Genes,enzymes and membrane proteins of the nitrate respiration system ofEscherichia coli

Summary A method was devised to isolate mutants carrying deletions through several genetic loci (chlD ⁺ andchlA ⁺) which are involved in the membrane-bound nitrate respiratory complex ofEscherichia coli. Specific transducing phages were used to reintroduce these genes. Comparisons of membrane fractions from these transduced strains showed five membrane proteins that are necessary for the formation of an active nitrate respiration system. Two particular bacterial genes (chlD ⁺ andchlA ⁺) were shown to control these five membrane proteins.Three of the proteins specified bychlA ⁺, appear to be constitutively controlled and always present in the membrane ofE. coli irrespective of growth conditions, while the other two proteins, specified bychlD ⁺, appear to be induced byanaerobic growth in the presence of nitrate.     

Distribution of Cadmium,Lead, Nickel,and Strontium in Imbibing Maize Caryopses

Seed germination is tolerant to heavy metals apparently because the seed coat is impermeable to metal ions. However, it is not clear whether the seed coat is a universal barrier for all metals. In addition, depending on their physical and chemical properties, a distribution of various metals may differ within an imbibing caryopsis, and therefore they produce dissimilar effects on seed germination. The toxic effects of Cd(NO₃)₂, Pb(NO₃)₂, Ni(NO₃)₂, and Sr(NO₃)₂ were estimated from the germination rates of maize (Zea mays L.) caryopses following two-day incubation with these salts. The distribution of heavy metals and Sr was studied by histochemical methods based on the formation of colored complexes with dithizone (Cd and Pb), dimethylglyoxyme (Ni), and sodium rhodizonate (Sr). Although the metals under study did not affect maize radicle protrusion, they inhibited seed germination in the following order: Cd > Ni ≈ Pb > Sr. Cd and Pb accumulated mainly in the seed coat cells, but Sr and Ni in the embryo cells and in the cells of endosperm (Sr) and scutellum (Ni). Although Cd was found only in the seed coat, it was the strongest inhibitor of seed germination. Apparently, due to high toxicity, Cd exerted its inhibitory effect at the concentrations too low for histochemical assay. In spite of easy translocation across the seed coat of imbibing caryopses, Sr did not considerably inhibit radicle protrusion and seed germination, apparently because of its low toxicity and predominant localization in the apoplast of embryo and endosperm cells.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 635–640.Original Russian Text Copyright © 2005 by Seregin, Kozhevnikova.     

Silver toxicity in a nitrogen-fixing Cyanobacterium

Summary Exposure ofNostoc muscorum to combinations of Ag + Cr, Ag + Ni, Ag + PB and Ag, Cr, Ni and Cr alone brought about a complex pattern of inhibition of growth, NO₃, NH₄ and¹⁴CO₂ uptake and nitrogenase activity. The sensitivity hierarchy of different parameters to metal combinations may be given as: (a) inhibition of NH₄ uptake and nitrogenase was almost identical for Ag + Ni, Ag + Cr and Ag + Pb; (b) the order of inhibition of growth and NO₃ uptake was Ag + Cr > Ag + Ni > Ag + Pb; (c) the combination of Ag + Pb was least toxic for growth, nitrogenase, NO₃ and NH₄ uptake, but most toxic for¹⁴CO₂ uptake; (d) the toxicity hierarchy for all parameters together was¹⁴C0₂ > nitrogenase > NH₄ uPtake > NO₃ uptake > growth. Hence, carbon fixation may be employed for biological monitoring of metal toxicity in a laboratory microcosm and possibly in aquatic ecosystems.     

