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.     

鱼腥蓝细菌PCC7120铁吸收机制

铁离子是鱼腥蓝细菌PCC7120进行呼吸作用、光合作用和固氮作用中相关酶的重要辅基之一,缺铁将严重影响蓝细菌的生存.富氧的生态环境中铁通常以不溶的Fe3+形式存在,不易被细胞吸收利用.低铁条件下,鱼腥蓝细菌PCC7120分泌能螯合铁离子的嗜铁素,通过外膜上相应的转运体将嗜铁素-铁复合物转运到细胞内.综述了近年来在嗜铁素的种类及其生物合成途径、铁吸收系统的组成和功能等方面的最新进展,分析了铁吸收系统的调控机制,为进一步开展鱼腥蓝细菌铁吸收机制的研究提供依据.     

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.     

Cell Envelope Components Influencing Filament Length in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

Heterocyst-forming cyanobacteria grow as chains of cells (known as trichomes or filaments) that can be hundreds of cells long. The filament consists of individual cells surrounded by a cytoplasmic membrane and peptidoglycan layers. The cells, however, share a continuous outer membrane, and septal proteins, such as SepJ, are important for cell-cell contact and filament formation. Here, we addressed a possible role of cell envelope components in filamentation, the process of producing and maintaining filaments, in the model cyanobacterium Anabaena sp. strain PCC 7120. We studied filament length and the response of the filaments to mechanical fragmentation in a number of strains with mutations in genes encoding cell envelope components. Previously published peptidoglycan- and outer membrane-related gene mutants and strains with mutations in two genes (all5045 and alr0718) encoding class B penicillin-binding proteins isolated in this work were used. Our results show that filament length is affected in most cell envelope mutants, but the filaments of alr5045 and alr2270 gene mutants were particularly fragmented. All5045 is a dd-transpeptidase involved in peptidoglycan elongation during cell growth, and Alr2270 is an enzyme involved in the biosynthesis of lipid A, a key component of lipopolysaccharide. These results indicate that both components of the cell envelope, the murein sacculus and the outer membrane, influence filamentation. As deduced from the filament fragmentation phenotypes of their mutants, however, none of these elements is as important for filamentation as the septal protein SepJ.     

鱼腥藻7120遗传转化的研究进展

鱼腥藻7120作为模式生物被广泛用于光合、固氮、进化、代谢等基本生命现象的研究。近几年, 对其基因工程的研究使人们看到它在医药、环保、能源等方面的应用潜力, 但表达效率低是其发展的瓶颈。为了提高其表达效率, 研究者从鱼腥藻7120的载体(包括启动子、复制子、选择标记基因等)的改进、目的基因的优化(密码子和SD序列)、宿主的改善、转化方法的改变等方面进行了大量探索, 除了用于功能基因的研究, 已经有几十个外源基因在鱼腥藻7120中表达。除了研究载体, 诱变鱼腥藻7120形成有利于外源基因表达的突变体和摸索转基因蓝藻最佳生长条件和表达条件, 可能是新的发展方向。     

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.     

Isolation of Plasma and Thylakoid Membranes from the Heterocystous Cyanobacterium Anabaena sp. PCC 7120

具异型胞蓝细菌 Anabaena sp.PCC 7120质膜和类囊体膜的分离纯化 李斌 徐冬一 赵进东*