Investigation of C-phycoerythrin from the cyanobacterium Pseudanabaena W 1174

A cyanobacterium which produces high amounts of C-phycoerythrin was classified as a new Pseudanabaena strain. This strain (number W 1173 of our collection) has been cultivated for 6 years without changing its properties. It resembles Pseudanabaena catenata (strain B 1464-1) morphologically but differs in the pigmentation. Contrary to strain B 1464-1, no chromatic adaptation was observed with strain W 1173. It was found that phycoerythrins from both strains differ in the following properties: isoelectric points, number of bilin chromophores, and immunochemical properties. Besides native C-phycoerythrin (PEI, _max = 558 nm), a degradation product (PEII, _max = 544 nm and 562nm) has been found in crude extracts from strain W 1173. Criteria for integrity of C-phycoerythrin were discussed which are essential if this biliprotein is used as taxonomic character.Abbreviations PE C-Phycoerythrin - SDS sodiumdodecylsulfate Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Effect of light quality on the C-phycoerythrin production in marine cyanobacteria Pseudanabaena sp. isolated from Gujarat coast, India

The isolated cyanobacterium containing biopigments like chlorophyll-a, phycoerythrin, phycocyanin, and carotenoid was cultured under different quality of light modes to ascertain biomass and pigment productivity. On the basis of 16S rRNA gene sequence, the isolate was identified as Pseudanabaena sp. Maximum biomass concentration obtained in white-, blue-, and green-light was 0.82, 0.94, and 0.89 g/L, respectively. It was observed that maximum phycoerythrin production was in green light (39.2 mg/L), ensued by blue light (32.2 mg/L), while phycocyanin production was maximum in red light (10.9 mg/L). In yellow light, pigment production as well as the growth rate gradually declined after 12 days. Carotenoid production decreased in blue-, white-, and red-light after 15 days, while in green light it had increased gradually. The present communication suggests that Pseudanabaena sp. can be used for commercial production of phycoerythrin when grown under green light.     

The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.     

Preparation of highly purified 3 alpha- and 3 beta-hydroxysteroid dehydrogenases from Pseudomonas sp.

A method is described for preparing highly purified 3α- and 3β-hydroxysteroid dehydrogenases (EC 1.1.1.50 and EC 1.1.1.145, respectively), essentially uncontaminated with one another, from extracts of a steroid-induced Pseudomonas species. These enzymes are suitable for the microanalysis of 3α-hydroxy-, 3β-hydroxy-, and 3-ketosteroids.     

Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp.

Summary Versatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugants were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 × 10^–4 transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 m NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation. Correspondence to: K. Sode     

An improved crystal structure of C-phycoerythrin from the marine cyanobacterium <Emphasis Type="Italic">Phormidium</Emphasis> sp. A09DM

C-Phycoerythrin (PE) from Phormidium sp. A09DM has been crystallized using different conditions and its structure determined to atomic resolution (1.14 Å). In order for the pigment present, phycoerythrobilin (PEB), to function as an efficient light-harvesting molecule it must be held rigidly (Kupka and Scheer in Biochim Biophys Acta 1777:94–103, 2008) and, moreover, the different PEB molecules in PE must be arranged, relative to each other, so as to promote efficient energy transfer between them. This improved structure has allowed us to define in great detail the structure of the PEBs and their binding sites. These precise structural details will facilitate theoretical calculations of each PEB’s spectroscopic properties. It was possible, however, to suggest a model for which chromophores contribute to the different regions of absorption spectrum and propose a tentative scheme for energy transfer. We show that some subtle differences in one of these PEB binding sites in two of the 12 subunits are caused by crystal contacts between neighboring hexamers in the crystal lattice. This explains some of the differences seen in previous lower resolution structures determined at two different pH values (Kumar et al. in Photosyn Res 129:17–28, 2016).     

