A Novel Expression Vector for the Cyanobacterium, Synechococcus PCC 6301

A cyanobacterial expression vector was constructed using ribulose-1,5-bisphosphatecarboxylase/oxygenase (RuBisCO) promoter and terminator sequencesderived from Synechococcus PCC 6301. The recombinant plasmid,designated pARUB19, has an ampicillin-resistant (Ap^R) gene asa selectable marker and four unique restriction sites to allowthe insertion of foreign genes. Using this vector, the luciferasegene from the firefly, Photinus pyralis, was introduced intoSynechococcus PCC 6301 cells. The luciferase expression vectorcould be maintained stably in the host cells. Light productionof luciferin/luciferase was detected in the transformants. Luciferaseamounted to 1.2% of the total soluble protein. This plasmidmay facilitate higher levels of foreign gene expression in SynechococcusPCC 6301.     

Nucleotide Sequence of a Putative Sensory Kinase Gene from the Cyanobacterium, Anacystis nidulans 6301

The nucleotide sequence of a 2,146 bp portion of the Anacystisnidulans (Synechococcus PCC6301) genome has been determined.This region contains an open reading frame (ORF) of 392 codons,whose predicted protein sequence shows partial homology to thoseof E. coli phoM and envZ. Hence ORF392 is suggested to be asensory kinase gene in 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 cyanobacteriumSynechococcus sp. PCC7002. The plasmid consists of 4,809 bpand has at least four open reading frames that potentially encodepolypeptides of 50 or more amino acids. We found that a palindromicelement, the core sequence of which is G(G/A)CGATCGCC, is over-representednot only in plasmid pAQ1 but also in the accumulated cyanobacterialgenomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942,vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBLdatabases. It suggests that this sequence might mediate generearrangement, thus increasing genetic diversity, since recombinationevents are frequent in cyanobacteria.     

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 numbers 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.     

Genetic diversity in cyanobacterial symbionts of thalloid bryophytes

Two species of thalloid liverworts, Blasia pusilla and Caviculariadensa, form stable symbioses with nitrogen-fixing cyanobacteria.Both bryophytes promote the persistence of their cyanobacterialassociations by producing specialized gemmae, which facilitatethe simultaneous dispersal of the host and its nitrogen-fixingsymbionts. Here the genetic diversity of cyanobacterial symbiontsof Blasia and Cavicularia is examined. The results indicatethat the primary symbionts of both bryophytes are closely relatedand belong to a specific group of symbiotic Nostoc strains.Related strains have previously been reported from hornwortsand cycads, and from many terricolous cyanolichens. The evolutionaryorigins of all these symbioses may trace back to pre-Permiantimes. While the laboratory strain Nostoc punctiforme PCC 73102has been widely used in experimental studies of bryophyte–Nostocassociations, sequence-identical cyanobionts have not yet beenidentified from thalloid liverworts in the field. Key words: Blasia pusilla, bryophyte, Cavicularia densa, Nostoc, tRNA^Leu(UAA) intron, specificity, symbiosis Received 27 May 2007; Revised 15 November 2007 Accepted 21 December 2007     

Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation

Summary The phycobilisome rod linker genes in the two closely related cyanobacteria Synechococcus sp. PCC 6301 and Synechococcus sp. PCC 7942 were studied. Southern blot analysis showed that the genetic organization of the phycobilisome rod operon is very similar in the two strains. The phycocyanin gene pair is duplicated and separated by a region of about 2.5 kb. The intervening region between the duplicated phycocyanin gene pair was cloned from Synechococcus sp. PCC 6301 and sequenced. Analysis of this DNA sequence revealed the presence of three open reading frames corresponding to 273, 289 and 81 amino acids, respectively. Insertion of a kanamycin resistance cassette into these open reading frames indicated that they corresponded to the genes encoding the 30, 33 and 9 kDa rod linkers, respectively, as judged by the loss of specific linkers from the phycobilisomes of the insertional mutants. Amino acid compositions of the 30 and 33 kDa linkers derived from the DNA sequence were found to deviate from those of purified 33 and 30 kDa linkers in the amounts of glutamic acid/glutamine residues. On the basis of similarity of the amino acid sequence of the rod linkers between Synechococcus sp. PCC 6301 and Calothrix sp. PCC 7601 we name the genes encoding the 30, 33 and 9 kDa linkers cpcH, cpcI and cpcD, respectively. The three linker genes were found to be co-transcribed on an mRNA of 3700 nucleotides. However, we also detected a smaller species of mRNA, of 3400 nucleotides, which would encode only the cpcH and cpcI genes. The 30 kDa linker was still found in phycobilisome rods lacking the 33 kDa linker and the 9 kDa linker was detected in mutants lacking the 33 or the 30 kDa linkers. Free phycocyanin was found in the mutants lacking the 33 or the 30 kDa linkers, whereas no free phycocyanin could be found in the mutant lacking the 9 kDa linker.Abbreviations PCC Pasteur Culture Collection - UTEX University of Texas Culture Collection The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank Nucleotide Sequence Databases under the accession number M94218     

Functional Analysis of Fructose-1,6-Bisphosphatase Isozymes (fbp-I and fbp-II Gene Products) in Cyanobacteria

Synechococcus PCC 7942 contains two fructose-1,6-bisphosphataseisozymes (FBPase-I and FBPase-II), while Synechocystis PCC 6803has only one (FBPase-I) in spite of the occurrence of two FBPaseisozyme genes [Tamoi et al. (1998) Biochim. Biophys. Acta 1383:232]. We now demonstrate that disruption of the gene encodingFBPase-II (fbp-II) with a kanamycin resistance gene cartridgedoes not affect cell growth, Chl content, or CO² assimilationin Synechococcus PCC 7942, and disruption of the gene encodingFBPase-I (fbp-I) is a lethal mutation in both cyanobacteria.Accordingly, it is clear that FBPase-I is necessary to sustainphotosynthesis and gluconeogenesis in cyanobacteria. (Received September 10, 1998; Accepted December 10, 1998)     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Toxicity of a heterologous leucyl-tRNA (anticodon CAG) in the pathogen Candida albicans: in vivo evidence for non-standard decoding of CUG codons

Plasmids containing derivatives of the Saccharomyces cerevisiae leucyl-tRNA (tRNA3 ₃ ^Leu ) gene that vary in anticodon sequence were constructed and transformed into the pathogen Candida albicans and S. cerevisiae. C. albicans could readily be transformed with plasmids encoding leucyl-tRNA genes with the anticodons CAA and UAA (recognizing the codons UUG and UUA) and expression of the heterologous tRNA^Leu could be demonstrated by Northern RNA blotting. In contrast, no transformants were obtained if the anticodons were UAG (codons recognized CUN, UUR) and CAG (codon CUG), indicating that the insertion of leucine at CUG codons is toxic for C. albicans. All tRNA^Leu-encoding plasmids transformed S. cerevisiae with equally high efficiencies. These results provide in vivo evidence that non-standard decoding of CUG codons is essential for the viability of C. albicans.     

Toxicity of a heterologous leucyl-tRNA (anticodon CAG) in the pathogen Candida albicans: in vivo evidence for non-standard decoding of CUG codons

Plasmids containing derivatives of the Saccharomyces cerevisiae leucyl-tRNA (tRNA3 ₃ ^Leu ) gene that vary in anticodon sequence were constructed and transformed into the pathogen Candida albicans and S. cerevisiae. C. albicans could readily be transformed with plasmids encoding leucyl-tRNA genes with the anticodons CAA and UAA (recognizing the codons UUG and UUA) and expression of the heterologous tRNA^Leu could be demonstrated by Northern RNA blotting. In contrast, no transformants were obtained if the anticodons were UAG (codons recognized CUN, UUR) and CAG (codon CUG), indicating that the insertion of leucine at CUG codons is toxic for C. albicans. All tRNA^Leu-encoding plasmids transformed S. cerevisiae with equally high efficiencies. These results provide in vivo evidence that non-standard decoding of CUG codons is essential for the viability of C. albicans.     

