Identification of Extracellular Proteases Induced by Nitrogen-Limited Conditions in the Thraustochytrids Schizochytrium aggregatum ATCC 28209.

Thraustochytrids have attracted attention due to the high contents of useful lipids and growth rate. Genus Schizochytrium is commonly used for docosahexaenoic acid (DHA) production, while a strain, which produces a high amount of squalene, has been reported in the genus Aurantiochytrium. These organisms are heterotrophic, and Schizochytrium degrades the extracellular macromolecules, e.g., proteins and polysaccharides, as the nutrients. However, the extracellular lytic enzymes are not well-studied yet. Here, we investigated the induction of extracellular proteases of Schizochytrium aggregatum ATCC 28209. A casein-hydrolytic activity was induced in the nitrogen-limited conditions, and that was also detected by zymography after fractionation by non-heat denatured SDS-PAGE. The proteinous band corresponding to the protease activity was analyzed by MALDI-TOF mass spectrometry after digestion with trypsin. The molecular mass data of the protein fragments were compared to the protein database of S. aggregatum ATCC 28209 in the Joint Genome Institute, and we found that the molecular masses of the six peptides were matched with the prediction from the sequence of a protein, ID 63992, which was annotated as a peptidase S8 family protein. Interestingly, we found that a paralogous protein, ID 99856, was encoded by a gene flanking at the downstream of the gene for ID 63992, and the expression of both genes was similarly induced under the nitrogen-limited conditions. These findings may provide us a key to disclose the induction mechanisms of the extracellular lytic enzymes and the function of the proteolytic enzyme for the nutrition acquisition in thraustochytrids.

Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade.

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.

Complete sequence and structure of the genome of the harmful algal bloom-forming cyanobacterium Planktothrix agardhii NIES-204T and detailed analysis of secondary metabolite gene clusters.

Planktothrix species are distributed worldwide, and these prevalent cyanobacteria occasionally form potentially devastating toxic blooms. Given the ecological and taxonomic importance of Planktothrix agardhii as a bloom species, we set out to determine the complete genome sequence of the type strain Planktothrix agardhii NIES-204. Remarkably, we found that the 5S ribosomal RNA genes are not adjacent to the 16S and 23S ribosomal RNA genes. The genomic structure of P. agardhii NIES-204 is highly similar to that of another P. agardhii strain isolated from a geographically distant site, although they differ distinctly by a large inversion. We identified numerous gene clusters that encode the components of the metabolic pathways that generate secondary metabolites. We found that the aeruginosin biosynthetic gene cluster was more similar to that of another toxic bloom-forming cyanobacterium Microcystis aeruginosa than to that of other strains of Planktothrix, suggesting horizontal gene transfer. Prenyltransferases encoded in the prenylagaramide gene cluster of Planktothrix strains were classified into two phylogenetically distinct types, suggesting a functional difference. In addition to the secondary metabolite gene clusters, we identified genes for inorganic nitrogen and phosphate uptake components and gas vesicles. Our findings contribute to further understanding of the ecologically important genus Planktothrix.

The Trait Repertoire Enabling Cyanobacteria to Bloom Assessed through Comparative Genomic Complexity and Metatranscriptomics.

Water bloom development due to eutrophication constitutes a case of niche specialization among planktonic cyanobacteria, but the genomic repertoire allowing bloom formation in only some species has not been fully characterized. We posited that the habitat relevance of a trait begets its underlying genomic complexity, so that traits within the repertoire would be differentially more complex in species successfully thriving in that habitat than in close species that cannot. To test this for the case of bloom-forming cyanobacteria, we curated 17 potentially relevant query metabolic pathways and five core pathways selected according to existing ecophysiological literature. The available 113 genomes were split into those of blooming (45) or nonblooming (68) strains, and an index of genomic complexity for each strain's version of each pathway was derived. We show that strain versions of all query pathways were significantly more complex in bloomers, with complexity in fact correlating positively with strain blooming incidence in 14 of those pathways. Five core pathways, relevant everywhere, showed no differential complexity or correlations. Gas vesicle, toxin and fatty acid synthesis, amino acid uptake, and C, N, and S acquisition systems were most strikingly relevant in the blooming repertoire. Further, we validated our findings using metagenomic gene expression analyses of blooming and nonblooming cyanobacteria in natural settings, where pathways in the repertoire were differentially overexpressed according to their relative complexity in bloomers, but not in nonbloomers. We expect that this approach may find applications to other habitats and organismal groups.IMPORTANCE We pragmatically delineate the trait repertoire that enables organismal niche specialization. We based our approach on the tenet, derived from evolutionary and complex-system considerations, that genomic units that can significantly contribute to fitness in a certain habitat will be comparatively more complex in organisms specialized to that habitat than their genomic homologs found in organisms from other habitats. We tested this in cyanobacteria forming harmful water blooms, for which decades-long efforts in ecological physiology and genomics exist. Our results essentially confirm that genomics and ecology can be linked through comparative complexity analyses, providing a tool that should be of general applicability for any group of organisms and any habitat, and enabling the posing of grounded hypotheses regarding the ecogenomic basis for diversification.

