Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Extracellular production of lipoxygenase from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on wheat protein

In this study, the lipoxygenase (ana-LOX) gene from Anabaena sp. PCC 7120 was successful expressed and secreted in Bacillus subtilis. Under the control of the P43 promoter, with a signal peptide from the B. subtilis 168 nprB gene, and facilitated by the molecular chaperone PrsA, the production of the recombinant ana-LOX (ana-rLOX) reached 76 U/mL (171.9 μg/ml) in the supernatant. The purified ana-rLOX was investigated for its effect on dough protein. Ana-rLOX treatment decreased free sulfhydryl groups, increased glutenin macropolymer content, promoted the formation of covalent bonds between gluten protein, and affected protein crosslinking. The results indicated that large aggregates involving gliadin and glutenin were formed. The glutenin macropolymer played a role in the formation of the dough network structure through the exchange of thiol disulfide bonds and the formation of hydrogen or hydrophobic bonds with other proteins.     

Protection from UV-B Damage of Mosquito Larvicidal Toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">israelensis</Emphasis> Expressed in <Emphasis Type="Italic">Anabaena</Emphasis> PCC 7120

A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed δ-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of B. thuringiensis subsp. israelensis as a mosquito biological control agent. Received: 20 November 2001 / Accepted: 31 December 2001     

In silico characterization and transcriptomic analysis of nif family genes from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.     

Reductive transformation of methyl parathion by the cyanobacterium<Emphasis Type="Italic"> Anabaena</Emphasis> sp. strain PCC7120

Organophosphorus compounds are toxic chemicals that are applied worldwide as household pesticides and for crop protection, and they are stockpiled for chemical warfare. As a result, they are routinely detected in air and water. Methods and routes of biodegradation of these compounds are being sought. We report that under aerobic, photosynthetic conditions, the cyanobacterium Anabaena sp. transformed methyl parathion first to o,o-dimethyl o-p-nitrosophenyl thiophosphate and then to o,o-dimethyl o-p-aminophenyl thiophosphate by reducing the nitro group. The process of methyl parathion transformation occurred in the light, but not in the dark. Methyl parathion was toxic to cyanobacteria in the dark but did not affect their viability in the light. Methyl parathion transformation was not affected by mutations in the genes involved in nitrate reduction in cyanobacteria.     

Exopolysaccharide production in <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 under different CaCl<Subscript>2</Subscript> regimes

Influence of various levels of CaCl₂ (0, 1, 10 and 100 mM) on exopolysaccharide production has been investigated in the cyanobacterium Anabaena 7120. At the concentration of 1 mM CaCl₂, growth was found to be stimulatory while 100 mM was sub lethal for the cyanobacterial cells. Estimation of EPS content revealed that EPS production depends on the concentration of calcium ions in the immediate environment with maximum being at10 mM CaCl₂. A possible involvement of alr2882 gene in the process of EPS production was also revealed through qRT-PCR. Further, FTIR-spectra marked the presence of aliphatic alkyl-group, primary amine-group, and polysaccharides along with shift in major absorption peaks suggesting that calcium levels in the external environment regulate the composition of EPS produced by Anabaena 7120. Thus, both quantity and composition of EPS is affected under different calcium chloride concentrations presenting possibilities of EPS with novel unexplored features that may offer biotechnological applications.     

Transcription of the extended <Emphasis Type="Italic">hyp</Emphasis>-operon in <Emphasis Type="Italic">Nostoc</Emphasis>sp. strain PCC 7120

Background  

The maturation of hydrogenases into active enzymes is a complex process and e.g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N₂-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hyp-genes in Nostoc sp. strain PCC 7120.     

Characterization of a DUF820 family protein Alr3200 of the cyanobacterium <Emphasis Type="BoldItalic">Anabaena</Emphasis> sp. strain PCC7120

The hypothetical protein ‘Alr3200’ of Anabaena sp. strain PCC7120 is highly conserved among cyanobacterial species. It is a member of the DUF820 (Domain of Unknown Function) protein family, and is predicted to have a DNase domain. Biochemical analysis revealed a Mg(II)-dependent DNase activity for Alr3200 with a specific activity of 8.62×10⁴ Kunitz Units (KU) mg^?1 protein. Circular dichroism analysis predicted Alr3200 to have ~40% β-strands and ~9% α-helical structures. Anabaena PCC7120 inherently expressed Alr3200 at very low levels, and its overexpression had no significant effect on growth of Anabaena under control conditions. However, Analr3200 ⁺, the recombinant Anabaena strain overexpressing Alr3200, exhibited zero survival upon exposure to 6 kGy of γ-radiation, which is the LD₅₀ for wild type Anabaena PCC7120 as well as the vector control recombinant strain, AnpAM. Comparative analysis of the two recombinant Anabaena strains suggested that it is not the accumulated Alr3200 per se, but its possible interactions with the radiation-induced unidentified DNA repair proteins of Anabaena, which hampers DNA repair resulting in radiosensitivity.     

