Modulation of carbonic anhydrase activity in two nitrogen fixing cyanobacteria, Nostoc calcicola and Anabaena sp

The activity of enzyme carbonic anhydrase (CA) was investigated in two diazotrophic cyanobacteria, Anabaena sp. (ARM 629) and Nostoc calcicola, in the presence of CO2/NaHCO3 and different inhibitors. The CA activity increased when the cells were pretreated with a high concentration of CO2/NaHCO3 and then transferred to ambient level CO2. Maximum activity of CA was observed after 8 h of incubation in light on transfer of cells from high Ci to ambient level CO2, and was low when incubated in dark. Addition of the photosynthetic inhibitor DCMU brought about a differential reduction in CA activity, depending on the carbon source (NaHCO3/CO2). CA inhibitors--ethoxyzolamide (EZ) and acetazolamide (AZ)--inhibited the enzyme activity in both the genera, but the extent of inhibition was greater in Anabaena sp. than in N. calcicola. Such a variation in extent of inhibition/stimulation of CA activity being different in the two genera reflects differences in their inherent potential and genetic background. The relevance of such cyanobacterial strains as CO2 sinks is also discussed.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Inorganic carbon acquisition systems in cyanobacteria

This minireview focuses on the mechanism of inorganic carbon uptake in cyanobacteria and in particular the two CO₂-uptake systems and two bicarbonate transporters recently identified in Synechocycstis PCC 6803, and their presence in other cyanobacterial strains. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and characterization of a marine Anabaena sp. capable of rapid growth on molecular nitrogen

A marine filamentous cyanobacterium capable of rapid growth under N₂-fixing conditions has been isolated from the Texas Gulf Coast. This organism appears to be an Anabaena sp. and has been given the strain designation CA. Cultures grown on mineral salts medium bubbled with 1% CO₂-enriched air at 42°C show a growth rate of 5.6±0.1 generations per day with molecular nitrogen as the sole nitrogen source. This growth rate is higher than any other reported in the literature to date for heterocystous cyanobacteria growing on N₂. Under similar growth conditions, 7.5 mM NH₄Cl yields a growth rate of 6.6±0.1 generations per day while 7.5 mM KNO₃ allows for a growth rate of 5.8±0.4 generations-day. Nitrogen-fixation rates, as measured by acetylene reduction, show maximum activity values in the range of 50–100 nmoles ethylene produced/minxmg protein. These values compare favorably with those obtained from heterotrophic bacteria and are much higher than values reported for other cyanobacteria. Growth experiments indicate that the organism requires relatively high levels of sodium and grows maximally at 42°C. Because of its high growth rate on N₂, this newly isolated organism appears ideal for studying nitrogen metabolism and heterocyst development among the cyanobacteria.     

3种水稻土中7株固氮蓝细菌的分离与特征

【背景】蓝细菌是水生和陆地生态系统中生物固氮的主要贡献者。【目的】增加对稻田土壤固氮蓝细菌的了解,获得用于进一步研究的可培养固氮蓝细菌菌株。【方法】选择3种具有不同固氮能力的水稻土,采用BG11-N培养基分离培养固氮蓝细菌菌株,对新分离菌株进行形态特征观察,通过基因组DNA的nifH基因扩增明确其固氮潜力,进一步采用乙炔还原法和~(15)N_2示踪法定量测定其固氮能力,通过基因组DNA的16SrRNA基因序列比对进行鉴定。【结果】在光照培养条件下,采用BG11-N培养基共分离纯化得到自养菌株7株,细胞呈圆形或椭圆形、单列、无分枝、丝状和念珠状,在固体培养基上形成团垫状菌落。新分离菌株在BG11-N培养基中生长状况良好,以基因组DNA为模板可扩增出nifH基因,乙炔还原法和~(15)N_2示踪法测定结果显示具有较高固氮能力,同时具有铁载体生成能力。结合16S rRNA基因序列比对和形态特征,7株菌被初步鉴定隶属于念珠藻科(Nostocaceae)。【结论】从水稻土中分离到在稻田生物固氮中发挥重要作用的蓝细菌(念珠藻科)菌株,可培养固氮蓝细菌菌株固氮能力较高,兼具铁载体生成能力,可作为进一步深入研究的微生物资源,具有潜在的研究应用价值。     