Complete Genome Sequence of a Coastal Cyanobacterium, Synechococcus sp. Strain NIES-970.

Members of the cyanobacterial genus Synechococcus are abundant in marine environments. To better understand the genomic diversity of marine Synechococcus spp., we determined the complete genome sequence of a coastal cyanobacterium, Synechococcus sp. NIES-970. The genome had a size of 3.1 Mb, consisting of one chromosome and four plasmids.

Rapid ecotoxicological bioassay using delayed fluorescence in the marine cyanobacterium Cyanobium sp. (NIES-981).

The use of delayed fluorescence intensity as an endpoint for rapid estimation of the effective concentration (ECx) has been reported as an alternative to standard growth inhibition (at 72 h after exposure) in some algal species including Pseudokirchneriella subcapitata. In marine algae, although an approach of bioassaying using delayed fluorescence measurements has not been performed yet, its development would provide many benefits for marine environmental risk assessment. In this study, we selected marine cyanobacterium Cyanobium sp. (NIES-981) as our test algal species and demonstrated that this species is valid for the standard growth inhibition test based on criteria provide by Organization for Economic Co-operation and Development guidelines. Furthermore, standard inhibition tests and shorter period test using DF were performed in NIES-981 using five chemicals (3,5-DCP, simazine, diflufenican, K2Cr2O7, and CuSO4), and their EC50 and low-toxic-effect values (EC10, EC5, and NOEC) were determined from two dose-response curves. Based on comparisons of the two dose-response curves and the EC50 values, we conclude that DF intensity is useful as an endpoint for rapid estimation of EC50 in NIES-981.

Complete Genome Sequence of Cyanobium sp. NIES-981, a Marine Strain Potentially Useful for Ecotoxicological Bioassays.

Cyanobium sp. NIES-981 is a marine cyanobacterium isolated from tidal flat sands in Okinawa, Japan. Here, we report the complete 3.0-Mbp genome sequence of NIES-981, which is composed of a single chromosome, and its annotation. This sequence information may provide a basis for developing an ecotoxicological bioassay using this strain.

Complete Genome Sequence of Cyanobacterium Leptolyngbya sp. NIES-3755.

Cyanobacterial genus Leptolyngbya comprises genetically diverse species, but the availability of their complete genome information is limited. Here, we isolated Leptolyngbya sp. strain NIES-3755 from soil at the Toyohashi University of Technology, Japan. We determined the complete genome sequence of the NIES-3755 strain, which is composed of one chromosome and three plasmids.

Complete genome sequence of cyanobacterium Fischerella sp. NIES-3754, providing thermoresistant optogenetic tools.

Cyanobacterial phytochrome-class photosensors are recently emerging optogenetic tools. We isolated Fischerella sp. strain NIES-3754 from hotspring at Suwa-shrine, Suwa, Nagano, Japan. We determined complete genome sequence of the NIES-3754 strain, which is composed of one chromosome and two putative replicons (total 5,826,863bp containing no gaps). We identified photosensor genes of 5 phytochromes and 9 cyanobacteriochromes, which will facilitate optogenetics of thermophile.

Complete genome sequence of cyanobacterium Nostoc sp. NIES-3756, a potentially useful strain for phytochrome-based bioengineering.

To explore the diverse photoreceptors of cyanobacteria, we isolated Nostoc sp. strain NIES-3756 from soil at Mimomi-Park, Chiba, Japan, and determined its complete genome sequence. The Genome consists of one chromosome and two plasmids (total 6,987,571 bp containing no gaps). The NIES-3756 strain carries 7 phytochrome and 12 cyanobacteriochrome genes, which will facilitate the studies of phytochrome-based bioengineering.

Comparison of the terrestrial cyanobacterium Leptolyngbya sp. NIES-2104 and the freshwater Leptolyngbya boryana PCC 6306 genomes.

The cyanobacterial genus Leptolyngbya is widely distributed throughout terrestrial environments and freshwater. Because environmental factors, such as oxygen level, available water content, and light intensity, vary between soil surface and water bodies, terrestrial Leptolyngbya should have genomic differences with freshwater species to adapt to a land habitat. To study the genomic features of Leptolyngbya species, we determined the complete genome sequence of the terrestrial strain Leptolyngbya sp. NIES-2104 and compared it with that of the near-complete sequence of the freshwater Leptolyngbya boryana PCC 6306. The greatest differences between these two strains were the presence or absence of a nitrogen fixation gene cluster for anaerobic nitrogen fixation and several genes for tetrapyrrole synthesis, which can operate under micro-oxic conditions. These differences might reflect differences in oxygen levels where these strains live. Both strains have the genes for trehalose biosynthesis, but only Leptolyngbya sp. NIES-2104 has genetic capacity to produce a mycosporine-like amino acid, mycosporine-glycine. Mycosporine-glycine has an antioxidant action, which may contribute to adaptation to terrestrial conditions. These features of the genomes yielded additional insights into the classification and physiological characteristics of these strains.

Complete Genome Sequence of Microcystis aeruginosa NIES-2549, a Bloom-Forming Cyanobacterium from Lake Kasumigaura, Japan.

Microcystis aeruginosa NIES-2549 is a freshwater bloom-forming cyanobacterium isolated from Lake Kasumigaura, Japan. We report the complete 4.29-Mbp genome sequence of NIES-2549 and its annotation and discuss the genetic diversity of M. aeruginosa strains. This is the third genome sequence of M. aeruginosa isolated from Lake Kasumigaura.

Draft Genome Sequence of the Toxic Bloom-Forming Cyanobacterium Aphanizomenon flos-aquae NIES-81.

Aphanizomenon flos-aquae is a toxic filamentous cyanobacterium that causes water blooms in freshwaters across the globe. We present the draft genome sequence of the A. flos-aquae strain NIES-81, which was determined by 454 pyrosequencing technology. The draft genome is ~5.7 Mb, containing 5,802 predicted protein-coding genes and 58 RNA genes, with a G+C content of 38.5%.

Characterization of the subdomains in the N-terminal region of histidine kinase Hik33 in the cyanobacterium Synechocystis sp. PCC 6803.

Histidine kinase Hik33 responds to a variety of stress conditions and regulates the expression of stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. However, the mechanisms of response and regulation remain unknown. Generally, a histidine kinase perceives a specific signal via its N-terminal region. Hik33 has two transmembrane helices, a periplasmic loop, and HAMP and PAS domains in its N-terminal region, all of which might be involved in signal perception. To investigate the functions of these subdomains in vivo, we expressed a chimeric histidine kinase (Hik33n-SphSc) by fusing the N-terminal region of Hik33 with the C-terminal region of a sensory histidine kinase that is activated under phosphate-deficient conditions, SphS. Hik33n-SphSc responded to several stimuli that are perceived by intact Hik33 and regulated expression of the phoA gene for alkaline phosphatase, which is normally regulated under phosphate-deficient conditions by SphS. We introduced genes for modified versions of Hik33n-SphSc into Synechocystis and monitored expression of phoA under standard and stress conditions. Hik33n-SphSc lacking either the transmembrane helices or both the HAMP and PAS domains had no kinase activity, whereas Hik33n-SphSc lacking the HAMP or the PAS domain enhanced expression of phoA. Moreover, variants of Hik33n-SphSc, in which the membrane-localizing region was replaced by those of other histidine kinases, also responded to stress conditions. Thus, transmembrane helices, regardless of sequence, appear to be essential for the function of Hik33, while the HAMP and PAS domains play important roles in regulating kinase activity in vivo.