3′-terminal conserved loops of 16S rRNAs from the cyanobacterium Synechococcus an PCC 6301 and maize chloroplast differ only in two bases

The sequence of the 3′-terminal 43 nucleotides of 16S ribosomal RNA from the cyanobacterium Synechococcus AN PCC 6301 has been determined. This sequence is almost identical with the 3′-terminal sequence of 16S ribosomal RNA from maize chloroplasts. The evolutionary implications of these observations are discussed.     

A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization,cloning and targeted mutagenesis

DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301. This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized. RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium. A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor. This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA. Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase. pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J.Bact. 159, 36–41, and 1984b, Gene 31, 109–116).     

Donation of Electrons from Cytosolic Components to the Intersystem Chain in the Cyanobacterium Synechococcus sp. PCC 7002 as Determined by the Reduction of P700+

Cells, of Synechococcus sp. PCC 7002 showed a low oxidationlevel of P700 under a far-red light at 6 W m^–2 which inducednearly complete oxidation of P700 in spinach leaves, and a strongerfar-red light was required to observe the oxidation of P700.DCMU did not affect the level of P700⁺² but 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneinduced the oxidation of P700 under far-red light, indicatingthat the low oxidation level of P700 was due to the donationof electrons to P700⁺² from the cytosolic respiratory donorsthrough the intersystem chain at the plastoquinone pool. Theelectron transfer from the cytosolic donors to the intersystemchain was inhibited by HgCl₂ but not by antimycin A. The reductionof P700⁺ in Synechococcus cells, after illumination by strongfar-red light was mostly accounted for by the electron flowto the inter system chain from the respiratory donors (t     

A novel small stable RNA, 6Sa RNA,from the cyanobacterium Synechococcus sp. strain PCC6301

We isolated a novel RNA species from the unicellular cyanobacterium Synechococcus PCC6301 and determined its gene sequence. This novel RNA was termed 6Sa RNA from its length (185 nt). Cross-hybridization of 6Sa RNA to other related microorganisms suggests that its existence is restricted to the Synechococcus genus or related organisms. A high level of accumulation of this RNA was observed by Northern analysis, indicating that 6Sa RNA is stable in cells. Computer-aided prediction of the 6Sa RNA secondary structure also supports its stability.     

Cloning and Characterization of the nrtA Gene That Encodes a 45-kDa Protein Involved in Nitrate Transport in the Cyanobacterium Synechococcus PCC 7942

A 45-kDa protein in the cytoplasmic membrane of the cyanobacteriumSynechococcus PCC 7942 is involved in the active transport ofnitrate [Omata et al. (1989) Proc. Natl. Acad. Sci. USA 86:6612]. The gene coding for this protein (designated herein asnrtA) has been cloned and sequenced. The nrtA gene encodes aprotein of 443 amino acids with a calculated molecular weightof 48424. The deduced amino acid sequence of the protein is46.5% homologous to that of a 42-kDa cytoplasmic membrane proteinthat is synthesized under carbon-limited conditions in SynechococcusPCC 7942. (Received July 16, 1990; Accepted December 5, 1990)     

Expression of the Escherichia coli glutamate dehydrogenase gene in the cyanobacterium Synechococcus PCC6301 causes ammonium tolerance

The unicellular cyanobacterium Synechococcus PCC6301 lacks a hybridisable homologue of the strongly conserved gdhA gene of E. coli that encodes NADP-specific glutamate dehydrogenase. This is consistent with the failure to find this enzyme in extracts of the cyanobacterium. The E. coli gdhA gene was transferred to Synechococcus PCC6301 by transformation with an integrative vector. High levels of glutamate dehydrogenase activity, similar to those found in ammonium grown E. coli cells, were found in these transformants. These transformed cyanobacteria displayed an ammonium tolerant phenotype, consistent with the action of their acquired glutamate dehydrogenase activity as an ammonium detoxification mechanism. Minor differences in colony size and in growth at low light intensity were also observed.