Effects of lime-induced inorganic carbon reduction on the growth of three aquatic macrophyte species

Lime application to aquatic systems may be an effective means of stressing macrophyte growth and promoting changes in species assemblage by inducing temporary dissolved inorganic carbon (DIC) limitation of productivity. Shoot and root growth response to lime (as Ca(OH)₂) application was investigated for three macrophyte species (Elodea canadensis, Stuckenia pectinata, and Vallisneria americana) grown in experimental outdoor mesocosms. Lime was applied to mesocosms at three treatment levels to maintain pH for 1 week at 9.8–10.0 (1.64 mM), 10.3–10.5 (at the bicarbonate–carbonate equivalence point; 3.00 mM), and 10.8–11.0 (4.34 mM). pH recovered to control levels in all treated mesocosms 20 days after lime application. After treatment, HCO₃⁻ and DIC declined by 66, 93, and 93% and 60, 89, and 87%, respectively, versus increasing lime application. Concentrations remained lower in treated mesocosms versus the control throughout post-treatment. Differential growth response was observed in the 1.64 and 3.00 mM treatments, suggesting species-specific tolerances to both DIC concentration and form. V. americana was most sensitive to lime as the 1.64 mM treatment resulted in 54% shoot growth suppression versus the control and shoot plus root biomass loss in the 3.00 and 4.34 mM treatments. S. pectinata and E. canadensis exhibited net shoot and root growth (although significantly lower than controls) in both the 1.64 and 3.00 mM treatments and complete growth suppression in the 4.34 mM treatment. Selective control and shifts in species assemblage may be possible by adjusting lime concentration in relation to compensation point and needs to be investigated under field conditions.     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

Ecophysiological responses of the euhalophyte Suaeda salsa to the interactive effects of salinity and nitrate availability

Effects of salinity and nitrate nitrogen (NO₃⁻-N) on ion accumulation and chlorophyll fluorescence were monitored for two populations of Suaeda salsa grown from seeds in a greenhouse experiment. One population inhabits the intertidal zone and the other occurs on inland saline soils. Ion contents in soils and in leaves of the two populations were also investigated in field. In the greenhouse, seedlings were exposed to a NaCl concentration of 0.6 and 35.1 ppt, with 0.1 or 5 mM NO₃⁻-N treatments for 20 days. The contents of Na⁺ and Cl⁻ were higher, but NO₃⁻ was lower in soils of the intertidal zone than at the inland site. In the field, ion concentrations and the estimated contribution of these ions to osmotic potential in leaves showed no difference between the two populations, except that the estimated contribution of Na⁺ to osmotic potential in leaves of the intertidal population was lower than that in the inland population. In the greenhouse, in contrast, the concentration of Cl⁻ was lower, but NO₃⁻ concentration and the estimated contribution of NO₃⁻ to osmotic potential were higher, in the leaves of plants from the intertidal zone. Salinity had no effect on the maximal efficiency of PSII photochemistry (Fv/Fm) and the actual PSII efficiency (ΦPSII). The results indicated that S. salsa from the intertidal zone was better able to regulate Cl⁻ to a lower level, and accumulate NO₃⁻ even with low soil NO₃⁻ concentrations. Tolerance of the PSII machinery to high salinity stress may be an important characteristic for the studied species supporting growth in highly saline environments.     

