Characterization of dnaJ multigene family in the cyanobacterium Synechococcus elongatus PCC 7942

The genome of the cyanobacterium Synechococcus elongatus PCC 7942 contains four dnaJ homologs, which are classified into three types based on domain structure. Among these, dnaJ1, dnaJ2, and dnaJ3 are essential for normal growth, and hence we analyzed them with a view to characterizing their specificity. Expression analysis indicated that dnaJ2, which encodes type II DnaJ protein, exhibited typical responses to heat and high-light stresses. Their localization and ability to prevent aggregation of luciferase were also diverse, suggesting a possible functional differentiation of these proteins. Since the expression of dnaJ1, which belongs to conserved type I DnaJ, down-regulated under heat stress, the unique structure of DnaJ2 may be involved in stress responses of S. elongatus. Based on phylogenetic analysis, the diverse dnaJ family was assumed to have evolved its own specific functions in each cyanobacterial species.     

Expression analysis of multiple dnaK genes in the cyanobacterium Synechococcus elongatus PCC 7942

We analyzed the stress responses of three dnaK homologues (dnaK1, dnaK2, and dnaK3) in the cyanobacterium Synechococcus elongatus PCC 7942. A reporter assay showed that under stress conditions the expression of only the dnaK2 gene was induced, suggesting a functional assignment of these homologues. RNA blot hybridization indicated a typical stress response of dnaK2 to heat and high-light stress. Primer extension mapping showed that dnaK2 was transcribed from similar sites under various stress conditions. Although no known sequence motif was detected in the upstream region, a 20-bp sequence element was highly conserved in dnaK2; it was essential not only for the stress induction but also for the basal expression of dnaK2. The ubiquitous upstream localization of this element in each heat shock gene suggests its important role in the cyanobacterial stress response.     

Carbon-free production of 2-deoxy-scyllo-inosose (DOI) in cyanobacterium Synechococcus elongatus PCC 7942

Owing to their photosynthetic capabilities, there is increasing interest in utilizing cyanobacteria to convert solar energy into biomass. 2-Deoxy-scyllo-inosose (DOI) is a valuable starting material for the benzene-free synthesis of catechol and other benzenoids. DOI synthase (DOIS) is responsible for the formation of DOI from d-glucose-6-phosphate (G6P) in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics such as neomycin and butirosin. DOI fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source is necessary for high-yield DOI production. We constructed DOI-producing cyanobacteria toward carbon-free and sustainable DOI production. A DOIS gene derived from the butirosin producer strain Bacillus circulans (btrC) was introduced and expressed in the cyanobacterium Synechococcus elongatus PCC 7942. We ultimately succeeded in producing 400 mg/L of DOI in S. elongatus without using a carbon source. DOI production by cyanobacteria represents a novel and efficient approach for producing benzenoids from G6P synthesized by photosynthesis.     

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.     

Role of the GlgX protein in glycogen metabolism of the cyanobacterium, Synechococcus elongatus PCC 7942

The putative glgX gene encoding isoamylase-type debranching enzyme was isolated from the cyanobacterium, Synechococcus elongatus PCC 7942. The deduced amino acid sequence indicated that the residues essential to the catalytic activity and substrate binding in bacterial and plant isoamylases and GlgX proteins were all conserved in the GlgX protein of S. elongatus PCC 7942. The role of GlgX in the cyanobacterium was examined by insertional inactivation of the gene. Disruption of the glgX gene resulted in the enhanced fluctuation of glycogen content in the cells during light-dark cycles of the culture, although the effect was marginal. The glycogen of the glgX mutant was enriched with very short chains with degree of polymerization 2 to 4. When the mutant was transformed with putative glgX genes of Synechocystis sp. PCC 6803, the short chains were decreased as compared to the parental mutant strain. The result indicated that GlgX protein contributes to form the branching pattern of polysaccharide in S. elongatus PCC 7942.     

Cloning and characterization of the secY gene from the cyanobacterium Synechococcus PCC7942.

The secY gene product is an essential component of the Escherichia coli cytoplasmic membrane, which mediates the protein translocation across the membrane. We found a gene homologous to secY in the genome of the cyanobacterium Synechococcus PCC7942. The deduced amino acid sequence, 439 amino acids long, shows 43% homology with that of the E. coli secY. The hydrophobic profile suggests that the Synechococcus SecY protein is an integral membrane protein containing ten membrane-spanning segments, which are closely related to the E. coli counterpart. The SecY protein may participate in the protein translocation across the cytoplasmic or thylakoid membrane in Synechococcus PCC7942.     

