Isolation and characterization of a calmodulin-like protein from the cyanobacterium Nostoc sp. PCC 6720

A 21-kDa novel polypeptide which possesses characteristics normally considered to be diagnostic of the calmodulin present in eukaryotic cells was isolated from the cyanobacterium Nostoc sp. PCC 6720. The major technique employed in the isolation of the polypeptide was ion-exchange chromatography on a Mono Q column. The 21-kDa polypeptide was shown: to activate pea NAD kinase in vitro, in a Ca²⁺ requiring reaction; to react with polyclonal antibodies raised against spinach calmodulin, but not with those raised against bovine brain calmodulin; and to exhibit a Ca²⁺ dependent shift in migration during SDS-PAGE.Abbreviations ATCC American Type Culture Collection - DCPIP 2,6-dichlorophenylindophenol - PBS Phosphate buffered saline     

Construction of insertion mutants of Synechocystis sp. PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex

The gene encoding subunit IV of the cytochrome b₆/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa. The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostos sp. PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g. Chlorella protothecoides, Nicotiana tabacum). Transformation of Synechocystis sp. PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants. The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies. Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp. PCC 6803.Abbreviations aph aminoglycoside 3-phosphotransferase - cpDNA chloroplast DNA - km kanamycin - PSI photosystem I - PSII photosystem II     

Transformation and gene replacement in the facultatively chemoheterotrophic,unicellular cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6714 by electroporation

The unicellular cyanobacterium Synechocystis sp. PCC6714 can grow not only under photoautotrophic conditions, but also under chemoheterotrophic conditions if glucose is added to the medium. This makes it useful for the study of many aspects of bioenergetic mechanisms. In contrast to its closely related strain Synechocystis sp. PCC6803, which cannot grow chemoheterotrophically, Synechocystis PCC6714 is not naturally transformable. To enable gene transfer in this strain, we established a method for the introduction of self-replicating IncQ plasmids and for gene replacement using electroporation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of phytochelatin synthase-like protein encoded by alr0975 from a prokaryote, Nostoc sp. PCC 7120

Phytochelatins (PCs) are well known as the heavy metal-detoxifying peptides in higher plants, eukaryotic algae, fungi, and nematode. In contrast, neither PCs nor PC synthase genes have ever been identified in any prokaryotes. The genome sequences for the cyanobacterium Nostoc sp. PCC 7120 were recently completed and allowed us to identify a gene encoding a PC synthase-like protein, termed alr0975. The predicted product of alr0975 contains the conserved N-terminal domain but not the variable C-terminal domain found in eukaryotic PC synthases. The recombinant alr0975 protein strongly catalyzed the first step of PC synthesis, in which glutathione (GSH) is converted to gamma-glutamylcysteine (gamma-EC), although the protein only weakly catalyzed the second step of PC synthesis, namely the transfer of gamma-EC moiety to an acceptor GSH molecule to form PC(2). These results suggest alr0975 protein may be a more primitive form of the PC synthases found in eukaryotes.     

Phycobilisomes of wild type and pigment mutants of the cyanobacterium Synechocystis PCC 6803

Mutants affected in their pigment content and in the structure of their phycobilosomes (PBS) were isolated in the cyanobacterium Synechocystis PCC 6803 by enriching a population with the inhibitor p-hydroxymercuribenzoate. Three of these mutants, PMB 2, PMB 10 and PMB 11, with original phenotypes, are described. Applying several criteria of analysis (77K absorption and fluorescence, protein electrophoretic patterns, electron microscopy), it was possible to assign the component polypeptides to each substructure of the phycobilisome. The model structure obtained fits with those described in other species PMB 10 and PMB 11, completely lacking PC, are the first source of pure PBS cores available, in which no contamination by residual PC can be feared, and are thus particularly interesting for further biochemical studies. The capacity of genetic transformation of Synechocystis PCC 6803 by chromosomal DNA makes this system very convenient for the analysis of the regulation of synthesis of the PBS constituents.Abbreviations PSI, PSII photosystems I, II - PBS phycobilisomes - PC phycocyanin - APC allophycocyanin - APC-B alophycocyanin B - PE phycoerythrin - PEC phycoerythrocyanin - WT wind type - Chl chlorophyll Present address: Service de Physiologie Microbienne Institut Pasteur, 28, rue du Docteur Roux, F-75724 Paris Cedex 15, France     

