Properties of the glyceraldehyde-3-P dehydrogenase in heterocysts and vegetative cells of cyanobacteria

Abstract Glyceraldehyde-3-P dehydrogenase (GAPDH) in heterocysts and vegetative cells of 3 N₂-fixing cyanobacteria was found to utilize both NAD⁺ and NADP⁺. The enzyme activity was enhanced by thiols (glutathione, reduced lipoic acid and dithiothreitol). GAPDH of the 3 cyanobacterial species was not activated by thioredoxin. Heterocysts have now been shown to possess all the enzymes of glycolysis and the tricarboxylic acid cycle to convert glyceraldehyde-3-phosphate (GAP) to oxoglutarate and glutamate. The GAPDH reaction is a major source for the generation of NADH, which is oxidized by a thylakoid-bound NADH:plastoquinone oxidoreductase in heterocysts.     

Cyanolichens can have both cyanobacteria and green algae in a common layer as major contributors to photosynthesis

Background and Aims

Cyanolichens are usually stated to be bipartite (mycobiont plus cyanobacterial photobiont). Analyses revealed green algal carbohydrates in supposedly cyanobacterial lichens (in the genera Pseudocyphellaria, Sticta and Peltigera). Investigations were carried out to determine if both cyanobacteria and green algae were present in these lichens and, if so, what were their roles.

Methods

The types of photobiont present were determined by light and fluorescence microscopy. Small carbohydrates were analysed to detect the presence of green algal metabolites. Thalli were treated with selected strengths of Zn²⁺ solutions that stop cyanobacterial but not green algal photosynthesis. CO₂ exchange was measured before and after treatment to determine the contribution of each photobiont to total thallus photosynthesis. Heterocyst frequencies were determined to clarify whether the cyanobacteria were modified for increased nitrogen fixation (high heterocyst frequencies) or were normal, vegetative cells.

Key Results

Several cyanobacterial lichens had green algae present in the photosynthetic layer of the thallus. The presence of the green algal transfer carbohydrate (ribitol) and the incomplete inhibition of thallus photosynthesis upon treatment with Zn²⁺ solutions showed that both photobionts contributed to the photosynthesis of the lichen thallus. Low heterocyst frequencies showed that, despite the presence of adjacent green algae, the cyanobacteria were not altered to increase nitrogen fixation.

Conclusions

These cyanobacterial lichens are a tripartite lichen symbiont combination in which the mycobiont has two primarily photosynthetic photobionts, ‘co-primary photobionts’, a cyanobacterium (dominant) and a green alga. This demonstrates high flexibility in photobiont choice by the mycobiont in the Peltigerales. Overall thallus appearance does not change whether one or two photobionts are present in the cyanobacterial thallus. This suggests that, if there is a photobiont effect on thallus structure, it is not specific to one or the other photobiont.     

FraC/FraD-dependent intercellular molecular exchange in the filaments of a heterocyst-forming cyanobacterium, Anabaena sp

The filamentous, heterocyst‐forming cyanobacteria are multicellular organisms in which two different cell types, the CO₂‐fixing vegetative cells and the N₂‐fixing heterocysts, exchange nutrients and regulators. In Anabaena sp. strain PCC 7120, inactivation of sepJ or genes in the fraC operon (fraC, fraD and fraE) produce filament fragmentation. SepJ, FraC and FraD are cytoplasmic membrane proteins located in the filament's intercellular septa that are needed for intercellular exchange of the fluorescent tracer calcein (622 Da). Transmission electron microscopy showed an alteration in the heterocyst cytoplasmic membrane at the vegetative cell‐heterocyst septa in ΔfraC and ΔfraD mutants. Immunogold labelling of FraD confirmed its localization in the intercellular septa and clearly showed the presence of part of the protein between the cytoplasmic membranes of the adjacent cells. This localization seemed to be affected in the ΔfraC mutant but was not impaired in a ΔsepJ mutant. Intercellular transfer of a smaller fluorescent tracer, 5‐carboxyfluorescein (374 Da), was largely impaired in ΔfraC, ΔfraD and double ΔfraC‐ΔfraD mutants, but much less in the ΔsepJ mutant. These results show the existence in the Anabaena filaments of a FraC/FraD‐dependent intercellular molecular exchange that does not require SepJ.     