Characterization of the non-photochemical quenching of chlorophyll fluorescence that occurs during the active accumulation of inorganic carbon in the cyanobacterium Synechococcus PCC 7942

Previous work has shown that the maximum fluorescence yield from PS 2 of Synechococcus PCC 7942 occurs when the cells are at the CO₂ compensation point. The addition of inorganic carbon (C_i), as CO₂ or HCO₃ ^–, causes a lowering of the fluorescence yield due to both photochemical (q_p) and non-photochemical (q_N) quenching. In this paper, we characterize the q_N that is induced by C_i addition to cells grown at high light intensities (500 mol photons m^–2 s^–1). The C_i-induced q_N was considerably greater in these cells than in cells grown at low light intensities (50 mol photons m^–2 s^–1), when assayed at a white light (WL) intensity of 250 mol photons m^–2 s^–1. In high-light grown cells we measured q_N values as high as 70%, while in low-light grown cells the q_N was about 16%. The q_N was relieved when cells regained the CO₂ compensation point, when cells were illuminated by supplemental far-red light (FRL) absorbed mainly by PS 1, or when cells were illuminated with increased WL intensities. These characteristics indicate that the q_N was not a form of energy quenching (q_E). Supplemental FRL illumination caused significant enhancement of photosynthetic O₂ evolution that could be correlated with the changes in q_p and q_N. The increases in q_p induced by C_i addition represent increases in the effective quantum yield of PS 2 due to increased levels of oxidized Q_A. The increase in q_N induced by C_i represents a decrease in PS 2 activity related to decreases in the potential quantum yield. The lack of diagnostic changes in the 77 K fluorescence emission spectrum argue against q_N being related to classical state transitions, in which the decrease in potential quantum yield of PS 2 is due either to a decrease in absorption cross-section or by increased spill-over of excitation energy to PS 1. Both the C_i-induced q_p (t _0.5<0.5 s) and q_N (t _0.51.6 s) were rapidly relieved by the addition of DCMU. The two time constants give further support for two separate quenching mechanisms. We have thus characterized a novel form of q_N in cyanobacteria, not related to state transitions or energy quenching, which is induced by the addition of C_i to cells at the CO₂-compensation point.Abbreviations BTP- 1,3-bis[tris(hydroxymethyl)-methylaminopropane] - Chl- chlorophyll - C_i- inorganic carbon (CO₂+HCO₃ ^–+CO₃ ^2–) - DCMU- 3-(3,4-dichlorophenyl)-, 1-dimethylurea) - F- chlorophyll fluorescence measured at any time in the absence of a saturating flash - F_o- chlorophyll fluorescence with only the weak modulated measuring beam on - F_M'- chlorophyll fluorescence during a saturating flash - F_M- maximum chlorophyll fluorescence, measured in the presence of WL and FRL at the CO₂-compensation point or in the presence of DCMU - F_V- variable fluorescence (= F_M'–F₀) - FRL- supplemental illumination with far red light - MB- modulated measuring beam of the PAM fluorometer - MV- methyl viologen - PAM- pulse amplitude modulation - PFD- incident photon flux density - PS 1, 2- Photosystems 1 and 2 - Q_A- primary electron-accepting plastoquinione of PS 2 - q_N- non-photochemical quenching of chlorophyll fluorescence - q_p- photochemical quenching of chlorophyll fluorescence; rubisco-ribulose bisphosphate carboxylase/oxygenase - SF- saturating flash (600 ms duration) - WL- white light illumination     

Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and CO2-enriched air

Mass spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ isotopes were used to compare the rates of gross O₂ evolution (E₀), O₂ uptake (U₀) and net O₂ evolution (NET) in relation to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonas reinhardtii cells grown in air (air-grown), in air enriched with 5% CO₂ (CO₂-grown) and by cells grown in 5% CO₂ and then adapted to air for 6h (air-adapted).At a photon fluence rate (PFR) saturating for photosynthesis (700 mol photons m^-2 s^-1), pH=7.0 and 28°C, U₀ equalled E₀ at the DIC compensation point which was 10M DIC for CO₂-grown and zero for air-grown cells. Both E₀ and U₀ were strongly dependent on DIC and reached DIC saturation at 480 M and 70 M for CO₂-grown and air-grown algae respectively. U₀ increased from DIC compensation to DIC saturation. The U₀ values were about 40 (CO₂-grown), 165 (air-adapted) and 60 mol O₂ mg Chl^-1 h^-1 (air-grown). Above DIC compensation the U₀/E₀ ratios of air-adapted and air-grown algae were always higher than those of CO₂-grown cells. These differences in O₂ exchange between CO₂- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells.For all algae, the rates of dark respiratory O₂ uptake measured 5 min after darkening were considerably lower than the rates of O₂ uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U₀ is discussed and the energy requirement of the inducable CO₂/HCO₃ ^- concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered.Abbreviations DIC dissolved inorganic carbon - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - E₀ rate of photosynthetic gross O₂ evolution - PCO photosynthetic carbon oxidation - PFR photon fluence rate - PS I photosystem I - PS II photosystem II - U₀ rate of O₂ uptake in the light - MS mass spectrometer     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

