Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup⁺ (S24 and CT2014) and one Hup⁻ (M11)Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H₂ uptake rates were higher in 1 and 2% H₂ in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H₂ uptake were observed both, due to host species as well as due toRhizobium strains. However, no host dependent complete repression of the expression of H₂ uptake activity was observed in nodules of any of the host species formed with Hup⁺ strains.     

Immunolocalization of the uptake hydrogenase in the marine cyanobacterium Lyngbya majuscula CCAP 1446/4 and two Nostoc strains

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.     

The role of the bidirectional hydrogenase in cyanobacteria

Cyanobacteria have tremendous potential to produce clean, renewable fuel in the form of hydrogen gas derived from solar energy and water. Of the two cyanobacterial enzymes capable of evolving hydrogen gas (nitrogenase and the bidirectional hydrogenase), the hox-encoded bidirectional Ni-Fe hydrogenase has a high theoretical potential. The physiological role of this hydrogenase is a highly debated topic and is poorly understood relative to that of the nitrogenase. Here the structure, assembly, and expression of this enzyme, as well as its probable roles in metabolism, are discussed and analyzed to gain perspective on its physiological role. It is concluded that the bidirectional hydrogenase in cyanobacteria primarily functions as a redox regulator for maintaining a proper oxidation/reduction state in the cell. Recommendations for future research to test this hypothesis are discussed.     

Evidence for nitrogenase-catalyzed hydrogen uptake in nitrogen-fixing filamentous blue-green algae

Abstract Two new strains of (heterocystous) Anabaena , one of them devoid of hydrogenase activity, were compared with respect to hydrogen-uptake activities. Evidence was obtained for a hydrogen-recycling pathway in the dark independent of hydrogenase, because a similar recycling activity is measured in both strains. This hydrogen uptake is dependent on substrates for nitrogenase (molecular nitrogen or acetylene) and can be abolished by oxygen treatment of filaments or by uncouplers. The findings are interpreted as nitrogenase itself being directly involved in the hydrogen-uptake mechanism.     

Heterotrophic glucose uptake and respiration in Lake Kinneret

The turnover times of glucose, averaged for 0–10 m in the upper waters of Lake Kinneret and measured by the addition of single or multiple concentrations of substrate, ranged from 23 to 188 hours and 1 to 87 hours respectively. Potential uptake rates (estimated as V_max) ranged from 0.095 to 1.94 µg glucose l^–1h^–1, while measured uptake rates varied from 0.09 to 1.1 µg glucose l^–1h^–1. Concentrations of dissolved carbohydrates and glucose averaged 0.71 mg glucose equivalents l^–1 and 39 µg glucose l^–1 respectively. No evident relationships between glucose cycling and any fractions of dissolved organic matter, phytoplankton biomass or primary productivity were found. Turnover times were generally most rapid immediately after the decline of the spring Peridinium bloom. The respiration percentage of incorporated glucose ranged from 25% to 61% with highest values during the summer months. Respiration may be influenced by the nature of the indigenous bacterial population as well as by temperature. Daily heterotrophic glucose carbon uptake was about 9% of the photosynthetic incorporation and could provide a bacterial yield of about 7 × 10⁴ ml^–1d^–1.     

Sodium stimulation of uptake hydrogenase activity in symbiotic Rhizobium

Initial observations showed a 100% increase in H₂-uptake (Hup) activity of Rhizobium leguminosarum strain 3855 in pea root nodules (Pisum sativum L. cv Alaska) on plants growing in a baked clay substrate relative to those growing in vermiculite, and an investigation of nutrient factors responsible for the phenomenon was initiated. Significantly greater Hup activity was first measured in the clay-grown plants 24 days after germination, and higher activity was maintained relative to the vermiculite treatment until experiments were terminated at day 32. The increase in Hup activity was associated with a decrease in H₂ evolution for plants with comparable rates of acetylene reduction. Analyses of the clay showed that it contained more Na⁺ (29 versus 9 milligrams per kilogram) and less K⁺ (6 versus 74 milligrams per kilogram) than the vermiculite. Analyses of plants, however, showed a large increase in Na⁺ concentration of clay-grown plants with a much smaller reduction in K⁺ concentration. In tests with the same organisms in a hydroponic system with controlled pH, 40 millimolar NaCl increased Hup activity more than 100% over plants grown in solutions lacking NaCl. Plants with increased Hup activity, however, did not have greater net carbon or total nitrogen assimilation. KCl treatments from 5 to 80 millimolar produced slight increased in Hup activity at 10 millimolar KCl, and tests with other salts in the hydroponic system indicated that only Na⁺ strongly promoted Hup activity. Treating vermiculite with 50 millimolar NaCl increased Na⁺ concentration in pea plant tissue and greatly promoted Hup activity of root nodules in a manner analogous to the original observation with the clay rooting medium. A wider generality of the phenomenon was suggested by demonstrating that exogenous Na⁺ increased Hup activity of other R. leguminosarum strains and promoted Hup activity of R. meliloti strain B300 in alfalfa (Medicago sativa L.).     

Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

Environmental factors affectingin vitro nitrogenase activity of cyanobacteria isolated from rice-fields

The nitrogen-fixing capacity of four cyanobacterial strains was tested in relation to heterotrophic ability, tolerance to combined nitrogen and adaptive capacity to changes in light intensity and pH. Strains (Anabaena variabilis UAM 202;Calothrix marchica UAM 214;Nodularia spumigena UAM 204,Nostoc punctiforme UAM 205) were isolated from the rice-fields of Valencia (Spain).C. marchica, was the only strain able to grow and to fix dinitrogen under heterotrophic conditions, with fructose and glucose. Fructose was the best substrate supporting growth and dinitrogen fixation in mixotrophy (growth in the light under conditions where CO₂ and organic carbon are assimilated simultaneously), photoheterotrophy (growth in the light on an organic compound in the absence of net CO₂ fixation) and heterotrophy (growth on an organic compound in the dark). Ammonium repressed nitrogenase more than nitrate. Full repression was observed only at concentrations of combined nitrogen higher than those usually found in rice-fields. Carbohydrates had a protective effect on nitrogenase against ammonium inhibition inC. marchica. All four strains showed increased nitrogenase activity when the light intensity was increased during assays. Variations of pH normally occurring in rice fields led to no important changes in nitrogenase activity inC. marchica. This fact, together with its potential for heterotrophic growth and tolerance to combined nitrogen, make this the most suitable of the four strains for inoculation experiments in rice fields.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Binding,uptake and expression of foreign DNA by cyanobacteria and isolated etioplasts

Discoveries of the uptake and expression of various Escherichia coli plasmids by the cyanobacterium Anacystis nidulans and isolated cumber etioplasts are reviewed. In particular, the binding and uptake of nick-translated ³²P-labeled plasmids and the expression of genes in the native plasmids are considered.Permeaplasts of A. nidulans 6301 and isolated EDTA-washed cucumber etioplasts exhibit binding and uptake of DNA that is unaffected by uncouplers of photophosphorylation or by dissipators of transmembrane proton graident. ATP inhibits both binding and udptake by permeaplasts or EDTA-washed etioplasts but the analog AMP-PNP (non-hydrolzable) is noninhibitory. With permeaplasts there is no effect of 20 mM Mg²⁺ (in the light) upon intake, whereas with EDTA-washed etioplasts, Mg²⁺ at the same concentration inhibits uptake as does 20 mM Ca²⁺.The transformation of A. nidulans 6301 to ampicillin-resistance by the plasmid pBR322 is much enhanced in permeaplasts. Indeed extracts of transformed cells catalyze the hydrolosis of the -lactam nitrocefin. Transfromation of A. nidulans to antibiotic resistance may also be achieved with the plasmids pHUB4 and pCH1. The effect of light on transformation of A. nidulans 6301 differs with different plasmids. In pBR322 transformants the expression of ribulose bisphosphate carboxylase-oxygenase (RuBisCO) is markedly elevated. In these transformants, the foreign plasmid replicates by a pathway involving chromosomal integration and dissociation.The plasmid pCS75, a derivative of pUC9 (and therefore of pBR322) containing a Pst1 insert carrying genes for the large and small (S) subunits of RuBisCO from A. nidulans, is taken up and expressed in EDTA-washed cucumber cotyledon etioplasts. Expression is evidenced by the hydrolysis of nitrocefin and immunoprecipitation of labeled S subunits of RuBisCO (utilizing etioplasts which have been labeled with ³⁵S-methionine after incubation with pCS75). The plasmid pUC9-CM carrying a cat gene is also expressed in cucumber etioplasts in a manner that demonstrates dependence both on the duration of etioplast washing by EDTA and plasmid concentration. Translation (as measured by ³⁵S-methionine incorporation) by EDTA-washed etioplasts increases with cotyledon greening. However the enhancement of translation by prior incubation of EDTA-washed plastids with pCS75 decreases to zero during 24hr of cotyledon greening. Results suggest that the expression of foreign DNA in plastids may depend critically upon their developmental state.Abbreviations AMP-PNP adenyl-5-yl imidodiphosphate - AP^r amplicillin resistance, cat-chloramphenicol acetyltransferase - RuBisCO ribulose bisphosphate carboxylase/oxygenase     