Interaction of the molecular chaperone HtpG with uroporphyrinogen decarboxylase in the cyanobacterium Synechococcus elongatus PCC 7942

Uroporphyrinogen decarboxylase (HemE) is important due to its location at the first branch-point in tetrapyrrole biosynthesis. We detected a complex formation between full-length polypeptides of HtpG and HemE by biochemical studies in vivo and in vitro. The interaction suppressed the enzyme activity, suggesting a regulatory role of HtpG in tetrapyrrole biosynthesis.     

High-Throughput Functional Analysis of the Synechococcus elongatus PCC 7942 Genome

Synechococcus elongatus PCC 7942 was the first cyanobacterialstrain to be reliably transformed by exogenously added DNA andhas become the model organism for cyanobacterial circadian rhythms.With a small genome (2.7 Mb) and well-developed genetic tools,PCC 7942 provides an exceptional opportunity to elucidate thecircadian mechanism through genetics. We describe a projectto create mutations in every locus of the genome, both to assayeach locus for its potential contribution to the circadian clockand to archive data for the cyanobacterial community. Cosmidclones that carry inserts of PCC 7942 DNA are saturated withtransposon insertions in vitro to provide sequencing templatesand substrates for mutagenesis of the PCC 7942 genome via homologousrecombination. We have mutagenized 53% of the chromosome from50 chromosome-bearing cosmids and identified the positions ofinsertions in 31 of those cosmids and the 46 kb plasmid, pANL.PCC 7942 mutants defective for 490 different genes have beenscreened for circadian phenotypes. Mutagenesis of three apparentlyessential loci, including clpPIIclpX, resulted in circadianphenotypes. We developed an effective antisense suppressionmethod to further the analysis of essential genes. When completed,the set of comprehensive mutations will provide the communitywith a unique resource whose impact will extend beyond circadianresearch.     

Spectroscopic properties of PSI-IsiA supercomplexes from the cyanobacterium Synechococcus PCC 7942

The cyanobacterium Synechococcus PCC 7942 grown under iron starvation assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 CP43' or IsiA light-harvesting complexes [Nature 412 (2001) 745]. Here we present a spectroscopic characterization by temperature-dependent absorption and fluorescence spectroscopy, site-selective fluorescence spectroscopy at 5 K, and circular dichroism of isolated PSI-IsiA, PSI and IsiA complexes from this cyanobacterium grown under iron starvation. The results suggest that the IsiA ring increases the absorption cross-section of PSI by about 100%. Each IsiA subunit binds about 16-17 chlorophyll a (Chl a) molecules and serves as an efficient antenna for PSI. Each of the monomers of the trimeric PSI complex contains two red chlorophylls, which presumably give rise to one exciton-coupled dimer and at 5 K absorb and fluoresce at 703 and 713 nm, respectively. The spectral properties of these C-703 chlorophylls are not affected by the presence of the IsiA antenna ring. The spectroscopic properties of the purified IsiA complexes are similar to those of the related CP43 complex from plants, except that the characteristic narrow absorption band of CP43 at 682.5 nm is missing in IsiA.     

Molecular characterization and redox regulation of phosphoribulokinase from the cyanobacterium Synechococcus sp. PCC 7942

We isolated and characterized a gene encoding phosphoribulokinase (PRK) from Synechococcus sp. PCC 7942. The isolated sequence consisted of a 999 bp open reading frame encoding 333 amino acid residues of PRK. The PRK contained a pair of cysteinyl residues corresponding to Cys16 and Cys55 of spinach PRK regulated by a ferredoxin-thioredoxin system. However, there were seventeen amino acid residues lacking between the two cysteinyl residues compared with those of the chloroplastic enzyme in higher plants. The recombinant PRK of Synechococcus sp. PCC 7942 accounted for about 6-13% of the total soluble protein in the Escherichia coli. The specific activity of the enzyme was 230 micro mol min(-1) (mg protein)(-1). The enzyme activity was completely inactivated by treatment with 5,5'-dithiobis (2-nitrobenzoic acid) (cysteinyl residue-specific oxidant) or was decreased by treatment with H(2)O(2), but was more tolerant to oxidation than that of chloroplast. The oxidized PRK was fully activated by treatment with excessive dithiothreitol. Furthermore, incubation with 3 mM ATP protected the oxidation of the enzyme by either 5,5'-dithiobis (2-nitrobenzoic acid) or H(2)O(2). These results suggest Synechococcus sp. PCC 7942 PRK can be regulated by reversible oxidation/reduction in vitro, but might be resistant to oxidative inactivation in vivo.     

