Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans.

The cyanobacteria Synechococcus sp. strain PCC 7942 and Synechococcus sp. strain PCC 6301 are very closely related and both have been designated by the binomial Anacystis nidulans. The only established difference between the two strains is the superior transformation properties of strain PCC 7942. Significant homology between the rRNA genes of these strains was demonstrated by the ability of an rRNA operon from strain PCC 6301, interrupted by a spectinomycin and streptomycin resistance marker, to transform strain PCC 7942 by recombining with and replacing an endogenous rRNA operon. Restriction fragment length polymorphism data indicated that the chromosomes of the two strains were conserved around the three psbA loci, the two rRNA operons, and the psbDI locus. However, multiple polymorphisms were detected downstream of the psbDII locus, identifying a DNA rearrangement such as an inversion, insertion, or deletion within the chromosome. Analysis of genome structure by pulsed-field gel electrophoresis of large NotI restriction fragments showed only two bands that were visibly shifted between the chromosomes of the two strains. These data support their very close genetic relationship and the feasibility of studying genes derived from strain PCC 6301 in the highly transformable PCC 7942 strain.     

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)     

Sequence Determinants of Circadian Gene Expression Phase in Cyanobacteria

The cyanobacterium Synechococcus elongatus PCC 7942 exhibits global biphasic circadian oscillations in gene expression under constant-light conditions. Class I genes are maximally expressed in the subjective dusk, whereas class II genes are maximally expressed in the subjective dawn. Here, we identify sequence features that encode the phase of circadian gene expression. We find that, for multiple genes, an ∼70-nucleotide promoter fragment is sufficient to specify class I or II phase. We demonstrate that the gene expression phase can be changed by random mutagenesis and that a single-nucleotide substitution is sufficient to change the phase. Our study provides insight into how the gene expression phase is encoded in the cyanobacterial genome.     

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.     

Insertional inactivation of genes to isolate mutants of Synechococcus sp. strain PCC 7942: isolation of filamentous strains.

We have developed a simple procedure for generating mutants of the cyanobacterium Synechococcus sp. strain PCC 7942 in which the site of the lesion can be readily identified. This procedure involves transforming Synechococcus sp. strain PCC 7942 with a library of its own DNA that was fully digested with Sau3A and ligated into the plasmid vector pUC8. The homologous integration of the recombinant plasmid into the genome will often result in the disruption of a gene and the loss of gene function. We have used this method to generate many mutants of Synechococcus sp. strain PCC 7942 which grow as multicellular filaments rather than as unicells. Since the gene harboring the lesion was tagged with pUC8, it was easily isolated. In this paper, we discuss the usefulness of this procedure for the generation of mutants, and we characterize one mutant in which the lesion may be in an operon involved in the assembly of lipopolysaccharides.     

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)     

Identification and characterization of NF1-related loci on human chromosomes 22, 14 and 2

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. The disease is characterized by a very high mutation rate (up to 1/10000 gametes per generation). NF1-related loci in the human genome have been implicated in the high mutation rate by hypothesizing that these carry disease-causing mutations, which can be transferred to the functional NF1 gene on chromosome arm 17q by interchromosomal gene conversion. To test this hypothesis, we want to identify and characterize the NF1-related loci in the human genome. In this study, we have localized an NF1-related locus in the most centromeric region of the long arm of chromosome 22. We demonstrate that this locus contains sequences homologous to cDNAs that include the GAP-related domain of the functional NF1 gene. However, the GAP-related domain itself is not represented in this locus. In addition, cosmids specific to this locus reveal, by in situ hybridization, NF1-related loci in the pericentromeric region of chromosome arm 14q and in chromosomal band 2q21. These cosmids will enable us to determine whether identified disease-causing mutations are present at the chromosome 22-associated NF1-related locus. Received: 18 December 1995 / Revised: 5 February 1996     

ACandida albicansGenome Project: Cosmid Contigs, Physical Mapping, and Gene Isolation

A new project to map the genome of the pathogenic fungus,Candida albicans,has been started. The entire genome was cloned as 5088 cosmids, stored in individual microtiter plate wells. DNA was prepared and fingerprinted using restriction digestion, fluorescent labeling, and analysis on an ABI sequencer. These data are being used to construct contigs of the genome. Simultaneously, a DNA pooling system has been set up, suitable for PCR-based isolation of cosmids containing any known gene. Ultimately, these approaches will lead to the creation of a physically based map of theC. albicansgenome, providing the means to localize precisely all the genes, act as a substrate for genome sequencing projects, and provide probes for future studies of genome rearrangement and comparative genomics.     

