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PubMed2002

Dynamic characteristics of Prochlorococcus and Synechococcus consumption by bacterivorous nanoflagellates.

We compared the characteristics of ingestion of Prochlorococcus and Synechococcus by the marine heterotrophic nanoflagellate Pseudobodo sp. and by a mixed nanoflagellate culture (around 3 microm in size) obtained from an open sea oligotrophic area. Maximum ingestion rate on Synechococcus (2.7 Syn flagellate(-1) h(-1)) was reached at concentrations of 5 x 10(5) Syn mL(-1) and decreased between 6 x 10(5) and 1.5 x 10(6) Syn mL(-1). In order to validate laboratory data, one set of data on Synechococcus grazing was obtained during a field study in the oligotrophic northeastern Mediterranean Sea. Ingestion rates by heterotrophic nanoflagellates were related to Synechococcus abundance in the water, and the feeding rate showed a clear diel rhythm with consumption being highest during the night, declining during the day hours, and being lowest at dusk. Ingestion rates on Prochlorococcus increased linearly for the whole range of prey density used (i.e., from 1 x 10(3) to 3 x 10(6) Proc mL(-1)), with maximum ingestion of 6.7 Proc flagellate(-1) h(-1). However, for prey concentrations in the range of 10(3)-10(5), which are usually encountered in aquatic systems, ingestion rates were significantly less than on Synechococcus. In our experiments, both Prochlorococcus and Synechococcus proved to be poor food items for support of nanoflagellate growth.

Animals↗

PubMed1995

The cyanobacterium Synechococcus sp. strain PCC 7942 contains a second alkaline phosphatase encoded by phoV.

A gene (phoV) encoding an alkaline phosphatase from Synechococcus sp. strain PCC 7942 was isolated by screening a plasmid gene bank for expression of alkaline phosphatase activity in Escherichia coli JM103. Two independent clones carrying the same alkaline-phosphatase-encoding gene were isolated. One of these clones (pKW1) was further analysed and the nucleotide sequence of a contiguous 3234 bp DNA fragment was determined. Two complete open reading frames (ORF1 and phoV) and an incomplete ORF3 were identified reading in the same direction. The deduced phoV gene product showed 34% identity to the alkaline phosphatase PhoA from Zymomonas mobilis, and the N-terminal part of the putative ORF3 protein exhibited 57% identity to a protein of unknown function from Frankia sp. Insertional inactivation of the Synechococcus PCC 7942 phoV gene failed, indicating an essential role for either the phoV or the ORF3 gene product. PhoV consists of 550 amino acid residues, resulting in a molecular mass of 61.3 kDa. To overexpress the Synechococcus PCC 7942 phoV gene in E. coli, plasmid pKW1 was transformed into a phoA mutant of E. coli (CC118). In E. coli strain CC118(pKW1) PhoV was expressed constitutively with high rates of activity, and was shown to be membrane associated in the periplasmic space. After partial purification of the recombinant PhoV, it was shown that, like other alkaline phosphatases, the Synechococcus PhoV had a broad pH optimum in the alkaline region and a broad substrate specificity for phosphomonoesters, required Zn2+ for activity, and was inhibited by phosphate. In contrast to several other alkaline phosphatases, PhoV was inhibited by Mn2+. Due to the lack of a Synechococcus PCC 7942 phoV mutant strain, the function of PhoV remains uncertain. However, the present results show that Synechococcus PCC 7942 has a second, probably phosphate-irrepressible, alkaline phosphatase (PhoV, 61.3 kDa) in addition to the phosphate-repressible enzyme (PhoA, 145 kDa) already described.

Alkaline Phosphatase↗

PubMed1995

Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp. PCC 7942.

High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp. strain PCC 7942 was obtained using the Escherichia coli trc promoter and lacI repressor. The petE gene of Anabaena sp. strain PCC 7937 encoding plastocyanin precursor protein and the E. coli uidA gene encoding beta-glucuronidase were initially placed under the control of the trc promoter and lacI repressor by cloning into the E. coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain. These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events. Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker. The influence of IPTG concentration and induction time on gene expression with the E. coli trc/lacI system in Synechococcus was determined using beta-glucuronidase as a reporter. The Anabaena PCC 7937 petE gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.

