Marine Cyanobacterium Synechococcus Sp Research Articles

Structure of synechobactins, new siderophores of the marine cyanobacterium Synechococcus sp. PCC 7002

The coastal marine cyanobacterium Synechococcus sp. PCC 7002 produces three amphiphilic siderophores, synechobactins A-C, under iron-limiting growth conditions. The synechobactins are comprised of a citric acid backbone linked to two 1, 3-diaminopropane units. The terminal amine of one diaminopropane is acetylated and hydroxylated forming one hydroxamate group. The terminal amine of the other diaminopropane is appended to one of a series of fatty acids and N-hydroxylated on the fatty acid amide forming the second hydroxamate linkage. Synechobactins A-C differ among themselves in the identity of the fatty acid residue as dodecanoic acid, decanoic acid, or octanoic acid, respectively. They are the first structurally elucidated siderophores from marine cyanobacteria and are related to schizokinen, a previously identified siderophore that lacks amphiphilic character, isolated from other bacteria including freshwater cyanobacteria.

The pivotal role of cyanobacteria in the evolution of cellulose synthases and cellulose synthase-like proteins

Cellulose synthase and other members of the family 2 glycosyltransferases are ubiquitous in all kingdoms of life. To date, no attempt has been made to construct a phylogeny that positions cellulose synthases in relation to other members of this family or to elucidate relationships within the cellulose synthase group. In this study, a sequence from the unicellular, marine cyanobacterium Synechococcus sp. PCC 7002 is shown to share a unique common ancestor of a clade consisting of cellulose synthases from Dictyostelium discoideum and Nostoc, as well as a plant grouping that includes CesA proteins and cellulose synthase-like (Csl) proteins G, E, B, D, and F. A branching order is established for Csl proteins that places CslG as ancestral to other members of the Csl/CesA clade. Sequences from Ciona intestinalis and Aspergillis fumigatus are shown to branch at the base of the Eukaryota/Cyanobacteria clade. These data suggest multiple independent transfers of cellulose synthases. The implications of these findings in relation to the evolutionary history of cellulose synthase are discussed.

Photophysiology of the marine cyanobacterium Synechococcus sp. WH8102, a new model organism

Synechococcus spp. constitute a major and ubiquitous component of marine ecosystems. The genome of one strain of this genus, WH8102, has recently been completely sequenced. Since it can also be genetically manipulated, this clone has the potential to become a new model organism; however, to date, it remains poorly characterised in terms of pigment composition, optical properties and photophysiology. It has a very high phycourobilin to phycoerythrobilin (PUB:PEB) ratio (ca. 1.95 at low light), and is therefore representative of Synechococcus populations found in oligotrophic areas of the ocean. We show here that this strain has a very wide growth irradiance range from 650 μmol photons m -2 s -1 continuous white light, with a maximum growth rate (μ max = 1.13 ± 0.02 d -1 ) at 207 pmol quanta m -2 s -1 (I max ). As cells acclimated to high light, drastic variations in the chlorophyll a (chl a), β-carotene and phycoerythrin (PE) contents were observed, reaching a quasi steady state around I max . In contrast, the zeaxanthin content remained approximately constant whatever the light level. Similarly, the carbon and nitrogen contents did not significantly vary with irradiance. Red and orange fluorescences, as measured by flow cytometry, were found to correlate well with chl a and PE contents, respectively. Spectrometric analyses of phycobilisome (PBS)-containing fractions from cells grown under different photon fluxes suggest a specific reduction of the PEII content relative to other phycobiliproteins (PBPs) during acclimation of the PBSs to high light.