Phytotoxic effects of Cu, Zn, Cd and Pb on in vitro regeneration and concomitant protein changes in Holarrhena antidysenterica

The nodal explants of in vitro shoots of Holarrhena antidysenterica L. were cultured on Murashige and Skoog's (MS) medium augmented with 15 μM N⁶-benzyladenine (BA) alone (control) or supplemented with different concentrations (1, 5, 10 and 20 mg dm⁻³) of CdCl₂, CuSO₄, Pb(NO₃)₂ and ZnSO₄. The maximum morphogenic response in terms of average shoot number (4.95 ± 0.17) was seen in control. ZnSO₄ proved to be less inhibitory in comparison to CuSO₄, Pb(NO₃)₂ and CdCl₂. None of the explants cultured on CdCl₂ containing medium induced multiple shoots. Maximum protein content [3.80 ± 0.04 mg g⁻¹(f.m.)] was observed in control and slightly less [3.50 ± 0.02 mg g⁻¹(f.m.)] in tissues exposed to 1 mg dm⁻³ of CuSO₄ and minimum [1.00 ± 0.02 mg g⁻¹(f.m.)] in Zn treated (20 mg dm⁻³) explants. SDS-PAGE analysis of the treated tissues revealed that two new polypeptides (29 and 20 kDa) in response to Cu and Zn treatment, respectively, have been synthesized.     

Reduction of N-oxides and sulfoxide by the same terminal reductase inProteus mirabilis

Proteus mirabilis can grow anaerobically on the fermentable substrate, glucose. When the glucose medium was supplemented with an electron acceptor, growth doubled. However, the organism failed to grow anaerobically on the oxidizable substrate glycerol unless the medium was supplemented with an external electron acceptor. Dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nicotinamide N-oxide (NAMO), and nitrate (NO₃) can serve this function. Cell-free extracts ofP. mirabilis can reduce these compounds in the presence of various electron donors. In order to determine whether the same or different terminal reductase(s) are involved in the reduction of these compounds, we isolated mutants unable to grow on glycerol/DMSO medium. When these mutants were tested on glycerol medium containing TMAO, NAMO, and NO₃ as electron acceptors, it was found that there were two groups. Group I mutants were unable to grow with DMSO, TMAO, and NAMO, while their growth was unaffected with NO₃. Group II mutants were unable to grow on any electron acceptor including NO₃. Enzyme assays using reduced benzyl viologen with both groups of mutants were in agreement with growth studies. On the basis of these results, we conclude that the same terminal reductase is involved in the reduction of DMSO, TMAO, and NAMO (group I) and that the additional loss of NO₃ reductase in group II mutants is probably owing to a defect in the synthesis or insertion of molybdenum cofactor.     

The effect of lead on photosynthesis and respiration in detached leaves and in mesophyll protoplasts of Pisum sativum

Photosynthesis and transpiration rate of detached leaves of pea (Pisum sativum L. cv. Iłowiecki) exposed to solution of Pb(NO₃)₂ at 1 or 5 mmol·dm⁻³ concentrations were inhibited. The higher concentration of this toxicant decreased photosynthesis and transpiration rates 2 and 3 times respectively, and increased respiration by about 20 %, as measured after 24 hours of treatment. Similarly to Pb(NO₃)₂, glyceraldehyde solution, an inhibitor of phosphoribulokinase, at 50 mmol·dm⁻³ concentration decreased the rates of photosynthesis and transpiration during introduction into pea leaves. The rate of dark respiration, however, remained unchanged during 2 hours of experiment. The potential photochemical efficiency of PS II (Fv/Fm) and the activity of Rubisco (EC 4.1.1.39) at 5 mmol·dm⁻³ of Pb(NO₃)₂ were lowered by 10 % and 20 % respectively, after 24 hours. Neither changes in the activity of PEPC (EC 4.1.1.31) or protein and pigment contents were noted in Pb-treated leaves. The photosynthetic activity of protoplasts isolated from leaves treated for 24 or 48 hours with Pb(NO₃)₂ at 5 mmol·dm⁻³ concentration was decreased 10 % or 25 %, whereas, the rate of dark respiration was stimulated by about 40 % and 75 %, respectively. The content of abscisic acid, a hormone responsible for stomatal closure, in detached pea leaves treated for 24 h with 5 mmol·dm⁻³ of Pb(NO₃)₂ solution was increased by about 3 times; a longer (48h) treatment led to further increase (by about 7 times) in the amount of this hormone. The results of our experiments provide evidences that CO₂ fixation in detached pea leaves, at least up to 24 hours of Pb(NO₃)₂ treatment, was restricted mainly by stomatal closure.     