Redox control of copper homeostasis in cyanobacteria

Copper is essential for all living organisms but is toxic when present in excess. Therefore organisms have developed homeostatic mechanism to tightly regulate its cellular concentration. In a recent study we have shown that CopRS two-component system is essential for copper resistance in the cyanobacterium Synechocystis sp PCC 6803. This two-component regulates expression of a heavy-metal RND type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to an excess of copper in the media. We have also observed that both operons are induced under condition that reduces the photosynthetic electron flow and this induction depends on the presence of the copper-protein, plastocyanin. These findings, together with CopS localization to the thylakoid membrane and its periplasmic domain being able to bind copper directly, suggest that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen.     

Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay

Impact of ultraviolet-B radiation in causing the damages to the DNA of the cyanobacterium, Anabaena strain BT2 has been investigated. Exposure of genomic DNA (in vitro) to UV-B radiation for 1 h did not cause any shift in the absorption peak (lambda(max)) but more than 30% increase in absorbance was noticed in comparison to untreated control DNA (no exposure to UV-B). This increase in absorbance in a way may be comparable to typical hypochromic effect but there was no decrease in absorbance following transfer of UV-B-treated DNA to fluorescent light or in the dark. That the damaging effect of UV-B radiation on native structure of DNA is indeed real was also evident from the PCR-based assay such as RAPD, rDNA amplification, and ARDRA. Template activity of UV-B-treated genomic DNA was drastically inhibited, there was no amplification in RAPD assay after prior exposure of DNA to UV-B for 60 min. Only one band of approximately 400 bp was observed even after 60 min of exposure which suggests that certain segment of DNA strand is resistant to UV-B effects. Similar to the effects on RAPD profile, amplification of rDNA was significantly inhibited following exposure of genomic DNA to UV-B. Our findings clearly demonstrate that UV-B does affect the DNA of cyanobacteria and the killings of these microbes might be due to the irreversible damages caused to DNA by this high energy radiation. It is felt that PCR assay may be conveniently used for screening the damages caused to DNA by UV-B radiation in cyanobacteria and other microorganisms.     

Biosynthesis of a linoleic acid allylic epoxide: mechanistic comparison with its chemical synthesis and leukotriene A biosynthesis

Biosynthesis of the leukotriene A (LTA) class of epoxide is a lipoxygenase-catalyzed transformation requiring a fatty acid hydroperoxide substrate containing at least three double bonds. Here, we report on biosynthesis of a dienoic analog of LTA epoxides via a different enzymatic mechanism. Beginning with homolytic cleavage of the hydroperoxide moiety, a catalase/peroxidase-related hemoprotein from Anabaena PCC 7120, which occurs in a fusion protein with a linoleic acid 9R-lipoxygenase, dehydrates 9R-hydroperoxylinoleate to a highly unstable epoxide. Using methods we developed for isolating extremely labile compounds, we prepared and purified the epoxide and characterized its structure as 9R,10R-epoxy-octadeca-11E,13E-dienoate. This epoxide hydrolyzes to stable 9,14-diols that were reported before in linoleate autoxidation (Hamberg, M. 1983. Autoxidation of linoleic acid: Isolation and structure of four dihydroxy octadecadienoic acids. Biochim. Biophys. Acta 752: 353–356) and in incubations with the Anabaena enzyme (Lang, I., C. Göbel, A. Porzel, I. Heilmann, and I. Feussner. 2008. A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120. Biochem. J. 410: 347–357). We also prepared an equivalent epoxide from 13S-hydroperoxylinoleate using a “biomimetic” chemical method originally described for LTA₄ synthesis and showed that like LTA₄, the C18.2 epoxide conjugates readily with glutathione, a potential metabolic fate in vivo. We compare and contrast the mechanisms of LTA-type allylic epoxide synthesis by lipoxygenase, catalase/peroxidase, and chemical transformations. These findings provide new insights into the reactions of linoleic acid hydroperoxides and extend the known range of catalytic activities of catalase-related hemoproteins.     

Diverse roles of the GlcP glucose permease in free-living and symbiotic cyanobacteria

Certain cyanobacteria can form symbiotic associations with plants, where the symbiont supplies the plant partner with nitrogen and in return obtains sugars. We recently showed that in the symbiotic cyanobacterium Nostoc punctiforme, a glucose specific permease, GlcP, is necessary for the symbiosis to be formed. Results presented here from growth yield measurements of mutant strains with inactivated or overexpressing sugar transporters suggest that GlcP could be induced by a symbiosis specific substance. We also discuss that the transporter may have a role other than nutritional once the symbiosis is established, i.e., during infection, and more specifically in the chemotaxis of the symbiont. Phylogenetic analysis shows that the distribution of GlcP among cyanobacteria is likely influenced by horizontal gene transfer, but also that it is not correlated with symbiotic competence. Instead, regulatory patterns of the transporter in Nostoc punctiforme likely constitute symbiosis specific adaptations.     

