Differential regulation of ssb genes in the nitrogen‐fixing cyanobacterium,Anabaena sp. strain PCC71201

Anabaena sp. PCC7120 possesses three genes coding for single‐stranded DNA‐binding (SSB) protein, of which ssb1 was a single gene, and ssb2 and ssb3 are the first genes of their corresponding operons. Regulation of the truncated ssb genes, ssb1 (alr0088) and ssb2 (alr7559), was unaffected by N‐status of growth. They were negatively regulated by the SOS‐response regulatory protein LexA, as indicated by the (i) binding of Anabaena LexA to the LexA box of regulatory regions of ssb1 and ssb2, and (ii) decreased expression of the downstream gfp reporter gene in Escherichia coli upon co‐expression of LexA. However, the full‐length ssb gene, ssb3 (all4779), was regulated by the availability of Fe²⁺ and combined nitrogen, as indicated by (i) increase in the levels of SSB3 protein on Fe²⁺‐depletion and decrease under Fe²⁺‐excess conditions, and (ii) 1.5‐ to 1.6‐fold decrease in activity under nitrogen‐fixing conditions compared to nitrogen‐supplemented conditions. The requirement of Fe²⁺ as a co‐factor for repression by FurA and the increase in levels of FurA under nitrogen‐deficient conditions in Anabaena (Lopez‐Gomollon et al. 2007) indicated a possible regulation of ssb3 by FurA. This was substantiated by (i) the binding of FurA to the regulatory region of ssb3, (ii) repression of the expression of the downstream gfp reporter gene in E. coli upon co‐expression of FurA, and (iii) negative regulation of ssb3 promoter activity by the upstream AT‐rich region in Anabaena. This is the first report on possible role of FurA, an important protein for iron homeostasis, in DNA repair of cyanobacteria.     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

Oxidative stress management in the filamentous, heterocystous, diazotrophic cyanobacterium, Anabaena PCC7120

Reactive oxygen species (ROS) are inevitably generated as by-products of respiratory/photosynthetic electron transport in oxygenic photoautotrophs. Unless effectively scavenged, these ROS can damage all cellular components. The filamentous, heterocystous, nitrogen-fixing strains of the cyanobacterium, Anabaena, serve as naturally abundant contributors of nitrogen biofertilizers in tropical rice paddy fields. Anabaena strains are known to tolerate several abiotic stresses, such as heat, UV, gamma radiation, desiccation, etc., that are known to generate ROS. ROS are detoxified by specific antioxidant enzymes like superoxide dismutases (SOD), catalases and peroxiredoxins. The genome of Anabaena PCC7120 encodes two SODs, two catalases and seven peroxiredoxins, indicating the presence of an elaborate antioxidant enzymatic machinery to defend its cellular components from ROS. This article summarizes recent findings and depicts important perspectives in oxidative stress management in Anabaena PCC7120.     

A membrane-associated Mn-superoxide dismutase protects the photosynthetic apparatus and nitrogenase from oxidative damage in the Cyanobacterium Anabaena sp. PCC 7120

We investigated the functions of a membrane-associated manganese superoxide dismutase (MnSOD) of the heterocystous cyanobacterium Anabaena sp. PCC 7120. The gene sodA encoding MnSOD was inactivated by interposon mutagenesis and it was confirmed by Southern hybridization and immunoblotting. The strain A17, lacking sodA, grew more slowly than the wild type, and the difference in growth rates between the two strains became larger with an increase in growth light intensity. More severe inhibition of growth of A17 was observed when the cells were grown in the absence of combined nitrogen. Complementation of A17 with a full copy of the sodA gene restored the wild-type phenotypes. Strain A17 produced more malondialdehyde than did the wild type, especially under high light intensity, indicating more lipid peroxidation in the absence of MnSOD. A17 was also more susceptible to photoinhibition by a high light, and it was shown that both PSII and PSI were more severely damaged by the photoinhibitory light in A17, suggesting that the MnSOD plays important roles in protection of both photosystems. Immunoblotting revealed that the MnSOD was present in vegetative cells and heterocysts. Light greatly stimulated nitrogenase activity in the wild type under both aerobic and anaerobic conditions, but stimulated nitrogenase activity in A17 only slightly in air. The results suggest that reactive oxygen species produced in heterocysts under aerobic conditions cause the inactivation of nitrogenase in the absence of MnSOD.     