The effects of heavy metal salts on the phytohormonal status and sex expression in marijuana

We studied the effects of heavy metal salts (Pb(NO₃)₂, CuSO₄, and ZnSO₄) on phytohormonal status and sex expression in various cultivars of marijuana (Cannabis sativa L.), a dioecious plant, grown on Knop nutrient medium. Pb(NO₃)₂ and ZnSO₄ were added to the medium at the concentration of 10⁻⁹ M, and CuSO₄, at the concentration of 10⁻¹⁰ M. Plant were grown under controlled conditions at luminescent illumination, 22–24°C, and 80% humidity. The contents of GA and zeatin were determined by HPLC. Copper and zinc salts induced plant feminization, and this effect was coupled with zeatin accumulation. Lead salts favored plant masculinization coupled with GA accumulation. Thus, a shift in sex expression in marijuana plants was correlated with the heavy metal action on the balance of phytohormones, GA and zeatin.     

Impact of environmental pollution on cyanobacterial proline content

This study describes the toxic effects of different prominent aquatic pollutants—heavy metals (Cd & Pb), pesticides (alphamethrin and deltamethrin) and salt (NaCl)—on the intracellular proline content in the cyanobacterium, Westiellopsis prolifica–Janet strain–NCCU331. Despite a reduction in growth (measured as chlorophyll a content), the intracellular proline content increased in the presence of heavy metals, pesticides and high salt concentration. The intracellular cyanobacterial proline accumulation was more pronounced under salt stress than in the presence of pesticides and heavy metals. We have also compared whether or not anionic components influence heavy metal toxicity. It was found that the chlorides of Cd and Pb were more toxic than the NO₃ and the order of toxicity was CdCl₂ > PbCl₂ > Cd (NO₃)₂ > Pb (NO₃)₂. Among pyrethroids, deltamethrin was more toxic than alphamethrin. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

The Effect of Copper and Lead Ions on Growth and Lipid Composition of the Fern Matteuccia sthruthiopteris

In the present study, the effect of copper (Cu²⁺) and lead (Pb²⁺) ions on the growth and lipid composition of various parts of the fern, Matteuccia sthruthiopteris, was examined. Plants were incubated in the presence or absence of 1, 10, 100 μM of Cu(NO₃)₂ or Pb(NO₃)₂. Cu²⁺ and Pb²⁺ ions at concentrations of 1 and 10 μM caused an increased growth of the roots and leaves. A higher concentration of Pb²⁺ did not show any effect on growth, whereas that of Cu²⁺ slowed down the growth of the whole plants. The roots accumulated more than 700 μg of Cu²⁺ and 400 μg of Pb²⁺ per 1 g dry weight when the plants were incubated with the higher concentrations of metals, whereas in the leaves the concentration of Cu²⁺ was much lower and did not exceed 12 μg/g dry weight. No accumulation of Pb²⁺ ions by leaves was detected. The lipid composition of photosynthetic leave tissues was shown to be affected by the presence of metal ions in the root medium at either concentration studied. Various changes in lipid classes were noted as responsive reactions of M. sthruthiopteris to the heavy metal ions in nutrient medium. Cu²⁺ ions decreased the content of total lipids, total phospholipids, and individual phosphatidylcholines and phosphatidylethanolamines, whereas Pb²⁺ ions caused a decrease in the content of total lipids and glycolipids. Changes in the lipid composition were more pronounced in the mature leaves than in the scrolls of the studied fern.     

