In-vitro translation of different mRNA-containing fractions of Chlamydomonas chloroplasts

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.Abbreviation SDS sodium dodecyl sulfate     

Amide metabolism of Chlamydomonas reinhardi

Chlamydomonas reinhardi can utilise the lower aliphatic amides (C₁–C₄) as nitrogen sources. Of these only acetamide can serve as a sole carbon source. The acetamide analogue F-acetamide kills cells after conversion to F-acetate and F-citrate. This conversion is controlled by exogenous ammonia and, in part, acetate levels. Only one enzyme and one active site are involved in acetamidase function. Enzymatic analysis indicates an increased substrate range as compared to the growth — supported range, indicating uptake, toxicity or metabolic control restrictions.Abbreviations TCA trichloroacetic acid - TAP tris-acetate-phosphate medium - MIC mimmum inhibitory concentration - BSA bovine serum albumin     

Polymorphism and Mendelian inheritance of photosystem II 23-kilodalton polypeptide

Soluble proteins from leaves of Nicotiana glauca Grah., N. langsdorffii Weinm., their reciprocal hybrids and amphiploid hybrid (N. glaucaxN. langsdorffii) were resolved by two-dimensional gel electrophoresis. Among a group of well-resolved polypeptides, in the isoelectric-point range of 5–5.5 and relative-molecular-mass (M_r) range of 18–23 kilodaltons (kDa), species-specific variation was observed. Polypeptides designated L and l are specific to N. langsdorffii, and G and g to N. glauca, while C is common to both species. Polypeptides L, G and C are localized in the chloroplasts and associated with thylakoid membranes. Polypeptide L is more acidic than polypeptide G, and both polypeptides have an M_r of 23 kDa. They were isolated from two-dimensional gels and their first 13 N-terminal amino-acid sequences were determined. These were found to be identical to the 13N-terminal amino acids of the photosystem II (PSII) 23-kDa polypeptide from spinach (T. Jansen et al. (1987) FEBS Lett. 216, 234–240) and, except for one change, to those from pea (R. Wales et al. (1989) Plant Molec. Biol., in press). Polypeptides G and L cross-react with antiserum against the PSII 23-kDa polypeptide from pea. Therefore, polypeptides G and L are extrinsic PSII 23-kDa polypeptides. They appear jointly and in equal amounts in the reciprocal hybrids. Since chloroplasts in Nicotiana are maternally inherited, these results demonstrate that polypeptides G and L are encoded by nuclear genes, are polymorphic variants of the PSII 23-kDa polypeptide, and are inherited in a Mendelian manner.Abbreviations kDa kilodalton - LS large subunit of Rubisco - M_r relative molecular mass - NEPHGE non-equilibrium pH gradient gel electrophoresis - PSII photosystem II - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SS small subunit of Rubisco     

Changes in phosphatase activity associated with cell wall defects in Chlamydomonas reinhardi

Experiments were performed to isolate mutants lacking alkaline phosphatase in Chlamydomonas reinhardi. Mutants with null enzyme activity were obtained. A cytological study of these mutants however revealed cell wall defects, suggesting that the loss of phosphatase activity in these strains is not due to the inactivation of the corresponding phosphatase structural gene but rather to the leakage of this enzyme as a consequence of the cell wall abnormality. Incidentally, this finding provides the basis of a convenient method for selecting easily cell wall mutants of Chlamydomonas.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique.     

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt⁺ or mt^-). It is shown that before mt⁺ and mt^- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt^- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt⁺ gametes constituting the outer surface of the clumps with the mt^- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt⁺ flagellar tips then deactivate and swimming is resumed. The original mt^- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt^- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

Cell-wall abnormalities of a Chlamydomonas reinhardtii mutant strain under suboptimal growth conditions

Sporangia were accumulated in autotrophically and mixotrophically growing cultures of the Chlamydomonas reinhardtii mutant strain ls entering the stationary phase. Such an accumulation of sporangia was never observed in stationary-phase cultures of wildtype strains. Sporangia harvested from stationary-phase cultures of the mutant strain ls released their zoospores after being resuspended in fresh culture medium. Liberation of zoospores was also observed during fixation of these sporangia with glutaraldehyde and OsO₄. Release of zoospores during fixation was prevented by pretreatment with 3 mol·l^–1 LiCl. Ultrastructural analyses of these LiCl-pretreated sporangia revealed that they contained abnormal sporangial walls: sporangia containing sporangia and sporangia surrounded by additional multilayered cell walls have been observed. Similar abnormal cell-wall structures were found in sporangia accumulated at the end of the dark period, when the mutant strain ls was grown photoautotrophically under a 12 h light-12 h dark regime with suboptimal aeration. When grown under optimal conditions, this particular mutant did not show any abnormal wall structures.This work has been supported by a grant from the Deutsche Forschungsgemeinschaft. The authors thank Mrs. C. Adami for the photographic work.     

The chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii

We have adapted the procedure for the isolation of PSII membranes from higher plants (D.A. Berthold et al., 1981, FEBS Lett. 134, 231–234) to the green algae Chlamydomonas reinhardtii. The chlorophyll (Chl)-binding proteins from this PSII preparation have been further separated into single Chl-binding polypeptides and characterized spectroscopically. Seven single polypeptides were shown to bind Chl a and Chl b. In particular, we demonstrate that polypeptides p9, p10 and p22, which had not been previously shown to bind Chl a and b, have characteristics similar to those of CP29, CP26 and CP24 from higher plants. We note, however, that p9 and p10 are phosphorylatable in C. reinhardtii, at variance with CP29 and CP26 from higher plants. Our data support the notion that the PSII antenna systems in C. reinhardtii and in higher plants are very similar. Therefore, studies on the organization and regulation of light-harvesting processes in C. reinhardtii may provide information of general relevance for both green algae and higher plants.Abbreviations Chl chlorophyll - IEF isoelectrofocusing - LHC light harvesting complex - MW molecular weight - PAGE polyacrylamide gel electrophoresis - PS photosystem - RC reaction centre - SDS sodium dodecylsulfate We thank Dr. J. Olive (Institut Jacques Monod, Paris, France) for the electron-microscopy analysis, C. de Vitry (Institut de Biologie Physico-Chimique, Paris, France) for the kind gift of a PSII RC preparation and P. Dainese and M.L. Di Paolo (Universitá di Padova, Padova, Italy) for helpfull discussions. Professor Strasser and Elizbeth Scwartz (Université de Genova, Genova, Switzerland) are thanked for assistance in taking low-temperature fluorescence emission spectra. Roberto Bassi was recipient of a short-term fellowship from the European Molecular Biology Organization fellowship, during the early phases of the work.     

Characterization of the LI818 polypeptide from the green unicellular alga Chlamydomonas reinhardtii

The LI818 gene from Chlamydomonas encodes a polypeptide that is related to the chlorophyll a/b-binding proteins (CAB) of higher plants and green algae. However, despite this relatedness, LI818 gene expression is not coordinated with that of cab genes and is regulated differently by light, suggesting a different role for LI818 polypeptide. We show here that, in contrast to CAB polypeptides, LI818 polypeptide is not tightly embedded into the thylakoid membranes and is localized in stroma-exposed regions. Moreover, during chloroplast development, LI818 polypeptide accumulates before CAB polypeptides. We also show that the LI818 polypeptide forms with certain chlorophyll a/c-binding proteins (CAC) from the haptophyte Isochrysis galbana and the diatom Cyclotella cryptica a natural group that is distinct from those constituted by CAB, CAC and the chlorophyll a/a-binding proteins (CAA). Such an association suggests a very ancient origin for this group of polypeptides, which predates the division of the early photosynthetic eukaryotes into green (chlorophyte), red (rhodophyte) and brown (chromophyte) algae. Possible roles for the LI818 polypeptide are discussed.     

Sexual agglutination factor from Chlamydomonas eugametos

Chlamydomonas eugametos gametes can sexually agglutinate via their flagellar surfaces whereas vegetative cells cannot. Therefore, flagellar glycoproteins, present in gamete cells but absent from vegetative cells, were investigated as prospective mt ^-agglutination factors. They were identified as periodic acid Schiff (PAS) stained bands separated in sodium dodecyl sulphate-polyacrylamide electrophoresis gels. Gamete-specific bands were determined by comparison with equivalent gels of vegetative flagella and by immunological techniques using antisera raised against isolated mt ^- gamete flagella. Four high molecular weight flagellar glycoproteins proved to be gamete specific (PAS-1.2, PAS-1.3, PAS-3 and PAS-4). They were extracted from flagella by 3 M guanidine thiocyanate, separated in a column of Sepharose 2B, and tested for in vitro agglutination activity on mt ⁺ gametes. A single peak of activity was found to be correlated with the presence of the PAS-1.2 band. It is shown that mt ^- agglutination activity is related to the concentration of this glycoprotein in flagellar membranes.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid Schiff - GTC guanidine thiocyanate - mt ^-/+ mating type plus or minus     

