Reversible hydrogenase in Anabaena variabilis ATCC 29413

The presence and localization of a reversible hydrogenase in non-N₂-fixing cells of the filamentous cyanobacterium Anabaena variabilis were investigated by in vitro activity measurements, native-PAGE/activity stain, SDS-PAGE/Western immunoblots, and immunogold localization. Reversible hydrogenase activity was induced approximately 100-fold by sparging the cell suspensions with a mixture of 99% argon and 1% CO₂ for 20–26 h. Native-PAGE/activity stain demonstrated the presence of an in vitro functional enzyme with an apparent molecular mass of 118 kDa. Native-PAGE/Western immunoblots, using polyclonal antisera directed against purified hydrogenase from the purple sulphur bacterium Thiocapsa roseopersicina, detected two native proteins with molecular masses of 118 and 133 kDa, respectively. SDS-PAGE/Western immunoblots confirmed the presence of a single polypeptide with a molecular mass of approximately 40 kDa in both induced and non-induced cells. Immunocytolocalization experiments using ultrathin sections again demonstrated the presence of hydrogenase in both induced and non-induced cells. A higher specific labeling was associated with the thylakoid regions, which, using an image analyzer, was calculated to be approximately 4 x higher per cell area compared to in the centroplasm. It is suggested that anaerobic incubation induces higher reversible hydrogenase activity, regulated mainly at the level of activating (pre)existing form(s) of inactive enzyme(s)/protein(s), maybe in combination with synthesis of additional subunit(s).     

Complete genome sequence of Anabaena variabilis ATCC 29413

Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Anabaena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40^° C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence.     

Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413

Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase. A. variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M. Competition experiments with other oxyanions suggested that the molybdate-transport system of A. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system in A. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strain Anabaena sp. PCC 7120 transported (99)Mo in a very similar way to A. variabilis.     

Role of Carbonic Anhydrase in Photosynthesis of Blue-Green Alga (Cyanobacterium) Anabaena variabilis ATCC 29413

The affinity for NaHCO₃ (CO₂) in photosynthesis of Anabaenavariabilis ATCC 29413 was much higher in the cells grown underordinary air (low-CO₂ cells) than in those grown in air enrichedwith 2–4% CO₂ (high-CO₂ cells) (pH 8.0, 25?C). Ethoxyzolamide(50 µM) increased the K_m(NaHCO₃ in low-CO₂ cells aboutnine times (from 14.3 to 125), while the maximum rate of photosynthesisdecreased about 20%. When high-CO₂ cells were transferred tolow-CO₂ conditions, carbonic anhydrase (CA) activity increased,while K_m(NaHCO₃) in photosynthesis decreased from 140 to 30µM within about 5 h. The addition of CA to the suspensionof both high- and low-CO₂ cells enhanced the rates of photosyntheticO₂ evolution under CO₂-limiting conditions. The rate of ¹⁴CO₂fixation was much faster than that of H¹⁴CO₃^– fixation.The former reaction was greatly suppressed, while the latterwas enhanced by the addition of CA. These results indicate thatthe active species of inorganic carbon utilized for photosynthesiswas free CO₂ irrespective of the CO₂ concentration given duringgrowth. It is suggested that CA plays an active role in increasingthe affinity for CO₂ in photosynthesis of low-CO₂ cells of thisblue-green alga. (Received January 24, 1984; Accepted October 22, 1984)     

High-affinity vanadate transport system in the cyanobacterium Anabaena variabilis ATCC 29413

High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 x 10(-9) M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri.     

Short-term effect of ammonia on nitrogenase activity of Anabaena variabilis (ATCC29413)

Abstract Intact filaments of the cyanobacterium Anabaena variabilis switch off nitrogenase activity very rapidly upon addition of NH₄Cl when incubated in an alkaline environment (pH 10.0) permitting a fast NH₃-influx into the cells. When assayed in cell-free extracts (prepared from ammonia-treated filaments), nitrogenase remains inhibited in the presence of an ATP-regenerating system. Furthermore, l -methionine- d,l -sulfoximine, an inhibitor of glutamine synthetase, added to the filaments, prevents inactivation of nitrogenase by ammonia, showing that ammonia is not the compound directly responsible for nitrogenase switch-off.     

Molybdate transport and its effect on nitrogen utilization in the cyanobacterium Anabaena variabilis ATCC 29413

Molybdenum is an essential component of the cofactors of many metalloenzymes including nitrate reductase and Mo-nitrogenase. The cyanobacterium Anabaena variabilis ATCC 29413 uses nitrate and atmospheric N2 as sources of nitrogen for growth. Two of the three nitrogenases in this strain are Mo-dependent enzymes, as is nitrate reductase; thus, transport of molybdate is important for growth of this strain. High-affinity transport of molybdate in A. variabilis was mediated by an ABC-type transport system encoded by the products of modA and modBC. The modBC gene comprised a fused orf including components corresponding to modB and modC of Escherichia coli. The deduced ModC part of the fused gene lacked a recognizable molybdate-binding domain. Expression of modA and modBC was induced by starvation for molybdate. Mutants in modA or modBC were unable to grow using nitrate or Mo-nitrogenase. Growth using the alternative V-nitrogenase was not impaired in the mutants. A high concentration of molybdate (10 microM) supported normal growth of the modBC mutant using the Nif1 Mo-nitrogenase, indicating that there was a low-affinity molybdate transport system in this strain. The modBC mutant did not detectably transport low concentrations of 99Mo (molybdate), but did transport high concentrations. However, such transport was observed only after cells were starved for sulphate, suggesting that an inducible sulphate transport system might also serve as a low-affinity molybdate transport system in this strain.     

Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.     

Purification and characterization of the photosystem I complex from the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

A photoactive photosystem I complex has been purified from the filamentous, nitrogen-fixing cyanobacterium Anabaena variabilis ATCC 29413. Cells were broken using glass beads, and the membrane fraction was solubilized with beta-dodecyl maltoside followed by two rounds of fast protein liquid chromatography on anion exchange columns. The polypeptide composition of the isolated complex was determined by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis and N-terminal amino acid sequencing of the fractionated proteins. The purified complex consists of at least 11 proteins, identified as the PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK, PsaL, and PsaN proteins. The spectrum of the flash-induced absorbance change measured between 670 and 830 nm shows that the purified complex contains 99 +/- 11 chlorophyll a molecules per P700, the primary donor in photosystem I. The kinetics of the rereduction of oxidized P700 following an actinic flash indicate that forward electron transfer from P700 to the FA/FB iron-sulfur center acceptors is functional in the isolated complex.     

In vitro activation of dinitrogenase reductase from the cyanobacterium Anabaena variabilis (ATCC 29413).

Nitrogenase of the heterocystous cyanobacterium Anabaena variabilis was inactivated in vivo (S. Reich, H. Almon, and P. B?ger, FEMS Microbiol. Lett. 34:53-56, 1986). Partially purified and modified (inactivated) dinitrogenase reductase (Fe-protein) of such cells was reactivated by isolated membrane fractions of A. variabilis or of Rhodospirillum rubrum, and acetylene reduction was measured. Reactivation requires ATP, Mg2+, and Mn2+. The activating principle is localized in the heterocyst and was found effective only when prepared from cells exhibiting active nitrogenase. It also restores the activity of modified Fe-protein from R. rubrum.     

H(2) photoproduction by batch culture of Anabaena variabilis ATCC 29413 and its mutant PK84 in a photobioreactor.

Hydrogen production by Anabaena variabilis ATCC 29413 and of its mutant PK84, grown in batch cultures, was studied in a photobioreactor. The highest volumetric H(2) production rates of native and mutant strains were found in cultures grown at gradually increased irradiation. The native strain evolved H(2) only under an argon atmosphere with the actual rate as high as the potential rate (measured in small vials under optimal conditions). In this case 61% of oxygenic photosynthesis was used for H(2) production. In contrast the mutant PK84 produced H(2) during growth under CO(2)-enriched air. Under these conditions at the maximum rate of H(2) production (10 mL h(-1) L(-1)), 13% of oxygenic photosynthesis was used for H(2) production and the actual H(2) production was only 33% of the potential. Under an atmosphere of 98% argon + 2% CO(2) actual H(2) production by mutant PK84 was 85% of the potential rate and 66% of oxygenic photosynthesis was used for H(2) production. Hydrogen production under argon + CO(2) by the mutant was strictly light-dependent with saturation at about 300 microE m(-2) s(-1). However, the rate of photosynthesis was not saturated at this irradiation. At limiting light intensities (below 250 microE m(-2) s(-1)) 33-58% of photosynthesis was used for H(2) production. Hydrogen evolution by PK84 under air + 2% CO(2) was also stimulated by light; but was not saturated at 332 microE m(-2) s(-1) and did not cease completely in darkness. The rate of oxygen photoevolution was also not saturated. A mechanism for increasing cyanobacterial hydrogen production is proposed.     

A mutant of the cyanobacterium Anabaena variabilis ATCC 29413 lacking cyanophycin synthetase: growth properties and ultrastructural aspects

The gene cphA encoding cyanophycin synthetase was interrupted in Anabaena variabilis ATCC 29413 by insertional mutagenesis. The mutant lacked cyanophycin granules and the polar nodules of heterocysts. The mutant grew as fast as the wild-type irrespective of the nitrogen source at low light intensity whereas growth on N(2) was somewhat reduced in high light. It is concluded that cyanophycin metabolism and polar nodules are not essential for aerobic N(2) fixation.     

Valine 77 of heterocystous ferredoxin FdxH2 in Anabaena variabilis strain ATCC 29413 is critical for its oxygen sensitivity

Ferredoxins are small iron sulfur proteins necessary for electron donation. FdxH1 and FdxH2 are associated with two different nif gene clusters where they transfer electrons for the reduction of nitrogenase complex. FdxH1 was observed to be stable towards oxygen, whereas, FdxH2 was relatively unstable. We had identified the amino acid involved in oxygen sensitivity of ferredoxin protein using protein modeling. The exchange of valine to leucine at position 77 was critical for ferredoxin proteins in relation to its oxygen sensitivity. This exchange leads to a longer side chain, which inhibits the accessibility of oxygen to the iron sulfur cluster. Site directed mutagenesis and in vitro experiments confirms that valine indeed is involved in the oxygen sensitivity. The exchange of leucine to valine in FdxH1 makes it oxygen unstable. Thus, from the above results we can conclude that the position of leucine at position 77 is critical for oxygen sensitivity of ferredoxin and protein modeling can be used to identify specific amino acids in other oxygen-sensitive proteins.