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.     

Maximum activity of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase of Anabaena sp. strain CA requires the product of the rbcX gene

Filamentous cyanobacteria of the genus Anabaena contain a unique open reading frame, rbcX, which is juxtaposed and cotranscribed with the genes (rbcL and rbcS) encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Plasmid constructions containing the genes from Anabaena sp. strain CA were prepared, and expression studies in Escherichia coli indicated that the product of the rbcX gene mimicked the ability of chaperonin proteins to facilitate the proper folding of recombinant RubisCO proteins. The purified recombinant Anabaena sp. strain CA RubisCO, much like the RubisCO enzymes from other cyanobacteria, was shown not to undergo inhibition of activity during a time course experiment, and the properties of this chaperoned recombinant protein appear to be consistent with those of the enzyme isolated from the native organism.     

Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features in the 1 Mb region from map positions 64% to 92% of the genome

The contiguous sequence of 1,003,450 bp spanning map positions 64% to 92% of the genome of Synechocystis sp. strain PCC6803 has been deduced. Computer analysis of the sequence predicts that this region contains at least 818 potential ORFs, in which 255 (31%) were either genes that had already been identified or their homologues, 84 (10%) were homologues to registered hypothetical genes, and 149 (18%) showed weak similarities to reported genes. The remaining 330 ORFs showed no apparent similarity to any reported genes or carried no significant protein motifs. The potential ORFs as a whole occupied 86% of the sequenced region, implying compact arrangement of genes in the genome. As to the structural RNA genes, one rRNA operon consisting of 5,028 bp and at least 11 species of tRNA genes were identified. It is noteworthy that 10 out of the 11 tRNA species showed significant sequence similarities to tRNAs reported in plant chloroplasts. As other notable unique sequences, three classes of IS-like elements each with characteristics typical of IS elements were identified, and a typical unit of WD(Trp-Asp)-repeats which have only been detected in the regulatory proteins of eukaryotes was identified within the large 5,079-bp ORF located at map position 69%.     

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.     

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.     

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56-69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O. ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P < 0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.     

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.     

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The ribulose bisphosphate carboxylase/oxygenase rbcL and rbcS genes of a carbon monoxide-oxidizing bacterium, Hydrogenophaga pseudoflava DSM 1084, were cloned and sequenced. The cloned rbcL and rbcS genes had open reading frames of 1422 and 351 nucleotides encoding RbcL and RbcS with calculated molecular masses of 52,689 and 13,541, respectively. The known active site residues in other RbcL proteins were conserved in the H. pseudoflava proteins. The H. pseudoflava RbcS protein lacked the 12-residue internal sequence found in the plant enzymes. The 2 genes were separated by a 134 bp intergenic region and cotranscribed as a 2.0 kb rbcLS mRNA. Novel two perfect 9 bp direct repeats overlapping with two dyad symmetries were found in the rbcLS promoter region.     

SoxD: an essential mediator of induction of anterior neural tissues in Xenopus embryos

Group I introns were reported for the first time in the large subunit of Rubisco (rbcL) genes, using two colonial green algae, Pleodorina californica and Gonium multicoccum (Volvocales). The rbcL gene of P. californica contained an intron (PIC intron) of 1320 bp harboring an open reading frame (ORF). The G. multicoccum rbcL gene had two ORF-lacking introns of 549 (GM1 intron) and 295 (GM2 intron) base pairs. Based on the conserved nucleotide sequences of the secondary structure, the PIC and GM1 introns were assigned to group IA2 whereas the GM2 intron belonged to group IA1. Southern hybridization analyses of nuclear and chloroplast DNAs indicated that such intron-containing rbcL genes are located in the chloroplast genome. Sequencing RNAs from the two algae revealed that these introns are spliced out during mRNA maturation. In addition, the PIC and GM1 introns were inserted in the same position of the rbcL exons, and phylogenetic analysis of group IA introns indicated a close phylogenetic relationship between the PIC and GM1 introns within the lineage of bacteriophage group IA2 introns. However, P. californica and G. multicoccum occupy distinct clades in the phylogenetic trees of the colonial Volvocales, and the majority of other colonial volvocalean species do not have such introns in the rbcL genes. Therefore, these introns might have been recently inserted in the rbcL genes independently by horizontal transmission by viruses or bacteriophage.     

