Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema.

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.     

Regulation of Nitrogenase Synthesis by Oxygen in Klebsiella pneumoniae

Klebsiella pneumoniae does not fix N₂ under aerobic conditions. The two protein components required for nitrogenase activity were studied during aeration of cells in nitrogen-free media. Component II of nitrogenase was inactivated more slowly in vivo than component I during aeration. The rate of loss of component II was less than the rate of component II synthesis during derepression. No inactive components were detected in cells that had been growing on NH₄⁺ and then aerated in nitrogen-free medium. This supports the hypothesis that O₂ somehow represses the formation of nitrogenase.     

Activation of Inactive Nitrogenase by Acid-Treated Component I

When Azotobacter vinelandii was derepressed for nitrogenase synthesis in a N-free medium containing tungstate instead of molybdate, an inactive component I was synthesized. Although this inactive component I could be activated in vivo upon addition of molybdate to the medium, it could not be activated in vitro when molybdate was added to the extracts. Activation occurred, however, when an acid-treated component I was added to extracts of cells derepressed in medium containing tungstate. Acid treatment completely abolished component I activity. Mutant strains UW45 and UW10 were unable to fix N(2). Both strains synthesized normal levels of component II but produced inactive component I. Acid-treated component I activated inactive component I in extracts of mutant strain UW45 but not mutant strain UW10. This activating factor could be obtained from N(2)-fixing Klebsiella pneumoniae, Clostridium pasteurianum, and Rhodospirillum rubrum.     

Role of molybdenum in dinitrogen fixation by Clostridium pasteurianum.

The role of Mo in the activity and synthesis of the nitrogenase components of Clostridium pasteurianum has been studied by observing the competition of Mo with its structural analogue W. Clostridial cells when fixing N2 appeared strictly dependent upon the available Mo, showing maximal N2-fixing activity at molybdate concentrations in the media of 10 muM. Cells grown in media with 3 times 10(-6) muM Mo, although showing good growth, had only 15% as much N2-fixing activity. In the presence of W the synthesis of both nitrogenase components, molybdoferredoxin and azoferredoxin, was affected. Attempts to produce nitrogenase in W-grown cells by addition of high molybdenum to the media in the presence of inhibitors of protein synthesis showed that Mo incorporation into a possible inactive preformed apoenzyme did not occur. Unlike other molybdoenzyme-containing cells, in which W either is incorporated in place of Mo to yield inactive protein or initiates the production of apoprotein, C. pasteurianum forms neither a tungsten substituted molybdoferredoxin nor an apoprotein. It is concluded that in C. pasteurianum molybdenum is an essential requirement for both the biosynthesis and activity of its nitrogenase.     

Immunological evidence for the capability of free-living Rhizobium japonicum to synthesize a portion of a nitrogenase component

Immunodiffusion tests conducted under aerobic conditions demonstrated that cross-reactive material to antiserum prepared against the MoFe protein component of nitrogenase from soybean nodule bacteroids was detectable in extracts of free-living Rhizobium japonicum cells cultured in a standard medium under: aerobic conditions; aerobic conditions with nitrate; aerobic conditions with ammonia; anaerobic conditions with nitrate; and anaerobic conditions with nitrate and ammonia. The most intense precipitin bands resulted from cross-section of the antiserum with extracts of cells cultured anaerobically with nitrate or anaerobically with ammonia and nitrate. Immunodiffusion experiments with crude bacteroid extract and purified MoFe protein revealed a greater number of precipitin bands in tests conducted under aerobic conditions than those conducted under anaerobic conditions. These results indicate that some of the cross-reactive material observed under aerobic conditions resulted from breakdown of the MoFe protein. Bacteroid extracts of nodules from plants supplied with ammonia exhibited only a trace of nitrogenase activity. The addition of an excess of the Fe protein component of nitrogenase, however, resulted in a 270-fold enhancement of activity indicating the presence of active MoFe protein in these extracts.Our experiments together with results published elsewhere provide evidence that the genetic information for synthesis of a part of the MoFe component of nitrogenase is carried by Rhizobium.     

Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii.

Mutants of Azotobacter vinelandii ATCC 12837 were isolated which could fix N2 in the presence of high tungsten concentrations. The most studied of these mutants (WD2) grew well in N-free modified Burk broth containing 10 mM W, whereas the wild type would not grow in this medium. WD2 would also grow in Burk N-free broth at about the same rate as the wild type. WD2 in broth containing W exhibited 22% of the whole cell acetylene reduction activity of the wild type in broth containing Mo and showed a lowered affinity for acetylene. Two-dimensional gel electrophoresis experiments showed that N2-fixing cells of WD2 from broth containing W or Mo did not produce significant amounts of component I of native nitrogenase protein. Electron spin resonance spectra of whole cells and cell-free extracts of WD2 from broth containing W lacked any trace of the g = 3.6 resonance associated with FeMoCo.     

Function of MoaB proteins in the biosynthesis of the molybdenum and tungsten cofactors

Molybdenum (Mo) and tungsten (W) enzymes catalyze important redox reactions in the global carbon, nitrogen, and sulfur cycles. Except in nitrogenases both metals are exclusively associated with a unique metal-binding pterin (MPT) that is synthesized by a conserved multistep biosynthetic pathway, which ends with the insertion and thereby biological activation of the respective element. Although the biosynthesis of Mo cofactors has been intensively studied in various systems, the biogenesis of W-containing enzymes, mostly found in archaea, is poorly understood. Here, we describe the function of the Pyrococcus furiosus MoaB protein that is homologous to bacterial (such as MogA) and eukaryotic proteins (such as Cnx1) involved in the final steps of Mo cofactor synthesis. MoaB reconstituted the function of the homologous Escherichia coli MogA protein and catalyzes the adenylylation of MPT in a Mg2+ and ATP-dependent way. At room temperature reaction velocity was similar to that of the previously described plant Cnx1G domain, but it was increased up to 20-fold at 80 degrees C. Metal and nucleotide specificity for MPT adenylylation is well conserved between W and Mo cofactor synthesis. Thermostability of MoaB is believed to rely on its hexameric structure, whereas homologous mesophilic MogA-related proteins form trimers. Comparison of P. furiosus MoaB to E. coli MoaB and MogA revealed that only MogA is able to catalyze MPT adenylylation, whereas E. coli MoaB is inactive. In summary, MogA, Cnx1G, and MoaB proteins exhibit the same adenylyl transfer activity essential for metal insertion in W or Mo cofactor maturation.     

Unformylated initiator tRNA is not a signal for the stringent control of RNA synthesis.

When Escherichia coli MRE 600 or Bacillus subtilis W 23 are grown in glucose-salt medium supplemented with purines, thymidine and glycine, trimethoprim stops the synthesis of protein by causing a specific lack of methionyl-tRNA. The synthesis of RNA is simultaneously restricted by the stringent control mechanism. Guanosine tetraphosphate (ppGpp) largely accumulates. The addition of methionine abolishes the level of ppGpp and relieves the inhibition of RNA synthesis. The aminoacylation of methionine-specific tRNAs was found to be completely restored. The methionyl-tRNA_f^Met however does not become formylated. These results indicate that unformylated initiator tRNA is not a sufficient condition for the accumulation of ppGpp and the onset of stringent control.     

FAST AND SLOW COMPONENTS IN AXONAL TRANSPORT OF PROTEIN

(a) After injection of labeled leucine into the eye of goldfish, radioactive protein rapidly accumulates in the contralateral optic tectum in the layer containing the synaptic endings of the optic fibers. This material reaches the tectum 6–12 hr after the isotope injection, a fact which indicates that the rate of transport is at least 40 mm per day. (b) This rapidly transported material has been shown to consist exclusively of protein, in which the label remains attached to leucine. (c) Inhibition of protein synthesis in the retina prevents the appearance of the transported protein in the tectum, but inhibition of protein synthesis in the tectum does not. Substances having some of the same properties as leucine, such as cycloleucine and norepinephrine, are not transported to the tectum. These experiments all indicate that the transported protein is synthesized in the retina. However, inhibition of retinal protein synthesis after this protein has been formed does not interfere with the transport mechanism itself. (d) The fast component consists of about 85% particulate material. It may be distinguished from a slowly moving component, transported at 0.4 mm per day, which contains about 5 times as much radioactivity as the fast component, and which consists of 60% particulate matter and 40% soluble protein.     

