Nitrogenase Proteins Research Articles

The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis.

Active Fe protein of nitrogenase was synthesised in a non-nitrogen fixing organism when Escherichia coli was transformed with a plasmid encoding only two nif-specific genes, nifH and nifM of Klebsiella pneumoniae. Hence proteins NifH and NifM are sufficient to produce active Fe protein in E. coli. K. pneumoniae strains carrying chromosomal nifW- and nifZ- mutations were constructed and shown to be significant C2H2-reducing activity and to grow on N-free plates. Nevertheless, derepressing cultures of the mutant strains had reduced levels of MoFe protein activity, and consequently significantly lower levels of nitrogenase activity, than the nif+ parent strain. NifW and NifZ therefore appear to be involved in the formation or accumulation of active MoFe protein, but are not essential for nitrogen fixation in K. pneumoniae under the conditions tested.

Mössbauer studies of solid thionin-oxidized MoFe protein of nitrogenase.

Recently Hagen et al. (Hagen, W. R., Wassink, H., Eady, R. R., Smith, B. E., and Haaker, H. (1987) Eur. J. Biochem. 169, 457-465) reported the observation of S = 7/2 EPR signals for thionin-oxidized nitrogenase MoFe protein. Here we have studied the protein from Azotobacter vinelandii and Klebsiella pneumoniae with Mössbauer and EPR spectroscopies, with the following results: when the MoFe protein is oxidized by addition of stoichiometric amounts (6-8 equivalents) of dissolved thionin, the well characterized P-cluster state Pox results. Pox has an as yet undetermined, but half-integer electronic spin; however, the state is EPR-silent. In contrast, oxidation by addition of a large excess of solid thionin powder, the method used by Hagen et al., yields mixtures with variable proportions of two oxidized P-cluster forms, namely the familiar Pox and the new state Pox(S = 7/2) observed by Hagen et al. The Mössbauer data suggest that Pox and Pox(S = 7/2) are isoelectronic. The two states, however, have distinct electronic structures; the Mössbauer spectra of Pox exhibit the characteristic trapped-valence Fe2+ site, whereas the spectra of Pox(S = 7/2) lack this feature. Hagen et al. have proposed two new P-cluster models. We conclude that one of the models is incompatible with the Mössbauer data and that the basic assumptions of the other model are not supported by the available data. Finally, the Mössbauer data show that either oxidation method puts the cofactor centers into the diamagnetic state Mox.

ChemInform Abstract: X‐Ray Absorption of Azotobacter vinelandii Vanadium Nitrogenase.

Evidence for the existence of a vanadium-containing nitrogenase has existed for more than half a century, but progress in understanding this enzyme has only come recently. In 1980, Bishop and co-workers proposed that an alternative nitrogen-fixing enzyme exists in Azotobacter vinelandii and subsequently proposed that vanadium was involved. In 1986, Robson et al. demonstrated clearly that the alternate nitrogenase from Azotobacter chroococcum, Acl*, contained vanadium instead of molybdenum. Hales et al. have shown the vanadium is also found in the Azotobacter vinelandii alternative component I, Avl'. The molybdenum and vanadium nitrogenase proteins are similar in many respects. Like the molybdenum enzyme, both Acl* and Avl' exhibit an EPR spectrum characteristic of a species with an S = 3/2 ground state; Avl' also contains the so-called P-clusters. Additionally Acl* has recently been shown to possess an N-methylformamide soluble cofactor, FeVco, analogous to the well-known iron-molybdenum cofactor FeMoco. Arber et al. have reported X-ray absorption spectra for the Acl* enzyme and interpreted the EXAFS as evidence for a V-Fe-S cluster. The local vanadium structure is proposed to resemble a recently synthesized cubane-like VFe/sub 3/S/sub 4/ cluster, and analogies are drawn with the EXAFS-derived structure reported for the molybdenum nitrogenases. The authors reportmore » herein an X-ray absorption spectroscopic study of A. vinelandii vanadium nitrogenase, Avl', which supports and extends the work of Arber et al.« less

Electron-transfer studies involving flavodoxin and a natural redox partner, the iron protein of nitrogenase. Conformational constraints on protein-protein interactions and the kinetics of electron transfer within the protein complex.

