A HupS-GFP fusion protein demonstrates a heterocyst-specific localization of the uptake hydrogenase in Nostoc punctiforme (original) (raw)
Related papers
2010
All diazotrophic filamentous cyanobacteria contain an uptake hydrogenase that is involved in the reoxidation of H 2 produced during N 2-fixation. In Nostoc punctiforme ATCC 29133, N 2-fixation takes place in the microaerobic heterocysts, catalysed by a nitrogenase. Although the function of the uptake hydrogenase may be closely connected to that of nitrogenase, the localization in cyanobacteria has been under debate. Moreover, the subcellular localization is not understood. To investigate the cellular and subcellular localization of the uptake hydrogenase in N. punctiforme, a reporter construct consisting of the green fluorescent protein (GFP) translationally fused to HupS, within the complete hupSL operon, was constructed and transferred into N. punctiforme on a self-replicative vector by electroporation. Expression of the complete HupS-GFP fusion protein was confirmed by Western blotting using GFP antibodies. The N. punctiforme culture expressing HupS-GFP was examined using laser scanning confocal microscopy, and fluorescence was exclusively detected in the heterocysts. Furthermore, the fluorescence in mature heterocysts was localized to several small or fewer large clusters, which indicates a specificity of the subcellular localization of the uptake hydrogenase.
Fems Microbiology Letters, 2010
All diazotrophic filamentous cyanobacteria contain an uptake hydrogenase that is involved in the reoxidation of H 2 produced during N 2-fixation. In Nostoc punctiforme ATCC 29133, N 2-fixation takes place in the microaerobic heterocysts, catalysed by a nitrogenase. Although the function of the uptake hydrogenase may be closely connected to that of nitrogenase, the localization in cyanobacteria has been under debate. Moreover, the subcellular localization is not understood. To investigate the cellular and subcellular localization of the uptake hydrogenase in N. punctiforme, a reporter construct consisting of the green fluorescent protein (GFP) translationally fused to HupS, within the complete hupSL operon, was constructed and transferred into N. punctiforme on a self-replicative vector by electroporation. Expression of the complete HupS-GFP fusion protein was confirmed by Western blotting using GFP antibodies. The N. punctiforme culture expressing HupS-GFP was examined using laser scanning confocal microscopy, and fluorescence was exclusively detected in the heterocysts. Furthermore, the fluorescence in mature heterocysts was localized to several small or fewer large clusters, which indicates a specificity of the subcellular localization of the uptake hydrogenase.
Applied and Environmental Microbiology, 2004
Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H 2 ), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O 2 , CO 2 , N 2 , and H 2 was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO 2 and O 2 . The amount of H 2 produced per molecule of N 2 fixed was found to vary with light conditions, high light giving a greater increase in H 2 production than N 2 fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H 2 produced per molecule of N 2 fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO 2 , caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 mol (mg of chlorophyll a) ؊1 h ؊1 to 9 mol (mg of chlorophyll a) ؊1 h ؊1 after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.
Journal of Bacteriology, 2014
Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H 2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H 2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (⌬hupL), and we determined how this would affect the amount of H 2 produced. The ⌬hupL mutant demonstrated virtually no nitrogenase activity or H 2 production when grown under N 2-fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O 2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N 2-fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H 2 produced by the nitrogenase.
Applied and Environmental Microbiology, 2014
We have investigated two approaches to enhance and extend H 2 photoproduction yields in heterocystous, N 2 -fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO 2 supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H 2 production over time. Our results demonstrated that alginate-entrapped ⌬hupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ⌬hupL mutant, and Calothrix strain 336/3 films were supplemented with N 2 by periodic treatments of air, or air plus CO 2 . These treatments restored the photosynthetic activity of the cells and led to a high level of H 2 production in Calothrix 336/3 and ⌬hupL cells (except for the treatment air plus CO 2 ) but not in the Anabaena PCC 7120 strain (for which H 2 yields did not change after air treatments). The highest H 2 yield was obtained by the air treatment of ⌬hupL cells. Notably, the supplementation of CO 2 under an air atmosphere led to prominent symptoms of N deficiency in the ⌬hupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O 2 from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.
Fems Microbiology Letters, 2009
In N2-fixing cyanobacteria, the reduction of N2 to NH3 is coupled with the production of molecular hydrogen, which is rapidly consumed by an uptake hydrogenase, an enzyme that is present in almost all diazotrophic cyanobacteria. The cellular and subcellular localization of the cyanobacterial uptake hydrogenase remains uncertain, and it is definitely strain dependent. Previous studies focused mainly on heterocystous cyanobacteria and used heterologous antisera. The present work represents the first effort to establish the subcellular localization of the uptake hydrogenase in a N2-fixing filamentous nonheterocystous cyanobacterium, Lyngbya majuscula CCAP 1446/4, using the first antiserum produced against a cyanobacterial uptake hydrogenase. The data obtained revealed higher specific labelling associated with the thylakoid membranes of L. majuscula, reinforcing the idea that the cyanobacterial uptake hydrogenase is indeed a membrane-bound protein. For comparative purposes, the localization of the uptake hydrogenase was also investigated in two distinct heterocystous cyanobacterial strains, and while in Nostoc sp. PCC 7120 the labelling was only observed in the heterocysts, in Nostoc punctiforme, the presence of uptake hydrogenase antigens was detected in both the vegetative cells and heterocysts, corresponding most probably to an inactive and an active form of the enzyme.
Nitrogen fixation and hydrogen uptake in four cyanobacteria
International Journal of Hydrogen Energy, 1995
Anabaena uariabilis, Nostoc spongiaeforme, Westieliopsis prolifica, and Nostoc sp. isolated from diverse local habitats fixed nitrogen under aerobic conditions, but the nitrogenase activity was greater in anaerobic conditions. Nitrogenase activity was highest in fluorescent light in the case of A. uariabilis, F+! pro/i&n and Nostoc sp., but that of N. spongiaeforme was higher in incandescent light. N. spongiaqforme showed high nitrogenase activity in blue light also, whereas the other three species had stronger activity in red light than in blue light. A concentration of H, up to 20% enhanced the nitrogenase activity, but 25% was inhibitory in all of the four organisms. AU the four strains were found capable of utilizing exogenous hydrogen. N. spongiaeforme showed higher H, uptake activity both under aerobic and anaerobic conditions and its capacity for chromatic adaptation may partly account for such behaviour. Addition of DCMU inhibited hydrogen uptake in A. uariabilis, W prolifica and Nosroc sp. but not in hi. sponyiaefbrm~