Purification and Characterization of Cr-containing Nitrogenase Component I

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but can undergo phenotypic reversal to Nif+ under Mo deficient conditions, was able to grow in Cr containing but Mo and NH3 deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW3 grown on the Cr containing medium was shown to contain Fe and Cr (atom ratio of Fe to Cr and Mo to Cr: 11.60 and 0.41) and to have 70% of the C2H2 and H+ reduction activity of MoFe protein from the wild type strain of Azotobacter vinelandii Lipmann. The Cr containing protein was different in subunit composition from that of MnFe protein purified from the mutant strain grown in the presence of Mn, but similar to that of MoFe protein, that is, it was a tetramer composed of two differentsubunits (α2β2). The preliminary results indicated that the Cr containing protein might be a nitrogenase component Ⅰ protein.     

Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii.

In Klebsiella pneumoniae, Mo accumulation appeared to be coregulated with nitrogenase synthesis. O2 and NH+4, which repressed nitrogenase synthesis, also prevented Mo accumulation. In Azotobacter vinelandii, Mo accumulation did not appear to be regulated Mo was accumulated to levels much higher than those seen in K. pneumoniae even when nitrogenase synthesis was repressed. Accumulated Mo was bound mainly to a Mo storage protein, and it could act as a supply for the Mo needed in component I synthesis when extracellular Mo had been exhausted. When A. vinelandii was grown in the presence of WO2-(4) rather than MoO2-(4), it synthesized a W-containing analog of the Mo storage protein. The Mo storage protein was purified from both NH+4 and N2-grown cells of A. vinelandii and found to be a tetramer of two pairs of different subunits binding a minimum of 15 atoms of Mo per tetramer.     

Role of ammona in regulation of nitrogenase synthesis and activity in Anabaena cylindrica

The regulation of nitrogenase biosynthesis and activity by ammonia was studied in the heterocystous cyanobacterium Anabaena cylindrica. Nitrogenase synthesis was measured by in vivo acetylene reduction assays and in vitro by an activity-independent, immunoelectrophoretic measurement of the Fe-Mo protein (Component I). When ammonia was added to differentiating cultures after a point when heterocyst differentiation became irreversible, FeMo protein synthesis was also insensitive to ammonia. Treating log-phase batch cultures with 100% O₂ for 30 min resulted in a loss of 90% of nitrogenase activity and a 50% loss of the FeMo protein. Recovery was inhibited by chloramphenicol but not by ammonia or urea. The addition of ammonia to log-phase cultures resulted in a decrease in specific levels of nitrogenase activity and FeMo protein that occurred at the same rate as algal growth and was independent of O₂ tension of the culture media. However, in light-limited linear-phase cultures, ammonia effected a dramatic inhibition of nitrogenase activity. These results indicate that nitrogenase biosynthesis becomes insensitive to repression by ammonia as heterocysts mature and that ammonia or its metabolites act to regulate nitrogen fixation by inhibiting heterocyst differentiation and by inhibiting nitrogenase activity through competition with nitrogenase for reductant and/or ATP, but not by directly regulating nitrogenase biosynthesis in heterocysts.     

Auxin-Induced Regulation of Protein Synthesis in Tobacco Mesophyll Protoplasts Cultivated In Vitro: II. Time Course and Level of Auxin Control

When protoplasts, previously cultivated in a medium lacking auxin, are transferred to complete medium, proteins whose synthesis is stimulated by the hormone become detectable after about 30 minutes and reach a constant level 2 to 4 hours after the beginning of hormonal treatment. In contrast, proteins whose level of synthesis is reduced by auxin, are only affected after 6 hours of treatment.

Short radioactive labelings in deficient medium followed by chases in complete medium show that auxin does not interfere with posttranslational processes.

Analysis of in vitro translation products of protoplast RNA shows that the time courses of auxin effects on protein synthesis and mRNA accumulation are perfectly superimposable. This allows us to exclude the possibility that auxin affects the translation process, but indicates that this hormone acts by regulating the concentration of the auxin-sensitive protein mRNAs.

     

Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii.

DNA sequencing of the region upstream from the Azotobacter vinelandii operon (modEABC) that contains genes for the molybdenum transport system revealed an open reading frame (modG) encoding a hypothetical 14-kDa protein. It consists of a tandem repeat of an approximately 65-amino-acid sequence that is homologous to Mop, a 7-kDa molybdopterin-binding protein of Clostridium pasteurianum. The tandem repeat is similar to the C-terminal half of the product of modE. The effects of mutations in the mod genes provide evidence for distinct high- and low-affinity Mo transport systems and for the involvement of the products of modE and modG in the processing of molybdate. modA, modB, and modC, which encode the component proteins of the high-affinity Mo transporter, are required for 99Mo accumulation and for the nitrate reductase activity of cells growing in medium with less than 10 microM Mo. The exchange of accumulated 99Mo with nonradioactive Mo depends on the presence of modA, which encodes the periplasmic molybdate-binding protein. 99Mo also exchanges with tungstate but not with vanadate or sulfate. modA, modB, and modC mutants exhibit nitrate reductase activity and 99Mo accumulation only when grown in more than 10 microM Mo, indicating that A. vinelandii also has a low-affinity Mo uptake system. The low-affinity system is not expressed in a modE mutant that synthesizes the high-affinity Mo transporter constitutively or in a spontaneous tungstate-tolerant mutant. Like the wild type, modG mutants only show nitrate reductase activity when grown in > 10 nM Mo. However, a modE modG double mutant exhibits maximal nitrate reductase activity at a 100-fold lower Mo concentration. This indicates that the products of both genes affect the supply of Mo but are not essential for nitrate reductase cofactor synthesis. However, nitrogenase-dependent growth in the presence or absence of Mo is severely impaired in the double mutant, indicating that the products of modE and modG may be involved in the early steps of nitrogenase cofactor biosynthesis in A. vinelandii.     

Reactivation of an Inactive Centromere Reveals Epigenetic and Structural Components for Centromere Specification in Maize

Stable maize (Zea mays) chromosomes were recovered from an unstable dicentric containing large and small versions of the B chromosome centromere. In the stable chromosome, the smaller centromere had become inactivated. This inactive centromere can be inherited from one generation to the next attached to the active version and loses all known cytological and molecular properties of active centromeres. When separated from the active centromere by intrachromosomal recombination, the inactive centromere can be reactivated. The reactivated centromere regains the molecular attributes of activity in anaphase I of meiosis. When two copies of the dicentric chromosome with one active and one inactive centromere are present, homologous chromosome pairing reduces the frequency of intrachromosomal recombination and thus decreases, but does not eliminate, the reactivation of inactive centromeres. These findings indicate an epigenetic component to centromere specification in that centromere inactivation can be directed by joining two centromeres in opposition. These findings also indicate a structural aspect to centromere specification revealed by the gain of activity at the site of the previously inactive sequences.     

Purification and Characteristics of Mn-containing Nitrogenase Component

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but undergo phenotypic reversal to Nif+ under Mo deficiency, was able to grow in Mo- and NH3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C2H2- and H+-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰprotein.     

