Rakesh Tuli

Nitrogen fixation by Plectonema boryanum has a photosystem II independent component

The non-heterocystous filamentous cyanobacterium Plectonema boryanum showed a several-fold decline in photosystem II dependent O2 evolution during the diazotrophic phase of growth. A sharp fall in the amounts of light-harvesting pigments and an uncoupling between the electron transfer from the photosystem II complex to the quinone acceptors may lead to the depression of light-dependent O2 evolution during the diazotrophic phase. Nitrogen fixation as well as CO2 fixation required light, but were partly supported independently of photosystem II. A stimulation of photosystem I and a depression of photosystem II occurred during the diazotrophic phase of a nitrogen-fixing culture of P. boryanum.

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31☆

Abstract In cell-free preparations of NH 4 + -grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N 2 -grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH 4 + -mediated repression. Glutamine synthetase activity in both N 2 -grown and NH 4 + -grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N 2 -grown cultures, a drastic decrease in the enzyme activity by exogenous NH 4 + can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K m values for NH 4 + , glutamate Mg 2+ , and ATP similar to those observed for the enzyme from N 2 - and NH 4 + -grown Anabaena , but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH 4 + -mediated modification to a less active form may be a means of regulation of glutamine synthetase in N 2 -fixing cultures of Anabaena .

A mobilizable shuttle vector for the cyanobacterium Plectonema boryanum

Summary: Plasmid pSUP5011 contains a pMB1 origin of replication, an origin of transfer (oriT), and genes encoding resistance to the antibiotics ampicillin, chloramphenicol and kanamycin. pSUP5011 was conjugally mobilized from Escherichia coli into the non-heterocystous, filamentous, nitrogen-fixing cyanobacterium Plectonema boryanum UTEX 594 in the presence of a helper plasmid, RP4. Transconjugant cyanobacteria selected for resistance to kanamycin, ampicillin and chloramphenicol showed a variety of DNA rearrangements in pSUP5011. One such plasmid continued to show characteristic rearrangements following subsequent transfers into the cyanobacterium. A stable plasmid useful as a shuttle vector was isolated.

Photosystem II Independent Carbon Dioxide Fixation in Plectonema boryanum During Photoautotrophic Growth Under Nitrogen Fixation Conditions

The non-heterocystous filamentous cyanobacterium Plectonema boryanum UTEX 594 grew rapidly microaerobically under nitrogen-starvation conditions in continuous high light intensity by conducting oxygenic photosynthesis and oxygen sensitive nitrogen-fixation in alternating cycles. During diazotrophic phase, the light harvesting pigment phycocyanin declined with a concomitant depression in light dependent oxygen evolution by the cyanobacterium. A substantial component of light dependent carbon dioxide fixation during diazotrophic phase was not inhibited by DCMU in spite of complete cessation of photosynthetic oxygen evolution. Endogenous-reductant dependent electron transfer to photosystem I during diazotrophic phase is postulated even during photoautotrophic growth.

Regulation of glutamine synthetase in the blue-green alga Anabaena L-31

In N2-grown cultures of Anabaena L-31, in which protein synthesis was prevented by chloramphenicol, presence of NH+4 caused a drastic decrease of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) activity indicating NH+4-mediated inactivation or degradation of the enzyme. The half-life of glutamine synthetase was more than 24 h, whereas that of nitrogenase (reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolysing), EC 1.18.2.1) was less than 4 h, suggesting that glutamine synthetase may not act as positive regulator of nitrogenase synthesis in Anabaena. Glutamine synthetase purified to homogeneity was subject to cumulative inhibition by alanine, serine and glycine. The amino acids, however, exhibited partial antagonism in this behaviour. Glyoxylate, an intermediate in photorespiration, virtually prevented the amino acid inhibition. Kinetic studies revealed inhibition of the enzyme activity by high Mg2+ concentration under limiting glutamate level and by high glutamate in limiting Mg2+. Maximum enzyme activity occurred when the ratio of glutamate to free Mg2+ was 0.5 to 1.0. The results demonstrate that the enzyme is subject to multiple regulation by various metabolites involved in nitrogen assimilation.