The kinetics of electron-transfer reactions involving flavodoxins from Klebsiella pneumoniae (KpFld), Azotobacter chroococcum (AcFld), Anacystis nidulans (AnFld) and Megasphaera elsdenii (MeFld), the free, MgADP-bound and MgATP-bound forms of the Fe protein component of nitrogenase from K. pneumoniae [Kp2, Kp2(MgADP)2 and Kp2(MgATP)2] and Na2S2O4 were studied by stopped-flow spectrophotometry. Kinetic evidence was obtained for the formation of binary protein complexes involving KpFldSQ (semiquinone) with either Kp2(MgADP)2 (KD = 49 microM) or Kp2(MgATP)2 (KD = 13 microM) but not with Kp2 (KD greater than 730 microM). The binding of 2MgATP or 2MgADP to Kp2 therefore not only shifts the midpoint potential (Em) of the [4Fe-4S] centre from -200 mV to -320 mV or -350 mV respectively but also changes the affinity of Kp2 for KpFldSQ. Thermodynamically unfavourable electron from Kp2(MgADP)2 and Kp2(MgATP)2 to KpFldSQ occurs within the protein complexes with k = 1.2 s-1 (delta E = -72 mV) and 0.5 s-1 (delta E = -120 mV) respectively. Although AcFldSQ is reduced by Kp2, Kp2(MgADP)2 and Kp2(MgATP)2 (k = 8 x 10(3), 2.4 x 10(3) and 9 x 10(2) M-1.s-1 respectively), protein-complex formation is weak in each case (KD greater than 700 microM). Electron transfer in the physiologically important and thermodynamically favourable direction from Kp2FldHQ (hydroquinone) and AcFldHQ to Kp2ox.(MgADP)2 (the state of Kp2 that accepts electrons from FldHQ in the catalytic cycle of nitrogenase) is rapid (k greater than 10(6) M-1.s-1). The second-order rate constants for the reduction of KpFldSQ, AcFldSQ, AnFldSQ and MeFldSQ by SO2.- (active reductant formed by the predissociation of S2O4(2-) ion) exhibited the linear free-energy relationship predicted by the Marcus theory of electron transfer.

Ultrastructural analysis of the rehydration of desiccatedNostoc commune HUN (cyanobacteria) with particular reference to the immunolabelling of NifH

Vegetative cells and heterocysts of the filamentous desiccation-tolerant cyanobacteriumNostoc commune HUN retain their ultrastructural organisation and the integrity of their intra- and extracellular membranes after two years of desiccation and subsequent rehydration. Immunogold-labelling of thin sections demonstrated the presence of NifH (Fe protein of nitrogenase) in vegetative cells and heterocysts within five minutes of the rehydration of dried colonies. Immunogold label accumulated in discrete areas vegetative cells within 5 minutes of the rewetting of cells, and after 30 minutes a conspicuous association of NifH protein with heterocyst ribosomes was detected. After longer periods of rehydration, the deposition of gold particles became more random within both cell types but occurred with a greater frequency in heterocysts. Up to 24 hours after the rewetting of cells, two morphologically-distinct forms of heterocyst could be discerned. NifH protein was detected through Western blotting (subunit Mr=33,800) in protein extracts from samples ofNostoc commune, collected in different parts of the world and including some which had been desiccated for periods of up to 10 years. The results are discussed in relation to the sequential recovery of metabolic functions, particularly nitrogen fixation, which occurs upon the rehydration of cells after their prolonged storage in the air-dry state.