Postmitochondrial regulation of apoptosis by bicarbonate

Ion homeostasis may play a role in the regulation of apoptosis. The current study has shown such a role for bicarbonate (HCO(3)(-)). In apoptosis triggered by ATP depletion, the proapoptotic molecule Bax translocated from the cytosol to mitochondria, followed by cytochrome c release from the organelle, caspase activation, and development of apoptotic morphology. Apoptosis was significantly ameliorated, when HCO(3)(-) was omitted from the incubation medium. The HCO(3)(-) dependence was also demonstrated for apoptosis induced by staurosporine in HeLa cells. Of significance, when HCO(3)(-) was reintroduced, apoptosis was restored. The Cl(-)/HCO(3)(-) exchanger inhibitor DIDS suppressed apoptosis in HCO(3)(-)-containing medium, further supporting a role for intracellular HCO(3)(-) in apoptosis regulation. We subsequently examined HCO(3)(-)-dependent steps in the apoptotic cascade. Translocation of Bax and cytochrome c was not suppressed by the omission of HCO(3)(-), suggesting HCO(3)(-) regulation at postmitochondrial levels. In vitro reconstitution of caspase activation using exogenous cytochrome c and cytosolic extracts was not HCO(3)(-) dependent. HCO(3)(-) was not required for the enzymatic activity of recombinant caspases either. In conclusion, the results have provided compelling evidence for HCO(3)(-) regulation of apoptosis. Such regulation takes place at postmitochondrial levels, downstream of Bax/cytochrome c translocation.     

Sulphated exopolysaccharides produced by two unicellular strains of cyanobacteria,Synechocystis PCC 6803 and 6714

The exopolysaccharides (EPS) of two unicellular strains of cyanobacteria Synechocystis PCC 6803 and 6714, formed labile, radial structures, uniformly distributed on the cell surface, and stainable by specific dyes for acidic polysaccharides. The two strains produced EPS at similar rates, which depended, along with the duration of the producing phase, on the incubation conditions. The exopolysaccharides from both strains were constituted of at least 11–12 mono-oses, probably forming several types of polymers. They contained about 15–20% (w/w) uronic derivatives and 10–15% (w/w) osamines. Proteins represented 20–40% of total weight. A most interesting feature was the presence of 7–8% (molar ratio) sulphate residues, a characteristic that is otherwise limited to exopolysaccharides produced by eukaryotic algae.Abbreviations EPS exopolysaccharides - KDO 3-deoxy-d-mannooctulosonate - LPS lipopolysaccharides     

Isolation and partial characterization of Fix− mutants from Rhizobium strain 32H1

Summary Eight ineffective mutant strains were isolated from N-methyl-N'-nitro-N-nitrosoguanidine mutagenized cultures of cowpea Rhizobium strain 32H1. Strains CR1, CR2, CR3, CR4, CR5 and CR6 induced more, but smaller, nodules than the wild type. With the exception of strain CR2, these mutant strains reduced less than 1% of the amount of acetylene reduced by the wild type, in both the free-living and symbiotic assays. Strain CR2 reduced acetylene in the free-living assay but not in the symbiotic assay. Strains CR7 and CR8 responded variably (5–20% of the wild type) in free-living and symbiotic acetylene reduction assays. Nodules also varied from small white to normal-sized pink nodules. The phenotypic characteristics of the mutant strains were consistant with all leguminous plants tested and were stable upon reisolation from nodules. Fully effective revertants were selected from 4 of the ineffective mutant strains by the use of the leguminous plant,Macroptilium lathyroides. Serology, patterns of resistance to anti-bacterial agents, phage-typing, and antibiotic resistance markers were used to confirm strain identification.Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service and the North Carolina Agricultural Research Service, Raleigh, North Carolina. Paper no. 8834 of the Journal Series of the North Carolina Agricultural Research Service at Raleigh.     