A single gene <Emphasis Type="Italic">all3940</Emphasis> (Dps) overexpression in <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 confers multiple abiotic stress tolerance via proteomic alterations

DNA-binding proteins (Dps) induced during starvation play an important role in gene regulation and maintaining homeostasis in bacteria. The nitrogen-fixing cyanobacterium, Anabaena PCC7120, has four genes annotated as coding for Dps; however, the information on their physiological roles is limiting. One of the genes coding for Dps, ‘all3940’ was found to be induced under different abiotic stresses in Anabaena and upon overexpression enhanced the tolerance of Anabaena to a multitude of stresses, which included salinity, heat, heavy metals, pesticide, and nutrient starvation. On the other hand, mutation in the gene resulted in decreased growth of Anabaena. The modulation in the levels of All3940 in Anabaena, achieved either by overexpression of the protein or mutation of the gene, resulted in changes in the proteome, which correlated well with the physiological changes observed. Proteins required for varied physiological activities, such as photosynthesis, carbon-metabolism, oxidative stress alleviation, exhibited change in protein profile upon modulation of All3940 levels in Anabaena. This suggested a direct or an indirect effect of All3940 on the expression of the above stress-responsive proteins, thereby enhancing tolerance in Anabaena PCC7120. Thus, All3940, though categorized as a Dps, is possibly a general stress protein having a global role in regulating tolerance to multitude of stresses in Anabaena.     

Modulation of biocidal activity of <Emphasis Type="Italic">Calothrix</Emphasis> sp. and <Emphasis Type="Italic">Anabaena</Emphasis> sp. by environmental factors

An investigation was undertaken to evaluate a set of cyanobacterial strains in terms of production of biocidal compounds exhibiting allelochemical and fungicidal properties. Two cyanobacterial strains — Anabaena sp. and Calothrix sp. were selected for further investigation, on the basis of their larger inhibition zones on the lawn of Synechocystis and Synechococcus sp. and two phytopathogenic fungi — Rhizoctonia bataticola and Pythium debaryanum. The diameter of the inhibition zone was largest when extracellular filtrates of the two cultures incubated at high light intensity (90–100 μmol photons m⁻² s⁻¹) and temperature (40 ± 2 °C) or grown in medium containing two-folds higher P (1.4 mg/L, as compared to 0.7 mg/L in BG 11 medium) were taken. A pH of 8 was the most optimal for both strains, in terms of growth and biocidal activity. Partial purification of ethyl acetate extract using TLC, followed by GLC revealed a single peak. This study highlights the importance of environmental factors in aggravating or reducing the toxic effects of these harmful cyanobacteria and their potential as a biocontrol agent.     

Paired cloning vectors for complementation of mutations in the cyanobacterium<Emphasis Type="Italic"> Anabaena</Emphasis> sp. strain PCC 7120

The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF _A ) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.     

<Emphasis Type="Italic">In vitro</Emphasis> DNA-binding properties of a manganese response regulator (ManR) from <Emphasis Type="Italic">Anabaena</Emphasis> sp.

ManR of Anabaena sp. PCC 7120 is a manganese response regulator. Two ManR molecules bind to the specific DNA sequences at the same time, which was demonstrated by our previous results. From size exclusion chromatography, ManR exits as monomer in solution. Therefore, cooperative interactions of ManR–ManR play a role in DNA binding of the ManR, suggesting that ManR molecules bind co-operatively to DNA. When serial deletions of N-terminal of the ManR were also carried out the mutant proteins, ManRC111, ManRC130 and ManRC158, had completely lost the in DNA binding activity. Mutants ManRC 196, ManRC206, ManRC221 and ManRC230, however, could specifically bind to DNA, indicating that the amino acid residues between Val₁₆ and Ile₇₈ of the N-terminal of ManR are necessary for the DNA binding activity of C-terminal domain.Revisions requested 20 Ocotober 2004/15 November 2004; Revisions received 10 November/13 December 2004     

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539–552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10²–10³ M⁻¹ s⁻¹ and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.     