Amino Acid Sequence↗

PubMed1988

Examination of Genetic Relatedness of Marine Synechococcus spp. by Using Restriction Fragment Length Polymorphisms.

The relatedness of several marine Synechococcus spp. was estimated by DNA hybridization. Strains isolated from various geographical locations and representing a diversity of DNA base compositions and phycobiliprotein profiles were compared by restriction fragment length polymorphisms for a number of genes. DNAs from two marine red algae and a cryptomonad alga (which exhibit a phycobiliprotein composition similar to that of the marine Synechococcus spp.) and Synechococcus strain PCC6301 (Anacystis nidulans) were also included in the comparison. Strains WH8008, WH8018, and WH7805 were shown to be very similar to one another, as were strains WH7802 and WH7803. Strains WH8110 and WH5701 were clearly unrelated to any of the other strains, and no marine Synechococcus isolate showed any similarity to the freshwater Synechococcus strain PCC6301 or the eucaryotic algae. The method is relatively straightforward and sensitive and uses a variety of basic molecular biology techniques. Its utility in ascertaining the genetic relatedness and diversity of marine Synechococcus spp. and possible extension to field studies are discussed.

Journal Article↗

PubMed2006

Culture isolation and culture-independent clone libraries reveal new marine Synechococcus ecotypes with distinctive light and N physiologies.

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.

Bacterial Typing Techniques↗

PubMed2005

Effect of nutrient addition and environmental factors on prophage induction in natural populations of marine synechococcus species.

A series of experiments were conducted with samples collected in both Tampa Bay and the Gulf of Mexico to assess the impact of nutrient addition on cyanophage induction in natural populations of Synechococcus sp. The samples were virus reduced to decrease the background level of cyanophage and then either left untreated or amended with nitrate, ammonium, urea, or phosphate. Replicate samples were treated with mitomycin C to stimulate cyanophage induction. In five of the nine total experiments performed, cyanophage induction was present in the non-nutrient-amended control samples. Stimulation of cyanophage induction in response to nutrient addition (phosphate) occurred in only one Tampa Bay sample. Nutrient additions caused a decrease in lytic (or control) phage production in three of three offshore stations, in one of three estuarine experiments, and in a lysogenic marine Synechococcus in culture. These results suggest that the process of cyanophage induction as an assay of Synechococcus lysogeny was not inorganically nutrient limited, at least in the samples examined. More importantly, it was observed that the level of cyanophage induction (cyanophage milliliter(-1)) was inversely correlated to Synechococcus and cyanophage abundance. Thus, the intensity of the prophage induction response is defined by ambient population size and cyanophage abundance. This corroborates prior observations that lysogeny in Synechococcus is favored during times of low host abundance.

Bacteriophages↗

PubMedDate not supplied

Characterization of cryptic plasmids from marine cyanobacteria and construction of a hybrid plasmid potentially capable of transformation of marine cyanobacterium, Synechococcus sp., and its transformation.

Among forty strains of marine cyanobacteria isolated in our laboratory, five strains had 1-3 different plasmids. The unicellular marine cyanobacterium, Synechococcus sp. NKBG 042902, contains at least three plasmids (pSY09, pSY10, and pSY11). However, these plasmids are cryptic. Therefore, a hybrid plasmid pUSY02 containing the 1.4-kb HindIII fragment of pSY11 and Escherichia coli plasmid pUC18 was constructed. The plasmid pUSY02 transformed both marine Synechococcus sp. NKBG042902-YG1116, which is a cured strain, and fresh water Anacystis nidulans R2 by dark incubation or Ca2+ treatment. However, the plasmid pSG111 constructed from the plasmid DNA of A. nidulans R2 failed to transform marine Synechococcus sp. Electroporation was also applicable to transformation of marine Synechococcus sp. and fresh water A. nidulans R2. The plasmid pUSY02 was rapidly introduced into marine Synechococcus sp.

Aspergillus nidulans↗

PubMed1997

Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301.

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10(-5)-10(-7) exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50% of larvae (LC50) being 2.1 x 10(5) and 1.3 x 10(5) cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth.

Animals↗

PubMed1997

A suicide vector for allelic recombination involving the gene for glutamate 1-semialdehyde aminotransferase in the cyanobacterium Synechococcus PCC 7942.

Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C5 pathway for the synthesis of delta-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5 microM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100 microM gabaculine, this enzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemLGR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemLWT had retained its sensitivity. Southern hybridisation using gene probes for hemL, ampr and cmr confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10 microM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine.

