Isolation and characterization of two sequence-specific endonucleases from Anabaena variabilis.
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ABSTRACT:
SUBMITTER: Murray K
PROVIDER: S-EPMC1164119 | biostudies-other | 1976 Nov
REPOSITORIES: biostudies-other
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Project description:Determination of the 5'-terminal sequences flanking all the individual cleavage sites for endonuclease AvaI in bacteriophage-lambda DNA has shown that this enzyme recognizes the hexanucleotide sequences: (Formula: see text), This sequence is cut as shown by the arrows to give single-stranded 5'-tetranucleotide protrusions (cohesive ends). Endonucleases SmaI, XhoI and XmaI recognize different symmetrical subsets of this sequence and provide independent evidence for the occurrence of these subsets at particular endonuclease-AvaI cleavage sites in the bacteriophage-lambda genome. Further evidence for this structure came from the demonstration that DNA fragments generated by endonuclease AvaI can be ligated to form a discrete set of larger molecules and from nearest-neighbor analysis which showed that cytosine residues occurred at the 3'-side of cleavage points. The observation that endonuclease AvaII recognized a subset of the sites recognized by AsuI [Hughes, Bruce & Murray (1979) Biochem. J. 185, 59-63[ led to the deduction that AvaII recognize the pentanucleotide sequence: (Formula: see text), and breaks internucleotide bonds at the positions indicated by the arrows.
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Project description:The filamentous cyanobacterium Anabaena variabilis fixes nitrogen in the presence of vanadium (V) and in the absence of molybdenum (Mo), using a V-dependent nitrogenase (V-nitrogenase) encoded by the vnfDGK genes. Downstream from these genes are two genes that are similar to the vnfEN genes of Azotobacter vinelandii. Like the vnfDGK genes, the vnfEN genes were transcribed in the absence of Mo, whether or not V was present. A mutant with an insertion in the vnfN gene lacked V-nitrogenase activity; thus, the vnfEN genes were essential for the V-nitrogenase system in A. variabilis. Growth and acetylene reduction assays with wild-type and mutant strains suggested that the V-nitrogenase reduced dinitrogen better than acetylene. The similarity of the vnfEN genes of A. variabilis and A. vinelandii was not strong. The vnfEN genes of A. variabilis showed greater similarity to the vnfDK genes just upstream than to the A. vinelandii vnfEN genes. Sequence comparisons provide support for the idea that if the vnf genes were transferred laterally among bacterial strains, the vnf cluster was not transferred intact. It appears likely that the structural genes were transferred before a duplication event led to the evolution of the vnfEN genes independently in the two strains. The divergence of the vnfEN genes from the vnfDK genes suggests that this duplication, and hence the transfer of vnf genes, was an ancient event.
| S-EPMC178215 | biostudies-other
Project description:Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein, releasing the active form of a membrane-anchored transcription factor (MTF) or a membrane-tethered signaling protein in response to an extracellular or intracellular signal. RIP is conserved from bacteria to humans and governs many important signaling pathways in both prokaryotes and eukaryotes. Proteases that carry out these cleavages are named intramembrane cleaving proteases (I-CLips). To date, little is known about I-CLips in cyanobacteria. In this study, five putative site-2 type I-Clips (Ava_1070, Ava_1730, Ava_1797, Ava_3438, and Ava_4785) were identified through a genome-wide survey in Anabaena variabilis. Biochemical analysis demonstrated that these five putative A. variabilis site-2 proteases (S2Ps(Av)) have authentic protease activities toward an artificial substrate pro-?(K), a Bacillus subtilis MTF, in our reconstituted Escherichia coli system. The enzymatic activities of processing pro-?(K) differ among these five S2Ps(Av). Substitution of glutamic acid (E) by glutamine (Q) in the conserved HEXXH zinc-coordinated motif caused the loss of protease activities in these five S2Ps(Av), suggesting that they belonged to the metalloprotease family. Further mapping of the cleaved peptides of pro-?(K) by Ava_4785 and Ava_1797 revealed that Ava_4785 and Ava_1797 recognized the same cleavage site in pro-?(K) as SpoIVFB, a cognate S2P of pro-?(K) from B. subtilis. Taking these results together, we report here for the first time the identification of five metallo-intramembrane cleaving proteases in Anabaena variabilis. The experimental system described herein should be applicable to studies of other RIP events and amenable to developing in vitro assays for I-CLips.