X-ray absorption of Azotobacter vinelandii vanadium nitrogenase

Evidence for the existence of a vanadium-containing nitrogenase has existed for more than half a century, but progress in understanding this enzyme has only come recently. In 1980, Bishop and co-workers proposed that an alternative nitrogen-fixing enzyme exists in Azotobacter vinelandii and subsequently proposed that vanadium was involved. In 1986, Robson et al. demonstrated clearly that the alternate nitrogenase from Azotobacter chroococcum, Acl*, contained vanadium instead of molybdenum. Hales et al. have shown the vanadium is also found in the Azotobacter vinelandii alternative component I, Avl'. The molybdenum and vanadium nitrogenase proteins are similar in many respects. Like the molybdenum enzyme, both Acl* and Avl' exhibit an EPR spectrum characteristic of a species with an S = 3/2 ground state; Avl' also contains the so-called P-clusters. Additionally Acl* has recently been shown to possess an N-methylformamide soluble cofactor, FeVco, analogous to the well-known iron-molybdenum cofactor FeMoco. Arber et al. have reported X-ray absorption spectra for the Acl* enzyme and interpreted the EXAFS as evidence for a V-Fe-S cluster. The local vanadium structure is proposed to resemble a recently synthesized cubane-like VFe/sub 3/S/sub 4/ cluster, and analogies are drawn with the EXAFS-derived structure reported for the molybdenum nitrogenases. The authors reportmore » herein an X-ray absorption spectroscopic study of A. vinelandii vanadium nitrogenase, Avl', which supports and extends the work of Arber et al.« less

Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations.

Azotobacter vinelandii was grown diazotrophically at different dissolved oxygen concentrations (in the range of 3 to 216 microM) in sucrose-limited continuous culture. The specific nitrogenase activity, measured on the basis of acetylene reduction in situ, was dependent solely on the growth rate and was largely independent of oxygen and sucrose concentration. FeMo (Av1) and Fe (Av2) nitrogenase proteins were quantified after Western blotting (immunoblotting). When the cultures were grown at a constant dilution rate (D, representing the growth rate, mu) of 0.15.h-1, the cellular levels of both proteins were constant regardless of different dissolved oxygen concentrations. The same was true when the organisms were grown at D values above 0.15.h-1. At a lower growth rate (D = 0.09.h-1), however, and at lower oxygen concentrations cellular levels of both nitrogenase proteins were decreased. This means that catalytic activities of nitrogenase proteins were highest at low oxygen concentrations, but at higher oxygen concentrations they increased with growth rate. Under all conditions tested, however, the Av1:Av2 molar ratio was 1:(1.45 +/- 0.12). Cellular levels of flavodoxin and FeS protein II were largely constant as well. In order to estimate turnover of nitrogenase proteins in the absence of protein synthesis, chloramphenicol was added to cultures adapted to 3 and 216 microM oxygen, respectively. After 2 h of incubation, no significant decrease in the cellular levels of Av1 and Av2 could be observed. This suggests that oxygen has no significant effect on the breakdown of nitrogenase proteins.

The role of MgATP hydrolysis in nitrogenase catalysis.

Kinetic studies on MgATP hydrolysis by nitrogenase of Azotobacter vinelandii were performed in the presence and in the absence of reducing equivalents. By measuring the ATPase activity of dye-oxidized nitrogenase proteins it can be excluded that reductant-independent ATPase activity is the result of futile cycling of electrons. The turnover rates of MoFe protein during reductant-dependent and reductant-independent ATPase activity, when measured with excess Fe protein, have approximately the same value, i.e. 5 s-1 at pH 7.4 and 22 degrees C, assuming the hydrolysis of four molecules of MgATP per turnover of MoFe protein. For Fe protein on the other hand, the maximum turnover rate during reductant-independent ATPase activity is only about 6% of that of reductant-dependent ATPase activity. While the reductant-dependent ATPase activity shows a sigmoidal dependence on the concentration of MgATP, the reductant-independent ATPase activity yields hyperbolic saturation curves. To account for these results it is proposed that the rate-limiting step during MgATP hydrolysis by oxidized nitrogenase is the rate of regeneration of active Fe protein. In the presence of reductant, the regeneration of active Fe protein is stimulated, explaining the higher ATPase activity of nitrogenase during substrate reduction.