Site in Cell-free Protein Synthesis Sensitive to Diphtheria Toxin

The effects of diphtheria toxin on cell-free protein synthesis in a bacterial system, and preparations obtained from animals that were sensitive and resistant to toxin were examined. In the presence of nicotinamide adenine dinucleotide (NAD), toxin inhibited the incorporation of amino acids by endogenous and synthetic polynucleotides in both rat liver and guinea pig liver cell-free systems that were exposed to 6 Lf units per ml of toxin. A cell-free system derived from Streptococcus faecalis was resistant to high concentrations of toxin. Dialyzed toxin-antitoxin floccules that are formed in the presence of NAD and the 105,000 x g supernatant fluid from rat liver contain NAD. Such floccules are also active in protein synthesis in the absence of added transferase I or II. An operational model presents the view that the intoxication complex is formed at the ribosomal level and occurs in two steps. First, the toxin molecule binds to transferase II and alters its stereospecific relationship to transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase II caused by the binding of diphtheria toxin allows NAD to bridge between transferase I and II, which then results in an inactivated complex. The sensitivity of the cell-free system derived from the normally resistant rat implies that in some cells the cell membrane serves as a permeability barrier to the toxin molecule. The resistance of bacterial cell-free protein synthesizing systems to diphtheria toxin may reflect basic differences between transferase enzymes from bacterial and mammalian sources.     

Hb switching in neonatal cultures. Increase of Hb A synthesis in presence of an erythroid potentiating activity (EPA)

The role of EPA (erythroid potentiating activity) on the growth and on the pattern of hemoglobin synthesis in erythroid colonies from human neonates was investigated. Conditioned medium from the Mo cell line was used as a source of EPA. The results have shown that the addition of Mo medium to cultures determined a significant enhancement of the number and size of BFU-E and an increase of beta chain synthesis. The acceleration of hemoglobin switching is not related to an amelioration of the maturation of the erythroid colonies when grown in the presence of Mo medium. The enhancement of Hb A synthesis induced by Mo medium can directly be related to its EPA, which may operate by two different mechanisms: (1) the recruitment of early erythroid progenitors already preprogrammed to synthesize prevalently beta chains, or (2) the modulation of beta and gamma gene activity in cord blood BFU-E. Some evidence suggests that the first mechanism does operate.     

Biochemical studies with a new cytotoxic immunosuppressive agent, 3-acetyl-5-(4-fluorobenzylidene)-2,5-dihydro-4-hydroxy-2-oxothiophen (I.C.I. 47776)

1. A new cytotoxic agent, 3-acetyl-5-(4-fluorobenzylidene)-2,5-dihydro-4-hydroxy-2-oxothiophen (I.C.I. 47776), strongly inhibits protein and nucleic acid synthesis and, to a smaller extent, respiration in lymph-node cells and Landschütz ascites-tumour cells in vitro. 2. The activity of I.C.I. 47776 in vitro declines as the pH of the medium is increased and is inversely proportional to the concentration of serum in the medium. 3. The compound has no effect on the incorporation of leucine by a cell-free preparation from Landschütz ascites cells containing ATP and phosphoenolpyruvate. 4. I.C.I. 47776 stimulates glycolysis in suspensions of Landschütz ascites cells in the presence of excess of glucose but has no effect on glycolysis in suspensions of rat lymph-node cells. 5. I.C.I. 47776 markedly depresses ATP concentration in ascites cells in the absence of glucose but has no effect on the ATP concentration in the presence of glucose. The inhibition of protein synthesis by I.C.I. 47776 in ascites cells is, however, only partially reversed by the addition of glucose. 6. The ATP concentration of rat lymph-node cells incubated with I.C.I. 47776 in the absence of glucose is also markedly depressed but the addition of glucose increases the ATP concentration only slightly. Further, glucose has no effect on the inhibition of protein synthesis in lymph-node cells by I.C.I. 47776. 7. It is suggested that I.C.I. 47776 inhibits protein and nucleic acid synthesis in cell suspensions indirectly by acting as a mitochondrial poison. 8. The relevance of studies on the activity of I.C.I. 47776 in vitro to its cytotoxic and immunosuppressive action in vivo is discussed.