Isolation of highly purified cyclodextrin glucanotransferase from Bacillus sp. 1070

Two chromatographic processes for purification of cyclodextringlucanotransferase (CGTase) from Bacillus sp. 1070 was carried out. The enzyme has been purified into 9.5 times on Butyl-Toyopearl and followed immobilized metal ion chromatography on Cu(II)-Iminodiacetic (IDA)-agarose. By the application of second purification scheme (chromatography on Butyl-Toyopearl and DEAE-Sephacel) the specific activity of CGTase has folded into 13.5 times. The purity of enzyme was shown to be approximately 90% by SDS-electrophoreses data. It was shown that isolated enzyme has two isoelectric points estimated as 5.1 and 5.3.     

Molecular cloning and physical mapping of the nitrogenase structural genes from the filamentous, non-heterocystous cyanobacterium Pseudanabaena sp. PCC7409

Abstract Sequences homologous to the structural genes for dinitrogenase ( nifD and nifK ) and nitrogenase reductase ( nifH ) have been cloned from the filamentous, non-heterocystous cyanobacterium Pseudanabaena PCC7409. The nifHDK ⁻ homologous sequences were shown to reside on a 6.5-kb Eco RI restriction fragment by using a restriction fragment encoding the Klebsiella pneumoniae nifHDK genes as a heterologous hybridization probe. This 6.5-kb restriction fragment was cloned from a λ gt.wes Eco RI library of the Paseudanabaena sp. PCC7409 genome. This fragment was subcloned into the plasmid vector pUC9 to generate plasmid pPSU20. A detailed physical map of the insert in plasmid pPSU20 was determined, and relative positions of the nifH, nifD , and nifK homologous sequences on this fragment were determined by hybridization analysis with gene-specific fragments derived from the corresponding Anabaena sp. PCC7120 genes. The results indicate that these genes are contiguous in Pseudanabaena sp. PCC 7409 and are arranged in the order nifH, nifD , and nifK . This arrangement resembles that observed for other non-heterocystous cyanobacteria but differs from that observed for Anabaena, Calothrix , and Nostoc species.     

Characterization of highly purified photosystem I complexes from the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017

Photochemically active photosystem (PS) I complexes were purified from the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017, and several of their properties were characterized. PS I complexes consist of 11 subunits, including PsaK1 and PsaK2; a new small subunit was identified and named Psa27. The new subunit might replace the function of PsaI that is absent in A. marina. The amounts of pigments per one molecule of Chl d' were 97.0 +/- 11.0 Chl d, 1.9 +/- 0.5 Chl a, 25.2 +/- 2.4 alpha-carotene, and two phylloquinone molecules. The light-induced Fourier transform infrared difference spectroscopy and light-induced difference absorption spectra reconfirmed that the primary electron donor of PS I (P740) was the Chl d dimer. In addition to P740, the difference spectrum contained an additional band at 728 nm. The redox potentials of P740 were estimated to be 439 mV by spectroelectrochemistry; this value was comparable with the potential of P700 in other cyanobacteria and higher plants. This suggests that the overall energetics of the PS I reaction were adjusted to the electron acceptor side to utilize the lower light energy gained by P740. The distribution of charge in P740 was estimated by a density functional theory calculation, and a partial localization of charge was predicted to P1 Chl (special pair Chl on PsaA). Based on differences in the protein matrix and optical properties of P740, construction of the PS I core in A. marina was discussed.     

Preparation of highly purified F-actin-depolymerizing factor of human serum

F-actin depolymerizing factor (ADF) of human serum was purified 250-400-fold to more than 98% purity with high reproducibility. The purification included (1) 30-50% (NH4)2SO4 precipitation, (2) ion-exchange chromatography on DEAE-Sepharose, (3) chromatofocusing on Polybuffer exchanger 94 and (4) affinity chromatography on ConA-Sepharose. The recovery of ADF was estimated to be 20-30% whereas the ADF activity yield was 5-17%. The lower activity yield was thought to be due-partly to proteolysis and partly to destabilization of highly purified ADF.