Recombinant PsbF from Synechococcus sp. PCC 7002 forms b︰b homodimeric cytochrome b559

All organisms with oxygenic photosynthesis contain two photosystems: photosystem I (PS I) and photosystem II (PS II). The minimal photosystem II particles which are photochemically active contain three subunits: D1, D2 and cytochrome b559 (Cyt b559). The function of Cyt b559 remains unclear. We have successfully overexpressed the psbF gene, encoding the β subunit of Cyt b559, from a marine cyanobacterium Synechococcus sp. PCC 7002 as a fusion gene and obtained a redox-active form of Cyt b559. When the N-terminal GST protein of the fusion gene product was removed with thrombin, the PsbF protein was still redox-active, suggesting that the recombinant PsbF can form dimer in Escherichia coli. The absorption spectra of either the oxidized form or the reduced form of both GST fusion protein and the purified PsbF dimer and the difference spectra between the two forms are the same as that of the Cyt b559 isolated from the higher plants. Redox titration analysis of recombinant PsbF showed that the mid-point redox potential of the recombinant Cyt b559 was approximately 50 mV, which is close to the low potential of Cyt b559. The results are helpful to the understanding of localization and function of Cyt b559 on thylakoid membranes.

Synergistic effect of high-light and low temperature on cell growth of the Delta12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002.

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Delta12 fatty acid desaturase mutant strains. Under a light intensity of 250 mumol m(-2) s(-1), wild-type cells could grow exponentially in a temperature range of 20-38 degrees C, but growth was non-exponential below 20 degrees C and ceased at 12 degrees C. The Delta12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25-38 degrees C but grew slowly at 22 degrees C, and no cell growth took place below 18 degrees C. Under a very high-light intensity of 2.5 mmol m(-2) s(-1), wild-type cells could grow exponentially in a temperature range of 30-38 degrees C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Delta12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 degrees C, but were killed at 30 degrees C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Delta12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 degrees C under the normal light intensity of 250 mumol m(-2) s(-1). These results suggest that growth of the Delta12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria.

Requirement of Nickel as an Essential Micronutrient for the Utilization of Urea in the Marine Cyanobacterium Synechococcus sp. PCC 7002.

Synechococcus sp. PCC 7002 cells appeared to be nitrogen limited when grown on urea without nickel in the growth medium; the amounts of chlorophyll a and phycobiliproteins decreased and glycogen accumulated to high levels. When nickel was added to the urea medium, the diagnostics of nitrogen limitation was not observed. The activity of urease was dependent on the presence of nickel no matter what nitrogen source was used for cell growth. The ureC gene encoding the α-subunit of urease was constitutively transcribed in cells grown on all nitrogen sources tested. The addition of nickel to the growth medium rapidly led to a 15-fold increase in urease activity in 2 hours. These results indicate that cells of Synechococcus sp. PCC 7002 require a nickel supplement (5 μM NiSO4 in the growth medium) to utilize urea efficiently, and that Synechococcus sp. PCC 7002 cells are nickel-starved under the laboratory conditions regularly used for this strain. In two fresh water strains of cyanobacteria, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, high levels of urease activity were detected without addition of nickel to the growth medium, suggesting that these fresh water strains have a high-affinity uptake system for nickel or that their nickel requirements are lower than Synechococcus sp. PCC 7002.

Floating cultivation of marine cyanobacteria using coal fly ash.

The aim of this study was to develop improved methodologies for bulk culturing of biotechnologically useful marine cyanobacteria in the open ocean. We have investigated the viability of using coal fly ash (CFA) blocks as the support medium in a novel floating culture system for marine micro-algae. The marine cyanobacterium Synechococcus sp. NKBG 040607 was found to adhere to floating CFA blocks in liquid culture medium. Maximum density of attached cells of 2.0 x 10(8) cells/cm2 was achieved using seawater. The marine cyanobacterium Synechococcus sp. NKBG 042902 weakly adhered to floating CFA blocks in BG-11 medium. Increasing the concentration of calcium ion in the culture medium enhanced adherence to CFA blocks.

A novel nitrate/nitrite permease in the marine Cyanobacterium synechococcus sp. strain PCC 7002.