Electron donors and the quinone involved in dimethyl sulfoxide reduction inEscherichia coli

Escherichia coli can use dimethyl sulfoxide (DMSO) as an electron acceptor during anaerobic growth on the oxidizable substrate, glycerol. During growth, the DMSO is reduced to dimethyl sulfide (DMS). For the reduction of DMSO, NADH, formate, lactate, reduced benzyl viologen, reduced methyl viologen, and dithionite can serve as electron donors. The terminal reductase and the dehydrogenases linking the various electron donors to the electron transport chain were found to be membrane bound. Chlorate-resistant mutants (chl) were unable to grow and reduce DMSO. However, in the case of thechlD mutant, growth and DMSO reduction can be restored by growth in the presence of high concentrations of molybdate. Mutants ofE. coli blocked in menaquinone (vitamin K₂) biosynthesis—menB, menC, andmenD—were unable to grow with DMSO as an electron acceptor, even though the terminal reductase is present in these mutants. Both growth and DMSO reduction could be restored in these mutants by growth in the presence of the menaquinone intermediates,o-succinylbenzoate and 1,4-dihydroxy-2-naphthoate, depending on the metabolic block of the mutant. Thus menaquinone is involved in electron transport during DMSO reduction.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Alleviation of heavy metal stress in Spilanthes calva L. (antimalarial herb) by exogenous application of glutathione

To evaluate the role of exogenous application of a phytochelating agent glutathione in increasing resistance against different heavy metals stress, nodal explants excised from 28-day-old in vitro seedlings of Spilanthes calva L. were cultured on Murashige and Skoog’s medium supplemented with 10 μM benzyl adenine and five different concentrations (1, 5, 50, 100, or 200 mg/l) of four heavy metals: As₂O₃, CuSO₄, ZnSO₄, or Pb(NO₃)₂. Data were recorded for percent survival, shoot number, and shoot length after 28 d of heavy metal treatment. All four heavy metals severely inhibited growth and morphogenesis. Pb proved most inhibitory whereas Zn was least effective. Pb was further selected to study the reversal effect of glutathione on morphogenesis. The addition of different concentrations (1, 5, 10, or 25 mg/l) of glutathione to media containing the Pb resulted in a significant improvement in almost all growth parameters. Inclusion of glutathione at 10 mg/l was optimum for maximum reversal of the negative effects of heavy metals on morphogenesis.     

Effect of lead on the physiological,biochemical and ultrastructural properties of Leucaena leucocephala

• Heavy metals are characterised by a relatively high density and cause genotoxic, cytotoxic and mutagenic effects on plants, animals and humans. Lead (Pb) is one of the heavy metals that causes toxicity to plants and animals.
• This experiment was conducted using a hydroponic technique to study the effects of Pb(NO₃)₂ on physiological, biochemical and ultrastructural characteristics in Leucaena leucocephala seedlings. Plants were grown in a growth chamber for 21 days in Hoagland’s solution supplemented with 0 (control), 25, 50, 100, 300, 500 and 700 µm Pb(NO₃)₂.
• Shoot heights as well as root lengths decreased significantly in Pb‐treated plants with 300, 500 and 700 µm . In Pb‐treated plants with high Pb concentrations, photosynthesis rate (P_N), stomatal conductance (g_s) and transpiration rate (E) decreased. Total protein and carbohydrate content in Pb‐treated plants with 300, 500 and 700 µm increased significantly in leaves. Moreover, in Pb‐treated plants with 300, 500 and 700 µm Pb(NO₃)₂, mesophyll cells had enlarged chloroplasts with disrupted thylakoid membranes associated with large starch grains. In contrast, Pb treatments with 25, 50 µm and 100 µm were not toxic to the plants. Thick sections of roots of Pb‐treated plants with 300, 500 and 700 µm Pb showed distinct changes in structure of epidermal and cortical cells. Moreover, thin sections of roots of Pb‐treated plants with 300, 500 and 700 µm Pb had thickened walls of xylem cells.
• These results will shed more light in understanding the effects of heavy metal stress on plants.

     

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases

Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes (phaC1 _Pre , phaC2 _Pre ) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved α/β-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the α/β-hydrolase-fold coding portions of phaC1 _Pre and phaC2 _Pre at the 3′ terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73–75% β-hydroxydecanoate (β-HD) and 25–27% β-hydroxyoctanoate (β-HO). Deletion mutants, Δpha7 and Δpha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55–60 mol% β-HD and 40–45 mol% β-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.     