Pyrolysis mass spectrometry analysis of free-living and symbiotic cyanobacteria

The potential of pyrolysis mass spectrometry to distinguish closely related cyanobacterial strains was assessed by using the technique to compare symbiotic cyanobacteria isolated from the hornwort Phaeoceros laevis and free-living cyanobacterial strains at the same field site. The same strains had previously been compared using polymerase chain reaction-based DNA fingerprinting techniques (West & Adams 1997, Appl. Environ. Microbiol. 63: 4479–4484). Many of the strains were grouped identically by the two techniques, although there were some differences, possibly resulting from the ability of these cyanobacteria to develop a range of specialised cell types having different chemical compositions to the vegetative cells. Although growth conditions were chosen to suppress cellular differentiation, this may not always have been completely successful. With careful control of growth conditions pyrolysis mass spectrometry has considerable potential as an additional tool for the phenetic comparison of cyanobacterial strains. It has the advantage that analysis is directly derived from whole cells, and hence is simpler and cheaper than DNA-based methods, although it does require the growth of axenic strains. The technique may be particularly useful in the study of some of the more cryptic unicellular and non-heterocystous filamentous cyanobacterial groups, in which the lack of cellular differentiation should minimise any variability in the chemical composition of cells.     

Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana

Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H₂ ranged from 0.04 to 5.0 μmol H₂ (g fresh weight nodule)⁻¹ h⁻¹. The H₂ uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed K_m for H₂ of 13.0 and 23.6 μM H₂, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.     

蓝藻Hox氢酶催化产氢的调控

蓝藻是唯一能通过光合作用产生清洁可再生燃料氢气的原核微生物。一些蓝藻具有催化产氢活性的镍-铁Hox氢酶(双向氢酶), 由于其巨大的应用潜力受到广泛的关注。但Hox氢酶在蓝藻产氢过程中调控途径尚不清楚。本文对蓝藻Hox氢酶的结构、生态分布和表达调控的研究进展进行了总结。简单介绍了作者近来对模式蓝藻Synechocystis sp. PCC 6803 hox操纵子中两个未知功能基因ssl2420和sll1225的研究结果。     

Organization of the hupDEAB genes within the hydrogenase gene cluster of Anabaena sp. strain PCC7120

A 15-kb DNA fragment containing a cluster of hup genes has been identified and cloned from Anabaena sp. strain PCC7120. These genes are located upstream of the hupL gene in the adjacent fragment in the Anabaena chromosome. Sequence analysis of a 3.5-kb HindIII fragment showed the sequence of hupEAB and a part of the hupD gene, all of which showed high sequence similarity with hyp genes of Escherichia coli and hup genes of several nitrogen-fixing bacteria. These genes are oriented in one direction, as are the hup genes of other organisms. Although the Anabaena hupDEAB genes are in the same cluster as the hypABCDE cluster of E. coli, the relative positions of the genes differ and there is no hupC in Anabaena on either side of hupA or hupB. Unlike several other organisms, hupD and hupE are not closely linked or translationally coupled in Anabaena, but are separated by an intergenic space of 453 bp. RT-PCR analysis of RNA obtained from vegetative cells and heterocysts of Anabaena showed that the hupB gene is expressed only in heterocyst-induced cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

A cytosolic ferredoxin-independent hydrogenase possibly mediates hydrogen uptake in Trichomonas vaginalis

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Effects of fertilization rate and crop rotation on diazotrophic cyanobacteria in paddy field

Application of chemical fertilizers at the recommended level (medium fertility) or lower stimulated growth of the diazotrophic cyanobacterial population and nitrogenase activity in a paddy field. High fertilizer levels proved to be inhibitory to nitrogen-fixing cyanobacteria indicating that indiscriminate use of chemical fertilizers for a longer period drastically disturbed the natural ecological balance. The rice–mustard–moong (RMM) crop rotation was observed to be more suitable for cyanobacterial nitrogen fixation than rice–wheat–maize rotation. The cropped plots had higher nitrogenase activity than fallow plots. The low fertility coupled with RMM rotation were found to be best suited for promoting nitrogen fixation by cyanobacteria to supply the rice plants. A top dressing of chemical nitrogenous fertilizer drastically suppressed the cyanobacterial nitrogenase activity (ARA) within 12 h; the magnitude of inhibition varied with respect to the cropping system. The inhibition was overcome by the 10th day and the ARA value reached the preapplication value or even higher in the case of low fertility and medium fertility level plots. A regression equation was established to predict nitrogen fixation in a given soil ecosystem.