The nrtP and narB genes, encoding nitrate/nitrite permease and nitrate reductase, respectively, were isolated from the marine cyanobacterium Synechococcus sp. strain PCC 7002 and characterized. NrtP is a member of the major facilitator superfamily and is unrelated to the ATP-binding cassette-type nitrate transporters that previously have been described for freshwater strains of cyanobacteria. However, NrtP is similar to the NRT2-type nitrate transporters found in diverse organisms. An nrtP mutant strain consumes nitrate at a 4.5-fold-lower rate than the wild type, and this mutant grew exponentially on a medium containing 12 mM nitrate at a rate approximately 2-fold lower than that of the wild type. The nrtP mutant cells could not consume nitrite as rapidly as the wild type at pH 10, suggesting that NrtP also functions in nitrite uptake. A narB mutant was unable to grow on a medium containing nitrate as a nitrogen source, although this mutant could grow on media containing urea or nitrite with rates similar to those of the wild type. Exogenously added nitrite enhanced the in vivo activity of nitrite reductase in the narB mutant; this suggests that nitrite acts as a positive effector of nitrite reductase. Transcripts of the nrtP and narB genes were detected in cells grown on nitrate but were not detected in cells grown on urea or ammonia. Transcription of the nrtP and narB genes is probably controlled by the NtcA transcription factor for global nitrogen control. The discovery of a nitrate/nitrite permease in Synechococcus sp. strain PCC 7002 suggests that significant differences in nutrient transporters may occur in marine and freshwater cyanobacteria.

The marine cyanobacterium Synechococcus sp. WH7805 requires urease (urea amidohydrolase, EC 3.5.1.5) to utilize urea as a nitrogen source: molecular-genetic and biochemical analysis of the enzyme.

Cyanobacteria assigned to the genus Synechococcus are an important component of oligotrophic marine ecosystems, where their growth may be constrained by low availability of fixed nitrogen. Urea appears to be a major nitrogen resource in the sea, but little molecular information exists about its utilization by marine organisms, including Synechococcus. Oligonucleotide primers were used to amplify a conserved fragment of the urease (urea amidohydrolase, EC 3.5.1.5) coding region from cyanobacteria. A 5.7 kbp region of the genome of the unicellular marine cyanobacterium Synechococcus sp. strain WH7805 was then cloned, and genes encoding three urease structural subunits and four urease accessory proteins were sequenced and identified by homology. The WH7805 urease had a predicted subunit composition typical of bacterial ureases, but the organization of the WH7805 urease genes was unique. Biochemical characteristics of the WH7805 urease enzyme were consistent with the predictions of the sequence data. Physiological data and sequence analysis both suggested that the urease operon may be nitrogen-regulated by the ntcA system in WH7805. Inactivation of the large subunit of urease, ureC, prevented WH7805 and Synechococcus WH8102 from growing on urea, demonstrating that the urease genes cloned are essential to the ability of these cyanobacteria to utilize urea as a nitrogen source.

Purification and characterization of phycocyanin from the marine cyanobacterium Synechococcus sp. IO9201

This paper describes a suitable method for the optimum extraction and isolation of phycocyanin from the cyanobacterium Synechococcus sp. IO9201 isolated from Caribbean waters. Phycocyanin from this microorganism was purified to homogeneity and some of its properties were investigated. The purification steps consisted of extraction, hydrophobic interaction chromatography and ion exchange chromatography. Freezing at −21°C-thawing at 4°C, using an alkaline buffer was the best method for extracting phycocyanin from Synechococcus sp. IO9201. The best extraction was obtained using butyl-sepharose resin for hydrophobic interaction chromatography and 0.05 M Tris-HCl (pH = 7) containing 10% ethanol for phycocyanin elution. Finally, phycocyanin was further purified by ion exchange chromatography using Q-sepharose and eluted with a complex isocratic system. The estimated molecular weight of the phycocyanin purified from Synechococcus sp. IO9201 was 102 000 daltons by gel filtration and the isoelectric point was 4.6. When analyzed by SDS-PAGE, Synechococcus sp. IO9201 phycocyanin migrated as two bands having an apparent molecular weight of 21 360 and 18 980 Da. The first band corresponds to β phycocyanin subunits, whereas the second corresponds to α phycocyanin subunits. So, this phycocyanin was characterized as ( α CPC β CPC) 3.

Characterization of the alternative sigma-factors SigD and SigE in Synechococcus sp. strain PCC 7002. SigE is implicated in transcription of post-exponential-phase-specific genes.