Alleles↗

PubMed2004

Complex formation between ferredoxin and Synechococcus ferredoxin: nitrate oxidoreductase.

The ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been shown to form a high-affinity complex with ferredoxin at low ionic strength. This complex, detected by changes in both the absorbance and circular dichroism (CD) spectra, did not form at high ionic strength. When reduced ferredoxin served as the electron donor for the reduction of nitrate to nitrite, the activity of the enzyme declined markedly as the ionic strength increased. In contrast, the activity of the enzyme with reduced methyl viologen (a non-physiological electron donor) was independent of ionic strength. These results suggest that an electrostatically stabilized complex between Synechococcus nitrate reductase and ferredoxin plays an important role in the mechanism of nitrate reduction catalyzed by this enzyme. Treatment of Synechococcus nitrate reductase with either an arginine-modifying reagent or a lysine-modifying reagent inhibited the ferredoxin-dependent activity of the enzyme but did not affect the methyl viologen-dependent activity. Treatment with these reagents also resulted in a large decrease in the affinity of the enzyme for ferredoxin. Formation of a nitrate reductase complex with ferredoxin prior to treatment with either reagent protected the enzyme against loss of ferredoxin-dependent activity. These results suggest that lysine and arginine residues are present at the ferredoxin-binding site of Synechococcus nitrate reductase. Results of experiments using site-specific, charge reversal variants of the ferredoxin from the cyanobacterium Anabaena sp. PCC 7119 as an electron donor to nitrate reductase were consistent with a role for negatively charged residues on ferredoxin in the interaction with Synechococcus nitrate reductase.

Anabaena↗

PubMed2003

Excitation wavelength dependence of the fluorescence kinetics in Photosystem I particles from Synechocystis PCC 6803 and Synechococcus elongatus.

The excitation-wavelength dependence of the excited-state dynamics of monomeric and trimeric Photosystem I (PSI) particles from Synechocystis PCC 6803 as well as trimeric PSI particles from Synechococcus elongatus has been studied at room temperature using time-resolved fluorescence spectroscopy. For aselective (400 nm), carotenoid (505 nm), and bulk chlorophyll (approximately 650 nm) excitation in all species, a downhill energy-transfer component is observed, corresponding to a lifetime of 3.4-5.5 ps. For selective red excitation (702-719 nm) in all species, a significantly faster, an approximately 1-ps, uphill transfer component was recorded. In Synechococcus PSI, an additional approximately 10-ps downhill energy-transfer component is found for all wavelengths of excitation, except 719 nm. Each of the species exhibits its own characteristic trap spectrum, the shape of which is independent of the wavelength of excitation. This trap spectrum decays in approximately 23 ps in both monomeric and trimeric Synechocystis PSI and in approximately 35 ps in trimeric Synechococcus PSI. The data were simulated based on the 2.5 A structural model of PSI of Synechococcus elongatus using the Förster equation for energy transfer, and using the 0.6-1-ps charge-separation time and the value of 1.2-1.3 for the index of refraction that were obtained from the dynamics of a hypothetical PSI particle without red chls. The experimentally obtained lifetimes and spectra were reproduced well by assigning three of the chlorophyll-a (chla) dimers observed in the structure to the C708/C702RT pool of red chls present in PSI from both species. Essential for the simulation of the dynamics of Synechococcus PSI is the assignment of the single chla trimer in the structure to the C719/C708RT pool present in this species.

Biophysical Phenomena↗

PubMed2000

The IdiA protein of Synechococcus sp. PCC 7942 functions in protecting the acceptor side of Photosystem II under oxidative stress.

Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from Q(A), gave a decreased O(2) evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage.

Journal Article↗

PubMed1988

Expression of a gene encoding a novel ferredoxin in the cyanobacterium Synechococcus 6301.