Losses of inorganic carbon and nitrous oxide from a temperate freshwater wetland in relation to nitrate loading

We studied the export of inorganic carbon and nitrous oxide (N₂O) from a Danish freshwater wetland. The wetland is situated in an agricultural catchment area and is recharged by groundwater enriched with nitrate (NO₃ ^–) (1000 M). NO₃ ^– in recharging groundwater was reduced (57.5 mol NO₃ ^– m^–2 yr^–) within a narrow zone of the wetland. Congruently, the annual efflux of carbon dioxide (CO₂) from the sediment was 19.1 mol C m^–2 when estimated from monthly in situ measurements. In comparison the CO₂ efflux was 4.8 mol C m^–2 yr^–1 further out in the wetland, where no NO₃ ^– reduction occurred. Annual exports of inorganic carbon in groundwater and surface water was 78.4 mol C m^–2 and 6.1 mol C m^–2 at the two sites, respectively. N₂O efflux from the sedimenst was detectable on five out of twelve sampling dates and was significantly (P < 0.0001) higher in the NO₃ ^– reduction zone (0.35–9.40 mol m^–2 h^–1, range of monthly means) than in the zone without NO₃ ^– reduction (0.21–0.41 mol m^–2 h^–1). No loss of dissolved N₂O could be measured. Total annual export of N₂O was not estimated. The reduction of oxygen (O₂) in groundwater was minor throughout the wetland and did not exceed 0.2 mol 0₂ m^–2yr^–1. Sulfate (SO₄ ^––) was reduced in groundwater (2.1 mol SO₄ ^–– m^–2 yr^–1) in the zone without NO₃ ^– reduction. Although the NO₃ ^– in our wetland can be reduced along several pathways our results strongly suggest that NO₃ ^– loading of freshwater wetlands disturb the carbon balance of such areas, resulting in an accelerated loss of inorganic carbon in gaseous and dissolved forms.     

Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in CO2 removal processes

In this paper the utilization of the cyanobacteria Anabaena sp. in carbon dioxide removal processes is evaluated. For this, continuous cultures of this strain were performed at different dilution rates; alternatives for the recovery of the organic matter produced being also studied. A maximum CO₂ fixation rate of 1.45 g CO₂ L⁻¹ day⁻¹ was measured experimentally, but it can be increased up to 3.0 g CO₂ L⁻¹ day⁻¹ outdoors. The CO₂ is mainly transformed into exopolysaccharides, biomass representing one third of the total organic matter produced. Organic matter can be recovered by sedimentation with efficiencies higher than 90%, the velocity of sedimentation being 2 · 10⁻⁴ s⁻¹. The major compounds were carbohydrates and proteins with productivities of 0.70 and 0.12 g L⁻¹ day⁻¹, respectively. The behaviour of the cultures of Anabaena sp. has been modelized, also the characteristics parameters requested to design separation units being reported. Finally, to valorizate the organic matter as biofertilizers and biofuels is proposed.     

Isolation and characterization of hydrogenase-negative mutants of Azorhizobium caulinodans ORS571