The sigD and sigE genes, which encode two alternative sigma-factors from the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigD and sigE genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigD and SigE are group 2 sigma-factors. When stationary-phase cells were diluted into fresh growth medium, it was observed that the sigE mutant strain required longer times to re-establish exponential growth than the wild-type strain. By monitoring the growth rates in such dilution experiments, it was observed that the lag times for the mutant strain became progressively longer as the original cultures progressed towards stationary phase. Transcripts for the sigE gene initially increased and subsequently decreased as cells grew further into stationary phase. It was determined that a functional SigE protein is required for the expression of the starvation-induced protein DpsA/PexB. The results suggest that SigE is involved in the transcription of genes specifically expressed in the post-exponential phase.

A novel plasmid recombination mechanism of the marine cyanobacterium Synechococcus sp. PCC7002.

We describe a novel mechanism of site-specific recombination in the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The specific recombination sites on the smallest plasmid pAQ1 were localized by studying the properties of pAQ1-derived shuttle-vectors. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, functions as a resolution site for site-specific plasmid recombination. Furthermore, site-directed mutagenesis analysis of the element show that the site-specific recombination in the cyanobacterium requires sequence specificity, symmetry in the core sequence and, in part, the spacing between the elements. Interestingly, this element is over-represented not only in pAQ1 and in the genome of the cyanobacterium, but also in the accumulated cyanobacterial sequences from Synechococcus sp. PCC6301, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. Thus, these findings strongly suggest that the site-specific recombination mechanism based on the palindromic element should be common in these cyanobacteria.

Nucleotide sequence of plasmid pAQ1 of marine cyanobacterium Synechococcus sp. PCC7002.

We have determined the complete nucleotide sequence of pAQ1, the smallest plasmid of the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The plasmid consists of 4,809 bp and has at least four open reading frames that potentially encode polypeptides of 50 or more amino acids. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, is over-represented not only in plasmid pAQ1 but also in the accumulated cyanobacterial genomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. It suggests that this sequence might mediate gene rearrangement, thus increasing genetic diversity, since recombination events are frequent in cyanobacteria.

Identification of a cold-regulated RNA-binding protein from the marine cyanobacteriumSynechococcus sp. PCC7002

TherbpA gene of a marine cyanobacteriumSynechococcus sp. PCC7002 encodes an RNA-binding protein, which exhibited an affinity to poly (G) and poly (U) but not to poly (A) and poly (C). Although there are at least two homologous genes in this cyanobacterium, the RbpA protein was the only RNA-binding protein that was detected in the cell of this strain. This protein was apparently absent in the cells grown at 38 C but was abundant in the cells grown at 25 C.

Expression of two alternative sigma factors of Synechococcus sp. strain PCC 7002 is modulated by carbon and nitrogen stress.

The sigB and sigC genes, encoding two alternative sigma factors of the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigB and sigC genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigB and SigC are group 2 sigma factors. Starvation for either nitrogen or carbon caused an increase in sigB mRNA levels. Transcripts for the sigC gene initially increased but then decreased during nitrogen and carbon starvation. The SigC protein could not be identified in cyanobacterial extracts using antisera to Synechococcus sp. PCC 7002 SigA or RpoD from Bacillus subtilis. The ratio of the principal vegetative sigma factor, SigA, to SigB decreased during either nitrogen starvation or carbon starvation, and the levels of SigB also increased in the sigC mutant strain. These results imply that SigB and SigC play roles in modifying transcription in response to changes in carbon and nitrogen availability in this cyanobacterium.

Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp.

The eicosapentaenoic acid (EPA) synthesis gene cluster isolated from a marine bacterium, Shewanella putrefaciens strain SCRC-2738, was cloned and expressed in the marine cyanobacterium Synechococcus sp. A broad-host-range cosmid vector, pJRD215 (10.2 kb, Smr Kmr), was used to clone a 38 kb insert, pEPA, containing the EPA synthesis gene cluster, creating plasmid pJRDEPA (approx. 48 kb). This plasmid was transferred to the cyanobacterial host at a frequency of 2.2 x 10(-7). Cyanobacterial transconjugants grown at 29 degrees C produced 0.12 mg EPA (g dry weight)-1, whereas those grown at 23 degrees C produced 0.56 mg EPA (g dry weight)-1. The yield was further improved to 0.64 mg (g dry weight)-1 by incubation for 1 d at 17 degrees C. This is believed to be the first successful cloning and expression of such a large heterologous gene cluster in a marine cyanobacterium.