<Emphasis Type="Italic">Kretzschmaria varians</Emphasis> sp. nov., <Emphasis Type="Italic">Xylaria coremiifera</Emphasis> sp. nov. and <Emphasis Type="Italic">Xylaria umbonata</Emphasis> sp. nov. from Costa Rica

Kretzschmaria varians, a species apparently related to K. micropus, is described as new. It is distinguished primarily by having asci with 2 to 8 ascospores with inconstant germination slit length and remains of synnemata on stromata and surrounding substrate. Xylaria coremiifera, described here as new, bears small fragile coremia on pulvinate stromata and the surrounding substrate. Asci often have fewer than 8 ascospores, most frequently 4. Xylaria umbonata, described here as new, produces perithecia around a central umbo that appears to be the remains of a synnema. Ascospores have long spiralling germination slits.     

Trehalose-Producing Enzymes MTSase and MTHase in <Emphasis Type="Italic">Anabaena</Emphasis> 7120 Under NaCl Stress

Salt tolerance, a multigenic trait, necessitates knowledge about biosynthesis and function of candidate gene(s) at the cellular level. Among the osmolytes, trehalose biosynthesis in cyanobacteria facing NaCl stress is little understood. Anabaena 7120 filaments exposed to 150 mm NaCl fragmented and recovered on transfer to –NaCl medium with the increased heterocysts frequency (7%) over the control (4%). Cells failed to retain Na⁺ beyond a threshold [2.19 mm/cm³ (PCV)]. Whereas NaCl-stressed cells exhibited a marginal rise in K⁺ (1.1-fold) only at 30 h, for Na⁺ it was 130-fold at 48 h over cells in control. A time-course study (0–54 h) revealed reduction in intracellular Na⁺ beyond 48 h [0.80 mm/cm³ (PCV)] suggestive of ion efflux. The NaCl-stressed cells showed differential expression of maltooligosyltrehalose synthase (MTSase; EC 5.4.99.15) and maltooligosyltrehalose trehalohydrolase (MTHase; EC 3.2.1.141) depending on the time and the extent of intracellular Na⁺ buildup.     

Identification of Three <Emphasis Type="Italic">Superoxide dismutase</Emphasis> Genes from a <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

We report the characterization of three Superoxide dismutase (sod) genes isolated from a bacterium in the Geobacillus genus. We isolated the bacterium from high-temperature pond mud and used 16S rRNA gene sequence to confirm its identity in the Geobacillus genus. The three genes Mn-sod, Fe/Mn-sod, and Cu/Zn-sod were cloned and analyzed. Their open reading frames are Mn-sod: 615 bp encoding a 204 amino acid protein; Fe/Mn-sod: 1,236 bp encoding a 411 amino acid protein; Cu/Zn-sod: 522 bp encoding a 173 amino acid protein. When these sod genes were expressed in Escherichia coli, only Mn-SOD was able to be purified. The activity of the purified Mn-SOD we got was about 2,730 U/mg. Studies of this Mn-SOD showed that it was thermostable at 60°, had 70% activity at 80° after 2.5 h, and still had 30% activity at 90° after 2.5 h. Mn-SOD activity required the ion Mn²⁺. Based on gel electrophoresis, we deduced that this Mn-SOD was a homotetramer. No activity was detected after the other two genes (Fe/Mn-sod, Cu/Zn-sod) were expressed in Escherichia coli, but activities were detected when expressed in Pichia pastoris.     

Impairment of <Emphasis Type="Italic">ntc</Emphasis>A gene revealed its role in regulating iron homeostasis,ROS production and cellular phenotype under iron deficiency in cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120

Iron deficiency ends up into several unavoidable consequences including damaging oxidative stress in cyanobacteria. NtcA is a global nitrogen regulator controls wide range of metabolisms in addition to regulation of nitrogen metabolism. In present communication, NtcA based regulation of iron homeostasis, ROS production and cellular phenotype under iron deficiency in Anabaena 7120 has been investigated. NtcA regulates the concentration dependent iron uptake by controlling the expression of furA gene. NtcA also regulated pigment synthesis and phenotypic alterations in Anabaena 7120. A significant increase in ROS production and corresponding reduction in the activities of antioxidative enzymes (SOD, CAT, APX and GR) in CSE2 mutant strain in contrast to wild type Anabaena 7120 also suggested the possible involvement of NtcA in protection against oxidative stress in iron deficiency. NtcA has no impact on the expression of furB and furC in spite of presence of consensus NtcA binding site (NBS) and ?10 boxes in their promoter. NtcA also regulates the thylakoid arrangement as well as related photosynthetic and respiration rates under iron deficiency in Anabaena 7120. Overall results suggested that NtcA regulates iron acquisition and in turn protect Anabaena cells from the damaging effects of oxidative stress induced under iron deficiency.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.