Marine Diatom Cylindrotheca Research Articles

Acyl carrier protein-derived sequence encoded by the chloroplast genome in the marine diatom Cylindrotheca sp. strain N1

The chloroplast genome of chromophytic and rhodophytic algae differs from the plastid genome of plants and green algae in that it encodes the gene for the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase. Hybridization studies indicated that there was a second region of chloroplast DNA from the marine diatom Cylindrotheca sp. strain N1 that strongly hybridized to a previously isolated Cylindrotheca fragment that contained the rbcS gene and flanking sequences. Subsequent determination of the oligonucleotide sequence of this second chloroplast DNA fragment, however, indicated that hybridization was due to identical sequences 3' to the previously cloned Cylindrotheca chloroplast rbcL rbcS genes. Sequences derived from the 5' end of the second chloroplast DNA fragment contained a short open reading frame of 80 amino acids which was found to be highly homologous to bacterial acyl carrier protein and nuclear-encoded acyl carrier protein from plants. Amino acid residues in the environment of Ser-36 of the Escherichia coli protein, which is bound to a 4'-phosphopantetheine moiety, are virtually identical in the Cylindrotheca deduced sequence and all other sources of this protein. Unlike plant acyl carrier-deduced amino acid sequences, there was no leader peptide sequence found for the presumptive Cylindrotheca protein, consistent with the location of this DNA fragment on the chloroplast genome of this organism. DNA encoding the putative acyl carrier protein gene and rbcS thus represent two genes that are chloroplast-encoded in the chromophytic marine diatom Cylindrotheca, a significant departure from the organization of such genes in plants.

Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.

Cloning and expression of the chloroplast-encoded rbcL and rbcS genes from the marine diatom Cylindrotheca sp. strain N1

Both the rbcL and rbcS genes, encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase, have been found to be encoded by chloroplast DNA in the marine diatom Cylindrotheca sp. N1. The rbcS gene in this diatom was found to be adjacent to the rbcL gene by a combination of: (i) Southern-blotting analyses, using heterologous probes; (ii) examination of recombinant proteins synthesized in Escherichia coli, directed by cloned rbcL/rbcS genes; and (iii) synthesis of enzymatically active heterologous Rubisco protein in vivo by recombinant DNA procedures using large subunits of Anacystis nidulans and small subunits of Cylindrotheca sp. N1. It appears that two copies of rbcL and rbcS genes are encoded by the chloroplast DNA of this diatom.

Properties of malate dehydrogenase isolated from the marine diatom Cylindrotheca fusiformis

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the marine diatom Cylindrotheca fusiformis. A typical experiment provided 353-fold purification with 26% yield. The purified enzyme migrated as a single polypeptide band on SDS-polyacrylamide gel electrophoresis and with M r of 38 500. Whereas the M r of the holoenzyme was estimated to be 311 000 by gel filtration, suggesting that the enzyme consists of eight subunits with similar M r . The holoenzyme molecule shows a Stokes radius of 60 Å. The malate dehydrogenase exhibits pH optima at pH 6.0 for oxaloacetate reduction and pH 10.0 for malate oxidation. It is relatively stable in buffers with pH ranged from 6.0 to 8.5. The enzyme was sensitive to elevated temperature and the activation energy was calculated as 5.64 kcal/mol. The amino-acid composition of the enzyme was determined and the partial specific volume of the enzyme were calculated to be 0.736. The N-terminal sequence of the subunit polypeptide was determined to be Arg-Lys-Val-Ala-Val-Leu-Gly-Ala-Gly-Gly-Gly-Ile-Gly-Gln-Pro-Leu-Ser-Leu-Leu-Leu-Lys-Leu-Ser-, near identical sequences were reported for the malate dehydrogenases isolated from Escherichia coli, watermelon, and porcine heart.

Orientation of pigments and pigment-protein complexes in the diatom Cylindrotheca fusiformis. A linear-dichroism study

The orientation of pigments and pigment-protein complexes of the marine diatom Cylindrotheca fusiformis was studied by linear dichroism at 77 K. The technique of polyacrylamide gel squeezing was used to orient the diatom intact cells, their isolated thylakoid membranes and the three pigment-protein complexes: chlorophyll ac-fucoxanthin, chlorophyll ac and PS I complexes. The data indicate that specific orientation of various pigments exists at all structure levels. Tentative assignments of various features of the linear-dichroism spectra to the major photosynthetic pigments are presented. The orientation of the three pigment-protein complexes with respect to the thylakoid membrane plane and the major axis of the cell is also discussed.