Identification of trimethylation at C-terminal lysine of pilin in the cyanobacterium Synechocystis PCC 6803

Various post-translational modifications (PTMs) of pilin in Synechocystis sp. PCC 6803 have been proposed. In this study, we investigated previously unidentified PTMs of pilin by mass spectrometry (MS). MALDI-TOF MS and TOF/TOF MS showed that the molecular mass of the C-terminal lysine of pilin was increased by 42 Da, which could represent acetylation (ΔM = 42.0470) or trimethylation (ΔM = 42.0106). To discriminate between these isobaric modifications, the molecular mass of the C-terminal tryptic peptide was measured using 15T Fourier transform ion cyclotron resonance (FT-ICR) MS. The high magnetic field FT-ICR provided sub-ppm mass accuracy, revealing that the C-terminal lysine was modified by trimethylation. We could also detect the existence of mono- and di-methylation of the C-terminal lysine. Cells expressing a pilin point mutant with glutamine replacing the C-terminal lysine showed dramatically reduced motility and short pili. These findings suggest that trimethylation of pilin at the C-terminal lysine may be essential for the biogenesis of functional pili.     

Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Summary. Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells. Supplementary material to this paper is available in electronic form at Correspondence and reprints: Department of Biology, CB1137, Washington University, St. Louis, MO 63130, U.S.A.     

Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome

A physical map of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome has been constructed with restriction endonucleases PmeI, SwaI, and an intron-encoded endonuclease I-CeuI. The estimated size of the genome is 2.7 Mb. On the genome 49 genes or operons have been mapped. Two rRNA operons are separated by 600 kb and transcribed oppositely.     

Diversity of DNA methylation pattern and total DNA restriction pattern in symbiotic Nostoc

Attempts were made to use total DNA restriction patterns and the response of purified DNA to treatment with restriction endonucleases to characterize several symbiotic Nostoc strains which had been isolated from different host plants cultivated in Italy. Among 27 restriction endonucleases tested, several did not cut any DNA and no significant variation in the susceptibility of the genomes to DNA restriction was seen among the strains. Therefore the Nostoc strains could not be separated into groups based on their different susceptibilities to the action of restriction endonucleases. However, in studies of total DNA restriction patterns, the restriction endonucleases BfrI and HpaI gave unique band patterns for each cyanobacterial isolate. Different profiles were even found in strains isolated from host plants belonging to the same species. The results do not support any definition of symbiotic Nostoc genomic groups or species and show that a tight specificity between the host plant and the cyanobacterium might not exist in the symbiotic associations involving Nostoc.     

Assessment of the metal removal capability of two capsulated cyanobacteria, Cyanospira capsulata and Nostoc PCC7936

Two capsulated, exopolysaccharide-producing cyanobacteria, Cyanospira capsulata and Nostoc PCC7936, were tested with regard to their metal removal capability by using copper as model metal. The experiments, carried out with the sole cyanobacterial biomass suspended in distilled water and confined into small dialysis tubings, showed that C. capsulata biomass is characterized by the best efficiency in metal removal, with a q_max (maximum amount of copper removed per biomass unit) of 96 ± 2 mg Cu(II) removed per g of protein in comparison with the value of 79 ± 3 of Nostoc PCC7936 biomass. The experimental data obtained with both cyanobacterial biomass best fit the Langmuir sorption isotherm. The sorption of copper started from the first minutes of contact with the metal and attained the equilibrium state, when no more copper removal was evident, after 5 and 6 hours, for C. capsulata and Nostoc PCC7936, respectively. The best efficiency in Cu(II) removal was obtained at pH 6.1–6.2, while the presence of Mg²⁺ or Ca²⁺ reduced copper removal capability of both species to 60–70% of their q_max. The results showed that the biomass of C. capsulata and Nostoc PCC7936 possesses a high affinity and a high specific uptake for copper, comparable with the best performances shown by other microbial biomass, and suggest the possibility to use the capsulated trichomes of the two cyanobacteria for the bioremoval of heavy metals from polluted water bodies.     