Carbon and Nitrogen Mineralization of Lead Treated Soils in the Eastern Mediterranean Region,Turkey

The aim of this study was to determine the first effect of lead on microbial activity in soil. The study was carried out in the soil samples from four different radish (Raphanus sativus L. var. radicula, Brassicaceae) fields along the highway in a district (Kadirli, Osmaniye) of the Eastern Mediterranean Region, Turkey. After the calculation of Pb contents, the Pb amounts of the soil samples were brought up to 50 and 100 mg Pb kg^?1 by treatment with Pb(NO ₃ ) ₂ , and the samples for the carbon and the nitrogen mineralization were incubated under controlled conditions (28°C, constant moist). The carbon mineralization was determined by a CO ₂ respiration method for 30 days. The nitrogen mineralization was observed in vitro for 6 weeks. The untreated group was statistically different from the 50 and 100 mg Pb kg^?1 treatments in the aspect of the C(CO ₂ ) outlet during mineralization (P ≤ 0.05), but difference between the 50 and 100 mg Pb kg^?1 treatments was not significant. NH ₄ -N and NO ₃ -N contents of each soil were shown differences between across treatments. Based on these results, it is possible to conclude that the addition of 50 and 100 mg Pb kg^?1 provided a toxic effect threshold for the microbial activity into 30 days.     

The effects of lead,nickel, and strontium nitrates on cell division and elongation in maize roots

The effects of Pb, Sr, and Ni nitrates on the root growth, its cell division and elongation were studied. Two-day-old maize seedlings were incubated on the 35 μM Ni(NO₃)₂, 10 μM Pb(NO₃)₂, or 3 mM Sr(NO₃)₂ in the presence or absence of 3 mM Ca(NO₃)₂. Metal toxicity was evaluated after the inhibition of root growth for the first and second days of incubation in comparison with the roots kept on water or Ca(NO₃)₂ solution. The contents of metals were determined in the apical (the first centimeter from the tip) and basal (the third centimeter from the kernel) root parts by voltamperometry and atomic-absorption spectrophotometry. We measured the length of the meristem, the length of the fully elongated cells, counted the mitotic index (MI) in the meristem and the number of meristematic cells in the cortex row; we also calculated duration the cell cycle. In the absence of Ca(NO₃)₂, the metal content in the apical root region was higher than in basal one. In the presence of Ca(NO₃)₂, we observed reverse ratio most pronounced in the case of Pb and Sr. All metals tested markedly reduced MI in the cortex, which was determined by the increase in the cell cycle duration and accompanied by the meristem shortening. These metals affected differently cell division and elongation: Ni inhibited mainly cell division and to a lesser degree their elongation, whereas Sr and Pb affected both cell division and elongation; only Sr treatment resulted in the increased length of the fully elongated cells. In the presence of Ca, all studied growth indices changed less than in the absence of Ca, which was manifested in the less severe suppression of the root growth and was in agreement with the lower accumulation of the metals in the root tips. Possible causes for the heavy metal action on growth are discussed in connection with the specificity of their transport and accumulation.     

Experience on the neutron activation of natural/enriched Re,Sm, and Ho nuclides in a reactor for the production of radiotherapeutic radionuclides