LytM factor Alr3353 affects filament morphology and cell–cell communication in the multicellular cyanobacterium Anabaena sp. PCC 7120

Heterocyst‐forming cyanobacteria are organized as multicellular filaments of tightly interacting, functionally specialized cells. N₂‐fixing heterocysts differentiate from vegetative cells under nitrogen limitation in a semi‐regular pattern along the filament. Diazotrophic growth requires metabolite exchange between neighboring cells within the filament. This exchange occurs via cell–cell junction complexes that span the gap between the plasma membranes and thereby cross the septal peptidoglycan through an array of uniform nanopores formed by AmiC‐type cell wall hydrolases. We investigated how the lytic hydrolase AmiC1 (Alr0092) from Anabaena sp. PCC 7120, whose activity needs to be tightly controlled to avoid cell lysis, is regulated by the LytM factor Alr3353. Inactivation of alr3353 resulted in significantly fewer nanopores and as a consequence, a lower rate of fluorescent tracer exchange between cells. The mutant was not able to grow with N₂ as sole nitrogen source, although heterocysts were formed. Alr3353 localized mainly to fully developed intercellular septa of vegetative cells. The purified protein bound to peptidoglycan and enhanced the hydrolytic activity of AmiC1 in vitro. Our data show that the LytM factor Alr3353 regulates nanopore formation and cell–cell communication by directly interacting with AmiC1.     

Multiple modes of iron uptake by the filamentous,siderophore‐producing cyanobacterium,Anabaena sp. PCC 7120

Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore‐ and non‐siderophore‐producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore‐producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self‐secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe′) via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore‐ and non‐siderophore‐mediated iron uptake. While assimilation of Fe′ and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe′ reduction and uptake is advantageous for low‐density cultures, while at higher densities siderophore uptake is preferred.     

Biochemical characterization of a membrane-bound manganese-containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120

The filamentous cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) possesses two genes for superoxide dismutase (SOD). One is an iron-containing (FeSOD) whereas the other is a manganese-containing superoxide dismutase (MnSOD). Localization experiments and analysis of the sequence showed that the FeSOD is cytosolic, whereas the MnSOD is a membrane-bound homodimeric protein containing one transmembrane helix, a spacer region, and a soluble catalytic domain. It is localized in both cytoplasmic and thylakoid membranes at the same extent with the catalytic domains positioned either in the periplasm or the thylakoid lumen. A phylogenetic analysis revealed that generally the highly homologous MnSODs of filamentous cyanobacteria are unique in being membrane-bound. Two recombinant variants of Anabaena MnSOD lacking either the hydrophobic region (MnSOD(Delta 28)) or the hydrophobic and the linker region (MnSOD(Delta 60)) are shown to exhibit the characteristic manganese peak at 480 nm, an almost 100% occupancy of manganese per subunit, a specific activity using the ferricytochrome assay of (660 +/- 90) unit mg-1 protein and a dissociation constant for the inhibitor azide of (0.84 +/- 0.05) mm. Using stopped-flow spectroscopy it is shown that the decay of superoxide in the presence of various (MnSOD(Delta 28)) or (MnSOD(Delta 60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 x 10(6) m-1 s-1 (pH 10) and 6 x 10(7) m-1 s-1 (pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria.     

Activities of two dissimilar thioredoxins from the cyanobacterium Anabaena sp. strain PCC 7120.

Thioredoxin is a small redox protein that functions as a reducing agent and modulator of enzyme activity. A gene for an unusual thioredoxin was previously isolated from the cyanobacterium Anabaena sp. strain PCC 7120 and cloned and expressed in Escherichia coli. However, the protein could not be detected in Anabaena cells (J. Alam, S. Curtis, F. K. Gleason, M. Gerami-Nejad, and J. A. Fuchs, J. Bacteriol. 171:162-171, 1989). Polyclonal antibodies to the atypical thioredoxin were prepared, and the protein was detected by Western immunoblotting. It occurs at very low levels in extracts of Anabaena sp. and other cyanobacteria. No antibody cross-reaction was observed in extracts of eukaryotic algae, plants, or eubacteria. The anti-Anabaena thioredoxin antibodies did react with another unusual thioredoxin-glutaredoxin produced by bacteriophage T4. Like the T4 protein and other glutaredoxins, the unusual cyanobacterial thioredoxin can be reduced by glutathione. The Anabaena protein can also activate enzymes of carbon metabolism and has some functional similarity to spinach chloroplast thioredoxin f. However, it shows only 23% amino acid sequence identity to the spinach chloroplast protein and appears to be distantly related to other thioredoxins. The data indicate that cyanobacteria, like plant chloroplasts, have two dissimilar thioredoxins. One is related to the more common protein found in other prokaryotes, and the other is an unusual thioredoxin that can be reduced by glutathione and may function in glucose catabolism.     

Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A region of the genome of the filamentous, nitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 that contains a cluster of genes involved in nitrate assimilation has been identified. The genes nir, encoding nitrite reductase, and nrtABC, encoding elements of a nitrate permease, have been cloned. Insertion of a gene cassette into the nir-nrtA region impaired expression of narB, the nitrate reductase structural gene which together with nrtD is found downstream from nrtC in the gene cluster. This indicates that the nir-nrtABCD-narB genes are cotranscribed, thus constituting an operon. Expression of the nir operon in strain PCC 7120 is subjected to ammonium-promoted repression and takes place from an NtcA-activated promoter located 460 bp upstream from the start of the nir gene. In the absence of ammonium, cellular levels of the products of the nir operon are higher in the presence of nitrate than in the absence of combined nitrogen.     

AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses

Alkyl hydroperoxide reductase (AhpC) is known to detoxify peroxides and reactive sulfur species (RSS). However, the relationship between its expression and combating of abiotic stresses is still not clear. To investigate this relationship, the genes encoding the alkyl hydroperoxide reductase (ahpC) from Anabaena sp. PCC 7120 were introduced into E. coli using pGEX-5X-2 vector and their possible functions against heat, salt, carbofuron, cadmium, copper and UV-B were analyzed. The transformed E. coli cells registered significantly increase in growth than the control cells under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml^?1), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. Enhanced expression of ahpC gene as measured by semi-quantitative RT-PCR under aforementioned stresses at different time points demonstrated its role in offering tolerance against multiple abiotic stresses.     

Characterization of two naturally truncated, Ssb-like proteins from the nitrogen-fixing cyanobacterium, Anabaena sp. PCC7120

Single-stranded (ss) DNA-binding (Ssb) proteins are vital for all DNA metabolic processes and are characterized by an N-terminal OB-fold followed by P/G-rich spacer region and a C-terminal tail. In the genome of the heterocystous, nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC 7120, two genes alr0088 and alr7579 are annotated as ssb, but the corresponding proteins have only the N-terminal OB-fold and no P/G-rich region or acidic tail, thereby rendering them unable to interact with genome maintenance proteins. Both the proteins were expressed under normal growth conditions in Anabaena PCC7120 and regulated differentially under abiotic stresses which induce DNA damage, indicating that these are functional genes. Constitutive overexpression of Alr0088 in Anabaena enhanced the tolerance to DNA-damaging stresses which caused formation of DNA adducts such as UV and MitomycinC, but significantly decreased the tolerance to γ-irradiation, which causes single- and double-stranded DNA breaks. On the other hand, overexpression of Alr7579 had no significant effect on normal growth or stress tolerance of Anabaena. Thus, of the two truncated Ssb-like proteins, Alr0088 may be involved in protection of ssDNA from damage, but due to the absence of acidic tail, it may not aid in repair of damaged DNA. These two proteins are present across cyanobacterial genera and unique to them. These initial studies pave the way to the understanding of DNA repair in cyanobacteria, which is not very well documented.     

Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon

A second nitrogen fixation (nif) operon in the cyanobacterium (blue-green alga) Anabaena (Nostoc) sp. strain PCC 7120 has been identified and sequenced. It is located just upstream of the nifHDK operon and consists of four genes in the order nifB, fdxN, nifS, and nifU. The three nif genes were identified on the basis of their similarity with the corresponding genes from other diazotrophs. The fourth gene, fdxN, codes for a bacterial type ferredoxin (Mulligan, M. E., Buikema, W. J., and Haselkorn, R. (1988) J. Bacteriol. 167, 4406-4410). The four genes are probably transcribed as a single operon, but are expressed at a lower level than the nifHDK operon, and only after a developmentally induced DNA rearrangement occurs that excises a 55-kilobase pair element from within the fdxN gene (Golden, J. W., Mulligan, M. E., and Haselkorn, R. (1987) Nature 327, 526-529; Golden, J. W., Carrasco, C. D., Mulligan, M. E., Schneider, G. J., and Haselkorn, R. (1988) J. Bacteriol. 170, 5034-5041). The promoter for the nifB operon was located by primer extension. Comparison of the nifB 5'-flanking sequence with the nifH 5'-flanking sequence did not reveal any consensus base pairs that would define a nif promoter for Anabaena. The operon contains two instances of 7-base pair directly repeated sequences: seven copies of the repeated sequence are found between the nifB and fdxN genes and six copies are found between the nifS and nifU genes. The function of these repeats is unknown.     

Identification, genetic analysis and characterization of a sugar-non-specific nuclease from the cyanobacterium Anabaena sp. PCC 7120

A nuclease that could be recovered from the supernatant of cultures, as well as from cell-free extracts, of the cyanobacterium Anabaena sp. PCC 7120 was identified as a 29 kDa polypeptide by its ability to degrade DNA after electrophoresis in DNA-containing SDS-polyacrylamide gels. Some clones of a gene library of strain PCC 7120 established in Escherichia coli were found to produce the 29 kDa nuclease. The nucA gene encoding this nuclease was subcloned and sequenced. The deduced polypeptide, NucA, had a molecular weight of 29,650, presented a presumptive signal peptide in its N-terminal region and showed homology to the products of the nuc gene from Serratia marcescens and the NUC1 gene from Saccharomyces cerevisiae. The NucA protein from Anabaena itself, or from the cloned nucA gene expressed in E. coli, catalysed the degradation of both RNA and DNA, had the potential to act as an endonuclease, and functioned best in the presence of Mn2+ or Mg2+. An Anabaena nucA insertional mutant was generated which failed to produce the 29 kDa nuclease.     

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min^?1 mg protein^?1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min^?1 mg protein^?1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (?arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.