Membrane lipid composition of the unusual cyanobacterium Gloeobacter violaceus sp. PCC 7421, which lacks sulfoquinovosyl diacylglycerol

Gloeobacter violaceus sp. PCC 7421 is an unusual cyanobacterium with only one cellular membrane, which lacks the thylakoid membranes found in other oxygenic photosynthetic organisms. The cell membrane lipids in G. violaceus sp. PCC 7421 are monogalactosyl diacylglycerol, digalactosyl diacylglycerol, phosphatidyl glycerol and phosphatidic acid in the molar proportion of 51, 24, 18 and 4% respectively. This lipid composition resembles that of the cell membrane from other cyanobacteria, but completely lacks sulfoquinovosyl diacylglycerol. This lack of sulfoquinovosyl diacylglycerol is exceptional for a photosynthetic membrane. The membrane lipids are esterified to 14:0, 16:0, 16:1, 18:0, 18:1, 18:2 and α18:3 fatty acids. Received: 28 December 1995 / Accepted: 26 April 1996     

ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp. PCC 7002

Phycobilisomes (PBS) are the major light-harvesting, protein-pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phycobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities.     

Distinct roles of CpcG1 and CpcG2 in phycobilisome assembly in the cyanobacterium Synechocystis sp. PCC 6803

Structural role of the second copy of the rod–core linker CpcG, which was found by genome analysis, was studied in Synechocystis sp. PCC 6803 by gene disruption and fractionation of phycobilisome (sub)complexes. Disruption of cpcG2 (sll1471) resulted in a marked decrease in phycocyanin content both in the background of wild-type and cpcG1 (slr2051)-disruptant. The unique phycocyanin rod–CpcG2 complex without the major allophycocyanin components was isolated from the cpcG1-disruptant. By fluorescence analysis, it was proposed that CpcG2 protein connects the rods with a minor allophycocyanin component, to support energy transfer to Photosystem I.     

Isolation and identification of a new antibacterial entity from the Antarctic cyanobacterium Nostoc CCC 537

The present study was aimed at the isolation, purification and structural elucidation of an antibacterial entity/lead molecule from the Antarctic cyanobacterium Nostoc CCC 537. A methanolic extract of the cyanobacterium was bioassayed with Enterobacter aerogenes as a target. The extract was purified by TLC, and the most active band was subjected to HPLC. The fraction (retention time 15.7 min) designated as the active principle was antibacterial towards Gram positive Mycobacterium tuberculosis H37Rv, Staphylococcus aureus ATCC 25923, Gram negative Salmonella typhi MTCC 3216, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25992, Enterobacter aerogenes MTCC 2822 and multi-drug resistant strains of Escherichia coli GS 2003/01, 02, 03. Based on UV, IR, ¹H NMR, EIMS, and ESIMS data, the structure of the active principle is proposed as 4-[(5-carboxy-2-hydroxy)-benzyl]-1,10-dihydroxy-3,4,7,11,11-pentamethyloctahydrocyclopenta<a>naphthalene (Mr 428, Mp 243–249°C). This intracellular biomolecule is similar to anthraquinone and indane derivatives of a diterpenoid. The rate of production of the active principle currently corresponds to 1.70 mg g⁻¹ biomass dry weight. The inherent property of Nostoc sp. to synthesise niche-specific biomolecules/lead molecules may be exploited for future drug development.     

Identification of genes controlled by the manganese response regulator, ManR, in the cyanobacterium, Anabaena sp. PCC 7120

A computational search was carried out to identify additional binding sites for the manganese response regulator, ManR, in the genome of Anabaena sp. PCC 7120. This approach predicted ManR binding sites: the promoter regions of the genes of all3575-alr3576 and the gene of alr5134 from Anabaena sp. PCC 7120. Electrophoretic mobility shift assays confirmed that the ManR of Anabaena sp. PCC 7120 specifically bound to the promoter regions of all3575-alr3576 and alr5134.