Altered expression of the chloroplast ATP synthase through site-directed mutagenesis in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The chloroplast ATP synthase gates the flow of protons out of the thylakoid lumen. In Chlamydomonas reinhardtii deletion of any of the genes for the ATP synthase subunits, or misfolding of the peptides results in photosynthetic membranes devoid of the enzyme (Lemaire and Wollman, J Biol Chem 264:675–685, 1989). This work examines the physiologic response of an algal strain in which the epsilon subunit of the chloroplast ATP synthase has been truncated. Removal of 10 amino acids from the C-terminus of the peptide results in a sharp decrease in the content of the enzyme, but does not result in its exclusion from the thylakoid membranes. The ATP synthase of this mutant strain has a higher rate of ATP hydrolysis than the wild-type enzyme. This strain of C. reinhardtii exhibits reduced growth in the light, dependence on acetate, and a low threshold for the onset of photoinhibition. The role of the ATP synthase in regulating the proton concentration of the lumen is discussed. This work was supported in part by a grant from the National Science Foundation (MCB0110232).     

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28–30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28–30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSII photosystem II Dedicated to Prof. Erwin Bünning on the occasion of his 80^th birthday     

The cell wall of Chlamydomonas reinhardtii gametes: Composition,structure and autolysin-mediated shedding and dissolution

The cell walls of Chlamydomonas gametes are multilayered structures supported on frameworks of polypeptides extending from the plasma membrane. The wall-polypeptide catalogue reported by Monk et al. (1983, Planta 158, 517–533) and extended by U.W. Goodenough et al. (1986, J. Cell Biol. 103, 405–417) was re-evaluated by comparative analysis of mechanically isolated cell walls purified from several strains. The extracellular locus of wall polypeptides was verified by in vivo iodogen-catalysed iodination and by autolysin-mediated elimination of the bulk of these polypeptides from the cell surface. Three (w15, w16, w17) and possibly four (w14) polypeptides were located to the most exterior aspect of the wall because of their susceptibility to Enzymobeadcatalysed iodination and their retention by a cell-wall-less mutant. The composition of shed walls stabilised with ethylenediaminetetraacetic acid during natural mating and kinetic analysis of the dissolution of walls purified from a bald-2 mutant demonstrated the rapid and specific destruction of polypeptide w3. Differential solubilisation of wall polypeptides occurred after loss of w3. Wall dissolution, characterised by the generation of fishbone structures from the W2 layer, gave as many as four additional polypeptides. Charged detergents and sodium perchlorate extracted a comparable range of polypeptides at room temperature from mechanically isolated walls, i.e. components of the W4–W6 layers, hot sodium dodecyl sulphate solubilised framework polypeptides, while reducing agent was required to solubilise the W2 layer. A model of wall structure is presented.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Imazaquin and chlorsulfuron resistance and cross resistance in mutants of Chlamydomonas reinhardtii

Summary Chlorsulfuron and/or imazaquin resistant mutants of Chlamydomonas reinhardtii strain CW15 have been obtained and shown to have actolactate synthase (ALS) with altered sensitivity to one or both of these herbicides. Herbicide resistance in the three mutants described is allelic, and resistance appears to result from a dominant or semidominant mutation in a single, nuclear gene. Imazaquin and chlorsulfuron resistant ALS from imazaquin and chlorsulfuron resistant mutants, together with single-gene Mendelian inheritance of these phenotypes, suggests that ALS is the sole site of action of the two herbicides in Chlamydomonas. A high degree of cross resistance between the two herbicides was found in only one mutant. This mutant (IM-13) was selected for resistance to imazaquin and has a high level of in vitro resistance to both imazaquin (270-fold increased I₅₀) and chlorsulfuron (900-fold increased I₅₀). In another mutant selected for resistance to imazaquin (IMR-2), hyper-sensitivity to chlorsulfuron was found. A mutant selected for resistance to chlorsulfuron (CSR-5), had a substantial degree of resistance of chlorsulfuron (80-fold increased I₅₀), but not to imazaquin (7-fold increased I₅₀).     

Extracellular deamination of l-amino acids by Chlamydomonas reinhardtii cells

When grown in the light and in a Tris-acetate phosphate medium, cells of Chlamydomonas reinhardtii Dang. can use the following l-amino acids as a sole nitrogen source: asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine, whereas, in the absence of acetate, the cells only used l-arginine. The utilization system in the acetate medium consisted of an extracellular deaminating activity induced by l-amino acids; it took between 10 to 30 h before the system appeared in cells previously grown with ammonium. This deaminase activity was nonspecific, required an organic carbon source for its de-novo synthesis, and was sensitive to high ammonium concentration and light deprivation.Abbreviations HPLC high-performance liquid chromatography - TAP Tris-acetate-phosphate This work was supported by a grant of the CAICYT, Spain. The secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.To whom correspondence should be addressed.     

