the nitrogenase proteins (8, 9, 11).Earlier work from our laboratory demonstrated that the Hsp60 family chaperonins are commonly induced general-stress proteins in response to heat, salinity, and osmotic stresses in Anabaena strains (2, 4). Our recent work elucidated a major role of the cpn60 gene in the protection from photosynthesis and the nitrate reductase activity of N-supplemented Anabaena cultures (21). In this study, we integrated and constitutively overexpressed an extra copy of the groESL operon in Anabaena to evaluate the importance and contribution of GroEL chaperonin to the physiology of Anabaena during optimal and stressful conditions.Anabaena sp. strain PCC7120 was photoautotrophically grown in combined nitrogen-free (BG11⁻) or 17 mM NaNO₃-supplemented (BG11⁺) BG11 medium (5) at pH 7.2 under continuous illumination (30 μE m⁻² s⁻¹) and aeration (2 liters min⁻¹) at 25°C ± 2°C. Escherichia coli DH5α cultures were grown in Luria-Bertani medium at 37°C at 150 rpm. For E. coli DH5α, kanamycin and carbenicillin were used at final concentrations of 50 μg ml⁻¹ and 100 μg ml⁻¹, respectively. Recombinant Anabaena clones were selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin or in BG11⁻ liquid medium containing 12.5 μg ml⁻¹ neomycin. The growth of cyanobacterial cultures was estimated either by measuring the chlorophyll a content as described previously (18) or the turbidity (optical density at 750 nm). Photosynthesis was measured as light-dependent oxygen evolution at 25 ± 2°C by a Clark electrode (Oxy-lab 2/2; Hansatech Instruments, England) as described previously (21). Nitrogenase activity was estimated by acetylene reduction assays, as described previously (3). Protein denaturation and aggregation were measured in clarified cell extracts containing ∼500 μg cytosolic proteins treated with 100 μM 8-anilino-1-naphthalene sulfonate (ANS). The pellet (protein aggregate) was solubilized in 20 mM Tris-6 M urea-2% sodium dodecyl sulfate (SDS)-40 mM dithiothreitol for 10 min at 50°C. The noncovalently trapped ANS was estimated using a fluorescence spectrometer (model FP-6500; Jasco, Japan) at a λ_excitation of 380 nm and a λ_emission of 485 nm, as described previously (29).The complete bicistronic groESL operon (2.040 kb) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ608815","term_id":"222354886","term_text":"FJ608815"}}FJ608815) was PCR amplified from PCC7120 genomic DNA using specific primers (Table ​(Table1)1) and the amplicon cloned into the NdeI-BamHI restriction sites of plasmid vector pFPN, which allows integration at a defined innocuous site in the PCC7120 genome and expression from a strong cyanobacterial P_psbA1 promoter (6). The resulting construct, designated pFPNgro (Table ​(Table1),1), was electroporated into PCC7120 using an exponential-decay wave form electroporator (200 J capacitive energy at a full charging voltage of 2 kV; Pune Polytronics, Pune, India), as described previously (6). The electroporation was carried out at 6 kV cm⁻¹ for 5 ms, employing an external autoclavable electrode with a 2-mm gap. The electroporation buffer contained high concentrations of salt (10 mM HEPES, 100 mM LiCl, 50 mM CaCl₂), as have been recommended for plant cells (23) and other cell types (27). The electrotransformants, selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin by repeated subculturing for at least 25 weeks to achieve complete segregation, were designated AnFPNgro.

TABLE 1.

Plasmids, strains, and primers used in this study

Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120

DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N₂ fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.