Glutamine synthetases from rice: Purification and preliminary characterization of two forms in leaves and one form in roots☆

Abstract A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GS I ) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chromatography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GS I and the second type of leaf glutamine synthetase (GS II ) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GS I were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 μmol P / i mg protein/min) and transferase (1324 and 1156 μmol γ-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same K m values for glutamate (2.17 m m ), Mg 2+ (4.5 and 5.0 m m ), ATP (286 μ m ), NH 4 + (210 and 135 μ m ), and ADP (3.8 and 5.3 μ m ). In contrast, leaf GS II did not bind to ADP-Sepharose and had much higher K m values for glutamate (8.3 m m ), Mg 2+ (15 m m ), NH 4 + (684 μ m ), and ADP (33 μ m ).

Cluster analysis of genes for nitrogen fixation from several diazotrophs.

Hierarchical clustering and similarity coefficients of pairwise alignments of the published nucleotide sequences of 27nifH genes suggest thatnif genes are as ancient as the archaebacteria and clostridia. The positions ofnifHl ofMethanococcus thermolithotrophicus, nifH3 ofClostridium pasteurianum, nifH3 ofAzotobacter vinelandii andnifH ofFrankia suggest that a variety of lateral transfers may have occurred during evolution ofnifH gene. The genes for type 3 nitrogenase ofA. vinelandii may have diverged early from methanogens and clostridia. A high similarity coefficient with the derived amino acid sequence of type 3 nitrogenase suggests the presence of a functionally similar enzyme inC. pasteurianum. The type 2 nitrogenase genenifH2 of azotobacters seems to have originated recently from the genenifHl for conventional type I nitrogenase. RhizobialnifH genes comprise two closely related but discrete clusters that are in consonance with the plasmid or chromosomal location ofnif genes. The chromosomal and plasmid locatednifH of rhizobia seem to have evolved independently but contemporaneously.

Analysis of codon usage in genes for nitrogen fixation from phylogenetically diverse diazotrophs

SummaryA cluster analysis based on codon usage in genes for biological nitrogen fixation (nif genes) grouped diazotrophs into three distinct classes: anaerobes, cyanobacteria, and aerobes. In thenif genes ofKlebsiella pneumoniae there was no evidence for selection pressure in favor of highly translatable codons. However, in the nitrogen regulatory operonglnAntrBntrC of enteric bacteria the stoichiometrically high level of glutamine synthetase may be facilitated by the presence of efficiently translatable codons inglnA. Thenif genes of the cyanobacteriumAnabaena showed codon selection in favor of translational efficiency. Computation of codon adaptation indices for expression in heterologous systems indicated that the reading frames most suitable for expression ofnif genes inEscherichia coli, Bacillus subtilis, andSaccharomyces cerevisiae were present in azotobacters, clostridia, and cyanobacteria, respectively. In codon-usage-based cluster analysis, type 3 nitrogenase genes ofAzotobacter vinelandii grouped along with type 1 and type 2 genes. This is in contrast to the nucleotide sequence-based multiple alignment in which type 3 nitrogenase genes ofA. vinelandii have been reported to cluster with entirely unrelated diazotrophs such as methanogens and clostridia. This may be indicative of lateral transfer ofnif genes among widely divergent taxons. The chromosomal- and plasmid-locatednif genes of rhizobia also cluster separately in nucleotide sequence-based analysis but showed similar codon usage. These analyses suggested that the phylogeny ofnif genes drawn on the basis of nucleotide sequence homology was not masked by the taxon-specific pressure on codon usage.

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.

Characterization of a small endogenous plasmid from the Cyanobacterium Plectonema boryanum

The nonheterocystous filamentous CyanobacteriumPlectonema boryanum strain UTEX 594 contains at least two plasmids. A small 145 kb plasmid was cloned in pBR322. It has no homology with the bigger resident plasmid or with chromosomal DNA. A small fraction of the plasmid is present in the form of multimers or concatemers. Copy number and hybridization patterns of the plasmid were similar under dinitrogen-fixing and non-fixing conditions. Restriction site mapping of the plasmid was done to enable its use in the development of cyanobacterial cloning vectors. It is among the smallest natural plasmids reported from bacteria.