Bottlenose dolphins exchange signature whistles when meeting at sea

The bottlenose dolphin, Tursiops truncatus, is one of very few animals that, through vocal learning, can invent novel acoustic signals and copy whistles of conspecifics. Furthermore, receivers can extract identity information from the invented part of whistles. In captivity, dolphins use such signature whistles while separated from the rest of their group. However, little is known about how they use them at sea. If signature whistles are the main vehicle to transmit identity information, then dolphins should exchange these whistles in contexts where groups or individuals join. We used passive acoustic localization during focal boat follows to observe signature whistle use in the wild. We found that stereotypic whistle exchanges occurred primarily when groups of dolphins met and joined at sea. A sequence analysis verified that most of the whistles used during joins were signature whistles. Whistle matching or copying was not observed in any of the joins. The data show that signature whistle exchanges are a significant part of a greeting sequence that allows dolphins to identify conspecifics when encountering them in the wild.     

Extracellular polypeptides ofAnabaena L-31: Evidence for their role in regulation of heterocyst formation

Extracellular polypeptides released by both N₂-grown [peptide I] and NO₃-grown [peptide II]Anabaena L-31 have molecular weight of approximately 3,500 but have distinctly different amino acid composition. Acid hydrolysis of the peptide I fraction (obtained by separation on Sephadex G-25) yielded ten amino acids whereas that from peptide II fraction yielded only 3 amino acids. On addition to a freshly inoculated N₂-grown culture, the peptide I fraction stimulated pro-heterocyst and to a lesser extent heterocyst differentiation, whereas the peptide II fraction strongly inhibited differentiation. The inhibitory effect of polypeptide II fraction could not be relieved by methionine sulphoximine, which by itself enhances differentiation, but was greatly relieved by addition of the peptide I fraction. The data suggest but does not prove, thatAnabaena L-31 synthesises “inducer” or “inhibitor” peptides which could possibly control pattern formation.     

Utilization of the cyanobacteria Anabaena sp. CH1 in biological carbon dioxide mitigation processes

Before switching totally to alternative fuel stage, CO₂ mitigation process has considered a transitional strategy for combustion of fossil fuels inevitably. In comparison to other CO₂ mitigation options, such as oceanic or geologic injection, the biological photosynthetic process would present a far superior and sustainable solution under both environmental and social considerations. The utilization of the cyanobacteria Anabaena sp. CH1 in carbon dioxide mitigation processes is analyzed in our research. It was found that an original developed photobioreactor with internal light source exhibits high light utilization. Anabaena sp. CH1 demonstrates excellent CO₂ tolerance even at 15% CO₂ level. This enables flue gas from power plant to be directly introduced to Anabaena sp. CH1 culture. Double light intensity and increased 47% CO₂ bubble retention time could enhance CO₂ removal efficiencies by 79% and 67%, respectively. A maximum CO₂ fixation rate of 1.01 g CO₂ L⁻¹ day⁻¹ was measured experimentally.     

The 2.0A resolution structure of the catalytic portion of a cyanobacterial membrane-bound manganese superoxide dismutase

Cyanobacteria are shown to be unique in containing membrane-bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0A resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OH(x)) a five-coordinated trigonal bipyramidal geometry is formed, with OH(x) and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OH(x). The ligands including OH(x) are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn cdots, three dots, centered Mn distance (18.4A) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented.     

Cellular and molecular remodelling of a host cell for vertical transmission of bacterial symbionts

Various insects require intracellular bacteria that are restricted to specialized cells (bacteriocytes) and are transmitted vertically via the female ovary, but the transmission mechanisms are obscure. We hypothesized that, in the whitefly Bemisia tabaci, where intact bacteriocytes (and not isolated bacteria) are transferred to oocytes, the transmission mechanism would be evident as cellular and molecular differences between the nymph (pre-adult) and adult bacteriocytes. We demonstrate dramatic remodelling of bacteriocytes at the developmental transition from nymph to adulthood. This transition involves the loss of cell–cell adhesion, high division rates to constant cell size and onset of cell mobility, enabling the bacteriocytes to crawl to the ovaries. These changes are accompanied by cytoskeleton reorganization and changes in gene expression: genes functioning in cell–cell adhesion display reduced expression and genes involved in cell division, cell motility and endocytosis/exocytosis have elevated expression in adult bacteriocytes, relative to nymph bacteriocytes. This study demonstrates, for the first time, how developmentally orchestrated remodelling of gene expression and correlated changes in cell behaviour underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends.     

