Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

Evaluation of four fresh-water unicellular cyanobacteria as potential hosts for mosquitocidal toxins

Summary For biocontrol of mosquitoes, mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus have been cloned into a number of cyanobacteria. However, little is known about the persistence of such recombinant cyanobacteria in mosquito larval habitats. Four fresh water unicellular cyanobacteria, Synechococcus PCC6301, PCC7425, PCC7942 and Synechocystis PCC 6803, were evaluated under laboratory conditions related to mosquito breeding environments. Results indicated that Synechococcus PCC6301 was potentially the most suitable organism for use in the natural mosquito habitat as it could tolerate a wide range of temperatures, salinities, and biological and chemical insecticides. Moreover, strain PCC6301 could be ingested and digested by Culex quinquefasciatus larvae and could support the development of larvae to full insect maturity.     

General distribution of the nitrogen control gene ntcA in cyanobacteria.

The ntcA gene from Synechococcus sp. strain PCC 7942 encodes a regulatory protein which is required for the expression of all of the genes known to be subject to repression by ammonium in that cyanobacterium. Homologs to ntcA have now been cloned by hybridization from the cyanobacteria Synechocystis sp. strain PCC 6803 and Anabaena sp. strain PCC 7120. Sequence analysis has shown that these ntcA genes would encode polypeptides strongly similar (77 to 79% identity) to the Synechococcus NtcA protein. Sequences hybridizing to ntcA have been detected in the genomes of nine other cyanobacteria that were tested, including strains of the genera Anabaena, Calothrix, Fischerella, Nostoc, Pseudoanabaena, Synechococcus, and Synechocystis.     

Glucosylglycerate: a secondary compatible solute common to marine cyanobacteria from nitrogen-poor environments

The synthesis and accumulation of compatible solutes represent an essential part of the salt acclimation strategy of microorganisms. Glucosylglycerol is considered to be the typical compatible solute among marine cyanobacteria. However, genes that encode enzymes for the synthesis of glucosylglycerol were not detected in the genome sequences of marine picoplanktonic Prochlorococcus strains. Instead, we noticed the presence of genes that putatively encode for glucosylglycerate (GGA) synthesis among Prochlorococcus and most other closely related marine picocyanobacteria. Recombinant proteins from Prochlorococcus marinus SS120 and Synechococcus sp. PCC 7002 exhibited glucosyl-phosphoglycerate synthase (GpgS) activity, and GpgS is a key enzyme of GGA synthesis. GGA accumulation was found to be salt- as well as nitrogen-regulated in the coastal strain Synechococcus sp. PCC 7002. Moreover, GGA was also detected in all picoplanktonic Prochlorococcus and Synechococcus strains harbouring gpgS genes, especially under N-limiting conditions. These results suggest that marine picocyanobacteria acquired the capacity to synthesize the negatively charged compound GGA during their evolution. Our results establish GGA as the fifth most widespread compatible solute among cyanobacteria. Additionally, GGA appears to replace glutamate as an anion to counter monovalent cations in marine picocyanobacteria from N-poor environments.     

Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity.

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.     

Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity.

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.     

Algae in mosquito breeding sites and the effectiveness of the mosquito larvicide Bacillus thuringiensis H-14

The presence of cyanobacteria generally decreased the effectiveness of Bacillus thuringiensis H-14 (BTI) as a mosquito larvicide. The effect was more pronounced when the mosquito larvae were exposed to BTI in the presence of several cyanobacterial strains. No synergistic or antagonistic effect between the -endotoxin from BTI and the hepatotoxin from cyanobacteria was seen. Neurotoxic cyanobacterial strains caused very fast paralysis in mosquito larvae; the decreases in the effectiveness of BTI when tested in combination with a neurotoxic strain might be due to the effect of this paralytic action on the feeding rate of the mosquito larvae.     

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.     

Amino acid transport in taxonomically diverse cyanobacteria and identification of two genes encoding elements of a neutral amino acid permease putatively involved in recapture of leaked hydrophobic amino acids.

The activities of uptake of thirteen 14C-labeled amino acids were determined in nine cyanobacteria, including the unicellular strains Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803; the filamentous strain Pseudanabaena sp. strain PCC 6903, and the filamentous, heterocyst-forming strains Anabaena sp. strains PCC 7120 and PCC 7937; Nostoc sp. strains PCC 7413 and PCC 7107; Calothrix sp. strain PCC 7601 (which is a mutant unable to develop heterocysts); and Fischerella muscicola UTEX 1829. Amino acid transport mutants, selected as mutants resistant to some amino acid analogs, were isolated from the Anabaena, Nostoc, Calothrix, and Pseudanabaena strains. All of the tested cyanobacteria bear at least a neutral amino acid transport system, and some strains also bear transport systems specific for basic or acidic amino acids. Two genes, natA and natB, encoding elements (conserved component, NatA, and periplasmic binding protein, NatB) of an ABC-type permease for neutral amino acids were identified by insertional mutagenesis of strain PCC 6803 open reading frames from the recently published genomic DNA sequence of this cyanobacterium. DNA sequences homologous to natA and natB from strain PCC 6803 were detected by hybridization in eight cyanobacterial strains tested. Mutants unable to transport neutral amino acids, including natA and natB insertional mutants, accumulated in the extracellular medium a set of amino acids that always included Ala, Val, Phe, Ile, and Leu. A general role for a cyanobacterial neutral amino acid permease in recapture of hydrophobic amino acids leaked from the cells is suggested.     

Calcium carbonate formation by Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807

Precipitation of CaCO3 catalyzed by the growth and physiology of cyanobacteria in the genus Synechococcus represents a potential mechanism for sequestration of atmospheric CO2 produced during the burning of coal for power generation. Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807 were tested in microcosm experiments for their ability to calcify when exposed to a fixed calcium concentration of 3.4 mM and dissolved inorganic carbon concentrations of 0.5, 1.25 and 2.5 mM. Synechococcus sp. strain PCC 8806 removed calcium continuously over the duration of the experiment producing approximately 18.6 mg of solid phase calcium. Calcium removal occurred over a two-day time period when Synechococcus sp. strain PCC 8807 was tested and only 8.9 mg of solid phase calcium was produced. Creation of an alkaline growth environment catalyzed by the physiology of the cyanobacteria appeared to be the primary factor responsible for CaCO3 precipitation in these experiments.     

Ploidy in cyanobacteria

A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. Synechococcus PCC 7942 and Synechococcus WH7803 were found to contain 3-4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, Synechocystis PCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the 'Kazusa' strain more than 20 years ago. The results reveal that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.     

Nonribosomal peptide synthetase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC

Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.     

Cyanophycin production in a phycoerythrin-containing marine synechococcus strain of unusual phylogenetic affinity

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.     

Analysis of the hli gene family in marine and freshwater cyanobacteria

Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 micromol photon m(-2) s(-1) and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean (Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light (Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. [Nostoc] strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.     

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/).