ChemInform Abstract: EPR Study of the Vanadium‐Iron Protein of Nitrogenase from Azotobacter vinelandii.

ChemInform Abstract: EPR Study of the Vanadium‐Iron Protein of Nitrogenase from Azotobacter vinelandii.

The vanadium-iron protein of vanadium nitrogenase from Azotobacter chroococcum contains an iron-vanadium cofactor.

N-Methylformamide extracts of acid-treated precipitated VFe protein of the V-nitrogenase of Azotobacter chroococcum are yellow-brown in colour and contain vanadium, iron and acid-labile sulphur in the approximate proportions 1:6:5. E.p.r. spectra of the extracts exhibit a weak signal with g values near 4.5, 3.6 and 2.0 characteristic of an S = 3/2 metal-containing centre. The N-methylformamide extracts activated the MoFe protein polypeptides from mutants of nitrogen-fixing bacteria unable to synthesize FeMoco, the active centre of Mo-nitrogenase. The active hybrid protein exhibited the characteristic substrate-reducing phenotype associated with the VFe protein except that it could not reduce N2 to NH3. The above data are interpreted as demonstrating the existence of an iron- and vanadium-containing cofactor, FeVaco, within the VFe protein. It is suggested that nitrogen fixation requires specific interactions between FeVaco or FeMoco and their respective polypeptides. The biosynthesis of these cofactors is discussed.

Regulation of nitrogenase synthesis in histidine auxotrophs of Klebsiella pneumoniae with altered levels of adenylate nucleotides.

A histidine auxotrophic (hisA) mutant of Klebsiella pneumoniae is phenotypically Nif- when grown with 20 micrograms of histidine ml-1 but Nif+ when supplied with histidine at 100 micrograms ml-1. Reversion to Nif+ at 20 micrograms of histidine ml-1 occurs phenotypically by the addition of 2-thiazolyl-DL-alanine or genetically by mutation in hisG; 2-thiazolyl-DL-alanine inhibits and hisG encodes phosphoribosyl phosphotransferase, the first enzyme of the histidine biosynthetic pathway which consumes ATP. Physiological studies of the hisA mutant JS85 showed that after removal of NH4+ from a culture of the mutant grown with 20 micrograms of histidine ml-1, synthesis of nitrogenase polypeptides occurred at a rate similar to that in the wild type for about 3 h and acetylene reduction activity reached about 10% of the fully derepressed wild-type level. Shortly thereafter the concentration of intracellular adenylates decreased; in particular, ATP fell to about 10% of normal levels. Also, nitrogenase proteins (nifHDK products) and the nifJ gene product stopped being synthesized. These effects were not due to impairment of growth or protein synthesis by histidine starvation. Inhibition of phosphoribosyl phosphotransferase with 2-thiazolyl-DL-alanine restored nitrogenase activity and synthesis, indicating that the effect of the hisA mutation on nif expression was probably a consequence of lowered energy resources that occurred during anaerobic N starvation. The loss of ATP was not associated with nitrogenase synthesis or activity, since hisA nifA and hisA nifH double mutants underwent a loss of ATP in derepressing conditions. Transcription from the nifL, nifN, and nifH promoters was examined in hisA strains with Mu d(Ap lac) fusions in these nif genes. Transcription was not significantly influenced under conditions where adenylates were decreased in concentration. Also nif mRNA apparently accumulated in cultures unable to synthesize nitrogenase, suggesting that translational control of nif gene product synthesis occurs under unfavorable energetic conditions.

