Abstract
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).
Similar content being viewed by others
References
ArnonDI, McSwainBD, TsujimoteHY, WadaK (1974) Photochemical activity and components of membrane preparations from blue-green alga. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin. Biochim Biophys Acta 357: 231–245
AsadaY, KawamuraS (1984) Hydrogen evolution by Microcystis aeruginosa in darkness. Agric Biol Chem 48: 2595–2596
BotheH, KentemichT, HepingD (1991) Recent aspects on the hydrogenase-nitrogenase relationship in cyanobacteria. In: PolsinelliM, MaterassiR, VincenziniM (eds) Nitrogen fixation. Kluwer Academic, Dordrecht, pp 367–375
EisbrennerG, DistlerE, FloenerL, BotheH (1978) The occurrence of the hydrogenase in some blue-green algae. Arch Microbiol 118: 177–184
ErnstA, BögerP (1985) Glycogen accumulation and the induction of nitrogenase activity in the heterocyst-forming cyanobacterium Anabaena variabilis. J Gen Microbiol 131: 3147–3153
EwartGD, SmithGD (1989a) Purification and properties of soluble hydrogenase from the cyanobacterium Anabaena cylindrica. Arch Biochem Biophys 268: 327–337
EwartGD, SmithGD (1989b) An activity stain for the detection of cyanobacterial hydrogenase in polyacrylamide gels. Biochem Int 19: 89–97
EwartGD, SmithGD (1989c) Immunochemical analysis of the soluble hydrogenase from the cyanobacterium Anabaena cylindrica. Biochim Biophys Acta 997: 83–89
HouchinsJP (1984) The physiology and biochemistry of hydrogen metabolism in cyanobacteria. Biochim Biophys Acta 768: 227–255
HouchinsJP, BurrisRH (1981) Occurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120. J Bacteriol 146: 209–214
JanssonE, MartelA, LindbladP (1993) Ornithine cycle in Nostoc PCC 73102: stimulation of in vitro ornithine carbamoyl transferase activity by addition of arginine. Curr Microbiol 26: 75–78
KentemichT, BahnwegM, MayerF, BotheH (1989) Localization of the reversible hydrogenase in cyanobacteria. Z Naturforsch [C] 44: 384–391
KentemichT, CasperM, BotheH (1991) The reversible hydrogenase in Anacystis nidulans is a component of the cytoplasmatic membrane. Naturwissenschaften 78: 559–560
LambertGR, SmithGD (1980) Hydrogen metabolism by filamentous cyanobacteria. Arch Biochem Biophys 205: 36–50
LambertGR, SmithGD (1981) The hydrogen metabolism of cyanobacteria (blue-green algae). Biol Rev 56: 589–660
LindbladP (1992) Ornithine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase, and the effects of external carbon and ornithine on nitrogenase activity and citrulline synthesis. Physiol Plant 84: 275–282
LindbladP, GuerreroMG (1993) Nitrogen fixation and nitrate reduction. In: HallDO, ScurlockJMO, Bolhár-NordenkampfHR, LeegoodRC, LongSP (eds) Photosynthesis and production in a changing environment. A field and laboratory guide. Chapman & Hall, London, pp 299–312
LindbladP, SellstedtA (1990) Occurrence and localization of an uptake hydrogenase in the filamentous heterocystous cyanobacterium Nostoc PCC 73102. Protoplasma 159: 9–15
LindbladP, SellstedtA (1991) Immunogold localisation in TEM using uptake hydrogenase as an example. Microsc Anal 26: 29–31
MuthE (1988) Localization of the F420-reducing hydrogenase in Methanococcus voltae cells by immuno-gold technique. Arch Microbiol 150: 205–207
NiviereV, BernadacA, ForgetN, FernandezVM, HatchikianCE (1991) Localization of hydrogenase in Desulfovibrio gigas cells. Arch Microbiol 155: 579–586
OostJvan der, CoxRP (1989) Hydrogenase activity in nitrategrown cells of the unicellular cyanobacterium Cyanothece PCC 7822. Arch Microbiol 151: 40–43
OostJvan der, KanneworffWA, KrabK, KraayenhofR (1987) Hydrogen metabolism of three unicellular nitrogen-fixing cyanobacteria. FEMS Microbiol Lett 48: 41–45
PapenH, KentemichT, SchmüllingT, BotheH (1986) Hydrogenase activities in cyanobacteria. Biochimie 68: 121–132
PetersonGL (1983) Determination of total protein. Methods Enzymol 91: 95–119
PetersonRB, WolkCP (1978) Localization of an uptake hydrogenase in Anabaena. Plant Physiol 61: 688–691
RaiAN, BorthakurM, SoderbackE, BergmanB (1992) Immunogold localization of hydrogenase in the cyanobacterial-plant symbioses Peltigera canina, Anthoceros punctatus and Gunnera magellanica. Symbiosis 12: 131–144
RippkaR, DruellesJ, WaterburyJB, HerdmanM, StanierRY (1979) Generic assignment, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1–61
RohdeM, FürstenauU, MayerF, PrzybylaAE, PeckJrHD, LeGallJ, ChoiES, MenonNK (1990) Localization of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris using immunoelectron microscopic procedures. Eur J Biochem 191: 389–396
RowellP, KerbyNW (1991) Cyanobacteria and their symbionts. In: DilworthMJ, GlennAR (eds) Biology and biochemistry of nitrogen fixation. Elsevier, Amsterdam, pp 373–407
SerebriakovaL, ZorinNA, GogotovIN (1992) Hydrogenase activity of filamentous cyanobacteria. Microbiology [Russian] 61: 107–112
ZorinNA (1986) Redox properties and active center of phototrophic bacteria hydrogenases. Biochimie 68: 97–101
ZorinNA, LindbladP (1993) Localization of hydrogenase in the purple sulphur bacterium Thiocapsa roseopersicina. Arch Microbiol 160: 96–100
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Serebriakova, L., Zorin, N.A. & Lindblad, P. Reversible hydrogenase in Anabaena variabilis ATCC 29413. Arch. Microbiol. 161, 140–144 (1994). https://doi.org/10.1007/BF00276474
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00276474