A gene was discovered in the cyanobacterium Synechococcus 6301 that encodes a protein highly related to members of the [2Fe-2S] ferredoxin family found in chloroplasts and cyanobacteria. It follows a cluster of seven genes encoding subunits of the cyanobacterial ATP synthase complex. It is transcribed as a monocistronic mRNA of 408 nucleotide residues. Transcription starts at a site 55 bp upstream of the initiator methionine codon. Transcriptional initiation and termination signals with sequences similar to those found in Escherichia coli are not present. Comparison of the predicted sequence of the ferredoxin protein with those of other cyanobacterial and plant ferredoxins shows an average sequences identity of about 40%. Twelve amino acid residues are invariant, including the four cysteine residues that provide ligands for the [2Fe-2S] cluster. The deduced Synechococcus ferredoxin protein sequence has a C-terminal extension of eight amino acid residues relative to most other 2Fe-2S ferredoxins except for those from halobacteria, which also have a C-terminal extension. The sequence of the Synechococcus protein is most closely related to ferredoxins from the two complex cyanobacteria Chlorogloeopsis fritschii and Mastigocladus laminosus. The deduced protein sequence is not that of the major soluble ferredoxin that has been isolated from Synechococcus 6301 and is reported in the accompanying paper [Wada, Masui, Matsubara & Rogers (1988) Biochem. J. 252, 571-575]. So it appears to be a novel [2Fe-2S] ferredoxin and Synechococcus 6301 contains at least two [2Fe-2S] ferredoxins, which may have different roles in vivo.

Amino Acid Sequence↗

PubMed1988

Properties and structure of the soluble ferredoxin from Synechococcus 6301 (Anacystis nidulans). Relationship to gene sequences.

Photoautotrophic cultures of the unicellular cyanobacterium Synechococcus 6301 (Anacystis nidulans) possessed a single [2Fe-2S] ferredoxin with a midpoint redox potential of -385 mV. Determination of the amino acid sequence of the ferredoxin showed that it consisted of 98 residues, with methionine and tryptophan both absent, and with only the four cysteine residues that are required to co-ordinate the iron-sulphur cluster. Comparisons with other ferredoxin sequences showed that most resemblance was to those from filamentous cyanobacteria, with up to 87% homology. There was less resemblance to the ferredoxins of unicellular cyanobacteria, with 25 differences when compared with that from another Synechococcus sp. However, the sequence of Synechococcus 6301 ferredoxin was identical with that derived for a gene sequence for a putative ferredoxin from the genotypically closely related Synechococcus 7942 (Anacystis nidulans R2). In contrast, the sequence showed substantial differences from that corresponding to a putative ferredoxin gene from Synechococcus 6301 reported by Cozens & Walker [(1988) Biochem. J. 252, 563-569].

Amino Acid Sequence↗

PubMed2002

Carbon sequestration in Synechococcus Sp.: from molecular machines to hierarchical modeling.

The U.S. Department of Energy recently announced the first five grants for the Genomes to Life (GTL) Program. The goal of this program is to "achieve the most far-reaching of all biological goals: a fundamental, comprehensive, and systematic understanding of life." While more information about the program can be found at the GTL website (www.doegenomestolife.org), this paper provides an overview of one of the five GTL projects funded, "Carbon Sequestration in Synechococcus Sp.: From Molecular Machines to Hierarchical Modeling." This project is a combined experimental and computational effort emphasizing developing, prototyping, and applying new computational tools and methods to elucidate the biochemical mechanisms of the carbon sequestration of Synechococcus Sp., an abundant marine cyanobacteria known to play an important role in the global carbon cycle. Understanding, predicting, and perhaps manipulating carbon fixation in the oceans has long been a major focus of biological oceanography and has more recently been of interest to a broader audience of scientists and policy makers. It is clear that the oceanic sinks and sources of CO(2) are important terms in the global environmental response to anthropogenic atmospheric inputs of CO(2) and that oceanic microorganisms play a key role in this response. However, the relationship between this global phenomenon and the biochemical mechanisms of carbon fixation in these microorganisms is poorly understood. The project includes five subprojects: an experimental investigation, three computational biology efforts, and a fifth which deals with addressing computational infrastructure challenges of relevance to this project and the Genomes to Life program as a whole. Our experimental effort is designed to provide biology and data to drive the computational efforts and includes significant investment in developing new experimental methods for uncovering protein partners, characterizing protein complexes, identifying new binding domains. We will also develop and apply new data measurement and statistical methods for analyzing microarray experiments. Our computational efforts include coupling molecular simulation methods with knowledge discovery from diverse biological data sets for high-throughput discovery and characterization of protein-protein complexes and developing a set of novel capabilities for inference of regulatory pathways in microbial genomes across multiple sources of information through the integration of computational and experimental technologies. These capabilities will be applied to Synechococcus regulatory pathways to characterize their interaction map and identify component proteins in these pathways. We will also investigate methods for combining experimental and computational results with visualization and natural language tools to accelerate discovery of regulatory pathways. Furthermore, given that the ultimate goal of this effort is to develop a systems-level of understanding of how the Synechococcus genome affects carbon fixation at the global scale, we will develop and apply a set of tools for capturing the carbon fixation behavior of complex of Synechococcus at different levels of resolution. Finally, because the explosion of data being produced by high-throughput experiments requires data analysis and models which are more computationally complex, more heterogeneous, and require coupling to ever increasing amounts of experimentally obtained data in varying formats, we have also established a companion computational infrastructure to support this effort as well as the Genomes to Life program as a whole.