Integration of molecular and physiological models to explain time of anthesis in wheat

Background and Aims

A model to predict anthesis time of a wheat plant from environmental and genetic information requires integration of current concepts in physiological and molecular biology. This paper describes the structure of an integrated model and quantifies its response mechanisms.

Methods

Literature was reviewed to formulate the components of the model. Detailed re-analysis of physiological observations are utilized from a previous publication by the second two authors. In this approach measurements of leaf number and leaf and primordia appearance of near isogenic lines of spring and winter wheat grown for different durations in different temperature and photoperiod conditions are used to quantify mechanisms and parameters to predict time of anthesis.

Key Results

The model predicts the time of anthesis from the length of sequential phases: 1, embryo development; 2, dormant; 3, imbibed/emerging; 4, vegetative; 5, early reproductive; 6, pseudo-stem extension; and 7, ear development. Phase 4 ends with vernalization saturation (VS), Phase 5 with terminal spikelet (TS) and Phase 6 with flag leaf ligule appearance (FL). The durations of Phases 4 and 5 are linked to the expression of Vrn genes and are calculated in relation to change in Haun stage (HS) to account for the effects of temperature per se. Vrn1 must be expressed to sufficient levels for VS to occur. Vrn1 expression occurs at a base rate of 0·08/HS in winter ‘Batten’ and 0·17/HS in spring ‘Batten’ during Phases 1, 3 and 4. Low temperatures promote expression of Vrn1 and accelerate progress toward VS. Our hypothesis is that a repressor, Vrn4, must first be downregulated for this to occur. Rates of Vrn4 downregulation and Vrn1 upregulation have the same exponential response to temperature, but Vrn4 is quickly upregulated again at high temperatures, meaning short exposure to low temperature has no impact on the time of VS. VS occurs when Vrn1 reaches a relative expression of 0·76 and Vrn3 expression begins. However, Vrn2 represses Vrn3 expression so Vrn1 must be further upregulated to repress Vrn2 and enable Vrn3 expression. As a result, the target for Vrn1 to trigger VS was 0·76 in 8-h photoperiods (Pp) and increased at 0·026/HS under 16-h Pp as levels of Vrn2 increased. This provides a mechanism to model short-day vernalization. Vrn3 is expressed in Phase 5 (following VS), and apparent rates of Vrn3 expression increased from 0·15/HS at 8-h Pp to 0·33/HS at 16-h Pp. The final number of leaves is calculated as a function of the HS at which TS occurred (TS^HS): 2·86 + 1·1 × TS^HS. The duration of Phase 6 is then dependent on the number of leaves left to emerge and how quickly they emerge.

Conclusions

The analysis integrates molecular biology and crop physiology concepts into a model framework that links different developmental genes to quantitative predictions of wheat anthesis time in different field situations.     

The ManR specifically binds to the promoter of a Nramp transporter gene in Anabaena sp. PCC 7120: a novel regulatory DNA motif in cyanobacteria

The Anabaena sp. PCC 7120 ManR and a homologous protein of MntH were identified by BLAST search. Recombinant ManR protein was overexpressed in Escherichia coli and purified by an immobilized metal (Ni) affinity chromatography. Electrophoretic mobility shift assays revealed that ManR specifically bound to the promoter region of the mntH gene. Site-directed mutagenesis experiments demonstrated that the specific recognition site for ManR is TATGAAAAGAATATGAGAA, which is composed of two direct repeats of the consensus sequence (T/A)ATGA(G/A)A(A/G). This is a novel regulatory DNA motif in cyanobacteria, indicating that the expression of mntH was regulated by a two-component Mn(2+)-Sensing System containing ManR in Anabaena sp. PCC 7120. To date, this specific pathway of regulating mntH expression has only been found in cyanobacteria.     

Daphnia vertical distribution and the presence of toxic cyanobacteria

Daphnia can alter its vertical position in the water column in response to chemical cues from predators. In this study we tested the hypothesis that a Daphnia pulex clone with little evolutionary exposure to cyanobacteria would move away from patches of cyanobacteria (Anabaena affinis and A. flos-aquae) which contain potent endotoxins. Daphnia was censused at 2 h intervals for 6 h in laboratory columns in which there was a steep vertical gradient of cyanobacteria. Data were analyzed by repeated-measures ANOVA. Control (no Anabaena) and experimental columns showed no significant differences in Daphnia distributions. Daphnia in experimental columns frequently moved into areas with dense concentrations of Anabaena and stayed there for long periods of time. Our results show that this D. pulex clone does not exhibit a rapid (within 6 h) avoidance response to toxic Anabaena.     