In recent years, much effort has been concentrated on the use of Β-emitting radionuclides for the treatment of various cancers. The reports suggested the application of¹⁸⁶Re and¹⁵³Sm as radiotherapeutic radionuclides for the treatment of palliative widespread skeletal métastases, whereas¹⁶⁶Ho was suggested as an agent for radiation synovectomy. Hence, a study on the production of¹⁸⁶Re,¹⁵³Sm, and¹⁶⁶Ho radionuclides was carried out by neutron activation of the appropriate target materials using a Pakistan Atomic Research Reactor (PARR-1) at a neutrons flux of 1 x 10⁴ n/cm² s. These radionuclides were then converted to appropriate radiopharmaceuticals for their use on animals and patients. The targets of natural Re (metal), natural Sm₂O₃, enriched Sm₂O₃ (99.06%), Sm(NO₃)₃ (solid), Sm(NO₃)₃ (liquid), and Ho₂O₃ were irradiated in the PARR-1. After irradiation, the purity of these radionuclides were checked by a multichannel analyzer, Canberra series 85 (MCA) coupled with HPGe detector and then measured in radioisotope calibrator Capintec ionization chamber model CRC-5RH. The effect of the irradiation time and amount of target material was investigated on the production yields of the radionuclides. The results showed an increase in the specific activity of Re with an increase in the irradiation time from 1 to 72 h, whereas a decrease in the specific activity was observed with increase in the amount of Re from 10 to 100 mg. Similar results were obtained for¹⁵³Sm and¹⁶⁶Ho radionuclides. The results further indicated that the specific activity of powder target was much less than the liquid targets for¹⁵³Sm. Their conversion to the appropriate radiotherapeutic radiopharmaceuticals were also carried out by investigating the experimental conditions and acceptable quality of¹⁸⁶Re-HEDP and¹⁵³Sm-EDTMP complexes were prepared. These complexes were then used on animals and patients which showed good performance.     

Type 1 fimbriation is negatively regulated by cyclic AMP and its receptor proteinvia conjugative plasmid F inEscherichia coli K-12

Expression of fimbriation was studied inEscherichia coli K-12 CA8000 HfrH, and itscya, crp and MS2 resistant mutants. The cells of cya⁺ crp⁺ parent strain were observed to be flagellated bacilli, lacking fimbriae, unable to agglutinate erythrocytes and deficient in ability to produce surface pellicle during growth in stationary culture. The cells ofcya andcrp mutants were observed to be cocci or coccobacilli devoid of flagella, having haemagglutinating activity, fimbriated and capable of producing surface pellicle in stationary cultures. The fimbriation and haemagglutinating activities were lower incya mutants grown with cAMP supplementation. Thecya andcrp mutants produced relatively small, smooth and compact colonies consisting mostly of fimbriated cells, like those of earlier described Fimσ mutants. Thecya ⁺ crp⁺ MS2 resistant mutant produced large sized colonies like those of parent but was deficient in conjugal donor ability. It resembledcya andcrp mutants in haemagglutinating and fimbriation properties. Thecya andcrp mutants have been earlier shown to be deficient in several Tra functions including conjugal donor ability. It is concluded thatEscherichia coli K-12 cells express fimbriation when Tra functions of F-plasmid carried in them are not expressed either due to deficiency of active cAMP-receptor protein complex or mutation in F-plasmid or when F-plasmid is absent.     

Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k)

 A method is presented to isolate mutants of Chlorella sorokiniana with defects in NO₃ ⁻ metabolism. Three nitrite-reductase (NIR; E.C.1.7.7.1)-deficient mutants were obtained from 500 pinpoint-colony-forming clones. The final screening was performed using NO₃ ⁻, NO₂ ⁻ or NH⁺ ₄ as N-source. The mutants isolated absorb NO₃ ⁻ with rates close to those measured for the wild type and they excrete NO₂ ⁻ into the medium. The ratio between NO₃ ⁻ uptake and NO₂ ⁻ excretion was 1:1. The sensitivity of NO₃ ⁻ uptake to NH⁺ ₄ was reduced in the mutant strains as it was in the N-starved wild type of Chlorella. Nitrate reductase (NR; EC 1.6.6.1) expression and NR activity were slightly reduced compared to the wild type due to feedback regulation in the mutant strains. No NIR protein was found in the three mutants. However, NIR activity was obtained (50% of the wild-type) for one mutant strain. The NIR-deficient mutants and the already available NR-deficient mutants will be promising tools for investigations of the nitrate assimilation pathway on the molecular level and for studies searching for signaling of C and N metabolism by inorganic N-compounds. Received: 8 October 1999 / Accepted: 25 January 2000