Cyanamide: a new substrate for nitrogenase

1. (1) Cynamide (N≡C−NH 2) has been shown to be a substrate for purified Mo-nitrogenases of Klebsiella pneumoniae and Azotobacter chroococcum, with apparent K m values near 0.8 mM. 2. (2) Reduction products were CH 4, CH 3NH 2 and NH 3 formed by pathways requiring 6 or 8 electrons: N≡CNH 2 + 6e + 6H + → CH 3NH 2 + NH 3 ; N≡CNH 2 + 8e + 8H + → CH 4 + 2NH 3 3. (3) Acetylene reduction and hydrogen evolution were inhibited more than 75% by cyanamide (10 mM). Cyanamide also inhibited total electron flux at nitrogenase protein component ratios (Fe/MoFe) near 10. 4. (4) Cyanamide was also a substrate for the recently isolated Va-nitrogenase of A. chroococcum, but with an apparent K m of 2.6 mM showed weaker binding and an 8-fold lower V max than did either Mo-nitrogenase. 5. (5) The component ratios of nitrogenase proteins favouring CH 4 formation was 3.5 Fe/MoFe protein and 1 Fe/VaFe protein.

SQUID measurement of metalloprotein magnetization. New methods applied to the nitrogenase proteins

New techniques have been developed to exploit the sensitivity of a commercial SQUID susceptometer in the study of the magnetization of metalloproteins. Previous studies have ignored both the slow relaxation (hours) of spin I = 1/2 nuclei and residual ferromagnetic impurities in sample holders. These potential sources of noise were at or below the sensitivity of previous instruments. With these noise sources under control, one can now decrease the protein concentration by a factor of ten. In addition careful characterization of the frozen magnetization sample, including the use of a multi-instrument holder for combined study of the magnetization sample with Mössbauer spectroscopy, is required for reliable interpretation of the data in the face of paramagnetic impurities common to metalloprotein samples. Many previous magnetic studies of metalloproteins have been carried out in the Curie region. Saturation magnetization studies down to 1.8 K and up to 5 T can determine zero-field splitting parameters in addition to the spin and exchange coupling parameters measured in previous studies at lower fields and higher temperatures. Applications of these techniques to the study of the nitrogenase proteins of Azotobacter vinelandii are presented as examples.

Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein of nitrogenase.

Nitrogenase is composed of two separately purified proteins called the Fe protein and the MoFe protein. In Azotobacter vinelandii the genes encoding these structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). The MoFe protein contains an ironmolybdenum cofactor (FeMo cofactor) whose biosynthesis involves the participation of at least five gene products, nifQ, nifB, nifN, nifE, and nifV. In this study an A. vinelandii mutant strain, which contains a defined deletion within the nifH (Fe protein) gene, was isolated and studied. This mutant is still able to accumulate significant amounts of MoFe protein subunits. However, extracts of this nifH deletion strain have only very low levels of MoFe protein acetylene reduction activity. Fully active MoFe protein can be reconstituted by simply adding isolated FeMo cofactor to the extracts. Fe protein is not necessary to stabilize or insert this preformed FeMo cofactor into the FeMo cofactor-deficient MoFe protein synthesized by the nifH deletion strain. Extracts of the nifH deletion strain can carry out molybdate and ATP-dependent in vitro FeMo cofactor biosynthesis provided Fe protein is added, demonstrating that they contain the products encoded by the FeMo cofactor biosynthetic genes. These data demonstrate that the Fe protein is physically required for the biosynthesis of FeMo cofactor in A. vinelandii.

Electron paramagnetic resonance study of the vanadium-iron protein of nitrogenase from Azotobacter vinelandii

Electron paramagnetic resonance study of the vanadium-iron protein of nitrogenase from Azotobacter vinelandii

Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii

A strategy has been formulated for the site-directed mutagenesis of the Azotobacter vinelandii nifDK genes. These genes encode the alpha and beta subunits of the MoFe protein of nitrogenase, respectively. Six mutant strains, which produce MoFe proteins altered in their alpha subunit by known single amino acid substitutions, have been produced. Three of these transversion mutations involve cysteine-to-serine changes (at residues 154, 183, and 275), two involve glutamine-to-glutamic acid changes (at residues 151 and 191), and one involves an aspartic acid-to-glutamic acid change (at residue 161). All three possible phenotypic responses are observed within this group- i.e., normal, slow, and no growth in the absence of a fixed-nitrogen source. Two-dimensional gel electrophoresis indicates that all mutants accumulate normal levels of the subunits of both nitrogenase component proteins. Whole-cell and crude-extract acetylene-reduction activities indicate substantial levels of Fe protein activity in all strains. In contrast, MoFe protein activities do not parallel the diazotrophic growth capability for all strains. Two strains appear to exhibit altered substrate discrimination. Such analyses should aid in the identification of metallocluster-binding sites and subunit-subunit interaction domains of the MoFe protein and also provide insight into the mechanistic roles of the various prosthetic groups in catalysis.