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2^**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb ⁶³) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k ²³, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.Abbreviations Chl-P chlorophyll-protein - CM Carlsberg Monoclonal - Da dalton - LHC light-harvesting complex - PAGE polyacrylamide gel electrophoresis - PSI, II photosystem I, II - PSI-200 PSI containing LHCI polypeptides - SDS sodium dodecyl sulphate     

Purification and molecular properties of ferredoxin-glutamate synthase from Chlamydomonas reinhardii

Ferredoxin-glutamate synthase (EC 1.4.7.1) from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity, with a specific activity of 10.4 units mg^-1 protein, by a method which included chromatography on diethylaminoethyl sephacel and hydroxylapatite, and ferredoxin-sepharose affinity treatment. The enzyme is a single polypeptide chain of M _r 146000 dalton which shows an absorption spectrum with maxima at 278, 377 and 437 nm, and an A₂₇₆/A₄₃₇ absorptivity ratio of 7.0. The anaerobic addition of dithionite results in the loss of the absorption peak at 437 nm, which is restored upon reoxidation of the enzyme with an excess of 2-oxoglutarate, alone or in the presence of glutamine. This indicates the presence in the enzyme of a flavin prosthetic group, which is functional during the catalysis. The ferredoxin-glutamate synthase can be assayed with methyl viologen, chemically reduced with dithionite, but it is unable to use reduced pyridine nucleotide. Azaserine, 6-diazo-5-oxo-norleucine, bromocresol green and p-hydroxymercuribenzoate are potent inhibitors of this activity, which, on the other hand, is stable upon heating at 45°C for 10 min.Abbreviations DEAE-sephacel diethylaminoethyl sephacel - Fd ferredoxin - GOGAT glutaniate synthase (glutamine: -ketoglutarate aminotransferase) - SDS sodium dodecyl sulfate     

Protein-DNA interaction within one cloned chloroplast DNA replication origin of Chlamydomonas

Summary A partially purified algal protein mixture which supports in vitro DNA replication consists of soluble proteins and proteins extracted from thylakoid membrane. The membrane extract is essential for the specific initiation of replication at a displacement loop (D-loop) site previously mapped by electron microscopy. D-loop site and its flanking sequences have been cloned and sequenced. In this study, fragment-retention assays using various subclones of the sequenced region indicate that some proteins in the membrane extract bind strongly and specifically with a 494 bp restriction fragment which partially overlaps the D-loop site. Protein gel analyses of the protein-DNA complex identify three DNA-binding polypeptides with apparent molecular weights of 18, 24 and 26 kDa, respectively. Treatment with chloramphenicol, an inhibitor of chloroplast protein synthesis, for 1 h has no obvious effect on the contents of the 24 or 26 kDa polypeptides but significantly reduces the content of the 18 kDa polypeptide in the membrane extract.     

Light dependence of sexual agglutinability in Chlamydomonas eugametos

The mating activity of mating-type plus gametes of Chlamydomonas eugametos depends on light. Cells lost their ability to agglutinate with mating-type minus gametes after a dark period of 30 min. They regained their agglutinability after 10 min exposure to light. Other mating reactions, such as tipping and flagellar tip activation, were not dependent upon light. Since cycloheximide and tunicamycin did not affect the light-induced activation of flagellar agglutinability, no protein synthesis or glycosylation is involved in this process. Equal amounts of biologically active agglutination factor could be extracted from cells placed either in light or in darkness. A minor portion of the active material was found to be located on the flagellar surface of illuminated cells. No active material was found on the flagellar surface of dark-exposed cells, whereas their cell bodies contained the same amount of active material as the cell bodies of illuminated cells. Since a light-induced flow of agglutination factors from the cell body to the flagella could not be detected and dark-exposed cells could be slightly activated by amputation or fixation by glutaraldehyde, we propose that light affects flagellar agglutinability by an in-situ modification of the agglutination factor on the flagella. When mt ⁺ and mt ^- strains were crossed and the progeny examined for light-sensitivity, it was apparent that this phenomenon is not mating type-linked.Abbreviations and symbols FTA flagellar tip activation - mt ^+/- mating type plus or minus - WGA wheat-germ agglutinin     

Sex-specific binding and inactivation of agglutination factor in Chlamydomonas eugametos

Gametes of opposite mating type (mt ⁺ and mt ^-) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using ³⁵S-labeled flagella and the isolated mt ^-agglutination factor. It is shown that not only isolated flagella, but also the mt ^-agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt ^-agglutination factor in determining the sexual agglutinability of mt ^-gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt ^-agglutination factor can be completely inactivated.Abbreviations Mt ^+/- mating type plus or minus - PAS periodic-acid Schiff-reagent - Hepes 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - HMC buffer Hepes buffer (10 mM. pH 7.2, containing 1 mM MgCl₂ and 1 mM CaCl₂)     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen