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The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains two psbD genes encoding the D2 protein of the photosystem II reaction center: psbDI, which is cotranscribed as a discistronic message with psbC (the gene encoding CP43, a chlorophyll-a binding protein), and psbDII, which is monocistronic. Northern blot analysis of psbD transcripts showed that the two genes responded differently when wild-type cells were shifted from moderate to high light intensity. Whereas psbDII transcripts increased 500% relative to unshifted control cells, psbDI-psbC transcripts remained unchanged. The beta-galactosidase activities expressed from translational fusions between the psbD genes and the Escherichia coli lacZ reporter gene displayed responses similar to those seen in the RNA. D2 protein levels in thylakoid membranes from wild-type cells increased to 250% of those of the unshifted control cells 12 h after a shift to high light intensities. In contrast, in a mutant strain (AMC016) that carries an inactive psbDII gene, D2 levels decreased by 50% under identical conditions. These results suggested that induction of psbDII gene expression by light can serve as a supplementary system for maintaining a functional photosystem II reaction center at high light intensity. This hypothesis was corroborated by mixed-culture experiments, in which AMC016 cells competed poorly with wild-type cells at high light intensity. These data suggest for the first time that differential expression of members of a cyanobacterial gene family serves to maintain a functional PSII reaction center under diverse environmental conditions.
Mol Gen Genet 1992 Mar
PMID:Light-regulated expression of the psbD gene family in Synechococcus sp. strain PCC 7942: evidence for the role of duplicated psbD genes in cyanobacteria. 137 52

The interaction between homologous DNA sequences, distant from each other in the chromosome, was examined in the cyanobacterium Synechocystis PCC 6803. Most of the rbcL gene encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) was duplicated in the genome by a targeted insertion of a 3'-truncated gene copy into the psb A-I locus. Both rbcL genes, in the psb A-I region and at the rbc locus, were non-functional; The former due to the 3' truncation, and the latter due to a deletion in the 5'-region (creating a 5' truncation) and a mutation associated with an insertion of the Rhodospirillum rubrum rbc gene, yielding a high-CO2-requiring mutant ('cyanorubrum'). The 3' and the 5' truncated rbcL genes were linked to chloramphenicol and kanamycin resistance markers, respectively. Decreasing the kanamycin selective pressure concomitantly with exposure of the double resistance mutant to air, resulted in air-growing colonies. Analysis of their genomes, Rubisco proteins, and their ultrastructure revealed: 1) Reconstitution of a full-length cyanobacterial rbcL gene at the rbc locus; 2) simultaneous synthesis of the cyanobacterial (L8S8) and R. rubrum (L2) enzymes in meroploids containing both mutated and reconstituted rbcL genes; 3) reappearance of carboxysomes. Our results indicate extensive recombinatorial interactions between the homologous sequences at both loci leading to reconstitution of the cyanobacterial rbcL gene.
Mol Gen Genet 1992 Nov
PMID:Restoration of the wild-type locus in an RuBP carboxylase/oxygenase mutant of Synechocystis PCC 6803 via targeted gene recombination. 146 99

The enzyme acetohydroxy acid synthase (AHS), which catalyses the first common step in the biosynthesis of isoleucine, leucine and valine, has been demonstrated to be present in Spirulina platensis in two isoenzymic forms. The complete nucleotide sequences of the genes ilvX and ilvW encoding these two enzymes have been determined. Sequence analysis revealed the presence of two open reading frames, of 1836 and 1737 nucleotides for ilvX and ilvW, respectively. The predicted amino acid sequences of the two isoenzymes, compared with the Synechococcus PCC 7942 AHS enzyme and the large subunits of the Escherichia coli AHSI, II, III isoenzymes, revealed a notable degree of similarity. A small subunit has not been identified for either of the S. platensis AHS isoenzymes. Analysis by Northern blot hybridization demonstrated that the ilvX and ilvW genes are transcribed to give mRNA species of approximately 2.15 kb and 1.95 kb, respectively.
J Gen Microbiol 1992 Jul
PMID:Molecular characterization of the genes encoding acetohydroxy acid synthase in the cyanobacterium Spirulina platensis. 151 71

The iron-stress-induced genes isiA and isiB have been cloned and sequenced from the marine unicellular cyanobacterium Synechococcus sp. PCC 7002. These genes code for a photosystem II chlorophyll-binding protein and flavodoxin respectively. The genes form a dicistronic operon that is transcriptionally activated under iron-stress conditions to produce an abundant monocistronic message containing isiA and a much less abundant dicistronic message that also contains isiB. The arrangement of these genes, their transcriptional control and the relative abundance of the monocistronic and dicistronic messages produced under iron stress parallels the pattern shown by the freshwater cyanobacterium Synechococcus sp. PCC 7942. The genes for the corresponding proteins found under iron-replete conditions, CP-43 and ferredoxin, have also been cloned and sequenced. Northern blot analysis indicates that both of these genes are constitutively expressed under both iron-stress and iron-replete conditions.
J Gen Microbiol 1992 Aug
PMID:An iron stress operon involved in photosynthetic electron transport in the marine cyanobacterium Synechococcus sp. PCC 7002. 152 3