Substrate-binding lipoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in the transport of nitrate and nitrite

Of the four genes (nrtABCD) required for active transport of nitrate in the cyanobacterium Synechococcus sp. strain PCC 7942, nrtBCD encode membrane components of an ATP-binding cassette transporter involved in the transport of nitrite as well as of nitrate, whereas nrtA encodes a 45-kDa cytoplasmic membrane protein, the biochemical function of which remains unclear. Characterization of the nrtA deletional mutants showed that the 45-kDa protein is essential for the functioning of the nitrate/nitrite transporter. A truncated NrtA protein lacking the N-terminal 81 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, was shown to bind nitrate and nitrite with high affinity (Kd = 0.3 microM). Immunoblotting analysis using the antibody against the 45-kDa protein revealed a 48-kDa precursor of the protein, which accumulated in the cyanobacterial cells treated with globomycin, an antibiotic that specifically inhibits cleavage of the signal peptide of lipoprotein precursors. These findings indicated that the nrtA gene product is a nitrate- and nitrite-binding lipoprotein. The N-terminal sequences of putative cyanobacterial substrate-binding proteins suggested that lipoprotein modification of substrate-binding proteins of ATP-binding cassette transporters is common in cyanobacteria.     

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.     

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.     

The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants

The photosynthetic gene rbcL has been lost or dramatically altered in some lineages of nonphotosynthetic parasitic plants, but the dynamics of these events following loss of photosynthesis and whether rbcL has sustained functionally significant changes in photosynthetic parasitic plants are unknown. To assess the changes to rbcL associated with the loss of functional constraints for photosynthesis, nucleotide sequences from nonparasitic and parasitic plants of Scrophulariales were used for phylogeny reconstruction and character analysis. Plants in this group display a broad range of parasitic abilities, from photosynthetic ("hemiparasites") to nonphotosynthetic ("holoparasites"). With the exception of Conopholis (Orobanchaceae), the rbcL locus is present in all parasitic plants of Scrophulariales examined. Several holoparasitic genera included in this study, including Boschniakia, Epifagus, Orobanche, and Hyobanche, have rbcL pseudogenes. However, the holoparasites Alectra orobanchoides, Harveya capensis, Harveya purpurea, Lathraea clandestina, Orobanche corymbosa, O. fasciculata, and Striga gesnerioides have intact open reading frames (ORFs) for the rbcL gene. Phylogenetic hypotheses based on rbcL are largely in agreement with those based on sequences of the nonphotosynthetic genes rps2 and matK and show a single origin of parasitism, and loss of photosynthesis and pseudogene formation have been independently derived several times in Scrophulariales. The mutations in rbcL in nonparasitic and hemiparasitic plants would result in largely conservative amino acid substitutions, supporting the hypothesis that functional proteins can experience only a limited range of changes, even in minimally photosynthetic plants. In contrast, ORFs in some holoparasites had many previously unobserved missense substitutions at functionally important amino acid residues, suggesting that rbcL genes in these plants have evolved under relaxed or altered functional constraints.     

Sustained net CO2 evolution during photosynthesis by marine microorganisms

BACKGROUND: Many aquatic photosynthetic microorganisms possess an inorganic-carbon-concentrating mechanism that raises the CO2 concentration at the intracellular carboxylation sites, thus compensating for the relatively low affinity of the carboxylating enzyme for its substrate. In cyanobacteria, the concentrating mechanism involves the energy-dependent influx of inorganic carbon, the accumulation of this carbon--largely in the form of HCO3(-)-in the cytoplasm, and the generation of CO2 at carbonic anhydrase sites in close proximity to the carboxylation sites. RESULTS: During measurements of inorganic carbon fluxes associated with the inorganic-carbon-concentrating mechanism, we observed the surprising fact that several marine photosynthetic microorganisms, including significant contributors to oceanic primary productivity, can serve as a source of CO2 rather than a sink during CO2 fixation. The phycoerythrin-possessing cyanobacterium Synechococcus sp. WH7803 evolved CO2 at a rate that increased with light intensity and attained a value approximately five-fold that for photosynthesis. The external CO2 concentration reached was significantly higher than that predicted for chemical equilibrium between HCO3- and CO2, as confirmed by the rapid decline in the CO2 concentration upon the addition of carbonic anhydrase. Measurements of oxygen exchange between water and CO2, by means of stable isotopes, demonstrated that the evolved CO2 originated from HCO3- taken up and converted intracellularly to CO2 in a light-dependent process. CONCLUSIONS: We report net, sustained CO2 evolution during photosynthesis. The results have implications for energy balance and pH regulation of the cells, for carbon cycling between the cells and the marine environment, and for the observed fractionation of stable carbon isotopes.