Multiple rpoD-Related Genes of Cyanobacteria

Genomes of many eubacterial strains have been shown to encode for multiple rpoD-related genes. In this report, we describe the identification of the multiple rpoD-related genes of cyanobacterial strains. DNAs of three cyanobacterial strains, Anabaena sp. PCC7120, Synechococcus sp. PCC7942, and Synechocystis sp. PCC6803, were examined by Southern hybridization, using a synthetic probe designed for detecting rpoD or rpoD-related genes. Four or five hybridization signals were found in each DNA. Four DNA regions of Synechococcus sp. PCC7942 corresponding to the hybridization signals were cloned and partially sequenced. The sequence data indicate the presence of genes, named rpoDl, rpoD2, rpoD3, and rpoD4, whose products are highly similar to the basic structure of the principal σ factors of eubacterial strains. The rpoDl gene showed the greatest similarity to the sigA gene of Anabaena sp. PCC7120.     

Nucleotide sequence and homology comparison of two genes of the sulfate transport operon from the cyanobacterium Synechocystis sp. PCC 6803

The genome of Synechocystis sp. PCC 6803 contains an operon with homology to the sulfate permease of other prokaryotes. We used antibodies raised against cytoplasmic membrane protein to find three genes with strong homology to sbpA, orf81 and cysT genes of the cyanobacterium Synechococcus sp. PCC 7942, Escherichia coli, Salmonella typhymurium and Marchantia polymorpha. It is likely that the permease genes are expressed and the proteins are inserted into the cytoplasmic membrane.     

Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942.

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp.     

Assignment of 82 Known Genes and Gene Clusters on the Genome of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803

We have previously constructed the physical map of a cyanobacterium,Synechoystis sp. strain PCC6803 on the basis of restrictionand linking clone analysis. Since a total of 82 genes and geneclusters have been isolated from this strain, most of whichare involved in oxygenic photosynthesis, portions of their sequenceswere amplified by the PCR method and assigned on the physicalmap of the genome by hybridization with restriction fragments,ordered clones, which were obtained from cosmid and libraries,and long PCR-products. An exception was the gene psbG2 whichwas mapped on an extra-chromosomal unit of 45 kb. Since geneticmaps of some of genes assigned above, especially those for photosynthesis,have been reported for two other cyanobacterial strains, Anabaenasp. PCC7120 and Synechococcus sp. PCC7002, gene organizationswere compared among the three strains. However, no significantcorrelation was observed, suggesting that rearrangement of genesoccurred in the respective strains during or after establishmentof the species.     

CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production

Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0 μg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria.     

The first protein map of Synechococcus sp. strain PCC 7942

The first protein map was developed of Synechococcus sp. strain PCC 7942, a model organism for studies of photosynthesis, prokaryotic circadian rhythms, cell division, carbon-concentrating mechanisms, and adaptive responses to a variety of stresses. The proteome was analyzed by two-dimensional gel electrophoresis with subsequent MALDI-TOF mass spectroscopy and database analysis. Of the 140 analyzed protein spots, 110 were successfully identified as 62 different proteins, many of which occurred as multiple spots on the gel. The identified proteins participate in the major metabolic and cellular processes in cyanobacterial cells during the exponential growth phase. In addition, 14 proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Synechococcus sp. strain PCC 7942. These results may be helpful for the annotation of the recently sequenced genome of this cyanobacterium, as well as for biochemical and physiological studies of Synechococcus.     