Hydrogenase-negative (Hup^-) mutants of Azorhizobium caulinodans ORS571 were isolated by means of Tn5 mutagenesis. The colony test used for screening for Hup^- strains was based on the absence of reduction of triphenyltetrazolium chloride with hydrogen. Suspensions from cultures of the mutant strains grown under derepressing conditions did not use hydrogen with methylene blue or oxygen as the hydrogen acceptor. The mutants were shown to carry single Tn5 insertions at different locations in the A. caulinodans genome. Molar growth yields (corrected for poly--hydroxybutyrate formation) in chemostat cultures of the mutants were similar to those of the wild type. Molar growth yields of the mutants were not increased by passing additional hydrogen through chemostat cultures, which is in agreement with the hydrogenase-negative phenotype of the mutants. H₂/N₂ ratios (mol H₂ formed per mol N₂ fixed) were calculated from the hydrogen content of the effluent gas and the N-content of the bacterial dry weight. Low H₂/N₂ ratios (between 1.2 and 1.9) were found in both energy-limited (oxygen or succinate) cultures and in cultures limited by the supply of an anabolic substrate (Mg²⁺). ATP/2e values (mol ATP used at the transport of 2e to nitrogen or H⁺) were calculated from the H₂/N₂ ratios and the molar growth yields of nitrogen-fixing and ammonia-assimilating cultures. ATP/2e values were between 7 and 11. It was concluded that the calculated ATP/2e values comprise not only 4 mol ATP used at the transport of 2e through nitrogenase but also energy equivalents needed for reversed electron flow from NADH to the low-potential hydrogen donor used by nitrogenase.     

Evolutionary aspects of intestinal bicarbonate secretion in fish

Experiments compared intestinal HCO3- secretion in the intestine of marine teleost Gulf toadfish, Opsanus beta, to representatives of early chondrostean and chondrichthyan fishes, the Siberian sturgeon, Acipenser baerii, and white-spotted bamboo shark, Chiloscyllium plagiosum, respectively. As seen in marine teleosts, luminal HCO3- concentrations were 10-fold plasma levels in all species when exposed to hyperosmotic conditions. While intestinal water absorption left Mg2+ and SO4(2-) concentrated in intestinal fluids up to four-fold ambient seawater concentrations, HCO3- was concentrated up to 50 times ambient levels as a result of intestinal HCO3- secretion. Reduced luminal Cl- concentrations in the intestine of all species suggest that HCO3- secretion also occurs via Cl-/HCO3- exchange in chondrostean and chondrichthyan fishes. Sturgeon began precipitating carbonates from the gut after only 3 days at 14 per thousand, a mechanism utilized by marine teleosts to reduce intestinal fluid osmolality and maintain calcium homeostasis. Analysis of published intestinal fluid composition in the cyclostome Lampetra fluviatilis reveals that this species likely also utilize intestinal HCO3- secretion for osmoregulation. Analysis of existing cyclostome data and our results indicate that intestinal Cl-/HCO3- exchange plays an integral role in maintaining hydromineral balance not only in teleosts, but in all fish (and perhaps other animals) with a need to drink seawater.     

Isolation and characterization of stable mutants ofStreptomyces peucetius defective in daunorubicin biosynthesis

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced byStreptomyces peucetius. In this study we report isolation of stable mutants ofS. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, ε-rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed bydnrE (mutants SPAK and SPMAG),dnrS (SPFS and SPRHO) anddoxA (SPDHC) gene products.     

Contribution of N2 fixing cyanobacteria to rice production: availability of nitrogen from 15N-labelled cyanobacteria and ammonium sulphate to rice

This study investigate the potential contribution of nitrogen fixation by indigenous cyanobacteria to rice production in the rice fields of Valencia (Spain). N₂-fixing cyanobacteria abundance and N₂ fixation decreased with increasing amounts of fertilizers. Grain yield increased with increasing amounts of fertilizers up to 70 kg N ha^-1. No further increase was observed with 140 kg N ha^-1. Soil N was the main source of N for rice, only 8–14% of the total N incorporated by plants derived from ¹⁵N fertilizer. Recovery of applied ¹⁵N-ammonium sulphate by the soil–plant system was lower than 50%. Losses were attributed to ammonia volatilization, since only 0.3–1% of applied N was lost by denitrification. Recovery of ¹⁵N from labeled cyanobacteria by the soil–plant system was higher than that from chemical fertilizers. Cyanobacterial N was available to rice plant even at the tillering stage, 20 days after N application. This revised version was published online in June 2006 with corrections to the Cover Date.