Fingerprinting of freshly separated and cultured cyanobionts from different Azolla species using morphological and molecular markers

Differentiation of freshly separated (uncultured) and cultured cyanobionts of Azolla species was carried out employing morphological markers and profiles generated using SDS-PAGE and PCR–RFLP of 16S rRNA. The cell dimensions of vegetative cells, heterocysts and heterocyst frequency (25–28%) of the freshly separated cyanobionts were distinctly higher than that those recorded for the cultured cyanobionts (7–10%). The SDS-PAGE profiles of whole cell proteins of cultured cyanobionts (comprising 28–30 bands) and those of freshly separated cyanobionts (consisting of 6–10 bands) exhibited distinct differences and unique bands. AluI was able to discriminate freshly separated cyanobionts from cultured cyanobionts of same species of Azolla. The profiles of cyanobionts (freshly separated and cultured) of A. rubra (RU6503) generated using the restriction enzyme AluI were distinctly different from other cyanobionts and unique bands were observed in both cyanobionts. The cultured cyanobionts from A. microphylla (MI4018), A. filiculoides (FI1001) and A. pinnata (PP7001) showed the presence of a distinct band of 450, 622 and 307 bp, respectively. Three common bands of 500, 400 and 275 bp were recorded in the AluI restriction profiles of all the freshly separated cyanobionts. 16S rRNA PCR–RFLP analyses confirmed the existence of primary and secondary cyanobionts in the leaf cavities of different Azolla species. These techniques can be utilized for discriminating between freshly separated and cultured cyanobionts of Azolla and provide reliable fingerprints for these strains.     

Synonymous codon usage in Thermosynechococcus elongatus (cyanobacteria) identifies the factors shaping codon usage variation

Analysis of synonymous codon usage pattern in the genome of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 using multivariate statistical analysis revealed a single major explanatory axis accounting for codon usage variation in the organism. This axis is correlated with the GC content at third base of synonymous codons (GC3s) in correspondence analysis taking T. elongatus genes. A negative correlation was observed between effective number of codons i.e. Nc and GC3s. Results suggested a mutational bias as the major factor in shaping codon usage in this cyanobacterium. In comparison to the lowly expressed genes, highly expressed genes of this organism possess significantly higher proportion of pyrimidine-ending codons suggesting that besides, mutational bias, translational selection also influenced codon usage variation in T. elongatus. Correspondence analysis of relative synonymous codon usage (RSCU) with A, T, G, C at third positions (A3s, T3s, G3s, C3s, respectively) also supported this fact and expression levels of genes and gene length also influenced codon usage. A role of translational accuracy was identified in dictating the codon usage variation of this genome. Results indicated that although mutational bias is the major factor in shaping codon usage in T. elongatus, factors like translational selection, translational accuracy and gene expression level also influenced codon usage variation.     

Alternative pathways for phosphonate metabolism in thermophilic cyanobacteria from microbial mats

Synechococcus sp. represents an ecologically diverse group of cyanobacteria found in numerous environments, including hot-spring microbial mats, where they are spatially distributed along thermal, light and oxygen gradients. These thermophiles engage in photosynthesis and aerobic respiration during the day, but switch to fermentative metabolism and nitrogen fixation at night. The genome of Synechococcus OS-B′, isolated from Octopus Spring (Yellowstone National Park) contains a phn gene cluster encoding a phosphonate (Phn) transporter and a C–P lyase. A closely related isolate, Synechococcus OS-A, lacks this cluster, but contains genes encoding putative phosphonatases (Phnases) that appear to be active only in the presence of the Phn substrate. Both isolates grow well on several different Phns as a sole phosphorus (P) source. Interestingly, Synechococcus OS-B′ can use the organic carbon backbones of Phns for heterotrophic growth in the dark, whereas in the light this strain releases organic carbon from Phn as ethane or methane (depending on the specific Phn available); Synechococcus OS-A has neither of these capabilities. These differences in metabolic strategies for assimilating the P and C of Phn by two closely related Synechococcus spp. are suggestive of niche-specific constraints in the evolution of nutrient assimilation pathways and syntrophic relationships among the microbial populations of the hot-spring mats. Thus, it is critical to evaluate levels of various P sources, including Phn, in thermally active habitats and the potential importance of these compounds in the biogeochemical cycling of P and C (some Phn compounds also contain N) in diverse terrestrial environments.