Role of signal peptides in targeting of proteins in cyanobacteria

Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen.     

Circadian orchestration of gene expression in cyanobacteria

We wanted to identify genes that are controlled by the circadian clock in the prokaryotic cyanobacterium Synechococcus sp. strain PCC 7942. To use luciferase as a reporter to monitor gene expression, bacterial luciferase genes (luxAB) were inserted randomly into the Synechococcus genome by conjugation with Escherichia coli and subsequent homologous recombination. The resulting transformed clones were then screened for bioluminescence using a new developed cooled-CCD camera system. We screened approximately 30,000 transformed Synechococcus colonies and recovered approximately 800 clones whose bioluminescence was bright enough to be easily monitored by the screening apparatus. Unexpectedly, the bioluminescence expression patterns of almost all of these 800 colonies clearly manifested circadian rhythmicity. These rhythms exhibited a range of waveforms and amplitudes, and they also showed a variety of phase relationships. We also found bioluminescence rhythms expressed by cyanobacterial colonies in which the luciferase gene set was coupled to the promoters of several known genes. Together, these results indicate that control of gene expression by circadian clocks may be more widespread than expected thus far. Moreover, our results show that screening organisms in which promoterless luciferase genes have been inserted randomly throughout the genome by homologous recombination provides an extremely sensitive method to explore differential gene expression.     

Nitrite-specific active transport system of the cyanobacterium Synechococcus sp. strain PCC 7942

Studies on the nitrite uptake capability of a mutant of Synechococcus sp. strain PCC 7942 lacking the ATP-binding cassette-type nitrate-nitrite-bispecific transporter revealed the occurrence of a nitrite-specific active transport system with an apparent Km (NO2-) of about 20 microM. Similar to the nitrate-nitrite-bispecific transporter, the nitrite-specific transporter was reversibly inhibited by ammonium in the medium.     

Purification and characterization of a Synechococcus sp. strain PCC 7942 polypeptide structurally similar to the stress-induced Dps/PexB protein of Escherichia coli

A stable DNA/protein complex having an apparent molecular mass of approximately 150 kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.     

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120

The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp, encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO2. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp.     

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 micromol photons m(-2) s(-1). A clpP1 inactivation strain (delta clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of delta clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in delta clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.     

Circadian rhythm of the cyanobacterium Synechocystis sp. strain PCC 6803 in the dark

The cyanobacterium Synechocystis sp. strain PCC 6803 exhibited circadian rhythms in complete darkness. To monitor a circadian rhythm of the Synechocystis cells in darkness, we introduced a PdnaK1::luxAB gene fusion (S. Aoki, T. Kondo, and M. Ishiura, J. Bacteriol. 177:5606-5611, 1995), which was composed of a promoter region of the Synechocystis dnaK1 gene and a promoterless bacterial luciferase luxAB gene set, as a reporter into the chromosome of a dark-adapted Synechocystis strain. The resulting dnaK1-reporting strain showed bioluminescence rhythms with a period of 25 h (on agar medium supplemented with 5 mM glucose) for at least 7 days in darkness. The rhythms were reset by 12-h-light-12-h-dark cycles, and the period of the rhythms was temperature compensated for between 24 and 31 degrees C. These results indicate that light is not necessary for the oscillation of the circadian clock in Synechocystis.     

Characterization of RNA-binding protein genes in cyanobacteria

A number of genes that encode RNA-binding proteins belonging to the RNP family have been identified in cyanobacteria. All of them are predicted to encode small proteins with a single RNA recognition motif, containing both the RNP1 and RNP2 conserved motifs, and a short auxiliary motif which in many cases contains an abundance of glycine residues. Mutagenesis experiments to characterize the function of some of these gene products are being carried out. In Synechococcus sp. PCC 7942, interruption of the rbpA gene results in slower growth with an altered pigment composition.     

Tabulation of thirty-one putative new genes from cyanobacteria

In the context of other research cyanobacterial DNA sequences were obtained from genomic clones selected from libraries at random. Sequences from Synechococcus PCC 6301, Calothrix PCC 7601 and Calothrix D253 are now available from the GenBank/EMBL/DDBJ databases (accession number Z47089 to Z47128, Z47129 to Z47149 and Z47150 to Z47197, respectively) and have been searched for similarity to known sequences. Thirty-one putative new genes (encoding putative products with at least 40% identity over at least 50 amino acids, or the converse) are listed along with one sequence from Synechococcus PCC 6301 that had been isolated previously.     

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.     

Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942

The heat shock protein ClpB is essential for acquired thermotolerance in cyanobacteria and eukaryotes and belongs to a diverse group of polypeptides which function as molecular chaperones. In this study we show that ClpB is also strongly induced during moderate cold stress in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. A fivefold increase in ClpB (92 kDa) content occurred when cells were acclimated to 25 degrees C over 24 h after being shifted from the optimal growth temperature of 37 degrees C. A corresponding increase occurred for the smaller ClpB' (78 kDa), which arises from a second translational start within the clpB gene of prokaryotes. Shifts to more extreme cold (i.e., 20 and 15 degrees C) progressively decreased the level of ClpB induction, presumably due to retardation of protein synthesis within this relatively cold-sensitive strain. Inactivation of clpB in Synechococcus sp. increased the extent of inhibition of photosynthesis upon the shift to 25 degrees C and markedly reduced the mutant's ability to acclimate to the new temperature regime, with a threefold drop in growth rate. Furthermore, around 30% fewer delta clpB cells survived the shift to 25 degrees C after 24 h compared to the wild type, and more of the mutant cells were also arrested during cell division at 25 degrees C, remaining attached after septum formation. Development of a cold thermotolerance assay based on cell survival clearly demonstrated that wild-type cells could acquire substantial resistance to the nonpermissive temperature of 15 degrees C by being pre-exposed to 25 degrees C. The same level of cold thermotolerance, however, occurred in the delta clpB strain, indicating ClpB induction is not necessary for this form of thermal resistance in Synechococcus spp. Overall, our results demonstrate that the induction of ClpB contributes significantly to the acclimation process of cyanobacteria to permissive low temperatures.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.