Studies on the role of HtpG in the tetrapyrrole biosynthesis pathway of the cyanobacterium Synechococcus elongatus PCC 7942

In cyanobacterium Synechococcus elongatus PCC 7942, we observed that htpG-overexpression caused remarkable growth inhibition. In addition, subcellular fractionation experiments showed that HtpG was localized in the membrane fraction. To understand its function in cyanobacteria, we carried out yeast two-hybrid screening to identify specific proteins interacting with HtpG, and found out, HemE, uroporphyrinogen decarboxylase. When compared to the wild-type strain, the htpG-null mutant and -overexpressing strains exhibited higher and lower cytosolic HemE activity, based on the coproporphyrin production, respectively. These results strongly suggest that HtpG is involved in the regulation of tetrapyrrole biosynthesis through interacting with HemE protein.     

Reaction mechanism of phosphoribulokinase from a cyanobacterium,Synechococcus PCC7942

The dependence of the activity of phosphoribulokinase isolated from a cyanobacterium, Synechococcus PCC7942, on Mg²⁺ showed that its real substrates were Mg-ATP and free D-ribulose 5-phosphate. On the basis of results of kinetic inhibition studies and previously reported result of affinity chromatography, an ordered bi bi mechanism in which Mg-ATP binds before ribulose 5-phosphate is proposed. The K_m values for ATP and D-ribulose 5-phosphate were 0.09 and 0.27 mM, respectively. K_i values of ADP and D-ribulose 1,5-bisphosphate were 0.32 and 10.0 mM, respectively. Inhibition constants K_i1 and K_i2 for 6-phosphogluconate were 9.3 and 0.49 mM. K_ia was 0.13 mM. New kinetics on PRK gave higher control coefficient than the kinetics on Spinach PRK did in the model with PRK activity from 175 to 1000 µmol min^–1 mg^–1 chl.     

The endogenous redox rhythm is controlled by a central circadian oscillator in cyanobacterium Synechococcus elongatus PCC7942

Photosynthesis Research - The intracellular redox and the circadian clock in photosynthetic organisms are two major regulators globally affecting various biological functions. Both of the global...     

Phase determination of circadian gene expression in Synechococcus elongatus PCC 7942

The authors analyzed the upstream regulatory region of purF, a gene that is expressed in a minority phase that peaks at dawn (class 2 circadian phasing) in Synechococcus elongatus, to determine whether specific cis elements are responsible for this characteristic expression pattern. Fusions of various promoter-bearing fragments to luciferase reporter genes showed that normal class 2 phasing of purF expression was correlated with promoter strength. No specific cis element that is separable from the promoter was responsible for determining phase. Very weak promoter activity of unstable phasing was mapped to a 50-bp segment. Inclusion of sequences that flank this minimal promoter either upstream or downstream increased the promoter strength and stabilized the phase in class 2, but neither segment was individually necessary. Because the data suggested a role for the overall promoter context rather than a specific "phase element," the authors proposed that DNA topology is important in the phase determination of circadian gene expression in S. elongatus. To test this hypothesis, they fused the well-characterized DNA topology-dependent Escherichia coli fis promoter to luciferase and showed that it acts as a class 2 promoter in S. elongatus.     

Engineering Synechococcus elongatus PCC 7942 for Continuous Growth under Diurnal Conditions

Synechococcus elongatus strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs'' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium, Synechococcus elongatus PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.     

Nutrient-associated elongation and asymmetric division of the cyanobacterium Synechococcus PCC 7942

While tightly regulated, bacterial cell morphology may change substantially in response to environmental cues. Here we describe such changes in the cyanobacterium Synechococcus sp. strain PCC7942. Once maintained in stationary phase, these rod-shaped organisms stop dividing and elongate up to 50-fold. Increase in cell length of a thymidine-auxotroph strain upon thymidine starvation implies that inhibition of DNA replication underlies cell elongation. Elongation occurs under conditions of limiting phosphorus but sufficient nitrogen levels. Once proliferative conditions are restored, elongated cells divide asymmetrically instead of exhibiting the typical fission characterized by mid-cell constriction. The progeny are of length characteristic of exponentially growing cells and are proficient of further proliferation. We propose that the ability to elongate under conditions of cytokinesis arrest together with the rapid induction of cell division upon nutrient repletion represents a beneficial cellular mechanism operating under specific nutritional conditions.