Isotopic hybrids of nitrogenase. Mössbauer study of MoFe protein with selective 57Fe enrichment of the P-cluster.

Previous Mössbauer and EPR studies of the MoFe protein (approximately 30 Fe and 2 Mo) of nitrogenase have revealed the presence of two unique clusters, namely, the P-clusters (presumably of the Fe4S4 type) and the molybdenum- and iron-containing cofactors (or M-clusters). Mössbauer components D (approximately 10-12 Fe) and Fe2+ (approximately 4 Fe) represent subsites of the P-clusters while component S (approximately 2 Fe) appeared to belong to a separate, unidentified cluster. In order to refine the analyses of Mössbauer spectra, we have constructed an isotopic hybrid of the Klebsiella pneumoniae protein which contains 57Fe-enriched P-clusters and 56Fe-enriched M-clusters. The highly resolved 57Fe Mössbauer spectra of this hybrid show that component S behaves spectroscopically like the P-cluster sites D and Fe2+ in oxidized and reduced MoFe protein. This suggests that S is a subset of the P-clusters rather than a different cluster type. The present study shows, for the first time, that the Debye-Waller factors of different P-cluster subsites have a different temperature dependence. Thus, the Fe2+/D absorption ratio is 4.0:10.0 at 4.2 K and 4.0:11.6 at 173 K. We propose that the reduced MoFe protein contains two pairs of P-clusters: one pair containing one Fe2+ and three D-sites and the other one Fe2+, two D, and one S-site. We have argued previously that the oxidized P-clusters occur in pairs as well.

Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre.

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 X 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 X 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 X 10(5) less than k less than 3 X 10(6) M-1.s-1 and protein conformation change 6 X 10(2) less than k less than 6 X 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.

Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown at constant growth rate in a chemostat with different molar ratios of sucrose to ammonium (C/N) in the influent media. Both compounds were consumed at essentially the same ratios as were present in the influent media. At low (C/N)-ratios, the cultures were ammonium-limited. At increased (C/N)-ratio ammonium-assimilating cultures additionally began to fix dinitrogen. The (C/N)-ratio at which nitrogenase activity became measurable, increased when the ambient oxygen concentration was increased. Immunoblotting revealed the appearance of nitrogenase proteins when the activity became detectable. Nitrogenase activity as determined either by acetylene reduction or by total nitrogen fixation gave constant relative activities of 1:3.8 (mol of N2 fixed per mol of acetylene reduced) under all sets of conditions used in this investigation. In spite of the oxygen dependent variation of the (C/N)-ratio, nitrogenase became active when the ammonium supply was less than about 14 nmol of ammonium per g of protein. This suggests that oxygen was not directly involved in the onset of dinitrogen fixation.

Transcriptional activity of heterocysts isolated from Anabaena variabilis

Nitrogenase activity, RNA synthesis, and protein synthesis were measured in heterocysts of Anabaena variabilis. Heterocysts labelled in situ for 4 h with [14C]uracil accumulated label in rRNA and tRNA to the same specific activity as RNA from vegetative cells. With isolated heterocysts, however, assimilation of [3H]uracil into RNa occurred at about 10% the rate in vegetative cells, and ceased 90 min after isolation. Pulse-chase experiments indicated that heterogeneous, high-molecular-weight RNA synthesized during the first 30 min of incubation was turned over during a 2 h chase, howver there was no accumulation of label in rRNA and tRNA as was seen with heterocysts labelled in situ and with vegetative cells.