Complete nucleotide sequence of the freshwater unicellular cyanobacterium Synechococcus elongatus PCC 6301 chromosome: gene content and organization

The entire genome of the unicellular cyanobacterium Synechococcus elongatus PCC 6301 (formerly Anacystis nidulans Berkeley strain 6301) was sequenced. The genome consisted of a circular chromosome 2,696,255 bp long. A total of 2,525 potential protein-coding genes, two sets of rRNA genes, 45 tRNA genes representing 42 tRNA species, and several genes for small stable RNAs were assigned to the chromosome by similarity searches and computer predictions. The translated products of 56% of the potential protein-coding genes showed sequence similarities to experimentally identified and predicted proteins of known function, and the products of 35% of the genes showed sequence similarities to the translated products of hypothetical genes. The remaining 9% of genes lacked significant similarities to genes for predicted proteins in the public DNA databases. Some 139 genes coding for photosynthesis-related components were identified. Thirty-seven genes for two-component signal transduction systems were also identified. This is the smallest number of such genes identified in cyanobacteria, except for marine cyanobacteria, suggesting that only simple signal transduction systems are found in this strain. The gene arrangement and nucleotide sequence of Synechococcus elongatus PCC 6301 were nearly identical to those of a closely related strain Synechococcus elongatus PCC 7942, except for the presence of a 188.6 kb inversion. The sequences as well as the gene information shown in this paper are available in the Web database, CYORF (http://www.cyano.genome.jp/).     

Dipoid-Specific Genome Stability Genes of S. cerevisiae: Genomic Screen Reveals Haploidization as an Escape from Persisting DNA Rearrangement Stress

Maintaining a stable genome is one of the most important tasks of every living cell and the mechanisms ensuring it are similar in all of them. The events leading to changes in DNA sequence (mutations) in diploid cells occur one to two orders of magnitude more frequently than in haploid cells. The majority of those events lead to loss of heterozygosity at the mutagenesis marker, thus diploid-specific genome stability mechanisms can be anticipated. In a new global screen for spontaneous loss of function at heterozygous forward mutagenesis marker locus, employing three different mutagenesis markers, we selected genes whose deletion causes genetic instability in diploid Saccharomyces cerevisiae cells. We have found numerous genes connected with DNA replication and repair, remodeling of chromatin, cell cycle control, stress response, and in particular the structural maintenance of chromosome complexes. We have also identified 59 uncharacterized or dubious ORFs, which show the genome instability phenotype when deleted. For one of the strongest mutators revealed in our screen, ctf18Δ/ctf18Δ the genome instability manifests as a tendency to lose the whole set of chromosomes. We postulate that this phenomenon might diminish the devastating effects of DNA rearrangements, thereby increasing the cell''s chances of surviving stressful conditions. We believe that numerous new genes implicated in genome maintenance, together with newly discovered phenomenon of ploidy reduction, will help revealing novel molecular processes involved in the genome stability of diploid cells. They also provide the clues in the quest for new therapeutic targets to cure human genome instability-related diseases.     

PHOSPHORUS BIOAVAILABILITY MONITORING BY A BIOLUMINESCENT CYANOBACTERIAL SENSOR STRAIN 1

Phosphorus (P) is widely considered to be the main nutrient limiting the productivity of freshwater phytoplankton, but an assessment of its bioavailability in natural samples is highly complex. In an attempt to provide a novel tool for this purpose, the promoter of the alkaline phosphatase gene, phoA, from Synechococcus sp. PCC 7942 was fused to the luxAB luciferase genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus sp. PCC 7942 genome to yield strain APL, which emitted light when inorganic P concentrations fell below 2.3 μM. Light emission of P‐deprived cells decreased rapidly upon inorganic P readdition. The reporter was demonstrated to be a sensitive tool for monitoring the bioavailability of both inorganic and organic P sources. In water samples taken from a natural freshwater environment (Lake Kinneret, Israel), the luminescence measured correlated with total dissolved phosphate concentrations.