A putative transcriptional activator of the Crp/Fnr family from the marine cyanobacteriumSynechococcus sp. WH7803

Sequence analysis immediately downstream (3′) of a genomic region ofSynechococcus sp. WH7803, encoding a periplasmic phosphate-binding protein (PstS), revealed an open reading frame encoding a protein of 224 amino acids. The protein exhibited considerable sequence similarity to a family of bacterial transcriptional activators that includes Crp and Fnr and consequently the ORF is designatedptrA (putativetranscriptional activator). Upstream ofptrA a 18 nucleotide sequence representing a putative Pho box was found. Homologues of theptrA gene were detected in several other phycoerythrin-containing marineSynechococcus strains. This represents the third cyanobacterial member of this class of DNA-binding protein to be described and the first such homologue in the ecologically important marineSynechococcus group. The location ofptrA immediately downstream frompstS may implicate this protein in global phosphate regulation.

Characterization of the genes encoding a phosphate-regulated two component sensory system in the marine cyanobacterium Synechococcus sp. WH7803

An oligomer probe was designed to detect the presence of a putative phoB gene in the genome of the marine, phycoerythrincontaining cyanobacterium Synechococcus sp. WH7803. A 2.2 kb PstI fragment, identified using this probe, was cloned and the complete nucleotide sequence determined. The fragment contained two open reading frames encoding polypeptides which display all the sequence features expected of the response regulator and histidine protein kinase elements of a two component sensory system. Northern analysis confirmed that transcription of these genes was induced by phosphate limitation. On the basis of the sequence similarities and the regulation of their transcription by the availability of inorganic phosphate (Pi) these open reading frames were designated as phoB and phoR, respectively.

Identification of iron Superoxide dismutase and a copper/zinc Superoxide dismutase enzyme activity within the marine cyanobacterium Synechococcus sp. WH 7803

Three constitutive forms of Superoxide dismutase activity have been demonstrated in the cyanobacterial marine picoplankter Synechococcus sp. WH 7803 using polyacrylamide gel activity staining techniques. A protein which gave a positive non-haem iron stain on native polyacrylamide gels exhibited N-terminal similarity to both the iron Superoxide dismutase and the manganese Superoxide dismutase of Escherichia coli. The metal prosthetic group of each of the three activity bands was characterised by analysing their differential sensitivities to 5 mM H 2O 2, 2 mM cyanide and 2 mM of the copper chelator diethyldithiocarbamate. Three distinct Superoxide dismutase activities were observed, an iron Superoxide dismutase, a copper/zinc Superoxide dismutase and a third form which has not been identified. Growth of Synechococcus cells in ASW medium containing no added iron resulted in no alteration in the activity of the iron Superoxide dismutase. Growth of cultures in the absence of copper or zinc resulted in differential changes in the activities of the copper/zinc Superoxide dismutase and the unidentified Superoxide dismutase.

Effects of intensity and quality of light on phycocyanin production by a marine cyanobacterium Synechococcus sp. NKBG 042902

Among 150 strains, including marine cyanobacteria isolated from coastal areas of Japan and a freshwater cyanobacterium from the IAM collection, Spirulina platensis IAM M-135, the marine cyanobacterium Synechococcus sp. NKBG 042902 contained the highest amount of phycocyanin (102 mg/g dry cell weight). We have proposed that the cyanobacterium could be an alternative producer for phycocyanin. The effects of light intensity and light quality on the phycocyanin content in cells of Synechococcus sp. NKBG 042902 were investigated. When the cyanobacterium was cultured under illumination of 25 ώmol m−2 s−1 using a cool-white fluorescent lamp, the phycocyanin content was highest, and the phycocyanin and biomass productivities were 21 mg 1−1 day−1 and 100 mg 1−1 day−1 respectively. Red light was essential for phycocyanin production by this cyanobacterium. Phycocyanin and biomass production were carried out by the cyanobacterium cultures grown under only red light (peak wavelength at 660 nm) supplied from light-emitting diodes (LED). Maximum phycocyanin and biomass productivities were 24 mg 1−1 day−1 and 130 mg 1−1 day−1 when the light intensity of the LED was 55 ώmol m−2 s−1.