Algorithms↗

PubMed1998

Effects of UV-A (320 to 399 Nanometers) on Grazing Pressure of a Marine Heterotrophic Nanoflagellate on Strains of the Unicellular Cyanobacteria Synechococcus spp.

In the open ocean, where turbidity is very low, UV radiation may be an important factor regulating interactions among planktonic microorganisms. The effect of exposure to UV radiation on grazing by a commonly isolated marine heterotrophic nanoflagellate, Paraphysomonas bandaiensis, on two strains of the cyanobacteria Synechococcus spp. was investigated. Laboratory cultures were exposed to a range of irradiances of artificially produced UV-B (290 to 319 nm) and UV-A (320 to 399 nm) for up to 10 h. At a UV-B irradiance of 0.19 W m, but not 0.12 W m, grazing mortality of Synechococcus spp. and nanoflagellate-specific grazing rates were reduced compared to mortality and grazing rates with UV-A treatment. Within 6 h of exposure, UV-A alone suppressed grazing mortality at irradiances as low as 3.02 W m. The extent to which grazing mortality and nanoflagellate-specific grazing rates were suppressed by UV-A increased with both irradiance and duration of exposure. Over a 6-h exposure period, differences in grazing mortality were largely attributable to differential survival of nanoflagellates. Over a longer period of exposure, there was impairment by UV-A alone of nanoflagellate-specific grazing rates. Rates of primary productivity of Synechococcus spp. were also reduced by UV-A. The extent to which Synechococcus productivity was reduced, compared to the reduction in Synechococcus grazing mortality, depended on the duration of UV-A exposure. These results support the hypothesis that UV-A alone influences the composition and biomass of marine microbial communities by affecting predator-prey interactions and primary production.

Journal Article↗

PubMed1990

Nucleotide sequence and further characterization of the Synechococcus sp. strain PCC 7002 recA gene: complementation of a cyanobacterial recA mutation by the Escherichia coli recA gene.

The nucleotide sequence and transcript initiation site of the Synechococcus sp. strain PCC 7002 recA gene have been determined. The deduced amino acid sequence of the RecA protein of this cyanobacterium is 56% identical and 73% similar to the Escherichia coli RecA protein. Northern (RNA) blot analysis indicates that the Synechococcus strain PCC 7002 recA gene is transcribed as a monocistronic transcript 1,200 bases in length. The 5' endpoint of the recA mRNA was mapped by primer extension by using synthetic oligonucleotides of 17 and 27 nucleotides as primers. The nucleotide sequence 5' to the mapped endpoint contained sequence motifs bearing a striking resemblance to the heat shock (sigma 32-specific) promoters of E. coli but did not contain sequences similar to the E. coli SOS operator recognized by the LexA repressor. An insertion mutation introduced into the recA locus of Synechococcus strain PCC 7002 via homologous recombination resulted in the formation of diploids carrying both mutant and wild-type recA alleles. A variety of growth regimens and transformation procedures failed to produce a recA Synechococcus strain PCC 7002 mutant. However, introduction into these diploid cells of the E. coli recA gene in trans on a biphasic shuttle vector resulted in segregation of the cyanobacterial recA alleles, indicating that the E. coli recA gene was able to provide a function required for growth of recA Synechococcus strain PCC 7002 cells. This interpretation is supported by the observation that the E. coli recA gene is maintained in these cells when antibiotic selection for the shuttle vector is removed.

Amino Acid Sequence↗

PubMed1996

The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.

Acclimatization↗