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Project description:An endonuclease, AsuI, was isolated from extracts of Anabaena subcylindrica on the basis of gel-electrophoretic analysis of digests of bacteriophage-lambda DNA with the paritally purified extracts. The enzyme requires Mg2+, but no other cofactors. Endonuclease AsuI recognizes the interrupted tetranucleotide sequence: (Formula: see tex), and breaks the phosphodiester bonds indicated by the arrows to leave single-stranded trinucleotide projections at the 5'-termini of the DNA fragments.
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Project description:For the filamentous cyanobacterium Anabaena variabilis to grow without combined nitrogen, certain cells differentiate into heterocysts that fix N2, while vegetative cells perform photosynthesis. Much remains unknown on how heterocysts differ from vegetative cells in terms of carbon and energy metabolisms. Microarrays were used to investigate gene transcription patterns in vegetative cells, heterocysts, and filaments of N2-fixing phototrophic, mixotrophic, and heterotrophic cultures.
2013-11-01 | GSE46076 | GEO
Project description:BackgroundWhen the filamentous cyanobacterium Anabaena variabilis grows aerobically without combined nitrogen, some vegetative cells differentiate into N2-fixing heterocysts, while the other vegetative cells perform photosynthesis. Microarrays of sequences within protein-encoding genes were probed with RNA purified from extracts of vegetative cells, from isolated heterocysts, and from whole filaments to investigate transcript levels, and carbon and energy metabolism, in vegetative cells and heterocysts in phototrophic, mixotrophic, and heterotrophic cultures.ResultsHeterocysts represent only 5% to 10% of cells in the filaments. Accordingly, levels of specific transcripts in vegetative cells were with few exceptions very close to those in whole filaments and, also with few exceptions (e.g., nif1 transcripts), levels of specific transcripts in heterocysts had little effect on the overall level of those transcripts in filaments. In phototrophic, mixotrophic, and heterotrophic growth conditions, respectively, 845, 649, and 846 genes showed more than 2-fold difference (p < 0.01) in transcript levels between vegetative cells and heterocysts. Principal component analysis showed that the culture conditions tested affected transcript patterns strongly in vegetative cells but much less in heterocysts. Transcript levels of the genes involved in phycobilisome assembly, photosynthesis, and CO2 assimilation were high in vegetative cells in phototrophic conditions, and decreased when fructose was provided. Our results suggest that Gln, Glu, Ser, Gly, Cys, Thr, and Pro can be actively produced in heterocysts. Whether other protein amino acids are synthesized in heterocysts is unclear. Two possible components of a sucrose transporter were identified that were upregulated in heterocysts in two growth conditions. We consider it likely that genes with unknown function represent a larger fraction of total transcripts in heterocysts than in vegetative cells across growth conditions.ConclusionsThis study provides the first comparison of transcript levels in heterocysts and vegetative cells from heterocyst-bearing filaments of Anabaena. Although the data presented do not give a complete picture of metabolism in either type of cell, they provide a metabolic scaffold on which to build future analyses of cell-specific processes and of the interactions of the two types of cells.
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Project description:We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.
| S-EPMC2556551 | biostudies-literature
Project description:For the filamentous cyanobacterium Anabaena variabilis to grow without combined nitrogen, certain cells differentiate into heterocysts that fix N2, while vegetative cells perform photosynthesis. Much remains unknown on how heterocysts differ from vegetative cells in terms of carbon and energy metabolisms. Microarrays were used to investigate gene transcription patterns in vegetative cells, heterocysts, and filaments of N2-fixing phototrophic, mixotrophic, and heterotrophic cultures. Hybridizations used NimbleGen expression array chips (Product no. A4385-00-01, platform accession no GPL15883) designed against the 5,657 ORFs encoded in the A. variabilis genome (GenBank accession no. CP000117). Each ORF was represented by seventeen 60-mer oligonucleotides. Each oligonucleotide was present in four internal replicates. The twenty-seven microarray data files were normalized against each other. Expression array data were analyzed using ArrayStar 3.0 (DNASTAR, Madison, WI).
2013-11-01 | E-GEOD-46076 | biostudies-arrayexpress
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