The gene (ppc) encoding phosphoenolpyruvate carboxylase (PEPCase) in the cyanobacterium Anabaena sp. PCC 7120 has been isolated, characterized and its nucleotide sequence determined. Heterologous hybridization using the Synechococcus sp. PCC 7942 ppc gene as a probe of an Anabaena genomic DNA library identified an 8.2 kb HindIII DNA fragment that contained a 3.08 kb open reading frame encoding the cyanobacterial PEPCase. Deletion analysis of the 8.2 kb DNA fragment was used to determine sequences required for expression of enzyme activity in Escherichia coli cells. Primer extension data have been used to identify the cyanobacterial transcription initiation site and the position of the ppc start codon. Comparisons of the Anabaena deduced amino acid sequence with the Synechococcus sp. PCC 6301, E. coli and higher-plant ppc sequences have also been performed and the data are discussed with respect to conservation of specific regions of the protein.
J Gen Microbiol 1992 Apr
PMID:Identification, characterization and sequence analysis of the gene encoding phosphoenolpyruvate carboxylase in Anabaena sp. PCC 7120. 158 4

It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N2. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca2(+)-dependent protease activity, were not impaired in heterocyst formation and grew on N2 as sole nitrogen source.
Mol Gen Genet 1991 Jan
PMID:Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis. 190 Mar 47

Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Kmr) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Kmr gene into an AvaII restriction site of the gene psbD-1. All others harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.
Mol Gen Genet 1989 Mar
PMID:Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: selection of mutants defective in photosynthesis. 249 63

The ndhC and ORF159 genes of the maize plastid DNA (ptDNA) were sequenced and maize ORF159 was used to screen a library of genomic DNA of the blue-green alga Synechocystis sp. PCC 6803. The cyanobacterial gene homologous to ORF159 (ORF157) was isolated and sequenced. In sequencing the region upstream of ORF157, reading frames with homology to the ndhC and psbG genes of maize ptDNA were identified. The ndhC and psbG genes overlap in the ptDNAs of maize, tobacco and Marchantia polymorpha, but are separated by a noncoding spacer in Synechocystis. Northern blot analysis showed that the ndhC, psbG and ORF157/159 genes are cotranscribed in maize and Synechocystis. The three genes occur in the same order in ptDNA of maize, tobacco, and M. polymorpha as in Synechocystis 6803. The amino acid sequences of the NDH-C, PSII-G and the ORF157/159 proteins deduced from the maize genes are 65%, 52% and 53% homologous to those of Synechocystis. However, the cyanobacterial and higher plant NDH-C protein sequences are only 23% homologous to the mitochondrial NDH-3 protein. Protein products of in vitro transcription/translation of the Synechocystis transcription unit had apparent molecular masses of 6 kDa (NDH-C), 25 kDa (PSII-G) and 22 kDa (ORF157) on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis. If these are components of an NADH dehydrogenase, cyanobacteria appear to resemble mitochondria more than they do Escherichia coli and Rhodopseudomonas capsulata with regard to this enzyme complex.
Mol Gen Genet 1989 Mar
PMID:Characterization of the ndhC-psbG-ORF157/159 operon of maize plastid DNA and of the cyanobacterium Synechocystis sp. PCC6803. 249 64

An analysis of restriction endonuclease cleavage of DNA isolated from cyanophages that infect Anabaena and Nostoc species of cyanobacteria has provided evidence for counter-selection of restriction endonuclease sites. These include sites containing subsequences which are methylated by host (Anabaena PCC 7120) methylase(s) akin to the dam and dcm enzymes of Escherichia coli. Other sites which are counter-selected have no common sequence structure. The latter include those of the endogenous restriction endonucleases of the host, but other absent sequences are not attributable to isoschizomers of any known Anabaena or Nostoc restriction endonuclease. The cyanophages differ in their tolerance to DNA methylation. Isolates A-4L, AN-13 and AN-23 do not tolerate adenosine methylation in the GATC sequence whereas two cyanophages, A-1L and AN-10 (which are related) do tolerate dam-like methylation of this sequence. In addition, A-1L allows cytosine methylation at GGCC sequences, but AN-10 has counter-selected these sequences and the remaining sites are not methylated. Analysis of native and cloned A-4L DNA suggests that counter-selection has occurred against all sequences which would be methylated by the host at either adenosine or cytosine nucleotides.
J Gen Virol 1988 Mar
PMID:An analysis of restriction endonuclease sites in cyanophages infecting the heterocystous cyanobacteria Anabaena and Nostoc. 283 36

A procedure was developed for the isolation of heterocysts from cyanobacterial filaments without recourse to mechanical disruption of the vegetative cells. DNA was then extracted from purified heterocysts by heating with 2% (w/v) SDS at 70 degrees C for 10 min. Following purification, this DNA was used for treatment with a range of restriction endonucleases and the results compared with DNA isolated from vegetative cells. Both heterocyst and vegetative DNAs from Anabaena PCC 7120 and Anabaena CA (ATCC 33047) were cut by XbaI, HindIII, EcoRI, ClaI, HpaII and MspI. However, none of the DNAs were cut by XhoI, SalI or MboI, indicating that the DNA from both organisms is methylated, but that no gross changes in methylation occur during heterocyst formation. Treatment of the DNAs with the former enzymes, followed by separation of the fragments by agarose gel electrophoresis, resulted in most cases in patterns of bands, which allowed a limited comparison of heterocyst and vegetative DNAs. No major differences were seen between the heterocyst and vegetative DNAs of either organism, implying that there are unlikely to be extensive rearrangements or major loss of DNA during heterocyst differentiation.
J Gen Microbiol 1988 Nov
PMID:Isolation and restriction analysis of DNA from heterocysts and vegetative cells of cyanobacteria. 285 26


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