The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Subcellular distribution, exonuclease activity and heterogeneity of the enzymes.

Four DNA polymerases from the marine diatom Cylindrotheca fusiformis, polymerases A, B, C and D, were further differentiated by their subcellular localization, presence of deoxyribonuclease activity, apparent heterogeneity and molecular weights. Polymerases A, B and D occur in significant amounts in the soluble fraction, suggesting that they were originally localized in the nuclei, whereas polymerase C predominates in the chloroplasts. A mitochondrial DNA polymerase was also isolated and characterized by ion-exchange chromatography. Polymerase D has an associated nuclease activity which prefers denatured DNA and Mg2+, and has a pH optimum higher than that for polymerase activity. Co-elution from a DEAE-Sephadex column and co-sedimentation in glycerol density gradients of deoxyribonuclease and polymerase D activity suggest a molecular association. Polymerases A, B and C are devoid of nuclease activity. Glycerol-gradient-sedimentation analysis showed that all DNA polymerase fractions are heterogeneous at low ionic strengths, with the appearance of a single homogeneous activity of 0.5M-KCl. Estimated molecular weights of 100000, 82000 and 120000 for polymerases A, B and C respectively were obtained from sedimentation analysis and gel filtration. Polymerase D was estimated to have a molecular weight of about 100000 as determined by sedimentation analysis alone.

Isolation and characterization of cytoplasmic and chloroplastic ribosomes and their ribosomal RNAs from the diatom Cylindrotheca fusiformis.

The cytoplasmic and chloroplast ribosomes from the marine diatom Cylindrotheca fusiformis were isolated and characterized. The cytoplasmic ribosomes sedimented in sucrose at 84S and dissociated into subunits of 64S and 42S in the absence of Mg2+. It contained ribosomal RNAs with molecular weights of 1.31 X 10(6) and 0.70 X 10(6). The chloroplast ribosomes sedimented at 70S only in the presence of high Mg2+ concentrations (25-100 mM). No stable subunits were routinely observed and at very high levels of Mg2+ (greater than 100 mM) the 70S species was converted to a form sedimenting at 55S. At 4 degrees C ribosomal RNAs with molecular weights of 1.1 X 10(6) and 0.40 X 10(6) were detected on polyacrylamide gel electrophoresis. When the RNAs were resolved at room temperature the large molecular weight component disappeared while RNA with molecular weights of 0.65 X 10(6) and 0.53 X 10(6) were observed. Apparently the large chloroplast RNAs dissociated into two pieces of unequal molecular weight. These properties of the diatom's chloroplast ribosomes are very similar to those of the counterparts in unicellular green algae, which suggests that both types of algae have a common phylogenetic ancestor.

Photorespiration in diatoms III. Glycolate:Cytochrome c reductase in the diatom Cylindrotheca fusiformis

A glycolate (lactate): cytochrome c reductase is described in the marine diatom Cylindrotheca fusiformis. This reductase appears to be a measure of the algal glycolate dehydrogenase operating via the mitochondrial electron transport system. It shows a preference for the L-isomer of lactic acid over the D-isomer and an apparent K m for L-lactate of 33.5·10 −4 M (standard deviation, 1.1·10 −4 M). Its affinity for glycolic acid, an apparent K m of 13.4·10 −4 M (standard deviation, 2.1·10 −4 M), is significantly greater.

Composition of the nucleic acids of some algae.

AS part of a study on the chemical composition of algae1, the composition of the nucleic acids of various algae was investigated in order to determine whether there were differences between different groups of marine and freshwater forms. The following algae were studied : the freshwater diatoms Navicula pelliculosa (Breb.) Hilse and Nitzschia palea (Kutz.) W. Sm. ; the marine diatom Cylindrotheca gracilis (Breb.) Grun. ; the freshwater blue-green alga Anacystis nidulans (Richt.) Dr. and Daily ; and the freshwater green alga Chlorella pyrenoidosa Chick.